Ponzoni, Maurilio

Abstract Introduction: Multiple studies have demonstrated that diffuse large B-cell lymphoma (DLBCL) can be divided into subgroups based on their biology. However, these biological subgroups overlap clinically. While R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) remains the standard of care for treating patients with DLBCL, predicting which patients will not benefit from such therapy is important so that alternative therapy or clinical trials can be considered. Most of the studies stratifying patients select biomarkers first, then explore how these biomarkers can stratify patients based on outcome. We explored the potential of using machine learning to first group patients with DLBCL based on survival, then isolating the biomarkers necessary for predicting these survival subgroups. Methods: RNA was extracted from tissue paraffin blocks from 379 R-CHOP treated patients with de novo DLBCL, and from 247 patients with extranodal DLBCL. A targeted hybrid capture RNA panel of 1408 genes was used for next generation sequencing (NGS). Sequencing was performed using an Illumina NextSeq 550 System platform. Ten million reads per sample in a single run were required, and the read length was 2 × 150 bp. An expression profile was generated from the sequencing coverage profile of each individual sample using Cufflinks. A machine learning system was developed to classify patients into four groups based on their overall survival. This machine learning approach based on Naïve Bayesian algorithm was also used to discover the relevant subset of genes with which to classify patients into each of the four survival groups. To eliminate the underflow problem commonly associated with the standard Naïve Bayesian classifiers, we applied Geometric Mean Naïve Bayesian (GMNB) as the classifier to predict the survival group for each patient. Results: Using machine learning, patients were first divided into two groups: short survival (S) and long survival (L). To refine this model, we used the same approach and divided the patients in each group into two subgroups, generating four groups: long survival in the long group (LL), short survival in the long group (LS), long survival in the short group (SL), and short survival in the short group (SS). The hazard ratio for this model was 0.174 (confidence interval: 0.120-0.251), and P-value <0.0001. After defining these four groups, a machine learning algorithm was used to discover the biomarkers from the expression data of the 1408 genes from NGS data. To reduce the effects of noise and avoid overfitting, we employed a 12-step cross validation to obtain a robust measure. For an individual gene, a generalized Naïve Bayesian classifier was constructed on the training of one of the 12 subsets and tested on the other 11 testing subsets. This allowed us to limit the prediction process to 60 genes for each separation step. Using the selected biomarkers, we classified the patients in the original set (379 patients) into LL, LS, SL, and SS groups and then evaluated the survival pattern of these groups. As shown in Fig. 1A, the selected biomarkers predicted survival as expected in the overall survival groups prior to biomarker selection. For additional validation of the system, we used the selected biomarkers to classify a completely new set of 247 samples of patients with extranodal DLBCL. As shown in Fig. 1B, these selected biomarkers successfully predicted the overall survival in this group of patients with an HR of 0.530 (confidence interval: 0.234-1.197, P=0.005). This classification correlated with cell of origin classification, TP53 mutation status, MYC expression, and IRF4 expression. However, in a multivariate analysis, only TP53 mutation was independent in predicting prognosis (P=0.005) and age (below or over 60) (P=0.01) along with the survival grouping (P<0.000001). Conclusions: Using a novel machine learning approach with the expression levels of 180 genes, we developed a model that can reliably stratify patients with DLBCL treated with R-CHOP into four survival subgroups. This model can be used to identify patients who may not respond well to R-CHOP to be considered for alternative therapy and clinical trials. Figure 1 Figure 1. Disclosures Hsi: AbbVie Inc, Eli Lilly: Research Funding. Ferreri: Ospedale San Raffaele srl: Patents & Royalties; BMS: Research Funding; Pfizer: Research Funding; Beigene: Research Funding; Hutchison Medipharma: Research Funding; Amgen: Research Funding; Genmab: Research Funding; ADC Therapeutics: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; PletixaPharm: Membership on an entity's Board of Directors or advisory committees; x Incyte: Membership on an entity's Board of Directors or advisory committees; Adienne: Membership on an entity's Board of Directors or advisory committees. Piris: Millenium/Takeda, EUSA, Jansen, NanoString, Kyowa Kirin, Gilead and Celgene.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Winter: BMS: Other: Husband: Data and Safety Monitoring Board; Actinium Pharma: Consultancy; Janssen: Other: Husband: Consultancy; Agios: Other: Husband: Consultancy; Gilead: Other: Husband: Consultancy; Epizyme: Other: Husband: Data and Safety Monitoring Board; Ariad/Takeda: Other: Husband: Data and Safety Monitoring Board; Merck: Consultancy, Honoraria, Research Funding; Novartis: Other: Husband: Consultancy, Data and Safety Monitoring Board; Karyopharm (Curio Science): Honoraria.

Abstract Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities (MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2–targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL-MYC/BCL2-DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC/BCL2/TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. Implications: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg.

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity of B-cell lymphoma. Cell-of-origin (COO) classification of DLBCL is required in routine practice by the World Health Organization classification for biological and therapeutic insights. Genetic subtypes uncovered recently are based on distinct genetic alterations in DLBCL, which are different from the COO subtypes defined by gene expression signatures of normal B cells retained in DLBCL. We hypothesize that classifiers incorporating both genome-wide gene-expression and pathogenetic variables can improve the therapeutic significance of DLBCL classification. To develop such refined classifiers, we performed targeted RNA sequencing (RNA-Seq) with a commercially available next-generation sequencing (NGS) platform in a large cohort of 418 DLBCLs. Genetic and transcriptional data obtained by RNA-Seq in a single run were explored by state-of-the-art artificial intelligence (AI) to develop a NGS-COO classifier for COO assignment and NGS survival models for clinical outcome prediction. The NGS-COO model built through applying AI in the training set was robust, showing high concordance with COO classification by either Affymetrix GeneChip microarray or the NanoString Lymph2Cx assay in 2 validation sets. Although the NGS-COO model was not trained for clinical outcome, the activated B-cell–like compared with the germinal-center B-cell–like subtype had significantly poorer survival. The NGS survival models stratified 30% high-risk patients in the validation set with poor survival as in the training set. These results demonstrate that targeted RNA-Seq coupled with AI deep learning techniques provides reproducible, efficient, and affordable assays for clinical application. The clinical grade assays and NGS models integrating both genetic and transcriptional factors developed in this study may eventually support precision medicine in DLBCL.

Abstract To describe the demographic and tumor related characteristics and outcomes for patients with primary T-cell central nervous system lymphoma (TPCNSL). A retrospective series of patients with TPCNSL was compiled from twelve cancer centers and seven countries. This study involved 35 male and 10 female patients with a median age of 60 years (range 3–84). Twenty (44%) had Eastern Cooperative Oncology Group performance status (PS) of 0 or 1. Twenty six (58%) had involvement of a cerebral hemisphere and sixteen (36%) had lesions of deeper sites in the brain. Two patients had primary spinal cord lesions and one had meningeal disease only. Serum lactate dehydrogenase (LDH) was elevated in 7 of the 22 cases (32%) and cerebrospinal fluid (CSF) protein was elevated above normal in 19 of the 24 cases (79%) with available data. The median disease specific survival (DSS) for all patients was 25 months (95% confidence interval (CI) 11–38 months). The two and five-year DSS were 51 % (CI 35–66 %) and 17 % (CI 6–34 %) respectively. Univariate and multivariate analyses were conducted for the following factors: age (≤ 60 vs. > 60 years), PS (0 or 1 vs. 2, 3 or 4), involvement of deep structures of the central nervous system (no vs. yes), and methotrexate (MTX) use in the primary treatment (yes vs. no). Only PS and MTX use were significantly associated with better outcome with hazard ratios (HR) of 0.2 (CI 0.1–0.4) and 0.4 (CI 0.2–0.8) respectively. This is the largest series ever assembled of TPCNSL. The presentation and outcome appear similar to that of PCNSL of B cell origin. PS 0 or 1 and administration of MTX are associated with better survival. TPCNSL does not appear to require a different therapeutic management approach than B-cell PCNSL.

Abstract Context and objective Approximately 5-10% of diffuse large B-cell lymphoma (DLBCL) patients carry MYC gene translocations (MYC-translocation+) with a poor prognosis after standard chemotherapy. MYC-translocation+ DLBCL patients carrying BCL2 translocations (MYC+/BCL2+ double-hit lymphoma) have a worse survival. The efficacy of adjuvant radiotherapy in this setting is unknown. The purpose of this study is to evaluate the efficacy of radiotherapy as a part of the therapeutic regimen for patients with MYC-translocation+ DLBCL. Patients and methods From the International DLBCL R-CHOP consortium program, we selected 581 patients with de novo DLBCL treated with standard R-CHOP immunochemotherapy (diagnosed and treated from 2000 to 2010). We excluded patients with transformed DLBCL, primary mediastinal, cutaneous, testicular or central nervous system large B-cell lymphomas, patients with HIV infection, and patients not treated with R-CHOP. The median follow-up was 54.9 months. Fluorescence in situ hybridization assessing MYC was performed for all the patients (n=581) and results were correlated with available clinical data to identify clinicopathologic features associated with MYC translocation, and to evaluate the prognostic significance of MYC translocations regarding overall survival (OS, from the time of diagnosis to death from any cause) and progression-free survival (PFS, from the time of diagnosis to relapse or death from any cause). In the MYC-translocation+ DLBCL group, 38 patients received chemotherapy alone and 21 patients received chemotherapy with adjuvant radiation therapy. The clinicopathologic features and survival of MYC-translocation+ DLBCL patients treated with (n=21) and without radiotherapy (n=38) after immunochemotherapy were compared to in order to evaluate the radiotherapy efficacy and other confounding factors. Results MYC translocations were detected in 59 DLBCL patients (10.2%). Patients with MYC-translocation+ DLBCL more often had bulky tumors, involvement of multiple extranodal sites, and poorer OS (hazard ratio [HR]: 2.0, 95% confidence interval [CI]: 1.20 - 3.35, P= .0083) and PFS (HR: 1.96, 95% CI: 1.22 - 3.13, P= .0005) independent of the International Prognostic Index score. Poor survival was primarily attributable to patients with MYC+/BCL2+ double-hit DLBCL who were predominantly of germinal center B-cell subtype. Among MYC-translocation+ DLBCL patients, a better survival was achieved in patients who received radiotherapy (for OS, HR: .32, 95% CI: .15 - .71, P= .0049; for PFS, HR: .35, 95% CI: .17 - .73, P= .0043). Conversely, radiotherapy abolished the adverse impact of MYC translocations. In addition, radiotherapy was associated with better survival in the subset of patients with MYC+/BCL2+ double-hit lymphoma (P = .017 for OS, and P = .05 for PFS). However, owing to the common use of radiotherapy as consolidation therapy, the favorable prognoses in the group of patients who received radiotherapy could also be attributed to limited-stage disease and more frequent complete remission (CR) after first-line treatment and therefore these factors confound interpretation of the data. To address these issues, we evaluated radiotherapy efficacy in separate patient groups: patients who achieved CR, non-CR (PR/SD/PD) patients, and patient with stage I/II, or stage III/IV disease. The efficacy of radiotherapy appeared more apparent in patients with advanced disease who did not achieve CR after first-line chemotherapy. Multivariate analysis after adjustment for stage and IPI score validated that radiotherapy significantly improved OS (HR: .28, 95% CI: .10 - .81, P= .018) and PFS (HR: .32, 95% CI: .13 - .80, P= .015) of MYC-translocation+ DLBCL patients. Conclusions The presence of MYC translocations in DLBCL is an important biomarker that facilitates prognostic prediction and treatment stratification independent of the IPI score. For chemoresistant MYC-translocation+ DLBCL patients, radiotherapy seems to be an effective adjuvant regimen likely due to the higher frequency of extranodal involvement and bulky disease in MYC-translocation+ DLBCL patients. Our results provide a rationale for larger scale studies to assess the potential role of radiotherapy in the management of MYC-translocation+ DLBCL patients, particularly patients with MYC+/BCL2+double-hit DLBCL. Disclosures: Winter: Millenium: Research Funding; Novartis : Research Funding; Pfizer (Wyeth): Research Funding; Seattle Genetics: Research Funding; Spectrum: Research Funding; Janssen (Pharmacyclics): Research Funding; Spectrum (Allos): Consultancy; Sanofi Aventis: Consultancy; Tgen: Consultancy; AMBIT Biosciences (Spouse): Research Funding; Celgene (Spouse): DSMB, DSMB Other, Research Funding; Ariad Pharmaceuticals (Spouse): Research Funding; Novartis (Spouse): Consultancy, Research Funding; Amgen (Spouse): Consultancy, Research Funding; Astellas (Spouse): Research Funding; Caremark/CVS: Consultancy; Pfizer (Spouse): Consultancy; Sanofi Aventis (Spouse): DSMB, DSMB Other; Bristol Myers Squibb (Spouse): DSMB, DSMB Other; UptoDate, Inc.(Spouse): Patents & Royalties.

Abstract Abstract 2638 We recently demonstrated that over 30% of cases with splenic marginal-zone lymphoma (SMZL) express distinctive immunoglobulin (IG) receptors that utilize a single polymorphic variant of the IGHV1-2 gene (IGHV1-2*04) and also exhibit restricted antigen-binding site motifs and precise targeting of somatic hypermutation (SHM). On these grounds, we proposed the existence of molecular subtypes of SMZL defined by immunogenetic analysis of the IG receptors with implications for selection by specific (super) antigenic element(s) in the development of at least a major subset of SMZL. In order to gain insight as to whether antigen involvement is relevant only prior to the malignant transformation or if it continues to affect the SMZL clone itself leading to intraclonal diversification (ID) through ongoing SHM, we conducted a large-scale subcloning study of rearranged IG heavy variable genes, in a total of 471 subcloned sequences from 22 SMZL cases. The analysis was intentionally biased towards cases expressing IGHV1-2*04 receptors that exhibit a series of distinctive immunogenetic features, including biased usage of the IGHD3-3 and IGHD3-10 genes, unusually long heavy complementarity-determining region 3 (VH CDR3) and minimally/borderline mutated status (identity to the germline in the range of 97–99.6%). Hence, the study group included 16 IGHV1-2*04 cases and 6 cases utilizing other IGHV genes. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and a median of 21 (9–46) colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences from the same sample. Overall, 15/22 cases (68%) carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, of which 12 utilized the IGHV1-2*04 gene whereas the remaining 3 utilized other IGHV genes. The high frequency of ID within the IGHV1-2*04 group, ranging from limited to (often) pronounced, is noteworthy in view of the generally low level of SHM among IGHV1-2*04 receptors. Detailed analysis of the distribution and molecular features of CMs revealed: (i) restricted ID patterns, in the sense of identical mutations in certain VH positions among subclones of different cases; (ii) “hotspots” of ID, i.e. particular codons exhibiting intense ongoing mutational activity (especially notable in this respect was codon 39 in VH FR2); (iii) a predominance of conservative amino acid changes, characterized by somatically introduced amino acid belonging to the same biochemical category as the mutating amino acid; and (iv) limited diversification within VH CDR3. Additionally, in 7/12 IGHV1-2*04 cases with CMs, identified ID patterns delineate distinct “clusters” of subcloned sequences with unique as well as shared mutations and closely similar if not identical VH CDR3s (including identical VH CDR3 length), pointing to “branching” of the malignant clone into distinct subclones, perhaps able to evolve along related yet distinct pathways. In conclusion, our study indicates that the SHM mechanism may continuously operate in certain subsets of SMZL, especially those expressing IGHV1-2*04 receptors. Although the precise timing of interactions with antigen(s) and their functional implications for SMZL evolution will likely remain difficult to define accurately, the results reported here suggest a role for persistent antigenic stimulation, at least for certain immunogenetically defined subsets of SMZL. Disclosures: Mollejo: Red Temática de Investigación Cooperativa en Cancer (RETICC): Research Funding; Asociación Española contra el Cancer: Research Funding.

Abstract Context and objective Approximately 5-10% of diffuse large B-cell lymphoma (DLBCL) patients carry MYC gene translocations (MYC-translocation+) with a poor prognosis after standard chemotherapy. MYC-translocation+ DLBCL patients carrying BCL2 translocations (MYC+/BCL2+ double-hit lymphoma) have a worse survival. The efficacy of adjuvant radiotherapy in this setting is unknown. The purpose of this study is to evaluate the efficacy of radiotherapy as a part of the therapeutic regimen for patients with MYC-translocation+ DLBCL. Patients and methods From the International DLBCL R-CHOP consortium program, we selected 581 patients with de novo DLBCL treated with standard R-CHOP immunochemotherapy (diagnosed and treated from 2000 to 2010). We excluded patients with transformed DLBCL, primary mediastinal, cutaneous, testicular or central nervous system large B-cell lymphomas, patients with HIV infection, and patients not treated with R-CHOP. The median follow-up was 54.9 months. Fluorescence in situ hybridization assessing MYC was performed for all the patients (n=581) and results were correlated with available clinical data to identify clinicopathologic features associated with MYC translocation, and to evaluate the prognostic significance of MYC translocations regarding overall survival (OS, from the time of diagnosis to death from any cause) and progression-free survival (PFS, from the time of diagnosis to relapse or death from any cause). In the MYC-translocation+ DLBCL group, 38 patients received chemotherapy alone and 21 patients received chemotherapy with adjuvant radiation therapy. The clinicopathologic features and survival of MYC-translocation+ DLBCL patients treated with (n=21) and without radiotherapy (n=38) after immunochemotherapy were compared to in order to evaluate the radiotherapy efficacy and other confounding factors. Results MYC translocations were detected in 59 DLBCL patients (10.2%). Patients with MYC-translocation+ DLBCL more often had bulky tumors, involvement of multiple extranodal sites, and poorer OS (hazard ratio [HR]: 2.0, 95% confidence interval [CI]: 1.20 - 3.35, P= .0083) and PFS (HR: 1.96, 95% CI: 1.22 - 3.13, P= .0005) independent of the International Prognostic Index score. Poor survival was primarily attributable to patients with MYC+/BCL2+ double-hit DLBCL who were predominantly of germinal center B-cell subtype. Among MYC-translocation+ DLBCL patients, a better survival was achieved in patients who received radiotherapy (for OS, HR: .32, 95% CI: .15 - .71, P= .0049; for PFS, HR: .35, 95% CI: .17 - .73, P= .0043). Conversely, radiotherapy abolished the adverse impact of MYC translocations. In addition, radiotherapy was associated with better survival in the subset of patients with MYC+/BCL2+ double-hit lymphoma (P = .017 for OS, and P = .05 for PFS). However, owing to the common use of radiotherapy as consolidation therapy, the favorable prognoses in the group of patients who received radiotherapy could also be attributed to limited-stage disease and more frequent complete remission (CR) after first-line treatment and therefore these factors confound interpretation of the data. To address these issues, we evaluated radiotherapy efficacy in separate patient groups: patients who achieved CR, non-CR (PR/SD/PD) patients, and patient with stage I/II, or stage III/IV disease. The efficacy of radiotherapy appeared more apparent in patients with advanced disease who did not achieve CR after first-line chemotherapy. Multivariate analysis after adjustment for stage and IPI score validated that radiotherapy significantly improved OS (HR: .28, 95% CI: .10 - .81, P= .018) and PFS (HR: .32, 95% CI: .13 - .80, P= .015) of MYC-translocation+ DLBCL patients. Conclusions The presence of MYC translocations in DLBCL is an important biomarker that facilitates prognostic prediction and treatment stratification independent of the IPI score. For chemoresistant MYC-translocation+ DLBCL patients, radiotherapy seems to be an effective adjuvant regimen likely due to the higher frequency of extranodal involvement and bulky disease in MYC-translocation+ DLBCL patients. Our results provide a rationale for larger scale studies to assess the potential role of radiotherapy in the management of MYC-translocation+ DLBCL patients, particularly patients with MYC+/BCL2+double-hit DLBCL. Disclosures: Winter: Millenium: Research Funding; Novartis : Research Funding; Pfizer (Wyeth): Research Funding; Seattle Genetics: Research Funding; Spectrum: Research Funding; Janssen (Pharmacyclics): Research Funding; Spectrum (Allos): Consultancy; Sanofi Aventis: Consultancy; Tgen: Consultancy; AMBIT Biosciences (Spouse): Research Funding; Celgene (Spouse): DSMB, DSMB Other, Research Funding; Ariad Pharmaceuticals (Spouse): Research Funding; Novartis (Spouse): Consultancy, Research Funding; Amgen (Spouse): Consultancy, Research Funding; Astellas (Spouse): Research Funding; Caremark/CVS: Consultancy; Pfizer (Spouse): Consultancy; Sanofi Aventis (Spouse): DSMB, DSMB Other; Bristol Myers Squibb (Spouse): DSMB, DSMB Other; UptoDate, Inc.(Spouse): Patents & Royalties.

Abstract Abstract 634 We systematically explored the immunoglobulin (IG) gene repertoire in 337 cases with splenic marginal-zone lymphoma (SMZL), by far the largest series yet. To resolve classification uncertainties, we included in the analysis only cases with a diagnosis of SMZL based on spleen histopathological findings or cases fulfilling the 2008 SBLG criteria (Matutes et al. Leukemia 2008). We here report that the IG heavy variable (IGHV) gene repertoire in SMZL is remarkably biased, with only three genes accounting for 45.8% of cases (IGHV1-2, 24.9%; IGHV4-34, 12.8%; IGHV3-23: 8.1%, respectively), significantly extending previous similar observations. Particularly for the IGHV1-2 gene, strong biases became evident at the level of utilization of different alleles, since 79/86 rearrangements (92%) utilized allele *04 vs. only 7/86 rearrangements (8%) that utilized allele *02. This is noteworthy, taking into consideration that these two alleles differ in a single nucleotide, leading to a single amino acid change in framework region (FR)-3. The repertoire biases became more pronounced when the analysis was focused on 171 rearrangements from 163 cases classified as SMZL based on splenic histopathology, according to the 2008 WHO criteria. Within this subgroup, 56/171 cases (32.7%) utilized IGHV1-2*04. Noticeably, only 1/17 cases with a diagnosis of splenic diffuse red pulp lymphoma utilized IGHV1-2*04 (p<0.02 for comparison to SMZL). The IGHV1-2*04 rearrangements carried significantly longer heavy complementarity-determining region-3 (VH CDR3) than all other cases (median, 22 vs. 17 amino acids, respectively; p<0.001). In addition, 52/79 IGHV1-2*04 cases (65.8%) employed one of the IGHD3-3, IGHD3-9 or IGHD3-10 genes. In 28/32 IGHV1-2*04/IGHD3 rearrangements, the IGHD gene was utilized in the same reading frame, leading to VH CDR3s with common “IGHD-derived” amino acid (AA) motifs. Using bioinformatics tools previously applied to CLL, biased associations of IGHV, IGHD and IGHJ genes with stereotyped VH CDR3s were identified in 25/345 sequences (7.2%). Noticeably, only 10/28 IGHV1-2*04/IGHD3-3 rearrangements with “IGHD-derived” VH CDR3 amino acid motifs could be assigned to “stereotyped” clusters. Despite exhibiting restricted usage of the IGHV1-2*04 and IGHD3-3 genes leading to great overall VH domain similarity, the remaining cases did not fulfill the established criteria for VH CDR3 “stereotypy”, as defined in other lymphoid malignancies, namely CLL. Based on somatic hypermutation (SHM) analysis, the sequences were divided into three groups: (i) truly unmutated (100% germline identity, GI): 46/345 sequences (13.3%); (ii) minimally/borderline mutated (97-99.9% GI): 130/345 sequences (37.7%); and (iii) significantly mutated (<97% identity): 169/345 sequences (49%). At the individual gene level, the distribution of rearrangements of IGHV genes according to SHM status varied significantly. In particular, 56/79 IGHV1-2*04 rearrangements (71%) were predominantly “borderline mutated”, whereas the majority (>67%) of rearrangements utilizing the IGHV3-23, IGHV3-30 and IGHV3-7 genes were “significantly mutated”; finally, IGHV4-34 gene rearrangements were evenly distributed to the three mutational subgroups. Shared (“stereotyped”) AA changes were identified for IGHV1-2*04 rearrangements, with certain FR2 and FR3 codons emerging as “hotspots” for recurrent, conservative AA changes. In conclusion, we demonstrate that more than 30% of cases with a histopathological diagnosis of SMZL on the spleen express IGHV1-2*04 receptors with unusually long VH CDR3s, biased usage of the IGHD3-3 gene, leading to shared “IGHD-derived” VH CDR3 motifs, and very precise molecular features of SHM. The biased expression of a distinctive germline-encoded VH specificity might be considered as evidence for heavy chain dominance in the clonogenic IG receptors in SMZL. These findings allude to selection by specific (super)antigenic element(s) in the pathogenesis of at least a major subset of SMZL. In addition, they raise the intriguing possibility that certain subtypes of SMZL could derive from progenitor cell populations adapted to particular antigenic challenges through cellular selection of VH domain specificities. Disclosures: No relevant conflicts of interest to declare.

Abstract While multiple myeloma (MM) is almost invariably preceded by asymptomatic monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM), the alterations of the bone marrow (BM) microenvironment that establish progression to symptomatic disease are circumstantial. Our aim was to identify changes in the BM microenvironment eliciting angiogenesis in MM. We have found that in Vk*MYC mice harboring oncogene-driven plasma cell proliferative disorder, the progression from asymptomatic to symptomatic MM was characterized by substantial modifications of the BM microenvironment, including a switch from a T helper type 1 (Th1) to a Th2 response, and accumulation of CD206+Tie2+ macrophages, which eventually favored angiogenesis and increased in microvessel density (MVD). Notably, MVD was also increased at diagnosis in the BM of MGUS and SMM patients that subsequently progressed to MM when compared with MGUS and SMM that remained quiescent. These findings suggest a multistep pathogenic process in MM, in which the immune system may contribute to angiogenesis and disease progression. They also suggest investigating MVD in asymptomatic patients as prognostic factor for the progression and outcome of this disease. Disclosures No relevant conflicts of interest to declare.