ALBERT

Description: Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using FFPE-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 was significantly increased in RT-LN compared to CLL-LN. (mean ± SEM in RT vs. CLL, 30.6 ± 4.7 vs. 11.5 ± 2.8, p 〈 0.001). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line 'Karpas299'. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response. Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. Disclosures Parikh: Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

Description: Background: CLL patients (pts) treated with novel targeted therapies continue to experience disease progression. Approximately 7-15% of relapsed CLL treated with ibrutinib or venetoclax developed Richter's syndrome (RS), an aggressive high grade lymphoma transformation. Standard chemotherapy has limited efficacy in RS. We hypothesized that checkpoint blockade would re-establish anti-tumor immune response in progressive CLL/RS and here we aim to update the clinical data and correlative analysis of MC1485: a phase 2 study of PD-1 blocking antibody pembrolizumab in relapsed/refractory CLL and RS. Methods: Relapsed/refractory CLL including RS enrolled in the CLL arm of MC1485 trial was reported here. The primary end point of this study is overall response rate (ORR). Pts with prior allogeneic stem cell transplant were excluded. Pembrolizumab 200 mg was administered intravenously every 3 weeks. NCI CTCAE v4.0 and IWCLL 2008 criteria were used for non-hematological adverse events (AE) and for CLL grade hematological AE. Tumor expression of PD-1 and PD-L1 were assessed with standard immunohistochemical (IHC) staining. Percentage expressions of individual antigens were analyzed with whole-slide scanning followed by image analysis with Image-Pro software (Media Cybernetics). Fluorescence in situ hybridization (FISH) was performed on available CLL/RS tissue sections to assess copy number of chromosome 9p region that contains PD-L1 and PD-L2. Results: 25 pts including 16 CLL and 9 RS (biopsy-proven large cell lymphoma) were enrolled in CLL arm. The median age was 69 years (46-81). Twelve (48%) pts had del(17p) or monosomy 17 or TP53 mutation. The median number of prior therapies was 4 (1-10). 96%, 72% or 48% of pts had received alkylator and anti-CD20 antibody, purine analog or anthracycline. Fifteen (60%) pts had prior ibrutinib and 12 (48%) progressed clinically while receiving ibrutinib, including 6 who progressed to RS and 6 who developed progressive CLL. The median number of pembrolizumab doses that pts received was 3 (1 to 21), administered over a median treatment duration of 11 weeks (1 to 56). Drug-related AE occurred in 21 pts (88%) with the most common ones being neutropenia in 9 (37%), cough in 7 (29%) and dyspnea in 6 (25%) pts. Drug-related grade 3 or above AE occurred in 9 (38%) pts with most common ones being thrombocytopenia (5, 21%), dyspnea (2, 8%), and fatigue (2, 8%). The most common immune-related AE was liver enzyme elevation (12%, 8% G3) and was reversible with therapy interruption or steroid therapy. Based on investigator assessment using the Revised Response Criteria for Lymphomas and IWCLL 2008 criteria, one RS pt had complete response (CR, 4%), 3 RS had partial responses (PR, 12%). Among 9 RS pts, 1 CR (11%), 3 PR (33%), 3 SD (33%) and 2 PD (22%) were observed. Thus the ORR in RS was 44%. No CLL pts achieved a CR/PR, 3 had SD and 9 had progressive disease (PD). The ORR of all pts was 16%. Of the 4 RS who had CR/PR, responses occurred early after 2 cycles of therapy and median duration of therapy for CR/PR pts was 8.3 months with data cut-off by June 30th 2016 (Figure 1A). The causes for therapy discontinuation in RS were PD (3, 33%), alternate therapy (2, 22%), and thrombocytopenia due to increased marrow CLL (1, 11%). Increased marrow CLL was observed in 2 RS pts with PR to RS phase of disease assessed by PET, thus the protocol was amended to allow addition of idelalisib/ibrutinib to control the underlying marrow CLL. For RS pts who had a CR/PR or SD, variable nodal responses were observed (Figure 1B). In particular, 6 RS who had prior ibrutinib experienced CR or PR or SD with nodal responses. After a median follow-up of 10.2 (1.9 to 16.1) months, the median overall survival (OS) for all pts was 10.7 months (95% confidence interval [CI], 5.4 to not reached). The 6 month OS rate for RS and CLL pts were 73% and 59%, respectively. Biomarker assessment using IHC analysis on 10 pts with available tumor/nodal tissues (6 RS and 4 CLL) showed an increased expression of PD-L1 (p = 0.02) and a trend for an increased expression of PD-1 (p = 0.1) in the group of pts with CR/ PR versus pts in the group with no clinical response. One out of 10 tested showed polysomic for chromosome 9p. Conclusion: Pembrolizumab had an acceptable safety profile in CLL and RS patients. We confirmed that pembrolizumab has substantial therapeutic activity in RS. Single-agent pembrolizumab does not appear to have clear activity in CLL. Disclosures Ding: Merck: Research Funding. Parikh:Pharmacyclics: Honoraria, Research Funding. Shanafelt:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding; Cephalon: Research Funding; Hospira: Research Funding.

Description: Anaplastic large cell lymphomas (ALCLs) represent a relatively common group of T-cell non-Hodgkin lymphomas (T-NHLs) that are unified by similar pathologic features but demonstrate marked genetic heterogeneity. ALCLs are broadly classified as being anaplastic lymphoma kinase (ALK)+ or ALK−, based on the presence or absence of ALK rearrangements. Exome sequencing of 62 T-NHLs identified a previously unreported recurrent mutation in the musculin gene, MSCE116K, exclusively in ALK− ALCLs. Additional sequencing for a total of 238 T-NHLs confirmed the specificity of MSCE116K for ALK− ALCL and further demonstrated that 14 of 15 mutated cases (93%) had coexisting DUSP22 rearrangements. Musculin is a basic helix-loop-helix (bHLH) transcription factor that heterodimerizes with other bHLH proteins to regulate lymphocyte development. The E116K mutation localized to the DNA binding domain of musculin and permitted formation of musculin–bHLH heterodimers but prevented their binding to authentic target sequence. Functional analysis showed MSCE116K acted in a dominant-negative fashion, reversing wild-type musculin-induced repression of MYC and cell cycle inhibition. Chromatin immunoprecipitation–sequencing and transcriptome analysis identified the cell cycle regulatory gene E2F2 as a direct transcriptional target of musculin. MSCE116K reversed E2F2-induced cell cycle arrest and promoted expression of the CD30–IRF4–MYC axis, whereas its expression was reciprocally induced by binding of IRF4 to the MSC promoter. Finally, ALCL cells expressing MSCE116K were preferentially targeted by the BET inhibitor JQ1. These findings identify a novel recurrent MSC mutation as a key driver of the CD30–IRF4–MYC axis and cell cycle progression in a unique subset of ALCLs.

Description: Key Points Pembrolizumab was first shown to be clinically active in CLL patients with RT. PD-1 and PD-L1 expression in tumor microenvironment are promising biomarkers to select RT patients for PD-1 blockade.

Description: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. It has a strong genetic basis, showing a ~ 8-fold increased risk of CLL in first-degree relatives. Genome-wide association studies (GWAS) have identified 41 risk variants across 41 loci. However, for a majority of the loci, the functional variants and the mechanisms underlying their causal roles remain undefined. Here, we examined the genetic and epigenetic features associated with 12 index variants, along with any correlated (r2 ≥ 0.5) variants, at the CLL risk loci located outside of gene promoters. Based on publicly available ChIP-seq and chromatin accessibility data as well as our own ChIP-seq data from CLL patients, we identified six candidate functional variants at six loci and at least two candidate functional variants at each of the remaining six loci. The functional variants are predominantly located within enhancers or super-enhancers, including bi-directionally transcribed enhancers, which are often restricted to immune cell types. Furthermore, we found that, at 78% of the functional variants, the alternative alleles altered the transcription factor binding motifs or histone modifications, indicating the involvement of these variants in the change of local chromatin state. Finally, the enhancers carrying functional variants physically interacted with genes enriched in the type I interferon signaling pathway, apoptosis, or TP53 network that are known to play key roles in CLL. These results support the regulatory roles for inherited noncoding variants in the pathogenesis of CLL.

Description: Background The CLL international prognostic index (CLL-IPI) is a validated staging system for newly diagnosed CLL patients that combines age, clinical stage, serum Beta-2 microglobulin, TP53, and IGHV mutation status into a single score and stratifies CLL patients into four prognostic risk groups with a c-statistic=0.72. Next-generation sequencing identified ~60 putative driver genes recurrently mutated in CLL, and genome-wide association studies (GWAS) identified 41 single nucleotide polymorphisms (SNPs) associated with CLL risk. We previously showed that a polygenic risk score (PRS) of the weighted average of the number of risk alleles among these 41 SNPs is associated with risk of both CLL and MBL. We and others have also shown that the tumor mutational load (TML), i.e., the total number of somatically mutated driver genes, is associated with time to first treatment (TTT). Herein, we examine whether TML and PRS add prognostic value beyond CLL-IPI for disease progression in a cohort of CLL and high-count MBLs. Methods We used the Mayo Clinic CLL resource to identify newly diagnosed (0.01%, and by those identified in 1000 Genomes Project, ExAC and/or ESP6500 databases, unless present in known mutation hotspots or COSMIC. After filtering high/moderate impact mutations, we calculated the TML. Peripheral blood DNA was genotyped on Illumina Omni Express array; after extensive quality control metrics, we calculated the germline PRS as the weighted average of the number of risk alleles among the CLL susceptibility SNPs. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) to test for associations with TTT. We analyzed the low/intermediate risk groups and the high/very high risk groups in stratified analyses and tested for interactions between CLL-IPI and TML or PRS. Survival curves are displayed using the Kaplan-Meier method using P-values from the log-rank test; the PRS is displayed by tertile categories. Results Of 166 patients (120 CLL, 46 MBL), 67% were male and the median age at diagnosis was 62 years (range: 28-85). The median follow-up was 5.3 years, and 73 were treated for progressive disease as defined by the 2018 IWCLL criteria. Low risk CLL-IPI comprised 51%, intermediate risk 27%, high risk 18%, and very-high risk 4%. The most commonly somatically mutated genes were TP53 (8%), CHD2 (8%), BIRC3 (8%), SF3B1 (7%), and NOTCH1 (7%). We found that 42%, 32%, and 26% of patients had 0, 1, or 2+ genes with somatic mutations, respectively. The median PRS was 8.3 (range: 6.1-11.3) which is consistent with our prior PRS study on CLL/MBL and higher than 7.53 found in 7983 controls from our prior work. When we modeled TML, PRS, sex, CLL/MBL status and CLL-IPI together with the joint effects of CLL-IPI with TML and PRS, both interactions were significant in the model (P=0.008 for TML*CLL-IPI and P=0.014 for PRS*CLL-IPI), with c-statistic=0.81 (CI:0.77-0.85). When we stratified by CLL-IPI, TML was associated with shorter TTT in the low/intermediate risk (N=129, continuous HR=2.36 per TML category, CI: 1.61-3.47, P=1.3x10-5, Figure 1.A), but there was no evidence of an association in the high/very-high risk (N=37, continuous HR=1.12 per TML category, CI: 0.70-1. 78, P=0.64, Figure 1.B), after adjusting for PRS, sex, and CLL/MBL status. However, the PRS was associated with a shorter TTT in the high/very-high risk (continuous HR=1.77 per SD, CI: 1.00-3.12, P=0.048, Figure 1.D), with no evidence of an association in the low/intermediate risk (continuous HR=0.95 per SD, CI: 0.95-1.29, P=0.74, Figure 1.C), after adjusting for TML, sex, and CLL/MBL status. Conclusions In our study, TML was prognostic among the low/intermediate CLL-IPI risk groups while the PRS was prognostic among the high/very-high CLL-IPI risk groups. Sequencing CLL driver genes and genotyping relevant germline SNPs at time of diagnosis may provide incremental value over the CLL-IPI in predicting TTT among patients with CLL/MBL. Disclosures Parikh: MorphoSys: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Research Funding; Ascentage Pharma: Research Funding; Acerta Pharma: Research Funding. Ding:Merck: Research Funding; DTRM Biopharma: Research Funding. Cerhan:Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB.

Description: Introduction Despite continuous improvement of clinical outcome in multiple myeloma (MM), disease relapse remains a major challenge, leading to progressively shorter remissions and fewer treatment options. Strategies attempting to counteract this challenge include recent efforts resulting in an increase in the availability of novel promising anti-MM agents and targeting specific genetic profiles of the disease. In this context, we aim to develop predictive models of sensitivity and resistance to novel compounds by connecting an ex vivo high-throughput drug screen with genetic, transcriptomics, FISH, and clinical features. Methods Twenty compounds (afatinib, afuresertib, belinostat, buparlisib, cobimetinib, CPI-0610, crenolanib, dinaciclib, dovitinib, JQ1, LGH447, osimertinib, OTX015, panobinostat, romidepsin, selinexor, sunitinib, trametinib, venetoclax, and vorinostat) were selected based on overall promising anti-MM activity from an ex vivo high throughput drug screen with a panel of 79 single agents incubated for 24 hours. The area under the curve (AUC) was used to rank order the ex vivo responses for each compound and the lowest and highest quartile samples were identified for further analysis. Clinical data and FISH data, including t(11;14), t(4;14), t(14;16), del(17p), +1q, monosomy 13, and MYC rearrangement, were collected. Targeted DNA sequencing was performed using a 2.3 Mb custom capture panel covering 139 MM-relevant genes. mRNA-sequencing was performed and differential gene expression analysis in the highest and lowest quartile identified subsets of markers positively and negatively associated with the AUC response for a given compound. An additional unbiased selection of markers using lasso techniques was performed, resulting in predictive generalized linear models (GLM) for each agent. Responses from the remaining intermediate samples were estimated with the predictive models, with overall predictive ability assessed by correlating predicted AUCs with their actual counterparts. Results Our integrative analysis was performed on 50 primary patient samples (36% untreated and 64% relapsed MM). Venetoclax, dinaciclib, romidepsin, panobinostat, osimertinib, belinostat and selinexor were the most active compounds in the cohort. Interestingly, LGH447, dovitinib, selinexor, JQ1, OTX-015, cobimetinib, and trametinib showed increased activity in relapsed MM when compared to untreated samples (Wilcoxon Test; p0.7). Five (25%) of these compounds displayed a remarkably accurate prediction model in both training (highest and lowest quartiles) and validation (intermediate quartiles) samples (r〉0.8). Conclusions The GLM data integration approach enabled the establishment of effective predictive models, identifying FISH, transcriptomics, and mutations of putative driver genes important in anti-MM agent responsiveness. In addition, the resulting dataset is promising for future research focusing on the discovery of novel mechanisms of action and establishing markers of sensitivity and resistance to novel compounds. We are currently increasing our dataset and seek to create an omnibus approach that predicts responses to multiple anti-MM agents simultaneously. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Stewart:Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy; Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy.