Utility Of Non-Invasive Monitoring Of Circulating Tumor DNA At Diagnosis, Interim Therapy, and Relapse Of DLBCL Using High-Throughput Sequencing Of Immunoglobulin Genes

Recent studies have shown limited utility of routine surveillance imaging for patients with diffuse large B-cell lymphoma achieving remission after R-CHOP (Thompson et al, ASCO 2013 #8503; Ghesquieres et al, ICML 2013 #226). Separately, response assessment using early interim PET-CT during R-CHOP has suboptimal accuracy. We hypothesized that non-invasive tumor burden monitoring using peripheral blood could have utility for diagnosis and monitoring of DLBCL.

We prospectively profiled two cohorts of patients with DLBCL at various stages in their disease, including at diagnosis, during various therapies, after achieving remission, and upon relapse or progression. Cohort1 patients were treated with curative intent at Stanford University with blood collected at multiple time points during follow-up. Cohort2 patients participated in a multi-center Austrialian Phase 2 study (ALLG NHL21), where patients with poor risk DLBCL as identified by residual disease on interim PET/CT after 4 R-CHOP cycles received early treatment intensification using R-ICE/ASCT; blood samples were collected before the 1^st and after the 4^thR-CHOP cycles. Tumor-derived clonotypic IgH and IgK sequences were defined in diagnostic tissues grossly involved with DLBCL, and then tracked using high-throughput sequencing of immunoglobulin genes (Faham et al 2012 Blood).

We successfully defined for 77% (30/39) of patients the tumor-specific immunoglobulin sequences required for non-invasive surveillance, with calibration success correlating with input tissue mass. Using 194 assays on 79 blood specimens from these patients, we detected disease in 75% of patients at diagnosis of DLBCL, in 18% at interim time-points during therapy, 0% during radiographic/clinical remissions, and 80% at relapse or disease progression. When considering radiographic/clinical follow-up, noninvasive monitoring had 0% false positive rate but 26% false negative rate, with the latter likely due to low tumor burden at time of phlebotomy. Disease burden detectable in the blood was significantly correlated between IgH and IgK assays, and between the cellular (leukocyte) and acellular (plasma) fractions (r=0.77, p=0.004). Strikingly, clonotypic IgH/IgK sequences were significantly more abundant in plasma than in circulating leukocytes, and identified exclusively in plasma in 5% of blood samples with available paired cellular/acellular fractions. This allowed more accurate disease detection and monitoring using plasma circulating tumor DNA than blood leukocytes, including in cases of radiographically defined isolated CNS involvement, and in histological transformation of FL. Within the limitations of this small study employing frequent surveillance imaging, we observed no significant lead time between detection of disease in the plasma and radiographic/clinical evidence of progression.

Non-invasive surveillance of circulating tumor DNA has utility for tumor burden at diagnosis and relapse of DLBCL using high-throughput sequencing of immunoglobulin genes, with superior detection in blood plasma. The current assay has limited sensitivity at early interim time points during therapy.

Kong:Sequenta Inc.: Employment. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.