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Dysregulation of immune-checkpoint receptors has been reported at diagnosis of chronic myeloid leukemia (CML), however, their role in the maintenance of remission after tyrosine kinase inhibitor (TKI) cessation is unclear. We assessed programmed cell death-1 (PD-1), T-cell immunoglobulin, and mucin-domain containing protein-3 (TIM-3), cytotoxic T-lymphocyte–associated protein-4 (CTLA-4), lymphocyte-activation gene-3 (LAG-3), and T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT) expression on T-cell subsets, regulatory T cells (T-regs), and natural killer (NK) cells at the time of TKI cessation in 44 patients (22 patients sustained treatment-free remission [TFR] and 22 experienced molecular relapse [MolR]). We confirmed our previous finding that absolute numbers of T-regs are increased in patients who experienced MolR compared with those who sustained TFR. The immune-checkpoint receptors PD-1, CTLA-4, LAG-3, and TIGIT on T or NK cells were not differentially expressed between the MolR and TFR groups. However, TIM-3 was consistently upregulated on bulk T cells (CD3+) and T-cell subsets including, CD4+ T cells, CD8+ T cells, and T-regs, in patients who relapsed in comparison with those who maintained TFR after discontinuation. Furthermore, gene expression analysis from publicly available data sets showed increased TIM-3 expression on CML stem cells compared with normal hematopoietic stem cells. These findings suggest that among the targetable immune-checkpoint molecules, TIM-3 blockade may potentially improve effector immune response in patients with CML stopping TKI, while concomitantly targeting leukemic stem cells and could be a promising therapeutic strategy for preventing relapse after cessation of TKI in patients with CML.

Tyrosine kinase inhibitor therapy revolutionized chronic myeloid leukemia treatment and showed how targeted therapy and molecular monitoring could be used to substantially improve survival outcomes. We used chronic myeloid leukemia as a model to understand a critical question: why do some patients have an excellent response to therapy, while others have a poor response? We studied gene expression in whole blood samples from 112 patients from a large phase III randomized trial (clinicaltrials gov. Identifier: NCT00471497), dichotomizing cases into good responders (BCR::ABL1 ≤10% on the International Scale by 3 and 6 months and ≤0.1% by 12 months) and poor responders (failure to meet these criteria). Predictive models based on gene expression demonstrated the best performance (area under the curve =0.76, standard deviation =0.07). All of the top 20 pathways overexpressed in good responders involved immune regulation, a finding validated in an independent data set. This study emphasizes the importance of pretreatment adaptive immune response in treatment efficacy and suggests biological pathways that can be targeted to improve response.

Refereed/Peer-reviewed Chronic myeloid leukemia is characterized by a single genetic abnormality resulting in a fusion gene whose mRNA product is easily detected and quantified by reverse-transcriptase polymerase chain reaction analysis. Measuring residual disease was originally introduced to identify patients relapsing after allogeneic stem cell transplantation but rapidly adopted to quantify responses to tyrosine kinase inhibitors. Real-time quantitative polymerase chain reaction is now an essential tool for the management of patients and is used to influence treatment decisions. In this review we track this development including the international collaboration to standardize results, discuss the integration of molecular monitoring with other factors that affect patients’ management, and describe emerging technology. Four case histories describe varying scenarios in which the accurate measurement of residual disease identified patients at risk of disease progression and allowed appropriate investigations and timely clinical intervention.

Mutation detection is increasingly used for the management of hematological malignancies. Prior whole transcriptome and whole exome sequencing studies using total RNA and DNA identified diverse mu-tation types in cancer-related genes associated with treatment failure in patients with chronic myeloid leukemia. Variants included single-nucleotide variants and small insertions/deletions, plus fusion transcripts and partial or whole gene deletions. The hypothesis that all of these mutation types could be detected by a single cost-effective hybridization capture next-generation sequencing method using total RNA was assessed. A method was developed that targeted 130 genes relevant for myeloid and lymphoid leukemia. Retrospective samples with 121 precharacterized variants were tested using total RNA and/or DNA. Concordance of detection of precharacterized variants using RNA or DNA was 96%, whereas the enhanced sensitivity identified additional variants. Comparison between 24 matched DNA and RNA samples demonstrated 95.3% of 170 variants detectable using DNA were detected using RNA, including all but one variant predicted to activate nonsense-mediated decay. RNA identified an addi-tional 10 variants, including fusion transcripts. Furthermore, the true effect of splice variants on RNA splicing was only evident using RNA. In conclusion, capture sequencing using total RNA alone is suitable for detecting a range of variants relevant in chronic myeloid leukemia and may be more broadly applied to other hematological malignancies where diverse variant types define risk groups. Refereed/Peer-reviewed

Purpose: Imatinib induces a durable response in most patients with Philadelphia chromosome–positive chronic myeloid leukemia, but it is currently unclear whether imatinib reduces the leukemic stem cell (LSC) burden, which may be an important step toward enabling safe discontinuation of therapy. In this article, we use mathematical models of BCR–ABL levels to make inferences on the dynamics of LSCs.Experimental Design: Patients with at least 1 BCR–ABL transcript measurement on imatinib were included (N = 477). Maximum likelihood methods were used to test 3 potential hypotheses of the dynamics of BCR–ABL transcripts on imatinib therapy: (i) monoexponential, in which there is little, if any, decline in BCR–ABL transcripts; (ii) biexponential, in which patients have a rapid initial decrease in BCR–ABL transcripts followed by a more gradual response; and (iii) triexponential, in which patients first exhibit a biphasic decline but then have a third phase when BCR–ABL transcripts increase rapidly.Results: We found that most patients treated with imatinib exhibit a biphasic decrease in BCR–ABL transcript levels, with a rapid decrease during the first few months of treatment, followed by a more gradual decrease that often continues over many years.Conclusions: We show that the only hypothesis consistent with current data on progenitor cell turnover and with the long-term, gradual decrease in the BCR–ABL levels seen in most patients is that these patients exhibit a continual, gradual reduction of the LSCs. This observation may explain the ability to discontinue imatinib therapy without relapse in some cases. Clin Cancer Res; 17(21); 6812–21. ©2011 AACR.

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CCR Translation for This Article from BCR–ABL Transcript Dynamics Support the Hypothesis That Leukemic Stem Cells Are Reduced during Imatinib Treatment

The BCR::ABL1 gene fusion initiates chronic myeloid leukemia (CML), however evidence has accumulated from studies of highly selected cohorts that variants in other cancer-related genes are associated with treatment failure. Nevertheless, the true incidence and impact of additional genetic abnormalities (AGAs) at diagnosis of chronic phase (CP)-CML is unknown. We sought to determine whether AGAs at diagnosis in a consecutive imatinib-treated cohort of 210 patients enrolled in the TIDEL-II trial influenced outcome despite a highly proactive treatment intervention strategy. Survival outcomes including overall survival, progression-free survival, failure-free survival and BCR::ABL1 kinase domain mutation acquisition were evaluated. Molecular outcomes were measured at a central laboratory and included major molecular response (MMR, BCR::ABL1 ≤0.1%IS), MR4 (BCR::ABL1 ≤0.01%IS) and MR4.5 (BCR::ABL1 ≤0.0032%IS). AGAs included variants in known cancer genes and novel rearrangements involving the formation of the Philadelphia chromosome. Clinical outcomes and molecular response were assessed based on the genetic profile and other baseline factors. AGAs were identified in 31% of patients. Potentially pathogenic variants in cancer-related genes were detected in 16% of patients at diagnosis (including gene fusions and deletions) and structural rearrangements involving the Philadelphia chromosome (Ph-associated rearrangements), detected in 18%. Multivariable analysis demonstrated that the combined genetic abnormalities plus the ELTS clinical risk score were independent predictors of lower molecular response rates and higher treatment failure. Despite a highly proactive treatment intervention strategy, first-line imatinib-treated patients with AGAs had poorer response rates. This data provides evidence for the incorporation of genomically-based risk assessment for CML.

Refereed/Peer-reviewed Cancer treatment is constantly evolving from a one-size-fits-all towards bespoke approaches for each patient. In certain solid cancers, including breast and lung, tumor genome profiling has been incorporated into therapeutic decision-making. For chronic phase chronic myeloid leukemia (CML), while tyrosine kinase inhibitor therapy is the standard treatment, current clinical scoring systems cannot accurately predict the heterogeneous treatment outcomes observed in patients. Biomarkers capable of segregating patients according to outcome at diagnosis are needed to improve management, and facilitate enrollment in clinical trials seeking to prevent blast crisis transformation and improve the depth of molecular responses. To this end, gene expression (GE) profiling studies have evaluated whether GE signatures at diagnosis are clinically informative. Patient material from a variety of sources has been profiled using microarrays, RNA sequencing and, more recently, single-cell RNA sequencing. However, differences in the cell types profiled, the technologies used, and the inherent complexities associated with the interpretation of genomic data pose challenges in distilling GE datasets into biomarkers with clinical utility. The goal of this paper is to review previous studies evaluating GE profiling in CML, and explore their potential as risk assessment tools for individualized CML treatment. We also review the contribution that acquired mutations, including those seen in clonal hematopoiesis, make to GE profiles, and how a model integrating contributions of genetic and epigenetic factors in resistance to tyrosine kinase inhibitors and blast crisis transformation can define a route to GE-based biomarkers. Finally, we outline a four-stage approach for the development of GE-based biomarkers in CML.

Myeloid neoplasms and acute leukemias derive from the clonal expansion of hematopoietic cells driven by somatic gene mutations. Although assessment of morphology plays a crucial role in the diagnostic evaluation of patients with these malignancies, genomic characterization has become increasingly important for accurate diagnosis, risk assessment, and therapeutic decision making. Conventional cytogenetics, a comprehensive and unbiased method for assessing chromosomal abnormalities, has been the mainstay of genomic testing over the past several decades and remains relevant today. However, more recent advances in sequencing technology have increased our ability to detect somatic mutations through the use of targeted gene panels, whole-exome sequencing, whole-genome sequencing, and whole-transcriptome sequencing or RNA sequencing. In patients with myeloid neoplasms, whole-genome sequencing represents a potential replacement for both conventional cytogenetic and sequencing approaches, providing rapid and accurate comprehensive genomic profiling. DNA sequencing methods are used not only for detecting somatically acquired gene mutations but also for identifying germline gene mutations associated with inherited predisposition to hematologic neoplasms. The 2022 International Consensus Classification of myeloid neoplasms and acute leukemias makes extensive use of genomic data. The aim of this report is to help physicians and laboratorians implement genomic testing for diagnosis, risk stratification, and clinical decision making and illustrates the potential of genomic profiling for enabling personalized medicine in patients with hematologic neoplasms.

Refereed/Peer-reviewed

Atypical BCR-ABL1 transcripts occur in around 3% of patients with chronic phase chronic myeloid leukemia (CML) [1]. Due to the rarity of each individual transcript type there are limited data on th...

Approximately half of patients with chronic myeloid leukemia (CML) in sustained deep molecular response who discontinue tyrosine kinase inhibitors (TKIs) remain in treatment-free remission (TFR). Some of these patients have measurable residual disease (MRD) by BCR-ABL1 mRNA testing, and most have detectable BCR-ABL1 DNA by highly sensitive methods. We used fluorescence-activated cell sorting and BCR-ABL1 DNA PCR to investigate the lineage of residual CML cells in TFR. Twenty patients in TFR for >1 year provided blood for sorting into granulocytes, monocytes, B cells, T cells, and NK cells. MRD was identified predominantly in the lymphoid compartment and never in granulocytes. B cells were more often BCR-ABL1 positive than T cells (18 vs 11/20 patients) and at higher levels (median 10−4.9 vs 10−5.7; P = 0.014). In 13 CML patients studied at diagnosis lymphocytes expressing BCR-ABL1 mRNA comprised a small proportion of total leukocytes. These data improve our understanding of TFR biology, since it is now clear that MRD in the blood of TFR patients need not imply the persistence of multipotent CML cells. Lineage-specific assessment of MRD could be explored as a means to improve the prediction of TFR Refereed/Peer-reviewed

With the focus of leukaemia management shifting to the implications of low-level disease burden, increasing attention is being paid on the development of highly sensitive methodologies required for detection. There are various techniques capable of identification of measurable residual disease (MRD) either evidencing as relevant mutation detection [e.g. nucleophosmin 1 (NPM1) mutation] or trace levels of leukaemic clonal populations. The vast majority of these methods only permit detection of a single clone or mutation. However, mass spectrometry and next-generation sequencing enable the interrogation of multiple genes simultaneously, facilitating a more complete genomic profile. In the present review, we explore the methodologies of both techniques in conjunction with the important advantages and limitations associated with each assay. We also highlight the evidence and the various instances where either technique has been used and propose future strategies for MRD detection. Refereed/Peer-reviewed

Refereed/Peer-reviewed Response to tyrosine kinase inhibitor (TKI) therapy in CML can be measured by haematological, cytogenetic and molecular criteria. Achieving haematological and cytogenetic response is still a desirable early measure of response. However measurement of the level of BCR-ABL1 transcripts in the blood by qRT-PCR, adjusted to the international scale (IS), is now the primary means of monitoring response. Achieving time-dependent molecular targets is a strong predictor of survival as well as indicating the longer-term prospects of achieving eligibility for treatment-free remission (TFR). Optimal response can be determined solely by the achievement and maintenance of time-dependent molecular response levels. Likewise, treatment failure is largely defined by a failure to achieve molecular milestones or loss of that molecular response, except in cases where treatment failure is evident by progression to advanced-phase disease. Treatment failure mandates a switch in therapy, usually to a more potent TKI, if possible. In between optimal response and treatment failure, there is a “warning category” where outcomes are generally inferior and therapeutic decisions are more complex. Recently, the importance of determining the dynamics of response, not just the BCR-ABL1 level at one specified time point, has been recognized, particularly when determining the probability of achieving sustained TFR in eligible patients.

Blast crisis (BC) predicts dismal outcomes in patients with chronic myeloid leukaemia (CML). Although additional genetic alterations play a central role in BC, the landscape and prognostic impact of these alterations remain elusive. Here, we comprehensively investigate genetic abnormalities in 136 BC and 148 chronic phase (CP) samples obtained from 216 CML patients using exome and targeted sequencing. One or more genetic abnormalities are found in 126 (92.6%) out of the 136 BC patients, including the RUNX1-ETS2 fusion and NBEAL2 mutations. The number of genetic alterations increase during the transition from CP to BC, which is markedly suppressed by tyrosine kinase inhibitors (TKIs). The lineage of the BC and prior use of TKIs correlate with distinct molecular profiles. Notably, genetic alterations, rather than clinical variables, contribute to a better prediction of BC prognosis. In conclusion, genetic abnormalities can help predict clinical outcomes and can guide clinical decisions in CML., In chronic myeloid leukaemia (CML), the drivers of blast crisis and resistance to tyrosine kinase inhibitors are not fully characterised. Here, the authors analyse a cohort of CML samples with genomic technologies and find that at least one driver alteration is associated with progression and worse prognosis.

Simple Summary This study provides new insights into the changing transcriptomic and epigenomic landscapes in chronic myeloid leukaemia (CML) patients who are receiving tyrosine kinase inhibitor (TKI) therapy (often life-long). Alternative splicing, vital for cellular homeostasis, is dysregulated in human cancers. Remarkably, we found abnormal splicing patterns despite molecular remission in peripheral blood cells of chronic-phase CML patients. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Abstract Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy individuals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled samples from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission samples from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis samples. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease.

Refereed/Peer-reviewed Chronic myeloid leukemia (CML) represents the disease prototype of genetically based diagnosis and management. Tyrosine kinase inhibitors (TKIs), that target the causal BCR::ABL1 fusion protein, exemplify the success of molecularly based therapy. Most patients now have long-term survival; however, TKI resistance is a persistent clinical problem. TKIs are effective in the BCR::ABL1-driven chronic phase of CML but are relatively ineffective for clinically defined advanced phases. Genomic investigation of drug resistance using next-generation sequencing for CML has lagged behind other hematological malignancies. However, emerging data show that genomic abnormalities are likely associated with suboptimal response and drug resistance. This has already been supported by the presence of BCR::ABL1 kinase domain mutations in drug resistance, which led to the development of more potent TKIs. Next-generation sequencing studies are revealing additional mutations associated with resistance. In this review, we discuss the initiating chromosomal translocation that may not always be a straightforward reciprocal event between chromosomes 9 and 22 but can sometimes be accompanied by sequence deletion, inversion, and rearrangement. These events may biologically reflect a more genomically unstable disease prone to acquire mutations. We also discuss the future role of cancer-related gene mutation analysis for risk stratification in CML.

Accurate quantification of minimal residual disease during treatment of chronic myeloid leukaemia guides clinical decisions. The conventional minimal residual disease method, RQ-PCR for BCR-ABL1 mRNA, reflects a composite of the number of circulating leukemic cells and the BCR-ABL1 transcripts per cell. BCR-ABL1 genomic DNA only reflects leukemic cell number. We used both methods in parallel to determine the relative contribution of the leukemic cell number to molecular response. BCR-ABL1 DNA PCR and RQ-PCR were monitored up to 24 months in 516 paired samples from 59 newly-diagnosed patients treated with first-line imatinib in the TIDEL-II study. In the first 3 months of treatment BCR-ABL1 mRNA values declined more rapidly than DNA. By 6 months the two measures aligned closely. The expression of BCR-ABL1 mRNA was normalized to cell number to generate an expression ratio. The expression of e13a2 BCR-ABL1 was lower than that of e14a2 transcripts at multiple time points during treatment. BCR-ABL1 DNA was quantifiable in 48% of samples with undetectable BCR-ABL1 mRNA, resulting in minimal residual disease being quantifiable for an additional 5-18 months (median 12 months). These parallel studies show for the first time that the rapid decline in BCR-ABL1 mRNA over the first 3 months of treatment is due to a reduction in both cell number and transcript level per cell, whereas beyond 3 months falling levels of BCR-ABL1 mRNA are predominantly due to depletion of leukaemic cells. Refereed/Peer-reviewed

usc Background: Some patients receiving a tyrosine kinase inhibitor (TKI) for the first-line treatment of chronic phase chronic myeloid leukemia (CML-CP) experience intolerable adverse events. Management strategies in-clude dose adjustments, interrupting or discontinuing therapy, or switching to an alternative TKI. Methods: This multicenter, single-arm, Phase IIIb study included CML-CP patients intolerant of, but responsive to, first-line treatment with imatinib or dasatinib. All patients were switched to nilotinib 300 mg bid for up to 24 months. The primary endpoint was achievement of MR4.5 (BCR-ABL transcript level of ≤ 0.0032% on the International Scale) by 24 months. Results: Twenty patients were enrolled in the study (16 imatinib-intolerant, 4 dasatinib-intolerant); which was halted early because of low recruitment. After the switch to nilotinib 300 mg bid, MR4.5 at any time point up to month 24 was achieved in 10 of 20 patients (50%) in the full analysis set. Of the non-hematological adverse events associated with intolerance to prior imatinib or dasatinib, 74% resolved within 12 weeks of switching tonilotinib 300 mg bid. Conclusion: Nilotinib 300 mg bid shows minimal cross intolerance in patients with CML-CP who have prior toxicities to other TKIs and can lead to deep molecular responses. Refereed/Peer-reviewed

Introduction: Treatment Free Remission (TFR) is the ultimate goal of therapy for most CML patients. Despite adopting consensus eligibility criteria of a sustained deep molecular response and more than 4 years of TKI therapy, the relapse rate after TKI cessation is still around 50%. More sensitive detection of residual leukaemia has the potential to improve our capacity to predict TFR outcomes for individual patients. Aim: To correlate droplet digital PCR (ddPCR) assay results with TFR outcome, especially in the setting of undetectable levels measured by qRT-PCR. Method: ddPCR was performed on blood samples from 51 TFR-eligible CML patients at the time of TKI cessation. 5 µg RNA per sample was used in 8 wells/sample using the BioRad QXDx BCR-ABL %IS kit on QX200 ddPCR system which yielded BCR-ABL1% (IS) directly. All these patients achieved MR4.5 that was sustained for ≥ 2 years. Results: 100% of patient were in MR4.5 via qRT-PCR at the time of stopping. 61% of the 51 patients evaluated relapsed within 12 months. Median duration of TKI therapy for the whole group was 5.8 years (range 2.2- 14 years). 20 patients achieved TFR success with a median follow up of 24 months (TFR group; sustained BCR-ABL1 0.1% (IS) after stopping; median time of relapse 3 months, range 1-10 months). A ROC curve analysis correlated TFR outcome with ddPCR results, with BCR-ABL1 level ≥0.003% via ddPCR at the time of stopping identified as an optimal cut-off. Kaplan-Meier analysis showed that 89% of the patients with ddPCR ≥0.003 relapsed after TKI cessation, whereas the ddPCR We next assessed other known predictors of TFR success relative to ddPCR results in a Cox proportional hazard model. We have previously demonstrated that the BCR-ABL1 halving time after commencing therapy is highly predictive of TFR. At a univariate level, transcript type (e13a2 versus e14a2, p=0.01), BCR-ABL1 halving time (p>0.0001), and mRNA quantitation by ddPCR ≥ 0.003% (p=0.02) were all significantly associated with clinical outcome. Other variables including gender, age, ELTS score, Sokal score, MR4.5 duration and TKI duration were not associated with clinical outcome in this cohort (Figure 1B). In the multivariate analyses (Figure 1C), ddPCR remained an independent predictor after adjusting for ELTS, TKI duration and MR4.5 duration. Interestingly, ddPCR was not an independent predictor after adjusting for BCR-ABL1 transcript type or halving time. Conclusion: QXDx ddPCR assay is a promising tool for molecular residual disease monitoring in CML, especially when the BCR-ABL1 is undetectable by conventional method. The CML patients with levels of detectable BCR-ABL1 ≥0.003% measured by ddPCR have a significantly higher probability of relapse compared to patients with lower levels of the transcript. The ≥0.003% BCR-ABL1 level cut-off value could be a potential tool to aid decision-making when attempting TKI discontinuation in CML. However, even though a measurable level of BCR-ABL1 above 0.003% via ddPCR identified patients at high risk of relapse after a TFR attempt, it does not rule out the possibility of TFR; and a negative ddPCR result does not exclude the risk of molecular relapse. ddPCR may be most useful where other TFR predictive factors including BCR-ABL1 transcript type and halving time are not available. In-kind support was received from Bio-Rad for this study. Figure 1 Figure 1. Disclosures Branford: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Cepheid: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hughes: Novartis: Honoraria, Research Funding; Incyte: Honoraria; BMS: Honoraria, Research Funding. Yeung: BMS: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria.

Chronic myeloid leukaemia (CML) is driven by the activity of the BCR-ABL1 fusion oncoprotein. ABL1 kinase inhibitors have improved the clinical outcomes for patients with CML, with over 80% of patients treated with imatinib surviving for more than 10 years. Second-generation ABL1 kinase inhibitors induce more potent molecular responses in both previously untreated and imatinib-resistant patients with CML. Studies in patients with chronic-phase CML have shown that around 50% of patients who achieve and maintain undetectable BCR-ABL1 transcript levels for at least 2 years remain disease-free after the withdrawal of treatment. Here we characterize ABL001 (asciminib), a potent and selective allosteric ABL1 inhibitor that is undergoing clinical development testing in patients with CML and Philadelphia chromosome-positive (Ph +) acute lymphoblastic leukaemia. In contrast to catalytic-site ABL1 kinase inhibitors, ABL001 binds to the myristoyl pocket of ABL1 and induces the formation of an inactive kinase conformation. ABL001 and second-generation catalytic inhibitors have similar cellular potencies but distinct patterns of resistance mutations, with genetic barcoding studies revealing pre-existing clonal populations with no shared resistance between ABL001 and the catalytic inhibitor nilotinib. Consistent with this profile, acquired resistance was observed with single-agent therapy in mice; however, the combination of ABL001 and nilotinib led to complete disease control and eradicated CML xenograft tumours without recurrence after the cessation of treatment. Refereed/Peer-reviewed

Background We previously reported a high incidence of mutated cancer-related genes at CML diagnosis in selected chronic phase patients with a poor outcome compared to those with a good outcome. We also found a novel class of variant associated with the formation of the Ph chromosome comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were termed 'Ph-associated events' and were more frequent in patients with poor outcome. However, the risk attributable to these mutational events at diagnosis has not been defined in unselected cohorts. Aim To assess the impact of genomic events in a cohort of consecutively treated patients at diagnosis of chronic phase CML. Methods A hybridization capture sequencing method targeting genes implicated in myeloid and lymphoid malignancies was applied to diagnostic RNA of patients enrolled in the TIDEL II trial. Patients were treated with upfront imatinib with active intervention, dose escalation or nilotinib switch, primarily for lack of time-dependent molecular milestones. Single base variants, small insertions/deletions, splice variants, gene fusions, and focal gene deletions were assessed with pre-defined criteria for pathogenicity. These were further classified as pathogenic mutations in cancer-related genes or Ph-associated events. Univariate and multivariate analyses were performed to evaluate the influence of mutational events and other key clinical and demographic variables on outcome at 4 years. Failure events were as defined by the ELN 2020 recommendations. Results 160/210 TIDEL II patients have so far been sequenced. 33 relevant mutations with variant allele frequencies ≥5% were identified in 9 genes in 25 patients (16%). ASXL1 was most frequently mutated (10% of all patients) and other recurrently mutated genes at diagnosis were RUNX1, BCORL1, IKZF1 and DNMT3A. Ph-associated events occurred in 25 patients (16%). Most of these (14/25 patients) involved fusions between genes on chromosomes 9 and 22 consistent with deletions adjacent to BCR and ABL1, or fusions between BCR or ABL1 and genes/regions on chromosomes other than 9 or 22. These were consistent with variant translocation and some were cytogenetically cryptic. Among these and other Ph-associated events were complex rearrangements involving inversions and large duplications. These were detected in 14/25 patients. Five patients had both cancer-related gene mutations and Ph-associated events, totalling 45 patients (28%) with at least 1 genomic event. Cancer-related mutations at diagnosis were associated with inferior progression-free survival (PFS) 82% vs 91% P=.03, and failure-free survival (FFS) 55% vs 83% P Independent predictors of all survival and molecular outcomes were assessed with univariate and multivariate modelling (Table). Candidate prognostic variables were age at diagnosis, sex, transcript, Sokal and ELTS scores and the genomic variables. The only independent predictor of PFS was mutations in cancer-related genes. Cancer-related gene mutations, Ph-associated events and the ELTS score were independent predictors of FFS, MMR and MR4. We evaluated whether genomic data could be additive to the ELTS risk score. Low risk ELTS patients with any mutational event had inferior outcomes: FFS 76% vs 86%, P=.07; MMR 75% vs 92%, P=.02; MR4 41% vs 75%, P=0.006. Similar findings were observed in Intermediate risk ELTS patients: FFS 22% vs 91%, P Conclusion Despite a proactive strategy for TKI switch and a higher imatinib starting dose, the presence of cancer-related gene mutations or Ph-associated events conferred inferior outcomes. Combining the ELTS score with any mutational event further differentiated patient outcomes, demonstrating the power of integrating genomic data with current risk stratification. Disclosures Shanmuganathan: Janssen: Other: travel expenses; Novartis: Honoraria, Other: Travel expenses; Gilead: Other: Travel expenses; Amgen: Other: travel expenses. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Branford:Bristol Myers Squibb: Honoraria; Cepheid: Honoraria, Membership on an entity's Board of Directors or advisory committees; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Purpose of Review: Identification of the BCR-ABL1 fusion oncogene in patients diagnosed with chronic myeloid leukemia (CML) led to the development of targeted therapy responsible for the dramatic survival benefits observed in the past two decades. However, despite these revolutionary findings, there remains marked disparity in patient outcomes. Why do some patients present de novo while others evolve to the more aggressive stages of CML? Why can select patients successfully discontinue therapy as part of a treatment-free remission attempt whereas others fail to meet specific molecular milestones? Recent Findings: BCR-ABL1 kinase mutations are only identified in approximately 50% of patients with poor responses and disease progression, suggesting the presence of alternative resistance mechanisms. Numerous institutions have identified the presence of additional genomic events in addition to BCR-ABL1 with the increasing availability of next-generation sequencing. Summary: We explore the potential pathways and events that may cooperate with BCR-ABL1 to answer these questions but also challenge the fundamental tenet that BCR-ABL1 is always the sole event initiating CML. Refereed/Peer-reviewed

Blast crisis of chronic myeloid leukemia is associated with poor survival and the accumulation of genomic lesions. Using whole-exome and/or RNA sequencing of patients at chronic phase (CP, n = 49), myeloid blast crisis (MBC, n = 19), and lymphoid blast crisis (LBC, n = 20), we found 25 focal gene deletions and 14 fusions in 24 patients in BC. Deletions predominated in LBC (83% of structural variants). Transcriptional analysis identified the upregulation of genes involved in V(D)J recombination, including RAG1/2 and DNTT in LBC. RAG recombination is a reported mediator of IKZF1 deletion. We investigated the extent of RAG-mediated genomic lesions in BC. Molecular hallmarks of RAG activity; DNTT-mediated nucleotide insertions and a RAG-binding motif at structural variants were exclusively found in patients with high RAG expression. Structural variants in 65% of patients in LBC displayed these hallmarks compared with only 5% in MBC. RAG-mediated events included focal deletion and novel fusion of genes associated with hematologic cancer: IKZF1, RUNX1, CDKN2A/B, and RB1. Importantly, 8/8 patients with elevated DNTT at CP diagnosis progressed to LBC by 12 months, potentially enabling early prediction of LBC. This work confirms the central mutagenic role of RAG in LBC and describes potential clinical utility in CML management. Refereed/Peer-reviewed

David M. Ross, Ilaria S. Pagani, Yazad D. Irani, Jade Clarson, Tamara Leclercq, Phuong Dang, Jennifer McLean, Verity A. Saunders, Lisa Carne, John Reynolds, David S. Ritchie, Deborah L. White, Susan Branford, Timothy P. Hughes, Agnes S. M. Yong

The management of CML in pregnancy is challenging with the need to balance disease control against potential teratogenic effects of TKI therapy. In this multi-center case-cohort study of 16 women in chronic phase, CML ceased TKI treatment pre- or post-conception during their first pregnancy. Thirteen patients were on imatinib; 9 ceased their TKI prior to conception and 7 ceased at pregnancy confirmation. Twelve patients had achieved either MMR or better at time of TKI cessation. Eleven women lost MMR during pregnancy and two patients lost CHR. Fourteen women reestablished MMR on TKI recommenced. The depth molecular response prior to conception appeared to correlate well with restoration of disease control on TKI recommencement though duration of MMR did not appear to be as important. While interruption of TKI treatment for pregnancy usually leads to loss of molecular response, loss of hematological response is uncommon and disease control is reestablished with resumption of therapy in the majority of women. Refereed/Peer-reviewed

The third-generation tyrosine kinase inhibitor (TKI) ponatinib shows activity against all common BCR-ABL1 single mutants, including the highly resistant BCR-ABL1-T315I mutant, improving outcome for patients with refractory chronic myeloid leukemia (CML). However, responses are variable, and causal baseline factors have not been well-studied. The type and number of low-level BCR-ABL1 mutations present after imatinib resistance has prognostic significance for subsequent treatment with nilotinib or dasatinib as second-line therapy. We therefore investigated the impact of low-level mutations detected by sensitive mass-spectrometry before ponatinib initiation (baseline) on treatment response in 363 TKI-resistant patients enrolled in the PONATINIB for Chronic Myeloid Leukemia Evaluation and Ph+ Acute Lymphoblastic Leukemia trial, including 231 patients in chronic phase (CP-CML). Low-level mutations were detected in 53 patients (15%, including low-level T315I in 14 patients); most, however, did not undergo clonal expansion during ponatinib treatment and, moreover, no specific individual mutations were associated with inferior outcome. We demonstrate however, that the number of mutations detectable by mass spectrometry after TKI resistance is associated with response to ponatinib treatment and could be used to refine the therapeutic approach. Although CP-CML patients with T315I (63/231, 27%) had superior responses overall, those with multiple mutations detectable by mass spectrometry (20, 32%) had substantially inferior responses compared with those with T315I as the sole mutation detected (43, 68%). In contrast, for CP-CML patients without T315I, the inferior responses previously observed with nilotinib/dasatinib therapy for imatinib-resistant patients with multiple mutations were not seen with ponatinib treatment, suggesting that ponatinib may prove to be particularly advantageous for patients with multiple mutations detectable by mass spectrometry after TKI resistance. Refereed/Peer-reviewed

AimsRT-qPCR is used to quantify minimal residual disease (MRD) in chronic myeloid leukaemia (CML) in order to make decisions on treatment, but its results depend on the level of BCR-ABL1 expression as well as leukaemic cell number. The aims of the study were to quantify inter-individual differences in expression level, to determine the relationship between expression level and response to treatment, and to investigate the effect of expression level on interpretation of the RT-qPCR result.MethodsBCR-ABL1 expression was studied in 248 samples from 65 patients with CML by determining the difference between MRD quantified by RT-qPCR and DNA-qPCR. The results were analysed statistically and by simple indicative modelling.ResultsInter-individual levels of expression approximated a normal distribution with an SD of 0.36 log. Expression at diagnosis correlated with expression during treatment. Response to treatment, as measured by the number of leukaemic cells after 3, 6 or 12 months of treatment, was not related to the level of expression. Indicative modelling suggested that interpretation of RT-qPCR results in relation to treatment guidelines could be affected by variation in expression when MRD was around 10% at 3 months and by both expression variation and Poisson variation when MRD was around or below the limit of detection of RT-qPCR.ConclusionsVariation between individuals in expression of BCR-ABL1 can materially affect interpretation of the RT-qPCR when this test is used to make decisions on treatment.

Context For patients with CML, achieving sustained DMR is a prerequisite for attempting treatment-free remission. However, biomarkers that predict DMR are unknown. Objective Identify biomarkers associated with DMR. Design Exploratory analysis of samples from a phase 3, randomized, open-label study (NCT00471497). Patients Whole blood samples collected prior to tyrosine kinase inhibitor (TKI) therapy from 112 patients in the nilotinib 300-mg twice-daily (n=33), nilotinib 400-mg twice-daily (n=32), or imatinib 400-mg once-daily (n=47) arms of ENESTnd were analyzed using RNA sequencing. Samples from poor responders (BCR-ABL1IS > 10% by 3 months of therapy) and good responders (BCR-ABL1IS Main outcome measures Using multivariate logistic regression, the association of clinical variables (e.g., Sokal risk score, TKI, age, sex) with responder status was assessed. Additionally, we applied penalized regression to clinical variables and gene expression (13,569 genes) in independent and combined (gene and clinical) models to develop a predictive model of responder status. Results By multivariate analysis, younger age ( Conclusions Our gene expression model differentiates patients who achieved DMR from those with poor treatment response at 5 years. This work could be used to develop therapeutic targets and facilitate DMR in patients who would otherwise be poor responders. Study sponsored by Novartis Pharmaceuticals Corporation. This abstract was accepted and previously published at the 61st ASH® Annual Meeting and Exposition.

Therapy-related myeloid neoplasms (T-MN) are poorly characterized secondary hematological malignancies following chemotherapy/radiotherapy exposure. We compared the clinical and mutational characteristics of T-MN (n = 129) and primary myelodysplastic syndrome (P-MDS, n = 108) patients. Although the somatic mutation frequency was similar between T-MN and P-MDS patients (93% in both groups), the pattern was distinct. TP53 mutations were more frequent in T-MN (29.5 vs. 7%), while spliceosomal complex mutations were more common in P-MDS (56.5 vs. 25.6%). In contrast to P-MDS, the ring sideroblasts (RS) phenotype was not associated with better survival in T-MN, most probably due to genetic association with TP53 mutations. SF3B1 was mutated in 96% of P-MDS with ≥15% RS, but in only 32% T-MN. TP53 mutations were detected in 92% T-MN with ≥15% RS and SF3B1 wild-type cases. Interestingly, T-MN and P-MDS patients with "Very low" or "Low" Revised International Prognostic Scoring System (IPSS-R) showed similar biological and clinical characteristics. In a Cox regression analysis, TP53 mutation was a poor prognostic factor in T-MN, independent of IPSS-R cytogenetics, disease-modifying therapy, and NRAS mutation. Our data have direct implications for T-MN management and provide evidence that, in addition to conventional disease parameters, mutational analysis should be incorporated in T-MN risk stratification.

Genomic events associated with poor outcome in chronic myeloid leukemia (CML) are poorly understood. We performed whole-exome sequencing, copy-number variation, and/or RNA sequencing for 65 patients to discover mutations at diagnosis and blast crisis (BC). Forty-six patients with chronic-phase disease with the extremes of outcome were studied at diagnosis. Cancer gene variants were detected in 15 (56%) of 27 patients with subsequent BC or poor outcome and in 3 (16%) of 19 optimal responders (P 5 .007). Frequently mutated genes at diagnosis were ASXL1, IKZF1, and RUNX1. The methyltransferase SETD1B was a novel recurrently mutated gene. A novel class of variant associated with the Philadelphia (Ph) translocation was detected at diagnosis in 11 (24%) of 46 patients comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were more frequent at diagnosis in patients with poor outcome: 9 (33%) of 27 vs 2 (11%) of 19 optimal responders (P 5 .07). Thirty-nine patients were tested at BC, and all had cancer gene variants, including ABL1 kinase domain mutations in 58%. However, ABL1 mutations cooccurred with other mutated cancer genes in 89% of cases, and these predated ABL1 mutations in 62% of evaluable patients. Gene fusions not associated with the Ph translocation occurred in 42% of patients at BC and commonly involved fusion partners that were known cancer genes (78%). Genomic analysis revealed numerous relevant variants at diagnosis in patients with poor outcome and all patients at BC. Future refined biomarker testing of specific variants will likely provide prognostic information to facilitate a risk-adapted therapeutic approach. Refereed/Peer-reviewed

Delayed diagnosis leading to accelerated-phase chronic eosinophilic leukemia due to a cytogenetically cryptic, imatinib-responsive TNIP1 – PDFGRB fusion gene

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2V617F Polycythaemia Vera (PV). This substitution affects a conserved cysteine residue in EGFR domain 2 and leads to the formation of a ligand-independent covalent receptor dimer, associated with increased transforming potential. Aberrant signalling from the EGFRC329R receptor is cell type-dependent and in the TF1.8 erythroid cell line expression of this mutant suppresses EPO-induced differentiation. Clonal analysis shows that the dominant JAK2V617F-positive clone in this PV patient harbors EGFRC329R, thus this mutation may contribute to clonal expansion. Somatic mutations affecting other ERBB and related receptor tyrosine kinases are observed in myeloproliferative neoplasms (MPN), and we show elevated EGFR levels in MPN samples, consistent with previous reports. Thus activation of this group of receptors, via multiple mechanisms, may contribute to clonal growth and survival of the JAK2V617F disease clone in MPN.

The TOPS trial evaluated high- (800 mg/day; n = 319) versus standard-dose (400 mg/day; n = 157) imatinib in patients newly diagnosed with Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase. Patients had a minimum follow-up of 42 months or discontinued early. Major molecular response (MMR) rates were similar between arms at (51.6 vs 50.2 % for 400 and 800 mg/day, respectively; P = 0.77) and by (75.8 vs 79.0 %; P = 0.4807) 42 months. There were no differences in event-free survival (EFS), progression-free survival(PFS), or overall survival (OS) between arms. The estimated rates of PFS on treatment and OS at 42 months were significantly higher in patients with MMR at 6, 12, and 18 months compared with those without MMR.Adverse events were more frequent with high-dose imatinib. Patients with B1 treatment interruption (vs [1) and those able to maintain imatinib C600 mg/day (vs\600 mg/day) in the first year of treatment had faster and higher response rates, but no improvement in EFS or PFS. Adherence to prescribed dose without interruption may be more important than initiation of therapy with higher doses of imatinib. Achievement of MMR correlated with longterm clinical outcomes.

Pegylated interferon (Peg-IFN) increases molecular response rates when used in combination with imatinib (IM) and dasatinib compared with tyrosine kinase inhibitor (TKI) monotherapy in de novo chronic phase chronic myeloid leukemia (CP-CML). (Preudhomme NEJM 2010, Hjorth-Hansen Leukemia 2016). The phase II Pinnacle (ALLG CML 11) study evaluated the tolerability and molecular response rate of nilotinib (NIL) with Peg-IFN alfa-2B (PegIntron, MSD) in CP-CML patients. Co-primary end points were MMR (BCR-ABL1 ≤ 0.1%) at 12 mths and MR4.5 (BCR-ABL1 ≤ 0.0032%) at 24 mths. Key secondary end points were survival and overall molecular response. Patients were screened for cardiac / vascular disease and associated risk factors at baseline (EKG, left ventricular ejection fraction, arterial duplex of carotids and lower limbs, blood HbA1c and lipid profiles). Those with uncontrolled vascular risk factors (diabetes, hypertension, dyslipidemia) or a history of vascular events were excluded. Eligible pts received NIL 300mg BID alone for the first 3 months (mths). PegIntron was then added at 30mcg/week in pts without persistent hematological toxicities, increasing to 50mcg/week as tolerated over the following mth. Combination therapy continued until 24 mths, when pts reverted to TKI monotherapy. Switching to IM 400-600mg QD was allowed for pts with persistent grade II or any grade III/IV toxicity from NIL. Sixty pts were enrolled from 12 Australian centres. Median age was 48.5 years (range 19-72); 45% were female. Sokal risk was low in 43% and high in 18%. Median follow up (FU) was 34 mths (24-60). Data is presented on an intention to treat basis. Eight pts (13%) did not commence Pegintron (2 due to persistent haem toxicities, 4 from GI disturbance, liver/pancreatic enzyme derangements, and 2 from pt preference). Considering Pegintron as a product of protocol assigned dose and duration, adjusted for time from study entry, 21 pts (35%) received >85% of their assigned dose, 13 (22%) received between 50-84% and 18 pts (30%) received 85% of their assigned dose versus those wwho took Adverse events (AE) are reported at a similar frequency compared to the interim analysis. Grade III/IV AEs attributed to NIL were increased lipase and neutropenia (each 12%), pancreatitis (6%), thrombocytopenia and rash (each 5%). Grade III/IV AEs attributed to Pegintron were neutropenia (10%), atrial fibrillation (6%), and myalgia, depression and rash (4% each). Three vascular revents occurred: one case each of ischaemic colitis, femoral artery occlusion, coronary artery disease. The former occurred on imaitnib and the latter 2 occurred after 2.5 and 4 years of niloitnib respectively; both patient have since switched to imatinib. Eighteen pts (30%) have withdrawn from study: 2 withdrew consent, 6 due to intolerance (diarrhoea, pancreatitis, GI upset, rash, high amylase and LFT derangements), 4 for failing to consistently achieve BCR-ABL MMR, 2 for loss of response; 4 pts withdrew for other reasons. Fig 1B shows BCR-ABL1 transcript levels over time. At 3 mths, 22 (37%) have achieved MMR, 23 (38%) had BCR-ABL between 0.1-1%, and 6 (10%) had BCR-ABL between 1-10%; 3 have already withdrawn. Six pts (10%) had BCR-ABL1 ≥10%; 3 subsequently achieved and maintained MMR, 1 has BCR-ABL Combination therapy with NIL and Peg-IFN leads to favourable rates of molecular responses that may be superior to NIL monotherapy (Table). While the majority of patients did not durably tolerate full dose Pegintron, there was minimal interference with TKI dose intensity. Such strategies may maximise achievement of deep molecular response, allowing a trial of TKI cessation and the benefit of treatment free remission to an increased number of patients. Disclosures Yeung: BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding. Shanmuganathan:Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Grigg:Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Other: Travel; Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees. Reynolds:AUSTRALASIAN LEUKAEMIA & LYMPHOMA GROUP (ALLG): Consultancy; Novartis AG: Equity Ownership; Novartis Australia: Honoraria; Alfred Health: Employment, Other: Biostatistician for trials funded by the Australian government and Abbvie, Amgen, Celgene, GSK, Janssen-Cilag, Merck, Novartis, Takeda, but sponsored by Alfred Health.. Harrup:Cooperative Trial Group for NeuroOncolog: Other: Collaborative Clinical Trials Group; Cancer Council of Tasmania: Membership on an entity's Board of Directors or advisory committees. Ross:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Mills:Novartis: Other: Speaker Fees; Specialised Therapeutics: Honoraria; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Conference Sponsorship; MSD: Membership on an entity's Board of Directors or advisory committees. Yong:BMS: Honoraria, Research Funding; Celgene: Research Funding; Novartis: Honoraria, Research Funding. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau. Branford:Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cepheid: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hughes:Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. OffLabel Disclosure: Pegylated interferon is not registered for use in chronic phase CML

Background: Achieving sustained DMR (variably described as ≥ MR4 or ≥ MR4.5) is an emerging treatment goal for patients with CML. DMR is associated with excellent long-term clinical outcomes and a higher likelihood of successful treatment-free remission (TFR) upon discontinuation of tyrosine kinase inhibitor (TKI) therapy. Biological predictors of patients likely to achieve DMR are unknown. Here, we present an exploratory analysis of gene expression signatures in order to predict DMR to TKI therapy, as well as understand the biological underpinnings that allow a DMR, based on patients treated with imatinib or nilotinib in the ENESTnd study (NCT00471497). Methods: ENESTnd is a phase 3, randomized, open-label study comparing nilotinib 300 mg twice daily (BID), nilotinib 400 mg BID and imatinib 400 mg once daily (QD) in patients with newly-diagnosed CML. To maximize the likelihood of defining predictive and biologically relevant gene signatures, samples from a group of poor responders (BCR-ABL1IS > 10% by 3 months of therapy) and good responders (BCR-ABL1IS < 0.01% by 12 months of therapy) were selected across all treatment arms. Whole blood samples collected prior to study treatment initiation were available from 112 such patients from the total 846 patients enrolled in ENESTnd, and were subjected to RNA sequencing. DMR was assessed using quantitative polymerase chain reaction transcript ratios standardized to the international scale (IS) and was defined as BCR-ABLIS ≤ 0.01% (MR4) or ≤ 0.0032% (MR4.5). For statistical analysis, responders were defined as patients having achieved DMR by 5 years, whereas non-responders were in the trial for ≥ 5 years without achieving DMR. Five years was selected to ensure that patients on both imatinib and nilotinib arms had adequate time to reach MR4.5. The association of clinical variables with responder status (good or poor) was assessed via a multivariate logistic regression model. Results: We correlated clinical variables (eg, Sokal risk score, TKI, age, sex) with responder status for 112 ENESTnd study patients who received 400 mg imatinib QD (n = 47), 300 mg nilotinib BID (n = 33), or 400 mg nilotinib BID (n = 32). Of the 112 patient samples, 70 were included in the analysis using MR4.5 as an endpoint, with 47 patients characterized as responders (imatinib: 16; nilotinib: 31), and 23 as non-responders (imatinib: 13; nilotinib: 10). Of the 112 samples, 42 were excluded from analysis because the patients discontinued the trial before 5 years and did not achieve MR4.5 (imatinib: 18; nilotinib: 24). Younger age (< 35 years) was associated with good response (p < 0.02) in a multivariate analysis. We developed a predictive model of responder status by applying penalized regression to clinical variables and gene expression (13569 genes) in independent (clinical or gene expression) and combined gene and clinical models. The best performing model used patients with MR4.5 vs poor responders, with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.87 (Figure; Table). Including clinical variables did not result in markedly different performance (AUC = 0.85). Significantly, both models outperformed a model that included clinical variables only (AUC = 0.65). Relaxing the definition of good responders to include patients with MR4 yielded similar results (Table). Detailed biomarker/pathway analysis to explore the biological pathways that separate good and poor response are underway. Conclusions: We present a gene expression model that distinguishes patients who achieved a DMR from those with a poor response to treatment at 5 years. The approach for sample selection optimized the chances of finding a biological and clinical signal and may be applicable to all CML patients initiating TKI therapy. This work could yield new therapeutic targets that could potentially turn a patient biologically determined to be a poor responder into a good responder who might even achieve a TFR. Disclosures Radich: Novartis: Other: RNA Sequencing; TwinStrand Biosciences: Research Funding. Larson:Novartis: Honoraria, Other: Contracts for clinical trials; Celgene: Consultancy; Agios: Consultancy. Kantarjian:Ariad: Research Funding; Pfizer: Honoraria, Research Funding; BMS: Research Funding; Takeda: Honoraria; Astex: Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Novartis: Research Funding; Cyclacel: Research Funding; Amgen: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Jazz Pharma: Research Funding. Deininger:Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Ascentage Pharma: Consultancy, Honoraria; TRM: Consultancy; Sangoma: Consultancy; Fusion Pharma: Consultancy; Adelphi: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy. Pinilla Ibarz:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Teva: Consultancy; TG Therapeutics: Consultancy; Bayer: Speakers Bureau; Sanofi: Speakers Bureau. DeAngelo:Abbvie: Research Funding; Takeda Pharmaceuticals: Consultancy; Jazz Pharmaceuticals Inc: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; GlycoMimetics: Research Funding; Celgene: Consultancy; Incyte: Consultancy; Amgen: Consultancy; Blueprint: Consultancy, Research Funding; Shire: Consultancy. Branford:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Qiagen: Consultancy, Honoraria; Cepheid: Consultancy, Honoraria. Sadek:Novartis: Employment. Chaturvedi:Novartis Pharmaceuticals: Employment. Sondhi:Novartis: Employment, Other: Stock; Sanofi: Other: Stock. Mishra:Novartis: Employment. Purkayastha:Novartis Pharmaceuticals: Employment. Shrestha:Novartis: Employment. Obourn:Novartis: Employment. Druker:Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; CureOne: Membership on an entity's Board of Directors or advisory committees; Beat AML LLC: Other: Service on joint steering committee; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Patient True Talk: Consultancy; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Patents & Royalties, Research Funding; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Celgene: Consultancy; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Gilead Sciences: Other: former member of Scientific Advisory Board; ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Monojul: Other: former consultant; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding.

Background Mutation of genes linked to hematologic cancer have recently been reported in CML and are associated with early progression and resistance (Reviewed in Branford, Kim Leuk 2019). The mutations comprise single nucleotide variants (SNVs) and small insertions/deletions (indels), plus gene fusions and large focal gene deletions. In 39 patients (pts) in blast crisis (BC), all had at least 1 cancer gene mutation, including fusions in 33%: partner genes MLL, RUNX1, IKZF1, MECOM and CBFB. 50% of the fusions were novel and some were present at chronic phase diagnosis. BCR-ABL1 mutations rarely occurred as the sole mutant. NGS offers critical information for resistance assessment. For many clinical purposes, targeted DNA sequencing (seq) using panels of specific disease related genes is the most cost effective screening choice. However, this strategy could miss relevant fusions and deletions. Aim To determine whether an RNA based approach is more informative than DNA for detecting a broad range of mutations. Method A hybridization capture NGS gene panel was developed to target 126 genes relevant for myeloid/lymphoid leukemia. In a pilot study, DNA and RNA derived from 5 leukemia cell lines with well characterized mutations, including fusions and deletions, were panel sequenced. An additional 6 cell lines were sequenced using RNA, plus 49 pt samples with RNA stored for up to 14.6 years: 45 at diagnosis and 4 at BC/resistance. Six of these pt samples had prior whole exome and/or whole transcriptome seq. We used total RNA that detected intronic splice region variants from pre-spliced RNA. SNVs/indels were called from DNA/RNA with FreeBayes. Manta called focal deletions from DNA. Known and novel RNA fusions and novel splice junctions were detected using the STAR aligner. Gene expression used edgeR. Results For the 5 cell lines with DNA versus (v) RNA seq, SNVs/indels were reliably called in RNA, with a strong positive correlation of mutant allele frequency: DNA v RNA, r = 0.93. Two TP53 small deletions of 26 and 46 bp were not called in RNA, but were instead detected as novel RNA splice junctions. Read counts were 5.2 fold higher for RNA than DNA at sites of clinically relevant mutants, consistent with enrichment of seq read depth proportional to expression. Overall, RNA revealed a higher number of relevant mutants than DNA: RNA = 49 v DNA = 37, Fig A-C. Notably, the functional effect of splice region disrupting mutants and large focal deletions were evident by novel RNA splicing, Fig D-F. In the total 11 cell lines tested with RNA, all 13 reported fusions were called, including BCR-ABL1 and RUNX1, MLL, ETV6 and CBFB fusions. For 7 cell lines with variants described in the COSMIC Cell Lines Project, 23/23 cancer gene SNVs/indels were called, plus 7 cancer gene SNVs/indels not reported. These were verified by DNA seq. 15 gene deletions were evident by atypical RNA splicing and verified by DNA seq: IKZF1, CDKN2A/B, PAX5, BTG1, RB1 and NCOR1. Five other cell lines had verified CDKN2A deletions that were evident by loss of gene expression, Fig G. Two BTG1 deletions were not detected. For the 6 pt samples re-sequenced by the RNA panel, 8/8 verified fusion transcripts were detected with a 31 fold enrichment of read counts. 11/11 cancer gene SNVs/indels were called and 3/4 gene deletions. The exception was a CDKN2A deletion not detected by novel splicing but evident as loss of expression, Fig G. Seven other cancer gene SNVs were found at low allele frequency, including a resistant BCR-ABL1 mutation at 1.7% in the oldest sample. Of the 43 diagnosis samples without prior NGS, BCR-ABL1 transcripts were detected in all. BCR-ABL1 genomic breakpoints were called at base pair resolution in 39, 91%. Two pts had mutated ASXL1 at diagnosis and both failed imatinib by 9 months with mutant BCR-ABL1. By gene expression analysis, all but 1 of the total 45 diagnosis samples clustered together. The exception was a pt who transformed to lymphoid BC at 6 months that clustered with the lymphoid cell lines and lymphoid BC pts, Fig H. Conclusion RNA gene panel seq demonstrated enhanced sensitivity and an increased yield of clinically relevant mutations compared with DNA panel seq. A single RNA assay has the capacity to detect SNV/indels, known and novel gene fusions, focal deletions and the likely functional effect of splice disrupting mutations. RNA panel seq is a valuable tool for the comprehensive assessment of mutations that drive CML treatment failure and drug resistance. Disclosures Branford: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Qiagen: Consultancy, Honoraria; Cepheid: Consultancy, Honoraria. Shanmuganathan:Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Scott:Celgene: Honoraria. Hughes:Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel; Novartis, Bristol-Myers Squibb, Celgene: Research Funding.

Dasatinib treatment leads to excellent molecular responses in chronic phase chronic myeloid leukemia (CP-CML). Pleural effusions, an adverse event related dasatinib treatment, may lead to intolerance and drug discontinuations. Strategies aimed at minimising this may improve outcomes. In the Phase III Dasision study, pleural effusion affected ~22% of patients after 4 years of dasatinib treatment at 100mg/d (Cortes et al, 2016 JCO 34(20) 2333-40). The elderly are at particular risk (Latagliata et al 2013 Hem Onc 31(2) 103-9), and there is suggestion that higher dasatinib trough (Cmin) levels may increase the risk of pleural effusions. The randomised OPTIM study (EHA 2014 abstract 5678) has previously reported that patients with Cmin >3nM benefited from dose reductions with preservation of molecular responses. The CML12 (DIRECT) study, run by the Australasian Leukaemia & Lymphoma Group (ALLG) with financial support from BMS, is a single arm phase II study with the aim of minimizing dasatinib related toxicity whilst preserving efficacy using a similar treatment schema to the OPTIM study. Here, we report results of a per-protocol interim analysis based on early molecular response (EMR; BCR-ABL1 ≤10% at 3 months) and MMR (BCR-ABL1 ≤0.1%) at 12 months, both key secondary endpoints of the study. The primary endpoint of the study- the cumulative incidence of pleural effusion at 24 months - is not yet evaluable. DIRECT initially only enrolled patients >60 years old, predicted to derive the greatest potential benefit from a reduction in toxicity. The protocol was amended after 34 pts were accrued to include patients >18 years old at the recommendation of the trial management committee. All patients started treatment with dasatinib 100mg/day. Dasatinib Cmin was taken at 7, 28 and 56 days after treatment commencement. All Cmin directed dose adjustments were made prior to assessment of BCR-ABL1 at 3 months. Patients sequentially dose reduced to 70mg/day, then to 50mg/day, for Cmin results >3nM. Doses As of June 2019, 71 patients (of 80 planned) from 14 centres have been enrolled, with a median follow up of 7 months (range 0-31). Median age was 64 years (range 21-86) and 48% were female. Sokal risk was low in 40% and high in 9.2%. The median dasatinib Cmin at days 7, 28, 56 and 90 were 4.9, 3.5, 3.5 and 2.7nM respectively (Table). At these time points, 83%, 59%, 73% and 44% of patients had Cmin ≥3nM. There was a trend to lower Cmin after protocol directed dose reduction. The number of patients remaining on 100mg/day after 1, 2 and 3 months of therapy were 11/69 (16%), 5/63 (8%) & 5/55 (9%) respectively. Molecular response data were available for 48 patients at 3 months and 22 patients at 12 months. At 3 months, BCR-ABL1 ≤ 10% was achieved by 46 of 48 patients (96%), of whom 13 (27%) had achieved MMR (27%). At 12 months, MMR was achieved by 20/22 patients (91%), of whom 7 have achieved MR4.5 (BCR-ABL1 Adverse events occurred in 91.5% of patients at all grades - the majority of which were mild. Grade III/IV events occurred in 36.6% and 1.4% of patients respectively. Six of the 71 patients have discontinued dasatinib treatment early, all due to adverse events / dasatinib intolerance. Detailed adverse event data is embargoed until primary endpoint analysis. No patient has progressed to accelerated or blast phase CML. The DIRECT study demonstrated the feasibility of using dasatinib Cmin levels to optimise dosing. Early molecular response rates are encouraging and predict for excellent achievement of long-term molecular response. Long term efficacy and safety data are awaited. Table Disclosures Yeung: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Grigg:Abbvie: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Other: Travel. Shanmuganathan:Novartis: Honoraria, Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Amgen: Other: Travel Support; Janssen: Other: Travel Support; Gilead: Other: Travel Support. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau. Branford:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Qiagen: Consultancy, Honoraria; Cepheid: Consultancy, Honoraria. Mills:Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Speaker Fees; Amgen: Other: Conference Sponsorship; Specialised Therapeutics: Honoraria; MSD: Membership on an entity's Board of Directors or advisory committees. Shortt:Celgene: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Astex: Research Funding; Amgen: Research Funding; Gilead: Speakers Bureau; Takeda: Speakers Bureau. Ross:Celgene: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Harrup:Cancer Council of Tasmania: Membership on an entity's Board of Directors or advisory committees; Cooperative Trial Group for NeuroOncolog: Other: Collaborative Clinical Trials Group. Hughes:Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel.

Background Mutated cancer genes in patients (pts) with TKI failure and blast crisis (BC) CML have recently been described. RUNX1 mutations, namely single nucleotide variants (SNVs) and indels, were the most frequently detected besides BCR-ABL1 [reviewed in Branford, Kim Leuk 2019]. They were found in ~18% of pts, although splice variants were rarely described. RNA splicing events were associated with focal deletion of IKZF1 and RUNX1 in TKI resistant pts that were identified by copy number analysis and RNAseq [Branford Blood 2018]. Novel splicing associated with mutation of cancer genes is an unexplored area of study in resistance. RNA sequencing can assess the functional effect of splice site variants, which lead to splicing errors due to the use of alternative or cryptic splice sites and cause alterations to protein function. Aim We determined whether novel splicing can identify cancer genes with potential altered function. Methods RNAseq analysis was performed for 48 pts at diagnosis and 33 at BC using a protocol that preserved intron-retaining precursor RNA. Coverage of intron-exon borders was sufficient to detect intronic splice region variants. The STAR aligner was used to bioinformatically collate unannotated RNA splice junctions. 54 cancer genes were assessed and aberrant splice events were filtered based on the number of samples in which a splice junction occurred. Manual inspection of the splice junctions was performed using the Integrative Genomics Viewer. This approach identified previously verified aberrant splicing associated with IKZF1 and RUNX1 deletions. Results Ten previously undetected novel splice junctions were revealed in 9/33 pts (27%) in BC within key tumor suppressor genes CDKN2A/B (5), RB1 (1), ATM (1), and RUNX1 (3). The aberrant splicing pattern of CDKN2A and RB1 (Fig A/B) in 6 pts suggested large deletions, as previously described in our cohort with IKZF1 and RUNX1 deletions. Breakpoints associated with deletions ranging from 53 to 181 Kb were detected in the 5 pts with CDKN2A aberrant splicing. Similarly, a 90 Kb deletion of exons 18-27 of the RB1 gene led to the aberrant splicing. The pts transformed to lymphoid BC (median 5 months). 4 of these 6 pts were tested at diagnosis and the deletions were not detected, indicating they were gained at resistance. The aberrant splicing patterns of ATM and RUNX1 did not predict large deletions. These were related to somatic SNVs at canonical splice sites in ATM and in 2 of the pts with RUNX1 aberrant splicing. A splice acceptor site SNV in ATM resulted in skipping of exon 61 (Fig C) and protein truncation. This novel SNV has not been reported in any population or somatic variant database. Two pts in myeloid BC at 28 and 48 months after diagnosis had an identical somatic RUNX1 mutation at the canonical splice donor site of exon 5. The SNV was not detectable prior to imatinib treatment in both pts. The splice site SNV led to activation of a cryptic splice site within exon 5 in both pts (Fig D), which predicted premature termination. While this mutation is novel, an adjacent intronic SNV occurs in familial platelet disorder, leading to activation of the same cryptic splice site. The atypical RUNX1 splicing of the 3rd patient was associated with retention of 55 bp of intron 6 as a cryptic exon (Fig E), leading to protein truncation. A deep intronic SNV identified at lymphoid BC at 6 months of imatinib was detected near the cryptic exon by RNAseq and verified as somatic by DNA Sanger sequencing. This was predicted to activate cryptic RNA splicing elements and lead to intron sequence retention in a RUNX1 transcript. We sequenced the diagnosis sample using an RNA-based gene panel method under development that provides enhanced sensitivity of variant detection. The same pattern of atypical splicing was observed and the intronic SNV was present at low level. The RUNX1 mutation at diagnosis may have contributed to early BC. To our knowledge this is the first report of a RUNX1 truncating variant in CML involving a cryptic exon. Conclusion Enhanced bioinformatic analysis of RNAseq data has revealed a high proportion of pts with truncating mutations in cancer genes indicated by novel RNA splicing (27% pts in BC). Using RNA-based sequencing allows an evaluation of the potential functional effect of variants that are not apparent by DNA-based mutation analysis. We suggest that future studies include RNA sequencing to detect a broader spectrum of mutations associated with TKI resistance. Disclosures Shanmuganathan: Gilead: Other: Travel Support; Janssen: Other: Travel Support; Amgen: Other: Travel Support; Bristol-Myers Squibb: Honoraria, Other: Travel Support; Novartis: Honoraria, Other: Travel Support. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Scott:Celgene: Honoraria. Hughes:Novartis, Bristol-Myers Squibb, Celgene: Research Funding; Novartis, Bristol-Myers Squibb: Consultancy, Other: Travel. Branford:Cepheid: Consultancy, Honoraria; Qiagen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau.

Introduction. The Australian Familial Haematological Cancer Study (AFHCS) was founded in 2004 when only one familial haematological malignancy (FHM) gene was known, RUNX1 causing thrombocytopenia and predominantly an MDS/AML HM phenotype. Today, there are over a dozen known FHM genes, mostly characterized as predisposing to myeloid malignancies. Within our AFHCS cohort of over 200 families, this is also true with a significant proportion of families with predisposition to myeloid malignancies solved with our identification of germline mutations in genes such as GATA2, RUNX1,CEBPA and DDX41. In contrast the majority of unsolved families have a predominance of lymphoid malignancies, most commonly non-Hodgkin lymphoma and chronic lymphocytic leukaemia. In several of these families we have found known pathogenic mutations in genes involved in regulation of the homologous recombination (HR) DNA damage repair (DDR) pathway (e.g. BRCA2 Y1710fs* and PALB2 K142*) that are previously characterised with regard to their known solid tumour predisposition. In the AFHCS families with these mutations we observe a mixture of hematological tumors of mostly lymphoid origin as well as non-hematological tumours. This led us to hypothesise that there is a subset of families for which the predisposition to malignancy includes a spectrum of different malignancies, i.e. pan-cancer predisposition. Further, we hypothesise that these are associated with mutations in genes with roles in HR DNA repair. In light of this we undertook a review of the phenotypic and genetic characteristics of families in our AFHCS cohort. Methods. Malignancy phenotype data, with regard to the different tumor types observed in each AFHCS family was annotated and aggregated. Covariance matrix analysis was then used to compare the number of times different tumor types were observed occurring together in a family pedigree. Genetic data: we curated whole exome sequence data for damaging mutations in genes with a known role in HR DNA damage repair. Results. A phenotype review of our cohort found 52% of assessable FHM families also had multiple types of solid tumour and/or individuals with diagnosis of multiple malignancies, indicative of a 'pan-cancer' phenotype. Covariance analysis showed that Hodgkin and non-Hodgkin lymphoma most often occurred together and also co-occurred with non-HM tumors breast, melanoma, colorectal, prostate and ovarian subtypes, implying a possible shared genetic etiology (Figure 1A). Assessment of AFHCS families for mutations in DDR genes including DNA repair (e.g. FANC/BRCA pathway genes) and chromosome cohesion (RAD21 and related) identified 18 variants segregating with malignancy including 5 frameshift or stopgain mutations. These high impact mutations included genes previously characterised in the context of autosomal recessive predisposition to hematological malignancy such as PALB2/BRCA2 family (Fanconi Anemia, see Figure 1B), NBN1 (Nijmegen Breakage Syndrome), DDX11 (Warsaw Syndrome) and others. A further 18 variants segregated in families where pan-cancer status is still under investigation. Conclusions. Phenotypic and genetic analysis of unsolved HM predisposed families have identified that familial clustering of lymphoma in actuality identifies families with pan-cancer germline genetic susceptibility, characterised by mutations in DNA damage response genes. Collectively these mutations may have a significant impact on our understanding of germline cancer predisposition, a finding supported by recent genetic population studies on sporadic cohorts, including pediatric cancer cohorts, opening the door for the application of rational therapies as evidenced by the BRCA/PARP inhibitor paradigm. However, as each individual gene mutation is rare, international collaborative studies will be essential for accurate measurements of risk and prognostication to assist individuals in the clinic. Disclosures Branford: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Qiagen: Consultancy, Honoraria; Cepheid: Consultancy, Honoraria. Scott:Celgene: Honoraria.