ALBERT

Description: Key Points Cases of cHL may express TCA on the neoplastic cells. TCA-cHL have nodular sclerosis histology and lack T-cell genotype, with worse outcome compared with TCA-negative cHLs.

Description: Multiple Myeloma (MM) is a clonal B-cell malignancy characterized by the accumulation of terminally differentiated plasma cell in the bone morrow, which accounts for 10% of all hematological cancers. Despite major advance of last decade, MM remains an incurable disease in need of new therapeutic approaches. Leflunomide was originally developed as an immunosuppressive drug capable of alleviating the severity of autoimmune disease and preventing allograft and xenograft rejection and it has been FDA approved to use in the treatment of rheumatoid arthritis (RA) since 1998. It rapidly converted in gastrointestinal tract and plasma to open ring metabolite A771726. In current study, we examine the effects of A771726 to the growth of MM cell lines in vivo and show that A771726 inhibits the proliferation of four different well-characterized MM cell lines (MM.1, U266, RPMI8226 and MDR10V) in a dose- and time- dependent manner, regardless their sensitivity to the conventional chemotherapies. Cell cycle analyses further demonstrate that G1-S phase transition is blocked by A771726 in those cells. Moreover, we find that, at higher concentration, A771726 is able to induce apoptosis in at least two of the cell lines tested as measured by an increase of the cells with subG1 DNA content, an increasing in caspase activity and cleavage of caspase substrate. Previous studies suggested that A771726 exerts its anti-proliferate function mostly by inhibiting de novo pyrimidine synthesis in lymphocyte and supplementation of the cultures with exogenous uridine could prevent A771726-induced cell cycle block by restoring the intracellular UTP and CTP lever. However, we find that additions of exogenous uridine can prevent neither A771726-induced cell cycle block nor apoptosis in the MM cell lines tested, suggesting a novel mechanism. It shall be noted that pharmacologically, the highest concentration of A771726 (200uM) used in our study is readily achievable in the plasma through current drug regiment of Leflunomide in RA patients. Since its approval, Leflunomide has been proved to be an effective antirheumatic drug with mild side effects. Given the results from current study, we are tempting to suggest that Leflunomide might also provide a safe alternative in the treatment of MM. The vast knowledge gained from clinical use of Leflunomide in last six year will greatly help us greatly in testing this hypothesis in a clinical setting in a near future.

Description: Introduction Ibrutinib (ibr), a first-in-class BTK inhibitor, has high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL) independent of high-risk FISH abnormalities (NEJM. 2015; 373:2425-37, NEJM. 2013; 369:32-42). However, about 25% of patients discontinue ibr therapy at a median of 20 months treatment and ~40% patients stop ibr due to disease progression (JAMA Oncol 2015; 1:80-7, Blood 2015; 125:2062-67). Among progressed patients, at least half developed Richter's transformation (RT). Treatment options following progression are limited, with mortality rates exceeding 75% and a short median overall survival of 3 months. As the use of ibr becomes more prevalent in CLL and other types of non-Hodgkin lymphoma (NHL), more patients are at risk to develop resistance (BJH 2015; 170:445-56). Strategies to prevent and treat ibr-relapsed patients by understanding mechanisms of resistance are critically needed and will support rational drug development and therapeutic approaches. Recent studies including ours have provided some insights into ibr-resistance. Both BTK and PLCG2 mutations have been shown to confer ibr-resistance (NEJM 2014; 370:2352-54, NEJM 2014; 370:2286-94). Additionally, TRAIL-R has also been associated with the drug resistance in several ibr-relapsed patients (Nat Comm 2016). However, there is still limited understanding in the setting of disease progression during ibr treatment, such as what risk factors predispose patients to relapse, whether other molecular or cytogenetic lesions are associated with disease progression, and how RT is related to CLL tumor cells in the blood. Materials & Methods To address these questions, we analyzed 9 CLL patients treated with ibr and relapsed at the University of Chicago between 2008-2016. Among 9 patients, 6 developed RT at progression. The median age was 66.3 yrs (range, 52-88) and the median number of therapies prior to ibr initiation was 2 (range, 1- 4). The median duration of response to ibr was 16 months (range, 2-30). Eight patients discontinued ibr therapy due to CLL progression or RT. Longitudinal samples including peripheral blood (PB), bone marrow (BM) and tissue were collected from each patient at time points prior to ibr initiation (pre-ibr), post-relapse and at the time of RT. When possible, samples were also collected during the responding phase. Samples were analyzed using both UCM-OncoPlus, a hybrid capture 1,212 cancer-associated Next generation sequencing (NGS) gene panel and Affymetrix SNP arrays (CytoScan and OncoScan) to assess mutations, indels, copy number variations (CNVs) and loss of heterozygosity. A custom algorithm was developed to calculate mutant clonal frequencies (MCFs) using an integrative approach combining allelic frequencies, tumor purity and CNV data. K-means clustering was performed to identify gene mutations belonging to the same clonal populations. An amplicon-based 17-gene CLL panel was further used to sequence BTK in greater depth (~10,000x) to confirm the presence of minor clones (1-5%) in some samples. Results & Conclusions To determine the risk factors associated with relapse, we compared all pre-ibr samples for recurrent molecular and cytogenetic abnormalities. Eight of nine patients were found to have TP53 mutations and/or del (17p), several other genetic lesions also seem to be enriched in our cohort compared to larger populations of treated or untreated CLL patients. The mutation profiles of matched pre-ibr and relapsed CLL PB/BM for each patient were then compared to identify relapse-specific alterations that might drive CLL progression. As expected, BTK mutations were found in 5 of 9 patients including previously reported C481S/R as well as a structurally novel T316A located in the SH2 domain. The findings confirm that mutated BTK is the most common mechanism responsible for disease progression on ibr. Furthermore, the emergence of minor BTK clones was detected in progressed patients. Analyses are ongoing to determine the clonal relationship between CLL leukemia in PB/BM and large cell transformation at the tissue site. So far, IGH gene rearrangement assay on three of five patients demonstrated that the CLL and RT are of the same B-cell origin. We are also in the process of performing cluster analyses to understand mutations that travel in the same malignant clones or sub-clones in each patient and updated results will be presented. Disclosures Smith: Celgene: Consultancy; Amgen: Other: Educational lecture to sales force; Genentech: Consultancy, Other: on a DSMB for two trials ; AbbVie: Consultancy; TGTX: Consultancy; Gilead: Consultancy; Portola: Consultancy; Pharmacyclics: Consultancy; Juno: Consultancy. Wang:Portola Pharmaceuticals: Honoraria, Research Funding.

Description: Background: Burkitt lymphoma typically involves the bone marrow in advanced stage disease. Pure leukemic presentation of Burkitt lymphoma (pure Burkitt leukemia) with negative radiographic findings is uncommon but considered clinically as stage IV disease. The WHO classification clearly distinguishes Burkitt leukemia (formerly classified as FAB-L3) from B lymphoblastic leukemia, as the former has a mature B-cell phenotype (expresses surface immunoglobulin) and characteristically harbors a MYC translocation with an immunoglobulin partner. There is limited prognostic information on patients with pure Burkitt leukemia (PBL) because they are typically combined with Burkitt lymphoma patients with widespread disease (WBL). Methods: We performed a multicenter retrospective analysis of newly-diagnosed Burkitt lymphoma cases involving the bone marrow at four academic medical centers from 2000-2013. Inclusion criteria included availability of clinical information and follow-up data, MYC rearrangement, and treatment with intensive chemotherapy (Hyper-CVAD or ALL-like regimens). Pediatric patients were excluded (age 〈 21 years). Diagnosis was established by the institutions' expert hematopathologist in conjunction with cytogenetic, flow cytometric, and radiographic findings. Patients were placed in the pure Burkitt leukemia (PBL) group if they had bone marrow involvement but were otherwise radiographically negative (i.e. PET negative). Kaplan-Meier analysis was used to examine the difference in overall survival between the WBL and PBL groups. We also determined prognostic factors associated with survival in univariate and multivariate Cox regression analyses. Results: We identified 51 patients with bone marrow involvement by Burkitt lymphoma. Of these patients, 24 were excluded because of a lack of clinical or follow-up information (15), pediatric age group (7), no treatment initiated (1), or misdiagnosed and given inadequate treatment (1). Of the 27 patients that met our inclusion criteria, 11 patients had pure Burkitt leukemia (PBL) and 16 had tissue involvement with bone marrow disease (WBL). The male-to-female ratio in the PBL group was 4.5:1 and in the WBL 4:1 (p=0.23). The median age for the PBL was 46 years vs 47 years in the WBL (p=0.34). CSF involvement was similar in both groups (30% PBL vs. 38.5% WBL; p=0.14); with elevated LDH levels in all patients in both groups (PBL median 3399 IU/L, WBL 2550 IU/L; p=0.81). In general, the WBL group had more complex karyotypes and a significantly greater number of cases with chromosome 1q abnormalities (5/7, 71%) compared to the PBL group (1/5, 20%). In the survival analysis, patients with PBL exhibited significantly better survival with a 5-year-overall survival of 79.5% (95% CI: 57.7-100%) vs. 18.4% (95% CI: 5.3-63.3%) in the WBL group (p=0.002). Conclusion: To the best of our knowledge, this is the largest series comparing adults with pure Burkitt leukemia and Burkitt lymphoma with widespread disease. Within the limitations of a retrospective analysis, the 5-year overall survival for the widespread Burkitt lymphoma (WBL) group is inferior compared to pure Burkitt leukemia (PBL) treated with intensive chemotherapy. Interestingly, the WBL group had more complex karyotypes compared to the PBL group. Figure 1. Overall Survival of Burkitt Leukemia versus Widespread Burkitt Lymphoma (BL). Figure 1. Overall Survival of Burkitt Leukemia versus Widespread Burkitt Lymphoma (BL). Disclosures No relevant conflicts of interest to declare.

Description: Background: Programmed death - ligand 1 (PD-L1) expression is a dominant immune escape mechanism across human cancers. Ubiquitous copy gains of chromosome 9p24.1 - a region containing the PD-L1 and PD-L2 loci - lead to PD-L1 expression on Hodgkin-Reed-Sternberg cells in classical Hodgkin lymphoma. PD-L1 gene alterations also occur in diffuse large B cell lymphoma (DLBCL), albeit less commonly. Hypothesizing that PD-L1 gene alterations identify DLBCLs in which potent anti-lymphoma immune responses have been activated, we aimed to characterize the immune landscape of PD-L1 gene-altered DLBCLs, describe the key clinical features of these patients, and determine the degree to which PD-L1 gene alterations predict for response to PD-1 blockade therapy in DLBCL. Methods: FISH was performed on 105 formalin-fixed, paraffin-embedded (FFPE) DLBCL specimens with DNA probes to PD-L1, a region centromeric to PD-L2, and centromere 9. Lymphoma-infiltrating T cell numbers (68 cases), HLA class I/II expression (74 cases), and PD-L1 protein expression (93 cases) were assessed by IHC. RNA sequencing (seq) was performed on FFPE samples utilizing the Illumina HiSeq 2000 platform. Differentially-expressed genes were identified between PD-L1-altered and not altered DLBCLs using a false discovery rate (FDR) of 0.05 and log2 fold change of 〉1.5. Whole exome seq (WES) was performed on DNA from DLBCL specimens and 22 matched blood samples using the Illumina HiSeq exome kit in collaboration with Theragen Etex Bio Institute. Results: PD-L1 gene alterations were identified by FISH in 28/105 samples (27% - 16% relative PD-L1 copy gains, 7% PD-L1 amplifications, 2% PD-L1 translocations, 2% chromosome 9 polysomy), were enriched in non-germinal center (GC) DLBCLs (75% non-GC vs 25% GC; p=0.001), and were associated with robust PD-L1 protein expression (Fig 1A-B). PD-L1 gene-altered DLBCLs were more heavily infiltrated by CD8+ T cells compared to PD-L1 not altered DLBCLs (22.6 vs 13.8 CD8+ T cells/hpf; p

Description: We investigated age-related EBV+ B-cell lymphoproliferations in the Western population. The clinical features, histology, immunophenotype, EBV-encoded RNA in situ hybridization, and clonality by PCR of T-cell receptor gamma and immunoglobulin genes were categorized in 122 EBV+ lesions as follows: (1) reactive lymphoid hyperplasia; (2) polymorphic extranodal or (3) polymorphic nodal lymphoproliferative disease (LPD); and (4) diffuse large B-cell lymphoma (DLBCL). Interphase FISH for IG and PAX5 gene rearrangements was performed on 17 cases of DLBCL. The overall median age was 75 years (range, 45-101 years; 67 men, 55 women), and 67, 79, 73, and 77 years, respectively, for groups 1 through 4. Sixteen of 21 cases of polymorphic extranodal LPD were classified as EBV+ mucocutaneous ulcer. PCR for immunoglobulin genes was polyclonal in reactive lymphoid hyperplasia (84%) and monoclonal in 33%, 63%, and 56% of polymorphic extranodal and nodal LPD cases and DLBCL, respectively. All groups showed restricted/clonal T-cell receptor responses (27%-70%). By FISH, 19% of DLBCLs showed IGH@ rearrangements, but PAX5 was unaffected. Disease-specific 5-year survival was 100%, 93%, 57%, and 25% for groups 1-4, respectively, and 100% for patients with EBV+ mucocutaneous ulcer. Disease volume was predictive of therapy response (P = .0002), and pathologic subtype was predictive of overall outcome (P = .001). Age-related EBV+ B-cell LPD encompasses a wider disease spectrum than previously recognized and includes both reactive and neoplastic conditions. Reduction in the T-cell repertoire may contribute to decreased immune surveillance.

Description: Background: B-cell receptor (BCR) signaling pathway is recognized as a crucial pathway for the pathogenesis of neoplastic B-cells. Inhibition of the BCR signaling and the downstream pathway is highly effective in B-cell malignancy through Bruton tyrosine kinase inhibition by ibrutinib. In addition to cell proliferation inhibition, ibrutinib disrupts cell adhesion between tumor and its microenvironment through unknown molecular mechanisms, resulting in peripheral lymphocytosis with accompanying lymphadenopathy reduction in patients who receive ibrutinib. Methods and materials: In an effort to elucidate the link between BCR signaling and cell adhesion phenotype, we first characterized ibrutinib sensitive and resistant mantle cell lymphoma (MCL) cell lines. We measured cell proliferation and cell growth, and correlated ibrutinib sensitivity with cell adhesion disruption. We then used RNA-sequencing to identify differential pathways between sensitive or resistant cell lines in response to ibrutinib treatment. We validated RNA-Seq findings using cell lines, as well as animal models and human primary MCL tumor tissues and cells. Results: We found that intrinsic sensitivities of MCL cell lines to ibrutinib correlated well with their cell adhesion phenotype. RNA-sequencing revealed that BCR and cell adhesion gene signatures were simultaneously down-regulated by ibrutinib in ibrutinib-sensitive but not ibrutinib-resistant cell lines. Among the differentially expressed genes in the BCR gene signature, we identified and validated that RAC2, a regulator of cell adhesion, was down-regulated at both RNA and protein levels by ibrutinib only in ibrutinib-sensitive cells. Physical association of RAC2 with BLNK, an early BCR pathway adaptor, was disrupted by ibrutinib uniquely in sensitive cells. RAC2 knockdown with siRNA impaired cell adhesion while RAC2 over-expression rescued ibrutinib-induced reduction in cell adhesion. In a xenograft mouse model, mice treated with ibrutinib demonstrated tumor growth retardation along with down-regulation in RAC2 protein expression. Using immunohistochemical staining, we demonstrated that RAC2 was expressed in ~65% primary MCL tumor tissues with majority of RAC2-positive tumors characterized as being the more aggressive subtypes. Finally, primary MCL cells treated with ibrutinib demonstrated reduced RAC2 that is accompanied by cell adhesion impairment. Conclusions: Our findings uncover a novel cross-talk between BCR signaling and cell adhesion. Ibrutinib inhibits cell adhesion via down-regulation of RAC2. Our study highlights the importance of RAC2 and cell adhesion in MCL pathogenesis and new drug development. Disclosures Wang: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Research Funding; AstraZeneca: Consultancy, Research Funding; MoreHealth: Consultancy; Pharmacyclics: Honoraria, Research Funding; Novartis: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Research Funding; Acerta Pharma: Honoraria, Research Funding.

Description: Programmed death-ligand 1 (PD-L1) expression on malignant cells is a dominant immune escape mechanism across a variety of human cancers. A unique genetic mechanism underlying PD-L1 upregulation has been uncovered in classical Hodgkin lymphoma (cHL), in which copy gains of the chromosomal region (9p24.1) containing the programmed death-1 (PD-1) ligands PD-L1 and PD-L2 are recurrently observed. While chromosome 9p24.1 copy-number alterations are ubiquitous in cHL, they also occur in diffuse large B-cell lymphoma (DLBCL), albeit with a lower incidence. Here, fluorescence in situ hybridization was used to identify DLBCLs harboring PD-L1 gene alterations, thereby enabling a characterization of the immunogenomic landscape of these lymphomas. Among 105 DLBCL cases analyzed, PD-L1 alterations were identified in 27%. PD-L1 alterations were highly enriched among non–germinal center DLBCLs and exhibited robust PD-L1 protein expression. These lymphomas were heavily infiltrated by clonally restricted T cells and frequently downregulated human leukocyte antigen expression. RNA sequencing of PD-L1–altered DLBCLs revealed upregulation of genes involved in negative T-cell regulation and NF-κB pathway activation, while whole-exome sequencing identified frequent mutations in genes involved in antigen presentation and T-cell costimulation. Many of these findings were validated in a large external data set. Interestingly, DLBCL patients with PD-L1 alterations had inferior progression-free survival following front-line chemoimmunotherapy; however, in the relapsed/refractory setting, PD-L1 alterations were associated with response to anti-PD-1 therapy. Collectively, our results indicate that PD-L1 alterations identify a unique biological subset of DLBCL in which an endogenous antilymphoma immune response has been activated, and that is associated with responsiveness to PD-1 blockade therapy.