Abstract: Approximately 20–25% of children with B-cell precursor ALL harbour the t(12;21) (p13;q22)/ETV6-RUNX1 fusion which has been associated, almost universally, with a favourable outcome. However, as some ETV6-RUNX1 patients relapse additional predictors of treatment failure are required. Although additional abnormalities involving 12p and 21q (AA) are known to occur in these patients, previous studies have produced conflicting results with respect to their prognostic impact. In order to investigate the prognostic relevance of AA in this subset of patients, we screened a large cohort of children by FISH with break-apart probes targeting the ETV6 (DakoCytomation, Denmark) and RUNX1 loci (centromeric - RP11-272A03 and telomeric - RP11-396G11, Sanger Institute, UK). Among 368 ETV6-RUNX1 patients treated on the MRC ALL97 childhood treatment trial, material was available for testing on 247 (67%). Sample availability was not related to sex or age, but those tested were more likely to have a white cell count (WCC) of & gt;50×109/L and hence had a borderline inferior event free survival (EFS) at 5 years: 86% (SE 2.2%) v 93% (2.4%), p=0.05. FISH testing with the ETV6 and RUNX1 probes was successful in 245 (99%) and 244 (99%) patients, respectively. In total, 202 (82%) patients harboured at least one of the following five AA: loss of the untranslocated ETV6 allele (ETV6del): 165 (67%); Gain of a normal chromosome 21 (+21): 57 (23%); Gain of a der(21)t(12;21) chromosome [+der(21)]: 38 (16%); deletions from the der(12)t(12;21) [der(12)del] : 20 (8%) and gain of the der(12) t(12;21) [+der(12)]: 18 (7%). In 74 (30%) cases two or more of these AA were observed. Evidence that the ETV6-RUNX1 clone had doubled to a near-tetraploid clone was seen in 9 (4%) cases. In the majority of ETV6del cases (158, 96%) the deletion resulted in the loss of the entire ETV6 probe which spanned a region of ~750kb. The proportion of ETV6-RUNX1 positive cells carrying each specific AA ranged from 6% to 100% but in the majority of cases comprised more than 75% cells: 72%, 54%, 63%, 100% and 39%, respectively. The frequency of the AA did not vary by sex, with the exception of +der(12) which was more common among females (12%) than males (4%) [p & lt;0.03]. Patients with +der(12), +der(21) or +21 were significantly older compared to other patients: mean age 5.9 years v 4.6; 5.4 v 4.6 and 5.3 v 4.6 respectively, all p & lt;0.03. No age association was seen for patients harbouring either of the deletions. Patients with +der(21) were significantly less likely to have a WCC & gt;50×109/L [1/38 (3%) v 54/206 (26%), p=0.001]. Although a similar trend was seen for patients with +21 it was not significant: [8/57 (14%) v 47/187 (25%), p=0.079] . During a median follow-up period of 7 years, a total of 41 (17%) adverse events were observed, including 34 (14%) relapses and 16 (6%) deaths. The EFS and OS figures at five years were 86% (2.2%) and 94% (1.5%) respectively. None of the AA predicted a higher risk of relapse or death, whether considered separately or together. None of the 20 patients with der(12)del suffered a relapse or died; but as the total number of relapses and deaths was small, this difference was of only borderline significance (p=0.046). We report for the first time that the spectrum of AA in ETV6-RUNX1 positive patients extends to the der(12) and that der(12) deletions may correlate with an excellent outcome. Altogether more than 80% of ETV6-RUNX1 patients harbour an AA involving 12p or 21q. This study, based on 247 patients treated on a single protocol and with 7 years follow-up, is the most comprehensive to date. Contrary to previous reports, we conclude that AA involving 12p and 21q are not predictors of relapse or death in the context of modern treatment protocols.

Abstract: Inactivation of the tumor suppressor gene, CDKN2A, can occur by deletion, methylation, or mutation. We assessed the principal mode of inactivation in childhood acute lymphoblastic leukemia (ALL) and frequency in biologically relevant subgroups. Mutation or methylation was rare, whereas genomic deletion occurred in 21% of B-cell precursor ALL and 50% of T-ALL patients. Single nucleotide polymorphism arrays revealed copy number neutral (CNN) loss of heterozygosity (LOH) in 8% of patients. Array-based comparative genomic hybridization demonstrated that the mean size of deletions was 14.8 Mb and biallelic deletions composed a large and small deletion (mean sizes, 23.3 Mb and 1.4 Mb). Among 86 patients, only 2 small deletions were below the resolution of detection by fluorescence in situ hybridization. Patients with high hyperdiploidy, ETV6-RUNX1, or 11q23/MLL rearrangements had low rates of deletion (11%, 15%, 13%), whereas patients with t(9;22), t(1;19), TLX3, or TLX1 rearrangements had higher frequencies (61%, 42%, 78%, and 89%). In conclusion, CDKN2A deletion is a significant secondary abnormality in childhood ALL strongly correlated with phenotype and genotype. The variation in the incidence of CDKN2A deletions by cytogenetic subgroup may explain its inconsistent association with outcome. CNN LOH without apparent CDKN2A inactivation suggests the presence of other relevant genes in this region.

Abstract: Splenic marginal zone lymphoma (SMZL) is generally an indolent disorder which has a median survival of 10 – 13 years, but a minority of patients pursue a more aggressive clinical course. A number of adverse prognostic factors including the level of haemoglobin, lymphocyte count and platelet count, B2 microglobulin, presence of a paraprotein, IgVH gene mutational status and p53 loss or mutation have been identified, but these have not been consistent among recent series. We have studied 89 patients with SMZL, diagnosed on the basis of lymphocyte morphology, immunophenotype and marrow and splenic histology, when available, and have evaluated the impact of presenting Hb, lymphocyte count, spleen size, presence of a paraprotein, cytogenetic abnormalities and IgVH gene mutational status on both the need for treatment and on overall survival. In 43 patients the diagnosis was made following a routine blood count performed for an incidental reason. The M:F ratio was 1:1.78, median age at presentation was 67.5 years (range; 49 – 91) and mean follow-up was 78.1 months. 63/89 (71%)patients presented with palpable splenomegaly, and a further 5 developed splenomegaly during the course of their disease. 42/89 (47%) patients required treatment of whom 29 (32%) underwent splenectomy. 36 patients have not required treatment, 25 have received an alkylating agent, 12 have received fludarabine and 4 were treated with rituximab.15/29 (17%) received chemotherapy for progressive disease post splenectomy. In six patients there was transformation to a high grade lymphoma. 14 patients (16%) died, 11 of whom had a disease-related death. 24/83 patients (29%) had a paraprotein, 67/87 (77%) had an abnormal karyotype of whom 39 (45%) had an abnormality of 7q. Using interphase FISH, 7/78((9%) showed trisomy 3 and 7/74(9%) had p53 loss. Of the patients with high grade transformation, 3/6 had p53 loss. 47/67 patients (70%) had mutated IgVH genes, and 15 cases utilised the VH1-02 gene segment. Only spleen size 〉 10cm (p=0.0002) and Hb 〈 100 gm/l (p=0.008) correlated with the need for treatment. Only unmutated IgVH genes (p=0.032) and the need for further therapy following initial treatment with splenectomy (p=0.034) correlated with overall survival.The median survival of unmutated cases was 113 months and was not reached for mutated cases.The median survival of patients for whom splenectomy has been their only treatment has not been reached, but was 110 months for patients requiring additional treatment post splenectomy. There is a need for larger, multi-centre studies to identify poor risk patients with SMZL.

Abstract: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a viable treatment option for many cancers but its clinical utility is limited due to the occurrence of graft-versus-host disease (GVHD). Understanding the impact of obesity on immune function has become increasingly important in the setting of the current obesity pandemic. We report here that obesity has a negative and selective impact on acute gut GVHD. Diet-induced obese (DIO) mice exhibited increased gut permeability, endotoxin translocation and radiation-induced gastrointestinal damage. After allo-HSCT, DIO recipients across strains and sex had markedly increased pro-inflammatory cytokines (IL-6, TNF), GVHD biomarker ST2, MHC class II expression and exhibited rapid mortality associated with severe acute gut pathology. This obesity-associated lethal acute gut GVHD was dependent on donor CD4 T cells and occurred even in minor MHC mismatch strain combination in which only a delayed skin chronic GVHD resulted in lean recipients. Pro-inflammatory cytokine blockade targeting both IL-6 and TNF ameliorated obesity-associated acute gut GVHD while maintaining graft-versus-tumor (GVT) effects. Microbiome assessment of DIO mice revealed markedly reduced microbiome diversity and decreased Clostridiaceae abundance. Additionally, DIO mice had a significant increase of GVHD-associated Akkermansia muciniphila before and after allo-HSCT compared to the controls. Extended antibiotic treatment of DIO mice protected from the endotoxin translocation, cytokine storm as well as gut GVHD pathology but did not protect later development of chronic skin GVHD. These results demonstrate that obesity alters the microbiome and imparts differential effects on GVHD following allo-HSCT with decreased survival and this inferior outcome can be pre-empted by combined pro-inflammatory cytokine blockade or antibiotic pretreatment. Disclosures Pai: Roche-Genentech: Employment. Holtan:CSL Behring: Consultancy; Incyte: Consultancy; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy. Ferrara:ViraCor: Consultancy; Incyte: Consultancy; Kamada: Consultancy; Mallinckrodt: Consultancy; Enlivex: Consultancy; Xenikos: Consultancy; CSL Behring: Consultancy; National Institutes of Health: Research Funding. Levine:Novartis: Honoraria; Kamada: Research Funding; bluebird bio: Consultancy; National Cancer Institute: Research Funding; Incyte: Consultancy, Research Funding; Ironwood: Honoraria; Biogen: Other: non-financial support; Viracor: Patents & Royalties: biomarker patent. Abedi:Abbie: Speakers Bureau; Gilead: Speakers Bureau; Celgene: Speakers Bureau; BMS: Speakers Bureau; Takeda: Speakers Bureau. Blazar:Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Abstract: INTRODUCTION Diffuse large B-cell lymphoma (DLBCL) represents several distinct clinical pathologic entities recently identified by molecular profiling. Treatment with anti-CD19 chimeric antigen receptor (CAR) T-cell therapies is now standard for many patients with relapsed/refractory (R/R) disease. Although antigen loss of CD19 represents a known cause of late relapses, the majority of CAR-T cell treatment failure occurs very soon after treatment at which time the impact of molecular subtype and other somatic mutations of DLBCL is undefined. We sought to determine impact of molecular features of DLBCL tumors on clinical outcomes in a cohort of patients with R/R disease who were treated with axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) in order to provide insight into the mechanism of response or resistance to CAR-T cell therapy. METHODS We collected clinical data and formalin-fixed, paraffin embedded (FFPE) biopsy specimens from 121 DLBCL patients at the time of R/R disease after prior treatment with standard chemoimmunotherapy across 12 US academic medical centers who subsequently received commercial CAR-T cell treatment. Whole exome and transcriptome sequencing was performed on all cases to measure gene expression and gene copy number alterations. Genetic analysis was done on 96 patients with pre-treatment biopsies that passed sequencing quality filters, and expression analysis on 93 patients. Progression-free and overall survival (PFS, OS) measured from the day of CAR-T cell infusion were estimated using the Kaplan-Meier method and compared with the log-rank test. RESULTS Baseline demographics and treatment details of the patient population are shown in the Table (Panel A). Best overall response was CR in 43% of patients and PR in 10% patients. PFS and OS were significantly different based on best response to treatment (P & lt;0,001 Figure, Panel B). At the time of R/R disease, the most commonly mutated genes were TP53 (25%), KMT2D (23%), CREBBP (23%), BCL2 (20%), BTG2 (12%), ARID1A (11%), CARD11 (11%), MYD88 (11%) and PIM1 (11%), (Panel C). Molecular subtyping based on the method of Wright, et al. revealed cases to be BN2 (N=16), A53 (N=13), EZB (N=14), MCD (N=13), N1 (N=4), ST2 (N=8) and unclassifiable (UC) in 28 cases. Cluster analysis as described by Chapuy et al. assigned cases to be C0 (N=6), C1 (N=18), C2 (N=14), C3 (N=27), C4 (N=17) and C5 (N=14). The impact of subgroups on of PFS are shown in Panels D and E. While not statistically significant different across all groups, there was a trend towards improved outcomes in C5/MCD as well as the C2/A53 subtypes and a trend towards inferior PFS in the C3/EZB subtypes. Inferior PFS was observed in patients with mutations in BCL2 (P=0.009) and MYC (P & lt;0.001), but not BTG2 (P=0.095), MYD88 (P=0.106), or CD79B (P=0.086). An unbiased model comprising mutations in MYC, BCL2, CDKN2A, and KLHL6 was strongly associated with a lack of response to CAR-T therapy and a poor prognosis (HR=3.55, P & lt;0.001, Panel F). Gene Set Enrichment Analysis (GSEA) identified a gene signature reflecting T-cell activation in the pre-treatment tumor biopsy as being associated with a higher likelihood of response to treatment (Panel G). CONCLUSIONS DLBCL patients whose tumors have molecular features that are predictive of inferior response to standard frontline treatment including the high-risk subgroups (C2/A53) and (C5/MCD) have favorable treatment outcomes with CAR-T cell therapy. In contrast, individual driver mutations including MYC and BCL2, CDKN2A, and KLHL6 are associated with inferior PFS with CAR-T cell therapy, while mutations in BTG2, MYD88, and CD79B are associated with a favorable PFS. In addition, gene expression analysis implicates a potential role for the microenvironment in modulating responses to CAR-T therapy. These findings suggest that predictive biomarkers for response to traditional chemoimmunotherapy and cellular immunotherapy are distinct. Our results provide insight into potentially targetable pathways for the development of rational treatment strategies that may augment response CAR-T cell therapy. Figure 1 Figure 1. Disclosures Hill: Celgene (BMS): Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Gentenech: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Other: Travel Support, Research Funding; Beigene: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Incyte/Morphysis: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. McKinney: Novartis: Research Funding; Nordic Nanovector: Research Funding; Molecular Templates: Consultancy, Research Funding; Kite/Gilead: Honoraria, Speakers Bureau; Incyte: Research Funding; Genetech: Consultancy, Honoraria, Research Funding; Epizyme: Consultancy; Celgene: Consultancy, Research Funding; BTG: Consultancy; Beigene: Research Funding; ADC Therapeutics: Consultancy, Speakers Bureau; Pharmacyclics: Consultancy; Verastem: Consultancy. Neff: Spring Discovery: Consultancy, Ended employment in the past 24 months; EUSA Pharma: Speakers Bureau; Enzyvant: Consultancy. Reshef: BMS, Regeneron, TScan, Synthekine, Atara, Jasper, Bayer: Consultancy; ilead, BMS, Precision, Immatics, Atara, Takeda, Shire, Pharmacyclics, Incyte: Research Funding; Bayer: Consultancy; Gilead and Novartis: Honoraria. Oluwole: Janssen: Consultancy; Pfizer: Consultancy; Curio Science: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding. Ghosh: Incyte: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; TG Therapeutics: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Genmab: Consultancy, Honoraria; Epizyme: Honoraria, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; ADC Therapeutics: Consultancy, Honoraria; Adaptive Biotech: Consultancy, Honoraria; AbbVie: Honoraria, Speakers Bureau; Karyopharma: Consultancy, Honoraria; Genentech: Research Funding. Chen: Actinium Pharmaceuticals: Other: Principal Investigator, SIERRA Trial, Actinium. Hernandez-Ilizaliturri: AbbVie: Other: Advisory Boards; Incyte: Other: Advisory Boards; Celgene: Other: Advisory Boards; BMS: Other: Advisory Boards; Pharmacyclics: Other: Advisory Boards; Amgen: Other: Advisory Boards; Kite: Other: Advisory Boards; Gilead: Other: Advisory Boards; Epyzime: Other: Advisory Boards. Shah: Lily: Consultancy, Honoraria, Research Funding; Miltenyi Biotec: Consultancy, Honoraria, Research Funding; Kite: Consultancy; Epizyme: Consultancy; Legend: Consultancy; Incyte: Consultancy; Umoja: Consultancy. Stephens: Epizyme: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Innate Pharma: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees; JUNO: Research Funding; Novartis: Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; CSL Behring: Consultancy; Celgene: Consultancy; Mingsight: Research Funding; Arqule: Research Funding. Patel: Janssen: Consultancy; Kite Pharma: Consultancy, Speakers Bureau; TG Therapeutics: Consultancy, Speakers Bureau; MEI Pharma: Consultancy; Abbvie: Consultancy; Genentech: Consultancy; Bristol Myers Squibb: Consultancy, Speakers Bureau; BeiGene: Consultancy; Morphosys: Consultancy; Pharmacyclics: Consultancy; AstraZeneca: Consultancy, Research Funding, Speakers Bureau; ADC Therapeutics: Consultancy; Lilly: Consultancy. Pagel: Gilead: Consultancy; Actinium Pharmaceuticals: Consultancy; Kite, a Gilead Company: Consultancy; Incyte/MorphoSys: Consultancy; AstraZeneca: Consultancy; Pharmacyclics/AbbVie: Consultancy; Epizyme: Consultancy; BeiGene: Consultancy; MEI Pharma: Consultancy. Hsi: AbbVie Inc, Eli Lilly: Research Funding. Goy: Genentech/Hoffman la Roche: Research Funding; AbbVie/Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Consultancy, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Medscape: Consultancy; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Physicians' Education Resource: Consultancy, Other: Meeting/travel support; Xcenda: Consultancy, Honoraria; Genomic Testing Cooperative: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Leadership role; LLC(Targeted Oncology): Consultancy; Hoffman la Roche: Consultancy; Vincerx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Rosewell Park: Consultancy; Infinity/Verastem: Research Funding; MorphoSys: Honoraria, Other; Elsevier PracticeUpdate: Oncology: Consultancy, Honoraria; Xcenda: Consultancy; Bristol Meyers Squibb: Membership on an entity's Board of Directors or advisory committees; Vincerx pharma: Membership on an entity's Board of Directors or advisory committees; OncLive Peer Exchange: Honoraria; COTA (Cancer Outcome Tracking Analysis): Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Leadership role; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Elsevier's Practice Update Oncology, Intellisphere, LLC(Targeted Oncology): Consultancy; Incyte: Honoraria; AbbVie/Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Michael J Hennessey Associates INC: Consultancy; Novartis: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Karyopharm: Research Funding; Bristol Meyers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Phamacyclics: Research Funding; Constellation: Research Funding; Hackensack Meridian Health, Regional Cancer Care Associates/OMI: Current Employment. Ohgami: Stemline Therapeutics: Research Funding. Andreadis: CRISPR Therapeutics: Research Funding; GenMAB: Research Funding; Novartis: Research Funding; Roche: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Epizyme: Honoraria; Incyte: Honoraria; TG Therapeutics: Honoraria; Kite: Honoraria; Karyopharm: Honoraria; Atara: Consultancy, Honoraria; BMS: Research Funding; Merck: Research Funding. Thacker: Data Driven Bioscience: Current Employment. Rozzi: Data Driven Bioscience: Current Employment. Parker: Data Driven Bioscience: Current Employment. Happ: Data Driven Bioscience: Current Employment. Dave: Data Driven Bioscience: Current equity holder in publicly-traded company.