ALBERT

Description: Background: Diffuse large B-cell lymphoma (DLBCL) typically express monotypic surface immunoglobulin (sIg) light chain as part of the B-cell receptor (BCR) complex. Occasional cases are, however, sIg-negative and the clinical significance of this remains unclear. Distinct subtypes of DLBCL, including germinal center B-cell-like (GCB) and activated B-cell (ABC) type, exhibit differential dependence upon BCR-mediated survival signals. The role of activating mutations downstream of the BCR signaling complex (i.e. CD79A/B or CARD11), or in parallel costimulatory pathways (i.e. MYD88 mutations), are now well recognized in promoting such survival mechanisms. Surprisingly, however, less is known about the importance of sIg expression, or lack thereof, on various subsets of DLBCL or other aggressive large B cell lymphomas. Design: We sought to further characterize the clinicopathologic and genetic features of primary human surface Ig-negative DLBCL-not otherwise specified (NOS). All cases of large B-cell lymphoma that had flow cytometry performed at the University of Rochester from 2010-2013 were screened for evidence of a surface Ig-negative B-cell population. Additional available immunophenotypic data and/or clinicopathologic features were recorded. PCR heteroduplex analysis and sequencing of fresh frozen tumor samples was performed to identify putative compensatory activating mutations in MYD88 and CD79A /B, respectively. In addition, using multiplexed PCR and sequencing analysis we attempted to identify the presence or absence of functional immunoglobulin heavy chain (IGH)gene rearrangements. Results: Among 209 diagnostic specimens analyzed, 13 cases (6.2%) were found to contain a discrete population of CD19/CD20-positive B-cells lacking sIg light chain expression. These included 8 DLBCL-NOS, nearly all of which were GCB-type, including 6 out of 7 cases, or 83%, which were CD10-positive. This proportion is significantly greater than the 42% of all DLBCL-NOS that express CD10 (based on data from a large publicly available on-line database; p 〈 0.025, based on binomial probability). Furthermore, of the other high grade sIg-negative B cell lymphomas identified, 4 out of 5 were also CD10-positive. These included 2 cases of B-cell lymphoma, unclassifiable (BCL-U), intermediate between DLBCL and Burkitt lymphoma, and 2 cases of transformed follicular lymphoma (FL) - therefore also histologically and immunophenotypically consistent with derivation from a germinal center B-cell. The only exception was a non-GCB primary CNS lymphoma. Among 5 cases of sIg- DLBCL available for analysis, all were negative for the MYD88 L265P activating mutation, while only a single case contained an activating mutation in CD79A/B (CD79B Y196 ITAM mutation). In the same set, only 2 (of 5) cases produced amplicons by IGH PCR, and sequencing revealed only one of these had an intact reading frame. Conclusion: DLBCL-NOS lacking definitive sIg light chain expression are infrequent (less than 10%), but these tumors are predominantly GCB subtype. This observation lends support to the model that these tumors rely less on BCR-mediated survival signals, which may have implications for choosing targeted therapy. The mechanism by which these lymphoma survive despite the apparent lack of functional BCR is unknown, but compensatory activating mutations known to be frequent among ABC-type DLBCL appear to be lacking. Disclosures No relevant conflicts of interest to declare.

Description: Background: Recombination-activating gene (RAG) mediates recombination of the immunoglobulin heavy chain variable gene (IGHV) in immature B lymphocytes. Aberrant targeting of RAG to non-IGH sites in B cell acute lymphoblastic leukemia (B-ALL) contributes to the development of driver mutations and clonal evolution (Papaemmanuil et al. Nat. Genet. 2014). This finding suggests that patients whose ALL involves increased RAG-mediated clonal diversification would exhibit more aggressive disease. As an initial step toward addressing this hypothesis, we asked whether the extent of RAG-mediated diversification in a patient's leukemia is consistent across all cells or variable between subclones. To assess variation in RAG-mediated subclone diversification, we interrogated rearranged IGH sequences from diagnostic B-ALL specimens to identify early subclones (SC) and to quantify RAG-derived sub-subclones (SSC) from each. We hypothesized that if RAG activity is a consistent feature of the leukemia, all SC within a single patient will have a comparable extent of SSC evolution. Methods: Amplicon-based IGHV sequencing identified the number of clonal IGH SC and their RAG-derived SSC in 22 pre-treatment adult and pediatric patients with newly-diagnosed B-ALL. Analysis was performed on peripheral blood (PB) for all patients in addition to diagnostic bone marrow (BM) for 16 of the 22 patients studied. Ultra-deep sequencing of IGH utilized 500 ng genomic DNA (representing approximately 80,000 cells/specimen) for the MiSeqDx platform, generating ~300 bp reads surrounding the VDJ junctional region. The NCBI IgBlast sequence analysis tool assigned IGH VDJ identities according to germline reference. Methods for determining subclones (SC) of shared clonal lineage involved classifying reads with a common Jh identity and 6 shared upstream Dh-Jh junctional nucleotides (termed "6N_Jx") according to defined methods (Gawad et al. Blood 2012). Further evolved sub-subclones (SCC) - as evidence of ongoing RAG activity - were defined by unique junctional sequences upstream of the common Dh-Jh junction (termed the "NDN" region). SSC were quantified according to their relative proportions within each SC family. Results: VDJ-rearranged SC families were detected for 20 of the 22 patients studied (median 2/patient; range 1-7); further analysis to assess variation in RAG-mediated diversification was limited to the 18 cases with ≥ 2 SC. In 9 of these 18, numerous evolved SSCs were identified from at least one SC in the specimen. In 4/9, there were starkly distinct levels of RAG-mediated diversity observed between intrapatient SC families, with some clonal precursors giving rise to numerous SSCs (up to 2,200 SSC per SC) while others showed minimal-to-no RAG-derived evolution (Table 1). Fifteen of 16 patients with matched BM and PB diagnostic specimens had detectable SCs. Of these, 73% (11/15) shared the dominant SC, while in 27% (4/15) the dominant SC differed between sites. However, regardless of which SC predominated, the extent of SSC diversification within each SC was preserved between the BM and the PB, with similar evolution patterns observed regardless of disease site. There was no relationship between SC read frequency and number of SSCs. Conclusions: Using deep sequencing of a single IGHV locus, wide variation in the extent of subclone diversification was observed in 4 of 20 patients with B-ALL. These findings indicate that the degree of RAG-mediated heterogeneity in B-ALL can range from minimal to extensive among distinct subclones in a single patient. The data underscore the relevance of single cell investigation of tumor characteristics to improving our understanding of the mechanisms of clonal evolution in lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

Description: As we previously described, intra-peritoneal injection of single cell suspensions (including non-neoplastic cells) from 〉90% of primary B cell non-Hodgkin's lymphoma specimens results in lymphoma cell growth in the omentum of the NSG mouse strain (NOD-scid IL2rg null) (ASH Meeting on Lymphoma Biology 2014; abstract #144). To date, we have shown reproducible engraftment of 17/20 specimens (including 9 follicular, 5 marginal zone, 4 diffuse large B cell, 2 mantle cell). Molecular markers specific for each patient's tumor (including clone specific features of the IGK, IGH, BCL2 and BCL1 loci) provided verification that all 17 of the engrafted tumors contained the same clone identified in the original specimen. Each tumor was implanted and engrafted in at least 3 mice (range 3-41) and each had a characteristic and often distinctive time course of engraftment, propensity to disseminate, and histologic features, indicating that the behaviors of the xenografts reflected intrinsic properties of the original tumors. B cell engraftment required CD4+ T cells. B cells were purified by negative selection from three specimens with particularly robust engraftment, a FL (J1) and two MZL (M1 and W1). Depletion of non-B cells entirely ablated engraftment for all 3 tumors; mock depletion had no effect. Selective depletion of CD4+ but not CD8+ T cells ablated engraftment of both tumors assessed (J1 and M1). Finally, purified CD4+ cells (but not CD8+) fully reconstituted engraftment of purified B lymphoma cells from tumor M1. In addition, the xenograft model highlighted two unexpected features of these tumors: 1) The neoplastic B cells of some tumors were relatively resistant to engraftment compared to non-neoplastic B cells present in the same specimen. For 5 tumors (2 MZL and 3 FL), the number of human cells and the ratio of neoplastic to non-neoplastic B cells in the xenografts at two weeks were determined by performing bar-coded, ultra-deep sequencing on the entire mesentery including the omentum. For the 2 MZL, the fraction of B cells that was clonal was 〉 90% at 2 weeks ; in contrast, for 3 FL specimens, the fraction of B cells that were neoplastic dropped from 〉60% at time of implantation to less than 5%. These results indicate that neoplastic B cells do not necessarily have an engraftment advantage over non-neoplastic B cells. Where non-neoplastic cells can respond to proliferation/survival cues, neoplastic cells in the same FL specimens apparently resist the same signals. 2) We observed that B cells, including neoplastic B cells, have a striking propensity to differentiate into plasma cells when engrafted in the mice. In all specimens at the time of injection, neoplastic B cells comprised 〉60% of the cells and plasma cells were negligible. In contrast, at 2-4 weeks the number of CD20+ B cells fell to 20% of the cells in all xenografts. 4/17 xenografts (including 2 FL and 2 MZL tumors) assayed showed monoclonality of the plasma cells at two weeks (based on kappa:lambda light chain ratio). These results indicate that the neoplastic B cells of some tumors have a developmental plasticity in the xenograft that is not typically apparent in patients. In summary, a xenograft model reveals several features of low grade B cell lymphomas that are not apparent from static observations: 1) CD4+ T cells are essential to engraftment, suggesting that CD4+ T cells may be critical to lymphomagenesis; 2) neoplastic B cells of some tumors do not respond to the proliferation/survival cues to which non-neoplastic B cells are sensitive; 3) neoplastic B cells of some tumors differentiate into post-mitotic plasma cells, potentially eliminating these cells from the proliferative pool. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 146 Background: Cancers are characterized by genomic instability, and the resulting intra-clonal diversity is thought to underlie the evolution of more aggressive clinical course behavior and therapy resistance. Intra-clonal heterogeneity in follicular lymphoma (FL) has been documented qualitatively by analysis of mutations induced by Activation Induced Deaminase (AID) in IGH coding regions. AID also aberrantly targets non-coding regions of the genome causing numerous “passenger” mutations. To obtain a quantitative measure of genetic variation and to characterize population dynamics in FL, we performed ultra-deep “next generation” sequencing of selected non-coding regions known to be aberrantly targeted by AID in FL. Herein, we quantify and characterize AID-induced genetic variation in FL and relate the extent of variation to grade. Method: An ultra-deep sequencing approach, able to identify sequences present at 0.3% frequency, was developed to quantify sub-clonal populations within a tumor so that the extent of intraclonal heterogeneity can be compared between tumors. Genomic DNA was obtained from fresh/frozen specimens (10 FL grade 1/2, 2 FL grade 3, 3 hyperplastic lymph nodes and 1 epithelial cell line). H&E sections and flow cytometry data were examined to corroborate the diagnosis and fraction of tumor (〉50% in all cases). The ten sequenced (ABI-SoLID) regions were PCR amplicons of: 5'UTRs of BCL2, BCL6, MYC, PAX5, PIM1, RHOH, CD83 and SYK; IGH (mu enhancer-J6; clone-specific Vh-J6). Average mapped coverage was 〉 20,000-fold. To detect rare mutations while suppressing read noise, data were mapped using an algorithm (BFAST) designed to detect rare Single Nucleotide Variants (SNV); a novel recursive mapping pipeline enhanced detection of SNVs in heavily mutated regions. A novel filtering algorithm handled data as 34 base-pair “words” with confirmation based on partial assembly using independently obtained sequences from adjacent regions. Results: 656 mutations were detected in non-coding regions of the 12 FL specimens. FL specimens had more mutations in all 5' UTRs combined (range 14–193, median 47, n=12) than control specimens (range 2–7, n=4). The mutations were associated with AID-motifs (p