ALBERT

Description: Immune aplastic anemia (AA) is a life-threatening bone marrow failure syndrome in which the hematopoietic stem cells are destroyed, leading to pancytopenia. Although the exact biological process leading to AA remains largely unknown, bone marrow destruction is thought to be mediated by an autologous T cell response. We hypothesized that the autoimmune process in AA would create a T cell fingerprint unique to aplastic anemia. To decipher this signature, we collected an international, multi-centre cohort of 245 AA-samples from bone marrow and peripheral blood profiled with T-cell receptor beta (TCRβ) -sequencing. CD8+ T cell- and MNC-sorted samples from 153 clinically annotated AA patients were obtained from diagnosis, during remission and at relapse. To compare AA to similar diseases, we gathered 116 samples from other bone marrow failure syndromes, including MDS, PNH and hypoplastic LGL, and 45 samples from other autoimmune disorders. As healthy controls, we profiled 60 CD4+ and CD8+ T cells and utilized 786 MNC samples from public data repositories. To gain insight into T cell phenotypes, we also profiled 6 longitudinal samples with scRNA+TCRαβ-sequencing. As there are 1x1012-16 different TCRs and most of them are exclusive to individuals (private), we reasoned that by studying the most biologically interesting clonotypes from each individual, we could explain differences in disease severities, variation in treatment responses and pathogenesis. From all subjects, we selected private response clonotypes: highly expanded clonotypes (at least 1% of the total repertoire), convergent clonotypes (in which multiple nucleotide sequences converge to encode the same amino acid sequence) and from patients with AA, treatment-responding clonotypes (clonotypes that were suppressed/expanded after immune therapy). To analyse epitope-specificities of these clonotypes we leveraged TCRGP, our recently described Gaussian process method that can predict if TCRs recognize previously known epitopes. Clonotypes recognising viral epitopes (CMV, EBV and Influenza A) were enriched among private response clonotypes in comparison to the total repertoire (Fisher's exact test, p=2e-16), indicating that our filtering strategy indeed enriched for epitope-specific clonotypes. Of interest, the healthy donors' private response clonotypes showed more anti-viral clonotypes than did AA-patients (p=0.003), suggesting that in AA the epitope-specifities of private response clonotypes are not driven by common viral antigens. To identify specifities against unknown epitopes of the private response AA clones, we used an unsupervised learning strategy, GLIPH,that groups TCRs recognising the same epitope based on amino acid level similarities. Clonotypes in AA showed high convergence in their epitope-targets, as 1709 of 5744 (29.75%) clonotypes formed a single, potentially epitope-specific cluster that was not viral-specific. Similar analysis of control samples resulted in fewer clones clustering to the most prominent cluster (23.20%, p=1.63e-10), suggesting for a more homogenous target population within AA patients' clones. After showing sequence-level similarity of the private response clonotypes in AA, we aimed to link these pathological clonotypes to transcriptomes at the single-cell level using scRNA+TCRαβ-sequenced samples. The cells of the private response clonotypes showed multiple T cell phenotypes, but most cells (47.13%) in the bone marrow environment were recently activated CD8+ effector phenotype, marked by expression of GZMH, GNLY and PRF1. In comparison, the anti-viral clonotypes were mostly (37.3%) central memory phenotype (CCR7, TCF7). In serially sampled patients, anti-thymocyte globulin treatment suppressed private response clonotypes in a responding patient (55.03% of T cells to 12.79%), while the amount of these clonotypes increased in a non-responding patient (18.65% to 37.86%), where treatment mostly affected the viral-specific clonotypes. In summary, our data suggest that the private response clonotypes in immune AA patients may recognise a common antigen, which was not predicted to be viral. Further, at the single-cell level AA signature clonotypes are of effector phenotype and fluctuate following immunosuppressive treatment. Monitoring of these clonotypes throughout treatment may provide insight into disease biology and variation in treatment responses. Figure Disclosures Blombery: Janssen: Honoraria; Novartis: Consultancy; Invivoscribe: Honoraria. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Research Funding; Pfizer: Research Funding.

Description: Background: Marginal zone lymphoma (MZL) is an incurable but heterogeneous disorder for which there is no standard of care treatment in the relapsed/refractory (R/R) setting. The BTK inhibitor, ibrutinib (IB) is well tolerated with an overall response rate (ORR) of 48% as monotherapy in R/R MZL but rarely complete remission (Noy et al, Blood, 2017). The BCL-2 inhibitor, venetoclax (VEN) has also been evaluated in a small number of R/R MZL patients (pts) with evidence of activity and tolerability (Davids et al, JCO, 2017). We sought to evaluate the efficacy and safety of combination IB and VEN in pts with MZL based upon the rationale of (1) distinct mechanisms of action, (2) activity as monotherapies in MZL, and (3) acceptable non-overlapping toxicity profiles. We report preliminary results of the MZL cohort of an investigator-initiated phase II trial of combination IB and Ven (NCT02471391). Methods: Pts with MZL by WHO 2008 criteria who were R/R or treatment naïve but considered inappropriate for treatment with chemotherapy were enrolled. Pts commenced treatment with IB monotherapy at a dose of 560mg per day. After 4 weeks, VEN was added to IB treatment, in weekly step-wise dose escalation over 6 weeks to a target dose of 800mg per day in the initial cohort (n = 4), amended to 400mg per day, due to a reported IB-VEN drug interaction in a similar trial (NCT02910583). The primary endpoint was the complete remission rate (CRR) at 16 weeks. The secondary endpoints were to determine ORR and CRR, to determine minimal residual disease (MRD) elimination rates, to describe progression free survival (PFS), overall survival, duration of response, time to progression and frequency and severity of adverse events. Investigator-assessed response assessments, based on IWG criteria (Cheson et al, JCO, 2007), were performed with CT at 4, 16, 28, 40 and 56 weeks, PET/CT at weeks 16 and 56, and bone marrow (BM) aspirate and trephine, and MRD assessments by flow cytometry at all time-points. Results: Fourteen of 15 planned pts were enrolled at the time of data cut-off (May 2019), of whom 12 had reached the week 16 primary endpoint or discontinued and are reported here. Two remain on study for

Description: The BCL2 inhibitor venetoclax has complete response rates of up to 50% in chronic lymphocytic leukemia patients, but secondary resistance reflecting acquired mutations in BCL2 can lead to treatment failure. Blombery et al report that an unexpectedly large number of patients carry multiple BCL2 mutations with subclonal variation in their occurrence.

Description: Background and Aims The detection of sequence variants and copy number changes can improve diagnosis, inform prognosis and guide treatment in patients with bone marrow failure syndromes (BMFS). We aimed to establish and prospectively assess the impact of comprehensive genomic evaluation on diagnostic categorisation and clinical outcomes in patients with genomically uncharacterised BMFS. Methods Eligible patients were recruited from four participating institutions across Victoria, Australia. Inclusion criteria were (i) age 〉3 months (ii) clinicopathological diagnosis or suspicion of either acquired aplastic anaemia (AA), inherited BMFS, hypoplastic myelodysplastic syndrome (hMDS) or a BMFS with marrow hypoplasia/aplasia not able to be definitively categorised. Patients initially underwent 90-gene targeted sequencing (Peter MacCallum Cancer Centre PanHaem and Myeloid Amplicon next generation sequencing [NGS] panels) for rapid turnaround of accredited results for clinical decision-making. In addition, whole exome sequencing (WES), whole genome copy number analysis, NGS T-cell receptor β (TRB) repertoire assessment and longitudinal monitoring of selected mutations by digital droplet PCR (ddPCR) were performed. All patients received pre-test counselling and assessment. Genomic results were reviewed in centralised multidisciplinary case conferences including the treating clinician, molecular haematopathologists, medical scientists, clinical geneticists and genetic counsellors. Results 100 patients were enrolled. Median age was 25 years (range 3 months - 80 years); 39% were under 18 years. Detection of sequence variants or copy number abnormalities led to or confirmed a diagnosis of either an inherited or acquired BMFS in 36 patients. In 17 patients a diagnosis of an inherited BMFS was positively made by detection of pathogenic sequence variants or copy number changes in FANCA(1 patient [pt]), FANCM(1 pt), FANCI(1 pt), RAD51C(1 pt), HAX1(1 pt), SBDS(1 pt), DNAJC21(1 pt), RPS19(5 pts), RPL35A(1 pt), TERT(1 pt), TINF2(1 pt) and SAMD9L(1 pt). In five patients the clinical BMFS was considered undifferentiated without a clear candidate gene suspected on phenotypic features prior to genomic evaluation. Importantly, an established diagnosis of AA was altered to an inherited BMFS by genomic characterisation in two patients (SAMD9L, FANCA). In 19 patients pathogenic sequence variants or copy number changes were detected either leading to or confirming a diagnosis of an acquired BMFS (paroxysmal nocturnal haemoglobinuria, hMDS or AA). Pathogenic sequence variants were detected in TET2(n=5), RUNX1(n=4), ASXL1(n=3), PIGA(n=3), DNMT3A(n=3),CBL(n=2), and BCOR/IDH2/SF3B1/SRSF2/TP53/U2AF1(n=1 each). Sequencing-detected copy number abnormalities included loss of chromosome 7 (n=6), losses on chromosome 5q (n=2) and copy number loss of ETV6(n=2). Longitudinal monitoring of an acquired truncating RUNX1 mutation by ddPCR resulted in one patient undergoing allogeneic bone marrow transplant for a progressively rising allelic burden. There was a trend towards more restricted TRB diversity in patients with genomically-defined acquired BMFS versus inherited BMFS (normalised Shannon index ≤0.85, 36.4% vs 0%, p=0.09). Conclusion We have established and evaluated a model of comprehensive multimodal genomic characterisation and multidisciplinary care for 100 patients with BMFS. Our results demonstrate a significant contribution to diagnostic categorisation and patient care in this area of clinical need. Disclosures Lieschke: CSL Behring Australia: Consultancy. Tam:Janssen: Honoraria, Research Funding; Gilead: Honoraria; AbbVie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Travel funding; Pharmacyclics: Honoraria; Beigene: Honoraria, Other: Travel funding; Roche: Honoraria; Beigene: Honoraria, Other: Travel funding; Gilead: Honoraria; Roche: Honoraria; AbbVie: Honoraria, Research Funding.

Description: Venetoclax induces high rates of response (~80%), including complete remissions (CR) in patients with heavily pre-treated chronic lymphocytic leukemia (CLL) through inhibition of BCL2. Despite achieving deep and durable responses, most patients will eventually experience disease progression on treatment. The molecular mechanisms that mediate clinical resistance to venetoclax in vivo are largely unknown. From a cohort of 67 relapsed CLL patients (Anderson et al, Blood 2017; 129:3362-3370) treated with venetoclax on three early phase clinical trials, we performed focussed genomic evaluation in those with CLL-type progressions (as opposed to large cell Richter's transformation). Targeted amplicon next generation sequencing of a panel of 33 genes recurrently mutated in lymphoid malignancy was performed where suitable pre- and post-progression samples were available. Twenty-one patients experienced CLL progression after a median of 36 months (range 6 - 73). Fifteen patients had paired samples for detailed analyses. A single heterozygous nucleotide variant was detected in BCL2 (NM_000633.2:c.302G〉T, p.(Gly101Val)) in progression samples in 7 of 15 patients (Fig 1A). Further investigation using a highly sensitive (limit of detection 0.01%) and specific droplet digital PCR (ddPCR) assay indicated that the Gly101Val mutation was first detected at low variant allele fraction after 19-42 months on venetoclax, up to 25 months earlier than when standard disease progression criteria were met. The Gly101Val was not detected prior to venetoclax treatment in this cohort and was not detected in a series of samples from patients treated at our institution who had not received venetoclax (CLL [n=74], NHL [n=198], myeloma [n=103]) nor has it been described in cancer (COSMIC) or population (gnomAD) databases. To investigate whether Gly101Val directly causes resistance to venetoclax, we expressed it in two B-lineage cell lines (RS4;11 and KMS-PE-12). Gly101Val cells were ~30-fold less sensitive to venetoclax than cells expressing wild-type (WT) BCL2. The Gly101Val mutation conferred a selective advantage during continuous exposure to sublethal concentrations of venetoclax in 3-week cultures. The same phenomena was observed with primary patient Gly101Val mutant cells in both short-term survival assays and when cultured in a bone marrow stromal model (Thijssen et al, Haematologica 2015;100:302-6). On stroma, primary cells bearing the Gly101Val mutation demonstrated markedly increased resistance to venetoclax with concentrations higher than achievable clinically in vivo. In the absence of venetoclax, the Gly101Val mutant demonstrated preserved normal function by protecting cell lines from apoptosis induced by cytotoxics with similar effectiveness to WT BCL2. In binding assays, the capacity for venetoclax to compete in vitro with BIM for binding to the Gly101Val mutant was markedly reduced (~180-fold) compared to WT BCL2. This is most likely explained by the presence of a bulkier valine residue in a region juxtaposed to the venetoclax binding groove (Fig 1B). In cell-based assays, whilst venetoclax readily displaced BAX and BAK from WT BCL2 it was ineffective when these pro-apoptotic molecules were bound to the Gly101Val mutant. We observed that not all CLL cells at progression carried the Gly101Val mutation. One patient harbored distinct subclones with and without the BCL2 Gly101Val mutation at progression. The subclone with exclusively WT BCL2 was observed to have elevated BCL-xL by mass cytometry (CyTOF), while the Gly101Val clone had minimal BCL-xL expression. Together these data indicate that whilst the Gly101Val mutation is sufficient to enable clinical resistance to venetoclax, alternative mechanisms may also mediate resistance in the same patient. In conclusion, we have identified and functionally characterized a novel recurrent BCL2 mutation (Gly101Val) emerging in a cohort of patients with CLL-type progressions treated with venetoclax. The BCL2 Gly101Val impairs binding of venetoclax to BCL2, confers resistance to venetoclax in both patient leukemia cells and engineered cell lines, and provides a selective growth advantage over wild-type cells when maintained in the presence of the drug in vitro. This mutation provides new insights into the pathobiology of venetoclax resistance and provides a potential biomarker of impending clinical relapse. Figure 1 Figure 1. Disclosures Anderson: Walter and Eliza Hall: Employment, Patents & Royalties; AbbVie, Inc: Research Funding; Genentech: Research Funding. Gong:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Thijssen:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Birkinshaw:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Teh:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Xu:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Flensburg:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Lew:Walter and Eliza Hall: Employment, Patents & Royalties. Majewski:Abbvie: Patents & Royalties: I am an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Gray:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Tam:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BeiGene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding. Seymour:AbbVie: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Consultancy. Czabotar:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Huang:The Walter and Eliza Hall Institute of Medical Research: Other: Institutional funding for venetoclax including milestone and royalty payments.. Roberts:Walter and Eliza Hall: Employment, Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone and royalty payments related to venetoclax; AbbVie: Research Funding; Genentech: Research Funding; Janssen: Research Funding.

Description: Highly active BTK inhibitors (BTKis) and the BCL2 inhibitor venetoclax have transformed the therapeutic landscape for chronic lymphocytic leukemia (CLL). Results of prospective clinical trials demonstrate the efficacy of venetoclax to salvage patients with disease progression on BTKis, but data on BTKi therapy after disease progression on venetoclax are limited, especially regarding durability of benefit. We retrospectively evaluated the records of 23 consecutive patients with relapsed/refractory CLL who received a BTKi (ibrutinib, n = 21; zanubrutinib, n = 2) after stopping venetoclax because of progressive disease. Median progression-free survival (PFS) and median overall survival after BTKi initiation were 34 months (range,

Description: Richter syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) to a high-grade B-cell lymphoma and is associated with an aggressive clinical course and poor prognosis. Conventional treatment options for RS are generally associated with low response rates and limited durability making this entity an area of significant unmet therapeutic need. Immune checkpoint inhibitor therapy has shown promise in the treatment of some aggressive lymphoma subtypes. In RS, modest benefits have been reported in small phase two trials of anti-PD-1 monotherapy and in combination with ibrutinib, however larger scale studies are lacking (Ding et al Blood, 2017; Jain et al Blood, 2016). We sought to characterise the immune-evasion phenotype of RS focussing on potential genetic biomarkers which may inform the selection of patients who are most likely to benefit from immune-directed therapies. We first assessed the gene expression of immune-checkpoint molecules given their potential clinical relevance and ability to be targeted by available therapeutic agents. Given immunohistochemical (IHC) assessment of immune-checkpoint molecules is recognized to be associated with high inter-observer variability and there is a high correlation between gene expression of immune-checkpoint molecules and IHC, we performed gene expression quantification using the Nanostring nCounter Human Immunology V2 panel (Nanostring Technologies, USA). Nanostring analysis was performed on samples from 17 patients with histologically confirmed RS (DLBCL subtype) and compared to 73 cases of de novo (non-transformed) DLBCL. Significant differences in the gene expression of checkpoint molecules was observed between RS and DLBCL biopsies, including higher expression of LAG3, PD1 and TIGIT in RS (p=0.0001, logFC 1.9; p=0.0017, logFC 1.1 and p=0.0437, logFC 0.7 vs DLBCL, respectively). PD-L2 and TIM3 gene expression were both significantly lower in RS compared to DLBCL (p = 0.0059, logFC 0.8; p = 0.012, logFC 0.8). PDL1 and CTLA4 gene expression did not significantly differ between RS and DLBCL. We next assessed the gene expression of T- and NK- cell markers (including CD3, CD4, CD8, FOXP3 and CD56) and the ratios of these markers to malignant B-cells (CD19). We observed no significant difference between RS and DLBCL, consistent with a similar relative quantity of immune cell infiltration between the two entities. Significantly higher gene expression of CD39, a marker of CD8+ T-cell exhaustion, was observed in RS than DLBCL (p = 0.031; logFC 0.5). Additional immune-related genes were next assessed, including those involved in antigen presentation (e.g. B2M, HLA molecules, TAP), immunosuppressive cytokine generation (e.g. ARG1, IDO1) and apoptosis resistance (e.g. FAS) which showed no significant differences in expression between RS and DLBCL. To assess whether these findings were consistent across other transformed lymphoma subtypes, we compared RS to a cohort of transformed follicular lymphoma (tFL, n=16) and transformed marginal zone lymphoma (tMZL, n=25). LAG3 expression was significantly higher in RS compared to both tFL and tMZL (p=0.0002, logFC 2.7; p=0.019, logFC 1.7). PD1 expression was also significantly higher in RS than tFL but not tMZL (p=0.0045, logFC 1.7; p=0.39, logFC 0.4). Given the established association of copy number amplifications involving immune checkpoint molecules (e.g. PD-L1/PD-L2 on 9p24.1) representing a potential predictive biomarker of response in other lymphomas, we performed hybridization-based NGS with whole genome copy number assessment to evaluate immune checkpoint gene loci in the three cohorts. No significant focal amplifications were detected in RS samples with overexpressed immune-checkpoint molecules. In contrast, three patients in the DLBCL/transformed cohort had focal copy number amplifications involving PD-L1. No copy number amplification of LAG3 was observed in either RS or DLBCL. In summary, we have observed significantly increased gene expression of LAG3, PD1 and TIGIT in RS compared to de novo DLBCL. Combined with increased gene expression of the exhausted cytotoxic T-cell marker CD39, these data provide a strong biological rationale for pursuing LAG3 inhibition either alone or in combination with other immune checkpoint blockade to enhance anti-tumour T cell responses in this difficult-to-treat entity. CG/JL/YK co-first authors Disclosures Gould: NovoNordisk: Other: Travel funding - domestic flights to attend education, May 2018. Villa:Roche, Abbvie, Celgene, Seattle Genetics, Lundbeck, AstraZeneca, Nanostring, Janssen, Gilead: Consultancy, Honoraria. Tam:Abbvie, Janssen: Research Funding; Abbvie, Janssen, Beigene, Roche, Novartis: Honoraria. Neeson:Roche Genetech: Research Funding; Allergan: Research Funding; Juno/Celgene: Research Funding; Compugen: Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Seymour:Roche: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding. Dickinson:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Merck Sharpe and Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Blombery:Invivoscribe: Honoraria; Novartis: Consultancy; Janssen: Honoraria.