Detection of circulating tumour <scp>DNA</scp> in patients with aggressive B‐cell non‐Hodgkin lymphoma (2013)

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 circulating 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 circulating disease, they could eventually find a role in the treatment 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 formalin-fixed paraffin-embedded (FFPE) or frozen tissue were analysed using the Sequenta LymphoSIGHT™ method (Sequenta Inc., South San Francisco, CA, USA), as previously described (Faham et al, 2012a, 2012b). Briefly, genomic 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 clonotypes; 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 collected a sample of whole blood before the first cycle of therapy; when possible, we also collected a sample after the conclusion of therapy (only in the DFCI cohort). Seventeen patients were enrolled on this study (Table 1). None of 13 patients biopsied had bone marrow involvement. At least one dominant tumour clonotype could be established from the diagnostic biopsy for all 16 patients with sufficient 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 1, Fig 1A, B). The three patients in whom no DNA could be detected at diagnosis 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 fluorodeoxyglucose-avid disease at the time of sample collection. There was no apparent correlation between the level of circulating tumour DNA and clinical characteristics including histology, stage, burden of disease, or International Prognostic Index (The International Non-Hodgkin's Lymphoma Prognostic 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 outside 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 1, 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 cleared in all but one patient after curative-intent chemo-immunotherapy. This sequencing method may therefore represent 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 larger studies with longer follow-up. Nonetheless, our preliminary 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 traditionally thought to have a circulating component. 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. P.A.and Y.O. designed and performed the research, analysed the data, and wrote the paper. D.S.N. designed the research, analysed the data, and edited the paper. M.F. collected data, analysed the data, and edited the paper. C.C. analysed the data and edited the paper. M.K. analysed the data and edited the paper. L.W. collected data, analysed the data, and edited the paper. S.B. collected data and edited the paper. A.S.L. collected data and edited the paper. E.D.J. collected data and edited the paper. M.D. collected data, and edited the paper. C.J. collected data, analysed the data, and edited the paper. D.C.F. collected data and edited the paper. J.R.B. collected data, and edited the paper. N.H.F. collected data, and edited the paper. M.A.R. collected data, and edited the paper. S.N. collected data, and edited the paper. S.R. collected data, and edited the paper. A.Y. designed the research, collected data and edited the paper. A.S.F. designed the research, collected data and edited the paper. M.F., C.C., M.K. and L.W. are employees of Sequenta Inc. The authors have no other relevant conflicts of interest to disclose.