Topic: 20. Lymphoma Biology & Translational Research Background: Tafasitamab (tafa), an anti-CD19 immunotherapy that enhances antibody dependent cellular cytotoxicity (ADCC) and phagocytosis, achieved a 57.5% objective response rate in combination with lenalidomide in the phase 2 L-MIND trial (NCT02399085), and received accelerated US approval and conditional authorization in Europe for treatment of patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) ineligible for autologous stem cell transplant. DLBCL is a heterogeneous disease with subtypes characterized by cell of origin (COO) and/or driver mutation signatures; these are associated with differential response to standard immunochemotherapy. Identification of biomarkers predictive of response and more efficacious combination therapies with tafa may provide further benefit to patients with limited or incomplete responses. Aims: We performed a comprehensive characterization of DLBCL cell lines and examined whether tafa activity is associated with COO, specific molecular subtypes, and patients’ immune fitness. Methods: An extensive literature review was performed to assign DLBCL cell lines to COO subtypes—activated B-cell like (ABC) or germinal center B-cell like (GCB). Genomic profiles of DLBCL cell lines were downloaded from DepMap version 22Q1. The weights for the COO model were computed using a 15-gene expression signature (Scott et al. Blood. 2014) on frozen tissue data from Lenz et al (N Engl J Med. 2008) to score each DepMap DLBCL cell line. Additionally, key mutations associated with DLBCL molecular subtypes (MCD, N1, A53, BN2, ST2, EZB) defined in Wright et al (Cancer Cell. 2020) were compiled from DepMap data. Sensitivity data on DLBCL cell lines screened against BTK inhibitors (AVL-292, acalabrutinib, ibrutinib) were obtained from the PRISM repurposing dataset. Results: Genomic profiles of DLBCL cell lines included gene expression (n=30), copy number (n=21), mutation (n=32), and fusion data (n=30). Cell lines were characterized as COO subtypes ABC (n=4), GCB (n=21), and unclassified (n=5). Although there was general agreement between COO signature and the literature, we noted inconsistencies in 2 DLBCL cell lines. Interestingly, with exception of OCI-Ly3, all profiled cell lines had features of multiple molecular subtypes. In vitro, tafa ADCC was independent of DLBCL subtypes in the cell lines evaluated, whereas BTK inhibitor activity was enriched in cell lines carrying genetic alterations associated with MCD and N1 subtypes. In addition, tafa activity did not depend on TP53 status. To extend COO analyses in cell lines and account for impact of patient immune fitness on response, we tested tafa activity in an ex vivo ADCC assay using peripheral NK cells collected at baseline from patients in the L-MIND study. No strong correlation between tafa-mediated ADCC and COO was observed, although NK cells from patients with GCB tumors demonstrated more activity in this assay. Interestingly, patients with higher peripheral NK cell counts at baseline had increased PFS, suggesting that patients’ immune fitness contributes to response to tafa treatment. Summary/Conclusion: Consistent with L-MIND clinical data, tafa was active in both GCB and non-GCB DLBCL cell lines. Additionally, tafa activity was not associated with a specific molecular subtype or mutations in TP53. Biomarker analyses are ongoing in tafa clinical trials (topMIND [NCT04809467], frontMIND [NCT04824092], firmMIND [NCT05429268]) to confirm these findings and provide further insight into genomic and immune parameters that may play a role in response to tafa. Keywords: B cell lymphoma, Immunotherapy, Cancer, CD19

ventricular outflow tract (Figure 1F) distant from the tricuspid annulus.Limited ablation performed at this site did not alter accessory pathway conduction.Intracardiac echocardiographic imaging (Figure 1B) revealed the closest atrial tissue to this site was in the right atrial appendage.With atrial activation mapping during right ventricular pacing, the earliest atrial activation was in the right atrial appendage (Figure 1D) at a distance of 25.5 mm from the earliest ventricular signal (Figure 1E).The ventricular and atrial signals at this site were fused (Figure 1D).With radiofrequency ablation using 25 W initially and up-titrated to 40 W, ventricular pre-excitation was eliminated.The local EGM showed the initial fused atrial and ventricular signals had separated.After a waiting period greater than 30 minutes no further pre-excitation was observed.There was no VA conduction present after pathway elimination.Adenosine was given and resulted in complete AV block.Final ECG showed no preexcitation (Figure 1C).At one month follow up he had no recurrence of palpitations or tachycardia.Conclusion: Non-annular accessory pathways are rare in clinical practice.Of the limited case reports on right atrial appendage to right ventricle accessory pathways in structurally normal hearts, most had prior failed ablation attempts.We suspect this is likely due to under recognition of non-annular pathways.

Patients with tetralogy of Fallot (ToF) have an increased risk of reentrant ventricular arrhythmias (VAs) utilizing slowly conducting anatomic isthmuses (SCAIs). Isochronal late activation mapping (ILAM) is an alternative mapping technique that identifies deceleration zones (DZ) during sinus and paced rhythms. These areas have been shown to be candidate sites for reentrant ventricular arrhythmias.

Mantle cell lymphoma (MCL) is a rare, incurable lymphoma subtype characterized by heterogeneous outcomes. To better understand the clinical behavior and response to treatment, predictive biomarkers are needed. Using residual archived material from patients enrolled in the MCL3001 (RAY) study, we performed detailed analyses of gene expression and targeted genetic sequencing. This phase III clinical trial randomized patients with relapsed or refractory MCL to treatment with either ibrutinib or temsirolimus. We confirmed the prognostic capability of the gene expression proliferation assay MCL35 in this cohort treated with novel agents; it outperformed the simplified MCL International Prognostic Index in discriminating patients with different outcomes. Regardless of treatment arm, our data demonstrated that this assay captures the risk conferred by known biological factors, including increased MYC expression, blastoid morphology, aberrations of TP53 , and truncated CCND1 3 ′ untranslated region. We showed the negative impact of BIRC3 mutations/deletions on outcomes in this cohort and identified that deletion of chromosome 8p23.3 also negatively impacts survival. Restricted to patients with deletions/alterations in TP53 , ibrutinib appeared to abrogate the deleterious impact on outcome. These data illustrate the potential to perform a molecular analysis of predictive biomarkers on routine patient samples that can meaningfully inform clinical practice.

We assessed the concordance between immunohistochemistry (IHC) and gene expression profiling (GEP) for determining diffuse large B-cell lymphoma (DLBCL) cell of origin (COO) in the phase III PHOENIX trial of rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) with or without ibrutinib. Among 910 of 1114 screened patients with non-germinal centre B cell-like (non-GCB) DLBCL by IHC, the concordance with GEP for non-GCB calls was 82·7%, with 691 (75·9%) identified as activated B cell-like (ABC), and 62 (6·8%) as unclassified. Among 746 of 837 enrolled patients with verified non-GCB DLBCL by IHC, the concordance with GEP was 82·8%, with 567 (76·0%) identified as ABC and 51 (6·8%) unclassified; survival outcomes were similar regardless of COO or treatment, whereas among patients with ABC DLBCL aged <60 years, the overall and event-free survival were substantially better with ibrutinib versus placebo plus R-CHOP [hazard ratio (HR) 0·365, 95% confidence interval (CI) 0·147-0·909, P = 0·0305; HR 0·561, 95% CI 0·326-0·967, P = 0·0348, respectively]. IHC and GEP showed high concordance and consistent survival outcomes among tested patients, indicating centralised IHC may be used to enrich populations for response to ibrutinib plus R-CHOP.

Abstract Background The single‐arm DAWN trial (NCT01779791) of ibrutinib monotherapy in patients with relapsed/refractory follicular lymphoma (FL) showed an overall response rate (ORR) of 20.9% and a median response duration of 19.4 months. This biomarker analysis of the DAWN dataset sought to determine genetic classifiers for prediction of response to ibrutinib treatment. Methods Whole exome sequencing was performed on baseline tumor samples. Potential germline variants were excluded; a custom set of 1216 cancer‐related genes was examined. Responder‐ versus nonresponder‐associated variants were identified using Fisher's exact test. Classifiers with increasing numbers of genes were created using a greedy algorithm that repeatedly selected genes, adding the most nonresponders to the existing “predicted nonresponders” set and were evaluated with 10‐fold cross‐validation. Results Exome data were generated from 88 patient samples and 13,554 somatic mutation variants were inferred. Response data were available for 83 patients (17 responders, 66 nonresponders). Each sample showed 100 to >500 mutated genes, with greater variance across nonresponders. The overall variant pattern was consistent with previous FL studies; 75 genes had mutations in >10% of patients, including genes previously reported as associated with FL. Univariate analysis yielded responder‐associated genes FANCA, HISTH1B, ANXA6, BTG1, and PARP10, highlighting the importance of functions outside of B‐cell receptor signaling, including epigenetic processes, DNA damage repair, cell cycle/proliferation, and cell motility/invasiveness. While nonresponder‐associated genes included well‐known TP53 and CARD11, genetic classifiers developed using nonresponder‐associated genes included ATP6AP1, EP400, ARID1A, SOCS1, and TBL1XR1, suggesting resistance to ibrutinib may be related to broad biological functions connected to epigenetic modification, telomere maintenance, and cancer‐associated signaling pathways (mTOR, JAK/STAT, NF‐κB). Conclusion The results from univariate and genetic classifier analyses provide insights into genes associated with response or resistance to ibrutinib in FL and identify a classifier developed using nonresponder‐associated genes, which warrants further investigation. Trial registration: NCT01779791.

AbstractTo advance the use of circulating tumor DNA (ctDNA) applications, their broad clinical validity must be tested in different treatment settings, including targeted therapies. Using the prespecified longitudinal systematic collection of plasma samples in the phase 1/2a LYM1002 trial (registered on www.clinicaltrials.gov as NCT02329847), we tested the clinical validity of ctDNA for baseline mutation profiling, residual tumor load quantification, and acquisition of resistance mutations in patients with lymphoma treated with ibrutinib+nivolumab. Inclusion criterion for this ancillary biological study was the availability of blood collected at baseline and cycle 3, day 1. Overall, 172 ctDNA samples from 67 patients were analyzed by the LyV4.0 ctDNA Cancer Personalized Profiling Deep Sequencing Assay. Among baseline variants in ctDNA, only TP53 mutations (detected in 25.4% of patients) were associated with shorter progression-free survival; clones harboring baseline TP53 mutations did not disappear during treatment. Molecular response, defined as a >2-log reduction in ctDNA levels after 2 cycles of therapy (28 days), was achieved in 28.6% of patients with relapsed diffuse large B-cell lymphoma who had ≥1 baseline variant and was associated with best response and improved progression-free survival. Clonal evolution occurred frequently during treatment, and 10.3% new mutations were identified after 2 treatment cycles in nonresponders. PLCG2 was the topmost among genes that acquired new mutations. No patients acquired the C481S BTK mutation implicated in resistance to ibrutinib in CLL. Collectively, our results provide the proof of concept that ctDNA is useful for noninvasive monitoring of lymphoma treated with targeted agents in the clinical trial setting.

INTRODUCTION:This unplanned post-hoc analysis was based on data from the phase Ib DBL1002 study (NCT01569750) and evaluated the association between molecular biomarkers and clinical response to combined treatment with ibrutinib plus rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in diffuse large B-cell lymphoma (DLBCL) subtypes. METHODS:DLBCL subtyping was conducted using immunohistochemistry. Next-generation sequencing using immunoglobulin H primers assessed minimal residual disease (MRD). A quantitative assay evaluated Bruton's tyrosine kinase (BTK) occupancy by ibrutinib in peripheral blood mononuclear cells. Targeted DNA sequencing examined genetic variants by DLBCL subtype. Secreted protein expression was evaluated with a SomaLogic analyte panel. RESULTS:Among 21 patients with DLBCL (median age 53.5 years), 17 achieved a complete response (CR) and 4 a partial response (PR). Of the 11 subtyped patients, 9 had a CR (5/7 germinal center B-cell-like [GCB] and 4/4 non-GCB) and 2 had a PR (both GCB). Nine of 12 patients tested for MRD achieved early (cycle 2 day 1) MRD negativity; most had a CR. There was near-complete BTK occupancy at 4 h postdose. Mutation analysis (n = 19) revealed variants including CREBBP, KMT2D, LRP1B, BCL2, and TNFRSF14; only 1 CD79B and TP53 each; no CARD11 or MYD88. CONCLUSIONS:In this study, first-line ibrutinib plus R-CHOP benefited patients with DLBCL, with good overall response rate and early MRD negativity. With a caveat of small sample size, our results showed that a favorable genetic profile and younger patient age may be important to beneficial clinical outcome with ibrutinib plus R-CHOP in DLBCL.

Background Cancer associated copy number variation (CNV) events provide important information for identifying patient subgroups and suggesting treatment strategies. Technical and logistical issues, however, make it challenging to accurately detect abnormal copy number events in a cost-effective manner in clinical studies. Results Here we present CNV Radar, a software tool that utilizes next-generation sequencing read depth information and variant allele frequency patterns, to infer the true copy number status of genes and genomic regions from whole exome sequencing data. Evaluation of CNV Radar in a public multiple myeloma dataset demonstrated that CNV Radar was able to detect a variety of CNVs associated with risk of progression, and we observed > 70% concordance with fluorescence in situ hybridization (FISH) results. Compared to other CNV callers, CNV Radar showed high sensitivity and specificity. Similar results were observed when comparing CNV Radar calls to single nucleotide polymorphism array results from acute myeloid leukemia and prostate cancer datasets available on TCGA. More importantly, CNV Radar demonstrated its utility in the clinical trial setting: in POLLUX and CASTOR, two phase 3 studies in patients with relapsed or refractory multiple myeloma, we observed a high concordance rate with FISH for del17p, a risk defining CNV event (88% in POLLUX and 90% in CASTOR), therefore allowing for efficacy assessments in clinically relevant disease subgroups. Our case studies also showed that CNV Radar is capable of detecting abnormalities such as copy-neutral loss of heterozygosity that elude other approaches. Conclusions We demonstrated that CNV Radar is more sensitive than other CNV detection methods, accurately detects clinically important cytogenetic events, and allows for further interrogation of novel disease biology. Overall, CNV Radar exhibited high concordance with standard methods such as FISH, and its success in the POLLUX and CASTOR clinical trials demonstrated its potential utility for informing clinical and therapeutic decisions.

We analyzed potential biomarkers of response to ibrutinib plus nivolumab in biopsies from patients with diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and Richter's transformation (RT) from the LYM1002 phase I/IIa study, using programmed death ligand 1 (PD-L1) immunohistochemistry, whole exome sequencing (WES), and gene expression profiling (GEP). In DLBCL, PD-L1 elevation was more frequent in responders versus nonresponders (5/8 [62.5%] vs. 3/16 [18.8%]; p = 0.065; complete response 37.5% vs. 0%; p = 0.028). Overall response rates for patients with WES and GEP data, respectively, were: DLBCL (38.5% and 29.6%); FL (46.2% and 43.5%); RT (76.5% and 81.3%). In DLBCL, WES analyses demonstrated that mutations in RNF213 (40.0% vs. 6.2%; p = 0.055), KLHL14 (30.0% vs. 0%; p = 0.046), and LRP1B (30.0% vs. 6.2%; p = 0.264) were more frequent in responders. No responders had mutations in EBF1, ADAMTS20, AKAP9, TP53, MYD88, or TNFRSF14, while the frequency of these mutations in nonresponders ranged from 12.5% to 18.8%. In FL and RT, genes with different mutation frequencies in responders versus nonresponders were: BCL2 (75.0% vs. 28.6%; p = 0.047) and ROS1 (0% vs. 50.0%; p = 0.044), respectively. Per GEP, the most upregulated genes in responders were LEF1 and BTLA (overall), and CRTAM (germinal center B-cell–like DLBCL). Enriched pathways were related to immune activation in responders and resistance-associated proliferation/replication in nonresponders. This preliminary work may help to generate hypotheses regarding genetically defined subsets of DLBCL, FL, and RT patients most likely to benefit from ibrutinib plus nivolumab.

Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here we report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high-risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence indicates a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway can enhance histopathological grading and suggest immunoprevention strategies for intercepting the progression of PMLs to lung cancer.

A phase 1/2a study (LYM1002 [EudraCT 2014-005191-28]) of ibr (560 mg once daily) + nivo (3 mg/kg on a 14-day cycle) demonstrated acceptable safety and promising efficacy vs single-agent ibr in Richter’s transformation (RT), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL). We examined potential biomarkers of treatment (tx) response using archived biopsy samples. GEP was used for DLBCL subtyping and to assess proportions of 22 distinct immune cells. Exome data were generated from 72 formalin-fixed paraffin-embedded samples, and sequencing analysis was used to identify mutations in genes of interest and assess somatic mutation burden. The correlations of immune cell proportions and gene variants were evaluated by investigator-assessed responses in each histology and by ongoing responses in DLBCL patients (pts; progression-free survival [PFS] > 24 months, n = 7 vs not, n = 20). In pts with available GEP and response data, overall response rates were 29.6% (8/27) for DLBCL, 43.5% (10/23) for FL and 81.3% (13/16) for RT. Proportions of CD8 and follicular helper T cells, M1 macrophages, and resting dendritic cells were higher in DLBCL responders vs nonresponders, while the proportion of regulatory T cells was decreased. These subsets did not differ by response in FL although an increase of CD4 memory resting T-cells was noted in responders. The trend toward increased follicular helper T cell and resting dendritic cell proportions in DLBCL pts was also associated with longer survival (PFS > 24 months) vs not. Gene variant data and responder status were available for 26 pts with DLBCL, 26 with FL, and 17 with RT. Comparison between responders and nonresponders showed that DLBCL pts with RNF213 (4/10 [40.0%] vs 1/16 [6.2%]) and KLHL14 (3/10 [30.0%] vs 0/16) mutations were more likely to respond to ibr + nivo. Conversely, nonresponders were associated with variants in EBF1, ADAMTS20, AKAP9, SOCS1, TP53, and genes in BCR pathways such as TNFRSF14, MYD88, and NFKB1B. BCL2 mutation in FL (9/12 [75.0%] vs 4/14 [28.6%]) and ROS1 mutation in RT (0/13 vs 2/4 [50.0%]) were associated with response; both are involved in the NF-kB pathway. In DLBCL, the most frequent gene mutations were RNF213, NBPF1, and BCL2 in pts who had PFS > 24 months (3/7 [42.9%] each), and KMT2D (8/20 [40.0%]) and CSMD3 (8/20 [40.0%]) in pts who did not. Somatic mutation burden was lower in responders vs nonresponders, especially in germinal center B-cell-DLBCL, and in DLBCL pts with PFS > 24 months vs not. In conclusion, we report gene variations among DLBCL, FL, and RT pts associated with response or durable PFS with ibr + nivo. While ibr inhibits Bruton’s tyrosine kinase-dependent pathways, we identify alternative gene pathway variants that may impact tx outcomes. Immune cell infiltration into the microenvironment relates to differential tx response with this immune combination and is histology dependent. Citation Format: Brendan Hodkinson, Michael Schaffer, Joshua Brody, Wojciech Jurczak, Cecilia Carpio, Dina Ben-Yehuda, Irit Avivi, Rao Saleem, Muhit Özcan, John Alvarez, Rob Ceulemans, Nele Fourneau, Sriram Balasubramanian, Anas Younes. Phase 1/2a LYM1002 study of ibrutinib (ibr) + nivolumab (nivo): Exome and gene expression profiling (GEP) analyses by histology and responder status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4024.

Background Preclinical studies have shown synergistic antitumour effects between ibrutinib and immune-checkpoint blockade. The aim of this study was to assess the safety and activity of ibrutinib in combination with nivolumab in patients with relapsed or refractory B-cell malignant diseases. Methods We did a two-part, open-label, phase 1/2a study at 21 hospitals in Australia, Israel, Poland, Spain, Turkey, and the USA. The primary objective of part A (dose escalation) was to assess the safety of daily oral ibrutinib (420 mg or 560 mg) in combination with intravenous nivolumab (3 mg/kg every 2 weeks) to ascertain a recommended phase 2 dose in patients with relapsed or refractory high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma (del17p or del11q), follicular lymphoma, or diffuse large B-cell lymphoma. Dose optimisation was investigated using a modified toxicity probability interval design. The primary objective of the part B expansion phase was to establish the preliminary activity (the proportion of patients who achieved an overall response) of the combination of ibrutinib and nivolumab in four cohorts: relapsed or refractory high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma (del17p or del11q), follicular lymphoma, diffuse large B-cell lymphoma, and Richter's transformation. All participants who received at least one dose of treatment were included in the primary analysis and analyses were done by disease cohort. Findings Between March 12, 2015, and April 11, 2017, 144 patients were enrolled in the study. Three patients died before receiving study treatment; thus, 141 patients were included in the analysis, 14 in part A and 127 in part B. One dose-limiting toxicity (grade 3 hyperbilirubinaemia) was reported at the 420 mg dose in the diffuse large B-cell lymphoma cohort, which resolved after 5 days. The combination of ibrutinib and nivolumab led to overall responses in 22 (61%) of 36 patients with high-risk chronic lymphocytic leukaemia or small lymphocytic lymphoma, 13 (33%) of 40 patients with follicular lymphoma, 16 (36%) of 45 patients with diffuse large B-cell lymphoma, and 13 (65%) of 20 patients with Richter's transformation. The most common all-grade adverse events were diarrhoea (47 [33%] of 141 patients), neutropenia (44 [31%]), and fatigue (37 [26%]). 11 (8%) of 141 patients had adverse events leading to death; none were reported as drug-related. The most common grade 3-4 adverse events were neutropenia (40 [28%] of 141 patients) and anaemia (32 [23%]). The incidence of grade 3-4 neutropenia ranged from eight (18%) of 45 patients with diffuse large B-cell lymphoma to 19 (53%) of 36 patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma; incidence of grade 3-4 anaemia ranged from five (13%) of 40 patients with follicular lymphoma to seven (35%) of 20 patients with Richter's transformation. The most common serious adverse events included anaemia (six [4%] of 141 patients) and pneumonia (five [4%]). The most common grade 3-4 immune-related adverse events were rash (11 [8%] of 141 patients) and increased alanine aminotransferase (three [2%]). Interpretation The combination of ibrutinib and nivolumab had an acceptable safety profile and preliminary activity was similar to that reported with single-agent ibrutinib in chronic lymphocytic leukaemia or small lymphocytic lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma. The clinical response in patients with Richter's transformation was promising and supports further clinical assessment. Copyright (C) 2019 Elsevier Ltd. All rights reserved.

Introduction: Sequencing studies identified mutational drivers in diffuse large B cell lymphoma (DLBCL), capturing outcome difference in previously unrecognized patient subsets. However, the lack of routine-applicable genomic approaches limits translation of such information to the clinic. Currently, molecular prognostication consists in cell of origin (COO) determination by the Lymph2Cx NanoString assay. We recently developed two independent prognostic signatures incorporating genes reflecting the COO, the activation of pivotal oncogenic pathways, and the composition of tumor microenvironment (TME). We aimed this study at examining the prognostic strength of a model combining the performance of each signature and developing a comprehensive NanoString assay rapidly transferable to the clinic for prognostic purposes. Methods: The expression of the genes was measured by the NanoString nCounter Analysis System using customized probes for 73 genes, including 15 COO genes, 6 additional oncogenic genes (MYC, BCL-2, NFKBIA, PIK3CA, PTEN, STAT3), 47 TME genes, and 5 housekeeping genes. The analysis was performed on 175 newly diagnosed, nodal DLBCL, homogeneously selected from the RHDS0305 and DLCL04 trials. Patients had comparable clinical features and double-hit cases were excluded. Heatmaps, Kaplan–Meier survival estimator, tree-based survival model, and P values were produced by ‘R’ statistical software. Long-rank test was used to compare overall survival (OS) and progression-free survival (PFS) among groups. Multivariate analysis was constructed through the Cox proportional hazards regression model. Results: Based on the expression of the COO and oncogenic genes, a tree-based survival model stratified patients into subgroups showing significantly different survival, with MYC, BCL-2 and NFKBIA holding additional prognostic power based on their high (H) or low (L) expression. The TME panel identified a lower gene expression cluster (C3) with significantly worse survival than those at intermediate (C2) and higher expression (C1). Integration of COO-, TME-, and MYC/BCL-2/NFKBIA-based data produced a new survival risk categorization of DLBCL. The high-risk category, showing the worst outcomes, includes ABC/H/C1-2-3, ABC/L/C3 and GCB/H/C3 cases; the intermediate-risk category comprises ABC/L/C2, GCB/H or L/C1 and UN/H or L/C1 or C3 cases; whereas the low-risk category contains GCB/H or L/C2, GCB/L/C3, UN/H/C1 or C2 and UN/L/C2 or C3 cases, with longer survival. An unsupervised clustering analysis was also performed based on the expression of the entire 74-gene panel and stratified cases into four clusters with significantly different OS (p=0.011) and PFS (p=0.009). In particular, cluster 1 and 4 showed significantly worse survival than cluster 2 and 3 (Figure 1), and a multivariate Cox analysis indicated that the prognostic performance of the panel overcomes the IPI score. Finally, such model was also validated “in silico” using a gene expression profiling dataset (GSE10846 and GSE98588) relative to a cohort of 146 DLBCL patients uniformly selected according to R-CHOP treatment. Conclusions: This study supports the idea that DLBCL heterogeneity involves both tumor and TME, resulting in diverse transcriptional subtypes with distinct outcomes and, putatively, diverse biology. Our integrative analysis prompts the development of a new survival categorization outperforming current prognostic risk-assessment. Moreover, the applicability of a unique Nanostring-based assay to routine biopsies may facilitate the stratification of patients at diagnosis and their inclusion in future trials exploring novel therapeutic approaches.

Background: The cell of origin (COO) in diffuse large B-cell lymphoma (DLBCL) has prognostic importance. While the COO was originally classified into germinal center B-cell (GCB) or activated B-cell (ABC) subtypes by microarray analysis, routine use was not practical. Immunohistochemistry (IHC)–based methods are widely used with varying results due to lack of standardization. Several classification methods have been developed recently; understanding the concordance between these and existing methods is essential to their practical application. Therefore, we evaluated concordance between 3 commercial assays: a standardized Hans-based IHC method and 2 gene expression profiling (GEP) methods and compared these to the accepted microarray classification.Methods: 137 DLBCL-confirmed tumor samples were evaluated using a standardized Hans-based IHC method for GCB or non-GCB subtype, by a published microarray-based assay, a digital gene expression-based Lymphoma Subtyping Test (LST) and a next-generation sequencing-based assay (EdgeSeq COO). Subtype calls from the 3 GEP methods were harmonized to “GCB” or “non-GCB” and assessed for concordance.Results: Concordance between the Hans-based IHC assay and the microarray-based assay, LST assay and EdgeSeq assay was 79.6% (N=137), 80.0% (N=125) and 78.2% (N=64), respectively; the positive percent agreement (PPA) in non-GCB was 88.9%, 87.0% and 78.1%, respectively. Concordance for the Hans-based IHC assay versus GEP methods was especially high for direct GCB calls (91.0%, 88.3% and 78.1% for microarray, LST and EdgeSeq COO methods, respectively). The newly developed GEP assays performed well against the microarray GEP method, against which they were calibrated (concordance 93.7% and 87.5% and PPA 94.3% and 92.9%, respectively, for LST and EdgeSeq COO).Conclusion: These results demonstrated good consistency between various platforms for stratification of DLBCL into COO subtype classifications. Application of a standardized Hans-based IHC assay offers a robust, rapid and easily accessible platform to classify DLBCL into prognostically important subtypes.

Advances in Acute Myeloid Leukemia (AML) research have shown that the bone marrow microenvironment may distinctly vary across disease subtypes, and that this variation is associated with disease risk and response to conventional therapies. Novel therapies aimed at altering the tumor microenvironment, such as T-cell redirection, CAR-T and checkpoint inhibition, are emerging as promising treatment options for AML patients; however, there remains a critical need to determine how response to immune modulation may vary within different subsets of AML. Thus, in collaboration with the Beat AML Consortium, we carried out comprehensive mass cytometry profiling of patient bone marrow samples of nearly 100 Beat AML subjects and characterized their ex vivo response to several immune modulators.

Abstract Squamous cell carcinoma (SCC) of the lung is a leading cause of cancer mortality in the U.S. due to late-stage diagnosis and lack of effective treatments. Lung SCC arises in the epithelial layer of the bronchial airways and is often preceded by the development of premalignant lesions (PMLs). The molecular alterations involved in the progression of PMLs to lung SCC are not clearly understood as not all PMLs progress to carcinoma. We hypothesize that molecular characterization of PMLs and nonlesion areas will allow us to identify alterations associated with histology and lesion progression. We used mRNA sequencing to profile biopsies obtained from high-risk smokers undergoing lung cancer screening by auto-fluorescence bronchoscopy and CT at the Roswell Park Cancer Institute in Buffalo, NY. For each subject (n=49), a brushing of the airway field (normal fluorescing area) and endobronchial biopsies were collected over time in repeat locations with serial bronchoscopies. The discovery cohort, included 29 subjects, 197 biopsies, and 91 brushes, while the validation cohort included 20 subjects, 111 biopsies and 49 brushes. The mRNA-Seq data were aligned to hg19 using STAR, and gene/transcript levels were summarized using RSEM. Immune, stromal, and epithelial cell content were inferred using xCell. Biopsy molecular subtypes were discovered using consensus clustering in the discovery cohort and used to train a nearest centroid subtype predictor to assign subtypes in the validation cohort and the brushes. We identified four distinct molecular subtypes in the discovery cohort bronchial biopsies using genes (n=3936) co-expressed across the the discovery cohort brushes and biopsies and two additional RNA-seq lung SCC-related datasets. One of the four molecular subtypes is enriched (p<0.001) for samples with dysplasia histology, high basal cell content, and the classic SCC tumor subtype. These associations are replicated in the validation cohort. There is also significant concordance between the molecular subtypes defined in the biopsies and their corresponding brush from the normal-appearing airway. Genes associated with IFN-gamma signaling and T cell-mediated immunity were observed to be downregulated among lesions that remained stable or progressed to more severe histology within the high-grade subtype. Staining adjacent biopsies to those profiled revealed that decreased expression of genes related to T cell-mediated immunity is associated with decreased numbers of CD4+ and CD8+T cells within the lesions and the surrounding tissue. We have identified four molecular subclasses of premalignant lung SCC lesions that may associate with prognosis. Molecular classification of PMLs may lead to biomarkers of future disease progression that could be used to stratify patients into prevention trials and to monitor efficacy of the treatment. Additionally, the results suggest that personalized lung cancer chemoprevention that targets specific cancer-related pathways or the immune system may have potential therapeutic benefits. Citation Format: Jennifer E. Beane, Sarah Mazzilli, Ania Tassinari, Joshua Campbell, Christopher Moy, Michael Schaffer, Catalina Perdomo, David Jenkins, Mary Beth Pine, Gang Liu, Sherry Zhang, Hangqio Lin, Jessica Vick, Evan Johnson, Suso Platero, Christopher Stevenson, Marc Lenburg, Mary Reid, Samjot Dhillon, Avrum Spira. Bronchial premalignant lesions have distinct molecular subtypes associated with future histologic progression [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A05.

Matrix high-throughput screening (HTS) methods are increasingly employed to rapidly define potential therapeutic drug combinations. We used combination HTS to identify compounds showing synergistic anti-proliferative activity with ibrutinib, an irreversible, small-molecule inhibitor of Bruton's tyrosine kinase. The goal was to identify ibrutinib combinations with maximum synergistic effects in heme malignancy lines, particularly in non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL). Growth inhibition (GI) was used to measure cell viability; synergy scores characterized strength of synergistic interaction. Single-agent ibrutinib demonstrated varying degrees of activity across 30 cell lines evaluated. In DLBCL lines, TMD8 was the most sensitive to ibrutinib (GI(50)=0.001); combinations with BCL-2 inhibitor ABT-199, and PI3K inhibitors IPI-145 and GDC-0941 showed the strongest synergistic activity. Anti-proliferative synergies were also observed with BET bromodomain inhibitor (+)-JQ1, XPO1 inhibitor selinexor, and IRAK4 inhibitor, and confirmed using apoptosis assay. These findings are intended to inform and advance treatment of B-cell malignancies.

Purpose The Bruton's tyrosine kinase inhibitor ibrutinib has demonstrated clinical activity in B-cell malignancies. The DAWN study assessed the efficacy and safety of single-agent ibrutinib in chemoimmunotherapy relapsed/refractory follicular lymphoma (FL) patients. Methods DAWN was an open-label, single-arm, phase II study of ibrutinib in patients with FL with two or more prior lines of therapy. Patients received ibrutinib 560 mg daily until progressive disease/unacceptable toxicity. The primary objective was independent review committee-assessed overall response rate (ORR; complete response plus partial response). Exploratory analyses of T-cell subsets in peripheral blood (baseline/cycle 3) and cytokines/chemokines (baseline/cycle 2) were performed for available samples. Results Between March 2013 and May 2016, 110 patients with a median of three prior lines of therapy were enrolled. At median follow-up of 27.7 months, ORR was 20.9% (95% CI, 13.7% to 29.7%, which did not meet the 18% lower-bound threshold for the primary end point). Twelve patients achieved a complete response (11%; 95% CI, 5.8% to 18.3%). Median duration of response was 19.4 months (range, 1 to ≥ 33 months), with a median progression-free survival of 4.6 months and a 30-month overall survival of 61% (95% CI, 0.51% to 0.70%). Lymphoma symptoms resolved in 67%. Seven of 32 patients who experienced initial radiologic progression responded upon continuing therapy (pseudoprogression). The most common adverse events were diarrhea, fatigue, cough, and muscle spasms; 48.2% of patients reported serious adverse events. In patients who experienced a response, regulatory T cells were downregulated at C3D1 ( P = .02), and Th1-promoting (antitumor) cytokines interferon-γ and interleukin-12 increased ( P ≤ .035). Conclusion With an ORR of 20.9%, ibrutinib failed to meet its primary efficacy end point in chemoimmunotherapy in patients with relapsed/refractory FL, although responses were durable and associated with a reduction in regulatory T cells and increases in proinflammatory cytokines.