Abstract and Introduction

Abstract
Objective: Sacrectomy creates a large, complex tissue defect that presents a reconstructive challenge for plastic surgeons. Several myocutaneous flaps have been described for reconstruction following sacral tumor extirpation; however, current publications focus on the reconstructive options applicable to adults. We present a method of reconstruction following sacral tumor extirpation in a pediatric patient. Methods: The patient was 22 months old and in need of complex closure following low sacral amputation (S3-S4 osteotomy) and en bloc resection of a yolk sac tumor. Following tumor extirpation, the patient was left with a complex defect including extensive dead space, multiple exposed nerve roots, projection of the rectum into the wound, and inadequate soft tissue for primary closure. Results: Reconstruction with human acellular dermal matrix to address the risk of posterior rectal herniation and bilateral gluteal V to Y advancement flaps for obliteration of the dead space allowed for durable closure of the surgical defect. Conclusions: This represents the first case report documenting sacral resection and reconstruction with bilateral V to Y gluteal advancement flaps in a pediatric patient.

Introduction
Reconstruction of the soft-tissue defects resultant from total or partial sacrectomy is described in the adult population; however, discussion of the reconstructive options available to the pediatric population does not exist. Only 1 pediatric case of reconstruction with a myocutaneous flap closure has been reported through 2013.[1] The 2 most commonly documented options for myocutaneous flap reconstruction of large sacral wound defects include vertical rectus abdominis myocutaneous flaps and bilateral gluteal advancement flaps. Here, we present a pediatric patient with low sacral amputation (S3-S4 osteotomy) and en bloc resection of tumor followed by reconstruction with human acellular dermal matrix (HADM; AlloDerm, LifeCell Corporation, Branchburg, New Jersey) and bilateral V to Y gluteal advancement flaps.

Section 1 of 4

Next: Methods/Case Presentation

Methods/Case Presentation
An Asian female, age 17 months, was diagnosed with a yolk sac tumor after biopsy of a mass emanating from the sacrum and extending into the buttocks. The patient had a normal birth history, with appropriate achievement of developmental milestones including walking without assistance. She did not appear to have pain related to the mass. Determination of bowel or bladder effects was complicated by her age. She underwent bleomycin chemotherapy and had a good response with a reduction in the size of the tumor. It was felt that she could benefit from surgical resection. Magnetic resonance imaging and computed tomographic scans demonstrated that the tumor was located in the presacral region extending up to the S3 nerve roots and involving the distal sacrum and coccyx. At 22 months of age, the Neurosurgery and Orthopedic Surgery Oncology teams performed low sacral amputation and en bloc resection of the tumor through a posterior approach (Fig 1). The resultant defect was complex: extensive dead space was formed by bony resection, gluteal and other muscles were lateralized by mass effect, multiple exposed nerve roots filled the field, the rectum projected into the wound causing concern for hernia or pseudohernia, and overlying subcutaneous and skin was inadequate (Fig 2).

(Enlarge Image)

Figure 1. Yolk sac tumor with attached sacral bone measuring 6 cm (length) 4.5 cm (width) 4.3 cm (depth).

(Enlarge Image)

Figure 2. Intraoperative photographs. Patient is positioned prone with head oriented to the right. Hash marks on right buttock indicate area that was de-epithelialized and inset into wound (left). Tumor resection resulted in a complex defect with extensive dead space and projection of the rectum into the wound (right).

Results
The posterior rectal herniation was addressed by suturing a 4 cm by 3 cm piece of HADM circumferentially to the deep fascia and muscle edges surrounding the rectum. Bilateral gluteal V to Y advancement flaps were then designed on each buttock. The flap on the right buttock was partially deepithelialized on the medial aspect, dissected near the gluteal perforators, advanced, and inset deep into the wound to obliterate the dead space. The left buttock flap was then advanced to meet the right flap in a pants-over-vest fashion. The V advancements flaps were closed in a Y fashion (Fig 3). The cumulative surface area of the 2 flaps was 19 cm across by 8 cm in height, representing a significant size in this 11-kg pediatric patient. At the 4 week follow-up, the patient demonstrated durable wound reconstruction without signs of wound infection or dehiscence requiring reoperation (Fig 4).

(Enlarge Image)

Figure 3. Intraoperative photographs. Patient is positioned prone with head oriented to the right. 4 3 cm2piece of HADM was sutured circumferentially to the deep fascia and muscle edges to address posterior rectal herniation (left). Closure of the bilateral gluteal flaps in a pants-over-

vest fashion with V advancement flaps closed in a Y fashion (right). HADM indicates human acellular dermal matrix.

(Enlarge Image)

Figure 4. Postoperative photograph after complex wound closure of partial sacrectomy defect at 4 weeks.

Discussion
Resection of sacral tumors by a posterior approach results in large defects that are difficult to reconstruct and present several potential complications including wound dehiscence, impaired motor function, and parasacral herniation. In the adult population, the documented surface area of reconstruction has ranged from 189.8 cm2 to 245.7 cm2; our patient had a reconstructed surface area of 152 cm2.[2,3] The defect size relative to patient size created a unique challenge in selecting the appropriate means of closure. In comparison to primary closure, reconstruction with a myocutaneous flap results in a statistically significant reduction in wound infections and dehiscence requiring reoperation.[4,5] Several techniques for reconstruction with autologous tissue transfer have been reported including ventral rectus abdominis myocutaneous (VRAM) flaps, omental flaps, gluteal flaps, gluteal-thigh flaps, and free flaps.[14,6,7] Because our patient required a low sacral amputation, the entire resection was performed by a posterior approach. Therefore, the option of mobilizing a VRAM flap was not considered advantageous because it would result in donor site morbidity in an otherwise nonoperated field. Furthermore, Garvey et al[6]argue against utilizing a VRAM flap in partial sacrectomies below the S2-S3 level because of the risk of pedicle compression. The bilateral V to Y gluteal advancement flaps have the advantage of large tissue volume, proximity to the defect, and a blood supply that remains intact through the posterior approach.[1,7,8] With respect to the unique complications of reconstruction following partial sacrectomy, there is no statistically significant relationship between flap choice and postoperative

complications.[6] Patient's functional outcome and ambulatory status is primarily dependent on the type of surgical resection rather than on the means of reconstruction.[7,9] Although the gluteus maximus is the primary extensor of the hip, hypertrophy of the semitendinosus, semimembranosus, biceps femoris, and adductor magnus compensate in active extension of the hip for activities such as ambulation, climbing stairs, and standing from a seated position.[10] Finally, inadequate reconstruction following sacral resection may result in parasacral herniation of the rectum.[11] The sacral defect, soft-tissue dead space, and sacral nerve root resection resulting in pelvic floor atrophy contribute to this problem.[12,13] For this reason, we created a pelvic diaphragm using HADM, which is effective in preventing parasacral herniation.[1] Surgical management of complex sacral wounds in the pediatric patient population following tumor extirpation lacks documented literature. The 22-month-old female underwent resection and partial sacrectomy resulting in a significant defect requiring posterior rectal hernia repair with AlloDerm and complex wound closure with bilateral gluteal V to Y tissue rearrangement. Therefore, this represents the first case report documenting this method of complex sacral reconstruction, with HADM and bilateral V to Y advancement flaps, performed in a pediatric patient.

Previous Page
Section 4 of 4

Abstract
Pediatric severe ulcerative colitis that is resistant to current medical treatment can successfully be managed surgically with a colectomy, ileal pouch creation and pouchanal anastomosis. Key issues that should be considered and discussed before the pouch option can be offered include alternative surgical procedures, pouch function expectations, risk of surgical leak, pelvic sepsis, anastomotic strictures, acute and chronic pouch inflammation, Crohn's disease

of the pouch and risk of reduced fertility for females. A long-term risk is malignancy of the residual colonic tissue. The decision to proceed with a pouch or not poses a substantial emotional burden to the child and family. Despite the risk of surgical complications and pouch inflammatory and functional challenges, the overwhelming majority of children and their families are satisfied with their pouch surgery outcomes. Further study is needed to assess preoperative risk predictors, prevention and treatment of complications.

Background
Inflammatory bowel disease (IBD) has increased in incidence over the past few decades,[1] similar to numerous allergic and autoimmune conditions. One of every four patients presents in childhood.[2] The disease can significantly impact patients' quality of life, not only through symptoms, but also through growth restriction, puberty delay, impact on social and emotional development and compromised school attendance at the critical age period of 1020 years, where these milestones take place. In addition, 10 years after diagnosis, owing to persistent inflammation, the risk of colon cancer increases by 0.51% annually.[3] About half of the children with IBD have ulcerative colitis (UC) involving the mucosal layer of the colon. For these children, anti-inflammatory agents targeting the diseased mucosa are used. The goals of UC therapy are to control symptoms, restore growth and development and prevent complications, such as colon cancer. More recently, the use of mucosal healing has gained interest as an objective measure of therapy success and its ability to predict a better disease course.[4] Available medical treatments have been successful in meeting the therapy goals for most, but not all, patients. Colectomy may be necessary for intractable colitis (despite therapy with mesalamine, steroids, azathioprine, calcineurin inhibitors and anti-TNF agents), steroid dependent colitis or when significant dysplasia is present. Colectomy occurs in the setting of a hospitalized child with acute severe colitis, or in the outpatient setting. Eight out of 100 children with UC require surgery within a year of diagnosis,[5] and up to 26% within 5 years.[6] Before the use of

anti-TNF- therapy, more than 60% of children with acute severe colitis needed a colectomy within 1 year.[7] The overwhelming majority of these children go on to receive a pelvic pouch comprised of the small intestine. Despite the fact that most published data apply to adults with pouches, this review attempts to highlight some pertinent issues in the care of children who receive a pouch as part of their surgical management for UC, including the decision on and impact of the type of surgery, functional challenges, expectations and short- and long-term complications.

Anticipated Changes After Colectomy

The main colonic functions are absorption, storage, evacuation and maintaining homeostasis with its massive microbiome. Water, sodium, chloride, calcium, oxalate and vitamin K are absorbed in the colon. A key challenge after colectomy is dehydration and electrolyte loss. However, by the time bowel continuity is restored, commonly 3 months after pouch creation, the small bowel will usually have compensated for such losses. Salvaged carbohydrates fermented by the colonic microbiota to short chain fatty acids (SCFA) are reabsorbed through the colon. While healthy, the SCFA caloric contribution to the body is negligible. However, SCFA may modulate mucosal inflammation, lending basis to their use in pouchitis management. The ileocecal valve prevents reflux of the colonic contents into the ileum and regulates colonic filling. The implication of the absence of such a barrier in a pouch is unclear. Although the rectum has the largest capacity within the colon, in healthy individuals it is usually empty until shortly before defecation. Stools can be stored in any part of the colon, including the ascending colon.[8] The smaller volume that a pouch offers has obvious restrictions, leading to the frequent bowel movements a child is expected to have, even with a healthy pouch. Intermittent colonic contraction waves further prepulse the colonic contents distally. When the rectum is filled and its mechanoreceptors are stimulated, a reflex arc through the sacral spinal nerves mediates the defecation sequence that includes urge sensation, internal anal sphincter relaxation (through the rectoanal inhibitory reflex) and, when appropriate, a voluntary relaxation of the external anal sphincter. The angle between the rectum and anal canal is reshaped by posture (sitting or squatting) and pelvic floor muscle relaxation, allowing less resistance to outflow. The increased intrapelvic pressure (Valsalva or straining) and colonic peristalsis result in stool and air evacuation. Since the

pouch innervation and anatomy within the pelvis are different from the native colon, this may well affect the evacuation function for many patients.

Previous Page
Section 3 of 10

Next: Colectomy in Pediatric Ulcerative Colitis

Colectomy in Pediatric Ulcerative Colitis

The most common indications for colectomy in childhood are UC and familial adenomatous polyposis (FAP). Since the majority of children with UC have pancolitis or left-sided colitis that can extend to pancolitis, partial colectomy has little if any role in surgical therapy for UC. The ideal surgery for UC would remove all colonic mucosa in order to eliminate inflammation and its malignancy risk, minimize pelvis dissection and manipulation of pelvic organs, maintain bowel continuity with the anal canal, leave the sensorimotor apparatus of the bowel and anal sphincters intact and preserve the child's body image.

Surgical Considerations: Should All Children Receive a Pouch After Colectomy?

The surgical options after colectomy are permanent ileostomy or restoring bowel continuity with an ileoanal (straight) anastomosis (IAA) or an ileal pouchanal anastomosis (IPAA), also termed restorative proctocolectomy. It is important that the child and family spend sufficient time with the surgery team, including a stoma nurse, to understand the surgical options and postsurgical expectations. Ample information resources should be provided as early in the process as possible. A comprehensive patient and family guide is available through the United Ostomy Associations of America.[101] In addition, we have found that having the patient and family communicate directly with patients and families who have had the surgery can be extremely valuable. The decision to proceed with a colectomy in the first place is understandably quite difficult, more so in the patient who is not critically ill or hospitalized. The decision of which surgery to choose is a different challenge, as it commits the child/adolescent and parent to a new lifelong reality, either with bowel continuity (and its challenges) or permanent ileostomy (and its change in body image and social implications). It is particularly important to involve the

adolescent patient in this decision. Anecdotally, we have noted that generally the patient's and parent's decisions are in line. However, more knowledge remains to be gleaned about the dynamics of this decision-making process on the parent, child and adolescent's sides.

Permanent Ileostomy
The current surgical techniques, including stoma protective devices and dedicated 'stoma teams' at larger centers, make a permanent ileostomy a feasible and acceptable option for some children. The small intestine generally shows enough adaptation to compensate for fluid and mineral losses after a colectomy. Nevertheless, maintaining adequate hydration and mineral intake remains essential, especially for active children. The stoma output (volume, consistency) tends to be sensitive to components of the oral intake. Complications include skin irritation, obstruction, volvulus, dehiscence and associated nephrolithiasis.[9] A permanent ileostomy may offer some advantages over restoring continuity; possible fertility preservation, avoiding pouchitis (see below) and, surprisingly, few restrictions on activity. One of our patients, who is quite involved in outdoors activities, found the permanent ileostomy option more convenient than a pouch after weighing the expected stool frequency and the risk of pouchitis (see below) if he happened to be on a long hike. How does ileostomy compare with IPAA when it comes to quality of life? Although body image perception is thought to be poorer in adults with ileostomy, the quality of life is quite similar to IPAA patients.[10] The overwhelming majority of children and adults with UC opt to be in bowel continuity. Perhaps the main reasons for this choice are restoring a 'healthy' body image without the stoma and the desire to defecate normally similar to peers. Additionally, there is the burden of stoma care to children and their caregivers. Patients may generally underestimate the potential complications of surgery to restore bowel continuity. Two surgical options have been most commonly used: IAA or IPAA, commonly termed J-pouch, named for its shape (Figure 1). Other technically different pouches have been used, including the S and W pouches.

(Enlarge Image)

Figure 1. The J-pouch. Reproduced with permission from [68].

leoanal Anastomosis
Given the liquid stool consistency in the small bowel and the lack of a reservoir capacity after colectomy, a simple ileal anastomosis to the anal verge is expected to leave a patient with excess stool frequency and seepage. Seetharamaiah et al. published a large retrospective study assessing outcomes for 112 IAA and 91 IPAA in children.[11] Surgery indications were UC in 168 and FAP in 35 patients. Daytime and night-time stool frequencies were significantly higher with IAA patients 124 months after surgery. By 24 months, the mean number of daytime stools was 8.4 3.9 for IAA and 6.2 2.8 for IPAA. A similar trend was noted in a meta-analysis of pediatric studies.[12] Interestingly, by 24 months, 92% of IAA patients and 95% of IPAA patients were fully continent during day and nighttime, with the differences not being statistically significant.[11]

Ileal PouchAnal Anastomosis

The IPAA procedure has been performed for over 40 years.[13]Its original goal was to create a reservoir of two connected open ileal segments. At the time of colectomy, in a two-step approach, most surgeons opt to fashion a pouch with pouchanal anastomosis along with a diversion ileostomy, followed by an ileumpouch anastomosis a few months later. A pouchogram is done before the ileostomy reversal to screen for any leaks. At the time of ileostomy closure, the pouchanal anastomosis is evaluated for any evidence of stricture. Significant strictures may be due to ischemia or leakage at the anastomotic site. They may occur early, but typically do not present until 69 months following the operation. When present, they may be dealt with by using one of several different surgical techniques, such as simple dilation, a YV

stricturoplasty or diamond advancement flap. If the stricture is encountered prior to ileostomy closure, the closure may be delayed while the anoplasty heals. For malnourished or steroid-dependent children, or patients who receive infliximab within 8 weeks,[14] the following three-step approach is usually considered: colectomyileostomy, pouch creation with anal anastomosis and ileostomy closure and ileumpouch anastomosis. The first step leaves an ileostomy and a rectal residual (Hartman's) pouch. Within the residual pouch, the diseased mucosa may continue to be the source of blood and mucous despite the diversion ileostomy. Most surgeons proceed to the second step within a few months after the first. Overall, one-step procedures without a temporary 'protective' ileostomy pose higher risks of anastomotic leaks and pelvic sepsis.[15] In select pediatric patients, a one-step procedure may be safely completed, reducing the number of operations, operative time and incidence of anastomotic strictures.[16,17] Laparoscopic and laparoscopicassisted surgery may also be completed successfully.[1820] It is important to keep in mind that the connection of the ileal pouch to the anal canal is not technically feasible without some residual rectal 'cuff'; a small segment of the rectum (at least 0.5 cm), which must be maintained to preserve nervous enervation and sphincter continence (Figure 1). Less of this cuff is left behind with a handsewn anastomosis, while at least 12 cm of cuff is left behind after a double-stapled technique. While complications of the handsewn technique are higher in regard to anal sphincter function, the double-stapled technique is more prone to the development of symptomatic inflammation (known as cuffitis; Figure 2) or dysplasia and neoplasia. Because laparoscopic techniques may be challenging in adequately dividing the rectum near the dentate line because of the instrumentation available, an even longer rectal cuff may be left behind.[21,22] The residual rectal mucosa warrants future endoscopic monitoring of the pouch. Surgical outcomes are also related to the surgeon's experience, the number of pouches done and the technique (handsewn vs stapled pouchanal anastomosis).[23] Our own technique removes 5 cm of rectal mucosa while the patient is in a prone jack-knife position. This allows excellent visualization of the rectum. The patient is then

repositioned into lithotomy and the colectomy and partial proctectomy are completed laparoscopically with the J-pouch reconstruction done via a small pfannenstiel incision.

(Enlarge Image)

Figure 2. Cuff inflammation.

Children with IPAA (mean age: 12 years), who were followed for a mean of 3.7 years after surgery, were noted to have a similar quality of life, physical function and self-esteem compared with healthy children.[24] In another study, 26 out of 27 children with median age of 15 years described their health after IPAA as completely or fairly good.[25] The parents of younger children who received a pouch at a median age of 6.8 years, and were followed for a mean of 9 years, reported excellent outcomes.[26]

After the Colectomy, Before the Pouch: Deciding Whether to Continue on or Not
Naturally, before IPAA surgery is determined to be the desired course of action, realistic expectations, potential complications and risk to the patient must be considered. Of all complications, the patient's (and providers') perhaps most-feared complication is pouch failure and the need to revert to a permanent ileostomy, with or without pouch removal. Pouch failure occurs in 57% of adult patients within 10 years of its construction.[27,28] Predictors include early pelvic sepsis and need for transanal drainage.[29] Crohn's disease, highly associated with pouch complications, including failure,[30] can manifest with inflammation of the pouch, the afferent small bowel limb and other small bowel locations or fistula formation. Patients with known Crohn's disease are not commonly offered IPAA, although select patients with Crohn's colitis, no

extensive small bowel disease and no perianal disease can be considered.[31] For some patients with severe or intractable colitis, clarifying whether they have UC or Crohn's disease can be challenging.[32] When the type of IBD is not clear in adults, known as IBD Unclassified (IBDU), the outcomes do not appear to be different after IPAA than those in UC.[33] A change of diagnosis from IBDU or UC to Crohn's disease can occur after colectomy, based on a gross and microscopic exam of the resected colon and ileum. Should this change in diagnosis mean no IPAA? A prospective study of 153 adults with UC or IBDU did not show that any single or combination UCatypical histologic findings after colectomy (including giant cells, transmural inflammation and patchy disease) predicted pouch complications or Crohn's disease of the pouch.[34] On the other hand, a family history of Crohn's disease and anti-Saccharomyces cerevisiae IgA antibodies are independently associated with a post-IPAA diagnosis of Crohn's disease in adults with UC or IBDU.[35] Coukos et al. explored whether serology tests obtained after ileostomy closure and ileal anastomosis to IPAA are associated with pouch complications and Crohn's of the pouch in 142 adults, with a median follow-up of 86 months.[36] Complications occurred in 31% of all patients. Positive anti-S. cerevisiae IgG or anti-CBir1 markers were associated with a higher risk for developing any post-IPAA complication, particularly fistulae and Crohn's of the pouch. Since such risk factors raise the risk of pouch complications, they should be discussed with patients before proceeding. Further study is needed to define the ideal time for assessing serologic markers and their utility in decision making preoperatively.

Postoperative Complications
More than 50% of all patients develop at least one complication after pouch surgery, the most common of which is pouchitis.[3739] Approximately one out of ten children develop a stricture, leak or fistula. Certainly, multiple preoperative risk factors, including malnutrition, opportunistic infections and exposure to steroid, tacrolimus, cyclosporin and anti-TNF- agents, exist for an ill patient with UC. Prolonged preoperative steroid therapy may relate to poor

postoperative catch-up growth.[40] Infliximab increases post-IPAA complications in children, including infections and small bowel obstruction.[14] Calcineurin inhibitor use does not seem to increase early postoperative complications.[41] Ileus can be prolonged in patients with a protracted preoperative course or prolonged narcotic use. Anastomotic leaks can appear at multiple sites (Figure 3): peristomal, Hartman's pouch, proximal ileal pouch end, IPAA or ileoileal anastomosis site after ileostomy take-down. Leakage may result in abscess formation and lead to fistula development. Small bowel obstruction can similarly appear at any of the anastomotic sites or ileostomy. Portal vein thrombosis is a rare complication that has been reported in children after colectomy and IPAA.[42] Patients presenting with abdominal pain, fever and leukocytosis should be considered for this diagnosis, which may also be the presentation of a surgical leak or pouchitis. Adhesions can predispose patients to such complications as internal herniation and bowel strangulation.[14,40,41] Fistulas can develop and extend from the pouch to nearby structures. The presence of a fistula heralds pouch failure in 29% of adult patients.[43] While earlier-appearing fistulae may be related to the surgery itself, those appearing from 6 months to a year later may be caused by Crohn's of the pouch.

(Enlarge Image)

Figure 3. Pouch leaks. Pouch leaks at the tip of 'J' (A2), pouch body (A3) and pouchanal anastomosis, leading to pelvic sepsis (A4), pouchvaginal fistula (A5) and pre-sacral fluid collection (A1). Endoscopic view of a leak at the tip of 'J' (B).Reproduced with permission from [66].

In a small pediatric case series, a manometry study of the neorecto-anal function revealed similar maximum neorectal tolerated threshold volume and

preserved neorectoanal inhibitory reflex between children with IPAA with or without incontinence compared with healthy controls.[44] However, patients with IPAA and incontinence showed significantly lower maximum anal sphincter pressure at rest and during voluntary contraction, in addition to higher sensory threshold. Both findings suggest sphincter and sensory dysfunction as the causes of incontinence. In adult patients who receive an IPAA, there is evidence of increased infertility of up to 48 versus 15% for medically treated UC patients;[45] it is unclear how this risk is modified in children; further study is needed. Adenocarcinoma within the IPAA has been reported in adults with FAP and UC.[46] Presence of precolectomy neoplasia, which is quite uncommon in children with UC due to short duration of disease before surgery, is a risk factor for pouch neoplastic changes.[47] A pediatric retrospective review by Sarigol et al. found similar histologic findings in pediatric and adult pouches, with no dysplasia 5 years on from pouch creation in their pediatric patients.[48] The authors suggest screening pouchoscopy 5 years after pouch surgery, or 7 years from UC diagnosis, followed by more frequent pouchoscopy, should severe inflammation be present. Biopsies from the residual cuff area should be carefully obtained.

Pouchitis
Pouchitis is the most common complication of IPAA. Chronic pouchitis and Crohn's of the pouch are the two most common causes of pouch failure and removal. The term 'pouchitis' denotes symptoms resulting from pouch inflammation that can be demonstrated endoscopically and histologically. A pouch disease activity index, based on symptoms, endoscopic and histologic criteria, is commonly used to assess pouchitis.[49] Pouchitis symptoms can be quite debilitating, including daytime or nocturnal diarrhea, urgency, tenesmus, incontinence, pain and bleeding. When these symptoms are present without inflammation, a diagnosis of irritable pouch syndrome is suspected.[50] On endoscopic exam (known as a pouchoscopy), pouchitis appearance is

nonspecific and spans findings from edema to ulceration (Figure 4). Microscopically, the hallmark of pouchitis is the presence of polymorphonuclear cells, crypt abscesses and ulceration. Calprotectin has been shown to correlate with pouch inflammation in children.[51] The pediatric literature on pouchitis is quite meager and, therefore, much of what we know is from adult data or anecdotal experience. What is unique about pouchitis is that it does not occur commonly in patients who receive a pouch for familial polyposis.[11,52] This supports the idea that pouchitis is the end point of pouch flora interaction with the intestinal cells in a predisposed patient. Generally, pouchitis occurs within 6 months after ileostomy take-down, although it can also occur prior to the ostomy being closed. Risk factors for pouchitis include NOD2/CARD15 mutations, extensive UC, backwash ileitis, positive perinuclear antineutrophil cytoplasmic antibodies, primary sclerosing cholangitis, preoperative corticosteroid or calcineurin inhibitor exposure, preoperative thrombocytosis and use of NSAIDs.[41,50] Preoperative infliximab does not seem to increase the likelihood of chronic pouchitis.[14] There are different types of pouchitis: acute versus chronic (>4 weeks); infrequent (one to two acute episodes) versus relapsing (three acute episodes) versus continuous; and treatment-responsive versus treatment-refractory.[53]

(Enlarge Image)

Figure 4. Pouchitis.

The bacterial flora within the ileal pouch may differ between patients with UC versus FAP.[54] A role for pouch flora in pouchitis is also suggested by the successful use of antibiotics as the first-line of therapy. Limited placebocontrolled trials using antibiotics for pouchitis have been conducted in adults; none, however, have been completed in children. Generally, treating pouchitis

in children follows the practice for adults. Both metronidazole and ciprofloxacin have been shown to be effective for pouchitis.[50,55,56]Liquid metronidazole has poor palatability, which presents a challenge for its use with children. Although liquid ciprofloxacin is available, the warning for Achilles tendonitis and tendon rupture raises concerns about its use, especially with concomitant steroids. For patients who do not respond to monotherapy with metronidazole or ciprofloxacin, an alternative regimen combining antibiotics, topical antibiotics (enema form) and a sequential antibiotic regimen has been used.[8] The probiotic VSL#3 seems to be more effective compared with placebo for maintaining remission of chronic pouchitis in adults who achieved remission with antibiotics. However, it is unclear whether VSL#3 is useful in preventing pouchitis.[57] Fewer than one out of three patients with chronic pouchitis have an identifiable cause (Box 1 ). A unique pouchitis entity has been described comprising of: antibiotic-resistant pouchitis, positive autoimmune markers, association with immune-mediated disease and increased apoptotic cells on pouch histology.[58] When antibiotics are not helpful, anti-inflammatory agents can be used. In adults, mesalamines and steroids, both in topical and oral forms, have been used as second-line options for treating pouchitis.[8] Immunosuppressive and biological therapies have also been used (see Crohn's of the pouch). Chronic treatmentunresponsive pouchitis can severely compromise quality of life and remains one of the most common indications for pouch excision.

Crohn's After Pouch Creation

Between six and 13% of children with UC will have a diagnosis change to Crohn's disease after colectomy.[38,59] A Crohn's disease diagnosis can occur at the following different stages: when patients with known Crohn's disease undergo the surgery for severe Crohn's colitis; after colectomy, based on surgical specimen exam; and after pouch surgery (known as de novo Crohn's). Crohn's disease can appear within the pouch (CDP) and/or in any part of the gastrointestinal tract or perianal area. CDP was seen in 1315% of

children.[59,60] A comprehensive clinical review on CDP has been authored by Shen.[61] Crohn's disease behavior can be inflammatory, causing ulcerations within the pouch or proximal small bowel (Figure 5). This should not be confused with NSAID-induced ulcers or backwash ileitis.[27,62] Interestingly, ongoing primary sclerosing cholangitis is associated with prepouch significant ileitis.[63] CDP induces symptoms that are similar to chronic pouchitis. Fistulizing Crohn's disease can extend into the perianal area and nearby structures, including the vagina. This can be confused with surgery-related fistulae that may appear within a few months of surgery. Fibrostenosing CDP can compromise the pouch structure itself and evacuation dynamics. It is important to distinguish fibrostenosing CDP from a surgery-associated stricture, although in reality this may be difficult. De novo Crohn's of the pouch, which can occur weeks to years after surgery, carries a poorer prognosis, with higher pouch failure compared with early-appearing CDP.[62] CDP should be suspected if a patient develops de novo fistula more than 612 months after ileostomy take-down in the absence of postoperative leak, abscess and sepsis. The presence of histologic evidence of granulomas or pyloric gland metaplasia would also suggest a diagnosis of Crohn's disease. However, caution is suggested against obtaining tissue samples from the pouch suture line, since these may show foreign-body granulomas. Although immune modulators and anti-TNF agents have been used for CDP, the risk of needing surgery for pouch failure is high.[64,65]

(Enlarge Image)

Figure 5. Crohn's of the pouch.

Expert Commentary

In centers with ample expertise, children with medically intractable colitis are successfully managed with colectomy and IPAA. While similar complications appear in both adults and children, little is known about specific risks in children. There is a strong need for better predictors for pouch outcomes and complications that can further shape the discussion about alternative surgical options, such as permanent ileostomy.

Five-year View
More knowledge will be available on the role of the intestinal flora in pouch complications. Better predictive tools will be available to assess preoperative risk of pouchitis and Crohn's of the pouch.

Parotid Small Cell Carcinoma Presenting With Long-term Survival After Surgery Alone
Abstract and Introduction
Abstract
Introduction Primary involvement of the salivary glands in small cell carcinoma is rare, and has one of the worst prognoses of salivary gland neoplasms. However, it has been reported that some cases have a favorable outcome, although the prognostic factors are still under consideration. Multidisciplinary therapy was usually required to achieve long-term survival. Recently, a resemblance of some small cell carcinomas of the salivary gland to cutaneous Merkel cell carcinoma was suggested; the latter have the potential for spontaneous regression, which is related to a favorable clinical outcome. Case presentation We present a locoregional advanced parotid small cell carcinoma with multiple lymph node metastases in an 87-year-old Asian woman. The tumor was controlled by surgery alone, and nine-year diseasefree survival was achieved without any adjunctive therapy. To the best of our

knowledge, this is the longest reported follow-up of head and neck small cell carcinoma. Conclusion We believe this to be the first case of small cell carcinoma with involvement of the salivary glands reported in the literature with a good outcome after surgery alone without any adjunctive therapy.

Introduction
Primary involvement of the salivary glands in small cell carcinoma (SmCC) is rare, and tumors in the salivary glands account for less than 1% of all carcinomas of the parotid gland and 3.5% of all malignant tumors of minor salivary glands. This tumor has one of the worst prognoses of salivary gland neoplasms. The prognosis for patients with SmCC of the salivary glands has been reported to be more favorable than for those with SmCC of other sites.[1
4]

However, there is no doubt that parotid SmCC is a high-grade malignancy

that should be treated aggressively. Surgery, adjunctive radiation therapy and/or chemotherapy have been performed in most cases.[1] SmCCs of the salivary gland are classified into neuroendocrine types and ductal carcinomas. The neuroendocrine type can be classified further into Merkel-cell-like SmCC and pulmonary variants, based on cytokeratin 20 immunoreactivity with a dot-like staining pattern.[2] Recently, the resemblance of Merkel-cell-like SmCCs of salivary gland to cutaneous Merkel cell carcinoma has been suggested, and some of them have spontaneous regression potential related to a favorable clinical outcome.[1,2] In this article, we report a locoregional advanced parotid SmCC that had an unusual clinical course. The tumor was controlled by surgery alone, and nine-year diseasefree survival was achieved without any adjunctive therapy. We also studied the expression of an oncogene or tumor suppressor gene, and demonstrated the expression of mammary serine protease inhibitor(Maspin), which is an important tumor suppressor gene of salivary gland carcinomas.

Case Presentation

An 87-year-old Asian woman in good general health presented with a progressively enlarging mass located in her left preauricular region. Fine needle aspiration (FNA) cytology analysis performed in the clinic suggested malignant cells of uncertain origin, but the tumors showed a rapid and consistent regression after FNA. During six months of observation, the tumor regrew and the patient was referred to our hospital. A physical examination revealed a 55-mm tumor located in the parotid with extension to the retroparotid area, and swelling of her left laterocervical lymph node. A computed tomography scan showed a peripheral enhanced mass in her right parotid gland. Magnetic resonance imaging revealed low signal intensity on T1-weighted images and iso-high signal intensity on T2-weighted images. The images showed the lesion to be clearly circumscribed, with homogeneous hypo-iso signal intensity on T1-weighted images and heterogeneous high signal intensity on T2-weighted images. The tumor demonstrated circumscribed and heterogeneous enhancement on gadoliniumenhanced T1-weighted images (Figure 1). Computed tomography scans of her thorax and abdomen did not reveal involvement of other sites.

(Enlarge Image)

Figure 1. Magnetic resonance imaging scans. Magnetic resonance imaging shows this lesion to be clearly circumscribed, with (A) homogeneous hypo-iso signal intensity on T1-weighted images and (B) heterogeneous high signal intensity on T2-weighted images. (C) Axial and (D) coronal gadolinium-enhanced T1-weighted images show the presence of a circumscribed and heterogeneously enhanced tumor.

FNA was performed again and a cytological examination revealed a Papanicolaou classification of Class V, suggesting SmCC. A total parotidectomy and modified neck dissection were performed. On histopathology, the tumor showed diffuse growth with confluent necrosis in the

salivary gland. The tumor cells had scant cytoplasm and hyperchromatic nuclei without prominent nucleoli. Mitotic figures were frequently observed (Figure 2). Neoplasmic elements reveal positivity for cluster of differentiation 56, neuron specific enolase, synaptophysin and a dotted staining pattern with cytokeratin 20 (Figure 3). Based on our histopathological findings, a diagnosis of a Merkelcell-like SmCC of the parotid gland was made. Additional studies were positive for Maspin.

(Enlarge Image)

Figure 2. Histological features. (A) Diffuse growth with necrosis (lower right) of tumor cells is seen. Several residual ducts and glands are observed in the tumor. (B) The tumor shares the features of small cell carcinomas seen in other organs. It is composed of small- to mediumsized epithelioid cells with hyperchromatic, finely granular nuclei and scant cytoplasm. Mitotic figures are frequently seen.

(Enlarge Image)

Figure 3. Immunohistochemistry for cytokeratin 20. Most tumor cells express cytokeratin 20 with a characteristic dot-like pattern. (A) Original magnification 400; (B) original magnification 600.

Our patient's postoperative course was uneventful, and no postoperative radiotherapy was administered. The nine-year clinical follow-up, which is the longest follow-up of head and neck SmCC in our knowledge, revealed no locoregional recurrence or distant metastasis.

Discussion
The parotid gland tumor in our case appeared on histology to be a SmCC. No tumor formation was recognized in other locations; therefore, this was an extremely rare primary SmCC of the parotid gland. This case also has the longest follow-up to the best of our knowledge. SmCC can occur in any organ, although the vast majority occur in the lung. The prognosis for patients with SmCC of the salivary glands has been reported to be more favorable than for those with SmCC of the lung or larynx.[1,2,4] However, there is no doubt that parotid SmCC is a high-grade malignancy that should be treated aggressively. The main treatment for parotid SmCC is a surgical approach with partial or total parotidectomy. The association of radiotherapy with surgery has shown a decrease in relapses and an increase in survival. Seventy-five percent of local relapses occurred in cases where surgery had been the only treatment, whereas, when associated with radiotherapy, the rate of local relapse was 20%.[1,5] Our case was controlled by surgery alone, and nine-year disease-free survival was achieved without any adjunctive therapy despite the advanced nature of the disease.

Recently, two parotid SmCCs with unusual clinical courses were reported. Mulder et al. presented a primary SmCC of the parotid with massive local recurrence that regressed spontaneously.[6] Jorcano et al. also presented an advanced case where a complete response and long-term survival was achieved by radiotherapy alone.[5] Both cases involved Merkel-cell-like SmCC. The neuroendocrine type of SmCC can be subdivided into Merkelcell-like and pulmonary varieties on the basis of cytokeratin 20 immunoreactivity with a dotlike staining pattern.[7] It has been speculated that some salivary Merkel-cell-like SmCC are closely related biologically to cutaneous Merkel cell carcinoma, which is known to be less aggressive than extrasalivary SmCC.[8] One possibility for a favorable clinical outcome after initial treatment is the potential for spontaneous regression. We could find one only case of spontaneous regression of a salivary Merkel-cell-like SmCC,[6] but spontaneous regression of cutaneous Merkel cell carcinomas has been described in 20 cases.[9,10] Furthermore, 10% to 20% of all cases of metastasized cutaneous Merkel cell carcinomas present no obvious primary tumor.[10] It is not known if the primary lesion had regressed in these cases. The reasons for spontaneous regression remain unclear. Mulder et al. explained the mechanism of spontaneous regression as follows: "apoptotic events seem to play an important part, and a local T-cell mediated immune response triggered by surgical trauma might also be involved". The hypothesis that spontaneous regression occurs after surgery might help understand this unusual clinical course. The fact that spontaneous regression was observed after initial FNA also supports this hypothesis in our case. To understand the mechanism of spontaneous regression further, we studied the expressions of various oncogenes or tumor suppressor genes, and demonstrated the expression of Maspin. Maspin belongs to the serine protease inhibitor family and may be associated with a favorable prognosis in common salivary gland carcinomas such as adenoid cystic carcinoma, mucoepidermoid carcinoma and carcinoma ex pleomorphic adenoma.[11] Although Maspin expression in salivary SmCC might be important information, the relationship between Maspin and spontaneous regression is under consideration.

Conclusion
The mechanism of spontaneous regression in salivary SmCC is not fully understood, but our experience with this case leads us to suggest that some

cases have a favorable clinical outcome that is related to the potential for spontaneous regression.

Consent

Acute Mesenteric Ischemia and Duodenal Ulcer Perforation

A Unique Double Pathology

Abstract
Background: Acute mesenteric ischaemia and duodenal perforation are surgical emergencies with serious consequences. Patients presenting with acute mesenteric ischaemia alone face a high mortality rate as high as 60% whereas those presenting with peptic ulcer perforation the mortality rates range from 6-14%. There are very few reported cases of patients presenting with this dual pathology. Case presentation: We report a unique case of a 53 year old Italian lady who presented with acute mesenteric ischaemia and duodenal perforation. This is the first report of massive bowel ischaemia and duodenal perforation with no apparent underlying common pathophysiology leading to this presentation. Conclusion: Early management in the intensive care unit and appropriate surgical intervention maximised the patient's chances of survival despite the poor prognosis associated with her dual pathology. The rare pathology of the patient described can be explained by two possible hypotheses: peptic ulcer disease causing duodenal ulceration, which precipitated ischaemic infarction of the small bowel. The second hypothesis is the patient developed a stress related ulcer following ischaemic bowel infarction secondary to arterial thrombosis.

Background
Acute mesenteric ischemia (AMI) comprises a group of pathophysiologic processes that have a common end pointbowel necrosis. The survival rate has not improved substantially

during the past 70 years, and the major reason is the continued difficulty in recognizing the condition before bowel infarction occurs.[1,2] Clinical presentation is nonspecific in most cases and can be characterized by an initial discrepancy between severe abdominal pain and minimal clinical findings. In general, patients with AMI have an acute onset of symptoms and a rapid deterioration in their clinical condition. Complications such as ileus, peritonitis, pancreatitis, and gastrointestinal bleeding may also mask the initial signs and symptoms of AMI.[2] Acute mesenteric ischemia can be categorized into 4 specific types based on its cause. The most frequent cause is arterial emboli. They are responsible for approximately 40% to 50% of cases.[1,3] Most mesenteric emboli originate from a cardiac source. The second most common cause is acute mesenteric thrombosis accounts for 25% to 30% of all ischemic events.[4,5] Most of the reported cases of mesenteric ischemia due to arterial thrombosis occur with a background of severe atherosclerotic disease, the most common site near the origin of the Superior Mesenteric Artery.[6] Commonly, patients with this condition can tolerate major visceral artery obstruction because the slow progressive nature of atherosclerosis allows the development of important collaterals. The third major cause is non-occlusive mesenteric ischemia. The pathogenesis of is poorly understood but often involves a low cardiac output state associated with diffuse mesenteric vasoconstriction. Splanchnic vasoconstriction in response to hypovolemia, decreased cardiac output, hypotension, or vasopressors best explain the difference between this entity and other forms of AMI.[7,8]Mesenteric venous thrombosis is the least common cause of mesenteric ischemia, representing up to 10% of all patients with mesenteric ischemia and 18% of those with AMI. Most cases are thought to be secondary to other intra-abdominal pathologic conditions (such as malignancy, intraabdominal sepsis, or pancreatitis) or were classified as idiopathic.[9] Mesenteric venous thrombosis is usually segmental, with oedema and hemorrhage of the bowel wall and focal sloughing of the mucosa. Thrombi usually originate in the venous arcades and propagate to involve the arcuate channels. Hemorrhagic infarctions occur when the intramural vessels are occluded.[2] Involvement of the inferior mesenteric vein and large bowel is uncommon. The transition from normal to ischemic intestine is more gradual with venous embolism than with arterial embolism or thrombosis.

Case Presentation
A 53-year-old lady presented following a collapse at home. In the ambulance she became unresponsive with a temperature 33.4oC, heart rate of 95 beats per minute and un-recordable blood pressure. On arrival active resuscitation commenced and a brief history of three days of severe, intermittent abdominal pain, absolute constipation, and reduced urine output was noted. Prior to this recent deterioration the patient had been well, with no weight loss or change in appetite. She had a past medical history of hypertension, osteoporosis, a previous appendicectomy; and a 20-pack year smoking history. She denied any dyspeptic symptoms prior to her admission, and had never taken any antacids. Her medications on admission were regular beta blocker, regular long term bisphosphonates and paracetamol on as required basis. On inspection the patient appeared of a normal body habitus, with a BMI of 22 and examination revealed a peritonitic abdomen. Blood tests from admission revealed grossly elevated inflammatory markers (CRP>500mg/L), neutrophilia and acute kidney failure. Her blood gas showed metabolic acidosis with a high lactate (12.7mmol/L), and low base excess (21.8 mmol/L) and bicarbonate (6.9mmol/L). Due to her severe acute renal impairment no contrast was initially used for imaging. CT scan of her abdomen performed on admission revealed extensive free peritoneal fluid and also fluid adjacent to the right kidney in the retro peritoneum (Figure 1). There was no evidence of any free air, obstruction, perforation or abdominal aortic aneurysm, with no evidence for small bowel ischaemia. Furthermore an ascitic tap was performed which revealed a clear straw coloured fluid with a very high lactate dehydrogenase, and normal protein. It did not yield growth of any organisms.

(Enlarge Image)

Figure 1. CT scan of the abdomen showing free peritoneal fluid.

Despite optimal management in the intensive care unit the patient's inflammatory markers, creatinine and alanine aminotranserase all continued to rise, and urine output remained minimal. In light of her clinical deterioration, 12 hours after admission the patient underwent an urgent diagnostic laparoscopy. Intraoperatively approximately 3 litres of free intraperitoneal purulent bile stained fluid filled the abdomen, and multiple fibrinous exudates surrounded the entire small bowel. A 162 cm section of distal small bowel appeared ischemic, and an anterior duodenal (D1) perforation was identified. The procedure was converted to a laparotomy in view of the findings. The D1 perforation was repaired using an omental patch, and the ischemic bowel wrapped in warm saline-soaked packs, with minimal benefit. The distal 162cm of dead small bowel was resected, and because of her critical condition the

neodistal small bowel had a few viable slightly dusky patches left behind for a relook the following day. A washout was performed and a laparostomy using a saline bag was applied. One day post-operatively the patient was dialysed and kept intubated due to unresolving metabolic acidosis. During the second laparotomy another 50cm of small bowel appeared ischaemia and was resected. 2 meters of healthy small bowel was left in situ. An ileocaecal anastomosis was made, a washout performed, and the abdominal wound closed. Two weeks after her initial laparotomy the patient had an endoscopy for a nasojejunal tube insertion. The omental patch repair was performed during laparotomy for duodenal perforated ulcer. However, once endoscopy was performed few days after the first laparotomy for naso-jejunal feeding tube insertion, a small hole was identified near the D1 repair site and was clipped (Figure 2). A repeat endoscopy a week later showed closure of the duodenal defect and a 1cm healing ulcer. The patient made a slowly recovery and she was discharged few weeks later.

(Enlarge Image)

Figure 2. Endoscopic clipping of anterior duodenal perforation.

Histology showed haemorrhagic infarction in sections of small bowel with ischaemic changes throughout the submucosa and muscle coat, with normal appearing Mesenteric vessels.

Discussion and Conclusion

The unusual presentation of this patient raised an important question regarding her dual pathology: which occurred first? Did she have a perforated duodenal ulcer, causing sepsis and hypotension leading to small bowel ischemia, or did she suffer from ischaemic bowel, and subsequently developed a stress-related perforated ulcer? After performing a through literature search, we did identify a single case report that described a patient presenting with acute abdomen and the subsequent intervention revealed exactly the same double pathology of Small bowel ischaemia and duodenal ulcer. However the histopathology of the bowel in that particular case did show evidence of polyarteritis nodosa explaining the cause of the acute bowel ischaemia.[10] In order to explain this double pathology it is vital to note that in peptic ulcer disease the two major precipitating factors are Helicobacter pylori infection and non-steroidal anti-

inflammatory drugs (NSAIDs). Ulcer incidence increases with age and therapy with drugs such as corticosteroids, anticoagulants and bisphoshonates. Complications (bleeding, perforation, obstruction) can occur in patients with peptic ulcers of any aetiology. Perforation occurs in about 5% to 10% of patients with active ulcer disease.[11] With this background we are proposing two explanations for this pathology. The first is that the patient had been on long term bisphosphonates and this increased her risk for peptic ulcer disease. If we assume that the perforation of the duodenal ulcer occurred first, it was likely that it led to mesenteric venous thrombosis causing ischaemic infarction of the small bowel. The histology results favour this theory as the patient had segmental involvement of the small bowel with the sparing of the large bowel and this commonly present in acute ischaemia of the bowel that is associated with mesenteric venous thrombosis. The second hypothesis is that the patient developed a stress related duodenal ulcer post ischaemic bowel infarction and eventually this ulcer perforated. The cause of the ischaemia is likely due to arterial thrombosis with a background of severe atherosclerotic disease caused by the patient's long history of hypertension and smoking. Patients with this type of bowel ischaemia present later as they can tolerate major visceral artery obstruction because the slow progressive nature of atherosclerosis allows the development of important collaterals. The patient had 3 days history of feeling unwell and constipation with minimal urine output before she collapsed.

Consent
Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Series Editor of this journal.

RNA-based Determination of ESR1 and HER2 Expression and Response to Neoadjuvant Chemotherapy
Abstract and Introduction

Abstract
Background Hormone and human epidermal growth factor receptor 2 (HER2) receptors are the most important breast cancer biomarkers, and additional objective and quantitative test methods such as messenger RNA (mRNA)-based quantitative analysis are urgently needed. In this study, we investigated the clinical validity of RT-PCR-based evaluation of estrogen receptor (ESR1) and HER2 mRNA expression. Patients and methods A total of 1050 core biopsies from two retrospective (GeparTrio, GeparQuattro) and one prospective (PREDICT) neoadjuvant studies were evaluated by quantitative RT-PCR for ESR1 and HER2. Results ESR1 mRNA was significantly predictive for reduced response to neoadjuvant chemotherapy in univariate and multivariate analysis in all three cohorts. The complete pathologically documented response (pathological complete response, pCR) rate for ESR1+/HER2 tumors was 7.3%, 8.0% and 8.6%; for ESR1/HER2 tumors it was 34.4%, 33.7% and 37.3% in GeparTrio, GeparQuattro and PREDICT, respectively (P < 0.001 in each cohort). In the KaplanMeier analysis in GeparTrio patients with ESR1+/HER2 tumors had the best prognosis, compared with ESR1/HER2 and ESR1/HER2+ tumors [diseasefree survival (DFS): P < 0.0005, overall survival (OS): P < 0.0005]. Conclusions Our results suggest that mRNA levels of ESR1 and HER2 predict response to neoadjuvant chemotherapy and are significantly associated with long-term outcome. As an additional option to standard immunohistochemistry and gene-array-based analysis, quantitative RT-PCR analysis might be useful for determination of the receptor status in breast cancer.

Introduction
Hormone and human epidermal growth factor receptor 2 (HER2) receptors are central breast cancer biomarkers and constitute the dominant biological determinants of breast cancer.[1
3]

Currently, these markers are routinely evaluated for each patient by immunohistochemistry.

Major limitations of immunohistochemistry are the inter-observer and technical variability as well as the inability to generate quantitative data. An American Society of Clinical Oncology/American College of Pathologists analysis has stated that the

immunohistochemical assessment of hormone and HER2 receptors is inaccurate in up to 20%.[4,5] The evaluation of messenger RNA (mRNA) markers by quantitative RT-qPCR might be an interesting additional option. Several retrospective evaluations suggest that RNA-based determination of hormone receptors, HER2 as well as other markers is generally feasible. [6
11]

However, the current guidelines have emphasized that it would be essential to link the

RNA markers directly to the clinical outcome before this new technology can be used as a diagnostic test.[4] In the NEOpredict translational research program, we have validated an RNA-based approach for assessment of the receptor status by evaluation of ESR1 and HER2 mRNAs using predefined cut-offs[1,12] in a total of 1050 tumor samples from three neoadjuvant multicenter studies. As clinical end-points we use chemotherapy response and outcome after anthracyclinetaxane-containing neoadjuvant chemotherapy.

Patients and Methods

Clinical Studies
The bases for the sequential-validation strategy were three sequential neoadjuvant clinical multicenter studies by the German Breast Group and the Arbeitsgemeinschaft Gynkologische Onkologie Breast Study Group(consort diagram, Figure 1AC). The Ethics committee approval was obtained for all centers participating in the clinical studies and from the institutional review board of the Charit hospital. All samples were formalin-fixed paraffinembedded (FFPE) pretherapeutic core biopsies collected at baseline, before randomization, with written informed consent.

(Enlarge Image)

Figure 1. Consort statementflow chart of samples from the GeparTrio (A) and GeparQuattro (B) validation cohorts as well as the prospective PREDICT study cohort (C).

GeparTrio Study As a first cohort 262 core biopsies from patients treated with six cycles of docetaxel (Sanofi-Aventis, Frankfurt, Germany) (Taxotere), doxorubicin (Adriamycin) and cyclophosphamide (TAC), within the GeparTrio (NCT00544765)[1,14] or the GeparTrio Pilot[15] study, were used. For the current investigation, the inclusion criteria were six cycles of TAC irrespective of response after the initial two cycles of TAC, available pCR data, tissue samples with at least 30% tumor tissue, successful RNA isolation. The expression of HER2 mRNA had been analyzed in a previous project in 278 samples from GeparTrio with a focus on concordance between central and local pathology.[16] Based on the available tissue for further PCR analyses, 262 of those samples were included in the present study. GeparQuattro Study As a second validation cohort, we used samples from 474 patients enrolled in the prospective, phase III GeparQuattro trial (NCT 00288002).[17] The samples from all three chemotherapy arms (study arm A, B, C) were used, all other inclusion criteria were the same as in GeparTrio (Figure 1). A small number (n = 10) of locally HER2 positive cases were included by mistake which had been treated with trastuzumab. Tumors from ten additional patients were found to be HER2+ by RT-PCR, these tumors had been HER2 by local pathology assessment (and were therefore not treated with trastuzumab), resulting in a total of 20 HER2 positive tumors. It was decided to include these patients in the analysis of RNA-based molecular tumor types. Prospective PREDICT/GeparQuinto Study The PREDICT study was designed as a substudy of GeparQuinto [1820] for prospective validation of biomarkers in HER2 tumors in the neoadjuvant setting (Figure 1C). Between September 2009 and October 2010, a total of 314 samples were evaluated. The inclusion criteria were (i) HER2 patients who were randomly assigned to setting 1 and did not receive bevacizumab, (ii) available tumor sample, available pCR data. The biomarker analysis was carried out parallel to recruitment of the GeparQuinto study and was completed before the study outcome data were analyzed. A pathological complete response (pCR) was defined as the pathologically confirmed absence of residual invasive tumor in breast and lymph nodes at the end of chemotherapy (ypT0/Tis, ypN0), based on the histopathological reports, which were centrally reviewed. In GeparTrio survival analysis was carried out with a median follow-up time for disease-free

survival (DFS) of 55.43 months (range 1.4884.76) and for overall survival (OS) of 58.68 months (range 1.4896.49). All clinical data, including the immunohistochemical data were extracted from the study database and represent the local assessment. As the current clinical hormone-receptor status is based on the combination of ER and progesterone receptor (PR), we decided to use this combined definition for the immunohistochemistry-based status. For GeparTrio, HER2 status was determined centrally using immunohistochemistry and silver in-situ hybridization, as the testing of HER2 on core biopsies was not fully established at the time the study was conducted.

Sample Preparation and RNA Extraction

From each FFPE tissue block, a 10 m (GeparTrio and PREDICT samples) or a 5 m section (GeparQuattro samples) was cut. The presence of at least 30% tumor tissue was evaluated. The RNA isolation from FFPE sections was carried out using the robotic tissue preparation system and VERSANT tissue preparation reagents (both Siemens Healthcare Diagnostics, Tarrytown) as described earlier [2123]). Samples were defined to have sufficient RNA if the mean of the C t values of three reference genes CALM2, OAZ1 and RPL37A was below 33.4. For assessment of DNA contamination, a progestagen-associated endometiral protein gene-specific quantitative PCR without preceding reverse transcription was carried out. The samples were considered to be substantially free of DNA when C tvalues >38 were detected.

Gene Expression Analysis Using Reverse Transcription Quantitative PCR

Primer and probe sequences of the genes used for determination of molecular tumor types are given in reference.[24] Reference genes were selected by a model-based variance estimation approach.[25]Based on Affymetrix U133A gene expression, datasets from 379 breast cancer tumors (213 surgical specimen; 186 pretherapeutic biospies), we selected and verified RPL37A, CALM2 and OAZ1 as stably expressed reference genes. The gene expression was assessed by RT-qPCR using the SuperScript III PLATINUM OneStep Quantitative RT-PCR System with ROX (Invitrogen, Karlsruhe, Germany) according to manufacturer's instructions in 384-well plates in an ABI PRISM 7900HT (Applied Biosystems,

Darmstadt, Germany) (GeparTrio and GeparQuattro samples) or in 96-well plates in an Agilent Mx3005 (Agilent, Bblingen, Germany) (PREDICT samples) for 30 min at 50C, 2 min at 95C followed by 40 cycles for 15 s at 95C and 30 s at 60C. All PCR assays were carried out in duplicate in GeparTrio and in triplicate in GeparQuattro and PREDICT. The mean of the C t values for each gene was calculated. To assure accuracy of the assays, a standardized reference RNA (Stratagene qPCR Human Reference Total RNA, Agilent Technologies, Waldbronn, Germany) was tested for each gene in parallel to the FFPE samples. For exclusion of contamination, no-template-controls were assessed in parallel.

Normalization and Cut-offs for Molecular Tumor Classification

Normalization was carried out in an identical way in all cohorts using three reference genes. The relative gene expression levels for ESR1 and HER2 are given as C tvalues, whereas C t = 20 [C tGOIC t(mean of RPL37A, CALM2, OAZ1)]. The cut-off values were predefined based on the two previous studies including 274 independent breast tumor samples (for ESR1)[12] and 167 breast cancer samples (for HER2).[6] Since a different PCR platform was used in the PREDICT study, a constant target-specific shift in C t values between previous and current assay conditions occurred. The cut-offs from the GeparTrio and GeparQuattro studies were therefore transformed by addition of an offset. The offsets for ESR1 and HER2 were predetermined for this study by reassessment of the 167 samples from the previous study using the old and new assay conditions resulting in the cut-offs of 18.6 for HER2 mRNA and 14.5 for ESR1 mRNA. These cut-offs are, therefore, numerically different, but identical with regard to mRNA levels to the previously published cut-offs and the cut-offs used in GeparTrio and GeparQuattro. Variabilities of ESR1 and HER2 PCR assays were published previously.[6,21] Moreover, we have tested reproducibility in a subset of 25 samples from the PREDICT study in two different laboratories, with high correlations (ESR1 r = 0.998; HER2 r = 0.90) and high concordance of classification (100%) (supplementary Figure S1, availableatAnnals of Oncology online).

Statistical Analysis

Statistical analysis was carried out using MATLAB 7.5.0 (The MathWorks, Natick, MA), SPSS version 15.0 (SPSS Inc. Chicago, Illinois, USA), GraphPad PRISM 4 (GraphPad software, La Jolla, California), SAS 9.2 (SAS Institute Inc., Cary, NC) as well as the Rpackage. The probability of pCR as a function of gene expression parameters and/or clinical baseline parameters was determined by univariate and multivariate logistic regression analysis. Fisher's exact test was used to compare pCR rates in subgroups. Concordance between mRNA and immunohistochemistry/in situ hybridization was assessed with crosstables and Yule's Q. Survival analyses were carried out by the KaplanMeier and log-rank test. All tests were two-sided with significance levels set at 0.05.

Results
Baseline Clinical Data
An overview on the study patients is given in the consort diagram (Figure 1A C) and in supplementary Table S1,availableat Annals of Oncology online. The clinical parameters age, grade, and stage are comparable in the GeparTrio

and GeparQuattro cohort. In the PREDICT study the patients were younger, had more G3 tumors and the tumors were smaller than in the other two cohorts. RNA was isolated from a total of 1093 core biopsies, 1050 of those samples (96.1%) contained sufficient RNA and were included in the study.

Quantitative Assessment of ESR1 and HER2 mRNA Levels as Predictive Factor for Chemotherapy Response in the Three Cohorts
In univariate logistic regression, mRNA expression of ESR1 was predictive as a continuous parameter for a reduced response to neoadjuvant chemotherapy with an odds ratio (OR) of 0.74, 0.76 and 0.78 in the GeparTrio, GeparQuattro and PREDICT (all P-values <0.001) study. In multivariate analysis including established clinicopathological parameters, ESR1 mRNA was still significant in all the three cohorts with an OR of 0.78 (P = 0.003, GeparTrio), 0.76 (P < 0.001, GeparQuattro) and 0.88 (P = 0.050, PREDICT, Table 1). In contrast, HER2 mRNA levels as a continuous parameter were significantly predictive only in the PREDICT study in univariate analysis, and not significant in the other cohorts and in the multivariate analyses (Table 1).

Definition of Tumor Types Based on the mRNA Levels of ESR1 and HER2
We have defined four mRNA-based subtypes ESR1+/HER2, ESR1+/HER2+, ESR1/HER2+ and ESR1/HER2, using predefined cut-offs [6, 12] (Figure 2AC). In the GeparTrio cohort, 58% ESR1+/HER2, 10% ESR1+/HER2+, 9% ESR1/HER2+ and 23% ESR1/HER2 tumors were found (Figure 2A). In the GeparQuattro and the PREDICT study, the distribution of ESR1+/HER2 and ESR1/HER2 was very similar in both the studies (Figure 2B and C). In the GeparQuattro cohort, only 4.5% of tumor samples were HER2+ (see the methods section). According to the inclusion criteria of the prospective PREDICT study, only patients from the HER2 negative study arms of GeparQuinto were included and only two (0.6%) were found to be HER2+ by mRNA analysis.

(Enlarge Image)

Figure 2. RNA-based molecular typing based on the expression of ESR1 and HER2 in the GeparTrio (A), GeparQuattro (B) and the PREDICT cohort (C). pCR cases are indicated as blue spots. Please note that the cut-offs are nominally different in the PREDICT study, this is because the cut-offs have been adapted to a different PCR platform. The cut-offs represent identical mRNA levels in all the three cohorts.

The pCR rates were different in the four biological subgroups (Figure 3AC). In ESR1+/HER2 tumors, the pCR rate was 7.3% in GeparTrio, 8.0% in GeparQuattro and 8.6% in PREDICT. ESR1/HER2 tumors had a pCR rate of 34.4%, 33.7% and 37.3% in GeparTrio, GeparQuattro and PREDICT (P < 0.001 compared with ESR1+/HER2 tumors in all the three cohorts). ESR1/HER2+ tumors had a particularly high pCR rate of 43.5% (GeparTrio) and 50% (GeparQuattro). In GeparTrio, trastuzumab was not part of the neoadjuvant regimen, in GeparQuattro 50% of patients (10 of 20) in this study cohort had received trastuzumab, which might have contributed to the slightly higher pCR rate.

(Enlarge Image)

Figure 3. Different rates of pathological complete response (pCR) in the molecular tumor types in the GeparTrio (A), GeparQuattro (B), and the PREDICT cohort (C). The molecular tumor types have significantly different response rates to neoadjuvant chemotherapy, which are similar in the three cohorts. (*two-sided Fisher's test; **chi-square test).

Comparison of mRNA-based and Immunohistochemistry-based Subtypes

A high concordance was observed between RNA-based ESR1 status and immunohistochemistry-based hormone receptor status (ER and/or PR) of 92.7%, 90.3% and 91.4% in GeparTrio, GeparQuattro and PREDICT (supplementary Table S2, availableat Annals of Oncologyonline). For comparison of ESR1 mRNA status and ER status by immunohistochemistry, the concordance was 90.2%, 90.0% and 93.3% in the three studies (supplementary Table S3, availableat Annals of Oncologyonline). For HER2, the concordance between immunohistochemistry/in situ hybridization and RTqPCR was 95.8%, 96.6%, and 99.4% in the three studies (supplementary Table S4, availableat Annals of Oncologyonline). Supplementary Table S5,availableat AnnalsofOncology online, shows the pCR rates of the groups resulting from the combination of ER immunohistochemistry and mRNA status. Those tumors that were ER+ by both methods had a pCR rate of 9.8% (all the three cohorts combined), and those that were negative by both methods had a pCR rate of 37.8%. Tumors that were negative by immunohistochemistry and positive by mRNA had a low pCR rate of 12.9%, while tumors that were positive by IHC and negative by mRNA analysis had a pCR rate of 23%.

Survival Analysis of the RNA-based Tumor Types

As an additional outcome parameter, we carried out a KaplanMeier analysis for DFS and OS in the GeparTrio cohort comparing the RT-qPCR-based and immunohistochemistry-based molecular typing (Figure 4). Patients with ESR1+/HER2 tumors had the best DFS and OS, while patients with ESR1/HER2 and ESR1/HER2+ tumors had a comparably poor prognosis (Figure 4, DFS: P < 0.0005, OS: P < 0.0005). The prognostic impact of RTqPCR-based and IH-based assessment was very similar for the four molecular subtypes. In this cohort that had not been treated with neoadjuvant trastuzumab, patients with ESR1+/HER2+ tumors had a similar DFS as ESR1/HER2 tumors. For OS, the prognosis of the ESR1+/HER2+ tumors was better than the ESR1/HER2 tumors.

(Enlarge Image)

Figure 4. KaplanMeier survival analysis of disease-free survival (DFS; A and C) and overall survival (OS; B and D). Comparison of the molecular tumor types based on the determination of ESR1 and HER2 by RT-qPCR (A, and B) and the tumor types based on conventional immunohistochemistry of hormone receptors and HER2 (C and D) in GeparTrio.

Discussion
In this study, we have validated mRNA assessment of ESR1 and HER2 for prediction of response to neoadjuvant chemotherapy by analysis of three independent cohorts with a total of 1050 patients. The pCR rates of the individual subtypes are similar to those observed rates using immunohistochemistry in other neoadjuvant studies.[2630] The introduction of PCR-based molecular typing as an additional method is a promising approach to reduce the inherent variability of immunohistochemistry. It should be noted that both the approaches have their advantages: immunohistochemistry allows a direct visual control, but is only semiquantitative with a relevant observer-related and instrument-related bias, which might be partly improved by automated analysis platforms. RNAanalysis is quantitative, objective and offers many options for integrated quality control by use of tissue-specific and marker-specific positive and negative controls. Both the approaches depend on an enzymatic reaction as a central element; however, the reaction conditions are more stable in the standardized environment of a quantitative PCR cycler. The PCR analysis should be always linked to a histopathological quality control to ensure an adequate selection of tissue. Our analysis shows that both the methods have a high concordance and are similarly predictive. However, tumors that are ER by immunohistochemistry and ER+ by RT-PCR have a comparably low pCR rate of 12%. This might suggest that tumor biology could be better reflected by mRNA analysis; however, the small sample size of the discordant group limits the interpretation. In our investigation, we have used only ESR1 mRNA expression and have not included the PR. This decision was based on the report of the Early Breast Cancer Trialist's Collaborative Group that the ER is the only relevant determinant of response to endocrine therapy.[1] The concordance is similar if

ESR1 mRNA is either compared with ER immunohistochemistry or compared with HR status (combined ER and PR). Reproducibility of the RT-PCR assay has been tested with high correlations in a subset of 25 samples from PREDICT. In addition, the variability of the qRTPCR assays for ESR1 and HER2 used in this study has been reported in two separate publications.[6,21] For a very similar test, the Endopredict multigene assay that uses the identical platform, we have carried out a proficiency test in seven laboratories with a very good performance (Pearson's correlation r = 0.994; 100% concordance of classification).[31]Therefore, RNA-based molecular assays can be used in established molecular pathology laboratories. The RNA-based approach could be used for additional validations of those tumors that are hormone-receptor negative by immunohistochemistry but that have histological features that are highly suggestive for hormone-receptor positivity, such as tubular, lobular or grade 1 histology. Furthermore, standardized RNA-based testing may serve as an additional standardized entry criterion for clinical studies. Interestingly, our data suggest that ESR1 expression is predominantly regulated on a continuous scale and that the level of ESR1 expression is linked to the OR for chemotherapy response. In contrast, HER2 is significant for response if the predefined groups are used but it is not significant on a continuous scale. This might lead to the hypothesis that the underlying biological event for HER2 overexpression, the HER2 gene amplification, leads to distinct tumor groups rather than to a continuum of HER2 expression levels. The distribution pattern of HER2 mRNA expression in Figure 2 is suggestive of differences between ESR1+ and ESR1 tumors. In ESR1+ tumors, HER2 mRNA expression was generally higher and a continuum is observed with gradually increasing expression levels. In contrast, in ESR1 tumors there are distinct groups of HER2+ and HER2 tumors. The identical observation has recently been described by Pinhel et al.[32] This suggests that other pathways, such as the estrogen receptor pathway, might also influence HER2 expression

levels.[33,34] Considering the continuum of HER2 mRNA expression in hormonereceptor positive tumors, it is not clear if the cut-off levels for HER2 that are used in this study will also be valid in the context of trastuzumab therapy, in particular as recent reports have suggested discrepancies between mRNA and immunohistochemical HER2 determination.[35] The GeparTrio trial did not contain trastuzumab therapy and the analyses in GeparQuattro and PREDICT were focused on patients with HER2 tumors. It should be noted as a limitation of the study that the number of HER2+ cases was very low in the GeparQuattro and the PREDICT cohort, so that the high concordance rates between RNA and protein analysis are mainly caused by the large number of HER2 negative cases. Therefore, the role of HER2 mRNA expression should be further evaluated in tumor cohorts that have been treated with trastuzumab, this evaluation is currently ongoing in the HER2+ study arm of the GeparQuattro study. Taken together, it would be a major improvement for individualized therapy if oncologists and pathologists have the option to choose between different methods for biomarker analysis for the determination of receptor status. Based on the results of our study, it is possible to develop a certified standardized diagnostic test system for RNA-based receptor analysis that could be used for biomarker assessment in the local pathology laboratories. The integration of immunohistochemistry, mRNA-based analysis and clinical parameters could be used to reliably identify those patients who have an increased benefit from neoadjuvant chemotherapy.

Development and Pilot Implementation of a Locally Developed Trauma Registry

Abstract
Background Trauma registries (TRs) play an integral role in the assessment of trauma care quality. TRs are still uncommon in developing countries owing to awareness and cost. We present a case study of development and pilot implementation of "Karachi Trauma Registry"

(KITR), using existing medical records at a tertiary-care hospital of Karachi, Pakistan to present results of initial data and describe its process of implementation. Methods KITR is a locally developed, customized, electronic trauma registry based on open source software designed by local software developers in Karachi. Data for KITR was collected from November 2010 to January 2011. All patients presenting to the Emergency Department (ED) of the Aga Khan University Hospital (AKUH) with a diagnosis of injury as defined in ICD-9 CM were included. There was no direct contact with patients or health care providers for data collection. Basic demographics, injury details, event detail, injury severity and outcome were recorded. Data was entered in the KITR and reports were generated. Results Complete data of 542 patients were entered and analysed. The mean age of patients was 27 years, and 72.5% were males. About 87% of patients had sustained blunt injury. Falls and motor vehicle crashes were the most common mechanisms of injury. Head and face, followed by the extremities, were the most frequently injured anatomical regions. The mean Injury Severity Score (ISS) was 4.99 and there were 8 deaths. The most common missing variables in the medical records were ethnicity, ED notification prior to transfer, and pre-hospital IV fluids. Average time to review each chart was 14.5 minutes and entry into the electronic registry required 15 minutes. Conclusion Using existing medical records, we were able to enter data on most variables including mechanism of injuries, burden of severe injuries and quality indicators such as length of stay in ED, injury to arrival delay, as well as generate injury severity and survival probability but missed information such as ethnicity, ED notification. To make the data collection process more effective, we propose provider based data collection or making a standardized data collection tool a part of medical records.

Background
Trauma registries (TRs) are databases used to monitor and enhance the quality of trauma care and public health programs related to injury prevention and research.[13] The scope of a particular TR determines the amount of information captured through them and may vary from a "minimal dataset"

collected in emergency departments (ED) to a "comprehensive dataset" with information from pre-hospital care to rehabilitation.[48]While maintaining TR is a requirement of many trauma systems, standardization of variables is important to ensure outcome comparison in terms of patient and injury characteristic.[1,3,7,9] Trauma registries are well established in in many highincome countries (HIC) such as United States; have been used to promote injury prevention, change policies and to evaluate trauma system effectiveness.[10] In many instances, the registries are guided through the American College of Surgeons guidelines for selection of data points.[2,7,11] Ninety per cent of trauma- and injury-related deaths and disabilities occur in low-and middle-income countries (LMICs).[12] A significant number of these deaths can be averted through improvement in trauma care in these countries.[6,1316] However, because information on injuries and trauma from LMICs is most often based on routine health surveys, surveillance reports, police data and hospital-based case series, information about the process and quality of trauma care or clinical outcomes is lacking.[8,12,1720] Limited useful information on trauma care in LMICs underscores the importance of TRs in these settings. Examples of successful implementation of trauma registries in LMICs are also uncommon due to the cost of obtaining and maintaining a TR.[1,3,12,16] Currently available commercial TRs such as Collector, Trauma One and NTRACS are expensive products. For instance, Collector which has over 1500 clients in 10 countries, costs about 7500 USD for application and 2500 USD for yearly license. The cost of training and updates are in addition to maintenance, which makes it and other commercial products inaccessible for many LMICs. TRs in many of the developing countries are under-developed, incomplete and used for surveillance purposes.[3] A locally developed electronic trauma registry is thus needed to assess injury adjusted trauma outcomes and to test this software in a hospital setting.

The objective of this study is to describe the structure, process of development and pilot implementation of a locally developed, electronic trauma registry the "Karachi Trauma Registry" (KITR) - from Karachi, Pakistan using existing medical records. We also share the lessons learnt during the implementation in a low income country.

Methods
The Development of Electronic Trauma Registry
The development of electronic registry was a four step process (Figure 1) which was followed by pilot testing. The development began in December 2008 with finalization of variables by a team consisting of a general surgeon, emergency physician, and public health professionals with special interest in trauma outcome research. In the next step, the IT experts were consulted for software and application design. The variables were organized for calculation of survival probability as well as ensuring that all the stages in-hospital treatment were recorded with date, time and interventions. The development of the electronic registry (KITR) required multiple iterations between March-August 2009, and open source softwares were used during the programming. The first software version was pre-tested on 120 trauma cases in August- October 2009 to check the data entry, any errors, collation of data and back-hand calculators. Based on the findings of pre-test, further modifications were carried out. The final product was a Windows-XP based software which could be installed as a stand-alone database system on PC and required Pentium III or higher processor, with a hard disk storage capacity (RAM) of at least 1 GB. The registry was based on SQL Server 2005 and is also supported by SQL Server express, which provides storage, processing and controlled access of data. KITR required dot net (.Net) Framework 3.0 and Microsoft Excel 2007 for pivot table analysis but does not require an internet connection.

(Enlarge Image)

Figure 1. Development and implementation process Karachi Trauma Registry.

Data Handling and Derivation of Trauma Indices

To facilitate data entry, separate tabs for recording patient demographics, injury details, emergency evaluation, treatment, in-hospital course and discharge details were provided (Figure 2). Several dropdown menus and a checklist were provided to minimize free text entry as much as possible. The built-in spread sheets and calculators helped store, collate and analyse data. Details of insurance or payer information were, however, not a part of the registry. The software was password protected and security of the database was ensured by encryption at the server, which was also login sensitive. The KITR used International Statistical Classification of Diseases and Related Health Problems (ICD 9 - CM) and Abbreviated Injury Scaling (AIS) 2005[21] for standardization of definitions and injury scaling. The registry was capable of generating different trauma scores (Glasgow Coma Scale, Revised Trauma Score, Injury Severity Score) and probability of survival (Trauma Injury Severity Score - TRISS) score.[22]

(Enlarge Image)

Figure 2. Snapshot of KITR with dropdown menus and tabs.

Pilot Implementation
The pilot study was conducted over a three-month period (November 2010 to January 2011) in the ED of the AKUH in Karachi, Pakistan.

Setting
AKUH is a 650- bedded tertiary referral centre, with 50,000 annual ED visits and training programs in Emergency Medicine and Trauma Surgery among others. The hospital has a 24hours on-call trauma team comprising of Emergency physicians and residents from general surgery, orthopaedics, anaesthesia and neurosurgery. Some of the health information is available as electronic records such as triage list, admissions, laboratory, radiology,

discharge summaries etc. while the history and physical examination and progress notes are manually written in the files.

Case Definition
All trauma patients presenting to the ED with history of trauma within 24 hours, or transferred from other hospitals and coded as International Classification of Disease (ICD) injury codes (ICD-9-CM 800959.9) were included in this study. Isolated hip fractures and dead-on-arrival trauma patients were excluded. Since AIS and TRISS scores cannot be derived for poisoning, these cases were also excluded. The cases included Data Sources The data sources included medical records; doctors' and nurses' notes; laboratory, radiology, and operative reports and discharge summaries. Daily report of ED visits with age, primary complaint and disposition was obtained from the electronic health information system. The triage, admission, and ED discharge list were utilized to capture patients with injuries. Data Collection and Entry For this pilot study the medical records of trauma patients were reviewed by a research assistant trained in medical chart abstraction, ICD-9 injury codes, AIS and injury severity scoring. A form was used for data collection, which did not involve direct contact with patients or their attendants. The information consisted of details about the patient's demographics, injury event and mechanism, physiological parameters, investigations, severity of injury, operative and non-operative procedures, complications, discharge capacity and follow-up until patient's discharge from the hospital or death of the patient. Random checks of the medical charts were performed by the principal investigator for accuracy and completeness of data collection during the study to compare the actual information and that on the hard copies. All ICD codes and AIS scores were cross checked prior to data entry by the PI and errors were corrected. All Electronic records were cross checked for accuracy and discrepancies noted, however once data entry had taken place, no items were changed, modified, or corrected. Missing or incorrect items were listed as shown in the Table 1 . Reports Basic frequency tables were produced on the number of admissions, demographics, mechanism of injuries, ICD -9 coding of injuries, discharge disposition, length of stay, probability of survival and actual survival.

The pilot study protocol was approved by the Ethics Review Committee of the Aga Khan University.

esults
Cost of KITR Development and Pilot Testing
The development of KITR from concept to operational software took 23 months. The estimated cost for the development of the software was USD 9,600. This included the time of investigators (54% of estimated cost), the cost of software development (16% of estimated), and implementation cost (30% of estimated). The actual cost incurred was the implementation cost in the form of stipends of research assistant and miscellaneous expenditure.

Case Ascertainment and Item Completion

Triage and admission/discharge list indicated 946 cases; however, number of records within the case definition was 732 during the study period. The number of cases used for the registry was 542 (74%); reasons included nonavailability of charts for review (n = 176), patients still receiving care in hospital during study period (n = 3) or insufficient documentation of injuries to assign AIS scores (n = 10). Table 1 shows item completion and errors. Some

variables which were a part of the registry, were not documented in the medical charts; for instance ethnicity (95%), the amount of IV fluids administered in pre-hospital phase (94%), Safety Equipment (81%) and ED notification prior to arrival of patients (90%). These undocumented variables are entered as "unknown" in the KITR. For those patients who were transferred in, ED was notified in only 8.6% cases. Total 25 data points were found as erroneous. Errors in AIS and ICD included nine AIS scores (1.7% of all cases) and six ICD codes (1% of all cases) were corrected prior to data entry and other 10 items (Table 1) were recognized as wrong data entry at the time of verification of electronic data.

Time Burden
The mean time for data retrieval and entry was 29.5 minutes (range 1550 minutes) per case. Time for data abstraction and hard copy questionnaire completion was 14.5 minutes (range: 820 minutes) while the mean time for data entry was 15 minutes (range: 730 minutes) based on the number of entries. (Total time 29.5 minutes and a range of 1550 minutes) This time burden excludes the time taken for double checking the records or data entry in the registry.

Patient Characteristics and Injury Mechanism

Table 2 gives demographic details and distribution of injury severity scores (ISS). Mean age of the victims were 27 years (range: 189 years) and males represented a higher proportion of recorded cases in all age groups (n = 394; 72.6%). The most common mechanisms of injury were fall (37%), motor vehicle crash (33%), and gunshot injuries (7%). Miscellaneous injuries (16%) included sports injuries, assault with blunt object, bites and occupational injuries.

Injury Severity and Survival Analysis

Many patients presented with multiple injuries located in more than one anatomical region; therefore 1155 injuries were recorded in KITR from 542

cases. The most common injuries included head, face and upper extremity injuries (Figure 3).

(Enlarge Image)

Figure 3. Frequency of injuries according to anatomical region* (N = 1155). * Region according to Abbreviated Injury Scale.

As shown in Table 2, 82% of the patients in our sample had an Injury Severity Score of 9 categorized as mild, 9% had ISS: 915 classified as moderate injuries, 7% had ISS between 1625, and only 2% had ISS of >25 representing critical injuries. 2.6% of patients had a probability of survival of less than 50% (Table 3). Eight patients (1.47%) died; five of those who died had a probability of survival of <50%. Disability at the time of discharge was recorded as per clinicians' assessment in the medical charts. More than half of the patients (n = 287) had no disability at the time of discharge from the hospital, 245 (45.2%) had temporary disability, and 10 (1.84%) had permanent disability at the time of discharge.

Quality Indicators
The registry was capable of generating quality indicators, such as pre-hospital delay, ED length of stay, length of stay in hospital, disposition from ED as well as predicted and actual survival. Although pre-hospital time in 81% of cases was less than 4 hours (range: 10 minutes to 28 hours), the large variability of pre-hospital time can be attributed to inter-facility transfers. Over 80% of patients were either transferred to in-patient units or discharged from the ED in 8 hours.

Discussion
This paper describes the three main steps for trauma registry implementation in a developing country; a- the process of development of the registry; baffordability of its development and implementation and c- the challenges of the implementation of the software. The team of trauma experts and software developers took almost 2 years with a direct cost of USD: 9,600 to develop a functional trauma registry. The most critical test of the success of the effort was in the implementation of the registry in a real hospital based patient care scenario. There is limited literature on TRs in developing countries.[2325] Kampala Trauma Registry was developed to establish an injury surveillance system in Uganda.[23] This was a paper based data collection system and attempted to demonstrate the feasibility of a trauma registry in limited resource setting. There was no electronic software and survival analysis was based on Kampala Trauma score (KTS). Similarly, a pilot test of trauma registry was undertaken in Haiti, utilizing a paper form for data collection and Epi Info for data entry

and analysis.[24] The registry variables included mechanism of injury, Glasgow coma score, body region, treatment and investigations but did not anatomical injury scores. The Cape Town Trauma Registry was designed for middleincome setting with a spatial distribution of injury events using GIS mapping, for injury surveillance and control.[25] The above examples are registries with serve as injury surveillance systems and focus on systematic data collection and analysis, with intent to defining issues in implementing a trauma registry in a low income setting. Other examples from LMIC attempted survival outcome comparison with the US Major Trauma Outcome Study[26] or creation of a database to record a particular type of injuries.[27] A recent report from a highincome country in the Middle East described the process of converting a single centre registry into a multicenter database, which is hard to replicate in lowincome settings.[28] Similar to other settings, we found four critical success factors for the implementation of trauma registry in our hospital. 1- The fundamental importance of good patient records, patient identification and documentation of all relevant information cannot be overstated. In settings with a paper-based health information system, there would be a need for creating a process of patient identification, data collection and follow-up. The most effective strategy to identify patients post-hoc in our settings was the ED triage where a system of identifying and separating trauma patients was likely to lead to most capture. 2- Training of personnel and availability of technical support to the staff.[1,3,7] 3- A third prerequisite is sustainable funding, which is by far the most common reason for the lack of a long-term implementation plan for a registry.[1,3,7,12] 4- Finally, one of the most important factors which alone can impact these barriers is institutional buy-in from senior hospital management. This provides an impetus for enhancing the quality of trauma care, improves motivation and participation of the care providers, ensures confidentiality of data and protects from medico-legal aspects of providing care to the injured.[12,2325,29]

Data abstraction and case ascertainment from this pilot revealed some important factors which will impact the process of implementation at a larger scale. The coordinator based implementation model did not include direct contact with patients, attendants or health care providers. Potentially it may result in loss of information of some variables which are supposed to be a part of medical records, as in our experience. In those settings where electronic health records are not available, access to medical records can be difficult. The alternative method of provider based data collection may ensure a higher level of completeness but in high volume facilities this could be challenging and more expensive.

Limitations
The study was done in a single tertiary-care academic institution with a electronic health information system, trauma team and round-the-clock availability of computed tomography (CT) and other diagnostic modalities. This setting may not reflect the reality of all private or public tertiary-care centres in Pakistan or in other developing countries. Wider, multi centre implementation studies would be needed to improve the data collection system and the implementation process.

Conclusion
KITR is the first electronic trauma registry in Pakistan developed with local resources. This registry was able to generate surveillance data such as mechanism of injuries, burden of severe injuries and quality indicators such as length of stay in ED, injury to arrival delay, injury severity and survival probability. To make the data collection process more effective, provider based data collection or making a standardized data collection tool a part of medical records will be helpful.

Treatment of Sternal Wound Infection With Vacuum-assisted Closure

Abstract and Introduction

Abstract:
Introduction. Previous work has demonstrated the efficacy of vacuumassisted closure (VAC) in the treatment of poststernotomy local wound infections, compared to historical treatment protocol. The negative pressure has been found to protect wounds against contamination, prevent wound fluid retention, increase blood flow, and increase rates of granulation tissue formation. For this study, a retrospective analysis compared patients receiving VAC as definitive treatment versus bridging to delayed flap closure. Methods. Sixteen patients developed sternal wound infections after cardiac surgeries at the authors' institution from 2006 to 2008. Data was gathered regarding patient comorbidities, treatment method, and outcome. Study objectives included assessment of risk factors that warranted secondary surgical closure and examination of long-term followup where VAC was the definitive treatment modality. Results. Group A (n = 12) had VAC as the final treatment modality. Group B (n = 4) required myocutaneous flap closure. One patient in Group B passed away prior to flap surgery. Both groups had similar risk factors, except Group B had a higher risk of body mass index (BMI) > 35 that was near statistically significant (P = 0.085; odds ratio = 0.0, 95% CI = [0.0 1.21]). Group A required a shorter hospital stay on average. Long-term follow-up showed the majority of Group A had completely healed sternal wounds 2-3 years from initial cardiac surgery. Conclusions. Vacuum-assisted closure as definitive treatment modality is a successful, first line therapy for local superficial sternal wound infections. When deep infections occur, however, VAC as bridge-to-flap coverage is recommended over attempted secondary healing with VAC.

Introduction
Sternal wound infections after cardiac surgery is a concerning complication, increasing morbidity and mortality. Approximately 0.3% - 5.0% of median

sternotomy surgical approaches result in infection.[1]Mortality rates range in the literature between 14% - 47%.[2] Preoperative risk factors for sternal wound infections include diabetes mellitus, chronic obstructive airway disease, obesity, and smoking.[3]Postoperative risk factors include blood transfusions, surgical chest exploration, prolonged postoperative ventilation, and longer stay in the intensive care unit.[46] Microbiology of sternal wound infections is variable. Staphylococcus aureus is the most common pathogen (29%), followed by Staphylococcus epidermidis (22%), with a notable frequency ofPseudomonas aeruginosa, methicillin-resistant staphylococci and streptococci, facultative and aerobic gram-negative rods, and anaerobes.[7,8] Since the mid-1950s, when the median sternotomy became a common approach for intracardiac procedures, the poststernotomy wound infection has had few treatment solutions.[9] Superficial infections were treated by irrigation, debridement, and open dressing changes. Deep infections were more difficult to treat. One of the first treatment options for deep sternal wound infections was a closed mediastinal antibiotic irrigation system.[10] This was an improvement from prior treatment regimen of open wound healing, following initial debridement, with frequent dressing changes to promote granulation and secondary wound closure.[11] Later treatment options involved debridement of devitalized tissue, daily dressing changes, and eventual delayed definitive closure of the wound by vascularized flaps such as pectoralis muscle, rectus muscle, or omental transpositions.[12,13] In the 1990s, the advent of negative pressure devices improved management of pressure ulcers and chronic wounds.[14] Since then, vacuum-assisted closure (VAC) devices have revolutionized wound management, improving skin grafts, enhancing reepithelialization of skin graft donor sites, and allowing safe temporary closure of the abdomen.[1517] Negative pressure devices improve tissue healing through several proposed mechanisms, including an increase in local blood flow, reduction in tissue edema, removal of chronic wound fluid and

necrotic tissue, reduction in bacterial colonization rates, and wound size contraction.[18] Vacuum-assisted closure consists of a vacuum pump, polyurethane foam into which an evacuation tube is embedded, and a transparent adhesive dressing (KCI International, San Antonio, TX).[19] The reticulated polyurethane foam has a 400 m - 600 m pore size. The foam is cut and contoured to fit the size of the tissue defect. The foam is covered with an adhesive drape and connected through the evacuation tube to the vacuum pump. The suction generates a continuous vacuum, equally distributed in the foam. The negative pressure ranges from 0 - 200 mm Hg with typical therapeutic range from 75 mm Hg 125 mm Hg. The foam is changed every 23 days. Nevertheless, the literature lacks a large prospective multicenter trial. Longterm outcome, in general, is lacking. Questions remain regarding sternal stability and need for further treatment. In an attempt to improve upon the existing methods, this study aimed to evaluate VAC as an effective short-term treatment and durable long-term treatment for sternal wound infections. Furthermore, this study sought to investigate the utilization of VAC to lessen the need for further invasive treatment options, such as myocutaneous and/or omental flap coverage. The authors expected patients who responded to VAC as definitive treatment to have fewer co-morbid risk factors than patients who required secondary surgical closure. In addition, the authors proposed that the VAC technique as definitive treatment would reduce treatment time, hospital stays, and outpatient followup. Finally, the authors expected long-term followup to show complete healing with VAC as definitive closure, as well as high patient satisfaction.

Materials and Methods

Approval from the Institutional Review Board at University of California Davis Medical Center was obtained for this study with individual consent waived. Institutional Review Board dates of approval are from April 18, 2007 to May 31, 2009.

Between 2006 and 2008, 590 coronary artery bypass graft procedures and 376 heart valve repair/replacement procedures were performed at the authors' institution (UC Davis Medical Center). A total of 16 patients developed sternal wound infections and were treated with the VAC technique either as definitive therapy (Group A) or followed by a second procedure (Group B). Initially, the intended treatment for all patients was VAC only. In 4 cases, however, the wound defect was unresponsive to definitive VAC alone. These patients required myocutaneous and/or omental flap closure. Sternal wound infections were defined by positive results of microbial culture, persistent erythema and/or drainage from wound, or persistent pyrexia with neutrophilia. The VAC wound management protocol was initiated in 2006. Patient characteristics and wound culture data were obtained and compared to assess differences between groups. Superficial infections were defined as infection having depth of skin to subcutaneous tissue. Deep infections were defined, for the purpose of this study, as infection having a depth of muscle to bone. An attempt was made to follow up with patients by phone to obtain further information. Patients were asked about current wound size, further interventions at outside hospitals, satisfaction with the VAC device and overall sternal wound infection experience using a scale of 1 to 10, in which 1 = worst experience, and 10 = best experience.

tatistics
Two-sided Fisher's exact test was used to assess association between patient group, patient characteristics, and wound culture. Odds ratios (ORs) with 95% exact confidence intervals (CIs) were reported. Summary statistics are expressed as mean standard deviation (SD) (median; range). The two-sided exact Wilcoxon rank-sum test was used to compare groups A and B for VAC duration, hospital days, follow-up time, and satisfaction with the VAC device. A P-value < 0.05 was considered statistically significant.

Results
Between 2006 and 2008, 16 patients developed superficial and deep sternal wound infections out of 835 median sternotomy procedures. The 3-year incidence was 1.9% (95% CI = 1.1% - 3.1%). Of these 16 patients, 4 required

flap coverage. Table 1summarizes the treatment and follow-up methods of each patient with early mortality. Table 2 summarizes the role of VAC in the 2 study groups. Group A consists of 12 patients who received VAC as definitive therapy. Of these, 6 received formal operating room irrigation and debridement. The other 6 received bedside or clinic irrigation and debridement. None of these patients had delayed primary surgical closure of their wound, nor did they receive flap coverage. Figure 1 presents a patient from Group A with VAC for a draining sternal wound. Group B consists of 4 patients who required flap coverage of their sternal wounds. One of these patients died due to cardiovascular failure prior to delayed flap closure. The 3 remaining patients received VAC until their myocutaneous and/or omental flap coverage. Figure 2 presents a patient from Group B who received VAC as a bridge to eventual flap closure.

(Enlarge Image)

Figure 1. Group A patient with draining sternal wound.

(Enlarge Image)

Figure 2. Group B patient with vacuum-assisted closure as bridge to secondary surgical closure. A) preoperative, B) postdebridement, C) sternal fixation devices covered with bilateral pectoralis major muscle flaps, D) postoperative day 27.

The following data is presented as the mean standard deviation; median; range. Group A (43.8 32.6; 35.5; 10136) had fewer days of VAC usage

than Group B (175.8 161.8; 176; 21 - 330), which was not statistically significant (P = 0.162). Group A (43.3 35.7; 32; 14 - 40) had fewer hospital days for their cardiac condition or sternal wounds than Group B (74 47.9; 56.5; 40 - 143), but was not statistically significant (P = 0.074). As expected, Group A (3.7 2.0; 3; 0 8) had fewer months of outpatient followup than Group B (1613.9; 12; 436), which was statistically significant (P = 0.012). There were no complications as a result of the VAC device. All 16 patients survived 90 days after initial infection diagnosis. Table 3 compares patient characteristics between Group A and Group B. As expected, both groups had similar preoperative risk factors. The one risk factor that reached near statistical significance was BMI > 35, found in 4 out of 10 patients in Group A, and all 4 patients in Group B (P-value = 0.085; OR = 0.0, 95% CI = [0.0 1.21]). Table 4 compares the wound cultures between Groups A and B. As expected, both groups had similar microbial pathogens. The most common isolates were methicillin-resistant Staphylococcus aureus (6/16, 38%), coagulase negative Staphylococcus aureus (5/16, 31%), and methicillin sensitive Staphylococcus aureus (4/16, 25%). Half the wounds had polymicrobial cultures (8/16, 50%). Telephone surveys were conducted with results shown in Table 5 . Ten of the 14 (71%) surviving patients were reached. The 4 patients lost to follow-up were from Group A. One patient from Group A and 1 patient from Group B had passed away at the time of phone questionnaire. All treatment and follow-up were performed at the University of California Davis Medical Center (UCDMC). None of the patients reached had received additional treatment for their sternal wounds outside UCDMC. One patient from Group A received 4 weeks of followup by a local cardiothoracic surgeon to assess healing after discharge from UCDMC, as the patient lived more than 1 hour away from UCDMC. All patients from Group A reached by telephone had healed sternal wounds. One

patient in Group B still has a 1 cm draining wound for whom surgery was pending at the time of publication. Patients' satisfaction with the wound VAC was diverse (6.4 3.0; 7.5; 2 - 10). Group A (7.1 2.8; 7.5; 2 - 10) had higher satisfaction with VAC than Group B (4.7 3.1; 4; 2 - 8), but it was not statistically significant (P = 0.333). Patients who had a low satisfaction with VAC cited inability to sleep due to device noise, interference with mobility, and difficulty with maintaining a vacuum seal.

Discussion
This study examines patient characteristics for those whom VAC was the sole treatment modality compared to those for whom VAC was used as a bridge-to-flap coverage. The results suggest that VAC has been successful as both a sole and bridging therapy in patients with sternal wound infections. This reduced the need for additional surgery for a significant number of patients in this study. There were no VAC-related deaths or complications. The 2 patients who died had significant comorbid conditions. Early mortality was 0 for this cohort. These results are consistent with the literature. Superficial infections did not require delayed flap surgery, and patients did well with IV antibiotics, VAC therapy, and irrigation and debridement. The patients who did not respond to VAC therapy as sole therapy were more likely to have deeper infections, such as mediastinitis. These patients, for the most part, did well after surgical intervention. However, 1 patient in the surgical treatment group died, while another still requires additional surgical intervention over 3 years after initial sternal wound infection. The VAC therapy was effective in that it obviated the need for flap coverage. While operative debridement is an additional procedure, the morbidity associated with the procedure is minimal compared to flap coverage. Requiring a debridement in the operating room is not considered a failure of VAC treatment.

On patient follow-up, the satisfaction with the VAC was not as high as expected. Perhaps this is a function of poor patient understanding of sternal wound infections. When patients were informed of the significant morbidity and mortality associated with these infections, their appreciation grew. It should be noted that all 4 patients lost to follow-up were from the group receiving VAC as definitive treatment. Compared to the literature, both groups have many of the known independent risk factors for mediastinitis, which include obesity (OR 1.27 6.49),[20,21] diabetes mellitus (OR 2.6 5.82),[22,23]smoking (OR 1.8 3.27),[20,24] congestive heart failure (OR 1.33 3.36),[23,25] renal failure (OR 6.93),[26] peripheral vascular disease (OR 2.11 3.7),[23,27] coronary artery disease (OR 2.67 6.85),[26.28] and post-cardiac surgery re-exploration (OR 3.3 9.2).[28,29] Vacuum-assisted closure has proven effective in the management of a spectrum of sternal wounds. With superficial wounds, it has obviated the need for delayed flap closure, as well as reducing the discomfort of multiple daily dressing changes to 13 VAC changes a week. For deep sternal infections, VAC plays a role as dressing as the wound progresses toward healing, or as bridge-to-flap coverage. This study is consistent with numerous studies in the literature describing improved outcomes with VAC, either as a stand-alone treatment or compared to other treatment modalities[3038] Vacuum-assisted closure has been found to be more efficacious than continuous irrigation.[32] All patients in this study survived 90 days after initial infection diagnosis. Domkowski et al[33] also presented a low early mortality (3.4%), and similar to this study, included both superficial and deep infections. A quarter of the patients (4/16, 25%) used VAC as a bridge-to-tissue flap treatment; this is consistent with Doss et al,[34] who bridged 20% of their patients with sternal wound infections using VAC. Sjogren et al[37] noted a significantly improved outcomes of sternal wounds treated with VAC compared to conventional treatment in terms of mortality rate and first line treatment failure rate. This study had 4 transitions from VAC to eventual flap coverage. Song et al[12] and Hersh et al[39] validate the use of VAC as bridge-to-flap coverage successfully. It should be noted that a comparison group with conventional treatment, consisting of wet-todry dressing changes, was not feasible as it is no longer part of the treatment algorithm at the corresponding author's institution. Furthermore, the authors do not believe in the

application of skin graft or delayed primary closure in the setting of infection. A better understanding of VAC requires a larger multicenter randomized trial with an agreed-upon treatment protocol. These protocols, although present in the literature, lack significant consensus.

Limitations
This study compares patients who received VAC for sternal wound infections after cardiac surgery. The main limitations of the study include the small number of patients, the lack of randomization, and uneven treatment groups. However, this is expected in a relatively rare condition. Furthermore, all patients began VAC therapy with the intention of it being the sole treatment modality.

Conclusion
Vacuum-assisted closure has improved the morbidity and mortality associated with sternal wound infections. Patients require fewer surgical interventions, resulting in improved recovery. Vacuum-assisted closure as definitive treatment modality is a successful, first line therapy for local superficial sternal wound infections. When deep infections occur, however, VAC as bridge-to-flap coverage is recommended over attempted secondary healing with VAC.

Pertussis and Persistent Cough

Abstract and Introduction
Abstract
Background: Epidemiologic issues of testing, treatment, prevention, immunization, mandated reporting, and post-exposure prophylaxis do not often intrude on the Emergency Department management of the well-appearing adolescent or adult with a couple of weeks of cough. Objectives: Considering that waning immunity to pertussis, the only vaccinepreventable disease with increasing cases and deaths in the United States, is responsible for 1235% of such illness, such issues need to be considered.

Discussion: Mostly self-limited in adults, transmission of pertussis to infants places them at risk for hospitalization and death. Pertussis is highly contagious (80% transmission), and atypical presentations are the rule in adults (cough alone) and infants (apnea, bradycardia, poor feeding). Treatment in the first few weeks can impact the clinical course, and later treatment can still prevent transmission. Clinical features like paroxysmal cough, inspiratory "whoop," and post-tussive emesis have mildly increased predictive values and may be absent in adults. Testing is unreliable after 3 weeks of cough. Treatment reduces communicability within 5 days and is suggested without regard to test results within 3 weeks of cough onset for those aged > 1 year (within 6 weeks of cough for < 1 year). Reporting requirements are based on the clinical case definition: ([Cough 2 weeks] + [paroxysms OR whoop OR post-tussive emesis]). Lower reporting thresholds are appropriate during an outbreak or when vulnerable populations are at risk. Post-exposure prophylaxis is recommended for at-risk contacts. Tdap is encouraged for all adults. Conclusion: Practical recommendations consistent with the most current guidelines are offered.

Introduction
Adults or adolescents with persistent cough frequently present to Emergency Departments (EDs). Once life threats and significant illness have been ruled out, there are additional and potentially serious epidemiologic issues that are often not addressed.

Typical Case Scenario

Clinical Information
An adolescent or adult, without significant comorbidities, presents to your ED with 23 weeks of cough. She saw another provider about 10 days ago, was diagnosed with "bronchitis" and treated with an albuterol inhaler and a cough medicine containing hydrocodone. The cough continues, is non-productive, occasionally better with the inhaler and cough medicine, and has caused the

patient to vomit once or twice. There is no history of fever. She is afebrile and well appearing with an occasional cough. There is no respiratory distress; vital signs are normal and oxygen saturation is 99%. Lungs are clear, with no edema. The remaining physical examination is unremarkable. A chest X-ray study is negative. There is mild subjective improvement after nebulized albuterol + ipratroprium, and no change in the examination.

Diagnosis and Disposition

Your patient continues to look well. You diagnose "persistent bronchitis" and consider antibiotics (recognizing the limited evidence to support the use of antibiotics in "acute bronchitis" usually defined as 3 weeks of cough).[1] Possibly adding some steroids to go with the inhaler, you discharge the patient for follow-up with their primary care physician.

Discussion: Clinical and Epidemiological Issues: What About Adult Pertussis in This Patient?
Caused by Bordetella pertussis, this highly contagious respiratory illness (80% secondary attack rate) has four classic phases in children ( Table 1 ). Atypical pertussis occurs in adolescents, adults, and in infants under 1 year of age.[2] Although easier to diagnose, "classic" cases are currently less frequent than the atypical ones. Like our clinical scenario, adolescents and adults with pertussis often present with cough for only 23 weeks. Among general practitioners in France, pertussis was considered as infrequently as 6% of the time in adult patients presenting with cough of over 14 days duration, even though such patients have a pretest probability of pertussis of 1235%.[3
5]

The only clinical features that "modestly" increase the pretest probability of pertussis in

adults are post-tussive emesis (Likelihood Ratio [LR] = 1.8) and inspiratory whoop (LR = 1.9). Paroxysmal cough is common, less often reported, less severe, and non-specific (LR = 1.1).[5] Thirty-nine percent of pertussis polymerase chain reaction (PCR)-positive patients in one outbreak had only 12 weeks of cough (median 12 days) and none of the other casedefinition symptoms of pertussis (paroxysmal coughing, "whoop," or post-tussive emesis).[6]

Infants may have no paroxysms or cough at all, presenting with poor feeding, apnea, or bradycardia. Infants are the group at highest risk for serious illness or death.[7] Atypical infections occur in immunized adolescents and adults due to waning immunity and issues of vaccine efficacy. The original whole-cell pertussis vaccine was dramatically effective when it was introduced in the 1940s. However, cases have been increasing again since the 1980s as a result of waning immunity, which rarely lasts more than 12 years.[8] Childhood cases are still the least frequent. Forty-four percent of pertussis cases now occur in adolescents or adults, and infants under 1 year of age have the highest reported rates of pertussis.[9] The vaccine is poorly effective in the first year of life, and most infants receive little passive immunity from their mother. The waning immunity of adults in the home limits any benefit of herd immunity. Household members are the primary source of 76 83% of infant pertussis.[10] Pertussis "boosters" were not recommended until 2006, when the newly licensed Tdap (tetanus, diphtheria, and pertussis) vaccine replaced Td, with the specific goals of protecting adults and "reducing the reservoir of pertussis in the population at large".[11]

Increasing Incidence
Pertussis is the only vaccine-preventable disease with increasing numbers of cases and deaths in the United States (US).[12] For example, in 2010, 9143 cases of pertussis (including 10 infant deaths) were reported from California, the most since 1947.[13]

Diagnosis
The diagnosis of pertussis is a clinical one. Positive testing is not required for presumptive diagnosis or treatment. The clinical case definition for pertussis from both the Centers for Disease Control and Prevention (CDC) and the American College of Chest Physicians is the same: "cough illness lasting for 2 weeks and one of the following: paroxysms of coughing, inspiratory "whoop," or post-tussive vomiting, without another apparent cause.[12,14]

Differential Diagnosis
The differential diagnoses of pertussis include infections caused by other etiologic agents, including adenoviruses, respiratory syncytial virus, Mycoplasma pneumoniae, Chlamydia

pneumoniae, and otherBordetella species such as B. parapertussis, and rarely, B. bronchiseptica or B. holmseii.[12]

Complications
Infants. Unvaccinated or incompletely vaccinated infants under a year of age are the primary concern. Their immunity is weak, their presentations atypical, and they are at the highest risk for hospitalization and death (usually due to secondary bacterial pneumonia). More than half of infants <1 year of age who get pertussis are hospitalized.[11] Eighty-nine percent of Canadians admitted for pertussis were under 6 months of age, and most deaths were in those under 3 months.[15] Dehydration, poor feeding with malnutrition and weight loss, sleep disturbance, and encephalopathy also occur. Although prompt treatment is most beneficial, early clinical diagnosis is difficult. Apnea may be the only symptom, with minimal cough. The best protection for infants is prevention by vaccination of household contacts, plus the early treatment of suspected cases in the home, coupled with post-exposure therapy for the infant. Adults. Complications in adults are largely cough-induced and include urinary incontinence, herniated disc, sudden hearing loss, carotid dissection, inguinal hernia, fractured ribs, intracranial hemorrhage, and cough syncope. Hospitalization rates can range from 3% to 12%. Angina, encephalopathy, pneumonia, and death can occur, especially in the very elderly.[11]

Epidemiologic Issues: Testing, Treatment, Reporting, and Post-exposure Prophylaxis

Pertussis is rarely considered in the differential diagnosis of cough in adults and adolescents, so it is rarely reported.[3] Rossi-Foulkes et al. note that although board preparation materials for pediatricians cover pertussis extensively, similar tools for the Internal Medicine and Family Practice board examinations provide no information on the public health issues addressed here.[16] Search of a commonly used bank of 1839 emergency medicine review questions from the Council of Emergency Medicine Residency Directors found no reference to "pertussis" or "whooping cough."

Testing. Chest radiographs are negative. Commonly available tests (nasopharyngeal culture and PCR) are most sensitive early in the clinical course. PCR has optimal sensitivity during the first 3 weeks of cough, with increasing false negatives after the fourth week.[17] Culture (the "gold standard" with 100% specificity) has very low sensitivity after 2 weeks of cough (1545% within 21 days of onset of cough; 13% if 3 weeks) and results may take as long as 2 weeks to return.[11] The CDC recommends that PCR and culture be done together whenever testing is done. Technique is important for PCR testing, using either Dacron swabs or nasopharyngeal washings. Other tests (serology, direct fluorescent antibody, gel electrophoresis) are not well standardized and not "confirmatory." Only 44% of patients aged 15 years with clinical pertussis have laboratory evidence of infection.[18] Testing may be more likely positive in childhood pertussis cases. Children are often seen earlier in the course of a cough illness than adults, when tests are more accurate. Treatment. Most previously immunized adults or adolescents we see in the ED with prolonged cough will eventually recover without antibiotics. Their partial immunity generally results in milder illness than that seen in infants and young children. Treatment is most effective in lessening symptoms if offered early in the disease, during the first 12 weeks before coughing paroxysms occur (the time when pertussis is most difficult to diagnose). Extra vigilance during outbreaks or in families may aid in early diagnosis and effective early treatment. "Clinicians should strongly consider treating before test results if clinical history is strongly suggestive, or the patient is at risk for severe or complicated disease, e.g., infants".[19] A major value of later treatment is the ability of that intervention to eliminate B. pertussisfrom the nasopharynx and prevent transmission to more vulnerable populations. For that reason, the CDC recommends treatment at any time within 3 weeks of cough onset for those over a year of age, and within 6 weeks of cough onset for those younger. The period of communicability is reduced to 5 days after treatment with antibiotics. Coughing (symptomatic) household members of a pertussis patient should be treated as if they have pertussis.[12] Earlier treatment and prevention of transmission may reduce the considerable burden of adult pertussis: loss of work, prolonged symptoms, and multiple provider visits.[11] There are no proven treatments for pertussis-induced cough; steroids and beta-agonists are not effective.

Macrolide antibiotics eradicate B. pertussis within 5 days. Recommendations include azithromycin (for 5 days) and clarithromycin (7 days). These have fewer gastrointestinal side effects, easier dosing, and better compliance than erythromycin (which is recommended for 14 days). In infants <1 month of age, azithromycin is preferred due to concerns for infantile hypertrophic pyloric stenosis, which is associated with erythromycin. Trimethoprim/sulfamethoxazole for 14 days is an alternative for patients who cannot tolerate macrolides and who are not pregnant, nursing, or <2 months of age. Doses are standard, except for infants <6 months, for whom azithromycin is recommended at 10 mg/kg/day for 5 days. No work or school is recommended for patients with suspected pertussis until completion of at least 5 days of antimicrobial therapy.[12] Reporting. US physicians are legally required to report pertussis cases when clinically suspected. Recognizing the difficulties with testing, reporting requirements are based on the clinical case definition: "A cough illness lasting at least 2 weeks with one of the following: paroxysms of coughing, inspiratory 'whoop', or post-tussive vomiting, without other apparent cause (as reported by a health professional)." In an outbreak, "a case may be defined as a cough illness lasting at least 2 weeks".[9] Historically, physicians report a minority of suspected pertussis cases to health departments.[16] Of urgent care providers in Utah, only 19% knew that clinically diagnosed pertussis was reportable.[20]There is some increase in reporting during outbreaks, most from positive laboratory tests. Adults are rarely reported. Post-exposure Prophylaxis (PEP). Individuals with pertussis are most infectious during the initial catarrhal period and for the first 2 weeks of spasmodic cough, but can remain infectious for up to 6 weeks, especially in the case of non-immune infants. Post-exposure chemoprophylaxis (with the same antibiotic regimens used for treatment) is recommended within 3 weeks of exposure for close contacts of patients with clinically suspected pertussis who are at risk of severe disease and death. These include infants, pregnant women in their third trimester (because they will soon have contact with a newborn), and those that are immunocompromised (personal communication with S.W. Martin, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention; January 12, 2012). As with pregnant women, chemoprophylaxis

should be considered for other contacts who can secondarily expose high-risk individuals (e.g., contacts of the primary case who are caretakers of young infants).

Conclusion
Adult Pertussis: Summary and Practical Approach to Epidemiologic Concerns
Testing. Given the generally low sensitivity, testing is not recommended after 3 weeks of cough, and is most useful for public health surveillance and case finding during an outbreak. The CDC recommends treating anyone you test. Treatment . Early treatment may benefit the patient; later treatment reduces transmission. Treat within 3 weeks of cough if >1 year of age, within 6 weeks of cough onset if <1 year. Reporting and Post-exposure Prophylaxis. Reporting and PEP are the areas in which the greatest uncertainties exist with regard to appropriate clinician behavior in the context of current CDC recommendations and health department requirements. Reporting. Reporting is mandated for this difficult clinical diagnosis, yet few doctors do so, "almost never" for adults. CDC epidemiologists note that: Nonspecific symptoms, atypical presentation and difficulties associated with laboratory diagnosis make pertussis diagnosis challenging, especially in adults who often present late in the clinical course. Reporting decisions should be based on clinical presentation and available epidemiologic information (personal communication with S.W. Martin, Division of Bacterial Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention; January 12, 2012). The same pretest probability of pertussis (1235%) that may lower one's threshold for macrolide therapy in the persistently coughing adolescent or adult may not seem high enough to warrant reporting those same patients as "clinically suspected" pertussis, because up to 90% will not have the disease. Balancing the knowledge that up to 39% of adult pertussis patients may have cough only

against the difficulty of health department follow-up for all persistently coughing adults, the best solution may be to report suspected adult pertussis in those who clearly meet the reporting standard ([Cough 2 weeks] + [paroxysms OR whoop OR post-tussive emesis]), with a lower reporting threshold for those who may put others at risk (workers in child care, schools, and health care; caretakers of infants under a year of age). Reporting threshold should fall during an outbreak, because the false-positive rate will also fall with an increase in prevalence. I consider reporting adults: 1) in an outbreak; 2) with household transmission from a diagnosed case; or 3) with a "Super-classic" (my words) clinical case. Additionally, I will likely report those patients I choose to test, insuring health department follow-up on test results. Prophylaxis. See above. Recommendations for pertussis prophylaxis are analogous to those for influenza during the 20092010 pandemic: to "consider" prophylaxis for persons with increased risk for complications who were close contacts of "confirmed, probable, or suspected" cases, during that person's infectious period (for pertussis, 21 days of cough onset). In the absence of a doctorpatient relationship, most ED prophylaxis will likely consist of discharge instructions with that recommendation. Prevention. Give or recommend Tdap to all adults, regardless of age, who have not had it (regardless of the interval from their last Td), with particular emphasis on parents-to-be, health care workers, and caretakers of infants. This includes pregnant women in the third trimester (>20 weeks) or immediately postpartum for those not who have not had it previously.[21]

Recommendations
Consider adding these epidemiologic concerns related to pertussis and its serious consequences in infants to your approach to the persistently coughing adolescent or adult. What is their Tdap status? The best evidence is that Tdap likely protects for at least 10 years.[2224] Ask about associated symptoms of paroxysmal cough, whoop, and post-tussive emesis. Are they in close contact with vulnerable populations, or secondarily with others with such contact?

Consider discussing/recommending post-exposure prophylaxis for their vulnerable contacts. If you are going to treat "persistent bronchitis," use a macrolide. Consider pertussis in infants under a year of age with a cough illness, apnea, poor feeding, or bradycardia. Inquire about persistently coughing family members and their Tdap vaccination status. Consider adding pertussis PCR and culture to Respiratory Syncytial Virus or other testing you might do in such infants. Consider macrolide therapy in seriously ill infants at risk for pertussis. Offer or advise Tdap to adults or adolescents who have not received it, with particular emphasis on pregnant women and their families. Utilize your local health department. Give them a call. Health department concerns often center on preventing additional cases in the workplace or in schools and other groups of school-age children (clubs, teams). They are often contacted regarding issues of school exclusion, appropriate treatment, contact assessment, or prophylaxis recommendations. Opinions, practices, and resources will vary, and your local agency sets the standard. Look for changes and more specific guidance in an outbreak. Take advantage of their services: counseling, second opinion, case finding, free pertussis testing, and free immunizations (Tdap). They can give your patient or their teacher a call and take many problems off your hands. Consider reporting. Advise isolation for 5 days if treating.

30-yr Course and Favorable Outcome of Alveolar Echinococcosis Despite Multiple Metastatic Organ Involvement in a Nonimmune Suppressed Patient
Abstract and Introduction
Abstract

We report the 30-yr history of a well-documented human case of alveolar echinococcosis, with a lung lesion at presentation followed by the discovery of a liver lesion, both removed by surgery. Subsequently, within the 13 years following diagnosis, metastases were disclosed in eye, brain and skull, as well as additional lung lesions. This patient had no immune suppression, and did not have the genetic background known to predispose to severe alveolar echinococcosis; it may thus be hypothesized that iterative multi-organ involvement was mostly due to the poor adherence to benzimidazole treatment for the first decade after diagnosis. Conversely, after a new alveolar echinococcosis recurrence was found in the right lung in 1994, the patient accepted to take albendazole continuously at the right dosage. After serology became negative and a fluoro-deoxy-glucose-Positron Emission Tomography performed in 2005 showed a total regression of the lesions in all organs, albendazole treatment could be definitively withdrawn. In 2011, the fluoro-deoxy-glucose-Positron Emission Tomography showed a total absence of parasitic metabolic activity and the patient had no clinical symptoms related to alveolar echinococcosis.

The history of this patient suggests that multi-organ involvement and alveolar echinococcosis recurrence over time may occur in non-immune suppressed patients despite an apparently "radical" surgery. Metastatic dissemination might be favored by a poor adherence to chemotherapy. Combined surgery and continuous administration of albendazole at high dosage may allow alveolar echinococcosis patients to survive more than 30 years after diagnosis despite multi-organ involvement.

Introduction
Alveolar Echinococcosis (AE) caused by the metacestode of the "fox tapeworm" Echinococcus (E.) multilocularis is one of the most lethal helminthic diseases in humans.[1,2] Humans become infected through contact with eggs (oncospheres) present in the feces of the definitive hosts, most often foxes or dogs, but also wolves and cats, by handling the animals or by ingesting contaminated vegetables without cooking them. Only observed in the northern hemisphere, and especially in central Europe, Russia/Siberia, Central Asia, Western China, north of Japan, and Alaska, AE is also one among the rare parasitic diseases with a surgical treatment, because of its "tumor-like" progression. The only available antiparasitic chemotherapy, i.e. high doses of albendazole (ABZ) or mebendazole

(MBZ) given continuously, is only parasitostatic in this disease and the complete resection of the parasitic lesions is thus recommended whenever possible.[3,4] In most of the cases, the parasitic lesions are initially located in the liver and may then invade adjacent organs; however, true metastases may also be seen in any organ or tissue. Extra-hepatic locations were already present at diagnosis in 34% of cases in those cases recorded in the EurEchinoReg European registry from 1982 to 2000.[5] When the clinical symptoms at presentation are related to such extra-hepatic locations, the diagnosis of AE is difficult and very often it is confirmed after surgery on the pathological aspects of the lesions and/or evidence of its parasitic nature by PCR.[6,7] Except for lung metastases, AE metastatic dissemination is often associated with immune deficiency of the host.[8] "Multi-organ AE" usually qualifies cases with hepatic, pulmonary and cerebral locations;[9,10] metastases to more than 2 organs/tissues are extremely rare, either simultaneously or successively, in immune-competent subjects. The involvement of several organs is among the main causes of poor prognosis.[11,12] We report here the case of an immune-competent man with an AE discovered from a lung metastasis in 1981 who had 5 different locations of the disease, either simultaneously or successively, underwent 4 surgical operations on 6 different organs/tissues in 4 different locations, and who may nevertheless be considered cured from the disease after a unique follow-up of more than 30 years.

Case Report
The patient was a 38-year-old man who was admitted into a local hospital of the region of Franche-Comt, Eastern France, for the cure of an inguinal hernia. Systematic surgery preassessment disclosed by chance a lung nodule which evoked pulmonary cancer. Thoracotomy with right upper lobectomy of the lung was performed in January 1982 in Dijon University Hospital, France, and the fortuitously discovered "tumor" was totally removed. At the end of the operation, through the diaphragm, the liver appeared to be enlarged, with a modified structure, and the surgeon found a liver tumor with a very hard consistency and a necrotic content of 100 mL which was aspirated; the diagnosis of AE was thus evoked. Pathological examination of the lung "pseudo-tumor" and of the surgical biopsies taken after diaphragm incision, as well as the Computed Tomography (CT) images obtained after the operation (Figure 1), confirmed the diagnosis. As the patient wished to postpone the proposed liver resection, flubendazole was given to the patient at a daily dosage of 9.0

g[13] until 1985 when he was eventually transferred to Besanon University Hospital for radical liver resection and inclusion in chemotherapy trials. The patient gave his consent to be included in clinical research studies within the framework of the study "Immunogenetics of AE in humans" first, in 1994, then as part of the prospective follow-up of AE patients in the FrancEchino Registry in 2003; both prospective cohort studies were approved by the French legal ethical committee (CCPPRB/Franche-Comt: comit consultatif pour la protection des personnes en recherche biomdicale, for the region of Franche-Comt); written informed consent was obtained from the patient for publication of this report and any accompanying images.

(Enlarge Image)

Figure 1. Abdominal CT Scan (1982): alveolar echinococcosis heterogeneous lesion of the right lobe of the liver, including a large necrotic area (N) and a calcified area (C).

In May 1985, the specific serology tested by ELISA using crude larval Em (EmC), and purified Em2 as antigens[14] was positive but in favor of a stable disease, as well as the unchanged CT images which showed the well-limited parasitic mass in the right liver with partial necrosis and calcifications, and no involvement of the biliary tree and/or hepatic and portal vessels. A right hepatectomy with cholecystectomy was thus performed, which removed a 1.45 kg parasitic tumor and was considered to be curative. Flubendazole was replaced by MBZ at a daily dosage of 4.5 g from June to December 1985; then replaced by ABZ at a daily dosage of 400 mg twice a day for 1 month followed by 2 week-interruptions of treatment as recommended by the manufacturer at that time ('cyclic administration'). [3]MBZ and ABZ were given within the framework of WHO-coordinated therapeutic trials[15,16] and the patient had a regular prospective follow-up in Besanon University Hospital WHO-clinical reference Centre. ABZ treatment was spontaneously interrupted by the patient himself in June 1986, because of minor subjective side-effects (digestive discomfort) without any biological abnormalities. The previous treatment by MBZ was then resumed. In May 1988,

there were no clinical or biological abnormalities; ultra-sound examination only showed the expected enlargement of the left liver usually observed after right hepatectomy and the patient was considered to be cured. As serology (ELISA using crude larval antigen EmC, and purified Em2) was fully negative and liver and lung imaging remained normal, MBZ was thus stopped on May, 1988. In April 1991, the patient was admitted into the University Hospital/AE reference Centre with a 3-month long history of headache and right exophtalmia; visual acuity, ocular mobility and eye fundus examinations were normal. A re-increase in specific antibodies in the serum was disclosed although abdominal and thoracic imaging showed no recurrence of AE in the liver or adjacent organs, or in the lung. Cerebral CT-scan and Magnetic Resonance Imaging (MRI) disclosed 2 lesions: one in the orbit and one in the right frontal lobe of the brain. Both had a micro-polycystic hypodense structure, with scattered calcifications. The surgical treatment consisted of a subtotal resection of the lesion in the right frontal lobe, the adjacent dura, the invaded part of the eye lid and the eroded frontal bone. Brain and right orbital AE, with bone involvement was confirmed by the pathological examination of the lesions which showed necrotic and fibrous tissue filled with small cysts. Microscopically, typical E multilocularisgerminal layer and protoscoleces were observed as well as periparasitic epithelioid cells and the granulomatous infiltrate of macrophages, lymphocytes and giant cells. ABZ at a daily dosage of 800 mg, following the "cyclic" administration as before, was introduced again in June 1991, with recommendations of strict adherence to the treatment. At the end of 1991, the situation was clinically stable, with a marked regression of the remaining orbital and cerebral lesions. Once again, upon the patient's demand, because of digestive discomfort, in March 1992, ABZ was switched to MBZ; then the patient spontaneously interrupted his treatment in July 1992. ABZ was resumed in October 1992 when the patient complained of ptosis of the right eye and reduction of his visual acuity, despite unchanged images of the brain and orbital region. But once again the patient stopped his treatment in February 1993. At the beginning of 1994, thoracic CT-scan showed AE recurrence, as a 45 mm in diameterlesion in the right pulmonary apex (Figure 2). From that date, the patient accepted to take ABZ at an increased dosage (20 mg/day/kg) and continuously, as suggested by the recommendations of the WHO-Informal Working Group on Echinococcosis for severe AE

cases (1996 Guidelines). In February 1996, as AE seemed under control, the 52-yr old patient benefited from an elective left hip prosthesis in that same local hospital he had been admitted first in 1981. Because of an abnormal structure of the bone disclosed by the surgeon, a pathological examination was performed in Besanon University Hospital on bone sections. Despite abnormalities compatible with parasitic vesicles, formal AE diagnosis could not be ascertained since no typical germinal layer was observed; the inflammatory infiltrate could also be due to associated arthritis; and no PCR could be performed retrospectively on the lesions because of technical issues. Complete clinical and radiological assessment of possible metastatic locations of the disease did not disclose any other lesions. In 1999, the size of the pulmonary lesion had markedly decreased (to 21 mm in diameter) and in 2000 serology (Em2 and EmC ELISA) was completely negative. Since then, the patient has been in good health, except for overweight (height: 178 cm; weight: 100 kg; abdominal perimeter: 117 cm) and subjective complaints such as headaches and pain in his left hip. Adherence to ABZ treatment was good. Yearly imaging exams including ultrasound examination and CTscans did not show any changes in the images. A Fluoro-DeoxyGlucose (FDG)-Positron Emission Tomography (PET) combined with Computed Tomography (CT) performed in 2003 showed the absence of FDG uptake by the lesions in all organs, 1 h after FDG injection. Serology (Em2 and EmC ELISA) remained negative and ABZ was thus withdrawn definitively in April 2003. At last follow-up in 2011, there was no evidence of recurrence/relapse, as evidenced by negative PET-CT with conventional and delayed acquisition of the images 3 h after FDG injection (Figure 3), and by negative Em2+ ELISA serology (Bordier Affinity Products, Crissier, Switzerland).[17] At that time, serology was also assessed by E. multilocularis western-blot (LD Bio, Lyon, France)[18] in parallel on two sera: a sample frozen in 1987, and a freshly collected sample. With the serum sampled in 1987, two bands at 7 kDa and 2628 kDa were observed, while with the serum collected in 2011 only binding to 2628 kDa Echinococcus antigens was observed. Disappearance of the 7 kDa band thus confirmed imaging and ELISA serology results.

(Enlarge Image)

Figure 2. Thoracic CT Scan (1994): recurrent pulmonary lesion of alveolar echinococcosis after the right lobectomy performed in 1982.

(Enlarge Image)

Figure 3. Positron Emission Tomography combined with CT Scan (2011): no FluoroDeoxyGlucose uptake whatever the previously involved organ.

Discussion
In the 1970s, in the patients with AE, life expectancy was estimated to be reduced by 18.2 and 21.3 years for men and women respectively; by 2005 it was reduced by approximately 3.5 and 2.6 years, respectively.[19] Continuous ABZ has greatly contributed to prolonged

survival.[12,20] Presence of metastasis is generally considered of poor prognosis in AE; a recent study on 387 French patients confirmed that it was actually associated with higher AEspecific mortality.[12] Moreover, multiple extra-hepatic locations of AE are usually associated with immune suppression.[8] The history of the AE patient we are reporting suggests that multi-organ involvement and AE recurrence over time may occur in non-immune suppressed patients despite an apparently "radical surgery" which removed all visible AE lesions, and might be favored by a poor adherence to the benzimidazole chemotherapy. It also shows, however, that combined surgery and continuous administration of ABZ at high dosage may allow patients to survive more than 30 years after diagnosis despite multi-organ involvement. Occurrence of metastatic locations of AE lesions is one of the hallmarks which justify the similarities between AE and malignant tumors, as exemplified by the PNM classification which parallels the TNM classification of tumors.[21] Invasion of neighboring organs and tissues ("N" in the PNM classification) byE. multilocularis progression from the initial liver location results in secondary lesions in the right lung through the diaphragm, in peritoneal and retroperitoneal cavities, hepatic pedicle, round ligament, right kidney and adrenal gland, pancreas, stomach or spleen. True metastases ("M" in the PNM classification) are mainly observed in the lung (7% of cases),[5] more rarely brain (3%), spleen (1%)[5] or bones (less than 1%),[6,22] in the skin[23] muscle,[24] heart,[24,25] and in any possible anatomical locations.[2] The clinical presentation of our patient combined all main 3 locations; the brain location was associated with orbit, eye, and facial/cranial bone involvement, which is per se very rare and was for that reason published several years ago.[26] In addition, metastases occurred along a 13-year period, from the patient's inaugural presentation with a lung metastasis in 1981 to the last discovery of another lung metastasis in 1993; meanwhile, brain, eye and bone locations were disclosed. In otherwise non-immune suppressed patients, genetic factors leading to poor cellular immunity and a marked trend to immune tolerance are statistically associated with the severity of AE, which includes metastasis formation,[27,28] e.g. HLA B8, DR3, DQ2 haplotype; however, this 'at risk' haplotype was not present in our patient which actually was HLA A11, A10; B13, B41; DR7, DR13; DQ2, DQ3. The main risk factor for disseminated AE is actually immune suppression. First observations were reported after liver transplantation for AE in patients who were not treated by benzimidazoles after receiving the liver graft.[29] Increase in residual extra-hepatic lesions as

well as occurrence of brain or spleen metastasis were observed in a European series of 45 transplanted patients.[30] In the 1990s, such observations contributed to greatly reduce the indication of liver transplantation to treat AE.[3] Since then, AE occurrence in patients with kidney or heart transplantation has also been reported.[31,32] Rapidly progressing AE was also observed in AIDS[33,34] and it was suggested that pregnancy could be a tolerogenic situation which may have favored occurrence of brain metastasis.[35] Within the recent years, development of cancer chemotherapy and use of more potent immune suppressive drugs as well as immune modulating biological agents, especially anti-TNF compounds, have contributed to an increase in the number of disseminated and/or rapidly progressing "opportunistic AE" in patients with cancer, hematological disorders or chronic inflammatory diseases.[7,36,37] Our patient, however, all along his clinical course, did not present any overt spontaneous or disease/treatment-related- immune suppression. There is no reason either to think that he was infected by an unusually virulent strain of E. multilocularis: several patients were diagnosed with slowly progressing, non-disseminated AE within a 30 km-range from his residence; and recent genetic analyses of E. multilocularis in the patient's endemic area do not favor major differences which might be responsible for more or less aggressive potential of the various strains.[38] Absence of the highly specific E. multilocularis 1618 kDa band at Western Blot on the serum sampled in 1987, a few months before the first treatment withdrawal, was compatible with the negative ELISA results at that time, and thus with "inactive" lesions; however, "inactivity" was only temporary, as the following evolution well demonstrated. First introduced in the pharmacopeia at the end of 1970s, benzimidazoles, although they do not kill E. multilocularis larvae, are the only drug available to treat AE and, being able to prevent metacestode growth and provided they are taken for life, have markedly contributed to improve AE patients' prognosis.[11,12,19] Albendazole, taken at the appropriate dosage of at least 15 mg/kg/day according a continuous schedule, is currently considered as the drug of choice.[4] Bad adherence to treatment, initial treatment with flubendazole which was later proved inefficient in AE, iterative switches from ABZ to MBZ because of side-effects, as well as insufficient dosage of ABZ due to the "discontinuous" schedule of administration recommended during the 19801990s[3] may have been the main reasons for repeated dissemination of AE lesions in the reported patient's. Adherence to treatment is essential and

bad adherence, or ABZ withdrawal because of side-effects, have been shown to be crucial for lesion recurrence in patients with residual lesions after liver transplantation.[20] It is remarkable that, after 13 years of unreliable intake of the antiparasitic drugs tainted with successive extra-hepatic metastases of the disease, good adherence to ABZ treatment was followed by the inactivation of the lesions, negative PET-CT images and serology within 10 years, and a decision of definitive treatment withdrawal. The relative 'tolerance' of physicians in charge of the patient towards his bad adherence to treatment may also have been encouraged by the overconfidence of the surgeons about the complete resection of the initial lung and liver lesions. In addition, the absence of any reliability in the patient's allegations regarding last intake of the antiparasitic drug prevented them to measure ABZ sulfoxide plasma levels, which is, however, usually routinely performed in that reference center; MBZ measurements were not available when the patient switched to MBZ, which he did most of the time; and lower bioavailability of MBZ is well known.[3] Difficulties to assess completeness of the surgical resection and necessity to adjunct benzimidazoles even if resection was considered to be curative, were stressed as early as the 1980s[14,39] and were further highlighted by the observations of recurrence after liver transplantation.[30] Measurement of ABZ levels and proper interpretation of low levels of ABZ sulfoxide as evidence of bad adherence to treatment are also essential in the follow-up of AE patients.[4] It is fortunate that our patient is still alive 30 years after the diagnosis of AE and 9 years after antiparasitic drug withdrawal; this observation confirms the therapeutic effectiveness of ABZ, despite its absence of parasitocidal effect, and possible withdrawal of the drug in highly selected subjects. Absence of FDG uptake at PET-CT assessment and negative Em2+ ELISA serology in 2011 suggest an aborting evolution of the parasitic lesions whatever the involved organ or tissue; persistence of the 2628 kDa band at Western Blot is likely related to the persistence of non-viable parasitic tissue in various organs of the patient, as is often the case in AE patients with aborted lesions.[2] The current recommendations concern the "security margin" to be observed by the surgeons for the resection of AE lesions, similar to those recommended in oncological surgery, as well as the 2-yr ABZ continuous treatment which is mandatory in the so-called "radically operated on" patients.[4] Following these recommendations would have likely prevented the occurrence of the distant metastases and also radically changed our patient's quality of life.

Temporomandibular Joint Arthroplasty With Human Amniotic Membrane

A Case Report

Abstract and Introduction

Abstract
This case reports the usage of human amniotic membrane combined with a costochondral graft as an interpositional material in temporomandibular joint reconstruction for the first time in humans. Because of the favorable outcome 20 months postoperatively, it has to be considered as an approach bringing to light the antiadhesive potential of amniotic membrane. This case report must be regarded as initial spadework and should motivate other institutions to intensify their clinical research in this field. Because of the fact that currently used interpositional materials do not prevent the recurrence of temporomandibular joint ankylosis sufficiently, it is of great interest to establish a proper therapeutic intervention fulfilling these demands. Furthermore, the demonstrated antiadhesive properties of amniotic membrane highlight its multifaceted field of application. Nevertheless, further studies have to prove the findings reported in our case.

Introduction
Gap arthroplasty and interpositional arthroplasty are the most commonly used therapeutic options to treat TMJ (temporomandibular joint) ankylosis. The major complication of both interventions is the recurrence of ankylosis. To date, no surgical or nonsurgical procedure can guarantee nonrecurrence; therefore, its effective therapy posts a clinical challenge. On the basis of the unsatisfactory results experienced with the current treatment options, our aim is to find an adequate new way to deal with TMJ ankylosis.[15]

The Case
A 53-year-old, otherwise healthy woman presented herself because of pain in the left TMJ region and an insufficient mouth opening, which exhibited itself in the preceding weeks. Clinical examination revealed a maximum interincisal opening of 15 mm. In preliminary investigations, radiographic, computed tomographic, and magnet tomographic analyses were performed and a

neoplasm at the left condylar process, involving the whole joint cavity, was detected. To clarify the finding, a biopsy of the suspicious mass was performed and a giant cell tumor was diagnosed. After careful evaluation of the diagnosis, risks, and benefits of all possible treatment options, the woman was assessed to undergo radical excision of the tumor, including total resection of the condylar process plus the articular disk. On the basis of the positive outcome of the previously performed animal experiments and the informed consent of the patient, a costochondral graft combined with an allogeneic human amniotic membrane (HAM), was chosen to reconstruct the joint head. For this intention, ethical approval of the independent ethics committee was obtained.

Methods
Starting the operation, the occlusion was fixed with wire splints. In the next step, the costochondral graft was harvested without any complication from the right ninth rib and kept in a moist bandage during excision of the neoplasm. Through a submandibular and a preauricular approach, the tumor and the condylar process were totally resected. The margins of all frozen sections were clear. The harvested autologous costochondral rib graft was prepared and shaped before transplantation (Fig 1). Osteosynthesis of the cleaned, shaped, and adjusted rib graft to the mandibular ramus was ensured with 2 mini plates (Fig 2). The chondral part of the rib graft was placed facing the articular fossa of the temporal bone, forming a new condyle that ensured articular functions. The cryoconserved and moist allogeneic amniotic membrane was mounted onto the condylar part of the new joint head. Thereby, the epithelial side of the membrane was adjusted toward the joint cavity (Fig 3). Before closing the operation site, the patient's occlusion was reevaluated and adjusted. Intraoperatively, no surgical complications were noticed and also the anesthesia was well tolerated by the patient.

(Enlarge Image)

Figure 1. Positioning of the harvested rib graft.

(Enlarge Image)

Figure 2. Osteosynthesis of the rib graft with 2 mini plates.

(Enlarge Image)

Figure 3. Insertion of the amniotic membrane.

Previous Page
Section 3 of 5

Next: Results

Results
The postoperative treatment included intensive physiotherapy and regular follow-up appointments. Wound healing, mouth opening, and her general condition were evaluated on the 1st, 3rd, 5th, 7th, 14th, and 30th day and then every month postoperatively. A 20-month follow-up of the patient showed uneventful wound healing without complication. Ankylosis of the TMJ was avoided by long-term observation and the patient presented a maximum interincisal opening of 32 mm 8 months postoperatively (Fig 4). The correction of the ankylosis has to be considered as the consequence of an optimal interplay of the resection of the tumor, the successful reconstruction, aggressive physiotherapy, and good compliance by the patient.

(Enlarge Image)

Figure 4. Maximum interincisal mouth opening, 8 months postoperatively.

Discussion
Temporomandibular joint ankylosis is classified into true and false ankylosis. False ankylosis is the restricted movement of the joint due to extra-articular factors such as muscles, nerves, or the coronoid process. True or intra-articular TMJ ankylosis is the obliteration of the TMJ by fibrous tissue, mainly resulting from trauma, surgical intervention, infection, rheumatoid arthritis, or neoplasm, causing pain, insufficient mouth opening, and difficulty in mastication and speech.[1] The main treatment options for TMJ ankylosis are interposition arthroplasty and gap arthroplasty. There is some controversy in the literature regarding the most appropriate therapeutic intervention to use. Many systematic reviews tried to summarize the literature and to point out the best operative intervention, but no common consent could be established.[2,3,5] In gap arthroplasty, the condylar process is cut and no interpositional

material is inserted.[2,3] The complications of gap arthroplasty are anterior open-bite deformities, functional impairment, and frequently reankylosis. In interpositional arthroplasty, alloplastic or biological materials are transplanted between the cut mandibular head and the joint cavity.[1,3] Alloplastic materials, such as silicone or Teflon, achieve a preservation of the vertical height of the ramus, though allergic and foreign body reactions have been reported.[6] The facts that alloplastic materials are very expensive and must not be used in children, limit their utilization.[7] The disadvantage of biological materials such as fascia lata and fat graft is that they cannot preserve the vertical height of the mandibular ramus and in consequence, functional problems are common.[7] Beside this, Alloderm, an acellular dermal matrix, showed good results when used for TMJ reconstruction.[8] At present, the most frequently used interpositional materials are bone grafts such as metatarsal, sternoclavicular, or costochondral grafts.[9] Among these, the costochondral graft interposition has been extremely popularized.[9] In consequence, the use of an autologous costochondral graft combined with free fat graft, as it is reported to have the best long-term outcome, is considered the criterion standard.[6,10,11] Nevertheless costochondral grafts have an unpredictable growth potential and their extraction is always accompanied by donor site morbidity.[12,13]Furthermore, development and recurrence of TMJ ankylosis poses a problem with any interpositional material.[14] In conclusion, none of the currently utilized methods can prevent TMJ ankylosis and leads to completely satisfactory joint reconstruction. Out of these insufficient therapeutic possibilities, it is our aim to establish a new therapy option to handle TMJ ankylosis. Amniotic cells have been documented to express various surface markers associated with embryonic stem cells and are known to have pluripotent properties.[15,16] Production of anti-inflammatory factors, such as IL-1 and IL-2 receptor antagonists and IL-10, plus endostatin, all of which inhibit endothelial cell proliferation, angiogenesis, and tumor growth, is another property of amniotic cells.[17] Because of these properties, amniotic cells were used in the form of amniotic membranes for skin transplantation for the first time in 1910 and nowadays many disciplines are trying to use the beneficial effects of amniotic cells.[18,19] As HAM promotes wound healing and proven antiadhesive effects, it is used as a skin application in many surgical disciplines and in gynecology to prevent intrauterine adhesions.[10,20,21] Concerning oncology, HAM is stated to interfere with tumor angiogenesis, growth, and metastasis. Its best known, most auspicious, and the only routinely accepted application in Germany is ocular surface reconstruction.[10]As

HAM has been found to prevent fibrosis and to promote wound healing, it is considered to have the potential to decrease or even prevent TMJ ankylosis.[19,22,23] In the rabbit model, HAM has prevented fibrosis and reankylosis as an interpositional material.[1] The aim of this case was to build on the encouraging outcome of the animal study and to prove these findings. Because of the fact that we used HAM combined with a costochondral graft, the results cannot be directly linked to HAM. On the basis of our experience, the outcome was better than in our conventionally treated cases, but no solid evidence exists. Although no fibrotic intergrowth, an uneventful integration of HAM, and recovery of a normal mouth opening range can be reported in this case, this innovative therapy option has to be compared to the currently applied interventions. As this case describes the usage of HAM combined with a costochondral graft in TMJ reconstruction for the first time, it has to be considered as an approach bringing to light the great potential of amniotic membrane in this indication. Beyond a doubt, one case makes it hard to accept efficacy and safety. Nevertheless, the reported antiadhesive effect, preventing TMJ ankylosis 20 months postoperatively, gives hope to finding a proper interpositional material preventing TMJ ankylosis. In any event, further clinical studies with high validity need to be performed to verify these findings.

Hello. I am Dr. Maurie Markman from Cancer Treatment Centers of America in Philadelphia, Pennsylvania. I want to briefly discuss a very interesting abstract that was presented at the recent meeting of the Society of Gynecologic Oncology. The abstract was titled "NCCN (National Comprehensive Cancer Network) Treatment Guidelines for Ovarian Cancer: A Population-based Validation Study of Structural and Process Quality Measures."[1] This was a very interesting paper that has been discussed in various media presentations and newspapers around the country. It is important to point out that the paper, although it represents a very well-done study, does not conclude what some have suggested it concludes. By looking at a population of patients in the California Cancer Registry who were either treated by NCCN guidelines or not, the analysis demonstrated that the population treated according to NCCN guidelines had superior outcomes.

However, the conclusion that the patients had a superior outcome because they were treated by NCCN guidelines is simply not scientifically accurate. One can say that there is an association between the 2, but there is not a cause and effect here -- at least none that can be determined by this analysis. The issue of association versus causation is a very well-understood problem. Clearly, an individual doctor taking care of an individual patient will do what is best for that patient on the basis of the doctor's clinical judgment. For example, a patient may have considerable comorbidity or quite extensive disease, and the clinician may decide that it is not in the patient's best interest to follow certain guidelines. That patient, because of the comorbidity or the extensive disease, may have an inferior outcome compared with individuals who have less comorbidity and less extensive disease, and who are treated by NCCN guidelines. This does not mean that the treatment that followed NCCN guidelines was the cause of the superior outcome, but rather that there is an association between the 2. To conclude that patients will definitely have a superior outcome if guidelines are followed (and, quite frankly, which might not be in the patient's best interests because of comorbidity or the extensive nature of the disease) would not be good from a clinical perspective. So clearly, more work needs to be done. The analysis itself is very important. The issue of quality in the care of all cancer patients and in this particular setting of patients with ovarian cancer needs to be evaluated at the national level, the regional level, and the individual institutional level. One must be very cautious, when one sees an association between 2 factors, not to conclude that factor A caused factor B, because the end result may not have a positive effect on patient care. I would encourage you to read the paper when it is finally published, so that you can examine this issue in greater detail in terms of the impact that this analysis and these types of analyses should have on our thinking about the quality of care we provide our patients.

I thank you for your attention.

Autologous Bone Marrow Grafts Promising for Low Back Pain

Pauline Anderson

Apr 15, 2013

Print Email

Editors' Recommendations

MRI Overused for Low Back Pain, Study Shows

Low Back Pain: Osteopathic Manual Therapy Appears to Help

Low Back Pain Guidelines Aid in Management

Topic Alert
Receive an email from Medscape whenever new articles on this topic are available.

Add Back Pain to My Topic Alert

Drug & Reference Information

Lumbar Facet Arthropathy Lumbosacral Radiculopathy Low Back Pain and Sciatica

Fort Lauderdale, Florida Interest is growing in the potential for autologous bone marrow injections to treat lower back pain, one of the most common types of pain and among the most difficult to treat. At this year's American Academy of Pain Medicine (AAPM) 29th Annual Meeting, researchers here reported positive results at 2 years after such biocellular grafts, with additional anecdotal evidence for some patients up to 3 years after injection. For lead researcher D. Joseph Meyer, MD, PhD, a specialist in anesthesiology and pain medicine in Columbia, Missouri, the results are encouraging. "I think these preliminary observations are very intriguing and in my opinion, they're exciting," he said. Dr. Meyer hopes these new data will "spark some interest" among his colleagues to develop a prospective long-term study. Bone Marrow Aspirate For this study, researchers at the Columbia Interventional Pain Center and at the Bluetail Medical Group in Chesterfield, Missouri, collected data on 24 consecutive patients (17 men; average age, 45 years) who had had low back pain for an average of 4 years and had not responded to numerous treatment approaches. All had evidence of lumbar disc degeneration, as seen on MRI or computed tomography.

Dr. D. Joseph Meyer

From each patient, investigators obtained 60 mL of iliac bone marrow aspirate, which they then spun down and concentrated in a centrifuge device to obtain a 8-mL bone marrow aspirate concentrate (BMAC). They injected 1 to 1.5 mL of this concentrate into each

affected lumbar disc annulus under fluoroscopy, and then immediately injected another 0.5 to 1 mL outside the annulus. They treated no more than 2 discs in each patient. Patients also got a small amount of injected steroid. The whole process "literally took 20 minutes to an hour," said Dr. Meyer. During follow-up, none of the 24 patients experienced worsening of their pain, although just 12 received only the autologous treatment; the others had other therapies. Of the12 patients treated solely with disc BMAC, researchers had information on 11 at 5 to 12 months. Of these, 8 experienced significant improvement in their pain, reporting an average of 74% pain relief. They also experienced a significant improvement in being able to tolerate activity and/or a significant drop in medication use. Dr. Meyer also reported on the 8 patients at 2 years. Five of the 8 had significant pain relief (average 81% pain relief). The 3 who didn't respond in the first year still did not respond in the second year. Although not included in the poster, Dr. Meyer said that the 3 patients he and his colleagues have tracked out to the 3-year mark "are doing great." Limitations of the study include the fact that it was an uncontrolled retrospective case review from which no statistical conclusions can be drawn. Dr. Meyer believes these are the first published findings of this treatment approach in low back pain, which affects an enormous number of people. The pain is typically due to degenerative discs, although it's largely a clinical diagnosis. "In the vast majority of cases, there's obvious evidence of disc degeneration," said Dr. Meyer. "But there is no one test; it's more considered to be a diagnosis of exclusion." Treatment options for this type of pain include surgery and spinal fusion, but these interventions garner only mediocre results. Cellular grafts are the "latest evolutionary development" in the hunt to find an injection that cures low back pain, said Dr. Meyer. In the past, researchers have investigated injections of

fibrin, a natural "sealant" that helps in the healing process, and more recently, platelet-rich plasma. This new approach encompasses bone marrow cells and cells from the body's own fat stores, sources that have "multipotent abilities," said Dr. Meyer. Such treatments are being investigated for a variety of painful conditions in addition to low back pain, including degenerative joint conditions, arthritis, and sports injuries. Although he's optimistic about the study's implications, Dr. Meyer is also realistic. "I never use the word 'regenerate'," he said. "There's nothing statistically positive in the poster; these are observations." However, he said he hopes the results will help define the benefits of BMAC and spur further research. "I hope that as we study this in a more controlled prospective fashion we can refine the technique, and learn what is actually happening." Catabolic Steroid? After presentation of the study results, Robert Martin, MD, a sports medicine specialist, Coastal Spine and Pain Center, Jacksonville, and Orange Park, Florida, criticized the treatment approach for including a steroid. "We have been injecting steroids for years and years and years, and we know that it causes atrophy of tissue," he later told Medscape Medical News. "If you want an anabolic process, if you want tissue to strengthen and growth, you don't put a catabolic steroid in there."
Information from Industry

Rebif (interferon beta-1a): Update your knowledge Explore efficacy data

Catabolic steroids are reported to break down muscle mass, while anabolic steroids build muscle mass, he noted. In fact, added Dr. Martin, a steroid will "sabotage" the very reparative process the injection is supposed to stimulate.

It's great that the research team is pursuing an innovative treatment approach, "but my thinking is that if you're going to think outside the box, then get outside the box," said Dr. Martin. He also pointed out that the patients in the study could have pain in addition to discogenic pain. It's much more difficult to differentiate various types of pain for example, arthritis and musculo-tendon pain in patients with back pain than in those with, say, knee pain, he said. Dr. Meyer has disclosed no relevant financial relationships. American Academy of Pain Medicine (AAPM) 29th Annual Meeting. Abstract 203. Presented April 11, 2013.

Fort Lauderdale, Florida Young patients with chronic pain who take medications other than opioids to cope with their symptoms may experience cognitive adverse effects from these drugs, a new study has found. Although researchers expected that patients with chronic pain would have a higher anticholinergic cognitive burden than healthy persons, they were surprised to find that the highest burden was among those aged 30 to 39 years. "Physicians should be aware of the potential cholinergic effects of medicines they prescribe to patients and whether or not there's a cumulative effect to these medicines," said lead author Cady Block, a PhD candidate at the University of Alabama at Birmingham with an interest in the neuropsychology of pain. She presented the findings here at the American Academy of Pain Medicine (AAPM) 29th Annual Meeting. Common Medications The study included 30 patients with chronic pain who had an average age of 40.51 years; 62.10% were female, and 86.2% were white. The average

number of medications was 3.93, and the average pain intensity rating during testing was 4.76. No patients were using opioid analgesic medications. Patients were compared with 30 control patients (average age, 38.56 years; 56.70% female and 70.0% white). In this group, the average number of medications was 1.20 and the average pain intensity rating was 0.14. Common medications that have anticholinergic effects include those taken for pain (atropine, codeine), for sleep (alprazolam, amitriptyline), for mood (bupropion, haloperidol), for gastrointestinal discomfort (dimenhydrinate, loperamide), and for cardiac issues (atenolol, dipyridamole). These drugs block cholinergic transmission at muscarinic receptor sites. These receptors mediate the primary cognitive effects attributed to cholinergic pathways, including attention, learning, and short-term memory. Researchers used the Anticholinergic Cognitive Burden (ACB) Scale developed at the Indiana University Center for Aging Research. The scale identifies the severity of anticholinergic effects of prescription and over the counter (OTC) medications divided into "possible" (score of 1) and "definite" (score of 2 or 3 depending on the strength of the effect) categories. The easy-to-use scale, available online, "is a nice way to capture potential cognitive effects of these medicines, which as a variable are hard to account for sometimes in research," commented Block. This is the first time that the scale, which is typically applied in an older population, was used in patients with chronic pain, she said. Researchers also used several neuropsychological tests to measure psychomotor speed, attention/concentration, memory, and executive function. After adjustment for demographic characteristics, patients with chronic pain had a significantly higher ACB score (P < .01) than the controls. Those in the 30- to 39-year-old age group had the highest ACB scores. Whereas patients in this younger age group took about the same number of medications as older

persons (3.66 vs 4.02), the ones they took were more likely to have anticholinergic properties. The executive function of patients with chronic pain who have a higher ACB score appeared to be affected. On the Wisconsin Card Scoring Test-64, they made more perseverative errors than healthy controls (P = .05). It's typically older people who have neuropsychological deficits with the use of anticholinergic medications. As the body ages, the central nervous system becomes more sensitive to these medications. The study should serve as a reminder that when a patient needs a different medication, physicians might consider an option that doesn't increase the risk for cognitive dysfunction, said Block. Polypharmacy a Problem Polypharmacy among patients with chronic pain is a growing problem. These patients take medications not only for their pain but also for depression, anxiety, sleep problems, and adverse drug effects, such as constipation and drowsiness, said Block. As well, these patients might reach for OTC medications that can also have cholinergic effects. Block sees "a big role" for neuropsychological assessment in pain management, and the ACB scale could be a valuable addition to assessment. For example, she said, it could be used to assess how patients are doing before and after an intervention to find out what happens in the aging population as they are being treated for pain, and to assess pain in the rehabilitation population, she said.
Information from Industry

Rebif (interferon beta-1a): Update your knowledge Explore efficacy data

Keith Susko, MD, a specialist in pain relief and physical medicine in Fort Myers, Florida, found the study results "surprising" and novel.

"She's right on target with this problem with the anticholinergic drugs effecting cognition of patients, which has been overlooked. She has overturned some rather surprising findings here where there was actually a worse response in the younger age group of 30-39 years than in the elderly age group due to anticholinergic effects." There's not enough being done on the neuropsychology of pain, added Dr. Susko. The study was part of a larger IIS grant funded by Teva Pharmaceuticals. American Academy of Pain Medicine (AAPM) 29th Annual Meeting. Poster 221. Presented April 12, 2013. Fort Lauderdale, Florida For the first time, researchers have demonstrated the involvement of the spinal cord in chronic pain. Using resting-state functional MRI of participants in whom central sensitization had been induced and who were not experiencing pain, researchers showed the spread of functional connectivity in the spinal cord. Although this spinal cord component had been shown in animals, this was the first time that spinal involvement in pain has been shown functionally in the resting state in humans, said Brittney R. Reyes, research assistant in the laboratory that carried out the experiments. The lab is led by Sean Mackey, MD, PhD, professor, pain medicine, anesthesia, and chief, Pain Management Division, Stanford University, California. The new study helps contribute to the "whole picture" of chronic pain, rather than just the brain component, said Reyes. "This is the start of what will likely be a lot of spinal cord research," she told Medscape Medical News. "I think that we're going to find that the spinal cord is as important as the brain is in terms of pain." She presented the findings here during the American Academy of Pain Medicine (AAPM) 29th annual meeting.

Brittney R. Reyes

Whole Picture The study included 2 groups, each with 8 healthy volunteers. In the first group, researchers induced central sensitization by first applying heat for 5 minutes to the left lower forearm of each participant, after which they measured the area of mechanical hyperalgesia. They then applied a cream with capsaicin (a substance that blocks a chemical involved in transmitting pain signals to the brain) to the forearm for 30 minutes. When the cream was removed, they administered the heat again for another 5 minutes and then remeasured the area of mechanical hyperalgesia. "The spread in the mechanical hyperalgesia, or secondary hyperalgesia, was seen as a sign by us that we had induced central sensitization," said Reyes. In another nonsensitized group, researchers applied heat to the left forearm of each participant for 30 seconds and followed this with 40 seconds of rest, repeating this process 7 times. Participants in this group did not receive the capsaicin cream. All participants had 2 scans: a "heat pain" scan that was used to functionally define the dorsal horn and a "resting state" scan during which the participants were asked to lie in the scanner without completing a task. Participants in both groups reported having no pain before or during the resting-state scan.

Dr. Sean Mackey

The resting-state scans allowed the researchers to assess spontaneous lowfrequency fluctuations in signals in the spinal cord. They were looking for functional relationships between regions, or areas that act similarly, that may reflect direct or indirect relationships between regions. "The idea behind functional connectivity is that just because a subject isn't performing a task does not mean that communication between different regions or within the central nervous system ceases," said Reyes. The task-related heat pain scans from the nonsensitized group defined the region of interest. Researchers used this region of interest to extract time courses from the resting-state scans. "We put these time courses into our analysis as a regressor, and basically asked our model to look for areas in the spinal cord that acted similarly to this original time course." It's believed that areas that act similarly in the spinal cord indicate functional connectivity when the subject is at rest. Spreading Connectivity The analysis of resting-state scans showed that the functional connectivity in the nonsensitized group was limited to a specific area in the C6 region of the spinal cord. However, the functional connectivity in the sensitized group extended into adjacent spinal segments, spreading from C6 to C5 in regions of the dorsal horn. This was the spread that was measured the second time in this sensitized group.

"We found that central sensitization results in a spread in functional connectivity within the spinal cord, even when subjects report having no pain," said Reyes. "Given the incidence of hyperalgesia (hypersensitivity to pain) and allodynia (sensitivity to touch) experienced by patients with chronic pain, the implication for the role of the spinal cord is very important." The lab at Stanford is among the first to do functional imaging in the spinal cord, said Reyes. "It took a lot of technological advancements to get to this point because it's difficult to image the human spinal cord." After the presentation, Wally Smith, MD, professor, medicine, and chair, Division of Quality Health Care, Virginia Commonwealth University School of Medicine, Richmond, complimented the team for a study that he found was "elegant, complex, and meaningful." However, Dr. Smith asked whether functional connectivity is "a basic human trait" or whether the mechanism differs depending on the presence and type of disease. Senior author Dr. Mackey responded that this is not yet known.
Information from Industry

Do your relapsing MS patients need a support service they can call day or night? Learn about MS LifeLines

"These are just the first baby steps," he said. However, he added that the team will test patients with fibromyalgia to see whether they have the same type of enhanced synchrony and connectivity across spinal cord segments. Asked how long it takes for get central sensitivity, Dr. Mackey noted that it probably occurs rapidly, judging by the nature of the capsaicin model causing mechanical hyperalgesia outside of where the cream is applied. "I think that for the first time we're actually showing how and where it's occurring, and at least part of the mechanism behind it." This study was supported by the National Institutes of Health.

American Academy of Pain Medicine (AAPM) 29th Annual Meeting. Abstract 107. Presented April 12,

Drinking cup of beetroot juice daily may help lower blood pressure
April 15, 2013 Study Highlights:

Blood pressure decreased about 10 mm Hg in high blood pressure patients who drank a cup of beetroot juice daily. Beetroot juice contains dietary nitrate, which may help relax blood vessel walls and improve blood flow. Increasing intake of foods rich in dietary nitrate may be an affordable and attainable way to manage blood pressure, researchers said.

EMBARGOED UNTIL 3 p.m. CT/4 p.m. ET, Monday, April 15, 2013 DALLAS, April 15, 2013 A cup of beetroot juice a day may help reduce your blood pressure, according to a small study in the American Heart Association journal Hypertension. People with high blood pressure who drank about 8 ounces of beetroot juice experienced a decrease in blood pressure of about 10 mm Hg. But the preliminary findings dont yet suggest that supplementing your diet with beetroot juice benefits your health, researchers said. Our hope is that increasing ones intake of vegetables with a high dietary nitrate content, such as green leafy vegetables or beetroot, might be a lifestyle approach that one could easily employ to improve cardiovascular health, said Amrita Ahluwalia, Ph.D., lead author of the study and a professor of vascular pharmacology at The Barts and The London Medical School in London. The beetroot juice contained about 0.2g of dietary nitrate, levels one might find in a large bowl of lettuce or perhaps two beetroots. In the body the nitrate is converted to a chemical called nitrite and then to nitric oxide in the blood. Nitric oxide is a gas that widens blood vessels and aids blood flow. We were surprised by how little nitrate was needed to see such a large effect, Ahluwalia said. This study shows that compared to individuals with healthy blood pressure much less nitrate is needed to produce the kinds of decreases in blood pressure that might provide clinical benefits in people who need to lower their blood pressure. However, we are still uncertain as to whether this effect is maintained in the long term. The study involved eight women and seven men who had a systolic blood pressure between 140 to 159 millimeters of mercury (mm Hg), did not have other medical complications and were not taking blood pressure medication. The study participants drank 250 mL of beetroot juice or water containing a low amount of nitrate, and had their blood pressure monitored over the next 24 hours. Blood pressure is typically recorded as two numbers. Systolic blood pressure, which is the top number and the highest, measures the pressure in the arteries when the heart beats. Diastolic blood pressure, the bottom and lower number, measures blood pressure in the arteries between heart beats. Compared with the placebo group, participants drinking beetroot juice had reduced systolic and diastolic blood pressure even after nitrite circulating in the blood had returned to their previous levels prior to drinking beetroot. The effect was most pronounced three to six hours after drinking the juice but still present even 24 hours later. In the United States, more than 77 million adults have diagnosed high blood pressure, a major risk factor for heart diseases and stroke. Eating vegetables rich in dietary nitrate and other critical nutrients may be an accessible and inexpensive way to manage blood pressure, Ahluwalia said.

Getting people to eat more fruits and vegetables is challenging, but results of the study offer hop e, she said. In the U.K., the general public is told that they should be eating five portions of fruit or vegetables a day but this can be hard to do. Perhaps we should have a different approach to dietary advice. If one could eat just one (fruit or vegetable) a day, this is one more than nothing and should be viewed as positive. The USDA recommends filling half your plate with fruits and vegetables, and the American Heart Association recommends eating eight or more fruit and vegetable servings every day. Co-authors are Suborno M. Ghosh, B.Sc.; Vikas Kapil, M.A., M.B.B.S., M.R.C.P.; Isabel Fuentes-Calvo, Ph.D.; Kristen J. Bubb, Ph.D.; Vanessa Pearl; Alexandra B. Milsom,BSc PhD; Rayomand Khambata, B.Sc., Ph.D.; Sheiva Maleki-Toyserkani, B.Med.Sci.; Mubeen Yousuf, B.Med.Sci.; Nigel Benjamin, M.D., F.R.C.P.; Andrew J. Webb, Ph.D., M.R.C.P.; Mark J. Caulfield, M.D., F.R.C.P.; and Adrian J. Hobbs, B.Sc., Ph.D. Author disclosures are on the manuscript. The British Heart Foundation funded the study. The American Heart Association has tips on adding more fruits and vegetables to your diet. Follow @HeartNews on Twitter for the latest heart and stroke news. ### Statements and conclusions of study authors published in American Heart Association scientific journals are solely those of the study authors and do not necessarily reflect the associations policy or position. The association makes no representation or guarantee as to their accuracy or reliability. The association receives funding primarily from individuals; foundations and corporations (including pharmaceutical, device manufacturers and other companies) also make donations and fund specific association programs and events. The association has strict policies to prevent these relationships from influencing the science content. Revenues from pharmaceutical and device corporations are available atwww.heart.org/corporatefunding. Additional resources, including multimedia, are available in the right column of this link:http://newsroom.heart.org/news/drinking-cup-of-beetroot-juice-daily-may-help-lower-bloodpressure?preview=dd96e3d0b67c320d2457686f580fe9e0 For Media Inquiries: (214) 706-1173 Maggie Francis : (214) 706-1382 Carrie Thacker: (214) 706-1665; carrie.thacker@heart.org Julie Del Barto (broadcast): (214) 706-1330; Julie.DelBarto@heart.org For Public Inquiries: (800) AHA-USA1 (242-8721) heart.org and strokeassociation.org

uman Gene Patent Case Goes to Supreme Court

Decision could have wide-ranging implications for biotech industry, medical research, experts say Monday, April 15, 2013

Related MedlinePlus Page Genes and Gene Therapy

MONDAY, April 15 (HealthDay News) -- The question of whether human genes can be patented is at the center of a case to be heard Monday by the U.S. Supreme Court. The court's decision could have a profound effect on medical research in the country, efforts to fight diseases such as breast and ovarian cancer, and the multi-billion dollar medical and biotechnology industry, experts say. The U.S. Patent and Trademark Office has been granting patents on human genes for more than 30 years, according to the Associated Press. The current case stems from a 2009 lawsuit filed by the American Civil Liberties Union on behalf of breast cancer patients and health professionals challenging the validity of Myriad Genetics' patents on two genes associated with breast and ovarian cancer risk. BRCA1 and BRCA2 gene mutations are linked to increased risk of breast and ovarian cancer, and Myriad sells the only BRCA gene test, which gives them a monopoly on a highly profitable business, critics say. Opponents also say that allowing companies to patent human genes or parts of human genes will hinder potentially lifesaving research to fight diseases such as breast cancer. "What that means is that no other researcher or doctor can develop an additional test, therapy or conduct research on these genes," Karuna Jagger, executive director of Breast Cancer Action, told the AP. Myriad has the patent on a specific method of isolation and identification of specific BRCA mutations, explained Dr. Iuliana Shapira, director of cancer genetics at North Shore-LIJ Cancer Institute in Lake Success, N.Y. The way Shapira sees it, patents allow a temporary legal monopoly over the use of an invention, but genes are "not invented" by human intelligence and therefore cannot be patented. On the other hand, if scientists "edit" the gene, for example removing parts of it or introducing some parts and thus creating "a synthetic gene" -- something that does not exist in nature and has specific functions -- that type of "synthetic gene" is patentable, Shapira said. "Some synthetic genes are used by the biotechnology industry to make therapeutic antibodies, such as Rituxan (used to treat lymphoma) or Herceptin (used to treat breast cancer)," Shapira said. "These synthetic genes have patents that nobody disputes." Some of the opposition's concerns are overblown and some are simply incorrect, according to Mark Capone, president of Myriad Genetics Laboratories, Inc., a subsidiary of Myriad. "Myriad cannot, should not and has not patented genes as they exist in the human body on DNA," Capone told the AP. "This case is truly about isolated DNA molecules, which are synthetic chemicals created by the human ingenuity of man that have very important clinical utilities, which is why this was eligible for a patent." However, the ACLU contends that isolating the DNA molecules doesn't stop them from being DNA molecules, and that these molecules are not patentable. That position appears to have the support of the Obama administration, the AP reported. In court papers, Solicitor General Donald Verrilli said

artificially created DNA can be patented, while "isolated but otherwise unmodified genomic DNA is not patent-eligible." Dr. Barbara Pober, a professor of medical sciences at Quinnipiac University's Frank H. Netter MD School of Medicine, agreed that the Myriad genes should not have been patented. "Genetic sequencing in the laboratory, as performed by Myriad Genetics, does not involve sufficient alteration or modification of the gene to warrant the protections afforded to a patented product," she said. "In fact, patenting genes inhibits dissemination of genetic information to patients, as well as innovation and technology by eliminating the ability of both small and large laboratories to develop genetic tests," she added. As a result, consumers and hospitals pay more for tests and wait longer for the results, she said. Whatever the ruling, the implications will be significant in terms of shaping laws governing biotechnology and medical innovations, one expert said. "The intellectual framework that comes out of the decision could have a significant impact on other patents -- for antibiotics, vaccines, hormones, stem cells and diagnostics on infectious microbes that are found in nature," Robert Cook-Deegan, director for genome ethics, law and policy at Duke University, said in a statement, the AP reported. "This could affect agricultural biotechnology, environmental biotechnology, green-tech, the use of organisms to produce alternative fuels and other applications," he explained. Current research would come to a halt, others contend. Biotechnology Industry Organization said in a friend of the court brief that invalidating the current patent law "would chill a wide range of important activities that benefit society," according to The New York Times. Others are less convinced that the ruling will have an enormous impact, especially since the Myriad patents will expire over the next two years. Dr. James Evans, a professor of genetics and medicine at the University of North Carolina, Chapel Hill, told the Times that the significance of the nine justices' decision "will be much more ideological than it will be practical." SOURCES: Iuliana Shapira, M.D., director, cancer genetics, North Shore-LIJ Cancer Institute, Lake Success, N.Y., and investigator, Feinstein Institute for Medical Research in Manhasset, N.Y.; Barbara R. Pober, M.D., professor, medical sciences, Frank H. Netter M.D. School of Medicine, Quinnipiac University, Hamden, Conn.; April 15, 2013, New York Times and Associated Press HealthDay

ANCER: L'lectroporation irrversible traite et laisse les tissus sains intacts

Actualit publie le 15-04-2013 Society of Interventional Radiology

unenouvelle thrapie mini-invasive capable de traiter en toute scurit des cancers complexes, tout en laissant les tissus sains intacts savre un traitement prometteur pour les cancers difficiles, suggre cette tude prliminaire

Llectroporation irrversible (IRE),

prsente la 38e Runion scientifique annuelle de Society of Interventional Radiology.

Dfinie comme une nouvelle thrapie qui utilise des impulsions lectriques dune microseconde pour supprimer le cancer au niveau cellulaire sans endommager les tissus sains environnant s, elle savre, selon ces rsultats, particulirement bnfique dans le traitement du cancer du foie, du poumon, du pancras et d'autres cancers proches les vaisseaux sanguins, des nerfs et autres structures sensibles.
Le Pr Constantinos T. Sofocleous, auteur principal et radiologue interventionnel au Memorial SloanKettering Cancer Center de New York explique que le traitement apparat particulirement efficace chez les personnes atteintes d'un cancer qui s'est propag au-del de la tumeur primaire et qui n'ont pas de bonnes options de traitement. LIRE utilise de puissants champs lectriques pour crer de petits trous dans la membrane cellulaire pour perturber l'quilibre entre les molcules de l'intrieur et de l'extrieur de la cellule sans entraner dautres dommages cellulaires. IRE semble ce stade une thrapie idale pour le traitement des tumeurs situes proximit de structures sensibles. L'IRE cible prcisment les cellules cancreuses : Alors que d'autres traitements, comme la chirurgie, la radiothrapie ou l'ablation thermique, peuvent endommager les tissus sains situs proximit de la tumeur, l'IRE perfore prcisment les cellules cancreuses donc entrane moins de dommages aux vaisseaux sanguins, nerfs, canaux biliaires etc Dans son t ude, mene auprs de 25 patients atteints, prsentant au total 40 mtastases au foie, poumon, pancras, glande thyrode, prostate, utrus, muqueuse utrine, ovaires et rectum, lIRE prouve sa scurit dans le traitement des cancers mtastass ou diffus, du foie, du poumon, de la vessie et de la rgion pelvienne. La taille moyenne des tumeurs des participants tait d'environ deux centimtres. LIRE a t utilise en raison de la localisation des lsions, proximit de structures critiques, sur 30 sances de traitement et sans complications majeures. LIRE se situe la frontire des traitements de radiologie interventionnelle non chirurgicaux et ne ncessite que des incisions de la taille d'une pointe de crayon pour guider par imagerie de petits instruments pour les tumeurs cibles. La technique nentrane que des risques minimes et des temps d'arrt limits pour les patients. Le Dr Sofocleous conclut : Nous traitons

souvent les patients qui n'ont pas d'autres options thrapeutiques conventionnelles ou qui ont une sant telle que mme la chirurgie mini-invasive est trop dangereuse. Nous travaillons accrotre l'efficacit de l'IRE et souhaitons pouvoir bientt la tester vs d'autres traitements .
Source: Society of Interventiona

INFECTIONS bactriennes: Des nano-ponges pour absorber les toxines

Actualit publie le 16-04-2013 Nature Nanotechnology

Dveloppe par des ingnieurs de l'Universit de Californie San Diego, cette nanosponge savre capable de retirer en toute scurit un large ventail de toxines dangereuses de la circulation sanguine, y compris les toxines produites par le SARM, E. Coli, le venin de serpent ou dabeille. Ces travaux, passionnants, viennent dtre publis dans ldition du 14 avril de la revue Nature Nanotechnology.
Constitues d'un noyau polymre biocompatible envelopp dans une membrane de globule rouge naturel, la nanosponge, jusquici tudie chez la souris, peut neutraliser les toxines qui dtruisent

les cellules en perant des trous dans leurs membranes cellulaires et absorber diffrentes toxines porognes (ou qui augmentent la porosit) quelle que soit leur structure molculaire.
Le principe semble simple : Afin d'chapper au systme immunitaire et rester en circulation dans le sang, les nano-ponges sont enveloppes (cloaking) dans des membranes de globules rouges. L'quipe spare les globules rouges partir d'un petit chantillon de sang l'aide d'une centrifugeuse, puis met les cellules dans une solution qui les fait gonfler et clater, librant l'hmoglobine et permettant disoler les membranes. Celles-ci sont ensuite mlanges avec les nanoparticules sphriques jusqu' ce que les nanoparticules se revtent de la membrane de globule rouge. Une membrane de globule rouge peut permettre de produire des milliers de nanoponges, qui sont 3.000 fois plus petites qu'un globule rouge. Un taux de survie de 44% : Dans leur tude, les chercheurs montrent que les nano-ponges ont permis 89% des souris inocules avec SARM (Staphylococcus aureus rsistant la mticilline), de neutraliser la toxine alpha-hmolysine et de survivre des doses ltales. En fin dexprience, lutilisation de nano-ponges aprs administration de la dose ltale conduit un taux de survie de 44%. Ces nano-ponges denviron 85 nanomtres de diamtre reprsentent une manire innovante d'liminer les toxines du sang, explique Liangfang Zhang, professeur de nanognie l'UC San Diego et l'auteur principal de l'tude. Leur grand avantage, en regard de traitements spcifiques chaque toxine, est de pouvoir neutraliser les toxines causes par un large ventail d'agents pathognes, dont le SARM et d'autres bactries rsistantes aux antibiotiques. Des travaux, qui selon son auteur, pourraient mme conduire la disparition de thrapies spcifiques pour les morsures de serpents venimeux et les piqres d'abeilles, ce qui rendrait le traitement vital plus rapidement et facilement accessible. Ces nanoparticules dguises en globules rouges pourraient aussi tre aussi utilises pour dlivrer des mdicaments directement une tumeur. Mais la priorit des chercheurs,

face lmergence des antibiorsistances, reste aujourdhui le traitement anti -SARM.

that absorbs pore-forming toxins (Visuel Nanosponge@Zhang Research Lab)

Source: Nature Nanotechnology doi:10.1038/nnano.2013.54 online 14 April 2013 A biomimetic nanosponge

Clinical and cellular features in patients with primary autosomal recessive microcephaly and a novel CDK5RAP2mutation
Lina Issa1,2, Katrin Mueller3, Katja Seufert3, Nadine Kraemer1,2, Henning Rosenkotter4, Olaf Ninnemann1, Michael Buob4, Angela M Kaindl1,2* and Deborah J Morris-Rosendahl3,5*

*Corresponding authors: Angela M Kaindl angela.kaindl@charite.de - Deborah J Morris-Rosendahl deborah.morrisrosendahl@uniklinik-freiburg.de Equal contributors Author Affiliations
1

Institute of Cell Biology and Neurobiology, Charit University Medicine, Berlin, Germany Department of Pediatric Neurology, Charit University Medicine, Augustenburger Platz 1, Berlin 13353, Germany Institute of Human Genetics, Albert-Ludwigs University Medical Center Freiburg, Breisacherstr 33, Freiburg 79106, Germany Sozialpaediatrisches Zentrum, Klinikum, Ludwigsburg, Germany

Current Address: National Heart and Lung Institute, Imperial College London, London, UK

For all author emails, please log on. Orphanet Journal of Rare Diseases 2013, 8:59 doi:10.1186/1750-1172-8-59

The electronic version of this article is the complete one and can be found online at:http://www.ojrd.com/content/8/1/59 Received: Accepted: Published: 12 January 2013 29 March 2013 15 April 2013

2013 Issa et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background
Primary autosomal recessive microcephaly (MCPH) is a rare neurodevelopmental disorder that results in severe microcephaly at birth with pronounced reduction in brain volume, particularly of the neocortex, simplified cortical gyration and intellectual disability. Homozygous mutations in the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene CDK5RAP2 are the cause of MCPH3. Despite considerable interest in MCPH as a model disorder for brain development, the underlying pathomechanism has not been definitively established and only four pedigrees with three CDK5RAP2 mutations have been reported. Specifically for MCPH3, no detailed radiological or histological descriptions exist.

Methods/Results
We sought to characterize the clinical and radiological features and pathological cellular processes that contribute to the human MCPH3 phenotype. Haplotype analysis using microsatellite markers around the MCPH1-7 and PNKP loci in an Italian family with two sons with primary microcephaly, revealed possible linkage to the MCPH3 locus. Sequencing of the coding exons and exon/intron splice junctions of the CDK5RAP2 gene identified homozygosity for the novel nonsense mutation, c.4441C>T (p.Arg1481*), in both affected sons. cMRI showed microcephaly, simplified gyral pattern and hypogenesis of the corpus callosum. The cellular phenotype was assessed in EBV-transformed lymphocyte cell lines established from the two affected sons and compared with healthy male controls. CDK5RAP2 protein levels were below detection level in immortalized lymphocytes from the patients. Moreover, mitotic spindle defects and disrupted -tubulin localization to the centrosome were apparent.

Conclusion
These results suggest that spindle defects and a disruption of centrosome integrity play an important role in the development of microcephaly in MCPH3.

Keywords:
Microcephaly; CDK5RAP2 mutation; Cell division; CDK5RAP2 protein

Introduction
Primary autosomal recessive microcephaly (MCPH) delineates a genetically heterogeneous and rare subgroup of congenital microcephalies characterized by a pronounced reduction of brain volume, particularly of the neocortex, simplified gyral pattern and intellectual disability [1,2]. Homozygous mutations of the Cyclin-dependent kinase 5 regulatory subunit-associated protein 2 gene, CDK5RAP2 (OMIM*608201), were identified in 2005 as a cause for MCPH type 3 (MCPH3, OMIM#604804) [3]. To date, three different mutations have been identified: two in three Pakistani families and one mutation in a Somali patient: (i) a nonsense mutation in exon 4 (c.246T>A, p.Y82X), (ii) an A to G transition in intron 26 (c.4005 -15A>G, p.R1334SfsX5) introducing a new splice acceptor site, a frame shift and a premature stop codon, and (iii) a nonsense mutation in exon 8 (c.700 G>T, p.E234X) [3-5]. All three mutations have been proposed, but not shown, to lead to a truncated protein and a loss of CDK5RAP2 function. CDK5RAP2 is associated with the centrosome, microtubuli and Golgi apparatus, is enriched in neural progenitors within the ventricular and subventricular zone of the immature brain, can be also detected in glial cells and early neurons, and is strongly downregulated with brain maturation[6,7]. One current model for the microcephaly phenotype caused by CDK5RAP2 mutation

invokes a premature shift from symmetric to asymmetric neural progenitor cell divisions with a subsequent depletion of the progenitor pool and a reduction in the final number of neurons, and decreased cell survival [6,8]. Underlying mechanisms include a deregulation of the role of CDK5RAP2 in centrosome function, spindle assembly and/or response to DNA damage [6,8]. Despite considerable interest in MCPH as a neural stem cell defect and window into the control of neurogenesis in humans, the underlying pathomechanisms have not been definitively established and specifically for MCPH3, no detailed radiological descriptions of patients or functional analyses in patient samples have been reported to date. In the present study, we report a novel CDK5RAP2 mutation and describe for the first time in detail the clinical, radiological and cellular phenotype in two MCPH3 patients of European descent. We are thereby able to attribute the microcephaly phenotype in MCPH3 at least partially to a mitotic spindle defect and centrosome disorganization.

Material and methods

Patients
Informed consent was obtained from the parents of the patients for the molecular genetic analysis, the publication of clinical data, magnetic resonance images (MRI) and studies on immortalized lymphocytes (LCLs). DNA was extracted from EDTA blood samples using the Illustra BACC2 DNA extraction kit (GE Healthcare, Munich, Germany). Samples from microcephaly patients and controls were used in this study with approval from the local ethics committees of the Charit and the Freiburg University (approval nos. EA1/212/08 and 494/11, respectively).

Haplotype analysis using microsatellite markers

Six microsatellite markers were selected for each of the MCPH1 to 7 and PNKP loci, so that three markers were located on each side of each gene. The markers flanking the CDK5RAP2 gene were: CHLC.GGAA23B10, D9S258, D9S2152, D9S103, D9S116 and D9S1823. PCR was performed with 1 ng patient DNA and primer pairs in which the forward primer was always labeled with 6FAM. PCR fragments were resolved by capillary electrophoresis on an ABI 3100 sequencer. Fragment analysis was performed using GeneScan software (Applied Biosystems, Foster City). Haplotypes were constructed in the family by inspection of the microsatellite fragment lengths.

PCR and DNA sequencing

Thirty-eight coding exons of the CDK5RPAP2 gene and at least 50 bp of the intronic, exon-flanking sequence were analyzed by PCR (Taq Polymerase, Qiagen, Hilden, Germany), and cycle sequencing using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit Version 1.1 (Applied Biosystems, Darmstadt, Germany). Capillary electrophoresis was performed using an ABI 3100 sequencer (Applied Biosystems, Foster City, CA, USA). Sequence data were analyzed using SeqPilot DNA sequence analysis software (JSI, Kippenheim, Germany). The database sequence NM_018249 for the CDK5RAP2 gene was used as reference, and primers were developed in our laboratory (available on request).

Establishment of Ebstein-Barr virus-transformed lymphocytes and culture

Ebstein-Barr virus-transformed lymphocytes (LCLs) were established according to the protocol published by Neitzel et al. 1986 [9]. Non-adherent LCLs were cultured in RPMI 1640 with L-Glutamine (Invitrogen, Darmstadt, Germany) supplemented with 20% v/v fetal bovine serum (Invitrogen) and 1% v/v penicillin-streptomycin (Sigma-Aldrich, Taufkirchen, Germany).

Immunocytology
For fixation, cells were plated on Poly-L-lysine (Sigma-Aldrich) coated coverslips, cultured for 30 min in standard conditions, and incubated in 37C PFA 4% for 10 min prior to rinsing with phosphate buffered saline (PBS 1). Coverslips were further incubated at room temperature (RT) in staining buffer (0,2% gelatin, 0,25% Triton X-100 in PBS 1) for 20 min and subsequently in 10% donkey normal serum (DNS) in staining buffer for 30 min for blocking. Coverslips were incubated overnight at 4C with primary antibodies in the staining buffer containing 10% DNS followed by an incubation with the corresponding secondary antibodies for 2 h at RT. Nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI, 1:1000, Sigma-Aldrich). Fluorescently labeled cells were analyzed and imaged by a fluorescent Olympus BX51 microscope with the software Magnafire 2.1B (2001) (Olympus, Hamburg, Germany), and all images were processed using Adobe Photoshop. The anti-CDK5RAP2 antibody (HPA035820; 1:200) utilized in this study recognizes amino acids 13071390 of CDK5RAP2 which is unique for the human CDK5RAP2 protein sequence (accession no. NP_060719.4, Uniprot Q96SN8). Further primary antibodies were as follows: mouse anti--tubulin (T5326, SigmaAldrich, 1:5000), mouse anti-alpha-tubulin (T9026, Sigma-Aldrich; 1:1500), mouse anti-pericentrin (ab28144, Abcam; 1:1000), mouse anti-acetylated alpha-tubulin (T6793, Sigma-Aldrich; 1:1000), mouse anti-GM130 (610823, BD Biosciences; 1:1000).

Protein extraction procedure and Western blot

Protein extracts for Western blots were isolated from LCLs by homogenization in radio-immunoprecipitation assay (RIPA) buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich) and 1 protease inhibitor cocktail tablet per 10 ml RIPA buffer (Complete Mini; Roche Diagnostics, Mannheim, Germany), 20 min incubation on ice and centrifugation at 4C for 10 min at

3000 g and for 20 min at 16000 g. Protein concentrations were determined using a bicinchoninic acid (BCA) based assay, according to the instructions of the manufacturer (BCA Protein Assay Kit; Pierce Biotechnology, Rockford, IL, USA). Protein extracts (30 g per sample) were denaturated in Laemmli sample loading buffer at 95C for 5 min, separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred in transfer buffer in a semi-dry fashion using Trans-Blot SD Semi-Dry transfer cell (Bio-Rad, Munich, Germany) onto nitrocellulose membrane (Bio-Rad, Munich, Germany). The membranes were incubated for 1 h at RT in blocking buffer (TBS-T 1x with 5% bovine serum albumin (BSA)), rinsed three times with TBS-T (1x) for 8 min each at RT on a shaker and then incubated overnight at 4C with rabbit anti-CDK5RAP2 (1:200, HPA035820, Sigma-Aldrich; also verified with antibody from Abnova PAB17507, 1:200), mouse anti--tubulin (1:5,000) or mouse anti-CHK1 (1:1000, Sigma-Aldrich) antibodies. After incubation with the corresponding secondary antibodies donkey anti-rabbit (1:2000; Amersham Biosciences, Freiburg, Germany) and goat anti-mouse (1:10,000; Dako, Hamburg, Germany), the immunoreactive proteins were visualized using a technique based on a chemiluminescent reaction. The gel pictures were obtained with a Bio-Rad imager (Bio-Rad laboratories, Munich, Germany). Western blot experiments were run in triplicate.

Results
Phenotype of patients with MCPH3
The first son (Patient 1) was born prematurely to Italian parents who were third cousins (Figure 1A), at gestational week 35, with a birth weight of 2570 g (exact birth parameters not available). At the age of 3 months, he weighed 4530 g (370 g<3rd centile), was 53 cm long (3,9 cm <3rd centile, -4.1 SD), and had an occipital-frontal head circumference (OFC) of 33,5 cm (4,8 cm<3rd centile, 5.9 SD). Further progression of the OFC is shown in Figure 1B. A closed fontanel, a simian crease, an abdominal hernia and slightly increased muscle reflexes, but no pyramidal signs were observed. Skeletal scintigraphy ruled out craniosynostosis (premature closure of the fontanels) as a cause of microcephaly. The results of routine blood tests including a full blood count, electrolytes, liver, kidney and thyroid parameters, CK and tests for TORCH and metabolic diseases were normal. Clearly defined areas of hyperpigmentation were apparent on the medial side of the right leg, left ankle and left pectoral. Chromosome analysis revealed a normal male karyotype. The results of an ophthalmological investigation as well as ultrasound of the kidneys and hip joints were normal. At age 4,5 months, audio-acoustic emissions could not be detected, and brainstem audiometry revealed a slightly elevated absolute threshold of hearing of 3540 dB. However at age 1 year tests (BERA) were repeated, and his hearing was found to be normal. On cranial MRI, microencephaly, simplified gyral pattern, particularly frontally, and agenesis of the corpus callosum were apparent (Figure 1C, 58). On EEG, the oscillations were slower than expected for age, but epileptic discharges were absent. His initial short stature appeared to become less prominent with age, so that at age 9 years his height was average. At age 8 years the following tests were performed, all with normal results: full blood count, differential blood analysis, glucose, creatinine, CK, LDH, GOT, GPT, TSH, T4. An IQ test revealed intellectual disability (IQ 5069) with slight developmental delay in speech and motor functions, and a short concentration span. According to the Munich Functional Development test (MFE II) at age 5 years his speech and understanding were at an age of 2736 months and expressive speech was at an age of 2534 months. Moreover the boy suffered from a tic disorder manifesting as repetitive blinking, nodding or smacking of the lips. He had behavioral problems, with hyperactivity, bouts of rage and aggression, which were severe enough to necessitate short-term admission to a childrens psychiatric hospital at age 11 years. He was socially inept, easily upset and irascible. However, the behavioral problems responded well to treatment with risperidone.

Figure 1. Clinical features of MCPH3 patients with novelCDK5RAP2 gene mutation. (A) Pedigree of the Italian family: the parents were third degree cousins. (B) Development of the OFC of both patients, from age 2 months to 5 years in patient 1 (triangles) and birth to 3 years 10 months in patient 2 (dots) (centiles refer to WHO Child Growth Standards) [10,11]. The OFC of patient 2 was below 3.5 SD at birth and further decreased to about 6.4 SD by the age of 3 years and 10 months. (C) T1/2weighted magnetic resonance images (MRI) of patient 1 at age 2,5 months (58) and patient 2 at 3 months (912) compared to those of a healthy 3-year old boy. The reduced size of the brain with extra-axial spaces (5, 7, 911), sloping forehead (6, 7, 11), simplified gyral pattern frontally with shallow, wide sulci (5, 7, 9) and corpus callosum agenesis (6) and hypogenesis (10) are apparent.

The second son (Patient 2) was born in the 40th week of gestation with a birth weight of 3130 g (25th-50th centile), 49 cm long (25th50th centile) and OFC of 30 cm (2,1 cm <3rd centile, -3.5 SD). Apgar scores were 9/10/10. Full blood count, differential blood analysis, TORCH and newborn metabolic screening, CK, electrolytes, liver and kidney parameters were all normal. Shortly after birth, intermittent breathing pauses were observed, but never again observed thereafter. He had dysmorphic features including a sloping forehead, low set ears, a relatively high-arched palate and simian creases. Similar to his brother, he had relatively large, maplike areas of hyperpigmentation with well-defined borders: four on his inner right leg, five around the ankle, and one on his left pectoral. The further development of the OFC is shown in Figure 1B. Chromosome analysis and tests for lactate, LDH, GOT, GPT and AP at age six months were normal. All further investigations including electrocardiogram (ECG), ultrasound of the cranium, hips, kidneys, adrenals, bladder as well as an ophthalmological examination yielded normal results. Cranial MRI at the age of five months showed microencephaly, simplified gyral pattern particularly of the frontal lobes and corpus callosum hypogenesis (Figure 2C, 912). Moreover, an increased space in the posterior fossa, consistent with a megacisterna magna most probably secondary to mild cerebellar hypoplasia, could be visualized (Figure 2C, 10). At age 11 months, the patient had developed normally with respect to motor skills; however, two years later mild motor and intellectual developmental delay was noted with an index value of 56 for cognitive development on the Bayley Scales of Infant Development. Although he had relatively good speech development, especially considering that he was brought up to be bilingual, he had similar behavioral problems to his brother, with temper tantrums, problems with motivation and concentration and was not able to attend a regular nursery school. Neither of the patients had seizures by ages 11 years (Patient 1) and 6 years (Patient 2). The patients parents did not consent to the publication of photos of the patients. The clinical findings in both patients as well as those of previously published patients are summarized in Table 1.

Figure 2. Novel CDK5RAP2 gene mutation in patients with MCPH3.(A) Schematic representation of the domain structure of CDK5RAP2 showing the positions and the effects of CDK5RAP2 mutations that cause MCPH3. Nonsense mutation c.4441C>T is shown as a solid arrow and previously described CDK5RAP2 mutations as dotted arrows. (B) Predicted protein products that result from the CDK5RAP2 gene mutations. The star indicates the binding site of the anti-CDK5RAP2 antibody utilized in this study. (C) Electropherogram shows mutation c.4441C>T in patient 1 and the normal sequence in a control. Table 1. Summary of the clinical features in the patients described in this report (P1=patient 1, P2=patient 2), together with those from previous publications (=not reported)

A novel CDK5RAP2 mutation in a family with MCPH

Haplotype analysis using microsatellite markers revealed that both affected sons in the family were homozygous for a haplotype surrounding the MCPH3 locus, shared by the heterozygous parents, who are third degree cousins. Possible linkage consistent with compound heterozygosity in both sons was also suggested for the MCPH4, MCPH7 and PNKP loci; however, sequencing of theSTIL and PNKP genes did not reveal mutations. Sequencing of CDK5RAP2 showed that both affected sons were homozygous for the mutation c.4441C>T, which results in the nonsense mutation p.Arg1481*; both parents were heterozygous fo r the mutation (Figure 2). The resulting CDK5RAP2 protein is predicted to result in a truncation that affects the second SMC, the pericentrin, and the Golgi binding sites (Figure 2).

Cellular phenotype of patients with CDK5RAP2 gene mutation

We investigated the pathogenicity of the identified missense mutation in immortalized lymphocytes (LCLs) from the two patients with MCPH3 and from controls. In control LCLs, CDK5RAP2 localized to the centrosomes during each stage of the cell cycle (Figure 3). Consistent with studies in murine cells [12], centrosomal CDK5RAP2 levels were relatively low during interphase, increased in the subsequent prophase and remained high throughout mitosis until telophase, when signals dropped to interphase levels. CDK5RAP2 further accumulates at the Golgi apparatus [13], and we detected a partial colocalization with the cis-Golgi matrix protein GM130 in LCLs during inter- and prophase. In prometaphase, the Golgi apparatus begins to fragment and loses its pericentriolar location close to CDK5RAP2 (Figure 4). In metaphase and anaphase the fragments were still somewhat dispersed in the cytoplasm but some could already be detected in the proximity of the CDK5RAP2-positive centrosomes. During telophase, cytokinesis separates the two daughter cells, and reassembly of the Golgi apparatus occurs in the centrosomal region of each daughter cell. InCDK5RAP2 mutant LCLs, CDK5RAP2 levels were below detection levels when assessed through immunocytology and western blots using two antibodies that bind to different positions at the C-termini of full-length CDK5RAP2 (Figure 3). Since the Golgi domain described previously at the C-terminus [13] is predicted to be lost in our patients, we further analyzed the Golgi

integrity through immunostaining with GM130. GM130 immuno-signal clusters were apparent in interphase cells from patients. However, Golgi fragmentation appeared to occur earlier during mitosis and had disappeared by prophase (Figure 4).

Figure 3. CDK5RAP2 in immortalized lymphocytes and dispersion of centrosomal protein -tubulin in CDK5RAP2 mutant patients cells. CDK5RAP2 protein levels were below detection level in immortalized lymphocytes from both patients with the c.4441C>TCDK5RAP2 mutation when assessed by (A) Western blot and (B) immunocytology. Subcellular localization of CDK5RAP2 and -tubulin throughout the cell cycle in immortalized lymphocytes of ( C) controls and (D) MCPH3 patient 2. In controls, centrosomal CDK5RAP2 levels were weak during interphase, increased in the subsequent prophase and remained high throughout mitosis until telophase, when signals dropped to interphase levels. In the patient cells the alignment of the chromosomes at the spindle poles was less precise than in the control cells. Cells were stained with CDK5RAP2 (red), -tubulin (green) as a centrosomal marker, and DNA is stained with DAPI (blue). Scale bars 10 m. Western blot results reveal that total tubulin protein levels are similar in patients and controls (Additional file 1: Figure S1).

Figure 4. Golgi apparatus marker GM130 in CDK5RAP2 mutant patients cells. Subcellular localization of CDK5RAP2 and GM130 throughout the cell cycle in immortalized lymphocytes of ( A) control and (B) MCPH3 patient 2. Golgi fragmentation appeared to occur earlier during mitosis,already showing a dispersed signal during prophase in patient cells.

Additional file 1: Figure S1. Gamma tubulin in CDK5RAP2 mutant patient and control LCLs. (A, B) Total gamma tubulin protein levels detected via Western blots in immortalized lymphocytes of controls and MCPH3 patients did not differ significantly. Format: TIFF Size: 141KB Download file Because CDK5RAP2 impacts on human brain size and has been associated with progenitor proliferation, we next sought to examine the integrity of the centrosome and the establishment of the mitotic spindle apparatus in patients and controls. CDK5RAP2 colocalized with the centrosomal protein -tubulin throughout the cell cycle in control LCLs (Figure 3). In patient cells where CDK5RAP2 was below the detection level, we did not observe a complete loss of -tubulin from the centrosome nor a massive reduction of total -tubulin via western blot, but rather a more dispersed -tubulin staining around the centrosome (Figure 3). Pericentrin localization was normal in patient cells when compared to control cells (Figure 5). In addition, spindle defects with an increase of abnormal spindles with broad and unfocused poles of microtubuli (41% and 55% versus 9% of 100 counted metaphase LCLs of patient 1 and 2 versus control; One-way ANOVA, p<0.001) were detected in CDK5RAP2 mutant LCLs (Figure 6). There was a trend in patients towards an increase in multipolar spindles (4% and 11% versus 3.5% of 100 counted metaphase LCLs of patient 1 (not significant) and 2 (p<0.05) versus control; One-way ANOVA) and a decrease of spindle pole distance (5.4 m and

4.8 m versus 5.8 m of 100 counted metaphase LCLs of patient 1 (not significant) and 2 (p<0.05) ver sus control; One-way ANOVA) in CDK5RAP2mutant LCLs (Figure 6). Also several LCLs from patients showed lagging chromosomes, this was significantly increased in one of the patients (patient 2) and only showed a tendency to be increased in the other patient. CHK1 protein has been shown to be downregulated in Cdk5rap2 mutant cells [14]. Although slightly reduced in both patient cell lines as compared to the control, the difference in the concentration of CHK1 protein was not significant (Figure 7).

Figure 5. Pericentrin in CDK5RAP2 mutant patient and control cells. No difference was detected regarding the subcellular localization of pericentrin throughout the cell cycle in immortalized lymphocytes of ( A) control and (B) MCPH3 patients (here patient 2), however the staining in patients was weaker and the signal more diffuse.

Figure 6. Spindle defects in CDK5RAP2 mutant patients cells.Subcellular localization of CDK5RAP2 and -tubulin throughout the cell cycle in immortalized lymphocytes of (A) control and (B) MCPH3 patient 2. In controls, CDK5RAP2 is weak and centrosomal during interphase and shows abnormal spindle formation. In patients, spindle pole formation did not appear to be as precise as in the control cells, with the chromosomes not as uniformly positioned at the spindle poles. Cells were stained with CDK5RAP2 (red), -tubulin (green) as a centrosomal marker, and DNA is stained with DAPI (blue). Scale bars 10 m. (C) Quantification results of abnormal spindles (unfocused -tubulin staining at spindle poles), multipolar spindles and spindle pole distance.

Figure 7. CHK1 in CDK5RAP2 mutant patient and control LCLs. (A,B) Total CHK1 protein levels detected via Western blots in immortalized lymphocytes of controls and MCPH3 patients did not differ significantly.

Discussion
In the present study, we have identified the novel nonsense mutation c.4441C>T (R1481*) in the CDK5RAP2 gene in a homozygous constellation in two boys of Italian descent with primary microcephaly (Figures 1 and 2). We thereby, for the first time, provide detailed clinical and radiological information on MCPH3 patients of European descent. The siblings suffer from congenital microcephaly, intellectual disability, speech deficit, a tic disorder and severe behavioral problems. Further tests did not reveal any significant hearing impairment or epilepsy as a cause for the speech deficit. Therefore, although sensorineural hearing loss has been reported in two patients with mutations in CDK5RAP2[5,15], this is not a consistent finding in MCPH3. Both patients had microcephaly, simplified gyral pattern of the cerebral cortex, with shallow sulci anteriorly and deep sulci parietally and posteriorly,

and corpus callosum hypogensis on cMRI. There was no particular evidence of reduced white matter volume in the patients, despite the fact that CDK5RAP2is expressed in glial cells of the developing rodent brain. Why the white matter is not more severely affected in MCPH remains unclear. Future studies will need to address the question as to what extent white matter disease also contributes to brain size reduction in MCPH patients. It is unclear whether these clinical and radiological features are also present in the previously reported three pedigrees of Pakistani descent with MCPH due to homozygous CDK5RAP2 mutations. In addition to the brain, we recently reported that CDK5RAP2 is widely expressed in various organs of newborn mice and human fetuses with high CDK5RAP2 mRNA and protein levels in the thymus and the kidney [7]. Moreover, it has been reported that in the MCPH3 murine model Hertwigs anemia mice display defects in multiple organs including the thymus and also have a hematopoietic phenotype (hypoproliferative anemia, leucopenia, predisposition to hematopoietic tumors) [16]. In other MCPH subtypes, individual patients have been reported with short stature (especially in MCPH1 and MCPH5 [17-19]), early puberty, renal agenesis and polycystic kidneys[17]. As this point warrants further investigation in patients, we investigated the clinical phenotype of our patients in detail with respect to multi-organ involvement. Short stature was detected in both patients up to an age of two years, but thereafter normalized in patient 1 for whom detailed data were available. This normalization of height after infancy (in contrast to the pattern of head growth) is a disease feature that has been reported similarly in patients with ASPM gene mutations[18]. There was no evidence of further organ involvement or malignancy, specifically no anemia or leucopenia and no kidney or thymus abnormality. The three homozygous mutations in the CDK5RAP2 gene reported so far, 246T>A in exon 4, 700G>T in exon 8 and 4005 15A>G in intron 26, have been proposed, but not shown, to lead to truncated proteins of 82 (Y82*), 234 (E234*) and 1334 (R1334Sfs*5) amino acids, respectively, and a loss of CDK5RAP2 function (full length protein 1893 amino acids; Figure 1). While the first and second mutant protein should lack most of the CDK5RAP2 transcript except for the N-terminus including a part of the TuRC-binding domain or the N-terminus including the complete TuRC-binding domain and a part of the SMC-domain, respectively, the third protein should lack the C-terminus of CDK5RAP2, especially the c-terminal SMC domain as well as the pericentrin and the Golgi binding sites. The homozygous nonsense mutation reported here, 4441C>T in exon 30, is predicted t o lead to a truncated protein of 1481 amino acids (R1481*). The resulting CDK5RAP2 protein in our patients should lack parts of the second SMC domain as well as the pericentrin and the Golgi binding sites (Figure 1). No studies on patient specimen exist that shed light on the effect of the reported CDK5RAP2 gene mutations. We recently reported a high CDK5RAP2 expression in proliferating progenitors of the germinal matrix and early (not mature) neurons as well as glial cells in the neocortex of murine embryos and human fetuses [7]. This is in concordance with results of neuroimaging studies in MCPH patients due to non-CDK5RAP2 mutations demonstrating a reduced brain volume that affects especially the neocortex [17,18]. Based on results from in vivoand in vitro studies, the human MCPH phenotype is considered to be the result of a premature shift from symmetric to asymmetric neural progenitor-cell divisions (with a subsequent depletion of the progenitor pool) as well as of a reduction in cell survival [6,8]. To study the effect of the reported CDK5RAP2 gene mutation on cell proliferation in our patients, we studied EBV-transformed lymphocytes (LCLs) from both of our patients and from controls. Here, CDK5RAP2 localized to the centrosomes during each stage of the cell cycle in controls but was absent from patient cells, when assessed via immunocytology and western blots (Figure 3). The latter finding of CDK5RAP2 levels below detection levels in cells of our patients indicates that very little or no protein is present secondary to nonsense-mediated decay of the mutated transcript. In contrast to Cdk5rap2 shRNAi studies performed on mouse tissues [20], we detected a failure of the centrosomal protein -tubulin to localize properly at the centrosome, while total -tubulin protein levels were normal in patient cells (Figure 3, Additional file 1: Figure S1). Pericentrin, which interacts with CDK5RAP2 through defined protein domains [21], was not altered in its localization in patient LCLs (Figure 5). This result is in line with those of Buchman et al. 2010[21] who concluded from their studies in murine tissues that the centrosomal recruitment of pericentrin is not dependent upon Cdk5rap2, while the converse is true. Despite the predicted loss of the C-terminal Golgi domain in mutant CDK5RAP2, the Golgi apparatus could be visualized normally using immunostaining with GM130. However, Golgi fragmentation appeared to occur earlier during mitosis and had disappeared by prophase (Figure 4). Further, we observed an unfocused and disorganized mitotic microtubule assembly, a decrease in spindle pole distance and a trend towards more multipolar spindle poles as well as chromosome misalignment in patient cells (Figure 6). These results, although generated in lymphocytes and not neural progenitors, suggest that spindle defects and a disruption of centrosome integrity could play a role in the development of microcephaly in MCPH. On the other hand, they also underline the fact that, at least in the cells studied, despite a lack of normal CDK5RAP2, a centrosomal structure can still be formed, microtubuli can still be nucleated to the centrosomes and cells can still divide. Since microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1 [22] and Chk1-downregulation has been demonstrated in mutant Cdk5rap2 cells [14], we analyzed CHK1 levels in CDK5RAP2 mutant and control cells. Although slightly reduced in both patients, there was not a significant decrease in total CHK1 levels in patients cells (Figure 7). Brain size at birth is largely determined by the relative rates of proliferation and cell death. By highlighting the clinical, radiological and cellular phenotype of MCPH3 patients, we offer a further glimpse into how a disruption of the CDK5RAP2 gene may impact on

the development in humans. Further analysis of patient samples provides a means to investigate processes that cause MCPH and to verify mechanisms described in other model systems and in settings where animal models are neither sufficient nor satisfactory.

Competing interests
The authors declare no competing interests in the preparation or publication of the data in this manuscript.

Authors contributions
AM and DMR were responsible for the project conception and wrote the manuscript. LI, NK and ON performed the lymphocyte analysis, generated figures and proofread the manuscript. KM and KS performed genetic analysis and compiled clinical data. HR and MB attended the patients and provided clinical data. All authors read and approved the final manuscript.

Acknowledgements
The authors thank Julia Knig, Horst von Bernuth, Margret Oberreit, Angela Hbner, Katrin Khler, Marcus Lettau, Ottmar Janssen, Angela Steiert, Christine Zeschnigk, Ulrike Schneider, Elke Jantz-Schuble and Bernd Rsler for discussions and technical assistance. We are grateful to Dr. Wolfgang Brunk and Prof. Peter Winkler for supplying MR images of the patients.

Funding
This work was supported by the German Research Foundation (SFB665), the Sonnenfeld Stiftung and the Berliner Krebsgesellschaft e.V.

References
1. Kaindl AM, Passemard S, Gressens P: Autosomal recessive primary microcephalies (MCPH). Eur J Paediatr Neurol 2009, 13:458. PubMed Abstract | Publisher Full Text 2. Thornton GK, Woods CG: Primary microcephaly: do all roads lead to Rome? Trends Genet 2009, 25:501-510. PubMed Abstract | Publisher Full Text |PubMed Central Full Text 3. Bond J, Roberts E, Springell K, Lizarraga SB, Scott S, Higgins J, Hampshire DJ, Morrison EE, Leal GF, Silva EO, Costa SM, Baralle D, Raponi M, Karbani G, Rashid Y, Jafri H, Bennett C, Corry P, Walsh CA, Woods CG: A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size. Nat Genet 2005, 37:353-355. PubMed Abstract | Publisher Full Text 4. Hassan MJ, Khurshid M, Azeem Z, John P, Ali G, Chishti MS, Ahmad W: Previously described sequence variant in CDK5RAP2 gene in a Pakistani family with autosomal recessive primary microcephaly. BMC Med Genet 2007, 8:58. PubMed Abstract | BioMed Central Full Text |PubMed Central Full Text 5. Pagnamenta AT, Murray JE, Yoon G, Sadighi Akha E, Harrison V, Bicknell LS, Ajilogba K, Stewart H, Kini U, Taylor JC, Keays DA, Jackson AP, Knight SJ: A novel nonsense CDK5RAP2 mutation in a Somali child with primary microcephaly and sensorineural hearing loss. Am J Med Genet A 2012, 158A:2577-2582. PubMed Abstract | Publisher Full Text |PubMed Central Full Text 6. Kraemer N, Issa L, Hauck SC, Mani S, Ninnemann O, Kaindl AM: Whats the hype about CDK5RAP2? Cell Mol Life Sci 2011, 68:1719-1736. PubMed Abstract | Publisher Full Text 7. Issa L, Kraemer N, Rickert CH, Sifringer M, Ninnemann O, Stoltenburg-Didinger G, Kaindl AM: CDK5RAP2 Expression during murine and human brain development correlates with pathology in primary autosomal recessive microcephaly. Cereb Cortex 2012. Publisher Full Text

8. Megraw TL, Sharkey JT, Nowakowski RS: Cdk5rap2 exposes the centrosomal root of microcephaly syndromes. Trends Cell Biol 2011, 21:470-480. PubMed Abstract | Publisher Full Text |PubMed Central Full Text 9. Neitzel H: A routine method for the establishment of permanent growing lymphoblastoid cell lines. Hum Genet 1986, 73:320-326. PubMed Abstract | Publisher Full Text 10. de Onis M, Garza C, Onyango AW, Borghi E: Comparison of the WHO child growth standards and the CDC 2000 growth charts. J Nutr 2007, 137:144-148. PubMed Abstract | Publisher Full Text 11. de Onis M, Onyango AW, Borghi E, Garza C, Yang H: Comparison of the World Health Organization (WHO) Child Growth Standards and the National Center for Health Statistics/WHO international growth reference: implications for child health programmes. Public Health Nutr 2006, 9:942-947. PubMed Abstract | Publisher Full Text 12. Barrera JA, Kao LR, Hammer RE, Seemann J, Fuchs JL, Megraw TL: CDK5RAP2 regulates centriole engagement and cohesion in mice. Dev Cell 2010, 18:913-926. PubMed Abstract | Publisher Full Text | PubMed Central Full Text 13. Wang Z, Wu T, Shi L, Zhang L, Zheng W, Qu JY, Niu R, Qi RZ: Conserved motif of CDK5RAP2 mediates its localization to centrosomes and the Golgi complex. J Biol Chem 2010, 285:22658-22665. PubMed Abstract | Publisher Full Text |PubMed Central Full Text 14. Barr AR, Kilmartin JV, Gergely F: CDK5RAP2 functions in centrosome to spindle pole attachment and DNA damage response. J Cell Biol 2010, 189:23-39. PubMed Abstract | Publisher Full Text |PubMed Central Full Text 15. Moynihan L, Jackson AP, Roberts E, Karbani G, Lewis I, Corry P, Turner G, Mueller RF, Lench NJ, Woods CG: A third novel locus for primary autosomal recessive microcephaly maps to chromosome 9q34. Am J Hum Genet 2000, 66:724-727. PubMed Abstract | Publisher Full Text |PubMed Central Full Text 16. Lizarraga SB, Margossian SP, Harris MH, Campagna DR, Han AP, Blevins S, Mudbhary R, Barker JE, Walsh CA, Fleming MD: Cdk5rap2 regulates centrosome function and chromosome segregation in neuronal progenitors. Development 2010, 137:1907-1917. PubMed Abstract | Publisher Full Text |PubMed Central Full Text 17. Woods CG, Bond J, Enard W: Autosomal recessive primary microcephaly (MCPH): a review of clinical, molecular, and evolutionary findings. Am J Hum Genet 2005, 76:717-728. PubMed Abstract | Publisher Full Text |PubMed Central Full Text 18. Passemard S, Titomanlio L, Elmaleh M, Afenjar A, Alessandri JL, Andria G, de Villemeur TB, Boespflug-Tanguy O, Burglen L, Del Giudice E, Guimiot F, Hyon C, Isidor B, Megarbane A, Moog U, Odent S, Hernandez K, Pouvreau N, Scala I, Schaer M, Gressens P, Gerard B, Verloes A: Expanding the clinical and neuroradiologic phenotype of primary microcephaly due to ASPM mutations. Neurology 2009, 73:962-969. PubMed Abstract | Publisher Full Text 19. Kaindl AM, Passemard S, Kumar P, Kraemer N, Issa L, Zwirner A, Gerard B, Verloes A, Mani S, Gressens P: Many roads lead to primary autosomal recessive microcephaly.

Prog Neurobiol 2010, 90:363-383. PubMed Abstract | Publisher Full Text 20. Fong KW, Choi YK, Rattner JB, Qi RZ: CDK5RAP2 is a pericentriolar protein that functions in centrosomal attachment of the gamma-tubulin ring complex. Mol Biol Cell 2008, 19:115-125. PubMed Abstract | Publisher Full Text |PubMed Central Full Text 21. Buchman JJ, Tseng HC, Zhou Y, Frank CL, Xie Z, Tsai LH: Cdk5rap2 interacts with pericentrin to maintain the neural progenitor pool in the developing neocortex. Neuron 2010, 66:386-402. PubMed Abstract | Publisher Full Text 22. Tibelius A, Marhold J, Zentgraf H, Heilig CE, Neitzel H, Ducommun B, Rauch A, Ho AD, Bartek J, Kramer A: Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1. J Cell Biol 2009, 185:1149-1157. PubMed Abstract | Publisher Full Text |PubMed Central Full Text