IgH VDJ Research Articles

Abstract 6673: Simultaneous detection of IGH VDJ rearrangements and JH translocations via Ring-Seq, a novel multiplex PCR technology for targeted sequencing of translocations with novel gene partners or breakpoints

Abstract Introduction: Next-generation sequencing (NGS) of B cell receptor rearrangements is a leading method for clonality assessment and measurable residual disease (MRD) monitoring of B cell malignancies. Current methods rely upon multiplex PCR (mPCR) amplification of rearranged IGH, IGK or IGL loci via variable and joining gene targeting primers followed by NGS to assess rearrangements. Limitations include a need for reflex testing owing to somatic hypermutation (SHM) mediated loss of sensitivity and an inability to detect clinically relevant translocations of the IGH locus. Here we apply Ring-Seq, a novel technology for the targeted detection gene fusions with unknown partners, to simultaneously detect IGH VDJ rearrangements and translocations in a single reaction. We demonstrate detection of clinically relevant translocations and VDJ rearrangements from highly degraded gDNA. Methods: The method begins with a highly efficient circularization of DNA fragments followed by a multiplex inverse PCR of joining genes that preferentially amplifies breakpoint junction containing templates. Amplicon libraries are sequenced on the G4 platform and analyzed to detect translocations and VDJ rearrangements. We pooled gDNA from reference cell lines harboring marker IGH VDJ rearrangements and BCL1/2-JH translocations. Pooled reference gDNA was spiked into a background of healthy donor PBL gDNA to evaluate performance over a range of marker frequencies. To simulate performance with degraded materials, gDNA input was fragmented to ~200bp by sonication prior to analysis. Results: BCL1-JH and BCL2-JH translocations and marker IGH VDJ rearrangements were detected from 50ng pooled reference gDNA. Translocations and marker VDJ rearrangements were also detected in 50ng samples consisting of reference gDNA spiked at 10% and 1% frequency into a background of healthy donor PBL gDNA. Sequencing of healthy donor PBL gDNA yielded IGH repertoire features that were highly concordant with results obtained from BIOMED-2 mPCR analysis of the same samples. Conclusions: We have applied Ring-Seq to enable the simultaneous detection of B cell receptor rearrangements and JH translocations relevant to the diagnosis of MCL and FL while minimizing loss of sensitivity owing to SHM. Traditional mPCR often fails to detect JH translocations owing to variation in the breakpoint region. Consequently, suspected MCL and FL diagnostic samples are assessed for translocations by FISH, while separate material is submitted for NGS-based VDJ clonality analysis via traditional mPCR. Ring-Seq may streamline this process by consolidating separate tests into a single rapid NGS assay for the G4 platform. Looking ahead, we envision broad applicability of the technology to mPCR-based variant detection. Citation Format: Shannon Owens, Katherine Fichter, Richard Que, Sabrina Shore, Timothy Looney. Simultaneous detection of IGH VDJ rearrangements and JH translocations via Ring-Seq, a novel multiplex PCR technology for targeted sequencing of translocations with novel gene partners or breakpoints. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6673.

Immunoglobulin VDJ repertoires reveal hallmarks of germinal centers in unique cell clusters isolated from zebrafish (Danio rerio) lymphoid tissues.

DNA mutagenesis during antibody affinity maturation has potentially oncogenic or autoimmune outcomes if not tightly controlled as it is in mammalian germinal centers. Cold blooded vertebrates lack germinal centers, yet have a functional Ig gene mutator enzyme, Aicda. In fish there are clusters of Aicda+ cells encircled by pigmented 'melano-macrophages' and we test the hypothesis that these clusters are functionally analogous to germinal centers. Sequenced IgH VDJ repertoire libraries from individual isolated clusters showed evidence of B-cell clonal expansion and VDJ somatic hypermutation. Construction of Ig clonal lineage trees revealed that unlike surrounding lymphoid tissue, each cluster is dominated by a few B-cell VDJ clonotypes having hundreds of mutated variants. Recruitment of B-cells to the clusters appears to be ongoing, as there are additional Ig clones having smaller lineages. Finally, we show evidence for positive selection for replacement mutations in regions encoding the antigen contact loops, but not in the framework regions, consistent with functional antibody modification. Melano-macrophages appear to trap the Ag used for post-mutation B-cell selection, performing a role analogous to the follicular dendritic cells of mammalian germinal centers. These findings provide insights into the evolution of the affinity maturation process, the improvement of fish vaccines and possibly also the workings of atypical ectopic germinal centers generated in several human diseases.

Simultaneous Detection of B Cell Receptor Rearrangements and Joining Gene Translocations Via Ring-Seq, a Novel Multiplex PCR Technology for Targeted Next-Generation Sequencing of Translocations with Novel Gene Partners or Breakpoints

Introduction Next-generation sequencing (NGS) of B cell receptor rearrangements is a leading method for clonality assessment and measurable residual disease (MRD) monitoring of B cell malignancies. Current methods rely upon multiplex PCR (mPCR) amplification of rearranged IGH, IGK or IGL loci via variable and joining gene targeting primers followed by NGS of amplicons to assess clonality and determine the presence of marker rearrangements. Limitations of this approach include a need for reflex testing owing to somatic hypermutation (SHM) mediated loss of sensitivity and an inability to detect clinically relevant translocations involving immune receptor loci. Here we introduce Ring-Seq, a novel approach for the targeted detection of gene fusions, including those with unknown partners or breakpoints. Unlike traditional mPCR immune repertoire assays, the Ring-Seq protocol leverages highly efficient template circularization coupled with inverse PCR to enable targeted sequencing of VDJ rearrangements and translocations while avoiding loss of sensitivity due to variable gene SHM. Using the Singular Genomics G4TM sequencing platform, we apply Ring-Seq to simultaneously identify clinically relevant translocations and VDJ rearrangements of the IGH locus from highly degraded material. Methods The Ring-Seq method begins with a highly efficient intramolecular ligation of DNA fragments followed by a multiplex inverse PCR of joining genes that preferentially amplifies breakpoint junction containing fragments. Amplicon libraries are sequenced on the G4 platform via 2x150bp reads and analyzed to detect translocations and immune receptor rearrangements. We pooled gDNA from reference cell lines known to harbor marker IGH VDJ rearrangements (Ramos, CA-46) and BCL1-JH and BCL2-JH translocations (IVS-0010 and IVS-0030, Invivoscribe Cat #40880550 and 40881750 respectively). Pooled reference gDNA was spiked into a background of healthy donor peripheral blood leukocyte (PBL) gDNA to evaluate performance over a range of marker frequencies. To simulate performance with degraded materials, gDNA input was fragmented to ~200bp length by sonication (Covaris) prior to analysis. Results BCL1-JH and BCL2-JH translocations and marker IGH VDJ rearrangements were detected from 50ng pooled reference control gDNA. Translocations and marker VDJ rearrangements were also detected in 50ng samples consisting of fragmented reference control material spiked at 1% frequency into a background of fragmented healthy donor PBL. We observed a high on-target rate, enabling marker detection with less than 1M reads per sample in all conditions tested. Conclusions We have applied a novel technology for multiplex inverse PCR, Ring-Seq, to enable the simultaneous detection of B cell receptor rearrangements and clinically relevant JH translocations, including those involving non-canonical breakpoint regions. Unlike traditional mPCR approaches for B cell receptor sequencing, Ring-Seq avoids targeting the highly mutable variable gene region, thereby obviating the need for reflex testing due to SHM-mediated loss of sensitivity. We expect the approach to be particularly well suited to the future identification and monitoring of translocation-associated B cell malignancies such as mantle cell lymphoma.

Validation of Next Generation Sequencing-Based Clonality Assays for Diagnosis and Minimal Residual Disease Tracking in Lymphoid Malignancies

Introduction:B or T cell receptor (BCR/TCR) clonal rearrangements have served as important diagnostic markers and minimal residual disease (MRD) tracking markers to guide treatment decisions in lymphoid malignancies. Next generation sequencing (NGS)-based BCR/TCR clonality assessment allows easier sample preparation, higher sensitivity and simpler standardization than flow cytometry and polymerase chain reaction (PCR)-based assays. Here, we developed NGS-based BCR/TCR clonality assays to identify and track disease-associated clonotypes of IGH, IGK, IGL, TCRB and TCRG rearrangements and BCL1/2-IGH translocations in lymphoid malignant cells. Our studies validated the analytical performance of the assays using genomic DNA (gDNA) from cell lines and patient samples diagnosed with acute lymphoblastic leukemia (ALL), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), lymphoma and lymphoid blast phase chronic myeloid leukemia (BP-CML), as well as peripheral blood gDNA and circulating tumor DNA (ctDNA) from lymphoma patients.Methods:Our BCR/TCR clonality assays were based on two rounds of multiplex PCR followed by NGS. In the first-round PCR, the sequences of complementary determining region 3 (CDR3) in rearranged immune receptor genes were amplified by multiplex primers, and sample-specific index and NGS adapters were then added in the second-round PCR. Sequencing was performed on NovaSeq 6000 System and processed with customized bioinformatics pipelines. BCR clonality assays could identify IGH (V H-D H-J H or D H-J H), IGK (V κ-J κ, V κ-Kde and intronRSS-Kde) and IGL (V λ-J λ) rearrangements, as well as BCL1-IGH and BCL2-IGH translocations. TCR clonality assays could detect TCRB (V β-D β-J β) and TCRG (V γ-J γ) rearrangements. To evaluate the performance of our assays, we detected BCR or TCR clonality in gDNA from 4 cell lines and 40 clinical samples with ALL, MM, CLL and lymphoma, as well as paired chronic- and blast-phase samples of CML. Limit-of-detection (LOD) was estimated by clinical samples and cell lines with a background of peripheral blood gDNA from healthy donors. Linearity of detection was established with gDNA of cell lines spiked into normal gDNA to generate across orders of magnitude of clonal frequencies. To ensure consistent performance of the assays, we tested separate reactions and a single mixed reaction for IGH VDJ, IGH DJ, IGK and IGL. In addition, peripheral blood gDNA matching aforementioned lymphoma cases and ctDNA samples were also tested by our assays.Results:Both BCR clonality of B-cell lymphoid malignancies (B-ALL, MM, CLL and lymphoma) and TCR clonality of T-cell lymphoid malignancies (T-ALL and lymphoma) showed above 90% positive detection rate and mostly positive in more than one receptor gene. Chronic and blast phase samples from the same CML patient showed an identical dominant clonotype. The assays had high sensitivity, with LoD defined between 1 to 2 malignant cells in both BCR and TCR clonality assessment. Linearity was observed with clonal frequencies from 1 to 10 -6, which indicated consistence between observed and expected frequencies. The sequencing results with a single adjusted mixed reaction for IGH VDJ, IGH DJ, IGK and IGL were comparable to that with separate reactions, suitable for both diagnosed samples and MRD samples, suggesting the robustness of the assays. Our testing results also showed that peripheral blood gDNA of lymphoma patients carried identical clonotypes found in malignant tissues and ctDNA.Conclusion:We characterized and validated the performance of our NGS -based BCR/TCR clonality assays. We also demonstrated its potential application as a highly sensitive tool for diagnosis and MRD tracking for lymphoid malignancies, including ALL, MM, CLL, lymphoma and even CML at risk of lymphoid blast transformation. DisclosuresNo relevant conflicts of interest to declare.

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. DisclosuresNo relevant conflicts of interest to declare.

Genomic analysis of a second rainbow trout line (Arlee) leads to an extended description of the IGH VDJ gene repertoire

High-throughput sequencing technologies brought a renewed interest for immune repertoires. Fish Ab and B cell repertoires are no exception, and their comprehensive analysis can both provide new insights into poorly understood immune mechanisms, and identify markers of protection after vaccination. However, the lack of genomic description and standardized nomenclature of IG genes hampers accurate annotation of Ig mRNA deep sequencing data. Complete genome sequences of Atlantic salmon and rainbow trout (Swanson line) recently allowed us to establish a comprehensive and coherent annotation of Salmonid IGH genes following IMGT standards. Here we analyzed the IGHV, D, and J genes from the newly released genome of a second rainbow trout line (Arlee). We confirmed the validity of salmonid IGHV subgroups, and extended the description of the rainbow trout IGH gene repertoire with novel sequences, while keeping nomenclature continuity. This work provides an important resource for annotation of high-throughput Ab repertoire sequencing data.

Subclonal Variation of RAG-Mediated Diversification in B Cell Acute Lymphoblastic Leukemia

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.

Next-Generation Sequencing-Based Assay Shows High Clonal Characterization Success Rate for Plasma Cell Neoplasms, and Concordance with Flow Cytometry in Minimal Residual Disease Detection

IntroductionAfter therapy or stem cell transplantation, multiple myeloma patients achieving complete response (CR) or stringent complete response (sCR) can still have a significant risk of disease relapse, illustrating the importance of using highly sensitive methods for minimal residual disease (MRD) detection and prognostication. Two techniques used clinically for MRD detection include multiparametric flow cytometry (FC), which has a sensitivity down to 2-6 X 10-6 of cells, and next-generation sequencing (NGS)-based assay for detection of patient-specific clonal IGH VDJ gene sequences associated with the neoplastic plasma cells (PC). We determined the clonal characterization success rate of plasma cell neoplasm samples from a single institution in a clinical lab, using a commercially available NGS-based assay, Lymphotrack® (Invivoscribe, San Diego, CA). The characterized clonal sequences were used for MRD detection in subsequent monitoring samples, and the results were compared to concurrent FC findings.MethodsDNA was extracted from fresh marrow or formalin-fixed paraffin-embedded (FFPE) tissue, and amplified by PCR reactions using primers sets for IGH Leader, FR1, FR2, FR3 regions, and IGK. Sequencing was performed on the Illumina MiSeqTM Platform, and sequence analyses were performed using the Lymphotrack® software, and MSK-Lymphoclone, a software developed at our institution. Disease-associated clonal sequences were characterized based on predefined clonal calling criteria and stored. In subsequent samples sent for disease monitoring, a search for sequencing reads with high homology (>99%) to the patient-specific sequences was performed for MRD detection. 10-color FC for PC analyses were also performed on the same samples at our institution (Roshal M, et al. Blood Adv 2017;1(12):728-32), with a target minimum of 3 million cells for MRD analyses.ResultsOverall, clonal characterization was successful in 235/251 cases (93.6%), with no difference in number of sequencing reads between the successful and unsuccessful cases (p=0.24). Higher success rate was observed among cases with higher aspirate PC counts: ≥5% (95.6% success rate) and ≥10% (98.1% success rate). IGH FR1 and Leader primers together characterized 214/251 cases (85.3%), while the remaining cases required additional primers. The characterized clones showed high median somatic hypermutation (SHM) rate of 8.1% (range: 0.0-29.0%), as well as IGH V and J segment usage bias: V3 (50.2%), followed by V4 (20.3%); J4 (43.4%), followed by J6 (27.5%), concordant with prior literature. 187 samples from 124 unique patients were tested by the Lymphotrack® assay for monitoring purposes, of which the diagnostic clones were detected in 147/187 samples (78.6%), with no difference in number of sequencing reads between cases with and without detectable clone (p=0.35). Within the short median time interval of 9.5 months between the characterization and monitoring samples, most clonal sequences remained stable. In 2 cases, new clonal sequences emerged in subsequent samples. Overall, FC and Lymphotrack® showed high concordance rate for MRD detection (92.9%) (See figures). All discordant cases showed <5% PC by aspirate differential counts and CD138 immunostains. FC+/NGS- cases (9/184, 4.9%) showed abnormal PC comprising a median of 0.00095% of WBC by FC, while FC-/NGS+ cases (4/184, 2.2%) showed detectable clone at a median of 0.0405% of sequencing reads. Sampling differences might have contributed to the discrepancies. Additionally, in the FC+/NGS- cases, neoplastic subclones might be present at very low level in the characterization samples, below threshold for clonal calling, and therefore could not be specifically tracked in subsequent samples.ConclusionsOur study demonstrated high clonal characterization success rate for plasma cell neoplasms using the Lymphotrack® assay when multiple primers sets were used, and the assay showed concordance with FC in MRD detection for the majority of cases. MRD detection sensitivity can be limited by low sample concentration/volume. Furthermore, the presence of very low level neoplastic subclones in the characterization samples might hamper clonal calling and detection in subsequent samples. [Display omitted] DisclosuresHo:Invivoscribe, Inc.: Honoraria. Landgren:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Karyopharm: Consultancy. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria.

IG-MYC+ neoplasms with precursor B-cell phenotype are molecularly distinct from Burkitt lymphomas

AbstractThe WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue notes instances of Burkitt lymphoma/leukemia (BL) with IG-MYC rearrangement displaying a B-cell precursor immunophenotype (termed herein “preBLL”). To characterize the molecular pathogenesis of preBLL, we investigated 13 preBLL cases (including 1 cell line), of which 12 were analyzable using genome, exome, and targeted sequencing, imbalance mapping, and DNA methylation profiling. In 5 patients with reads across the IG-MYC breakpoint junctions, we found evidence that the translocation derived from an aberrant VDJ recombination, as is typical for IG translocations arising in B-cell precursors. Genomic changes like biallelic IGH translocations or VDJ rearrangements combined with translocation into the VDJ region on the second allele, potentially preventing expression of a productive immunoglobulin, were detected in 6 of 13 cases. We did not detect mutations in genes frequently altered in BL, but instead found activating NRAS and/or KRAS mutations in 7 of 12 preBLLs. Gains on 1q, recurrent in BL and preB lymphoblastic leukemia/lymphoma (pB-ALL/LBL), were detected in 7 of 12 preBLLs. DNA methylation profiling showed preBLL to cluster with precursor B cells and pB-ALL/LBL, but apart from BL. We conclude that preBLL genetically and epigenetically resembles pB-ALL/LBL rather than BL. Therefore, we propose that preBLL be considered as a pB-ALL/LBL with recurrent genetic abnormalities.

Biased IGH VDJ gene repertoire and clonal expansions in B cells of chronically hepatitis C virus–infected individuals

AbstractPatients chronically infected with hepatitis C virus (HCV) frequently develop mixed cryoglobulinemia (MC), a monoclonal expansion of immunoglobulin M (IgM)+ autoreactive B cells, and also have an increased B-cell lymphoma risk. Whether HCV infection also impacts the B-cell compartment and the B-cell receptor repertoire in patients not affected by MC or lymphomas is poorly understood. Flow cytometric analysis of peripheral blood B cells of 30 MC-negative HCV-infected patients and 15 healthy controls revealed that frequencies of class-switched memory B cells were increased in the patients, whereas frequencies of transitional and naive B cells were decreased. For 22 HCV+ patients and 7 healthy controls, we performed high-throughput sequencing of immunoglobulin heavy chain VDJ rearrangements of naive, mature CD5+, IgM+ memory, and class-switched memory B cells. An increased usage of several IGHV genes, including IGHV1-69 and IGHV4-59, which are closely linked to MC and HCV-associated lymphomas, was specifically seen among IgM+ memory B cells of the patients. Moreover, many, and partly very large, expanded clones were seen predominantly among IgM+ memory B cells of all HCV-infected patients analyzed. Thus, chronic HCV infection massively disturbs the B-cell compartment even in patients without clinically detectable B-cell lymphoproliferation and generates many large B-cell clones, especially among non–class-switched memory B cells. Because B-cell clones in MC and lymphomas derive from this B-cell subset, this establishes IgM+ memory B cells as a general target of lymphoproliferation in HCV+ patients, affecting apparently all patients.

FIRST LINE TREATMENT BY THE RIBVD REGIMEN ELICITS HIGH CLINICAL AND MOLECULAR RESPONSE RATES AND PROLONGED SURVIVAL IN ELDERLY MCL PATIENTS; FINAL RESULTS OF a LYSA GROUP TRIAL

Introduction: Eight R-CHOP21 cycles followed by rituximab maintenance is considered as the standard first-line treatment for elderly mantle cell lymphoma (MCL) patients. Complete response (CR) and undetectable minimal residual disease (uMRD) rates remain sub-optimal with tR-CHOP regimen (CR rate 30-35%, MR after 8 cycles 67%) and this translates to shorter response duration. Recently VR-CAP, integrating bortezomib to RCHOP has proved superiority to both R-CHOP and the R-BAC regimen (rituximab bendamustine cytarabine) has given also promising results. In this setting, we have explored the association rituximab-bendamustine-bortezomib and dexamethasone in the RiBVD regimen. Methods: In this prospective phase II study, all patients >65 years old with newly-diagnosed MCL were treated by the RiBVD regimen (inclusion criteria: AA stage II-IV, PS < 3, no active HIV, HBV or HCV infections, no renal or cardiac dysfunction, no diabetes). RiBVD was administered every 4 weeks: rituximab, 375 mg/m2 IV on day(D)1; bendamustine at 90 mg/m2 IV on D1 and D2; dexamethasone 40 mg IV on D2 and bortezomib1.3 mg/m2 subcutaneously on D1, 4, 8 and 11. Patients received a total of 6 cycles, if they responded (IWG criteria) after 4 cycles. No maintenance was delivered. MRD was centrally evaluated by RQ-PCR using patient specific IGH VDJ targets, and the FDG-PET response was evaluated visually according to Deauville criteria. The primary objective was to prolong PFS by 6 months (m) compared to the 18 m PFS reported for R-CHOP21. In order to define superiority of the RiBVD, PFS > 65% (H1) was required at 18 m. Treatment failure was considered if PFS at 18 m was 50%. With alpha and beta risks of 5% and 20%, respectively, 69 patients needed to be enrolled. Secondary objectives were to evaluate toxicity, known prognostic indices, response FDG-PET imaging and MRD. Results: Seventy four patients were enrolled; 80% (n = 58) had high MIPI score. ORR and CR were 84% (n = 62/74) and 75% (n = 56/74), respectively. After 6 cycles, 78% (n = 46/59) were FDG-PET negative; 87% (47/54) and 76% (35/46) had uMRD in blood (PB) and bone marrow (BM), respectively. With a median follow-up of 52 m the primary objective was reached (24 m PFS = 70%). Four years OS was 86.6% for uMRD patients at the end of treatment, compared to 28.6%, detectable MRD patients; p < 0.0001). Neither the MIPI score nor FDG-PET responses were predictive of OS. Toxicities were mainly hematologic with grade 3/4 neutropenia in 51% and thrombopenia in 36%. The principal grade 3/4 extra-hematologic toxicities were fatigue (19%), neuropathy (14%), cardiac (7%) or febrile neutropenia (5%). Keywords: bortezomib; mantle cell lymphoma (MCL); minimal residual disease (MRD).

Ex Vivo Modeling of Multiple Myeloma Provides Basis for Studying Treatment Combinations and Immunotherapy

BackgroundMultiple myeloma (MM) remains incurable despite treatment advances. While passive immunotherapy such as anti-CD38 antibodies is highly effective, active immunotherapy may provide long-lasting remissions by virtue of triggering memory. A phase 1 nivolumab study, an antibody targeting programmed death-1 (PD1), was unable to yield any responses in multiply relapsed MM patients. Conversely, preliminary trial data of lenalidomide combined with pembrolizumab, a different anti-PD1 antibody, found significantly higher response rates. These two differing outcomes reflect our limited understanding of checkpoint inhibition and immunotherapy in MM. There is a paucity of preclinical models to guide therapeutic studies. Cell lines and xenografted murine models are incapable of exploring active immunotherapy due to a lack of microenvironment and endogenous immune cell signals. Furthermore, malignant cells responsive to drugs in 2-dimensional (2D) cultures are known to display a more resistance in 3D. We have previously demonstrated that B-cell malignancies can be accurately studied using a 3D culture system of patient bone marrow mononuclear cells (BMCs) and can better inform translational trials. Herein we describe an ex vivo, 3D tissue culture model of patient-derived MM samples to more accurately test therapeutics including checkpoint inhibition using ipilimumab, a monoclonal antibody targeting cytotoxic T-lymphocyte antigen 4 (CTLA) which is crucial in co-stimulatory signaling of effector T-cells.MethodsA 3D extracellular matrix was created using matrigel in 12-well plates. BMCs were isolated from marrow aspirates of 5 MM patients at time of diagnosis and individually cultured. Each patient sample was tested for sensitivity against increasing concentrations of ipilimumab (1X, 3X, and 10X clinical doses) added into supportive medium. Plates were monitored visually by microscopy followed by harvest on day 21 using enzymatic degradation. Unique clonotypic heavy chain immunoglobulin rearrangement (IgH VDJ) from each sample was sequenced, validated and used for semi-quantitative PCR. Semi-QT PCR with clone-specific primers estimated malignant cell survival after harvest. Flow cytometry was used to define cell populations present in culture and to correlate with clonotypic PCR data. T-cell mediated activity was examined by reverse transcription of trizol-extracted, T-cell RNA after harvest.ResultsAll samples were successfully cultured, followed for 21 days and harvested. Flow cytometry confirmed presence of T-cell subsets, B-cells, NK cells and dendritic cells before and after culture in 3D. Minimal depletion of clonotypic cells was observed at 3x clinical levels of drug. At 10x simulated clinical therapeutic levels, 3 MM samples demonstrated >90% death of clonotypic MM cells while the other 2 demonstrated 62% and 72% death, respectively, compared to untreated control cultures. The extent to which the drug diffuses into the matrigel is as yet unknown. Flow cytometry of harvested cells suggest that T-cells demonstrate a modest shift toward CD4 and CD8 effector cells. Preliminary mechanistic data from one MM sample using trizol-extracted RNA and reverse transcriptase PCR harvested at 21 days from 3D culture suggests that anti-malignant, cytotoxic T-cell effect may be driven by granzyme B expression. Expanded data from the remaining samples will be presented.ConclusionsWe demonstrate that an ex vivo 3D tissue culture model of MM is both feasible and informative in studying immunotherapy. By culturing unselected BMCs which include stromal cells, immune cells and malignant populations, the 3D culture more closely mimics the tumor microenvironment with both the patient's immune system present as well as stromal supportive signals. In this study, we show that in the presence of active immune effector cells, ipilimumab has activity against patient-derived MM cells. The data suggests the importance of targeted cytotoxic T-cell activation as a primary mechanism of action. We have previously studied standard MM therapeutics such as cytotoxic chemotherapy, immunomodulatory drugs, and proteasome inhibitors in the same way. Consequently, this model is well positioned to study other immunotherapies such as other checkpoint inhibitors, cellular therapy, and combinations. Further testing with therapeutics targeting PD1/PDL1, and adenosine receptors are underway. DisclosuresVenner:Takeda: Honoraria; Celgene: Honoraria, Research Funding; J+J: Research Funding; Janssen: Honoraria; Amgen: Honoraria. Belch:Celgene: Honoraria; Janssen: Honoraria; Amgen: Honoraria; Takeda: Honoraria.

Absence of Evidence Implicating Hematopoietic Stem Cells As Common Progenitors for DLBCL Mutations

Background: Pre-leukemic hematopoietic stem cells (HSC) have been implicated in AML (Jan et al STM 2012) and also for several lymphoid leukemias including ALL, HCL, and CLL. Separately, relapse of ALL following CD19 CAR-T cell therapy has been associated with lymphomyeloid lineage switch. Finally, healthy persons with clonally expanded HSCs are at increased risk of hematologic malignancies including lymphomas, and in mouse DLBCL models we previously demonstrated the oncogenic sufficiency of BCL6 overexpression in HSC (Green et al 2014 Nat Comm). Nevertheless, the cellular origin of DLBCL in the majority of patients is not definitively known. We sought to investigate the presence of mutations found in DLBCL within matched HSCs.Methods: We deeply genotyped somatic mutations in diagnostic biopsy tissues of 16 patients with DLBCL using CAPP-Seq to a median sequencing depth of 1100x (Newman et al 2014 Nat Med; Scherer et al 2015 ASH). We then profiled each patient for evidence implicating HSCs using somatic mutation lineage tracing, in either direct or indirect fashion. For direct evaluation, we used highly purified, serially FACS-sorted HSCs from grossly uninvolved bone marrow (BM) (n=5; Fig 1a-b). For indirect assessment, we either profiled serial tumor biopsies (n=13), or interrogated sorted cells from terminally differentiated blood lineages (n=7), including peripheral CD3+ T cells, CD14+ Monocytes, and B cells expressing a light-chain discordant to that of tumor isotype. HSCs and differentiated lineages were then interrogated by direct genotyping, using 3 highly sensitive orthogonal quantitative methods, including Myd88 L265P droplet digital PCR (n=6), BCL6 translocation breakpoint qPCR (n=4), and DLBCL CAPP-Seq profiling of 268 genes (n=5). We used the theoretical limit of detection (LOD) genotyping performance for CAPP-Seq (0.001%, Newman et al 2016 Nat Biotech), and established analytical sensitivity of our custom MYD88 ddPCR via limiting dilution (~1%). These LODs met or exceeded the expected limit of sorting impurity by FACS (~1%). For 6 patients experiencing one or more DLBCL relapse, we deeply profiled 13 serial tumor biopsies by CAPP-Seq, and then assessed overlap in somatic mutations and VDJ sequences in biopsy pairs as additional indirect evidence implicating HSCs.Results: We obtained a median of ~2000 sorted HSCs and ~1700 sorted cells from differentiated lineages, and genotyped each population using one or more of the 3 direct genotyping methods described above. Three patients with sufficient cell numbers were profiled both by CAPP-Seq and either ddPCR (n=2) or qPCR (n=1). Surprisingly, we found no evidence implicating HSCs either directly or indirectly in any of the 16 patients, regardless of the assay employed or the cell types/lineages genotyped (e.g., Fig 1b). In 2 patients with MYD88 L265P mutations, we found evidence for MYD88+ B-cells with discordant light chains by ddPCR (~0.1%) potentially implicating common lymphoid precursors (CLPs), but found no evidence for similar involvement of T-cells or monocytes. In 6 DLBCL patients experiencing relapse, tumor pairs profiled by CAPP-Seq (median depth 957) shared 93% of somatic mutations (75-100%, Fig 1c). Such pairs invariably shared clonal IgH VDJ rearrangements (4/4, 100%), thus implicating a common progenitor arising in later stages of B-cell development, not HSCs.Conclusions: We find no evidence to implicate HSCs in the derivation of DLBCL. While formal demonstration of absence of pre-malignant HSCs in DLBCL would require overcoming practical and technical limitations (including number of available HSCs, sorting purity, and genotyping sensitivity), the pattern of shared somatic alterations at relapse makes this highly unlikely. We speculate that unlike lymphoid leukemias, the cell-of-origin for most DLBCLs reside later in B-lymphopoiesis, beyond CLPs. [Display omitted] DisclosuresNewman:Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy; Roche: Consultancy; Varian Medical Systems: Research Funding.

Integrated Analysis of IGHV Gene Status, Cell-of-Origin Signature and Genomic Features in Diffuse Large B-Cell Lymphoma

The molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) has been highlighted by gene expression profiling (GEP), dividing DLBCL into the following three main molecular subtypes with different clinical outcomes and responses to immunochemotherapy: the germinal center B-cell-like (GCB) subtype, the activated B-cell-like (ABC) subtype and the primary mediastinal B-cell lymphoma (PMBL) subtype. Despite frequent translocations involving the IGH (heavy chain) locus, B-cell receptor (BCR) expression is retained in almost all DLBCL, indicating that BCR signaling is crucial for the survival of malignant cells. Furthermore, DLBCL are characterized by recurrent somatic mutations that are supposed to be oncogenic drivers. Here, with the aim to more accurately define the DLBCL pathogenic subgroups, we report a detailed immunogenetic analysis of IGH rearrangements in a well-defined GEP DLBCL cohort and correlate these features with the somatic mutation status of recurrently mutated genes involved in lymphomagenesis. Methods: 204 DLBCL enrolled in the prospective LYSA LNH03 trial program were classified into molecular subtypes by GEP using Affymetrix arrays [82 ABC (40.2%), 77 GCB (37.7%), 29 (14.2%) unclassified and 16 PMBL (7.8%)]. The amplification of complete VDJ rearrangements was realized using BIOMED-2 protocols. Somatic hypermutation (SHM) characteristics were studied (mutation rate, glycosylation N-X-S/T sites, RGYW hotspots, composition of CDR3) and correlated with the molecular subtypes. To obtain a more comprehensive molecular portrait of the DLBCL cases, GEP and IGH VDJ analyses were correlated with the mutational status of a panel of 34 recurrently mutated genes in DLBCL such as MYD88, EZH2, CD79B, TNFAIP3, CARD11 and GNA13 (Dubois et al. Clinical Cancer Research 2016). Results: A total of 153/204 (75%) clonal VDJ rearrangements were successfully determined. Failures to detect clonotypic sequences were predominantly seen in PMBL (56%) and unclassified (31%) cases. The ABC subtype display a significantly lower SHM rate than the 3 other subtypes, especially in contrast to the PMBL subgroup that was characterized by a higher SHM rate (p<0.0001 for each). While IGHV gene usage generally reflected that of normal B cells, a marked increase in IGHV4-34 in ABC cases (30%) was confirmed and those cases tended to have less SHM. Acquisition of new N-glycosylation (N-Gly) sites, a hallmark of follicular lymphoma, was more prevalent in the GCB cases (48%) than in ABC (22%) (p=0.012) but was also observed in 5/7 (71%) PMBL cases with functional IGH rearrangement. This acquisition is correlated with a more frequent t(14;18)(q24;q32) in GCB subtype. As reported in FL, this suggests that lectin binding signals via surface Ig that contain N-Gly sites may provide an extrinsic antigen-independent signal in some DLBCL, and more specifically in t(14;18) DLBCL (Linley et al. Blood 2015 ). Finally, we correlated these immunogenetic features with the mutational status of some drivers genes frequently mutated in DLBCL. Within ABC subtype patients, MYD88 or TNFAIP3 mutated cases displayed a higher SHM rate (13.4% vs 8.4%, p <0.0001; 15.44% vs 11.13%, p=0.03), without any IGH family distribution bias. 9/56 (16%) GCB DLBCL harbored an EZH2 mutation. All expressed an IGHV3 rearrangement (100% vs 52% for the GCB-EZH2 wt, p=0.014). By contrast to MYD88 status, a similar IG SHM rate was observed in mutated and wt patients regarding EZH2, CARD11 or GNA13 genes. Conclusion: The IG characterization and the driver gene mutation analysis according to the cell of origin of the tumor allowed us to refine DLBCL subgroups. Our data suggest that there is a strong interaction between extrinsic mechanisms, including lectin-mediated antigen-independent activation or, in the IGHV4-34 cases, via N-acetyl lactosamine ligands, and intrinsic mechanisms to activate the BCR/NFKB pathway. These data could help us classify the different subgroups of DLBCL more accurately and, in particular, to identify new molecular features for BCR target therapy. DisclosuresHaioun:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Salles:Mundipharma: Honoraria; Janssen: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding.

Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients

Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis.

Minimal Residual Disease Monitoring By 8-Color Flow Cytometry in Mantle Cell Lymphoma Is Complementary to Q-PCR Monitoring and Will Facilitate Pre-Emptive Treatment: An EU-MCL and Lysa Study

Introduction: Mantle Cell Lymphoma (MCL) is characterized by frequent blood and bone marrow involvement. It has been demonstrated that use of Minimal Residual Disease (MRD) quantification in blood and/or bone marrow might be helpful in patient management. Gold standard MRD is based on Q-PCR clone specific amplification of IgH VDJ or IgH-BCL1 rearrangements, but these are relatively complex and time consuming and over half of the positive results are in a grey zone of borderline positivity. Flow cytometry (FCM) is more rapid and better adapted to individual patient management if quantitatively reproducible, but insufficiently sensitive when only 4 colors are used. We therefore developed a universal, 8-color, EuroFlow inspired, FCM strategy, which we compared with classical Q-PCR MRD in 61/97 patients included in (and 1 treated according to) the EU-MCL Younger and Elderly prospective trials who underwent Q-PCR MRD monitoring at Necker Hospital.Method: Q-PCR MRD from IgH VDJ (n=92) or BCL1-IgH (n=5) was performed prospectively from ficolled blood (PB) or bone marrow, from which residual material was cryopreserved in DMSO for FCM quantitation, using 10 antibodies labelled with 8 fluorochromes for positive and negative (CD45, CD19, CD5, LAIR1, CD11a, IGK, IGL, CD3, CD14 and CD56) gating, after diagnostic phenotyping of fresh material, using the same panel and a EuroFlow B lymphoid screening tube. Sensitivity of both techniques was at least 0.01% (1E-04). FCM was only considered positive if above 0.01%, whereas Q-PCR results were considered positive below quantifiable range (BQR) if borderline, above sensitivity, within Euro-MRD criteria for MRD positivity. BQR samples were separated based on the number of positive, triplicate samples. The objectives were to compare the two techniques and to determine their suitability for regular screening, with a view to pre-emptive treatment on molecular or phenotypic (MRD) relapse. Two patients were treated with Rituximab at MRD relapse, prior to clinical relapse, as proof of principle.Results: A total of 302 blood or bone marrow samples from 62 patients were quantified. Overall, 79% (42/53) of samples positive at or above 0.01% by PCR were also positive by FCM, compared to 29% (19/65) of those below 0.01%, but with at least 2 positive triplicates and virtually none of those with only 1 or no results above sensitivity (1%, 2/184). Quantification of the paired MRD results positive with PCR and/or FCM were significantly correlated (r2=0.74, P<0.0001).Amongst the 62 patients, 30 have relapsed and 19 have died. Nine relapsing patients (including one off protocol patient treated and monitored at initial and second MRD relapses) had sufficient MRD points to assess the capacity of PB Q-PCR or FCM to predict future clinical relapse sufficiently to justify pre-emptive treatment at MRD relapse. Clinical relapse was preceded by MRD relapse in 9/10 relapses by Q-PCR and 7/9 by FCM. Six of the 9 relapsing patients had achieved Q-PCR negativity in at least one PB sample. The mean latency for prediction by Q-PCR, when considering any increase in positivity to at least 2 positive triplicates as positive, was 11.3 months (range 1-24mths) and 5.4 months (0.5-11) when only results above 0.01% were considered positive. The equivalent latency by FCM was slightly shorter, at 6.5 months (0.5-21) Pre-emptive treatment of 2 patients at MRD relapse, prior to clinical relapse allowed re-establishment of molecular complete remission and a durable second remission in at least one with sufficient follow-up (Cf Fig.). [Display omitted] Conclusion: Eight color flow cytometry is a promising alternative to classical clone-specific Q-PCR strategies in monitoring therapy in MCL, with an excellent correlation (29/31, 94%) for MRD levels of at least 0.1% and acceptable correlation at 0.01-0.1% (13/22, 59%). While less sensitive at very low levels on cryopreserved material, FCM may clarify the clinical relevance of low-level borderline positivity; however it remains to be determined prospectively which technique will have greater prognostic value in patient management. FCM sensitivity will be improved by prognostic testing of fresh whole blood or bone marrow, and this pilot data clearly justifies such studies. Finally, MRD relapse precedes clinical relapse by several months, justifying pre-emptive treatment, monitored by prospective FCM and IgH Q-PCR within clinical trials. DisclosuresDreyling:Roche: Honoraria, Research Funding.

Frontline Therapy with the Ribvd Regimen Elicits High Clinical and Molecular Response Rates and Long PFS in Elderly Patients Mantle Cell Lymphoma (MCL); Final Results of a Prospective Phase II Trial By the Lysa Group

▪Background:Eight RCHOP/21 cycles followed by Rituximab maintenance is considered the standard of care for first line therapy in elderly MCL patients in Europe (1). However,complete clinical (CR) and molecular responses (MR) to therapy remain suboptimal(CR rate 30-35%, MR after 8 cycles 67 %). Regimen combining Rituximab and Bendamustine and more recently proteasome inhibitor Bortezomib (Velcade®) with CHOP regimen (VcR-CAP) demonstrated superior CR rates and PFS compared to R-CHOP. We have investigated the RiBVD regimen (Rituximab, Bendamustine,Velcade® and Dexamethasone) in a prospective phase II trial by the LYSA group.Aims of the study:The primary objective was to improve the median PFS by 6 months over the current 18 months PFS obtained with RCHOP when given without maintenance (ref Lenz, JCO 2005). The secondary objectives were to investigate the prognostic impact of molecular and FDG-PET responses on survival..Methods and Material:All patients aged 65 or older with a diagnosis of MCL were treated by the RiBVD regimen if they fulfilled the following inclusion criteria: AAstage II-IV, PS < 3, no other neoplasm, no active HIV or HBV or HCV infections, no renal (creatinin clearance > 20ml/mn) or cardiac dysfunction, no diabetes. The RiBVD regimen was administered every 4 weeks with Rituximab 375 mg/m² IV on day(D)1, Bendamustine 90 mg/m² IV on D1, 2, Dexamethasone 40 mg IV on D2 and Bortezomib (Velcade®) 1.3 mg/m² subcutaneously on D1, 4, 8 and 11. Primary prophylaxis with valacyclovir was mandatory for Herpes virus reactivation, but there was no recommendation for bacterial prevention. Patients were scheduled to receive a total of 6 cycles, if they achieved at least PR at 4 cycles. The IWG criteria, with and without FDG-PET, were used to define responses after 4 and 6 cycles. FDG-PET response was evaluated in each center with the five-point scale visual method of Deauville. Molecular responses (MR) were evaluated centrally by RQ-PCR using patient specific IGH VDJ targets as previously described (2).Results:Seventy six patients were recruited in one year (from November 2011 to December 2012), 74 were evaluable [2 patients were excluded because HBV positivity (n=1) or misdiagnosis (n=1)]. Fifty four samples were centrally reviewed for diagnosis, 45 were classic form of MCL, 9 pleomorphic variant and one patient had a DLBCL. Clinical characteristics of the 74 patients were: Median age 73 yo (64-83), sex ratio M/F = 2 (49/25), AAstage II/III-IV = 4/70, PS 0-1/2 = 63/11, MIPIscore low/intermediate/high= 3/19/50 (2 undetermined). The median follow-up is 21 months. Thirteen patients died because of lymphoma progression (n=7), cardiac arrest (n=2) or in one case each,pneumonia, Progressive Multifocal LeukoEncephalopathy, pancreatic carcinoma or of unknown causes. After 4 cycles the ORR was 86% (n=64) and CR/CRu 57% (n=42). After 6 cycles, the CR/CRu raised to 74% (n=55) the PR rate was 9% (n=7), 2 patients had a stable disease, 4 progressed and 5 had died during the treatment. The complete MR rate after 6 months on blood samples or bone marrow was respectively 83% (43/50) and 74 % (32/43). At 24 months, the PFS was 69% and the OS 80%. The MIPI score (high vs low/int) and the blood MR after 6 cycles were the only two statistically prognostic factors for PFS and OS. Toxicities were essentially hematologic with grade 3 or 4 neutropenia and thrombocytopenia in 51% and 36% of the cases, respectively. The main grade 3 or 4 extra-hematologic toxicities were fatigue (19%), neuropathy (14%), cardiac (7%) or febrile neutropenia (5%).Conclusion:The RiBVD regimen can be safely administered as first line therapy to elderly patients with MCL. Toxicities are mild and manageable. With 74% of CR/CRu, a 2 year PFS of 69% and 86% of patients achieving an MRD negative status in the blood after 6 cycles, without maintenance therapy, the RiBVD regimen is identified as a highly effective, well tolerated, first line treatment for elderly MCL patients.Reference : 1 - Kluin Nelemans HC et al, NEJM 2012, 2 - Gimenez E et al, BJH 2012 DisclosuresGressin:mundipharma: Consultancy. Off Label Use: Bendamustine and Bortezomib did not have authorization to their use in first line for MCL in France. Karlin:Janssen: Honoraria; celgene: Consultancy, Honoraria; Sandoz: Consultancy. Le Gouill:Mundipharma: Honoraria; Roche: Honoraria; Janssen: Consultancy.

Diversity of the murine antibody response targeting influenza A(H1N1pdm09) hemagglutinin

We infected mice with the 2009 influenza A pandemic virus (H1N1pdm09), boosted with an inactivated vaccine, and cloned immunoglobulins (Igs) from HA-specific B cells. Based on the redundancy in germline gene utilization, we inferred that between 72–130 unique IgH VDJ and 35 different IgL VJ combinations comprised the anti-HA recall response. The IgH VH1 and IgL VK14 variable gene families were employed most frequently. A representative panel of antibodies were cloned and expressed to confirm reactivity with H1N1pdm09 HA. The majority of the recombinant antibodies were of high avidity and capable of inhibiting H1N1pdm09 hemagglutination. Three of these antibodies were subtype-specific cross-reactive, binding to the HA of A/South Carolina/1/1918(H1N1), and one further reacted with A/swine/Iowa/15/1930(H1N1). These results help to define the genetic diversity of the influenza anti-HA antibody repertoire profile induced following infection and vaccination, which may facilitate the development of influenza vaccines that are more protective and broadly neutralizing.Importance: Protection against influenza viruses is mediated mainly by antibodies, and in most cases this antibody response is narrow, only providing protection against closely related viruses. In spite of this limited range of protection, recent findings indicate that individuals immune to one influenza virus may contain antibodies (generally a minority of the overall response) that are more broadly reactive. These findings have raised the possibility that influenza vaccines could induce a more broadly protective response, reducing the need for frequent vaccine strain changes. However, interpretation of these observations is hampered by the lack of quantitative characterization of the antibody repertoire. In this study, we used single-cell cloning of influenza HA-specific B cells to assess the diversity and nature of the antibody response to influenza hemagglutinin in mice. Our findings help to put bounds on the diversity of the anti-hemagglutinin antibody response, as well as characterizing the cross-reactivity, affinity, and molecular nature of the antibody response.

Striking Predictive Power For Relapse and Decreased Survival Associated With Detectable Minimal Residual Disease by IGH VDJ Deep Sequencing Of Bone Marrow Pre- and Post-Allogeneic Transplant In Children With B-Lineage ALL: A Subanalysis Of The COG ASCT0431/PBMTC ONC051 Study

We previously reported strong power to predict relapse and lower event free survival (EFS) associated with the presence of minimal residual disease (MRD) > 0.1% detected in bone marrow (BM) both pre- and post-transplant by standardized multi-channel flowcytometry in children with ALL enrolled on a randomized phase III Children's Oncology Group/Pediatric Blood and Marrow Transplant Consortium trial, ASCT 0431. We performed further analysis of this cohort to test whether the high level of sensitivity of MRD detection offered by deep-sequencing methods increases predictive power for relapse and event free-survival (EFS). Patients in the trial were children aged 1-21yrs who underwent TBI-based myeloablative HCT for high risk ALL in CR1 or CR2. BM samples were taken within 2 weeks of initiation of the preparative regimen (pre-HCT sample) and at 1,3, and 9-12 months after HCT. These samples were sent for central flow cytometry and banked.DNA was prepared from marrow specimens at each time point in a patient, and used as template in preparing IGH VDJ sequencing reactions. 20,000 B cell genomes were used as input for index specimen libraries, used to define IGH sequences greater than 5% frequency in the sample. For a large majority of patients the index sample was taken at diagnosis, prior to transplant, but in five patients this specimen was unavailable, so a specimen taken at relapse was used to define the tumor tagging sequences. 130 bp reads starting at the WGXG motif in the J segment and extending back across the CDR3 region into the V segment were collected in each library, to a coverage of at least 5X for the input genomes. Tumor tagging sequences were confidently identified in 64 patients. To assess MRD, we then prepared libraries from the equivalent of 100,000 B cell genomes at each time point during or after transplant, and screened the resulting data for the frequency of each tumor tracking sequence in the individual.The Kaplan-Meier estimate of EFS and the Gray estimate of cumulative incidence of relapse were calculated. Evidence of tumor in the pre-transplant sample had significantly increased 36-month cumulative incidence (CI) of relapse when compared with no evidence tumor (57% v. 4.4%; p=0.008). Similarly, evidence of tumor was associated with a significant increase in 36-month CI of relapse or death when compared with no evidence of tumor (70% v. 17%, p=0.0014; and 54% v. 17%, p=0.012). Evidence of tumor in the post-transplant sample (within 90 days of transplant) had significantly increased 36-month cumulative incidence (CI) of relapse when compared with no evidence tumor (75% v. 26%; p<0.001). Similarly, evidence of tumor was associated with a significant increase in 36-month CI of relapse or death when compared with no evidence of tumor (83% v. 33%, p=0.0014; and 59% v. 27%, p=0.012). ConclusionsPatients with no detectable leukemia by deep sequencing pre-HCT relapsed less that 5% of the time, a striking improvement compared to our previous ability to predict relapse with flow MRD (No detectable MRD by flow, 25% risk of relapse in the previous analysis). Detection of any tumor in the first 90 days after HCT by this method is also highly predictive of relapse and survival. Patients identified as high risk for relapse by this method may be candidates for interventions aimed at preventing relapse. Analysis including a direct comparison with flow MRD and post transplant chimerism, as well as definition of specific cut points for levels of disease detected by deep sequencing will be presented with this data. [Display omitted] [Display omitted] [Display omitted] Disclosures:Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Kalos:Novartis corporation: CART19 technology, CART19 technology Patents & Royalties; Adaptive biotechnologies: Member scientific advisory board , Member scientific advisory board Other. Cindy:Adaptive Biotechnologies: Employment. Williamson:Adaptive Biotechnologies: Employment. Grupp:Novatis: Research Funding.

Deep Sequencing Reveals Clonal Evolution Patterns and Mutation Events Associated With Relapse In B Cell Lymphomas

Roughly 30% of Diffuse Large B-cell Lymphoma (DLBCL) patients do not respond to standard treatment or relapse after initial therapy. Relapsed DLBCL treatments are limited and less effective, highlighting the need for new therapies. Unfortunately, our current understanding of the molecular mechanisms of DLBCL relapse is poor. We hypothesize that clonal heterogeneity may contribute to disease progression. Here, we sought to explore patterns of clonal heterogeneity and evolution during DLBCL relapse by exploiting unique features of B-cell lymphomas: VDJ recombination and somatic hypermutation (SHM); and to identify genetic events underling DLBCL relapse.We used high-throughput sequencing of PCR amplified IGH VDJ sequences in 17 diagnosis(D)-relapse(R) DLBCL pairs using Illumina MiSeq pair-end 2x150bp approach, which retrieved sequence information of almost the entire VDJ amplicon. Upon aligning reads to germline sequences we identified 0.46±0.16 million VDJs per tumor. The D and R tumors of all but one pair harbored the same major VDJ rearrangement, confirming they were clonally related regardless of the time to relapse (0.5-10 yr). We first compared VDJ clonal heterogeneity of each D and R tumor pair. Although the number of unique rearrangements per sample varied, there was no significant difference between D and R samples (8.0±9.2 vs 7.6±8.9 per 10K mapped reads, p=0.84, paired t-test). In addition to rearrangement, DLBCL VDJ sequences contain various degrees of SHM. Using SHM position information on each VDJ sequence, we delineated tumor subclonal VDJ population, and uncovered two distinct modes of DLBCL relapse by performing phylogenetic analysis of the subclonal VDJ population between the D and R samples of each pair. In scenario I (n=6), the major R subclones clustered in a separate branch from the D subclones (Divergent, Figure 1A), indicating that although R and D tumors were derived from the same B-cell (same VDJ recombination), their precursors diverged early, acquired different SHMs, and expanded separately. In the more frequent scenario II (n=10), the dominant D and R subclones clustered together, and shared the majority of the same SHMs (Linear, Figure 1B), suggesting the R tumor arose directly from the major D subclone following a linear fashion. Moreover, by taking into consideration of the subclone number, frequency and SHM degree, we compared the diversity of D tumor subclone structure (empirical entropy) between the divergent and linear groups, and observed that overall divergent D tumors had a more diverse pattern of subclones than that in linear D tumors (p=3e-06, t-test), indicating that subclonal heterogeneity of the D tumor could be used to predict relapse modes. [Display omitted] To further investigate these evolutionary patterns, we performed exome-seq on 8 pairs for which we had sufficient tissue (5 linear, 3 divergent). By comparing R to matched D sample, we found that linear mode R tumors gained roughly 4 times more coding-region single nucleotide variations (SNVs) than they lost (5.2±1.9 fold), while the divergent mode R tumors gained and lost similar number of SNVs (1.4±0.7 fold). These results are consistent with our VDJ phylogenetic analysis that tumors evolve divergently undergo evolution of their genomes in parallel and acquire different sets of mutations independently; whereas tumors evolve linearly only acquire additional mutations at relapse.Finally, exome-seq revealed potential molecular mechanisms of lymphomagenesis and relapse. First, 5 R samples had genetic lesions of CD58 or B2M, two genes involving in immune surveillance escape, suggesting that escaping immune surveillance via genetic alteration may be a common relapse strategy. Moreover, in all three divergent pairs, there were histone modifier mutations shared between D and R tumors, including mutations in MLL2, EP300, and SETDB1, suggesting that these mutations could act as early “drivers” or “facilitators” to establish aberrant epigenetic landscape in tumor initiating cells favoring malignant transformation.Altogether, our study for the first time provides important evidence that DLBCL relapse may result from multiple, distinct tumor evolutionary mechanisms, providing rationale for therapies for each mechanism. Moreover, this study highlights the urgent need to understand the driving roles of epigenetic modifier mutations in lymphomagenesis, and immune surveillance factor mutations in relapse. Disclosures:Martin:Teva: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Genentech: Speakers Bureau; Millennium: Research Funding; Seattle Genetics: Consultancy, Speakers Bureau.