ALBERT

Description: Diffuse large B cell lymphoma (DLBCL) comprises two distinct molecular subtypes: germinal center B cell (GCB) subtype and activated B cell (ABC) subtype. The pathogenesis of ABC-DLBCL is characterized by two processes - the activation of NF-KB and a block in terminal B-cell differentiation. However, in GCB-DLBCL only a few biologically relevant pathways have been identified, which has hampered the development of targeted therapies with specific efficacy in this subtype. Recurrent genetic alterations involved in PI3K-AKT signaling pathway have been identified in the patients with solid cancers, and dramatic responses have been observed to PI3K inhibitors in clinical trials. The prevalence and clinical significances of genetic alterations in PI3K-AKT pathway have not been well studied in DLBCL. Previous studies have reported that loss of PTEN expression was observed in almost half of GCB-DLBCL cases but were largely absent in the ABC-DLBCL. In addition, functional studies have recently shown that the inactivation of Gα13 signaling pathway genes may also activate AKT in germinal center driven lymphoma, raising the possibility that additional genes within these pathways are affected in GCB-DLBCL. Herein, we identified genetic alterations involved in the PI3K-AKT pathway and evaluated their clinical impact. We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL uniformly treated with R-CHOP at the BC Cancer Agency. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Mutation status was determined using deep targeted re-sequencing of the coding exons of 61 genes with a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips, and RNAseq. Immunohistochemical staining of phospho-AKT (pAKT) was performed on tissue microarrays (n=332). Cell-of-origin (COO) was assigned by gene expression (Lymph2Cx assay) in 323 cases - 183 GCB, 104 ABC and 36 unclassifiable. GISTIC analysis revealed several COO-specific peaks with copy number changes. Among them, focal 10q23.3 deletion including PTEN was detected in GCB-DLBCL (q-value=1.7e-7), but not in ABC-DLBCL. We also identified two focal amplification peaks in GCB-DLBCL containing microRNAs MIR17HG (13q31.1;q=1.01e-24) and MIR21 (17q23.3; q=1.45e-6), which are known to down-regulate PTEN and result in activation of PI3K-AKT signaling. Furthermore, we detected recurrent INPP4B deletion (4q21.23) in GCB-DLBCL (q= 0.004) only. Of note, the lipid phosphatase INPP4B has been shown to play a role as a tumor suppressor that controls the levels of PI3K lipid products leading to AKT activation and metastasis of some solid cancers. This is consistent with the observation that PTEN and INPP4B deletions were individually associated with increased pAKT protein expression in our cohort (p=0.015 and p

Description: Introduction: The standard first line treatment for diffuse large B-cell lymphoma (DLBCL) is rituximab (R) plus CHOP (R-CHOP). However, approximately 35-40% of patients (pts) relapse following such treatment, and outcomes with salvage therapy remain poor. Obinutuzumab (GA101; G) is a fully humanized, glycoengineered, type II anti-CD20 monoclonal antibody. It has demonstrated greater direct cell death induction and antibody-dependent cellular cytotoxicity/phagocytosis activity than R, and has shown activity and an acceptable safety profile when combined with CHOP (G-CHOP) in first-line treatment of pts with advanced DLBCL (Sharman et al. Leuk Lymphoma 2019). GOYA (NCT01287741) was a randomized, open-label, multicenter Phase III study that compared the efficacy and safety of G-CHOP with R-CHOP in pts with previously untreated DLBCL. In the primary analysis (median observation period: 29 months), G-CHOP did not significantly improve investigator (INV)-assessed progression-free survival (PFS) compared with R-CHOP (Vitolo et al. J Clin Oncol 2017). Here, we present results from the final analysis of GOYA. Methods: Pts were aged ≥18 years, had histologically documented, previously untreated, CD20-positive DLBCL, with adequate hematologic function, ≥1 bi-dimensionally measurable lesion, an ECOG performance status of ≤2, and were classified as being in an International Prognostic Index (IPI) risk group of high, high-intermediate, or low-intermediate risk. Low-risk pts with an IPI score of 1 (not due to age alone) or 0 with bulky disease (1 lesion ≥7.5cm) were also eligible. Pts were randomized (1:1) to 8 (21-day) cycles of G (1000mg i.v. on Days [D]1, 8 and 15, Cycle [C]1 and D1, C2-8) or R (375mg/m2 i.v. on D1, C1-8) in combination with 6 or 8 cycles of CHOP. Preplanned radiotherapy was allowed for bulky or extranodal disease. The primary endpoint was INV-assessed PFS. Secondary endpoints included independent review committee-assessed PFS (primary analysis only); overall survival (OS); complete response (CR) and overall response rate (ORR) with or without PET (according to modified Cheson 2007 criteria); event-free survival; disease-free survival; duration of response; time to next anti-lymphoma treatment; PFS according to cell of origin (COO; germinal center B cell [GCB] or activated B cell [ABC]), as an exploratory endpoint; and safety. Results: In total, 1418 pts were randomized in GOYA; of these, 704 pts who received G-CHOP and 710 who received R-CHOP were included in this final analysis (clinical cut-off date: January 31, 2018). Overall median follow-up was 47.7 months. Baseline characteristics were well balanced between the G-CHOP and R-CHOP arms. INV-assessed PFS was similar between G-CHOP and R-CHOP (5-year PFS, 63.8% vs 62.6%; stratified HR, 0.94; 95% CI: 0.78, 1.12; p=0.48; Table). There was no significant difference in 5-year OS between the G-CHOP and R-CHOP groups (77.0% vs 77.7%) or in CR or ORR (Table). In the subgroup analysis of pts with ABC, GCB and unclassified DLBCL, no significant reductions in risk of disease progression were observed (stratified HR for INV-PFS, 0.91 [95% CI: 0.61, 1.36], 0.80 [95% CI: 0.58, 1.12] and 1.10 [95% CI: 0.65, 1.88], respectively). No new safety signals were identified. Grade ≥3 adverse events (AEs; 75% vs 66%) and serious AEs (44% vs 38%) were more common with G-CHOP than R-CHOP. Grade ≥3 AEs of particular interest (≥2% of pts in either treatment arm) were more frequent in the G-CHOP arm: infusion-related reactions (10% vs 3%), neutropenia (57% vs 47%), infections (20% vs 16%), cardiac events (5% vs 3%), thrombocytopenia (6% vs 2%), and hemorrhagic events (3% vs 1%); secondary malignancies occurred in 3% and 2% of the R-CHOP and G-CHOP arms, respectively. AEs resulting in treatment withdrawal (12% [87/702] G-CHOP; 8% [58/701] R-CHOP) and AEs with fatal outcome (6% [43/702] G-CHOP; 4% [31/701] R-CHOP) were slightly more common with G-CHOP. The most common grade 5 AEs were pneumonia (n=5 both arms) and sepsis/septic shock (G-CHOP, n=7; R-CHOP, n=3). Conclusions: Consistent with the primary analysis, G-CHOP did not significantly improve INV-assessed PFS compared with R-CHOP in previously untreated pts with DLBCL. Furthermore, there was no significant difference in 5-year OS between the treatment arms. No unexpected safety signals were identified. Further investigation of outcomes is ongoing, including the prognostic value of COO and BCL2 positivity. Disclosures Sehn: F. Hoffmann-La Roche/Genentech: Consultancy, Honoraria, Research Funding; Lundbeck: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; Acerta: Consultancy, Honoraria; Acerta: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; F. Hoffmann-La Roche/Genentech: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Apobiologix: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; TEVA Pharmaceuticals Industries: Consultancy, Honoraria; Verastem: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Janssen-Ortho: Consultancy, Honoraria; Janssen-Ortho: Honoraria; Kite Pharma: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; TEVA Pharmaceuticals Industries: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Martelli:F. Hoffman-La Roche, Celgene, Janssen, Sandoz, Novartis, Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria; F. Hoffman-La Roche, Celgene, Janssen, Sandoz, Novartis, Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria. Trněný:Gilead Sciences: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; MorphoSys: Consultancy, Honoraria; Celgene: Consultancy; Incyte: Consultancy, Honoraria; F. Hoffmann-La Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Liu:Roche Pharma Development, Shanghai, China: Employment. Bolen:Genentech, Inc.: Employment; F. Hoffmann-La Roche: Equity Ownership. Knapp:F. Hoffmann-La Roche Ltd: Employment. Sahin:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Sellam:Roche: Employment, Equity Ownership. Vitolo:Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: GAZYVA (obinutuzumab) is a CD20-directed cytolytic antibody and is indicated for the following: in combination with chlorambucil, for the treatment of patients with previously untreated CLL; in combination with bendamustine followed by GAZYVA monotherapy, for the treatment of patients with FL who relapsed after, or are refractory to, a rituximab-containing regimen; in combination with chemotherapy followed by GAZYVA monotherapy in patients achieving at least a partial remission, for the treatment of adult patients with previously untreated stage II bulky, III or IV FL.

Description: Central nervous system (CNS) relapse carries a poor prognosis in diffuse large B-cell lymphoma (DLBCL). Integrating biomarkers into the CNS–International Prognostic Index (CNS-IPI) risk model may improve identification of patients at high risk for developing secondary CNS disease. CNS relapse was analyzed in 1418 DLBCL patients treated with obinutuzumab or rituximab plus cyclophosphamide, doxorubicin, vincristine, prednisone chemotherapy in the phase 3 GOYA study. Cell of origin (COO) was assessed using gene-expression profiling. BCL2 and MYC protein expression was analyzed by immunohistochemistry. The impact of CNS-IPI, COO, and BCL2/MYC dual-expression status on CNS relapse was assessed using a multivariate Cox regression model (data available in n = 1418, n = 933, and n = 688, respectively). High CNS-IPI score (hazard ratio [HR], 4.0; 95% confidence interval [CI], 1.3-12.3; P = .02) and activated B-cell‒like (ABC) (HR, 5.2; 95% CI, 2.1-12.9; P = .0004) or unclassified COO subtypes (HR, 4.2; 95% CI, 1.5-11.7; P = .006) were independently associated with CNS relapse. BCL2/MYC dual-expression status did not impact CNS relapse risk. Three risk subgroups were identified based on the presence of high CNS-IPI score and/or ABC/unclassified COO (CNS-IPI-C model): low risk (no risk factors, n = 450 [48.2%]), intermediate risk (1 factor, n = 408 [43.7%]), and high risk (both factors, n = 75 [8.0%]). Two-year CNS relapse rates were 0.5%, 4.4%, and 15.2% in the respective risk subgroups. Combining high CNS-IPI and ABC/unclassified COO improved CNS relapse prediction and identified a patient subgroup at high risk for developing CNS relapse. The study was registered at www.clinicaltrials.gov as #NCT01287741.

Description: Background Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma associated with poor outcomes. First-line treatment incorporates cytotoxic chemotherapy and rituximab, but there is no single standard of care regimen. In British Columbia (BC), between January 2003 and May 2013, R-CHOP was the preferred induction regimen. Based on results from the STIL-1 trial (Rummel et al, Lancet 2013) demonstrating improved CR rate and prolonged progression-free survival (PFS) of bendamustine and rituximab (BR) compared with R-CHOP, in June 2013, BR became the standard first-line therapy for all patients with MCL regardless of age. In both eras, fit patients generally ≤65 years of age responding to induction were eligible for high-dose BEAM and autologous stem cell transplantation (ASCT). Maintenance rituximab (MR) has been offered to responding patients post ASCT since 2004 and for transplant ineligible patients since 2012. The aim of this study was to assess the efficacy of BR as an induction regimen for MCL. Methods Patients with MCL treated with first-line BR were identified in the BC Cancer clinical and pathology databases as well as the Leukemia/BMT Program of BC transplant database. BR was initiated no later than December 2018. Treatment received was verified using the BC Cancer Provincial Pharmacy database, and clinical characteristics were verified using the BC Cancer Information System. Radiotherapy for localized disease, splenectomy, or a period of observation prior to systemic therapy were permitted. PFS and overall survival (OS) were calculated from the date of initiation of systemic therapy. In the subgroup of patients ≤65 years of age, results were compared to a historical cohort uniformly treated with R-CHOP. Baseline and treatment characteristics associated with PFS or OS (p65 years old, 9 underwent ASCT (age 66-71) and 92 did not. 63/69 patients received MR after ASCT, and 77/121 received MR after BR (without ASCT). Reasons for not receiving MR (6 after ASCT, 44 after BR) were 25 PD, 10 ASCT/BR toxicity, 7 pending, 3 patient preference, 3 early deaths, 2 physician preference. With a median follow-up of 2.4y (range 0.2 - 6.1) in living patients the 3y OS was 69.5% (95% CI 69.4-70.6) and 3y PFS 62.8% (95% CI 62.4-63.6). Baseline characteristics and outcomes were similar between patients ≤65y treated with BR vs. R-CHOP (Table 1, Figure 1A and 1B). 81/140 (58%) patients treated with R-CHOP underwent ASCT. In the subgroup of patients who underwent ASCT, outcomes calculated from the point of ASCT were not statistically different between BR vs. R-CHOP. In univariate analysis, age 〉65y, ECOG performance status 〉1, elevated LDH, blastoid/pleomorphic morphology, no ASCT, and no MR were associated with worse PFS and OS. In multivariate analysis including these variables as well as chemoimmunotherapy regimen, treatment with BR was not associated with PFS or OS. Conclusions In this population-based analysis, BR is an effective induction regimen for both transplant eligible and ineligible patients. ASCT is feasible in patients treated with BR induction. Even though BR was associated with a numerical improvement in PFS compared to R-CHOP in patients ≤65y, differences in PFS and OS were not statistically significant. Longer follow-up is necessary to fully understand the impact of BR in the frontline setting. We observed PD in 12% patients, suggesting that BR may not be an optimal induction regimen across all patients with MCL, particularly in those with aggressive or highly proliferative disease. Disclosures Villa: Roche, Abbvie, Celgene, Seattle Genetics, Lundbeck, AstraZeneca, Nanostring, Janssen, Gilead: Consultancy, Honoraria. Sehn:Morphosys: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria; Merck: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Merck: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Acerta: Consultancy, Honoraria; TG Therapeutics: Consultancy, Honoraria; Apobiologix: Consultancy, Honoraria; Janssen-Ortho: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; F. Hoffmann-La Roche/Genentech: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Verastem: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Acerta: Consultancy, Honoraria; F. Hoffmann-La Roche/Genentech: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Morphosys: Consultancy, Honoraria; TEVA Pharmaceuticals Industries: Consultancy, Honoraria; TEVA Pharmaceuticals Industries: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Janssen-Ortho: Honoraria; Celgene: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Lundbeck: Consultancy, Honoraria. Savage:BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria; Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding. Song:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Freeman:Seattle Genetics, Janssen, Amgen, Celgene, Abbvie: Consultancy, Honoraria. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Gerrie:Lundbeck, Seattle Genetics: Consultancy, Honoraria.

Description: Transformation to aggressive lymphoma is a critical event in the clinical course of follicular lymphoma (FL) patients. Yet, it is a challenge to reliably predict transformation at the time of diagnosis. Understanding the risk of transformation would be useful for guiding and monitoring patients, as well as for evaluating novel treatment strategies that could potentially prevent transformation. Herein, we review the contribution of clinical, pathological, and genetic risk factors to transformation. Patients with multiple clinical high-risk factors are at elevated risk of transformation but we are currently lacking a prognostic index that would specifically address transformation rather than disease progression or overall survival. From the biological standpoint, multiple studies have correlated individual biomarkers with transformation. However, accurate prediction of this event is currently hampered by our limited knowledge of the evolutionary pathways leading to transformation, as well as the scarcity of comprehensive, large-scale studies that assess both the genomic landscape of alterations within tumor cells and the composition of the microenvironment. Liquid biopsies hold great promise for achieving precision medicine. Indeed, mutations detected within circulating tumor DNA may be a better reflection of the inherent intratumoral heterogeneity than the biopsy of a single site. Last, we will assess whether evidence exists in the literature that transformation might be prevented altogether, based on the choice of therapy for FL.

Description: Introduction: Despite the improvement of outcome of aggressive B-cell lymphomas in the rituximab treatment era, central nervous system (CNS) relapse continues to pose a significant management problem. It remains a challenge to select patients (pts) in whom specific diagnostic procedures to identify CNS disease at diagnosis should be performed and to target a high risk group in whom a CNS prophylaxis strategy is warranted. The German High-Grade Non-Hodgkin Lymphoma Study Group (DSHNHL) recently proposed a new prognostic model incorporating the 5 IPI factors (age 〉 60 y, LDH 〉 N, stage 3 or 4, extranodal (EN) sites 〉 1) in addition to kidney/adrenal gland involvement to predict the risk of secondary CNS disease in pts with aggressive B-cell lymphoma. This model effectively stratified patients into 3 risk groups: low risk (0-1 factors, 2 y CNS relapse risk .6% (95% CI 0.0-1.2); intermediate risk (2-3 factors, 2 y CNS relapse risk 3.4 %( 95% CI 2.2-4.6)) and high risk (4-6 factors, 2 y CNS relapse risk of 10.2% (95% CI 6.3-14.1)) (Schmitz et al. Hematol. Oncol. 2013: 31, 047a). Herein, we sought to validate this model in an independent cohort of DLBCL treated with R-CHOP chemotherapy at the British Columbia Cancer Agency (BCCA). Methods: The DSHNHL dataset on which the model was initially developed was comprised of 2164 patients with aggressive B-cell lymphomas (n= 1735, 80.2% diffuse large B-cell lymphoma (DLBCL)), 18-80 years of age who were treated with rituximab with (CHO(E) P-like chemotherapy on prospective studies. The BCCA Lymphoid Cancer Database was screened to identify all patients with DLBCL treated with curative intent R-CHOP chemotherapy. Results: In total, 1597 patients were diagnosed with DLBCL at the BCCA and received at least one cycle of curative intent R-CHOP chemotherapy. The median follow-up for living patients was 4.2 years. Pts in the BCCA population-based DLBCL cohort were more likely to have poor risk features including PS 〉1, advanced age and a high IPI score (Table 1). Applying the 6 factor model, very similar risk groups were identified: low risk (0-1 factors 2 year CNS relapse risk .8% (95% CI 0.0-1.6%).; intermediate risk (2-3 factors 2 year CNS relapse risk 3.9% (95% CI 2.3-5.5%); and high risk (4-6 factors 2 y CNS relapse risk 12% (95% CI 7.9-16.1%) (Figure 1). The median time to CNS relapse was 6.7 months from the time of diagnosis in the BCCA group and was 7.2 months in the DSHNHL group highlighting that this event typically occurs early in the disease course. In both datasets kidney/adrenal involvement was highly associated with CNS relapse (2 year CNS risk BCCA 33%; 14% DSHNHL), the difference likely reflecting the higher risk pts in the BCCA population-based setting. Conclusions: We have validated the proposed DSHNHL prognostic model for CNS relapse in an independent dataset. The model identifies a high risk group in which diagnostic procedures to rule out CNS disease are highly recommended at diagnosis including MRI head, and cerebrospinal fluid analysis by cytology and flow cytometry and consideration of CNS-directed therapies. Kidney/adrenal involvement is consistently associated with a high risk of CNS relapse in the rituximab treatment era for which CNS prophylaxis should be incorporated into front-line therapy. Table 1 Clinical factor BCCA N=1597 DSHNHL N=2164 Age 〉 60 years * Median age 1035 (65%) 65 years (16-94) 974 (45%) 58 years (18-80) Median follow-up 4.6 years 2.9 years Male sex 915 (57%) 1244 (57.5%) PS 〉 1* 584 (37%) 247 (11%) Elevated LDH 1147 (53.0%) 737 (49.0%) EN 〉 1 396 (25%) 479 (22%) Stage 3 or 4 916 (57%) 1148 (53%) IPI * 0,1 2 3 4,5 463 (31%) 359 (24%) 350 (23%) 329 (22%) 1009 (47%) 523 (24%) 398 (18%) 231 (11%) Bulky disease 〉 7cm 636 (41%) 1027 (47.5%) * P〈 0.05 Figure 1 Figure 1. Disclosures Savage: F Hoffmann-La Roche: Other. Villa:F Hoffmann-La Roche: Other. Sehn:Roche: Research Funding. Pfreundschuh:Roche: Advisory Board Other, Research Funding. Gascoyne:Hoffman La-Roche: Research Funding. Connors:Seattle Genetics, Inc.: Research Funding; Roche: Research Funding.

Description: The molecularly defined cell-of-origin (COO) subtypes of diffuse large B-cell lymphoma (DLBCL), activated B-cell like (ABC) and germinal center B-cell like (GCB), have distinct underlying biology and outcomes in the R-CHOP treatment era. The recent advent of therapeutic agents that show subtype-specific activity has made the robust assignment of COO increasingly important for identifying patients that will respond to these agents. Although it has become common practice to use widely applicable immunohistochemistry-based algorithms to assign COO, these methods are reported to have widely variable concordance with COO as determined by gene expression profiling (GEP). Furthermore, using these IHC-based algorithms, the relationship between COO and treatment outcome to R-CHOP has been very inconsistent. We recently described an accurate and robust gene expression based assay for determining COO applicable to formalin-fixed paraffin-embedded (FFPE) biopsies. Here we sought to determine the prognostic significance of COO assigned using this assay and the relationship with other established prognostic factors. The Lymph2Cx assay, a 20 gene assay based on NanoString technology, was applied, as previously described (Scott et al Blood 2014;123:1214-17), to RNA extracted from the diagnostic FFPE biopsies (tumor content ≥ 60%) of 274 patients with de novo DLBCL. The median follow-up of living patients within this cohort, uniformly treated with R-CHOP at the BC Cancer Agency (Vancouver, Canada) was 6.1 years (range 1.2 – 13.2). Tissue microarrays of duplicate 0.6mm cores were stained for MYC and BCL2 (DAKO 124) to assess the proportion of tumor cells that expressed these proteins. Identification of MYC+/BCL2+ tumors (as described in Johnson et al J Clin Oncol 2012; 30: 3452-9; cut-offs: MYC ≥ 40%, BCL2 ≥ 50%) was performed independently by two expert hematopathologists (AM and PF) with consensus on discordant cases reached with a third hematopathologist (RDG). GEP data of sufficient quality was obtained from 271/274 (99%) of the biopsies, assigning the following COOs: 90 (33%) ABC, 153 (56%) GCB and 28 (10%) were unclassifiable. In comparison with the GCB group, the ABC group had a greater proportion of patients with stage III/IV disease (63% vs 45%, P=0.01) and higher IPI scores (P=0.03). The ABC subtype was associated with significantly inferior outcomes compared with the GCB group (Figure 1). This association was independent of IPI groupings (low (0-1), intermediate (2-3) and high (4-5)) in multivariate analyses. The prognostic significance of the COO was most evident in the intermediate IPI group (5 year time-to-progression (TTP) ABC 45% vs GCB 71%; log-rank P=0.001, HR 2.7 (95% CI 1.6 – 6.3)). It has been proposed that the prognostic power of COO is largely attributable to the greater proportion of MYC+BCL2+ cases within the ABC subtype (Hu et al Blood 2013; 121: 4021-31). We confirm that the proportion of tumors that are MYC+/BCL2+ by IHC is significantly higher in the ABC vs GCB subtype (57% vs 21%, P

Description: Background: Diffuse large B-cell lymphoma (DLBCL) is a highly heterogeneous neoplasm with 40% of patients experiencing treatment failure following immuno-chemotherapy (R-CHOP). Both cell-of-origin (COO) and presence of concurrent MYC/BCL2 rearrangements (DHIT) are significantly associated with distinct inferior outcome. Recently, next-generation sequencing (NGS) studies have uncovered distinct genetic subtypes, including a sizable ABC/GCB-independent group characterized by more frequent TP53 abnormalities. The patterns of TP53 mutations and the prognostic significance in DLBCL have been previously reported. However, such information is rarely available at the time of diagnosis as diagnosis of DLBCL for most patients is based on morphology and phenotype, assessed by immunohistochemistry (IHC). To bridge the gap between genotype and phenotype, we examined the TP53 mutational status and TP53 protein over-expression (IHC) in a large population-based DLBCL cohort uniformly treated with R-CHOP (Ennishi et al. Blood 2017 129:2760-2770). Methods: We analyzed 347 newly diagnosed de novo DLBCL cases uniformly treated with R-CHOP in British Columbia. Comprehensive clinical annotation was available through the BC Cancer Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of TP53 was performed using a Truseq Custom Amplicon assay (Illumina) on the Miseq platform. IHC staining for TP53 (DO7), TP21, COO (Hans) and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). COO classification was also performed using the Lymph2Cx assay (NanoString) (n=324). Strong TP53 expression (TP53+) was defined as high intensity (3/3) expression in 〉50% of the malignant cells. Results: TP53 mutations (p53mut) were present in 72 cases (22.2%) with 84% being missense and 64% localized to the DNA binding-motifs. There were 54 TP53+ tumors by IHC (17%), of which 51 (94%) had p53mut, with a sensitivity of 70% for detection of p53mut. All but one TP53+ with p53mut (50/51 cases) had missense mutations. All TP53+ with missense p53mut (85% of all missense p53mut) showed strong nuclear expression, one recurrent nonsense p53mut showed combined cytoplasm/nuclear TP53+, while all splice site and frameshift variants were TP53+-negative. All TP53+ cases were negative for TP21; 30 (56%) cases were GCB (Hans) while 24 (44%) cases were non-GCB (Hans) and only 4 (16%) cases were DHIT. Both p53mut and TP53+ were associated with poor overall survival (OS) (p=0.004 and p=0.007, respectively) and disease-specific survival (DSS) (p=0.003 and p=0.001, respectively). In multivariate analysis with IPI, COO (Hans) and DHIT status, both p53mut and TP53+ were independent predictors of OS (HR=0.6, 95%CI=0.4-0.9, p=0.008 and HR=0.6, 95%CI=0.4-0.9, p=0.011, respectively) and DSS (HR=0.6, 95%CI=0.4-0.9, p=0.01 and HR=0.5, 95%CI=0.3-0.8, p=0.004, respectively). These results were consistent when the Lymph2Cx was used to assign COO (n = 324). Importantly, patients with TP53+ tumors showed significantly poorer outcome (OS and DSS) when compared with patients with tumors negative for TP53 stratified by both Hans COO subtypes (p=0.001 and p