Citations

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 Abstract 769 Despite the many and diverse therapeutic approaches used to treat patients with mantle cell lymphoma (MCL), it remains an incurable disease. Recently, attention has turned into novel therapies targeting MCL-specific oncogenic pathways important for the growth and maintenance of the transformed phenotype. The chromosomal translocation t(11;14)(q13;q32) leading to cyclin-D1 over-expression is the hallmark of MCL. Constitute cyclin-D1 activation in B-lymphocytes maintains retinoblastoma protein in a phosphorylated state and promotes cell cycling, thus initiating the tumorigenesis process. Cyclin-D1 has been postulated as a putative target for therapeutic intervention, however its evaluation has been hampered by the incomplete understanding of the mechanism underlying this cyclin oncogenic function and by the lack of valid MCL models. To investigate these issues, we developed a combined cellular-genomics screening whereby responses to known cytotoxic compounds targeting cancer-related molecular pathways were correlated with genomic, gene expression and proteomic profiles of human MCL cells. Results showed that cyclin-D1 silencing had minimal antitumoral effects but significantly increased the therapeutic efficacy of several compounds, especially the BH3 mimetics that inhibited anti-apoptotic protein BCL-2. To further evaluate this finding we generated a MCL mouse model by transducing a tetracycline-regulatable cyclin-D1-expressing vector in murine pro-B cells, which allowed modulating cyclin-D1 expression levels. These mice generated lymphomas recapitulating most of the cellular, histopathological and molecular features of human MCL. Similar to the previous in vitro findings, cyclin-D1 inhibition in this model did not induce lymphoma regression, but sensitized cells to apoptosis. Analysis of the mechanisms underlying this therapeutic synergy identified a novel role for cyclin-D1 as a pro-survival molecule. Specifically, cyclin-D1 sequestrated the pro-apoptotic effector protein BAX in MCL cells, thereby favoring BCL2 anti-apoptotic function. Accordingly, therapeutic cyclin-D1 inactivation released BAX, thus sensitizing cells to apoptosis and inducing lymphoma regression. Interestingly, pharmacological blockade in vivo of cyclin-D1 with Roscovitine synergistically cooperated with the BH3 mimetic ABT-737 to effectively inhibit MCL tumor growth. In summary, our study reveals a novel role for cyclin-D1 in deregulating apoptosis in MCL cells and highlights the potential benefit of cyclin-D1 targeting, thus providing the rationale for the clinical evaluation of drugs targeting cell proliferation and survival pathways in MCL. Disclosures: Siebert: Abbott: Honoraria.

Abstract Splenic marginal zone lymphoma (SMZL) is an indolent B cell malignancy whose diagnosis is based on lymphocyte morphology, immunophenotype and marrow and/or splenic histology. Unlike other lymphomas, there is not a common chromosomal translocation specific for SMZL, and genetic prognostic factors are poorly defined. To investigate the pattern of genomic aberrations in SMZL, we applied comparative genomic hybridization to BAC microarrays (array CGH) to a well characterized series of 75 SMZL specimens. We applied two different 1 Mb-resolution BAC arrays: UCSF HumArray 3.2 and a novel array CGH platform developed at Univ. of Salamanca. These arrays allowed us to detect DNA copy number changes across the genome with high accuracy in 67 of 75 patient samples. Data were compared with our previous array CGH studies of 170 samples from different B-cell lymphoma subgroups. FISH studies for IGH, IGK and IGL translocations and 7q deletion were performed on tissue microarrays in 24 cases. Of the 67 samples, 19 (28%) showed a normal genomic profile. The median number of genomic aberrations per tumor was 2.2 (1.3 gains and 0.9 losses), which was lower than the rates detected in other lymphoma subgroups (diffuse large cell lymphoma, 6.4; mantle cell lymphoma, 6; follicular lymphoma, 4.5) and comparable to MALT lymphomas (2 abnormalities per tumor). SMZL cells showed a genomic pattern characterized by gain of chromosomes 3q24-q29 (18%), 6p (9%), 12q (9%), and 18q (4%) and loss of 7q32 (34%), 8p21-p23 (13%), 17p13 (10%) at P53 locus and 6q21-q27 (9%). Notably, no alterations of the P16/ARF (9p21) or MYC loci (8q24) were detected. Correlation of array CGH data with conventional cytogenetics, FISH and LOH studies revealed a high concordance. Detailed mapping of 7q deletions delineated a consensus region of loss of 3 Mb in 7q32. This 7q deletion was almost exclusive to SMZL, being observed in only 5 of 170 non-SMZL B-cell lymphomas (p=0.0000001). Four cases presented IG-translocation. Mutation of IGH was observed in 62% and correlated with a complex karyotype (61 vs. 13%; p=0,0008) whereas unmutated IGH correlated with the deletion of 7q (56 vs. 23%; p=0,01). Among the various genomic abnormalities, only the deletion of 8p or the presence of a complex karyotype correlated with inferior overall survival (OS) (median OS, 58 vs. 110 months, p=0,004; and 60 vs. 105 months, p=0,01; respectively). In summary, array CGH has defined a pattern of genomic aberrations in SMZL that differs from other B-cell lymphoma subgroups and that may predict overall survival. Because the deletion of 7q32 is the most distinctive genetic marker in SMZL, the identification of a putative tumor suppressor gene inactivated within the region of deletion seems mandatory.

AbstractObjectivesThe 2017 Workshop of the Society for Hematopathology/European Association for Haematopathology reviewed the role of molecular genetics in the diagnosis and biology of lymphoid neoplasms.MethodsThe Workshop Panel reviewed 82 cases.ResultsMolecular genetic testing reveals alterations that expand the spectrum of diseases such as DUSP22 rearrangement in ALK-negative anaplastic large cell lymphoma, large B-cell lymphoma with IRF4 rearrangement, MYD88 mutations in B-cell lymphomas, Burkitt-like lymphoma with 11q aberrations, and diagnostic criteria for high-grade B-cell lymphomas. Therapeutic agents and natural tumor progression may be associated with transcriptional reprogramming that lead to transdifferentiation and lineage switch.ConclusionsApplication of emerging technical advances has revealed the complexity of genetic events in lymphomagenesis, progression, and acquired resistance to therapies. They also contribute to enhanced understanding of the biology of indolent vs aggressive behavior, clonal evolution, tumor progression, and transcriptional reprogramming associated with transdifferentiation events that may occur subsequent to therapy.

Background:Tipifarnib is a potent and selective inhibitor of the enzyme farnesyltransferase (FT). FT catalyzes post‐translational attachment of farnesyl groups required for localization of signaling molecules to the inner cell membrane. CXCL12 is a chemokine that is essential for T cell homing to lymphoid organs and the bone marrow, and for the maintenance of immune cell progenitors.Aims:Herein we report preliminary efficacy, safety and biomarker data from a Phase 2 study of tipifarnib in angioimmunoblastic T‐cell lymphoma (AITL) and CXCL12+ peripheral T‐cell lymphoma (PTCL) patients (pts).Methods:This Phase 2 study (NCT02464228) is a multi‐institutional, single‐arm, open‐label trial initially designed as a two‐stage (11+7 pts) design to determine the efficacy, safety and biomarkers of tipifarnib in pts with relapsed/refractory (R/R) PTCL age >/ = 18 years and a performance status of 0–2. Based on initial findings, the study was amended to include a cohort of AITL (n = 12) and PTCL (n = 12) pts with the CXCL12 rs2839695 A/A genotype (CXCL12+ cohort). Pts received tipifarnib 300 mg administered orally twice daily on days 1–21 of 28‐day treatment cycles until progression of disease (PD) or unacceptable toxicity. The primary endpoint of the study is overall response rate (ORR). Ancillary studies are also ongoing to investigate the prognostic value of CXCL12 expression in pts who received tipifarnib and standard of care treatment.Results:As of 20 February 2019, 43 PTCL pts (20 AITL, 21 PTCL‐NOS, 1 ALK‐ ALCL, 1 gamma‐delta TCL) have been treated with tipifarnib, 19 pts in stages 1 and 2, and 24 pts in the ongoing AITL histology and CXCL12 cohort. Median number of prior treatment regimens was 3; 19 pts had a prior stem cell transplant. All pts had at least one treatment‐emergent adverse event (TEAE); 39 (90%) had at least 1 study drug‐related TEAE and 12 (28%) at least 1 drug related SAE. The most frequently observed drug‐related TEAEs of Grade ≥3 occurring in 10% or more of pts were blood and lymphatic system disorders, including neutropenia (44%), thrombocytopenia (37%), leukopenia (28%), anemia (21%) and febrile neutropenia (21%). There have been 14 deaths on study; none were related to study drug. Of 18 evaluable pts enrolled in Stages 1 and 2 of the trial, 3 partial responses (PR), 2 of them in pts with AITL histology, and 4 best responses of stable disease (SD) were observed. In the AITL cohort (11 evaluable pts), a 45% ORR and 73% clinical benefit rate (CBR; 3 CR, 2 PR and 3 SD) was observed. In the CXCL12+ cohort (n = 3 evaluable pts), 1 PR and 2 SD have been observed, with 4 pts pending cycle 2 response evaluation. CXCL12 expression correlated with favorable outcome to tipifarnib treatment. Pre‐treatment high CXCL12 expression had 90% sensitivity and 93% specificity to identify pts who experienced CR/PR/SD on study in a preliminary analysis of 24 pts. The prognosis of CXCL12 was also retrospectively investigated in 73 pts who received standard of care therapy (AITL N = 50, PTCL NOS = 23). A trend for poor prognosis (22 vs 40 months median OS from diagnosis, HR = 1.8, p = 0.09) was observed using the high CXCL12 subset using the cut‐off point that predicted clinical benefit under tipifarnib therapy. Fifty percent of AITL and 35% of non‐AITL samples overexpressed CXCL12.Summary/Conclusion:Preliminary activity of tipifarnib was observed in PTCL pts, particularly in those with tumors of AITL histology and high CXCL12 expression and enrollment in the CXCL12+ cohort continues.

Key PointsPhenotypic and genotypic profiling of MDM2 in DLBCL. MDM2 as a negative regulator of p53 tumor suppressor function.

The chromosomal translocation t (11;14)(q13;q32) leading to cyclin-D1 overexpression plays an essential role in the development of mantle cell lymphoma (MCL), an aggressive tumor that remains incurable with current treatment strategies. Cyclin-D1 has been postulated as an effective therapeutic target, but the evaluation of this target has been hampered by our incomplete understanding of its oncogenic functions and by the lack of valid MCL murine models. To address these issues, we generated a cyclin-D1–driven mouse model in which cyclin-D1 expression can be regulated externally. These mice developed cyclin-D1–expressing lymphomas capable of recapitulating features of human MCL. We found that cyclin-D1 inactivation was not sufficient to induce lymphoma regression in vivo; however, using a combination of in vitro and in vivo assays, we identified a novel prosurvival cyclin-D1 function in MCL cells. Specifically, we found that cyclin-D1, besides increasing cell proliferation through deregulation of the cell cycle at the G 1 –S transition, sequestrates the proapoptotic protein BAX in the cytoplasm, thereby favoring BCL2’s antiapoptotic function. Accordingly, cyclin-D1 inhibition sensitized the lymphoma cells to apoptosis through BAX release. Thus, genetic or pharmacologic targeting of cyclin-D1 combined with a proapoptotic BH3 mimetic synergistically killed the cyclin-D1–expressing murine lymphomas, human MCL cell lines, and primary lymphoma cells. Our study identifies a role of cyclin-D1 in deregulating apoptosis in MCL cells, and highlights the potential benefit of simultaneously targeting cyclin-D1 and survival pathways in patients with MCL. This effective combination therapy also might be exploited in other cyclin-D1–expressing tumors.