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Asciminib, a first-in-class allosteric inhibitor of BCR::ABL1 kinase activity, is now approved for the treatment of patients with chronic-phase chronic myeloid leukemia who failed 2 lines of therapy or in patients with the T315I mutation. Promising attributes include high specificity and potency against BCR::ABL1, activity against most kinase domain mutations, and potential for combination therapy with ATP-competitive tyrosine kinase inhibitors. Clinicians now have expanded third-line options, which in most cases will involve a choice between asciminib and ponatinib.

Asciminib is the first tyrosine kinase inhibitor (TKI) to Specifically Target the ABL Myristoyl Pocket (STAMP). In the ASCEMBL Phase 3 randomised study comparing asciminib to bosutinib in patients with CML in chronic phase (CP) after ³2 prior TKIs, asciminib demonstrated superior efficacy and tolerability vs bosutinib (major molecular response (MMR) rate at week 96: 37.6% vs 15.8%, treatment discontinuation 7.7% vs 26.3%) (Rea et al, EHA 2022). Here we present the real-world clinical outcomes of TKI-resistant/ intolerant CML patients in Australia who received asciminib through a MAP approved by Novartis.

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 treatment-free remission (TFR) rapidly becoming the ultimate goal of therapy in chronic myeloid leukemia (CML), there is a need to develop strategies to maximize sustained TFR by improving our understanding of its key determinants. Chronic-phase CML patients attempting TFR were evaluated to identify the impact of multiple variables on the probability of sustained TFR. Early molecular response dynamics were included as a predictive variable, assessed by calculating the patient-specific halving time of BCR-ABL1 after commencing tyrosine kinase inhibitor (TKI) therapy. Overall, 115 patients attempted TFR and had ≥12 months of follow-up. The probability of sustained TFR, defined as remaining in major molecular response off TKI therapy for 12 months, was 55%. The time taken for the BCR-ABL1 value to halve was the strongest independent predictor of sustained TFR: 80% in patients with a halving time of 21.85 days (last quartile) (P < .001). The e14a2 BCR-ABL1 transcript type and duration of TKI exposure before attempting TFR were also independent predictors of sustained TFR. However, the BCR-ABL1 value measured at 3 months of TKI was not an independent predictor of sustained TFR. A more rapid initial BCR-ABL1 decline after commencing TKI also correlated with an increased likelihood of achieving TFR eligibility. The association between sustained TFR and the time taken for BCR-ABL1 to halve after commencing TKI was validated using an independent dataset. These data support the critical importance of the initial kinetics of BCR-ABL1 decline for long-term outcomes.

There is currently no biomarker that reliably predicts treatment-free remission (TFR) in chronic myeloid leukaemia (CML). We characterised effector and suppressor immune responses at the time of tyrosine kinase inhibitor (TKI) cessation in patients from the CML8 and CML10 clinical studies. Natural killer (NK) cells with increased expression of activating NK receptors were higher in patients who achieved TFR. There was no difference in the proportion of CD4+ or CD8+ T cells. Furthermore, we found that FoxP3+ regulatory T cells (T reg) and monocytic myeloid-derived suppressor cells (Mo-MDSCs) were concomitantly decreased in TFR patients, suggesting that the effector and suppressor arms of the immune system work in concert to mediate TFR. A discovery cohort (CML10) was used to generate a predictive model, using logistic regression. Patients classified into the high-risk group were more likely to relapse when compared with the low-risk group (HR 7·4, 95% CI 2·9-19·1). The model was successfully validated on the independent CML8 cohort (HR 8·3, 95% CI 2·2–31·3). Effective prediction of TFR success may be obtained with an effector-suppressor score, calculated using absolute NK cell, T reg, and Mo-MDSC counts, at TKI cessation, reflecting the contribution of both immune suppressors and effectors in the immunobiology underlying successful TFR. Refereed/Peer-reviewed

The advent of pediatric inspired regimens has improved the outcome for younger adults with Acute Lymphoblastic Leukemia (ALL), however this comes at a considerable toxicity burden limiting its applicability in older adults. The advent of novel immunotherapies, such as Blinatumomab, an anti-CD19 targeting Bi-specific T-cell engager, has improved outcomes for adults with relapsed refractory and minimal residual disease (MRD) positive B-precursor (BCP) ALL. The Australasian Leukemia and Lymphoma Group (ALLG) undertook a phase 2 proof-of-concept study to explore the combination of Blinatumomab with reduced intensity chemotherapy in adults with newly diagnosed Ph- BCP ALL. Patients received an initial pre-phase of corticosteroids (Prednisolone 100mg/d, 5 days) followed by a low intensity chemotherapy debulking (Cyclophosphamide 150mg/m 2 BD D1-3, Vincristine 2mg IV D1, 11 and Dexamethasone 10mg/m 2 D1-4, D11-14). Following this patients received Blinatumomab at 9mcg/d days 1-7 and 28mcg/d days 8-28. A B cycle of Hyper-CVAD (Cytarabine 3g/m 2 BD for 4 doses and Methotrexate 1g/m 2 D1 with Methylprednisolone 50mg BD D1-3). Patients then received 3 alternating cycles of Blinatumomab (28mcg/d for 28 days) and B-cycles of Hyper-CVAD followed by 2-years of POMP maintenance in subjects not proceeding to allogeneic stem cell transplant (alloHSCT) which was at investigator discretion. MRD assessments by ASO-PCR were performed after the first B cycle, second B cycle and prior to maintenance therapy with an MRD response being a level of 10 -4 or less. This analysis is from a pre-specified interim analysis with a data cutoff of 29 th June 2021. 30 patients were enrolled with a median age of 51.7(range, 39.5 - 66.5 years) with 70% male subjects. ECOG performance score was 0 in 14 subjects, 1 in 12 and 2 in 4. High risk disease was identified in 5 subjects (1 t(4;11), 2 hypodiploid, 1 t(1;19) and 1 Ph-like). All patients attained a CR, with 28 at end of 1B and a further 2 at end of 2B. Of 26 patients evaluable for MRD, 70% had achieved an MRD response after cycle 1B and 83% at the end of cycle 2B. 15 patients have ceased study therapy; 6 patients died with progressive disease, 4 subjects exited to allogeneic stem cell transplant, 1 patient was withdrawn due to progressive disease, 1 had intolerable toxicity (peripheral neuropathy) and 1 at investigator discretion. There were no treatment related deaths. 15 remain on protocol in maintenance with the remainder having completed therapy. At data cut-off the median event free survival (EFS) was not reached (95% CI 8.3 months - NA) with an estimated 24 month EFS of 61.8% (95% CI 36.3 - 84.2%) (figure 1A), and similarly the median overall survival (OS) S was not reached (95% CI 21.0 months - NA) with an estimated 24 month OS of 68.6% (95% CI 41.5 - 85.1%)(figure 1B). This predicted EFS was greater than the pre-specified stopping rule of a 24-month EFS of 50%. 2 episodes of cytokine release syndrome (CRS) were recorded (1 grade 2, 1 grade 3) with the major toxicity being infective (53 episodes of sepsis, infection or febrile neutropenia) predominately related to chemotherapy cycles. 7 episodes of neurological toxicity were demonstrated (1 myelopathy and 4 peripheral neuropathy occurring during chemotherapy and 2 encephalopathy occurring during Blinatumomab administration). Overall, the combination of Blinatumomab with chemotherapy was tolerable and appeared efficacious with a high rate of remission and deep MRD responses observed. Responses appeared durable despite a low rate of alloHSCTin first remission. The major toxicity was infective and occurred in the context of chemotherapy cycles. Future developments from this protocol will emphasise further reduction in cytotoxic chemotherapy through incorporation of further novel agents to minimise this toxicity. Figure 1 Figure 1. Disclosures Fleming: Servier: Honoraria; Pfizer: Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau; Amgen: Honoraria, Research Funding, Speakers Bureau. Reynolds: Abbvie: Research Funding; Novartis AG: Current equity holder in publicly-traded company; Alcon: Current equity holder in publicly-traded company. Bajel: Abbvie, Amgen, Novartis, Pfizer: Honoraria; Amgen: Speakers Bureau. Yeung: BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding. Verner: Janssen-Cilag Pty Ltd: Research Funding. Wei: Abbvie, Amgen, Astellas, AstraZeneca, Celgene/BMS, Genentech, Janssen, MacroGenics, Novartis, Pfizer, and Servier: Honoraria; Novartis, Abbvie, Celgene/BMS: Speakers Bureau; Servier: Consultancy; Abbvie, Amgen, AstraZeneca, Celgene/BMS, Novartis, Servier and F. Hoffmann-La Roche: Research Funding; Abbvie, Amgen, Astellas, AstraZeneca, Celgene/BMS, Genentech, Janssen, MacroGenics, Novartis, Pfizer, and Servier: Membership on an entity's Board of Directors or advisory committees; Former employee of Walter and Eliza Hall Institute: Patents & Royalties: Prof. Andrew Wei is a former employee of the Walter and Eliza Hall Institute and is eligible for a fraction of the royalty stream related to Venetoclax. Greenwood: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Blinatumomab - usage in front-line therapy for Acute Lymphoblastic Leukaemia

Introduction Children with Down Syndrome (DS) frequently develop hematological malignancies including acute lymphoblastic leukemia (ALL), however, the genomic basis for the predisposition to DS-ALL remains unknown. DS-ALL is associated with increased rates of relapse, chemotherapy related toxicity and poor overall survival compared to non-DS patients. The P2RY8-CRLF2 (CRLF2r) gene fusion, the result of deletion of the pseudoautosomal region 1 (PAR1) on the X/Y chromosome (chr), has been identified in 60% of DS-ALL (+21) patients and 30% of iAMP21 (intrachromosomal amplification of chr21) ALL patients compared to only 5-16% of other B-ALL patients. The high mobility group nucleosome binding protein 1, encoded by HMGN1 on chr21 may play a role in DS-ALL leukemogenesis, due to its demethylase activity associated with transcriptional activation. Methods mRNA seq was performed on the blast cells of 76 ALL patients using the Universal Plus mRNA-seq with NuQuant kit. FusionCatcher, SOAPfuse and JAFFA were used to identify fusions and featureCounts used for mRNA expression of STAR (v2.7.3a) aligned BAM files. CRISPR/Cas9 gRNAs targeting P2RY8 intron 1 or CRLF2 5' UTR created the 320 KB (PAR1) deletion. Cas9/gRNAs were transduced into Jurkat cells ± HMGN1 overexpression, activated with doxycycline and stained with TSLPR (CRLF2/IL-7Rα dimer) for single cell sort and clonal expansion. Phosphorylated proteins were measured by intracellular flow cytometry and SYBR Green RQ-PCR was used for mRNA expression. Results Significantly higher HMGN1 expression was identified in CRLF2r ALL patients, compared to the control (BCR-ABL1+ patients. Fig. 1A; p To further understand the role of HMGN1 in CRLF2r development, HMGN1 was overexpressed (1.5-fold; HMGN1H) in Jurkat Cas9 cells to represent a trisomy level of expression. gRNAs targeting P2RY8 and CRLF2 were activated in cells ± HMGN1H with undirected repair. TSLPR surface and CRLF2 mRNA expression were higher in CRLF2r + HMGN1H cells compared to CRLF2r only cells (Fig. 1B,C; p=0.034 and CRLF2r + HMGN1H cells also demonstrated significantly increased pSTAT5 (MFI: 2359 ± 1; Fig. 1D), pAKT (MFI: 2339 ± 6; Fig. 1E) and pERK (MFI: 2478 ± 48; Fig. 1F) compared to CRLF2r only cells (MFI pSTAT5: 1910 ± 10; pAKT: 1727 ± 14; pERK: 1946 ± 6; all p Conclusion The P2RY8-CRLF2 (CRLF2r) gene fusionis prevalent in DS, +21 and iAMP21 ALL patients. Here, we demonstrate that CRLF2r is associated with high expression of HMGN1 (chr21)in ALL patient cells . Using CRISPR/Cas9 in an in vitro model, we demonstrate that enforced high expression of HMGN1 alters DSB repair mechanism, favoring PAR1 deletion and the subsequent formation of the P2RY8-CRLF2 gene fusion, with attendant higher expression of STAT5 target genes. Understanding the role of HMGN1 in the disproportionate number of DS-ALL patients who are diagnosed with CRLF2r has the potential to lead to novel therapeutic interventions, in this high-risk group of patients where efficacious therapeutic options are currently poor. Figure 1 Figure 1. Disclosures Yeung: BMS: Honoraria, Research Funding; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Pfizer: Honoraria. White: Novartis: Research Funding; BMS: Honoraria, Research Funding.

Neurofibromatosis type 1 (NF-1) is an autosomal dominant disorder affecting approximately 1:3000 individuals globally. While approximately 50% are familial, with over 3000 causative germline variants in the neurofibromatosis (NF1) gene identified, the remainder occur sporadically. These mutations lead to haploinsufficiency of NF1 and neurofibromin, a tumor suppressor and important negative regulator of RAS signaling. Children with NF-1 have a higher risk of developing juvenile myelomonocytic leukemia and acute myeloid leukemia, but rarely develop acute lymphoblastic leukemia (ALL). A 9-year-old male presented in 2015 with persistent migratory subcutaneous swellings and multiple bony aches with lytic lesions on bone imaging. He had a high white cell count with eosinophilia (WCC 43.4 x 10 9/L, eosinophils 23.87 x 10 9/L) with no circulating blasts, 10% marrow blasts (CD10+/CD19+/CD34+) and was CNS negative. Although previously undiagnosed, NF-1 was clinically suspected due to typical skin changes. He was diagnosed with iAMP21 ALL and NF-1 was confirmed with the identification of a germline NF1 donor splice site mutation (c.1845G>A:p.L615=). Bone marrow cells were sorted by flow cytometry on CD19 positivity and underwent transcriptomic sequencing. This revealed a P2RY8-CRLF2 gene fusion, with no other clinically relevant variants, while a custom Taqman low density array indicated high-risk B-ALL subtype Ph-like ALL. Multiplex ligation-dependent probe amplification (MLPA) confirmed iAMP21 and also identified IKZF1 exon 2-3 and BTG1 deletions. Treatment followed the high-risk B-ALL arm of the AEIOP-BFM ALL2009 protocol due to persistent end-consolidation MRD in addition to iAMP21 and the Ph-like phenotype. He relapsed three years later off treatment and was refractory to both salvage chemotherapy and blinatumomab. The iAMP21, P2RY8-CRLF2 gene fusion, IKZF1 exon 2-3 and BTG1 deletions remained detectable. Whole exome sequencing of CD19 positive samples from diagnosis, relapse and mesenchymal stem cells (germline control) was performed, identifying a NF1 c.7400dupT:p.L2467 frameshift (fs) mutation only at relapse. To understand the implications of NF1 p.L2467fs, the P2RY8-CRLF2 gene fusion was first transduced into the interleukin 3 (IL3) dependent murine pro-B cell line Ba/F3. P2RY8-CRLF2 alone is not transforming and is thought to be a secondary event in iAMP21 ALL, providing an ideal model to study the cumulative effect of the NF1fs. The NF1fs was then introduced to the P2RY8-CRLF2 cells by CRISPR/Cas9. A proliferation assay was performed without IL3 and demonstrated the P2RY8-CRLF2+NF1fs cell line was IL3 independent, indicative of leukemic transformation, whereas all other lines were not (vs Ba/F3, p = 0.001). Neurofibromin can be constitutively phosphorylated at the c-terminus, negatively regulating NF1-GAP activity, suppressing RAS signaling and inducing cell cycle arrest. Therefore, to demonstrate loss of function due to the c-terminus NF1 p.L2467fs and increased RAS signaling, western blotting for pERK was performed. Significant upregulation of pERK was confirmed in P2RY8-CRLF2+NF1fs in comparison to Ba/F3 control cells (p=0.007) (Figure 1). The MEK inhibitors trametinib and mirdametinib are in clinical trials for NF-1 patients and have shown efficacy in ALL models with RAS mutations. In a 3-day cell death assay, only P2RY8-CRLF2+NF1fs demonstrated sensitivity to trametinib (LD 50 P2RY8-CRLF2 = >6.4 µM, NF1fs = >6.4 µM, P2RY8-CRLF2+NF1fs =1.7µM; p < 0.001) and mirdametinib (LD 50 P2RY8-CRLF2 = >16 µM, NF1fs = >16 µM, P2RY8-CRLF2+NF1fs = 8.3 µM; p < 0.0001) (Figure 2). Here, we have demonstrated a LOF NF1fs mutation using an in-vitro model of ALL. Germline NF1 haploinsufficiency and a second hit NF1 mutation in ALL is limited to one report of monozygotic twins with neurofibromatosis. We propose that NF1 p.L2467fs caused bi-allelic LOF and therefore contributed to relapse in this patient. An understanding of the genomic complexities that lead to relapse may also inform personalized treatment strategies. While this patient subsequently achieved remission with inotuzomab and underwent successful stem cell transplantation, the sensitivity to MEK inhibitors is an exciting development for neurofibromatosis patients with ALL. Figure 1 Figure 1. Disclosures Yeung: Amgen: Honoraria; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. White: BMS: Honoraria, Research Funding; Novartis: Research Funding.

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.

Background HLA-matched sibling and unrelated donors are not always available for patients that are in need of an allogeneic haematopoietic stem cell transplant. Partially HLA-mismatched related (haploidentical) donors can be identified for the vast majority of patients but these transplants were historically complicated by excessive graft versus host disease (GVHD), nonrelapse mortality (NRM), and poor overall survival (OS). Haploidentical haematopoietic stem cell transplantation (Haplo HSCT) has become increasingly common over the last ten years, predominately due to the successful use of post-transplant cyclophosphamide (PTCy) in mitigating graft versus host disease (GVHD). Most studies using PTCy have been retrospective with various conditioning regimens and stem cell sources making interpretation of results difficult. ANZHIT-1 is a prospective Australian multi-centre phase II study of Haplo HSCT with defined conditioning and GVHD prophylaxis regimens. Methods: This is an ethics approved Phase II study (ACTRN: 12617000151336) conducted between 2015-2020 at six Australian allogeneic HSCT centres. All patients received peripheral blood Haplo HSCT from haploidentical family members. The reduced intensity regimen (RIC) was fludarabine, cyclophosphamide and 200cGy TBI and the myeloablative regimen (MAC) was fludarabine with IV busulfan 3.2mg/kg x 4. PTCy 50mg/kg D+3, D+4 was used with MMF (to D35) and a calcineurin inhibitor (CNI) as GVHD prophylaxis. CNIs were weaned and ceased by D+120 in patients who did not have evidence of GVHD. Choice of conditioning regimen was based on age and hematopoietic cell transplantation comorbidity index (HCT-CI) scores. Patients who had an HCTCI >3 or age >50yrs received RIC. Data collection was through the Australian Bone Marrow Transplant Recipient Registry. Survival was analysed using the Kaplan-Meier method. Results: Seventy eight patients were included in the study with a median follow up of 402.5 days (28-1259). There was a male predominance (66%). The median age was 53 years (range 18-69) and the median HCT-CI score was 1 (range 0-5). Thirty nine patients had acute myeloid leukaemia, 12 acute lymphoblastic Leukaemia, 14 Non Hodgkin lymphoma or Hodgkin lymphoma, 4 myelodysplastic syndrome and 9 other diagnoses. The majority received RIC (59%). Median neutrophil and platelet engraftment times were 18 days (range: 12-92) and 28 days (range: 13-262) respectively. Acute GVHD grade III-IV occurred in 14.1% of patients whilst chronic GVHD occurred in 20.5%. Moderate to severe cGVHD according to NIH consensus criteria occurred in 10.3% of patients. Overall survival probability at one year was 74.1% (95% CI: 52.9-86.8) for MAC recipients and 77.8% (95% CI: 61.6-87.8) for RIC recipients (Figure 1). Non relapse mortality (NRM) at D100 and 1 year were 2.2% and 8.7% respectively in the RIC group compared to 6.3% and 18.8% respectively in the MAC group. Causes of NRM included infection (n=5), GVHD (n=3) and VOD, respiratory failure and multi-organ failure in one patient each. Haemorrhagic cystitis and poor graft function were relatively common post-transplant morbidities but rarely contributed to NRM. Despite the use of peripheral blood stem cells and early weaning of CNIs, only 3/11 NRM deaths were due to GVHD. Relapse was the most common cause of death in the RIC group with 17.4% of patients relapsing at a median of 114 days (range 36-564) compared to only 6.3% at a median of 90.5 days (range 64-154) in the MAC group. Conclusion: This prospective Haplo HSCT trial utilising PTCY demonstrates encouraging overall survival rates. The RIC regimen had a promising toxicity profile but early relapse remains a concern despite early weaning of CNIs. In contrast, the MAC regimen is associated with a low relapse rate but NRM at one year remains significant. Further refinement of the conditioning and GVHD prophylaxis regimens will be incorporated in the follow-on study, ANZHIT-2, which will begin accrual in late 2020. Disclosures Bajel: Pfizer: Honoraria; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria; Astellas: Honoraria; Abbvie: Honoraria. Greenwood:Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hamad:Novartis: Honoraria; Abbvie: Honoraria.

Introduction Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of ALL associated with high relapse rates and poor survival. Rearrangements of Janus kinase 2 (JAK2r) are present in approximately 5% and 14% of pediatric and young adult Ph-like ALL cases respectively. The resultant JAK2 gene fusions drive leukemogenesis through constitutive activation of the JAK/STAT signaling pathway and are associated with very poor outcomes in patients with Ph-like ALL. All JAK inhibitors in clinical development are type I inhibitors, which bind in the ATP-binding site of JAK2. A phase II clinical trial is currently assessing the only FDA-approved JAK1/2 inhibitor, ruxolitinib in high-risk B-cell ALL cases harboring JAK2 alterations. The development of treatment resistance to targeted inhibitors in other diseases is well documented and often results in disease relapse. Elucidating mechanisms of ruxolitinib resistance in JAK2r ALL will inform approaches to monitor the emergence of resistance in ongoing clinical trials and enable the development of therapeutic strategies to overcome or avert resistance. Methods JAK2r B-ALL was modelled in the pro-B cell line, Ba/F3, by expressing the high-risk B-ALL fusion, ATF7IP-JAK2. Ruxolitinib resistance in three independent ATF7IP-JAK2 Ba/F3 cell lines was achieved following dose escalation to a clinically relevant dose of 1 μM ruxolitinib. Sanger sequencing of the RT-PCR amplified JAK2 fusion revealed each resistant line had acquired a different mutation within the JAK2 kinase domain. Therapeutic sensitives were assessed by staining with Fixable Aqua Dead Cell Stain (Invitrogen) and Annexin V, and analysis by flow cytometry. Alterations in signaling pathways were determined using phosphoflow cytometry and western blot analysis. Computational modelling of acquired JAK2 mutations and subsequent influence on ruxolitinib binding was performed using ICM-Pro (Molsoft L.C.C.). Results In addition to the identification of two known ruxolitinib resistant mutations, JAK2 p.Y931C and p.L983F, a novel p.G993A mutation was identified. All mutations localized to the ATP/ruxolitinib binding site and conferred resistance to multiple type-I JAK inhibitors, including ruxolitinib, BMS-911543, and AZD-1480 (Table 1). JAK2 p.G993A ATF7IP-JAK2 Ba/F3 cells were also resistant to the type-II JAK inhibitor, CHZ-868, which binds in an allosteric site of JAK2 in addition to the ATP-binding site. Ruxolitinib resistance correlated with sustained downstream STAT5 activation in the presence of 1 μM ruxolitinib compared with non-mutant ATF7IP-JAK2 Ba/F3 cells. Intracellular phosphoflow cytometry of ruxolitinib-resistant ATF7IP-JAK2 Ba/F3 cells confirmed constitutive activation of JAK/STAT signaling in the presence of 50 nM ruxolitinib, in contrast to non-mutant ATF7IP-JAK2 Ba/F3 cells. Computational modelling suggested that JAK2 p.L983F (Fig. 1D) sterically hinders ruxolitinib binding, while JAK2 p.Y931C may reduce ruxolitinib binding affinity by disruption of a critical hydrogen-bond (Fig. 1B). The novel JAK2 p.G993A mutation is predicted to alter DFG-loop dynamics by stabilizing the JAK2 activation loop (Fig1C). Conclusions This study demonstrates that the JAK2 ATP-binding site is susceptible to JAK inhibitor resistant mutations following ruxolitinib exposure in the setting of JAK2r ALL, highlighting the importance of monitoring the emergence of mutations within this region. In addition to previously described mutations we identified a novel JAK2 p.G993A mutation that conferred resistance to both type-I and type-II JAK inhibitors. The JAK2 p.G993A mutation was postulated to modulate the stability of a conserved domain. Understanding mechanisms of ruxolitinib resistance, as modelled here, has the potential to inform future drug design and the development therapeutic strategies for this high-risk cohort. Disclosures White: Amgen: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding.

Introduction Mantle cell lymphoma (MCL) is a mature B lymphoproliferative disorder with a frequently aggressive clinical course. Although the response rates in patients eligible for conventional chemoimmunotherapy are high, relapses are virtually inevitable, with a median overall survival (OS) of three to five years. For some patients allogeneic stem haematopoietic cell transplantation (alloHCT) is a potential therapeutic option. AlloHCT for MCL has been associated with long term overall survival (OS) of around 40%, with high rates of non-relapse mortality (NRM) of 20-40% and relapse rates of 20-30% (Urbano-Ispizua et al., Biol Blood Marrow Transplant 2015;21:1746, Robinson et al., Leukemia 2015;29:464). Whilst there is evidence of a graft-versus-lymphoma effect in MCL, it has not been shown to translate into improved OS. We performed a retrospective national registry study to examine alloHCT practice and outcomes for MCL in Australia and New Zealand in the modern era. Methods Data was collected through the Australasian Bone Marrow Transplant Recipient Registry (ABMTRR) for patients receiving an alloHCT for MCL between January 2009 and December 2019. This time range was chosen to capture the era of widespread rituximab use. Survival, relapse and toxicities of alloHCT were investigated, as well as transplant trends over time. Survival was analysed using the Kaplan-Meier method, with comparisons between the transplant periods 2009-2014 and 2015-2019 performed using the log-rank test. Cox proportional hazards regression was performed to determine significant risk factors for transplant outcome. The following risk factors were analysed for impact on outcomes: age, transplant before 2015, previous autologous HCT (autoHCT), remission status at transplant, use of myeloablative conditioning (MAC), haploidentical donor and use of T cell depletion (TCD). Results A total of 86 patients were included in the analysis. The median age was 56 (range 39-71). There was a male predominance with only 12% female patients. At the time of transplant, 51% were in complete remission, 26% had a partial response and 20% had stable or progressive disease (data missing in 3%). Sixty-seven percent had undergone previous autoHCT. The majority of donors were HLA-matched siblings (51%), followed by HLA-matched-unrelated (42%) and haploidentical (7%). Myeloablative conditioning was utilised in 14%, and T-cell depletion (TCD) in 24%. The median times to neutrophil engraftment (>0.5x109/L) and platelet engraftment (>20x109/L) were 16 and 20 days respectively. NRM at 1 and 5 years was 23% (95% confidence interval [95%CI] 10-39%) and 30% (95%CI 15-48%) respectively. The 100-day cumulative incidence of grade II to IV acute GVHD was 29%. The 3-year cumulative incidence of chronic GVHD was 27%. The median duration of follow up was 4.17 years (range 0-9.6 years). Median OS was 4.2 years, with an estimated 5-year OS of 47% (95%CI 35-58%) and 10-year OS of 23% (95%CI 8-43%) (figure 1). Five-year relapse free survival (RFS) was 38% (95%CI 26-50%) (figure 2). The cumulative incidence of relapse (CIR) was 20% at 1 year and 33% at 4 years, with a flattening of the curve after this point (figure 3). On multivariate analysis (MVA), the use of myeloablative conditioning (MAC) was associated with inferior RFS (hazard ratio 2.33; 95%CI 1.05-5.17; p=0.038) and OS (hazard ratio 3.11; 95%CI 1.39-7.00; p=0.006) (figure 4). No risk factors on MVA impacted NRM or CIR. Chronic GVHD was not associated with RFS or CIR. An average of nine alloHCTs were performed each year. There was an increase in the proportion of haploidentical transplants between 2009-2014 and 2015-2019 (4% to 10%). There was no significant change over time in OS, RFS or NRM, or in the use of MAC or TCD. Conclusion There has been no significant change in the rates of alloHCT for MCL in Australia and New Zealand over the past decade. Trends show an increasing utilisation of haploidentical donors. Overall outcomes were comparable to those previously published with favourable OS and durable remissions seen in a subset of patients. MAC was associated with inferior OS and RFS, however the cause for this is unclear given the lack of association with NRM or CIR. Ongoing reporting of alloHCT outcomes in MCL is important given the emerging role of novel therapies, such as Bruton tyrosine kinase inhibitors, bispecific T cells and CAR-T cell therapy. Figure 1 Disclosures Di Ciaccio: Jansen: Honoraria, Other: travel and accomodation grant. Greenwood:MSD: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hamad:Novartis: Honoraria; Abbvie: Honoraria.

Background and Objective 15% of pediatric and 40% of adult T-cell Acute Lymphoblastic Leukemia (T-ALL) patients fail conventional therapy highlighting the need for novel therapeutic strategies based on genomic alterations of individuals. We interrogated genomic alterations in Australian T-ALL patients for patterns of mutation and druggable targets. A subset of samples were used to establish patient-derived xenograft (PDX) models to evaluate novel therapies. Methods T-ALL patients' samples underwent next generation genomic analyses (128 total samples including diagnosis, refractory and relapse timepoints from 118 patients of all age groups). mRNA sequencing (mRNAseq) identified gene fusions and structural variants and assessed gene expression (n=101 patients). Fusions were called when identified by 2/3 predictors (FusionCatcher, SOAPfuse, JAFFA). Variant calling utilized GATK HaplotypeCaller and underwent several filtering steps to eliminate possible germline alterations and common SNPs. DNA copy number variations (CNVs) were detected via Multiplex Ligation-dependent Probe Amplification (MLPA: P202, P335, P383; n=64 patients). Establishment of PDX models from patient material (bone marrow or peripheral blood) is ongoing. Recapitulation of human disease was confirmed by mRNAseq in a subset of xenografts. Results Genomic fusion genes were identified in 46/101 samples (46%) by mRNAseq; the most common fusion identified was STIL-TAL1 (n=6). Increased expression of LCK and/or LAT (encoded proteins are involved in T-cell receptor (TCR) signal transduction) was observed in 100% of patients with the STIL-TAL1 gene fusion indicating TCR signaling pathways may be perturbed in this sub-group. Other common gene fusions were MLLT10-DDX3X (n=5) and KMT2A-MLLT4/AFDN (n=4). We also observed the previously reported fusions SET-NUP214, KMT2A-MLLT1, PICALM-MLLT10, NUP214-ABL1, several fusions involving TCR subunits as well as novel fusions involving KMT2A, NOTCH1, LMO1, ZEB2. Numerous nonsynonymous mutations were identified in 81/101 patients (80%) with mRNAseq data available (Figure 1). Broadly, the mutated genes encode proteins in the following categories: oncoproteins (NRAS, KRAS); tumour suppressors (TP53, CHEK2, BRCA1, BRCA2, PTEN), epigenetic regulators (EZH2, SETD5, DNMT3A); regulators of NOTCH signalling (NOTCH1, NOTCH2, FBXW7); transcription factors and regulators (IKZF1, KMT2A, EP400, SMARCD1, RUNX1, AFF1, AFF3); kinase and cytokine signal regulators (ATM, JAK2, JAK3, TYK2, FLT3, PTPN11). INDELS in clinically relevant genes were identified in 37/101 patients (37%) including alterations to: NOTCH1, PHF6, PTEN, STAT1, IL7R, CDKN2A, LYL1, WT1, JAK3, LEF1. The most common copy number alterations identified in our patient cohort were CDKN2A/B deletions (30/64 patients, 47%), PHF6 duplication (20/64 patients, 31%) and MLLT3 deletion (13/64 patients, 20%). In patients with CDKN2A/B deletions and additional CNVs, PHF6 duplication (n=6) and MYB duplication (n=4) were mutually exclusive. However, one patient without CDKN2A/B deletions harbored both MYB and PHF6 duplications. MLLT3 deletion always co-occurred with CDKN2A/B deletions (13/13 patients with MLLT3 deletion), but was never observed with PTEN deletion (0/7 patients with PTEN deletion). Patients with either CDKN2A/B deletions or PHF6 duplications frequently harbored NOTCH1 abnormalities: 13/32 patients (41%) and 8/21 patients (38%), respectively. PDX primagrafts investigated the engraftment latency and peripheral organ infiltration. Primagrafts established from patients harboring NUP214-SET1 or NUP214-ABL1 fusions engrafted at a slower rate (82 d and 91 d, respectively) than primagrafts from patients harboring a STIL-TAL1 fusion (45 d) or CDKN2A/B deletions (31 d and 48 d). Conclusions In our T-ALL cohort we demonstrate that the majority of cases harbor rearrangements, structural variations and duplication/deletion of genes associated with malignant transformation. We identified several co-occurring lesions as well as mutually exclusive genomic abnormalities. The top 20 mutated genes in our patient cohort differ to those reported for a pediatric cohort (Roberts et al 2019 Blood 134:649), indicating an association between patient age and genomic alteration. Secondary PDX models investigating novel targeted treatment strategies are ongoing. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. White:Bristol-Myers Squibb: Honoraria, Research Funding; Amgen: Honoraria.

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.

With the increasing adoption of treatment-free remission (TFR) as a goal for patients with chronic myeloid leukemia (CML), rigorous molecular monitoring has been recommended to ensure timely tyrosine kinase inhibitor (TKI) recommencement in the event of molecular relapse. The National Comprehensive Cancer Network (NCCN) has now incorporated TFR into its most recent guidelines, recommending monthly quantitative polymerase chain reaction monitoring of BCR-ABL1 for the first 12 months following TKI discontinuation.1 Molecular relapse, the trigger to restarting TKI, is currently defined as loss of major molecular response 2 (MMR; BCR-ABL1 ≤ 0.1% International Scale), and predominantly occurs in the first 6 months following TKI cessation.3,4 Delay in detection of molecular relapse may place patients at unnecessary risk of adverse outcomes, such as loss of complete hematological response. At a minimum, it delays the reachievement of MMR and deep molecular response (MR4; BCR-ABL1 ≤ 0.01% or MR4.5; BCR-ABL1 ≤ 0.0032%). Conversely, an overly rigorous monitoring schedule imposes unnecessary costs associated with a TFR attempt, adding to logistical difficulties experienced by some patients in accessing highly sensitive, standardized BCR-ABL1 testing,5 and may prevent some patients from stopping therapy. This is especially important in countries where funding of molecular testing is limited, and patients may be required to pay for additional tests. Refereed/Peer-reviewed

Introduction A majority of patients with diffuse large B cell lymphoma (DLBCL) will be cured with frontline chemoimmunotherapy, however a significant number of patients will relapse. Although autologous haematopoietic stem cell transplantation (autoHCT) may lead to sustained survival in some relapsing patients, long term survival with relapsed DLBCL is approximately 25% (Larouche et al., J Clin Oncol 2010;28(12):2094). Allogeneic HCT (alloHCT) is a potential treatment strategy in some DLBCL patients with relapsed disease. We performed a retrospective national registry study to examine alloHCT practice and outcomes for DLBCL in Australia and New Zealand in the modern era. Methods Data was collected through the Australasian Bone Marrow Transplant Recipient Registry (ABMTRR) for patients receiving an alloHCT for DLBCL between January 2009 and December 2019. Survival was analysed using the Kaplan-Meier method, with comparisons between the transplant periods 2009-2014 and 2015-2019 performed using the log-rank test. Both univariate and Cox proportional hazards regression were performed to determine significant risk factors for transplant outcome. The following risk factors were analysed for impact on outcomes: age, transplant before 2015, previous autoHCT, remission status at transplant, use of myeloablative conditioning (MAC), haploidentical donor (HD) and use of T cell depletion (TCD). Results A total of 154 patients were included in the analysis. The median age was 52 years (range 19-71) and 68% were male. Disease status at transplant was complete remission (CR) in 49%, partial response in 31% and stable or progressive disease in 17% (missing data in 3%). Fifty-five per cent had undergone a previous autoHCT. Approximately equal proportions of donors were HLA-matched siblings or matched unrelated (45% and 46% respectively) and 9% were HDs. MAC was utilised in 26%, and TCD in 24% (alemtuzumab in 3%, anti-thymocyte globulin in 21%) (data missing in 12%). The median times to neutrophil engraftment and platelet engraftment were 16 and 18 days respectively. Non-relapse mortality (NRM) at 1-year and 5-years was 20% (95%CI 7-30%) and 26% (95%CI: 13-38%). The 100-day cumulative incidence of grade II to IV acute graft versus host disease was 15% (95%CI 5-31%). The 3-year cumulative incidence of chronic graft versus host disease (cGVHD) was 56% (95%CI 46-65%) (figure 1). The median duration of follow up for the cohort was 3.98 years (range 0-9.64 years). Median overall survival (OS) post-transplant was 4.01 years, with 5-year OS of 47% (95%CI 38-56%) and 10-year OS of 40% (95%CI 26-54%) (figure 2). The 5-year relapse free survival (RFS) was 45% (95%CI 26-50%) (figure 3). The cumulative incidence of relapse (CIR) was 21% at 1 year and 32% at four years, however relapses were not seen after this point, suggesting a subpopulation with durable remissions (figure 4). On univariate analysis, TCD was associated with both reduced incidence of cGVHD (HR 0.35 95%CI 0.19-0.66, p=0.012) and increased NRM (HR 2.10 95%CI 0.88-4.99 p=0.043). These associations were maintained on multivariate analysis (MVA) (HR 0.29 95%CI 0.16-0.76, p=0.011; HR 2.19 95%CI 1.02-4.70, p=0.045) (figures 5, 6). TCD did not impact on RFS. The vast majority of TCD was given in unrelated donor alloHCTs. CR at time of transplant was associated with improved RFS on univariate analysis (HR 1.65 95%CI 1.04-2.64, p=0.034), however this association was not seen on MVA. No other analysed risk factors impacted OS, RFS, NRM, CIR or GVHD rates on either univariate or MVA. An average of 14 alloHCTs were performed each year, with a trend towards increasing annual numbers over time. There was a significant increase in the proportion of haploidentical transplants between 2009 and 2019 (p=0.003), though total numbers were low (n=14). There was no significant change over time for the use of MAC, TCD, nor in OS, RFS or NRM. Conclusion There has been an increase in the rates of alloHCT with HDs for DLBCL in Australia and New Zealand over the past decade. Survival and relapse rates are relatively favourable compared to the published literature, with sustained remissions observed (5 and 10-year OS of 47% and 40% respectively). TCD is associated with reduced cGVHD rates, as well as increased NRM. Ongoing reporting of alloHCT outcomes in DLBCL is important given the emerging role of novel therapies such as bispecific monoclonal antibodies and chimeric antigen receptor T cell therapy. Figure 1 Disclosures Di Ciaccio: Jansen: Honoraria, Other: travel and accomodation grant. Greenwood:Servier: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Spencer:Celgene, Janssen and Takeda: Speakers Bureau; AbbVie, Amgen, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Honoraria; Amgen, Celgene, Haemalogix, Janssen, Servier and Takeda: Research Funding; AbbVie, Celgene, Haemalogix, Janssen, Sanofi, SecuraBio, Specialised Therapeutics Australia, Servier and Takeda: Consultancy. Arthur:Royal North Shore Hospital: Current Employment. Hamad:Novartis: Honoraria; Abbvie: Honoraria.

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

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

We report a novel somatic mutation in the kinase domain of JAK2 (R938Q) in a high-risk pediatric case of B-cell acute lymphoblastic leukemia (ALL). The patient developed on-therapy relapse at 12 months, and interestingly, the JAK2 locus acquired loss of heterozygosity during treatment resulting in 100% mutation load. Furthermore, we show that primary ALL mononuclear cells harboring the JAK2 R938Q mutation display reduced sensitivity to the JAK1/2 ATP-competitive inhibitor ruxolitinib in vitro, compared to ALL cells that carry a more common JAK2 pseudokinase domain mutation. Our findings are in line with previous reports that demonstrate that mutations within the kinase domain of JAK2 are associated with resistance to type I JAK inhibitors. Importantly, given the recent inclusion of ruxolitinib in trial protocols for children with JAK pathway alterations, we predict that inter-patient genetic variability may result in suboptimal responses to JAK inhibitor therapy in a subset of cases. The need for alternate targeted and/or combination therapies for patients who display inherent or developed resistance to JAK inhibitor therapy will be warranted, and we propose that kinase-mutants less sensitive to type I JAK inhibitors may present a currently unexplored platform for investigation of improved therapies.

We present three cases of the rare hepatosplenic T-cell lymphoma (HSTCL); two patients suffering from Crohn disease who developed HSTCL on azathioprine without exposure to biologicals, and a third patient who had psoriasis treated using etanercept, cyclosporine and methotrexate. The evidence for an association between HSTCL and immunosuppressive drugs and biologicals is reviewed. We argue for improved pharmacovigilance processes to help determine the benefit to risk ratios for the use of these and other new agents.

Background - Conventional chemotherapy for adults with Acute Lymphoblastic Leukaemia (ALL) is associated with considerable treatment-related toxicity. Blinatumomab is a CD19/CD3 targeting bi-specific T-cell engager that has demonstrated promising efficacy in relapsed/refractory ALL, and in combination with chemotherapy in newly diagnosed patients. Preliminary studies also suggest less toxicity and good efficacy (CR/CRh 66%) when delivered as induction to older adults (median age 75) with newly diagnosed B-ALL (Advani, ASH 2018). Of responders, Minimal Residual Disease (MRD) negativity in this trial was achieved in 92%. Given this, the Australasian Leukaemia & Lymphoma Group (ALLG) has explored the potential for upfront Blinatumomab as induction for younger adults with Ph- B-lineage Acute Lymphoblastic Leukaemia, alternating with CNS-directed chemotherapy cycles. Aim - To assess response to therapy of the first 10 patients enrolled on this study by molecular MRD analysis as a surrogate measure of short-term efficacy of this combination. Method - The ALLG ALL8 study (ACTRN12617000084381) is a phase II proof-of-concept front-line study for patients fit for treatment with a Hyper-CVAD-like regimen (between 40-65 years) with newly diagnosed Ph- B-lineage ALL. Those with CNS positive disease are excluded. Patients receive a steroid pre-phase (Prednisolone 100mg daily for 7 days) followed by a disease debulking phase of cyclophosphamide 150mg/m2 BD day 1-3, vincristine 2mg day 1 & 11 and dexamethasone 10mg/m2 day 1-4 and 11-14. Patients then receive alternating cycles of Blinatumomab (at 9mcg/d for the first 7 days of cycle 1 followed by 28mcg/d until day 28) with B-cycles of Hyper-CVAD (Methotrexate 1g/m2 day 1, Cytarabine 3g/m2 BD day 2,3, Methylprednisolone 50mg BD day 1-3) (figure 1). All patients receive intrathecal prophylaxis with methotrexate, cytarabine and hydrocortisone prior to blinatumomab treatment blocks and day 1 and 8 of each B-cycle until a total of 8 doses were administered. At the completion of therapy, high-risk patients (MLL translocations, hypodiploid, complex karyotype or MRD positive at TP3) are recommend to proceed to allogeneic stem cell transplant while others receive 24 months of POMP maintenance. Minimal residual disease analysis was performed at a centralised EuroMRD accredited laboratory utilising Taqman probes and patient specific PCR primers targeted at immunoglobulin heavy chain or T-cell receptor (Ig/TCR) gene rearrangements. MRD positivity was defined as a detectable level of ≥ 1 x 10-4. Results - The following results are based upon the first 10 patients enrolled on ALL8. Median age of 54.7 years (range 43 to 66 years) and 70% were male. Median ECOG PS was 0 (range 0 - 2). 6 patients had an abnormal karyotype, 2 high-risk (one with hypodiploid karyotype, one with t(1;19). Median white cell count at diagnosis was 3.36 x 109/L (range 1.95 - 12.05 x 109/L). All patients (10/10) attained CR/CRi following induction therapy with no treatment related deaths. At the end of the induction phase (MRD TP1) 4 out of 10 had attained MRD negativity, 2 had detectable unquantifiable MRD and 4 were MRD positive. By the end of the 1st consolidation cycle (MRD TP2) 1 additional patient had become MRD negative and 1 MRD positive patient had become MRD unquantifiable. 1 patient was not evaluable at TP2 being withdrawn prior to this, 2 do not have TP2 available at this time (1 being MRD negative at TP1, 1 with unquantifiable low-level MRD). One patient had MRD increase between TP1 and TP2, this subject had a hypodiploid karyotype. Thus, by completion of 1st consolidation an aggregate of 7 out of 10 patients were MRD negative or had unquantifiable low-level MRD, 3 remaining MRD positive (with 1 MRD progression). Updated results will be presented at the meeting. Conclusion - Front-line therapy with Blinatumomab in combination with chemotherapy is feasible in adults and results in high levels of MRD response by the end of the first consolidation block with the majority of MRD negative responses attained after the first treatment block. Despite early incorporation of Blinatumomab into this treatment protocol, MRD progression was seen in one patient with high-risk cytogenetic abnormalities. Disclosures Fleming: Astellas: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria. Reynolds:Novartis Australia: Honoraria; Novartis AG: Equity Ownership; AUSTRALASIAN LEUKAEMIA & LYMPHOMA GROUP (ALLG): Consultancy; 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.. Nguyen:Australasian Leukaemia & Lymphoma Group: Employment. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Greenwood:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Verner:Janssen-Cilag Pty Ltd: Research Funding. Bajel:AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: travel funding. Wei:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Astellas: 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, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria, Research Funding; Janssen: Honoraria. OffLabel Disclosure: Blinatumomab is not currently approved for the front-line treatment of adults with Ph- B-ALL

Background Down Syndrome (DS) Acute Lymphoblastic Leukemia (ALL) patients have extremely poor outcomes with mortality rates four times greater than non-DS ALL patients within their first two years of diagnosis. They are more suspectible to treatment related toxicities and experience higher relapse rates compared to other ALL patients. Approximately 60% of DS-ALL patients harbor rearrangement of cytokine receptor like factor 2 (CRLF2r), specifically P2RY8-CRLF2, and/or the CRLF2 F232C activating mutation. These lesions are considered poor risk and currently no targeted therapy exist. How increased chromosome 21 gene dosage affect disease phenotype is not yet fully elucidated. However, the high mobility group nucleosome-binding domain-containing protein 1 (HMGN1) on chromosome 21, which competes with histone H1 to bind the nucleosome and results in gene activation may be a candidate for targeted therapy in DS-ALL. Methods We aimed to determine the role of HMGN1 in CRLF2r DS-ALL. To model CRLF2r DS-ALL, the trisomy 21 cell line, SET-2, was transduced with a retroviral vector encoding the CRLF2 F232C activating mutation. Gene knockdown of HMGN1 using CRISPR/Cas9 was performed in the SET-2 CRLF2r line and the non-trisomy-21, non-CRLF2 expressing Jurkat line. Individual knockdowns of another two genes on chromosome 21, DYRK1A and ERG were also performed. Knockdown of JAK2 was used as a control as it is critical for CRLF2 signaling. CellTiter-Glo was used to investigate proliferation of knockdown lines to test the hypothesis that HMGN1 is essential for CRLF2r DS-ALL cell proliferation. Lentiviral vectors encoding the P2RY8-CRLF2 fusion gene, CRLF2 F232C activating mutation or an overexpression construct of HMGN1 were transduced into BaF3 cells individually or in combination to test the hypothesis that overexpressing HMGN1 is associated with activation of CRLF2. Quantitative PCR (qRTPCR) for CRLF2 and flow cytometry for the CRLF2/IL7Rα receptor (TSLPR) were used to determine the effect of increased HMGN1 on CRLF2 expression. AnnexinV/7-AAD cell death assays were performed to determine if the effects of HMGN1 could be reduced by the demethylase inhibitor GSK-J4. Results Knockdown of HMGN1 resulted in an 80-90% decrease in HMGN1 protein expression in SET-2 CRLF2 and Jurkat lines compared to the Cas9 controls. While knockdowns of DYRK1A and ERG did not impair the proliferation of SET-2 CRLF2 cells, HMGN1 and JAK2 knockdowns led to a complete proliferation arrest over a period of 120hrs (p= Overexpression of either HMGN1 or P2RY8-CRLF2 alone in BaF3 cells did not result in cytokine independent transformation. However, cytokine independence was triggered in BaF3 cells when HMGN1 and P2RY8-CRLF2 were co-expressed (p= While there are no pharmacological inhibitors for HMGN1, Lane et al. (2014) have shown that the restoration of H3K27 methylation using the demethylase inhibitor GSK-J4 was able to prevent DS-ALL cells from repassaging. Therefore, we have employed the inhibitor GSK-J4 to determine if it can reduce cell survival in HMGN1 overexpressed BaF3 cells. Specific inhibition of BaF3 P2RY8-CRLF2 HMGN1 cells was evident by decreased cell viability at a concentration of 3.8µM compared to BaF3 P2RY8-CRLF2 or BaF3 HMGN1 lines (p= Conclusion These data support the hypotheses that HMGN1 has a significant role in DS-ALL cell proliferation and that overexpression of HMGN1 results in activation of P2RY8-CRLF2. This is the first report of a novel role for HMGN1 in P2RY8-CRLF2 activation and leukemic transformation in CRLF2r DS-ALL. Additionally, we show that HMGN1 is a potential candidate for the development of a pharmacological inhibitor for CRLF2r DS-ALL. Disclosures Yeung: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. White:BMS: Honoraria, Research Funding; AMGEN: Honoraria, Speakers Bureau.

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.

Introduction and Aim. Achievement of deep molecular response (DMR) is the prerequisite for treatment-free remission in chronic phase CML (CP-CML) patients (pts). Pts who fail to achieve early molecular response (BCR-ABL1 > 10% IS) at 3-months (mths), or have high ELTS score at diagnosis have inferior achievement of DMR. We and others have shown that the levels of NK-cell, T-cell, myeloid-derived suppressor cell, and neutrophils in the blood at diagnosis have an impact on DMR achievement. We hypothesized that Cluster of Differentiation (CD) (cell surface marker) gene expression might provide a surrogate marker to characterize immune cell composition. We aimed to identify pts who had a low probability of achieving DMR by 5 years (yrs) by combining 3-mths BCR-ABL1% and CD gene expression. This may enable clinicians to determine whether an individual patient is on a pathway towards DMR and potentially TFR or should be considered for a different therapeutic approach if TFR is the eventual goal. Methods. 119 blood samples from the imatinib-based TIDEL-II trial were subjected to transcriptomic microarray profiling. A total of 357 CD genes classified by the HUGO Gene Nomenclature Committee CD molecular gene group were assessed. We defined DMR as achieving MR4.5 (BCR-ABL1 < 0.0032%) at two consecutive time points. To construct a predictive model, the samples were randomly assigned to discovery and validation cohorts. Recursive partitioning and construction of a regression tree with tenfold cross-validation based on expression of 357 CD genes and 3-mths BCR-ABL1% were used as inputs in the discovery cohort. The performance was assessed based on accuracy of prediction of DMR by 5 yrs. The final model was validated using the independent validation cohort. All the analysis was performed using R statistical software. Results. Clinical variables (age, gender, ELTS, 3-mths BCR-ABL1%, MMR, and MR4.5) were well matched in the discovery (n=60) and independent validation cohort (n=59). The predictive model was constructed using the discovery cohort to reveal two risk groups: poor-risk (PR, 15% achieving MR4.5 at 5 yrs, n=19), and good-risk (GR, 88% achieving MR4.5 at 5 yrs, n=41) groups (Figure 1A-B). This model classified PR group by BCR-ABL1 ≥ 7.5% at 3 mths OR BCR-ABL1 < 7.5% at 3 months with high CD302 gene expression (≥7.9 log2 gene expression; top 15%) at diagnosis. GR group was defined as having both BCR-ABL1 < 7.5% and low CD302 gene expression ( We asked whether using the more conventional BCR-ABL1 10% cutoff instead of 7.5% in our model would give similar results, but the performance in predicting long-term DMR achievement was inferior: Pts predicted as PR with this criteria had ~2x higher achievement of DMR (e.g. 26% vs 14% using 3-mths BCR-ABL1 10% vs 7.5% cutoff respectively). ELTS score have been associated with the probability of DMR achievement. We compared the performance of ELTS in combination with 3-mths BCR-ABL1% by replacing CD302 gene expression with ELTS. The predictive accuracy was inferior. Pts with 3-mths BCR-ABL1 ≥7.5% OR BCR-ABL1 Conclusion. We have constructed a predictive model for DMR achievement for pts who receive optimised frontline imatinib therapy. This model performs better than combining ELTS and 3-mths BCR-ABL1%. We postulate that this predictive model could enable identification of poor risk pts at 3 mths who would benefit from intensified therapeutic approaches to obtain eligibility for TFR and potentially optimal clinical outcome. Disclosures Yeung: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Hughes:Novartis: Other: Advisory Board and Symposia, Research Funding; BMS: Research Funding.

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.

Eighty-six newly diagnosed Philadelphia-negative ALL pts were enrolled from 2012 to 2018, from 14 Australian centres; 82 pts were evaluable. Pts were stratified and treated as per the pediatric ANZCHOG Study 8 protocol based on BFM 2000. Response was assessed on day 33 and 79 by morphology, flow cytometry and RQ-PCR measurable residual disease (MRD) at a central lab according to EuroMRD criteria. Allogenic stem cell transplantation was permitted for high and very high-risk disease groups. Detailed genomic analysis was performed in 47 pts (to date), using whole transcriptome sequencing (mRNA Seq) and multiplex ligation-dependent probe amplification (MLPA) for recurrent ALL related gene deletions. Median age of the study was 24 (16 - 38) years; 28% were female; 59/82 (72%) had B-ALL. Median follow up was 36 (range 3-73) months. Induction mortality was 3.6%. CR rate at day 33 was 90.4% and day 79 (time point 2, TP2) 97.6%. Relapse free survival (RFS) at 2 years was 75.6% (95%CI 65.6 - 85.5%). CR rates at day 33 and day 79 were 90.4% and 97.6% respectively. The 2-year overall survival (OS) was 79.3% (18/82 events). In concordance with other studies, TP2 MRD predicted outcome in ALL06. MRD positive (pos) pts had a 2yr RFS of 68%, vs 98% in MRD negative (neg) pts (p=0.003). To date, 47 pts had mRNA Seq & MLPA; 11/47 pts had T cell ALL; 1/47 died during induction (2.1%). The median age of this subset was 21 (15-37) years, 23% were female and the RFS at 2 years was 73.97% (95%CI 65.6 - 91.44%). TP2 MRD remained predictive of outcome in this group with 2-year RFS in MRD pos pts 54% vs 95% in MRD neg pts (p=0.013, n=44). 13/47 pts have died with a 2-year OS of 73% (95%CI 62.7 - 90%). MPLA and mRNA Seq analysed independently of outcome data revealed 28/47 pts had genomic lesions categorise as High Risk (HR). These included fusions and structural genomic abnormalities involving KMT2A, IKZF1, IGH, ABL1, JAK, CRLF2, CDKN2A/B, PAX5, RAS and ZNF384. The remaining cases were classified as Standard Risk (SR) and included mainly hyperdiploid, T cell and ETV6-RUNX1 cases. Eleven of 13 pts who relapsed were genomic HR with poorer 2-RFS vs SR (59% vs 78.8%, p=0.023 respectively) (Fig 1.). We examined the relationship between genomics risk group and TP2 MRD, a known prognostic marker. Of the 22 pts who were MRD pos, 19 (86%) pts were in the HR genomics group. In contrast, for MRD neg pts, 13/22 were in the SR group (59%) (p=0.004 Fishers exact, Table 1). This demonstrates that the TP2 MRD positive group is strongly enriched for pts with HR genomics. Pts with HR genomics who were TP2 MRD pos had a 2 year RFS of 27% vs HR MRD neg or SR pts with a 2 yr RFS of 78% (p=0.001)(Fig. 2). Further, of the 13 deaths that were observed in this subset 9/13 (69%) fell within the group of pts with HR genomics/TP2 MRD+. The single induction mortality, for whom TP2 data was not available was also genomic HR. This is one of the first genomic surveys in a cohort of AYA pts, a group known for their inferior outcomes compared to children, treated on a pediatric inspired ALL protocol. Our overall outcomes compare favourably to other cohorts (EHA 2019 abstract 2416). In ALL06, genomic risk stratification based on previous published HR lesions, identified a HR cohort with significantly lower RFS and trend for inferior OS, vs a SR cohort. HR genomics was also associated with significantly higher rates of TP2 MRD positivity. Elucidation of targetable genomic lesions at the time of diagnosis may allow interventions to minimise MRD positivity and relapse in HR pts. Genomic information also improves understanding of underlying disease biology, providing targets for novel treatment discovery. Disclosures Yeung: Pfizer: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Greenwood:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Wei:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Astellas: 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, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria, Research Funding; Janssen: Honoraria. White:AMGEN: Honoraria, Speakers Bureau; BMS: Honoraria, Research Funding.

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

usc Background: BCR-ABL1 mutation analysis is recommended for chronic myeloid leukaemia patients. However, mutations may become undetectable after changing therapy, and it is unknown whether they have been eradicated. Methods: We examined longitudinal data of patients with imatinib-resistant mutations, which became undetectable by Sanger sequencing to determine whether mutations could reappear, and the related circumstances. Results: Identical imatinib- and nilotinib-resistant mutations reappeared following further therapy changes in five patients, and was associated with subsequent nilotinib resistance in four. Conclusion: The data suggest that some BCR-ABL1 mutations may persist at undetectable levels for many years after changing therapy, and can be reselected and confer resistance to subsequent inhibitors. Refereed/Peer-reviewed

Rising BCR-ABL1 transcripts indicate potential loss of imatinib response in CML. We determined whether the BCR-ABL1 doubling time could distinguish nonadherence from resistance as the cause of lost response. Distinct groups were examined: (1) acquired clinical resistance because of blast crisis and/or BCR-ABL1 mutations; and (2) documented imatinib discontinuation/interruption. Short doubling times occurred with blast crisis (median, 9.0 days; range, 6.1-17.6 days; n = 12 patients), relapse after imatinib discontinuation in complete molecular response (median, 9.0 days; range, 6.9-26.5 days; n = 17), and imatinib interruption during an entire measurement interval (median, 9.4 days; range, 4.2-17.6 days; n = 12; P = .72). Whereas these doubling times were consistently short and indicated rapid leukemic expansion, fold rises were highly variable: 71-, 9.5-, and 10.5-fold, respectively. The fold rise depended on the measurement interval, whereas the doubling time was independent of the interval. Longer doubling times occurred for patients with mutations who maintained chronic phase (CP: median, 48 days; range, 17.3-143 days; n = 29; P < .0001). Predicted short and long doubling times were validated on an independent cohort monitored elsewhere. The doubling time revealed major differences in kinetics according to clinical context. Long doubling times observed with mutations in CP allow time for intervention. A short doubling time for a patient in CP should raise the suspicion of nonadherence.

The use of the polymerase chain reaction (PCR) was a revolutionary step in molecular biology, allowing for small amounts of genetic material to be amplified and studied. The advent of real-time PCR was a further refinement that led to reliable quantification of RNA and DNA. This allowed response monitoring and the detection of minimal residual disease, which proved to have important correlations with outcome in certain malignancies. The technology is indispensable for physicians and pathologists caring for oncology patients. In this article we will review the applications of molecular technology in the diagnosis and management of malignancies. Using chronic myeloid leukaemia (CML) as an example, technical aspects and clinical correlations will be discussed, with emphasis on the importance of quality assurance and standardisation to allow for comparability of results across laboratories. We will also examine emerging technologies that allow for high throughput and rapid turnaround of specimens and speculate how these would affect outcomes in future health care. The established and emerging molecular technologies have applications in many fields of oncology.

Alfa Interferon, commonly used in chronic phase chronic myeloid leukemia (CML-CP) in the pre-imatinib era, was able to induce a cytogenetic response in a minority of patients (pts). Pegylated interferon (Peg-IFN) is better tolerated than IFN, and increases molecular response rates when used in combination with imatinib (IM) compared with IM monotherapy (Preudhomme NEJM 2010). 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 de novo CML-CP. Pts 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 28 mths (16-51). Data is presented on an intention to treat basis. Figure 1a shows BCR-ABL1 transcript levels over time. The co-primary end points are MMR (BCR-ABL1 ≤0.1% IS) AT 12 mths and MR4.5 (BCR-ABL1 ≤0.0032% IS) at 24 mths. At 12 mths, MMR and MR4.5 rates were 76.7% (95% CI 63.4-87%). and 43.4% (95% CI 30.1-57.3%), respectively. In 40 evaluable pts at 24 mths, MR4.5 was 50% (95% CI 29.9-70.1%). The median time to MMR and MR4.5 was 5.8 mths and 18 mths respectively for pts achieving these responses (Figs 1B & C). Six pts (10%) had BCR-ABL1 ≥10% at 3 mths - 2 of whom had multiple dose interruptions due to toxicity; 3/6 have since achieved MMR, 1 has BCR-ABL Dose intensities of NIL were assessed in 3 mth blocks. Median and lower quartile NIL dose intensity was 600mg/d for all 3 mth blocks up to mth 24, except for the lower quartile NIL dose of 567mg for the first 3 mths. 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, 22 pts (37%) received >90% of their assigned dose, 13 (22%) received between 50-90% and 25 pts (41%) received Grade III/IV adverse events (AE) attributed to NIL were increased lipase and neutropenia (each 12%), pancreatitis (6%), thrombocytopenia and rash (each 5%). Three thrombotic events occurred: ischemic colitis in a patient on IM monotherapy, femoral artery thrombosis in a 56yo man after 2.5 yrs of NIL, and coronary disease in a 51yo man after 4 yrs of NIL. Grade III/IV AEs attributed to Pegintron were neutropenia (10%), and myalgia, depression and rash (4% each); other common AEs included fatigue (35%), myalgia (23%), flu-like symptoms (21%) and depression (17%). Ten pts (13%) have withdrawn from study: 2 withdrew consent, 5 due to toxicity (pancreatitis, GI upset, rash, high amylase and fatigue), and 3 for failing to consistently achieve BCR-ABL 90% of assigned NIL/IM and Pegintron doses. This interim analysis suggests that combination therapy with NIL and Peg-IFN leads to favourable rates of molecular responses when compared with with NIL monotherapy (Table 1). While the majority of patients did not durably tolerate full dose Pegintron, there was minimal interference with TKI dose intensity. Longer term results, and impact upon treatment free remission outcome of this combination is awaited. Disclosures Yeung: Pfizer: Honoraria; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Specialised Therapeutics Australia: Honoraria; Amgen: Honoraria. Grigg:Takeda: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Shanmuganathan:Janssen: Honoraria; Royal Adelaide Hospital Research Fund: Other: Scholarship; Novartis: Honoraria, Other: Travel sponsorship; Bristol-Myers Squibb: Honoraria, Other: Travel sponsorship. Reynolds:Novartis: Equity Ownership, Other: former employee of Novartis AG and holds stock in the company. . Ross:BMS: Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Research Funding. Yong:Celgene: Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Research Funding. Branford:Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cepheid: Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: 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.

Introduction. Imatinib has revolutionised the treatment of chronic phase-chronic myeloid leukemia (CP-CML), with up to 70% of patients (pts) achieving major molecular response (MMR, BCR-ABL1 < 0.1% IS). Achievement of MMR by 2 years (yrs) is associated with an excellent prospect of long term survival. Currently, three baseline prognostic scoring systems - the Sokal, Hasford (Euro) and EUTOS risk scores - have all been used to identify pts with a poor response and/or an adverse prognosis in CP-CML. Recently, the EUTOS long-term survival (ELTS) score is shown to have strong predictive power for overall survival in CML pts. We have previously reported bioassays that have significant value for predicting MMR. Combinations of these biomarkers, together with clinical risk score, may provide a better indicator of high risk pts at the time of diagnosis. Aim. To identify high-risk pts by combining selected predictive bioassay, determine whether the ELTS score is more discriminating, and determine whether it provides additional predictive value when combined with the biomarker score. Methods. Bioassays including CRKL IC50 imatinib (White, Blood, 2005), OCT-1 Activity (OA)(White, JCO, 2010), leves of 39 plasma cytokines (Nievergall, Leukemia, 2016), expression of 20 most prognostic gene by qPCR TLDA (Kok, ASH abstract, 2015), ABCB1 gene expression (Eadie, Leukemia, 2016), KIR2DL5B genotype (Yeung, Blood, 2015), BIM and ASXL1 polymorphisms (Marum, Blood advances, 2017) were used in this study. High-risk by biomarker score (HR) was defined as pts who did not achieve MMR by 2 yrs. 210 TIDEL-II pts (frontline imatinib with early switch to nilotinib for failure to meet optimal time-dependent molecular targets) were used in this study (Yeung, Blood, 2015). Only 201 pts had ELTS scores. The Recursive Partitioning and Regression Trees (rpart) algorithm was used to identify important bioassays in predicting high-risk pts. Fisher's-exact test was used for statistical analysis. Results. In the TIDEL-II cohort, there were 21 high ELTS and 180 low/intermediate ELTS pts. Pts with high ELTS had significantly lower rates of MMR by 2 yrs compared to those pts with low/intermediate ELTS (57% vs 81%, p=0.02). We constructed a predictive model using multiple different bioassays as variables to predict high-risk pts. The rpart based model used in this analysis yielded four variables (IGFBP2 gene expression, KIR2DL5B genotype, OA, and MCP-1 cytokine plasma level) as most important for predicting high-risk pts. The accuracy of the model was 84%. Pts predicted as high-risk (HR, n=27) had significantly lower MMR achievement rate compared to those predicted as low-risk (LR), (26% vs 86%, n=183, p Conclusion. We developed a combined bioassays model that is predictive of MMR failure and adverse clinical outcomes for pts who receive optimised frontline imatinib therapy. This model performs well even without adding clinical parameters. Our model has additional predictive value when used together with the ELTS score, and can distinuguish HR pts within the low/intermediate ELTS group, as well as LR patients within the high ELTS category. Further confirmation of the predictive performance of this model, using a large independent patient cohort is now indicated. We postulate that this bioassay-based model could be used, in combination with ELTS, for identifying HR pts who would benefit from intensified therapeutic approaches to obtain optimal clinical outcome. Disclosures Yeung: Amgen: Honoraria; Pfizer: Honoraria; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Specialised Therapeutics Australia: Honoraria. Yong:Celgene: Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. White:BMS: Research Funding; Novartis: Honoraria, Research Funding. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background: For patients with chronic-phase chronic myeloid leukemia (CML), treatment-free remission (TFR) is increasingly becoming a goal of therapy. While the safety of TFR has been established [Mahon, Lancet Oncol 2010; Ross, Blood 2013], the ability to predict success following attempted TFR remains limited. Recent publication of Euro-Ski [Saussele, Lancet Oncol 2018], the largest tyrosine kinase inhibitor (TKI) cessation study to date, demonstrated that duration of MR4 (BCR-ABL1 Aim: To identify the predictors of TFR in a single academic centre. Methods: We performed a retrospective analysis of adult CML patients receiving their primary CML management at the Royal Adelaide Hospital between January 2008 and March 2018, reviewing both clinical and molecular data. Criteria for qualifying for a TFR attempt included a minimum of 3 years (yrs) of TKI therapy and 2 yrs of deep molecular response (DMR: BCR-ABL1 Results: A total of 298 patients were treated at our institution within the defined time frame and 280 patients qualified for inclusion into our retrospective analysis. TFR eligibility was attained in 114 patients and 96 (84%) attempted TFR. Table 1 details patient characteristics of patients attempting TFR. Of the 82 patients with >12 months of follow-up, 52% (n=43) remain off TKI at 12 months in MMR. Variables were assessed by univariate Cox proportional hazards regresssion analysis for their association with TFR. The most significant finding was that patients attempting TFR with e14a2 BCR-ABL1 transcripts were more likely to remain in TFR at 12 months (65%; n=24/37) in comparison to the e13a2 transcript (34%; n=10/29), p = 0.008. This advantage also translated to patients with both e14a2 and e13a2 transcripts when grouped with the e14a2 cohort and compared with e13a2 alone, p = 0.006. The negative effect of the e13a2 transcript was further confirmed on multivariate analysis (Figure 1a) as patients with either e14a2 or both transcript types were 2.24 times more likely to remain in TFR at 12 months compared with the e13a2 transcript, p=0.032. Patients with sustained MR4.5 >3.4 yrs prior to cessation were more likely to remain in TFR at 12 months (42 vs. 64%, p = 0.014). We postulated that the higher rate of TFR in patients with e14a2 might be due in part to the longer time in MR4.5 prior to cessation. The median duration of MR4.5 prior to stopping in the e14a2 cohort was 4.1 yrs (2.05 - 10.76) compared to 3.01 yrs (2 - 10.41) in the e13a2 group (Table 2). Cumulative incidence curves of all 280 patients in our analysis demonstrated that by 6 yrs of TKI therapy, 70% of patients with e14a2 transcripts achieved MR4.5 whereas only 52% of patients with e13a2 transcripts attained MR4.5; confirming that patients with e14a2 transcripts are more likely to achieve DMR earlier (Figure 1b). Furthermore by 8 yrs, 48% of patients with e14a2 transcripts became eligible for a TFR attempt compared with only 32% of e13a2 transcripts (Figure 1c). While patients with e13a2 transcripts eventually achieve the same frequency of MR4.5 as the e14a2 group, the earlier achievement of MR4.5 in e14a2 patients may have contributed to the difference in TFR success. Conclusion: The factors that we identified as most predictive for TFR success were duration of MR4.5 and the presence of the e14a2 transcript, which has not been described previously. We also observed earlier achievement of MR4.5 in the e14a2 cohort, consistent with other studies [Jain, Blood 2016]. These observations, taken together, raise important questions about the impact of transcript type on disease biology, drug sensitivity, and immunological response which warrant further investigation. Disclosures Shanmuganathan: Novartis: Honoraria, Other: Travel sponsorship; Janssen: Honoraria; Royal Adelaide Hospital Research Fund: Other: Scholarship; Bristol-Myers Squibb: Honoraria, Other: Travel sponsorship. Branford: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, Speakers Bureau; Cepheid: Honoraria; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Yong:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Research Funding. Hiwase:Celgene: Research Funding; Novartis: Research Funding. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria; Specialised Therapeutics Australia: Honoraria. Ross:BMS: Honoraria; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Research Funding. Hughes:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Takeda: Honoraria.

Introduction Mesenchymal stem cells (MSCs) isolated from donated tissue have been widely investigated as a treatment for acute graft versus host disease (GvHD), but with mixed results. Factors including MSC donor variability and the effects of prolonged MSC culture expansion may have contributed to inadequate outcomes. Induced pluripotent stem cells (iPSCs) can proliferate indefinitely without loss of pluripotency. The novel Cymerus™ manufacturing process facilitates a virtually limitless supply of well-defined and consistent MSCs from a single donation. Production is achieved by differentiating iPSCs into MSCs using proprietary clonogenic progenitor-based technology. This avoids both donor to donor variability and the need for excessive culture expansion once MSCs are formed. We are undertaking a Phase I clinical trial of Cymerus iPSC-derived MSCs (CYP-001) in steroid-resistant acute GvHD (NCT02923375). We believe this will be the first completed clinical trial involving iPSC-derived cells. Methods This is a multi-center, open label, dose escalation study to assess the safety, tolerability and efficacy of CYP-001 in adults with grade II-IV steroid-resistant acute GvHD, following allogeneic hematopoietic stem cell transplantation. All subjects had failed to respond to at least three days of steroid treatment (≥1 mg/kg/day), administered in accordance with standard management at each center. The first eight subjects enrolled in Cohort A received two intravenous (IV) infusions of CYP-001 one week apart, at a dose of 1 x 106 cells/kg, in addition to standard of care medications. After an independent data and safety monitoring board review, the next eight subjects entered Cohort B, in which the MSC cell dose was doubled. Primary evaluation was performed over eight study visits to day 100. Subjects then entered a follow-up phase of up to two years. Data for subjects in Cohort A with a minimum of six months follow-up are presented here. GvHD was staged and graded according to the 1994 Consensus Conference on Acute GvHD Grading. A Partial Response (PR) was defined as improvement in the severity of GvHD by at least one grade compared to baseline, while a Complete Response (CR) was defined as the absence of any GvHD signs or symptoms. The Overall Response (OR) rate was defined as the proportion of subjects showing either a PR or CR. The primary objective was assessment of the safety and tolerability of two infusions of CYP-001. The secondary objective was efficacy, assessed by best response to treatment, by Day 28 and Day 100 and overall survival at Day 28 and Day 100. Results Four males and four females, with an average age of 57 years (range: 45-66) were enrolled in Cohort A during 2017. At baseline, subjects had Grade II (n=3) or Grade III (n=5) steroid-resistant acute GvHD. One subject had skin, gastrointestinal (GI) and liver involvement; four subjects had skin and GI involvement; two subjects had GI involvement only; and one subject had skin involvement only. The treatment was well tolerated in all cases, and there were no treatment-related Serious Adverse Events (SAEs) reported. Three subjects experienced SAEs that were not considered to be study drug related: (i) febrile neutropenia, hypokalemia and parainfluenza, each of which resolved; (ii) a lower respiratory tract infection, which resolved; (iii) pneumonia, which was fatal. All eight subjects showed at least a PR. Four subjects achieved a CR by Day 100. In all four cases where a CR was achieved, it was then sustained until Day 100. The median GvHD grade at Day 100 was 0 (range: 0-II), compared to a median grade of III (range: II-III) at baseline. Disease progression (an increase in the severity of GvHD by at least one grade compared to baseline) was not observed in any subject at any study visit. Overall survival was 7/8 (87.5%) six months after the first infusion of CYP-001. The best response rates by Day 28 and Day 100 are summarized in Table 1, while the maximal response by individual subject is illustrated in Figure 1. Conclusion Infusion of CYP-001 at 1 x 106 iPSC-derived MSCs/kg was safe and well tolerated in this patient cohort. Treatment response and overall survival rates are encouraging compared to previously published outcomes. The Cohort B primary evaluation period is expected to be completed by September 2018, and progression to a Phase II trial in this clinically challenging disease will then be considered. Disclosures Bloor: AbbVie: Research Funding; Janssen: Research Funding. Radia:Mallinckrodt: Research Funding. Yeung:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria; Specialised Therapeutics Australia: Honoraria. Slukvin:Cynata Therapeutics Limited: Consultancy, Equity Ownership. Kelly:Cynata Therapeutics Limited: Employment, Equity Ownership. Rasko:Gilead: Honoraria; Abbvie: Speakers Bureau; Takeda: Speakers Bureau; International Society for Cellular Therapy: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Speakers Bureau; Cynata: Consultancy, Honoraria; bluebird bio: Honoraria, Other: Clinical trials ; Spark: Consultancy; FSHD Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Current Cure The Future Foundation: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria; Pfizer: Honoraria; GSK: Honoraria; Genea: Equity Ownership; IMAGO Biosciences: Consultancy; Rarecyte: Consultancy, Equity Ownership; Gene Technology Technical Advisory, OGTR, Australian Government: Other: Chair; Advisory Committee on Biologics, Therapeutics Goods Administration, Australian Government: Other: Past Chair.

The therapeutic intensification in De Novo Leukaemia (TIDEL)-II study enrolled 210 patients with chronic phase chronic myeloid leukemia (CML) in two equal, sequential cohorts. All started treatment with imatinib 600 mg/day. Imatinib plasma trough level was performed at day 22 and if 10% BCR-ABL1 at 3 months. Confirmed major molecular response was achieved in 64% at 12 months and 73% at 24 months. MR4.5 (BCR-ABL1 ≤0.0032%) at 24 months was 34%. Overall survival was 96% and transformation-free survival was 95% at 3 years. This trial supports the feasibility and efficacy of an imatinib-based approach with selective, early switching to nilotinib. This trial was registered atwww.anzctr.org.au as#12607000325404. Refereed/Peer-reviewed

Killer immunoglobulin-like receptors (KIRs) on natural killer (NK) cells have been shown to predict for response in chronic phase-chronic myeloid leukemia (CP-CML) patients treated with tyrosine kinase inhibitors. We performed KIR genotyping in 148 newly diagnosed CP-CML patients treated with a novel sequential imatinib/nilotinib strategy aimed at achievement of optimal molecular responses at defined time points. We found the presence of KIR2DL5B to be associated with inferior transformation-free survival and event-free survival and an independent predictor of inferior major molecular response (BCR-ABL1 ≤0.1%) and molecular response 4.5 (BCR-ABL1 ≤0.0032%). This suggests a critical early role for NK cells in facilitating response to imatinib that cannot be overcome by subsequent intensification of therapy. KIR genotyping may add valuable prognostic information to future baseline predictive scoring systems in CP-CML patients and facilitate optimal frontline treatment selection. Refereed/Peer-reviewed

Background: Discontinuation of tyrosine kinase inhibitor (TKI) treatment for chronic myeloid leukaemia (CML) patients in stable deep molecular response leads to treatment-free remission (TFR) in approximately 50% of cases. In most studies, monthly PCRs was performed for 12 months followed by 2-3 monthly testing thereafter. Around 80% of molecular relapses occur within the first 6 months after TKI cessation. The current recommendation for TKI recommencement is a single BCR-ABL1 value ≥0.1% IS (International scale), indicating loss of major molecular response (MMR). Not all institutions can offer monthly PCR monitoring due to financial constraints, particularly relevant in developing countries. For some patients, remaining on TKI is a cheaper alternative. Aim: To assess the safety of less frequent BCR-ABL1 monitoring for detection of loss of MMR for CML patients attempting TFR. Methods: We monitored 85 patients who ceased TKI with the aim of achieving TFR. Patients had a minimum of 24 months of sustained MR4 (n=3) or MR4.5 (n=82) prior to TKI cessation. At the time of TKI cessation, 64 patients were on imatinib (75%), 17 on nilotinib (20%) and 4 on dasatinib (5%). Forty of the patients were enrolled in the TWISTER study where the criteria for TKI recommencement was loss of MMR or 2 consecutively rising BCR-ABL1 positive values. The remaining patients were on a registry study and the trigger for TKI recommencement was loss of MMR. Results: TKI recommencement occurred in 49 of 85 patients. Median time to TKI recommencement was 4 months (range 2-28 months) at a median BCR-ABL1 value of 0.27% on the International Scale (IS), range 0.002-24% IS. Thirty-six of the 49 patients (73%) lost MMR prior to TKI recommencement; the median time to loss of MMR was 3 months (range 1 to 10 months). One patient lost MMR within the first month. Figure A demonstrates the time to loss of MMR in the 36 patients with PCR values ≥ 0.1%. Eighteen of the 36 patients (50%) lost MMR by the 3 month BCR-ABL1 assessment and 35 of 36 patients (97%) lost MMR by 6 months. The latest loss of MMR was at 10 months. Fourteen patients recommenced TKI at a BCR-ABL1 value of >1% and 1 recommenced at a value >10%. Clinician delay in TKI recommencement of 1 month resulted in a BCR-ABL1 rise from 0.84% to 24% with associated loss of complete hematological response. We propose monthly BCR-ABL1 testing between 2 and 6 months post TKI cessation followed by 2 monthly testing. Detection of a BCR-ABL1 value of ≥0.1% would trigger TKI recommencement. In the presence of a rising BCR-ABL1, which remains ≤0.1%, monthly monitoring should ensue in order to avoid hematological relapse. If this strategy were employed in this cohort of patients, only 1 patient would have had the trigger for TKI recommencement delayed by 1 month (estimated BCR-ABL1 at recommencement ~2.5%). This patient had loss of MMR in the first month post TKI cessation. If this molecular monitoring strategy was applied to patients in our cohort who had not lost MMR at TKI recommencement, we estimate that 1 other patient would have had TKI recommencement delayed by 1 month based on the average BCR-ABL1 doubling time of 1 log per month. A proportion of patients maintain low levels of BCR-ABL1 after TKI cessation and do not lose MMR. There were 2 such patients in our cohort. Conclusion: The critical time for molecular monitoring to trigger TKI recommencement is the first 6 months. A monthly monitoring strategy beginning 2 months after cessation would capture the majority of patients at loss of MMR. The data suggest that after 6 months, 2-monthly monitoring could follow. Monthly BCR-ABL1 testing can be re-introduced in the event of a positive result in those that ceased TKI with undetectable BCR-ABL1 or if there is a BCR-ABL1 result higher than the cessation value. This approach would reduce BCR-ABL1 testing by approximately 33% in the majority of cases while minimizing hematological relapse. Therefore this strategy would reduce the cost and inconvenience of molecular monitoring for a trial of TKI cessation, making the option of TFR available to some patients for whom it is otherwise not feasible. Disclosures Branford: Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy; Ariad: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Yeung:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Hughes:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group.

Introduction The emergence of next generation RNA sequencing (RNA-Seq) technologies will likely advance diagnostic, prognostic and therapeutic strategies for patients (pts) with various cancers.Novel fusions have recently been described in AML and solid tumors using RNA-Seq, and many were out-of-frame.It is not known whether novel fusions are generated at diagnosis (Dx) of CML and if so, their impact on treatment outcome. We used RNA-Seq coupled with whole exome sequencing to identify and characterize novel fusions at Dx of CML and at blast crisis (BC). A highly complex pattern of genomic rearrangements of chromosome (chr) 9 and 22 was found in some pts at Dx that generated novel fusions associated with multiple genomic breaks, multiple non-contiguous deletionsand inversion of genomic sequences, including BCR and ABL. Method RNA-Seqwas performed on Dx samples of chronic phase pts treated with first line TKI representing 2 extreme response groups: 14 pts with BC at a median of 6 months (mos), range 3-25 (group A, poor response), and 16 pts with rapid major molecular response by 3mosof imatinib (group B, optimal response). RNA-Seqwas also performed for 9 of 14 pts at BC (group C).The TruSeq Stranded Total RNA-RiboZero Gold Sample Prep Kit (Illumina) was used. This method enables computation of transcription direction and detection of genomic breaks from precursor RNA. Fusions were identified usingthe STAR algorithm and those detected in 4 normal controls were filtered out. Fusions with a high unique read count, supporting genomic breaks or detection atDxand BC for individual pts were prioritized for validation and their somatic status confirmed by RT-PCR.Correspondingwhole exome sequencingwas conducted for 30 samples. Copy number variation was detected usingSequenzaand exon level resolution ofdeletionswas achieved using an in-house sequence read normalization method. Results BCR-ABL fusions were detected by RNA-Seq in 29/30 pts at Dx and all pts at BC. In addition, novel fusions were identified in eachptgroup. GroupA(poor response). AtDx, 8 cytogenetically cryptic novel fusion transcripts were detected in 4/14 pts, Fig A pts1-4. All fusions involved genes or sequences onchr9 and/or 22 and all 4 pts had concomitant genomic inversion events. Fusion partners included inverted ABL intronic sequences and an inverted intergenic region on chr 22, potentially derived from the generation and activation of cryptic splice sites. BCR was a frequent fusion partner (5/8 fusion transcripts). Genomicdeletionswere detected adjacent to some fusions (3deletionsin 1pt),indicatingdeletionsmay have contributed to fusion formation, Fig B. All 4 pts with novel fusions and inversions had very rapid BC (within 5mosofDx). Group B (optimal response).AtDx, only 1/16 pts had a fusion detected in addition to BCR-ABL: TNRC6B (chr22)-NEK6 (chr9), Fig Apt5. Thisptalso had multiple non-contiguousdeletions: 2 each onchr9 and 22 associated with fusion formation, but no inversions, Fig B. Group C (BC). At BC, 3/9 pts gained fusions. No inversions were detected. Two pts had MLL fusions; MLL-BCAT1 (novel) and MLL-MLLT6. The MLL gene is a known fusion partner in acute leukemia, associated with poor prognosis. Both pts had sudden onset BC after a complete cytogenetic response. These fusions were supported by translocation events detected by cytogenetic analysis;t(11;12)(q23;p12) and t(11;17)(q23;q21). The thirdptgained an out-of-frame ANKRD11-UBQLN1 fusion at BC. Indeed, ANKRD11 expression was reduced by 3-fold at BC. Interestingly, thispthad a germline gain of function TP53 mutation. ANKRD11 is a p53 coactivator and loss of expression defined poor prognosis in breast cancer pts that harbored gain of function p53 mutations (Noll, 2012). The ANKRD11 fusion detected at BC in CML may have been selected with disease progression in the context of mutant p53. Conclusion We identified a subset of pts with novel fusions and inversion events at Dx involvingchr9 and 22. These inversions were detected among the pts studied with very rapid BC. The biological effects of the novel fusions remain to be determined. Our data support the presence of novel fusions, additional to BCR-ABL in CML and add a further layer of genetic heterogeneity associated with the Philadelphia translocation. Whether genomic inversions identify a small subset of CML pts with very poor prognosis requires expanded analysis. Disclosures Yeung: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding. Mueller:Ariad: Honoraria; Institute for Hematology and Oncology GmbH: Employment; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Dietz:Institute for Hematology and Oncology GmbH: Employment. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Hughes:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; 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:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding; Bristol Myers Squibb: Research Funding; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy.

Background: Philadelphia chromosome-positive (Ph+) CML is a myeloproliferative disease characterized by the presence of the abnormal Ph+ in hematopoietic cells. Imatinib, dasatinib and nilotinib are BCR-ABL TKIs commonly used in the treatment of CML-CP. Many patients on BCR-ABL TKI therapy will experience adverse events (AEs). Some of the more common AEs associated with first- and second-generation BCR-ABL TKIs include fluid retention, diarrhea, rash, musculoskeletal pain, nausea, vomiting, muscle cramps, and headache. Some patients will be unable to tolerate these AEs and will discontinue therapy. The current study aimed to assess the efficacy and safety of nilotinib in patients with CML-CP who are responsive to but intolerant of treatment with imatinib or dasatinib. Although the study was stopped early due to low recruitment, here we present results on cross-intolerance and molecular response in the patients who were switched from imatinib or dasatinib to nilotinib. Methods: Eligible adult patients had: Ph+ CML-CP associated with BCR-ABL quantifiable by real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR); received ≥3 months imatinib or dasatinib or both; were Results: The study was stopped early due to low recruitment; 20 patients were enrolled (mean age 53.9 years [range 31-77]; 14 female). 16 patients had received prior imatinib therapy, 4 patients prior dasatinib. Median nilotinib treatment duration was 494 days (mean 480, SD 167.6 days). At screening, 30% of patients were not in MMR, 45% had MMR and 25% had MR4.0. By month 3 and 24 of nilotinib treatment, 55% (11/20) and 65% (13/20) of patients, respectively, achieved at least a 1 log reduction in BCR-ABL levels. 35% (7/20) of patients achieved MR4.5 between baseline and month 3 of nilotinib treatment, and 50% (10/20) achieved MR4.5 at any time up to month 24. The proportion of patients with a molecular response at each visit up to month 15 is shown in the Figure. AEs during prior treatment with imatinib and dasatinib included gastrointestinal events (nausea, vomiting, diarrhea), superficial edema, myalgia, fatigue, rash, and headache, among others. 68% of AEs had resolved by month 3 of nilotinib treatment. Among the 13 evaluable patients on prior imatinib, 7 (54%) had resolution of all AEs during treatment with nilotinib; of 3 evaluable patients on prior dasatinib, 3 (100%) had AE resolution on nilotinib. Grade 3/4 AEs during nilotinib therapy occurred in 3 patients: diabetes mellitus, fatigue, neutropenia, pneumonia, osteoarthritis, and hyperuricemia. Conclusions: Although early termination of the study has not allowed for a robust analysis, these results suggest that nilotinib is effective and well tolerated in most patients intolerant of imatinib or dasatinib. During the first 3 months of switching to nilotinib, 55% of patients had achieved at least a 1 log reduction in BCR-ABL levels, and 35% of patients had achieved MR4.5. The cumulative rate of MR4.5 by 24 months was 50%. Achievement of this endpoint has been linked to favorable long-term outcomes, such as treatment-free remission. Furthermore, the majority of the AEs had resolved by month 3 of nilotinib therapy. This improved tolerance to nilotinib may result in improved treatment adherence. As such, although further study in a larger population is needed for confirmation, these results provide further evidence that nilotinib is a favorable option to establish a molecular response in patients intolerant of imatinib or dasatinib. Disclosures D'Rozario: BMS: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Branford:Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Ariad: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cepheid: Consultancy. Yeung:Ariad: Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Anderson:Novartis Pharmaceuticals: Employment. Gervasio:Novartis Pharmaceuticals: Employment. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The TIDEL-II trial used imatinib (IM) upfront in patients (pts) newly diagnosed with chronic myeloid leukaemia in chronic phase (CML-CP), and switched selected pts to nilotinib (NIL) on the basis of IM intolerance or failure to achieve time-dependent molecular response. We previously reported major molecular response (MMR; BCR-ABL ≤0.1% IS) at 12 months (mths) and transformation-free survival (TFS) at 3 years. This abstract reports the final analysis with minimum follow-up of 60 months. Patients were enrolled across 27 Australasian sites in 2 equal and sequential cohorts. All started treatment with IM 600mg OD and dose escalated to IM 800mg OD if IM trough levels were The study enrolled 210 pts with a median age of 49.7 years (range 16-81); 42% were female. Baseline demographics and outcomes were similar across 2 cohorts. Forty pts had day 22 IM trough At 60 mths, 75 (36%) pts had withdrawn from study, and 14 were lost to follow up (including 12 with outstanding data queries). Of the 121 pts (58%) who remained on TIDEL-II until 60 mths, 33 were on NIL (30 in MMR), 77 on IM (76 in MMR); treatment was unknown for 11 (10 in MMR). The median dose of IM was 600mg OD. Of the 51 pts who dose escalated to IM 800mg OD (31 for low IM trough level and 20 for failing to achieve molecular targets), only 9 remained on this dose until mth 60 (5 and 4 pts form the respective groups). Eight pts transformed to accelerated or blastic phase; 5 within the 1st year, 2 in the 3rd and 1 in the 5th year; 2/8 occurred after study withdrawal. There were 14 deaths, mostly due to cardiac events (n=5) or progressive leukaemia (n=6). In all, 13/210 pts (6%) had cardiac, cerebral or peripheral vascular disease, 9 while on NIL and 4 having only had IM. Pts failing to meet molecular targets (analysed according to the 1st target failed) at 3, 6 and 12 mths numbered 25 (12%), 23 (11%) and 30 (14%) respectively with 19, 16 and 20 switching to NIL (subsequent molecular outcomes, Table 2). Pts failing to achieve EMR had poor achievement of MMR and MR4.5 (44% and 8% respectively by 60 mths). Of the 11 EMR failure pts who achieved MMR, only 4 remained in MMR at 60 mths. Pts failing to achieve BCR-ABL≤ 1% at 6 mths had similarly poor outcomes, with MMR and MR4.5 being 52% and 13% respectively. In pts failing to achieve MMR by 12 months, MMR and MR4.5 by 60 mths were 93% and 33% respectively. Twenty pts switched to NIL for IM intolerance prior to 24 mths: 9/20 already in MMR, and 3/20 already in MR4.5 at time of switching. For the remaining pts, MMR and MR4.5 were achieved by 100% and 88% after switching. The TIDEL-II strategy of combining IM and NIL compares favourably with other upfront treatment strategies, with MR4.5 of 59% by 60 mths. IM dose escalation to 800mg OD for target failure was not well tolerated: only 18% of pts who dose escalated maintained this dose at 60 mths. However, this did not appear to be detrimental to overall outcomes, which were similar in the 2 cohorts. Pts switching to NIL for IM intolerance, and for failing to achieve MMR by 12 months after meeting prior goals, had high rates of MMR, superior to those who failed their 3 and 6 mth targets. Early identification of pts at high risk of EMR failure who might benefit from more intensive or experimental therapies may be necessary to further improve outcomes. Disclosures Yeung: Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding. White:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Research Funding. Branford:Novartis: Honoraria, Research Funding, Speakers Bureau; Qiagen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Otsuka: Research Funding; Ariad: Research Funding; Bristol Myers-Squibb: Honoraria; Cepheid: Consultancy. Butcher:Janssen: Consultancy; Roche: Consultancy; Novartis: Consultancy. Gottlieb:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Indee: Membership on an entity's Board of Directors or advisory committees. Arthur:Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Ross:Novartis Pharmaceuticals: Honoraria, Research Funding; BMS: Honoraria. Tam:Novartis: Honoraria. Mills:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Meeting attendance sponsorship. Hughes:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background ABL001 (Novartis) is an allosteric inhibitor that binds to the myristate binding pocket of Abl to hold Bcr-Abl in the inactive conformation. A Phase I multicentre trial of ABL001 in chronic myeloid leukemia (CML) is ongoing (Protocol CABL001X2101), and includes testing ABL001 in combination with the ATP-competitive Tyrosine Kinase Inhibitors (TKIs) imatinib (IM), nilotinib (NIL) and dasatinib (DAS) to determine if simultaneous targeting of the myristate and ATP binding pockets provides greater pharmacological inhibition of Bcr-Abl and promotes greater reduction in tumor burden. In vitro sensitivity to TKIs can be measured by assessing the phosphorylation status of the Bcr-Abl substrate CrkL. Using this method, high IM in vitro sensitivity at diagnosis (IC50IM Aim To investigate if the in vitro combination of ABL001 (at a pharmacologically-relevant concentration) with ATP-competitive inhibitors is more effective atBcr-Abl kinase blockade than IM, NIL or DAS as single agents. Methods Blood mononuclear cells (MNC) were isolated with informed consent either prior to TKI therapy (n=6 de novo chronic phase CML patients) or at TKI-switch (n=4 resistant, intolerant or non-compliant patients). For in vitro sensitivity, MNC were treated for 2 h with a pre-determined range of concentrations of IM, NIL and DAS -/+ 1.2 µM ABL001 (based on human PK parameters from Novartis, average peak plasma concentration on 40 mg BID = 578 ng/mL or 1.24 µM) or a much lower dose of 1.2 nM; or with ABL001 alone. Phosphorylated-Crkl (p-Crkl) levels were determined by western blot and the IC50 was calculated by densitometry. Results Kinase inhibition was markedly enhanced by addition of 1.2 µM ABL001 as compared to TKIs alone with mean IC50s without and with ABL001 of 1.52 µM ± 0.46 µM vs. 0.019 µM ± 0.012 µM (p=0.014), 79.1 nM ± 8.8 nM vs. 0.26 nM ± 0.22 nM (p0.6 µM, however, with the addition of just 1.2 nM ABL001, the IC50IM was reduced in all cases to Conclusions These data suggest that (1) in vitro kinase inhibition achieved with ABL001 in combination with IM, NIL or DAS is greater than that achieved with TKIs alone, even at a concentration 1000-fold lower than achievable on the current ABL001 dosing regimen; and (2) patients with high IC50IM who would be predicted to respond poorly to 600 mg IM have dramatically increased kinase inhibition with the addition of just 1.2nM ABL001. These results suggest that simultaneous targeting of themyristate and ATP binding pockets ofBcr-Abl may be more effective than targeting either site alone and provides a strong rationale for combining IM and ABL001, particularly in patients with low intrinsic sensitivity to IM. Figure 1 Addition of 1.2 nM or 1.2 µM ABL001 to ATP-competitive TKIs increases the degree of Bcr-Abl kinase inhibition achieved in vitro in CML mononuclear cells.Patient MNC were incubated for 2 h with (A) IM (B) NIL and (C) DAS + 1.2 nM (middle) or 1.2 µM (right) ABL001 and IC50 calculated by determining the concentration of TKI causing 50% reduction in p-CrkL. Solid dots represent diagnosis patients; open circles represent patients switching TKI for intolerance or resistance; Numbers are shown in parentheses; Black lines are means; error bars are SEM. The eight patients in (A) are shown in (D) paired with their response to 1.2 nM ABL001 (dotted line indicates the average IC50IM for 62 de novo CMLs in the TIDEL trial). Figure 1. Addition of 1.2 nM or 1.2 µM ABL001 to ATP-competitive TKIs increases the degree of Bcr-Abl kinase inhibition achieved in vitro in CML mononuclear cells.Patient MNC were incubated for 2 h with (A) IM (B) NIL and (C) DAS + 1.2 nM (middle) or 1.2 µM (right) ABL001 and IC50 calculated by determining the concentration of TKI causing 50% reduction in p-CrkL. Solid dots represent diagnosis patients; open circles represent patients switching TKI for intolerance or resistance; Numbers are shown in parentheses; Black lines are means; error bars are SEM. The eight patients in (A) are shown in (D) paired with their response to 1.2 nM ABL001 (dotted line indicates the average IC50IM for 62 de novo CMLs in the TIDEL trial). Disclosures Yeung: Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Research Funding. White:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hughes:Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Most patients with chronic myeloid leukemia (CML) treated with imatinib will relapse if treatment is withdrawn. We conducted a prospective clinical trial of imatinib withdrawal in 40 chronic-phase CML patients who had sustained undetectable minimal residual disease (UMRD) by conventional quantitative polymerase chain reaction (PCR) on imatinib for at least 2 years. Patients stopped imatinib and were monitored frequently for molecular relapse. At 24 months, the actuarial estimate of stable treatment-free remission was 47.1%. Most relapses occurred within 4 months of stopping imatinib, and no relapses beyond 27 months were seen. In the 21 patients treated with interferon before imatinib, a shorter duration of interferon treatment before imatinib was significantly associated with relapse risk, as was slower achievement of UMRD after switching to imatinib. Highly sensitive patient-specific BCR-ABL DNA PCR showed persistence of the original CML clone in all patients with stable UMRD, even several years after imatinib withdrawal. No patients with molecular relapse after discontinuation have progressed or developed BCR-ABL mutations (median follow-up, 42 months). All patients who relapsed remained sensitive to imatinib re-treatment. These results confirm the safety and efficacy of a trial of imatinib withdrawal in stable UMRD with frequent, sensitive molecular monitoring and early rescue of molecular relapse.

Recent studies have demonstrated that some patients with chronic myeloid leukemia (CML) can maintain remission after discontinuation of imatinib. A prerequisite is stable, undetectable BCR-ABL1. It is not known how many patients achieve this response or the factors associated with its achievement. We examined 423 de novo imatinib-treated patients to determine the cumulative incidence of achieving the discontinuation criteria as defined in the CML8 study (≥2 years of undetectable BCR-ABL1 [Stable MR(4.5)]), and predictive factors. After 8 years of imatinib, the cumulative incidence of Stable MR(4.5) was 36.5%. Therefore, 9% to 15% of first-line imatinib-treated patients would maintain remission after discontinuation. The BCR-ABL1 level at 3 months and factors at diagnosis were examined for association with Stable MR(4.5): Sokal risk, age, sex, and assigned imatinib dose. The only independent predictors were female sex (54.4% vs 27.2%; P = .018) and the 3-month BCR-ABL1 (P < .001). The highest cumulative incidence of Stable MR(4.5) after 8 years was 78.2% for patients with BCR-ABL1 ≤ 0.10%(IS) at 3 months (n = 38). Time to major molecular response (MMR) influenced the time to reach Stable MR(4.5) (P < .001), suggesting slower dynamics of response with a delayed MMR. The findings justify the focus on rapid reduction of BCR-ABL1 as a strategy to maximize potential suitability for imatinib discontinuation studies. The Iris trial was registered at http://www.clinicaltrials.gov as NCT00006343. The Tops trial was registered at http://www.clinicaltrials.gov as NCT00124748. The TIDEL I trial was registered at www.ANZCTR.org.au as ACTRN12607000614493. The TIDEL II trial was registered at www.ANZCTR.org.au as ACTRN12607000325404.