Exploring the Tumor Clonal Dynamics between Diffuse Large B-Cell Lymphomas and Bone Marrow Involvement By Deep Immunoglobulin Heavy Chain VDJ Sequencing

INTRODUCTION : The BM of patients with diffuse large B-cell lymphomas (DLBCL) can be involved as DLBCL (morphologically concordant), small B-cell lymphoma (SBCL) (morphologically discordant) or a monotypic B-lymphocyte population (MBCL) detected by flow cytometry (FC). The tumor clonal dynamics in the bone marrow (BM) of patients with DLBCL has not been studied and is poorly understood. We examined DLBCL and their paired BM, and comparatively analyzed their clonal evolution patterns and dynamics based on V(D)J usage and IGH somatic hypermutation profiles by deep sequencing.

METHODS : DLBCL and paired BM from 39 patients with DLBCL were included. In 14 of 39 cases, there was morphologic, immunophenotypic and/or cytogenetic (CG) evidence of BM involvement: 7 with DLBCL, 3 with SBCL, 3 with MBCL by FC only, and 1 with abnormal CG only. The IGH VDJ regions were PCR amplified using framework 1 (FR1) and/or FR2 primers and the amplicons generated from diagnostic DLBCL and BM were subjected to paired-end sequencing in separate flow cells, resulting in an average of ~0.75 million reads per sample. The V(D)J region with the highest count of read-pairs was defined as the major rearrangement combination. V(D)J usage and somatic hypermutations of all identifiable clones were analyzed. Clonal and subclonal comparisons were visualized by phylogenetic trees of the matched tumor and BM samples.

RESULTS : In all 14 cases in which there was prior morphologic, immunophenotypic and/or CG evidence of BM involvement by B-cell lymphoma, VDJ reads of various abundance related to the dominant clone in the original extra-medullary tumors (DoT) could be identified in the BM. They represented either tumor clones identical (DoBM) or divergent/ancestral (DiBM) to the DoT. All of these 14 cases showed tumor clones of the DiBM type in the BM. In 8 of these 14 cases, DoBM-type clones could also be detected, but DiBM-type clones dominated over the DoBM-type in 5 of these 8 cases. In 4 of the 10 cases which were involved by DLBCL or SBCL, the most abundant VDJ clones in the BM had distinctly different VDJ rearrangements compared to the DoT, implying that tumor clones unrelated to DoT represented the most abundant clones in the BM (UnrBM). Overall, DiBM- or UnrBM-type clones were the dominant clones in 90% (9/10) of cases with morphologic evidence of lymphoma involvement in BM. In 5 of 7 (71%) cases in which the BM showed involvement by DLBCL, i.e. morphologically concordant, only UnrBM- and/or DiBM-type clones were identified; in the remaining 2 cases, both DiBM and DoBM-type clones were present, with DiBM-type clone as dominant in 1 of the 2 cases. Minor clones similar to the DiBM- or UnrBM-type clones could be identified in a subset of the diagnostic extramedullary tumors.

Among the negative BM cases, deep sequencing detected lymphoma clones at a relatively low abundance in 16 of 25 cases (64%). In 14 of these 16 cases, DoBM-type clones were identified; 6 of these 14 also harbored DiBM-type clones. Two cases (2/16) had DiBM-type clones only. Notably, in two cases, the negative staging BM harbored minute DiBM- or UnrBM-type clones which persisted post-chemotherapy and resembled the dominant clone of the relapsed DLBCL, suggesting that this minute clone was chemo-resistant and may have served as a potential precursor for relapse.

CONCLUSIONS : Our studies indicate a complex tumor clonal dynamics in the BM of patients with DLBCL. Lymphoma involvement of the BM in patients with DLBCL, in most cases, represents expansion of tumor cells clonally divergent or unrelated from the dominant tumor clone in the primary tumor. In some instances, such tumor clones in the marrow can be traced back to the original diagnostic tumor. These findings underscore the extent of tumor heterogeneity between the primary DLBCL and the lymphoma in the BM that has not been previously recognized by routine pathologic examination. The frequent detection of low-abundance DoBM or DiBM-type clones in pathologically negative BM suggests that expansion of these clones depends on the appropriate microenvironment, which may be provided by the neoplastic lymphoid tissue but not the BM. In addition, minute clones of the DiBM- or UnrBM-type detected in the BM by deep VDJ sequencing but not by conventional diagnostic methods may be a harbinger of frank lymphoma development in BM or extramedullary relapse; thus, their detection may be of clinical significance.