correspondence
Detection of circulating tumour DNA in patients with
aggressive B-cell non-Hodgkin lymphoma
Current methods for detecting the presence of disease in
patients with diffuse large B cell lymphoma (DLBCL) or
mediastinal large B-cell lymphoma (MLBCL) rely primarily
on imaging methods, which are associated with significant
cost and radiation exposure. Very few patients with DLBCL
have evidence of circulating disease using flow cytometric
assays (Mancuso et al, 2010). This has so far precluded the
development of minimal residual disease (MRD) assessment
tools in those diseases, in contrast to tumours with a circu-
lating component, where MRD assays are emerging as
important methods (Ferrero et al, 2011). The availability of
high-throughput sequencing techniques now provides an
opportunity to probe for the presence of very small amounts
of circulating tumour genetic material in peripheral blood
(PB). If sequencing-based methods can reliably detect circu-
lating disease, they could eventually find a role in the treat-
ment and monitoring of patients with those tumours. We
present here a pilot study of a sequencing method designed
to examine whether tumour DNA is detectable in patients
with newly diagnosed DLBCL/MLBCL, and whether it
becomes undetectable after therapy.
Tumour samples obtained from PB samples and forma-
lin-fixed paraffin-embedded (FFPE) or frozen tissue were
analysed using the Sequenta LymphoSIGHTTM method
(Sequenta Inc., South San Francisco, CA, USA), as previ-
ously described (Faham et al, 2012a, 2012b). Briefly, geno-
mic DNA was extracted from tumour cells, peripheral blood
mononuclear cells (PBMCs) or plasma, amplified using
locus-specific primer sets for IGH and IGK rearrangements,
and sequenced. A frequency >5% in the diagnostic tumour
sample was considered to represent one of the tumour clo-
notypes; the frequency of the tumour clonotype(s) in PB
was calculated relative to the total number of reads in the
sample.
We approached consecutive patients with newly diagnosed
DLBCL or MLBCL who presented for care at Dana-Farber
Cancer Institute (DFCI). Independently, five patients with
DLBCL who had banked tumour and blood samples at the
M.D. Anderson Cancer Center (MDACC) were enrolled on a
similar study. Written informed consent was obtained from
all patients. All patients had a sample of tumour from their
diagnostic biopsy sent to Sequenta Inc. for analysis. We col-
lected a sample of whole blood before the first cycle of ther-
apy; when possible, we also collected a sample after the
conclusion of therapy (only in the DFCI cohort).
ª 2013 John Wiley & Sons Ltd
British Journal of Haematology, 2013, 163, 123–144
Seventeen patients were enrolled on this study (Table I).
None of 13 patients biopsied had bone marrow involvement.
At least one dominant tumour clonotype could be estab-
lished from the diagnostic biopsy for all 16 patients with suf-
ficient amounts of amplifiable DNA. The diagnostic biopsy
sample for one patient had <0.3 ng of DNA, and a dominant
tumour clonotype was not identified. Among the 16 patients
with an established tumour clonotype, circulating tumour
DNA could be detected pre-treatment in plasma in 11 of the
16 (69%, 90% confidence interval [CI] 45–87%), in PBMCs
in eight of the 16 (50%, 90%CI 28–72%), and in either in 13
of the 16 (81%, 90%CI 58–95%) (Table I, Fig 1A, B). The
three patients in whom no DNA could be detected at diag-
nosis all had stage I disease and had no evidence of disease
by positron emission tomography-computerized tomography
(PET-CT) after their diagnostic procedure at the time that
the study sample was drawn. Therefore, tumour DNA was
detected in all 13 (100%) of patients with evidence of fluoro-
deoxyglucose-avid disease at the time of sample collection.
There was no apparent correlation between the level of circu-
lating tumour DNA and clinical characteristics including his-
tology, stage, burden of disease, or International Prognostic
Index (The International Non-Hodgkin’s Lymphoma Prog-
nostic Factors Project, 1993). There was also no association
between the presence or level of circulating DNA and lactate
dehydrogenase elevation pre-treatment.
All patients were treated with chemo-immunotherapy; one
patient with MLBCL also received radiotherapy. Seven
patients from the DFCI cohort returned for collection of
post-therapy samples; the remaining received their care out-
side of DFCI and did not return for follow-up. At a median
follow-up of 269 (range, 220–281) days from the beginning
of therapy, none of the patients have relapsed. Among the
seven evaluable patients, six (86%, 90%CI 48–99%) had no
detectable circulating tumour DNA at the conclusion of
chemoimmunotherapy (Table I, Fig 1C).
Using a novel high-throughput sequencing technique, we
could determine the dominant tumour clonotype in 94% of
17 patients with DLBCL/MLBCL from routine diagnostic
tumour samples, and in 100% of those with adequate
amount of tumour material for analysis. Among those
patients, 81% had detectable circulating tumour DNA in
plasma or PBMCs; the proportion was 100% among the
patients with evidence of disease by PET-CT scan after their
diagnostic surgical biopsy. Furthermore, the tumour DNA
 124
Table I. Patient characteristics and tumour DNA results.

Clinical characteristics Tumour DNA measurements*

Correspondence

Dominant
tumour Post-
Center Bone Ki67 Treatment sequence treatment
patient Histology stage Age marrow IPI LDH (%) Treatment outcome identified Pre-treatment PB assay PB assay

DFCI 1 DLBCL IE (bowel) 43 Negative 0 Nl 50 RCHOP 9 6 CR Yes Negative† Negative

DFCI 2 DLBCL IIE (thyroid) 46 Negative 0 Nl 90 DA-REPOCH 9 6 IT chemo CR‡ Yes Positive in plasma and PBMCs Positive in
PBMCs only
DFCI 3 DLBCL IV (bowel) 36 Negative 2 Nl 30–40 RCHOP 9 6 CR Yes Positive in plasma only Negative
DFCI 4 MLBCL I 29 N/A 1 Hi 80–90 RCHOP 9 6 + IFRT PR by PET after Yes Positive in plasma only Negative
RCHOP§
DFCI 5 DLBCL IV (testis) 75 N/A 2 Nl 90 RCHOP 9 6 IT chemo CR (after three Yes Positive in plasma and PBMCs Negative
cycles)
DFCI 6 DLBCL I 66 Negative 1 Nl 80–90 RCHOP 9 6 CR (after three Yes Negative† N/A
cycles)
DFCI 7 DLBCL IE (bone) 31 N/A 0 Nl 80 RCHOP 9 6 + IFRT CR (after three Yes Positive in plasma and PBMCs N/A
cycles)
DFCI 8 DLBCL III 84 N/A 2 Nl 60–70 RCHOP 9 6 CR No N/A N/A
DFCI 9 DLBCL IV 72 Negative 2 Nl 80–90 RCHOP 9 6 No follow-up Yes Positive in PBMCs only N/A
DFCI 10 DLBCL II 53 Negative 0 Nl 60 RCHOP 9 6 CR Yes Positive in plasma and PBMCs Negative
DFCI 11 MLBCL II 61 Negative 2 Nl 40 RCHOP 9 6 CR Yes Positive in plasma only Negative
DFCI 12 DLBCL IE (spleen) 64 Negative 2 Nl >90 RCHOP 9 6 CR Yes Negative† N/A
MDACC 13 DLBCL III 52 Negative 1 Nl 10 RCHOP 9 6 CR Yes Positive in plasma and PBMCs N/A
MDACC 14 DLBCL IV (ovary) 31 Negative 2 Hi 90 RhCVAD 9 6 CR Yes Positive in plasma only N/A
MDACC 15 DLBCL IV (lung) 77 Negative 3 Hi 50 RCHOP 9 6 CR Yes Positive in PBMCs only N/A
MDACC 16 DLBCL IV (bone) 79 Negative 3 Hi 80 RCHOP 9 4 CR Yes Positive in plasma only N/A
MDACC 17 DLBCL III 68 Negative 3 Hi 80 RCHOP 9 6 CR Yes Positive in plasma and PBMCs N/A

IPI, International Prognostic Index (The International Non-Hodgkin’s Lymphoma Prognostic Factors Project, 1993); Nl, normal; Hi, High; DFCI, Dana-Farber Cancer Institute; MDACC, M.D.
Anderson Cancer Center; DLBCL, diffuse large B cell lymphoma; MLBCL, primary mediastinal B-cell lymphoma; N/A, not available; CR, complete remission; PR, partial remission; R, rituximab;
CHOP, cyclophosphamide, adriamycin, vincristine and prednisone; DA-REPOCH, dose-adjusted rituximab, etoposide, prednisone, vincristine, cyclophosphamide, adriamycin; hCVAD, hyperfraction-
ated cyclophosphamide, vincristine, adriamycin, dexamethasone; IFRT, involved field radiotherapy; IT, intrathecal; PBMCs, peripheral blood mononuclear cells.
*Measurements for all available samples consisted of separate plasma and PBMC assays. ‘Negative’ means that tumour sequences were detected in neither plasma nor PBMCs.
†No evidence of disease by positron emission tomography -PET/computerized tomography (PET-CT) after diagnostic biopsy/surgery at the time the pre-treatment sample was drawn.
‡Low level fluorodeoxyglucose-positive residual in thyroid with negative biopsy.
§This patient subsequently received involved field radiotherapy and achieved CR.

ª 2013 John Wiley & Sons Ltd

British Journal of Haematology, 2013, 163, 123–144
 Correspondence

(A)

(B)

Fig 1. DLBCL/MLBCL tumour clone sequences

in peripheral blood of patients. (A). Tumour
sequences pre-treatment in peripheral blood (C)
mononuclear cells (PBMCs) for each patient.
The patient number corresponds to that in
Table I; Patient 8 had no established tumour
clonotype. The number of sequences is
reported per million PBMCs. Values shown are
the mean of the maximum calibrating receptor
clones for IGH DJ (circles), IGH VDJ
(squares), or IGK (triangles). (B). Tumour
sequences pre-treatment in plasma for each
patient. The number of sequences is reported
per millilitre of plasma. (C). Changes in
peripheral blood mononuclear cells (left panel)
and plasma (right panel) tumour clone
sequences pre- and post-treatment. Values are
shown for each of the seven patients with both
pre- and post-treatment samples.

cleared in all but one patient after curative-intent chemo-

immunotherapy. This sequencing method may therefore rep-
resent a reliable dynamic tumour assessment. Our study is
based on a small number of cases, and leaves many questions
still unanswered, which await the maturation of ongoing lar-
ger studies with longer follow-up. Nonetheless, our prelimin-
ary findings recall the results obtained with minimal residual
disease (MRD) assays in diseases where MRD detection has
important clinical implications, and in which this sequencing
method has already shown promising results (Logan et al,
2011, 2012; Faham et al, 2012a,b; Harris et al, 2012). They
raise the possibility that MRD may become an evaluable and
actionable result in DLBCL and MLBCL, which are not tradi-
tionally thought to have a circulating component.
Acknowledgments
P.A. is supported by an ASCO/Conquer Cancer Foundation
Career Development Award. The blood and tissue samples
from the University of Texas M. D. Anderson Cancer Center
were obtained from the Lymphoma Tissue Bank supported
by the Lymphoma SPORE (CA136411) to A.Y. and S.S.N.,
the Cancer Center Support Grant CA16672, and the Fredrick
B. Hagemeister Research Fund.
Author contributions
P.A.and Y.O. designed and performed the research, analysed
the data, and wrote the paper. D.S.N. designed the research,

ª 2013 John Wiley & Sons Ltd 125

British Journal of Haematology, 2013, 163, 123–144
Correspondence

analysed the data, and edited the paper. M.F. collected data, Eric D. Jacobsen1
analysed the data, and edited the paper. C.C. analysed the data Matthew S. Davids1
and edited the paper. M.K. analysed the data and edited the Caron Jacobson1
paper. L.W. collected data, analysed the data, and edited the David C. Fisher1
paper. S.B. collected data and edited the paper. A.S.L. collected Jennifer R. Brown1
data and edited the paper. E.D.J. collected data and edited the Nathan H. Fowler2
paper. M.D. collected data, and edited the paper. C.J. collected M. Alma Rodriguez2
data, analysed the data, and edited the paper. D.C.F. collected Michael J. Wallace5
data and edited the paper. J.R.B. collected data, and edited the Sattva S. Neelapu2
paper. N.H.F. collected data, and edited the paper. M.A.R. col- Scott Rodig6
lected data, and edited the paper. S.N. collected data, and edited Anas Younes2
the paper. S.R. collected data, and edited the paper. A.Y. Arnold S. Freedman1
1
designed the research, collected data and edited the paper. A.S.F. Department of Medical Oncology, Dana-Farber Cancer Institute,
designed the research, collected data and edited the paper. Boston, MA , 2Department of Lymphoma and Myeloma, The University
of Texas, M.D. Anderson Cancer Center, Houston, TX , 3Department
of Biostatistics and Computational Biology, Dana-Farber Cancer
Conflict of interest
Institute, Boston, MA , 4Sequenta Inc, South San Francisco, CA ,
5
M.F., C.C., M.K. and L.W. are employees of Sequenta Inc. The Department of Diagnostic Radiology, The University of Texas, M.D.
authors have no other relevant conflicts of interest to disclose. Anderson Cancer Center, Houston, TX, and 6Department of Pathology,
Brigham and Women’s Hospital, Boston, MA, USA
Philippe Armand1*
E-mail: parmand@partners.org*
Yasuhiro Oki2*
Donna S. Neuberg3
*These two authors contributed equally to this work.
Malek Faham4
Craig Cummings4
Keywords: lymphomas, minimal residual disease, sequences
Mark Klinger4
Li Weng4
First published online 25 June 2013
Sangeetha Bhattar1
doi: 10.1111/bjh.12439
Ann S. LaCasce1

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