38 
HPV Detection Techniques
ZI-XUAN (ZOE) WANG  |  STEPHEN C. PEIPER
CONTENTS
HPV Biology
General Information of Human Papillomavirus
High-risk and Low-risk HPV Types
HPV and Cancer
HPV Vaccines
Mechanisms for HPV Oncogenesis
HPV Detection Methods
FDA-approved HPV Assays for Cervical Specimens
Detection Principles
HPV Testing Guidelines for Patient Screening and
Management
Additional HPV Detection Methods
Diagnostic Accuracy of HPV Screening Assays
Concluding Remarks
HPV Biology
GENERAL INFORMATION OF HUMAN
PAPILLOMAVIRUS
Human papillomaviruses (HPVs) are members of the Papillo-
maviridae family of viruses. They are small double-stranded
DNA viruses with a circular genome of approximately 8000
base pairs (bp) and an outer protein capsid, with no lipid
membrane/envelope.1 The HPV genome is composed of eight
genes, six of them expressed early in infection (so-called early
genes: E1, E2, E4, E5, E6, and E7) and two late (so-called late
genes: L1 and L2). There are over 120 types of HPV, which can
be divided roughly into a cutaneous group that is transmitted
through (skin) contact to cause common warts and a mucosal
group transmitted through sexual activities, including oral sex,
to trigger benign genital warts and malignancy in the anogeni-
tal tract (cervical, vulvar, vaginal, anal, and penile cancers) and
the upper aerodigestive tract (oropharyngeal cancer).2 The clas-
sification of HPV types, subtypes, and variants is determined
from the nucleotide sequence of the L1 gene, which is the most
conserved gene in the HPV genome and encodes the capsid
protein.3 Different types are established by sequence differences
of greater than 10%, subtypes by differences between 2% and
10%, and variants by differences of less than 2%. Studies of
HPV phylogeny suggest that these viruses are highly stable and
do not change host species, recombine, or alter their genomic
organization.
926
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HIGH-RISK AND LOW-RISK HPV TYPES
HPV is the most common sexually transmitted disease. There
is estimated to be 79 million infected people in the USA in
contrast to 41 million of all other sexually transmitted diseases
combined, according to the US Centers for Disease Control and
Prevention (CDC).4 There are approximately 40 HPV types that
are sexually transmitted. Among them, HPV types that are
oncogenic have been designated “high-risk” (HR) types, because
they are capable of inducing invasive carcinomas. The most
common 14 HR types are HPV 16, 18, 31, 33, 35, 39, 45, 51, 52,
56, 58, 59, 66, and 68, and they are included in the commercial
HPV screening panels. Additional HR types exist but are
uncommon and not screened for in routine clinical testing.
HPV types that are not oncogenic are designated as low-risk
types (6, 11, 40, 42, 43, 44, 54, 61, 70, 81, and CP6108). They
cause genital warts known as condyloma acuminata and are
rarely associated with invasive carcinoma (approximately 0.1%
for cervical cancer). Rarely, transmission of HPV6 and HPV11
from mother to new born baby can cause juvenile-onset recur-
rent respiratory papillomatosis in children less than 5 years.5
Cutaneous Group HPV
HPV types in the cutaneous group are not sexually transmitted.
They infect cutaneous squamous epithelium, resulting in
common warts (most commonly types 2, 4; also 26, 29, 57),
plantar warts (most commonly type 1; also 2, 4, 60, 63), flat
warts (most commonly types 3, 10, 28), and epidermodysplasia
verruciformis (most commonly types 5, 8, although multiples
have been reported).6
HPV AND CANCER
HPV in Cervical Cancer
Carcinoma of the cervix has a major impact on the health of
women globally as the second most common form of cancer
and the fifth highest cause of death. The vast majority of cases
occur in developing countries. In contrast, its incidence in
women ranks 8th and 12th in the USA and UK, respectively.
This decrease in incidence, as well as mortality, is attributable
to the development of screening programs using a novel tech-
nology at the time, the Papanicolaou smear (Pap smear). This
has resulted in a decrease of approximately 74% in incidence in
the USA over the past 50 years.
Harald zur Hausen postulated that HPV was involved in the
pathogenesis of cervical cancer7 and the virus was first identi-
fied in skin cancer.8 Professor zur Hausen and colleagues identi-
fied HPV16 and HPV18 in cervical cancer in the early 1980s.9
It is estimated that the majority of sexually active individuals

become infected with HPV, but that the infection is typically
transient and cleared by the immune system. Only persistent
infection over the course of years and decades by HR HPV will
result in the development of cancer.
HPV is the Cause of Cervical Cancer
It is now clear that HPV is the universal etiology of invasive
cervical cancer throughout the world. The International Bio-
logical Study on Cervical Cancer (IBSCC) reported the detec-
tion of HPV in 93% of cervical carcinomas in a cohort of 932
patients from 22 countries using a polymerase chain reaction
(PCR) assay for a 450 bp segment in the L1 open reading
frame.10 This raised the possibility of an alternative etiology for
cervical carcinoma in the 7% (n = 66) of patients lacking detect-
able HPV templates. A follow-up study11 was performed to
determine whether the absence of HPV DNA was genuine (i.e.,
these cases were true negatives) or false-negative results, which
could be explained by the disruption of the PCR primer
sequences (as could occur during viral integration), loss of the
L1 open reading frame, or the absence of tumor in the tissue
sampled. Since 450 bp exceeds the average length of DNA frag-
ments in tissues fixed in formalin and embedded in paraffin
(FFPE), the 66 negative samples from the initial study were
analyzed for the presence of a 100 bp segment in the E7 open
reading frame using specific primer pairs for 14 oncogenic HPV
types, as well as determining the ability of the DNA templates
to support the amplification of a control fragment of similar
length (β-globin) and the presence of tumor in the tissue ana-
lyzed. When corrected for adequacy of PCR amplification and
tumor content, the association of HPV with invasive cervical
carcinoma was predicted to be 99.7%. Therefore, on a global
basis, this represents the highest association of a cancer type
with a specific cause. This universal association of HPV as the
causal etiology of invasive cervical carcinoma provides a com-
pelling rationale for the development of a vaccine to eradicate
this common malignancy.
HPV16 and HPV18 are More Oncogenic
Not all HR HPV types are equal in their ability to induce malig-
nancies. In a cohort of 1739 patients with invasive squamous
cell carcinoma of the uterine cervix,12 1487 (85%) were infected
with one HPV type and 109 (6%) were infected with more than
one. Among patients infected with one type, 54.6% had HPV16;
11% had HPV18; 4.4% had HPV45; and 3.4% had HPV31.
Dual infection with HPV16 and HPV18 occurred in 36 (2.1%),
HPV16 plus another type in 36 (2.1%), and HPV18 plus another
type in 22 (1.3%). Many similar results have been published and
HPV16 and HPV18 are responsible for 60–70% of cases of
cervical carcinoma, and HPV31 and HPV45 are the cause of
another 10%, depending on the study design and geographic
locations.
HPV16, 18, and 45 are Found in Younger Women
with Cervical Cancer
HPV16 and HPV18 are also found in younger mean or median
age of women with cervical cancers in comparison to other HR
HPV types. A recent worldwide HPV screening and genotype
study with over 10 000 confirmed cervical cancer cases from 38
countries of different continents of the world showed HPV16
and HPV18 are the causes of 71% of cervical cancers.13 In the
same study, HPV45 was shown to cause a slightly higher per-
centage of overall cervical cancer than HPV31 (6% vs 4%).
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38  HPV Detection Techniques 927
When histologic types are grouped separately, HPV45 and
HPV31 were presented, respectively, in 5% and 4% of squa-
mous cell carcinomas but 12% and less than 1% of adenocar-
cinomas as well as of adenosquamous cell carcinomas. In this
study, HPV45 had the lowest mean age (46.8 years) of women
with cervical cancer, 3.2 years younger than HPV16 and 1.4
years younger than HPV18. Therefore, genotype HPV45 in
addition to HPV16 and HPV18 in clinical screening may have
added predictive value for patient outcome.
HPV in Head and Neck Cancer
The second most common malignancy caused by HPV is the
cancers of the upper aerodigestive tract in the mucosa of the
oral cavity, oropharynx, hypopharynx, larynx, sinonasal tract,
and nasopharynx. A review of worldwide cases of squamous
cell carcinoma of the head and neck reveals that HPV genomic
DNA has been detected in approximately 26%.14 The inci-
dence of oropharyngeal squamous carcinoma has increased in
the USA, primarily in white men between the ages of 40 and
55 years.15 These patients do not have a high exposure to
alcohol or tobacco, which are the recognized risk factors for
squamous cell carcinoma of the head and neck, but are posi-
tive for high-risk types of HPV. These cancers, which typically
occur in the tonsil and base of the tongue, are positive for
HPV16 in 60–72% of cases. Risk factors included a high
number of vaginal-sex partners and a high number of oral-
sex partners.16
HPV VACCINES
Two vaccines for HPV have been approved by the US Food and
Drug Administration (FDA) and are recommended for admin-
istration to adolescents to prevent diseases caused by this
virus.17–19 They are composed of non-infectious virus particles
containing products encoded by the L1 structural gene. The
bivalent vaccine prevents infection with HPV16 and HPV18,
which are responsible for approximately 70% of cervical carci-
nomas. The quadrivalent vaccine protects against infection with
HPV types 16 and 18 and HPV types 6 and 11, which are the
cause of approximately 90% of genital warts. It has also been
demonstrated that the quadrivalent vaccine prevents the devel-
opment of carcinomas of the anus, vagina, and vulva. The quad-
rivalent vaccine is the only one approved for administration to
males. These vaccines do not have an effect on existing disorders
induced by HPV. It is recommended that patients should receive
the vaccine prior to sexual activity, and vaccination is not rec-
ommended for patients over age 26.
HPV vaccination can potentially prevent about 21 000 cancer
cases and 360 000 genital warts each year, according to the
“Genital HPV Infection-Fact Sheet” posted on the CDC website.4
The breakdown of these neoplasms associated with HPV is
listed in Table 38-1.
MECHANISMS FOR HPV ONCOGENESIS
The HPV early genes are involved in functions that play a role
in controlling transcription and interaction with the regulatory
processes of the host cell. HPV exploits cellular “machinery” for
DNA replication because the genome does not encode proteins
for these functions. Therefore, it is essential for the virus to have
mechanisms to promote transcription of its genome and DNA
synthesis.20–22
928 PART 3  Special Techniques in Cytology
TABLE Estimated Annual Incidence of HPV-associated
38-1  Neoplasm
Estimated Annual
HPV-associated Neoplasm Incidence (USA)
Genital warts 360 000
Carcinoma of cervix 12 000
Carcinoma of vulva
Carcinoma of vagina
Carcinoma of penis
Anal carcinoma in women
Anal carcinoma in men
Oropharyngeal carcinoma in women
Oropharyngeal carcinoma in men
Data source: CDC website.4
a
Additional factors such as tobacco and alcohol use contribute to
these diseases.
E6 and E7 Oncoproteins Interact with Human
Tumor Suppressor Genes
Neither E6 nor E7 genes individually are sufficient to induce
immortalization; however, the combination of the E6 and E7
genes from high-risk strains, such as HPV16 and HPV18,
encode oncoproteins that are sufficient to immortalize nor-
mal keratinocytes,23 whereas those from low-risk types are
not.24 The mechanisms described below are summarized in
Fig. 38-1.
HPV E6 Oncoprotein Induces
the Degradation of P53
The E6 protein contains two zinc-finger domains. It first forms
a complex with a cellular ubiquitin ligase, designated the
E6-associated protein. This complex binds to P53, and the E6-
associated protein promotes the coupling of ubiquitin to lysine
residues in P53, leading to ubiquitin-mediated degradation of
P53 in the proteasome.25,26 The loss of P53 results in the absence
of a critical response to DNA damage, including arrest of cell
cycle progression at G1/S and G2M checkpoints. The promo-
tion of progression through the cell cycle favors replication of
the HPV genome, as well as proliferation and the accumulation
of genetic mutations that can lead to malignant transformation.
E6 also has pro-oncogenic effects that are independent of P53.
They include activation of telomerase, which may also be suf-
ficient for oncogenic transformation.27 This disrupts the normal
process of telomere shortening with successive cycles of DNA
replication, enabling the cell to avoid this mechanism that limits
the rounds of DNA synthesis. Since deletion of PDZ-binding
domains in E6 proteins from HR HPV results in loss of trans-
forming functions, it is possible that these types of interactions
are involved in enhancement of the Wnt signaling pathway and
disruption of scaffolds that support the assembly of signaling
complexes, such as the membrane-associated guanylate kinase,
WW and PDZ domain containing protein 1.28–30
E7 Mediates the Degradation of Rb
The E7 protein also contains zinc-finger domains and binds the
product of another key tumor suppressor gene, Rb.31 The Rb
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protein is phosphorylated at low levels in normal cells, and
regulates progression through the cell cycle. Hypophosphor-
ylated Rb binds the E2F transcription factor. When cell cycle
progression is activated, the association of cyclin D with Cdk4
kinase results in phosphorylation of Rb with loss of binding to
E2F.32 This activation of E2F triggers the transcription of genes
that promote DNA synthesis. Binding of E7 to Rb induces ubiq-
uitination and degradation. E7 also binds to and inactivates
2100 inhibitors of cyclin-dependent kinases (including Cdk4), which
results in phosphorylation, and inactivation, of Rb, leading to
500
cell cycle progression and proliferation.33–35 E7 proteins of low-
600 risk HPV types bind to Rb, but do not promote ubiquitin-
2800 mediated degradation. E7 also binds to two additional members
of the Rb family, P107 and P130, which are also negative regula-
1500
tors of E2F activity.36
1500a
HPV Infection in Episomal Stage
6700a
HPV infection is necessary, but not sufficient, for the develop-
ment of invasive squamous cell carcinoma. In fact, most infec-
tions are cleared through immunologic responses in 1–2 years.
The host target cell optimal for HPV infection is squamous
epithelium, initially localizing to the basal layer of cells.37,38
Since the HPV genome does not encode enzymes required for
DNA replication, it is dependent upon host machinery for viral
replication in an environment of progression through the
S-phase of the cell cycle, which is provided in the basal cell layer
and promoted through the actions of the E6 and E7 oncopro-
teins, as described above. The initial rounds of replication result
in 50–100 episomal viral genomes per cell,39 which are packaged
into viral particles following expression of the L1 capsid protein.
As infected cells progress through the maturation/differentiation
program of the squamous epithelium, infectious viruses are
released from desquamated cells. During the episomal (non-
integrated) phase of HPV infection, E6 and E7 oncoproteins are
not expressed at high levels due to suppression by E2 proteins.40
This model approximates the pathogenesis of low-grade squa-
mous intraepithelial lesions.
HPV Integration into the Human Genome
In contrast, high-grade squamous intraepithelial lesions have
active mechanisms that interrupt productive infection by dis-
rupting late events in the viral life cycle and introduce mecha-
nisms to intercalate into a pathological epidermal environment.
A major event in this process is viral integration, which is
observed in carcinomas associated with HPV16 and HPV18 in
approximately 80% and 100% of cases, respectively.41,42 HPV
genomes with partial deletions are preferentially integrated
into “fragile sites” in chromosomes. The E2 gene, which has
been shown to inhibit transcription of E6 and E7 RNA from
the viral promoter, is frequently deleted in integrated HPV
genomes.43 In this scenario, HPV E6 and E7 are expressed at
high levels and they promote the accumulation of mutations,
progression through the cell cycle, proliferation. Immor­
talization is now “hard-wired” throughout the maturation/
differentiation cycle of the squamous epithelium, leading fea-
tures of high-grade squamous epithelial lesions, with the
unscheduled presence of proliferating/dysplastic epithelial cells
in zones normally populated by maturing differentiated cells.
Therefore, HPV integration into the human genome represents
a crucial milestone for the progression from low-grade squa-
mous intraepithelial lesions to high grade and eventually to
cancer.
 38  HPV Detection Techniques 929

M
G2

G1
S

P
P53 STOP E2F Rb

HPV-encoded
oncoprotein

M
E6 P53
G2
E6AP
G1

P
HPV-encoded
S Rb oncoprotein

STOP E2F E7

B Degradation in proteasome

Figure 38-1  (A) Two tumor suppressor genes control cell cycle progression in normal cells. The P53 protein plays a major role in controlling
the G1–S transition in normal cells. Loss of P53 removes a negative regulator to cell proliferation. The retinoblastoma (Rb) protein is also a major
regulator of cell cycle progression. The binding of hypophosphorylated Rb protein to the E2F transcription factor results in inactivation. With loss
of Rb binding, E2F1 activates the transcription of target genes that promote the transition from G1 to S phase. These target genes include proteins
involved in DNA replication and chromosomal replication. (B) HPV-encoded oncoproteins inactivate tumor suppressor genes. HPVs infect basal
cells of squamous epithelium following micro-abrasions/trauma resulting in disruption of the epithelial barrier. Initially, HPV genome is episomal,
but subsequent integration into the host genome may occur. This results in upregulation of the E6 and E7 oncoproteins. The E6 oncoprotein binds
P53 and forms a complex with E6-associated protein ubiquitin-protein ligase (E6AP), also known as ubiquitin-protein ligase E3A (UBE3A). The
ubiquitination of P53 leads to degradation in the proteasome, with the loss of P53 regulation of G1–S transition. The E7 oncoprotein binds to the
hypophosphorylated Rb protein and targets it for ubiquitination and degradation. The degradation of Rb liberates the E2F1 transcription factor,
which promotes cell cycle progression and proliferation, as described for (A). Additional mechanisms may contribute to the oncogenic transforming
activities of the E6 and E7 oncoproteins in addition to inactivation of P53 and Rb.

HPV Detection Methods
HPV detection relies upon the detection of viral nucleic acid,
either DNA or RNA, in pathologic specimens. Since HPV
cannot be cultured, conventional viral culture methods are not
applicable. HPV infection, in general, does not elicit consistent
immune responses and, for this reason, serologic detection of
antibodies against HPV structural proteins is not reliable and
not used in clinical settings.44
The need for HPV detection can be divided into three cat-
egories, as below. The different detection method in each cat-
egory will be discussed separately.
1. HR HPV screening for cervical specimens from women
with abnormal cytology findings and women over 30,
regardless cytology results. In this setting, an HPV detec-
tion assay needs to be sensitive enough for the detection
of cervical intraepithelial neoplasia (CIN) 2 and above
but not too sensitive so low levels of HPV infection in
women with normal Pap results are not unnecessarily
pursued. Therefore, the terms clinical sensitivities and
clinical specificities are used in studies to compare differ-
ent screening assays.
2. HPV detection and genotyping for epidemiology studies
and vaccination effectiveness. A high analytical sensitivity

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930 PART 3  Special Techniques in Cytology
TABLE
38-2  FDA-approved HPV Cervical Cancer Screening Assays
Test Manufacturer Approval Date
Digene HC2 High- Digene Corporation 3/31/2003
risk HPV DNA Test Gaithersburg, MD, USA
Cervista™ HPV HR Third Wave Technologies 3/12/2009
Hologic, Madison , WI, USA
Cervista™ HPV16/18 Third Wave Technologies 3/12/2009
Hologic, Madison, WI, USA
cobas HPV Test Roche Molecular Systems 4/19/2011
Pleasanton, CA, USA
APTIMA HPV Assay Gen-Probe Incorporated, 10/28/2011
San Diego, CA, USA
APTIMA HPV16 Gen-Probe Incorporated, 10/12/2012
18/45 Genotype San Diego, CA, USA
HPV HR 14 panel: HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.
Data source: http://www.fda.gov/MedicalDevices/default.htm, accessed on July 5, 2013.
in detection and high analytical specificity in genotyping
are needed to achieve resolution and accuracy.
3. HPV detection in pathology specimens (FFPE) from
head and neck and other cancers to predict disease
courses and treatment responses. Quick turn around
time and the ability to be interpreted by a pathologist are
beneficial.
FDA-APPROVED HPV ASSAYS
FOR CERVICAL SPECIMENS
Routine screening in the USA and Europe is performed to
detect sexually transmitted “high-risk” HPV viruses. The Digene
Hybrid Capture I was the first FDA-approved HPV assay (1995).
The second generation of this assay, the Digene Hybrid Capture
II (HC2), which was initially approved by the FDA in 1999, is
the most widely used HPV screening test in clinical laboratories
in the USA, as a reflex test for patients with abnormal cytology
findings, atypical squamous cells of undetermined significance
(ASC-US). Extension of clinical use of the HC2 HPV DNA test
in conjunction with routine Pap smears for women over the age
of 30 was approved by the FDA in 2003. The sensitivity of this
assay was calibrated to detect high-grade CIN (CIN2 and CIN3)
lesions. Its threshold for detection is about 5000 copies of the
HPV genome and this level of viral load is highly concordant
with the presence of precancerous lesions and carcinoma.
Therefore, it is the gold standard for all subsequent HPV screen-
ing tests seeking FDA approval. The utility of the assay is in its
high negative predictive value; negative HPV results excluding
the likelihood of the development of invasive cervical carci-
noma in normal patients or those with ASC-US.
Two additional tests were approved by the FDA 10 years later,
in 2009: the Cervista HPV HR for detecting 14 HR HPV types
and the Cervista HPV 16/18 genotype assay from ThirdWave
Technologies (now Hologic). This marked the first FDA-
approved HPV genotyping assay. Roche Molecular Systems
developed a PCR-based assay that can simultaneously genotype
HPV16 and 18 and detect 14 HR HPV viruses, as well. This
assay was approved by the FDA in 2010. Gen-Probe Incorpo-
rated recently received FDA approvals for E6 and E7 messenger
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Detect DNA
or RNA What to be Detected Genotype
DNA HPV HR 13 types (HR No
14 panel but HPV 66)
DNA HPV HR 14 panel No
DNA HPV16 and 18 Yes 16 and 18
DNA HPV HR 14 panel Yes 16 and 18
RNA (E6 and E7) HPV HR 14 panel No
RNA (E6 and E7) HPV16 and 18/45 Yes 16 and 18/45
RNA (mRNA)-based detection of 14 HR viruses and genotyp-
ing of HPV16 and 18/45 in 2011 and 2012, respectively. These
FDA-approved assays are summarized in Table 38-2. Additional
details on detection principles for these FDA-approved assays
are described below.
DETECTION PRINCIPLES
HC2 High-risk HPV DNA Test
The HC2 HPV DNA Test is a signal amplification antibody
sandwich assay. It uses a probe cocktail of full-length genome
RNA probes to bind HPV DNA to form RNA/DNA hybrids that
are captured onto a microwell plate coated with antibodies that
recognize the structure of this hybrid complex. A second anti-
body conjugated to alkaline phosphatase that also binds the
RNA/DNA hybrid and alkaline phosphatase substrates are
added. If the clinical sample contains HPV DNA, a sandwich is
formed that includes alkaline phosphatase, which generates a
chemiluminescent signal above a background threshold. The
assay is often referred to as signal amplification, because mul-
tiple enzyme molecules are conjugated to one antibody and
multiple antibodies can bind to the same RNA/DNA hybrid
structure, resulting in an amplified signal to achieve the desired
sensitivity. The HC2 assay does not utilize a housekeeping gene
as an internal control to make certain that sufficient cervical
cells are present in the testing specimens to avoid false-negative
results. The HC2 HPV DNA assay is (FDA) approved for use
with ThinPrep liquid cytology medium and the Digene collec-
tion kit. It is not approved by the FDA for any other specimen
types, including SurePath medium.
Cervista HPV HR Assay
The Cervista HPV Assay from Hologic is another type of signal
amplification assay that uses Third Wave Invader Technology.
It uses three DNA sequence-specific probe pools, grouped on
their sequence similarity, in three individual microplate wells to
detect 14 HR HPVs. The probes in Pool 1 detect HPV types 51,
56, and 66, also called the A5/A6 HPV group. The probes in
Pool 2 detect HPV types 18, 39, 45, 59, and 68 (A7 group), and
those in Pool 3 detect HPV types 16, 31, 33, 35, 52, and 58 (A9

group). The Third Wave technology uses two serial isothermal
reactions. The primary reaction is to detect target-specific
DNA sequences and the secondary reaction is to produce fluo-
rescent signals. In the primary reaction, a target sequence-
specific oligonucleotide probe fused with a so-called “flap DNA
fragment” and another target-specific “invader” probe bind to
a DNA target sequence they recognize to form a “three-strand
DNA” structure that is uniquely recognized by the cleavase
enzyme. This enzymatic cleavage will result in the accumulation
of flap fragments when these probes cycle on and off the target
HPV DNA. In the second reaction, the flap DNA fragment
binds to a U-shaped DNA oligonucleotide cassette to form a
“three-strand DNA” structure again. The cleavase enzyme
cleaves this special structure to release fluorescent dye from
the quencher oligonucleotide in the U-shaped FRET cassette
to generate a fluorescent signal. Simultaneously, a fluorescent
signal to detect a control housekeeping gene, human histone 2
(H2be), is detected in another fluorescent channel. An invalid
result instead of negative will be issued if HPVs are negative
from all three pools when the signal for the housekeeping gene
does not cross a predefined threshold. This control process
avoids false-negative results. The same detection mechanism is
used for genotyping with HPV16 and HPV18 sequence-specific
probes.
Cobas HPV Test
The cobas HPV Test from Roche Molecular Systems is a real-
time PCR assay with automated sample extraction that indi-
vidually detects HPV16 and HPV18 genotypes as well as 12
other HR HPV types. The assay is a multiplex single tube reac-
tion with TaqMan probe chemistry. HPV16 DNA and HPV18
templates are each recognized by a specific probe labeled with
a unique fluorescent dye. The remaining 12 HPV types are
detected by a pool of TaqMan probes that are all labeled with
the same fluorescent dye. Therefore, the 14 HR HPV types are
detected in three different fluorescent channels, HPV16 or
HPV18 in the channel for the type-specific fluorochromes, and
the fluorochrome shared by the remaining 12 HPVs in the third
channel. The internal control gene, human β-globin, is detected
in the fourth channel to avoid false-negative results. The advan-
tage of this analytic strategy is the detection of 14 HR HPV
types and genotype 16 and 18 at the same time in a single reac-
tion. The assay is also only approved for cervical specimens
collected in ThinPrep cytology medium and Roche collection
medium.
Aptima HPV Assay
The Aptima HPV Assay from Gen-Probe is the fifth test for
HPV approved by the FDA, but the first assay to detect mRNA
(E6 and E7) instead of DNA. It uses magnetic microparticles
coated with sequence-specific probes to hybridize and capture
HPV E6 and E7 mRNA in the cell lysate. The signal amplifica-
tion is accomplished by transcription-mediated amplification
(TMA) with a reverse transcriptase enzyme (MMLV) and T7
RNA polymerase. Purified HPV mRNA strands are reverse-
transcribed into cDNA fragments that are fused with T7 pro-
moter sequences. T7 RNA polymerase recognizes these cDNA
fragments as templates to generate large amounts of HPV RNA
strands. A hybridization protection assay (HPA) is used for
detection. A chemiluminescent-labeled single-strand DNA
probe is hybridized to HPV RNA (synthesized by T7 polymer-
ase) to form DNA/RNA hybrids and the free labeled DNA
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38  HPV Detection Techniques 931
probes that did not hybridize are cleaved by enzymes. A lumi-
nescent signal is generated in the presence of HPV in the speci-
mens. A control human mRNA, which contains a poly-A tail, is
captured by oligo-dT and amplified by TMA. A labeled DNA
probe specific for this housekeeping gene is used for detection
and generates a different kinetic form of light. A similar mecha-
nism is used for the Aptima genotype (HPV16 and 18/45) assay.
ThinPrep collection medium and the Aptima Cervical Speci-
men Collection and Transport Kit are approved by the FDA for
sample collection using this test.
In the USA, the vast majority of clinical microbiology or
molecular diagnostic laboratories use one of above-mentioned
FDA-approved tests for HPV detection. In Europe and other
parts of the world, other HPV detection assays are used. A few
are briefly discussed below.
Other Commonly Used HPV Screening Assays
Additional HPV DNA-based commercial assays include the
Abbott RealTime High Risk HPV assay (Abbott, Wiesbaden,
Germany) that genotypes 16 and 18 and detects 12 HR HPV
types and the Reveal HPV Real-Time HPV Detection Kit
(GenoID, Budapest, Hungary) that detects 15 HR and 5 LR
HPV types. An additional mRNA-based assay is the PreTeck
HPV-Proofer (NorChip, Klokkarstua, Norway). It detects 5 HR
HPV types (16, 18, 31, 33, and 35), based on the expression of
their E6 and E7 mRNA.
HPV TESTING GUIDELINES FOR PATIENT
SCREENING AND MANAGEMENT
Two new sets of guidelines for cervical cancer screening have
been recommended in 2012, one by the US Preventative Serv-
ices Task Force45 and a second jointly by the American Cancer
Society, the American Society for Colposcopy and Cervical
Pathology, and the American Society for Clinical Pathology,46
that are in concurrence with the following:
1. Patients younger than 21 years should not be screened.
2. Patients between 21 and 29 years should be screened with
cytology alone every 3 years.
3. Patients between 30 and 65 should be screened with cytol-
ogy every 3 years, or with a combination of cytology and
HPV testing every 5 years.
4. Patients over 65 years should not be screened.
5. The screening approach described above should not be
altered for women who have received an HPV vaccine.
ADDITIONAL HPV DETECTION METHODS
Commercial Genotyping Assays
Multiple commercial assays for HPV genotyping are available
(Table 38-3). They are commonly used for clinical testing in
Europe, but their uses are limited for research in US.
PCR-based HPV Detection
Commonly used HPV primer sets for research includes PGMY,
My09/11, GP5+/6+, and SPF1/2. They all target the conserved
L1 region with consensus primers or primer pools to amplify a
broad spectrum of HPV types with different amplicon sizes
from 65 bp to 450 bp. Positive PCR results indicate the presence
of HPV. To further determine specific types, it is necessary to
hybridize the amplification products to specific probes, which
are typically fixed in solid state.
932 PART 3  Special Techniques in Cytology

TABLE
38-3  Commonly Used HPV Genotyping Assays
Total HPV
Assay Name Company Types Additional Information
Linear array HPV genotyping test Roche, Basel, Switzerland 37 Company did not breakdown the HR and LR
INNO-LiPA HPV genotyping extra Innogenetics NV, Ghent, Belgium 28 Automation is available
PapilloCheck Test Kit Greiner Bio-One, Vienna, Austria 24 18 HR and 6 LR
AID HPV-typing Kit Autoimmun Diagnostika, 15 Genotype HR HPV 16, 18, and 45, LR 6 and
Strasbourg, Germany 11. Group detection of other 10
Clart Human Papillomavirus 2 Genomica, Madrid, Spain 35 Company did not breakdown the HR and LR
IntelliPlex™ HPV DNA Genotyping Kit PlexBio, Taipei, Taiwan 50 16 HR and 34 LR
Hybribio 21 HPV GenoArray Hybribio Limited, Hong Kong, 21 15 HR and 6 LR
Diagnostic Kit China

Source: company websites.

HPV Detection in Formalin-fixed Paraffin-
embedded Specimens
HPV detection in oropharyngeal carcinomas is useful for clini-
cal management of these patients. The most commonly used in
the clinical setting is the antibody staining for a surrogate
marker human P16 protein, also known as cyclin-dependent
kinase inhibitor 2A (CDKIN2A). P16 slows the cell cycle by
inactivating cyclin-dependent kinases (cyclin D kinases CDK4/
CDK6) that phosphorylate the Rb protein and activate the E2F.
Oncogenic HPV infection/integration such as HPV16 and
HPV18 induces high-level expression of HPV E7, degradation
of Rb, and uncontrolled cell cycle progression as depicted in
Fig. 38-1. This process deregulates the feedback loop and
induces a high level of P16 expression. P16 immunohisto-
chemistry has shown excellent correlation with the direct
measures for detection of HPV.47 Thus, the immunohisto-
chemical detection of this surrogate biomarker for HPV infec-
tion fits logically into the molecular cascade of mechanisms
activated by the E6 and E7 oncoproteins.
PCR amplification of a 65 bp short amplicon in the L1 gene
using SPF1/2 primer sets to directly detect HPV is commonly
used for FFPE specimens in research settings. The primer
sequences can be found from the original paper.48 Since DNA
is damaged and fragmented in FFPE specimens, targeting longer
segments for amplification may yield false-negative results.
Diagnostic Accuracy of HPV
Screening Assays
FDA-approved HPV screening assays all have moderate analyti-
cal sensitivity, calibrated to the original HC2 assay to detect
about 5000 HPV genomes, to achieve an optimal balance
between clinical sensitivity (close to 100%) and specificity
(between 48% and 73% for cobas HPV for age 21–40 years) for
detecting CIN2/3 or worse.49 While all these assays have excel-
lent negative predictive value (close to 100%), their positive
predictive value for CIN3 or worse is minimal (cobas HPV
~8.5%). Therefore, adherence to the guideline for patient man-
agement is crucial to avoid excessive follow-up and colposcopy
procedures.
Concluding Remarks
Cytopathology is a screening technology that has high specifi-
city, but moderate sensitivity. It provides a subjective, yet accu-
rate, analysis that has relatively low throughput and requires
significant professional expertise at the level of cytopatholo-
gists and cytotechnologists. It is clear that this method requires
significant training and stringent quality control. In contrast,
HPV testing has high sensitivity, but moderate specificity. It
provides an objective analysis that is reproducible, robust, and
expensive. The molecular tests for HPV detection and geno-
typing are automated and essentially “turn key,” but there is a
significant investment in instrumentation and infrastructure,
including proficiency testing. While current guidelines in the
USA prescribe concurrent testing by cytopathology and HPV
detection, frontline screening by HPV analysis with confirma-
tion by cytopathology is gaining favor in some regions, such as
Europe.
Access the references list online at http://www.expert
consult.com

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