ALBERT

Description: Background Binding of E-selectin (E-sel) to sialyl Lex, the E-sel ligand, on the leukemic cell surface activates cell survival pathways and promotes chemotherapy resistance in AML. Higher expression of E-sel ligand is associated with relapse and poor survival. Uproleselan (GMI-1271), a novel E-selectin antagonist, disrupts cell survival pathway activation, enhances chemotherapy response and protects from toxicity such as mucositis with improved survival in vivo. Preclinical data support combination of uproleselan with chemotherapy improves response without additional toxicity. A phase 1/2 study (NCT02306291) of uproleselan added to chemotherapy (mitoxantrone, etoposide, cytarabine, MEC) in R/R AML showed promising outcomes at the recommended phase 2 dose (RP2D), including a CR/CRi rate of 41% and median OS of 8.8 m (95% CI 5.7-11.4). 11/16 (69%) evaluable patients were MRD negative (DeAngelo et al ASH 2018). Patients with sufficient expression of the appropriate E-selectin ligand (the target of the E-selectin inhibitor) exhibited higher CR/CRi rate and longer survival. Median OS for Leukemic blasts/E-sel ligand ≥10% vs leukemic blasts/E-sel ligand

Description: Introduction - AML is a complex group of malignancies, with heterogeneity in morphology, cytogenetics, molecular characteristics, aggressiveness and importantly, in its response to treatment and survival outcomes. Next generation sequencing by the Cancer Genome Atlas Research Network analysed 200 primary AML cases and identified 23 genes that display recurrent somatic mutations at varying frequency in AML (NEJM 368(22):2059-2074). Defects in DNA repair are frequently identified in treatment-related AML and inherited mutations in genes of DNA repair pathways predispose patients to myeloid malignancies. For example, biallelic mutations in FANC genes, which cause the recessive heritable bone marrow failure syndrome Fanconi Anaemia (FA) are associated with high risk of progression to AML and other cancers (Kutler et al.Blood, 101:1249-1256), suggesting a potential involvement of FANC gene mutations in AML pathogenesis. Methods - In this study we present a two-stage approach to gene discovery in AML: initial unbiased whole genome sequence (WGS) and whole exome sequence (WES) analysis of tumour DNA from a cytogenetically normal AML case at diagnosis and relapse, and corresponding germ-line DNA (prepared from mesenchymal stromal cells). Potential oncogenic mutations and changes associated with disease progression were identified. WES of a further 96 diagnostic AML samples further defined recurrent mutations and allowed identification of affected functional groups and networks in AML. Results – WGS and WES were performed on diagnosis, non-haematopoietic and relapse samples from an index AML patient. Somatic SNVs and indels unique to the tumour samples include a number of variants in genes previously reported as recurrently somatically mutated in AML including FLT3, WT1 and IDH2. Somatic mutations in genes not previously associated with AML were also identified including a mutation in FANCD2 (p.S1412N) present in the index AML tumour DNA at diagnosis and at relapse. Variants in genes recurrently mutated at low frequency in AML can also be disease drivers, however separating such genes from the background level of mutation in AML requires analysis across multiple samples, and sequencing studies to determine recurrence and/or mutations in proteins involved in the same functional pathway or complex. STRING-db v9.05 (Franceschini et al. NAR, 2013(41), Database issue) was used to identify a larger network of proteins, including and associated with the FANC genes, involved in homologous recombination-mediated DNA repair. Known somatic mutations from other AML studies were mapped onto this network; as shown in Figure 1 multiple genes in this extended network are affected by somatic mutation in AML suggesting a potential role in pathogenesis. Analysis of our WES data from diagnosis samples from a further 96 Australian AML cases identified an additional two somatic mutations in genes from the extended STRING-db v9.05 FANC network. In total we identified 18 mutations in the 16 classified FANC genes and 8 variants in the BLM complex as shown in Figure 2. Two of the germline FANC gene mutations, FANCM-Q13333fs and FANCD2-R926X, are known pathogenic mutations in FA. Patients with mutations in the 8 FANC genes of the core complex form a distinct subset from those with mutations in the other 8 FANC genes. 5 of the 8 patients with mutations in the BLM complex also form a separate group while BLM complex mutations are present in 2 patients that also have FANC mutations. For the two patients with acquired changes the allele frequency for these FANC mutations is greater than 25% suggesting an early origin in disease. Discussion. Our findings suggest that germline and somatic mutations affecting function of the FANC DNA repair pathway may be a recurrent abnormality in AML, potentially contributing to leukaemogenesis. FANC/BLM gene mutations frequently co-exist with mutations in DNMT3A and DNMT1; 46% of the patients with DNMT3A/DNMT1 mutations are also mutant for FANC or BLM complex genes representing significant over-representation (p = 0.021). Within the group of FANC and BLM patients there is also significant under-representation of FLT3-ITD mutations and mutations in N-RAS and K-RAS (p = 0.051), raising the possibility that defects in homologous DNA repair may favour cooperation with alternative signalling pathways. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Background CBF abnormalities remain a robust prognostic indicator of relatively favorable outcome in AML. Several groups have demonstrated greater responsiveness to HiDAC and studies at MD Anderson established the feasibility of omitting anthracycline when using fludarabine to favourably modulate the intracellular accumulation of cytarabine (Borthakur Cancer 2008). This study further explored that approach by assessing the clinical outcome and molecular responses of patients (pts) treated with fludarabine and HiDAC in the absence of anthracycline. Patients and Methods 52 eligible pts with previously untreated newly diagnosed CBF AML were enrolled from 13 centres between 02/05 and 06/09 (protocol registration ANZCTRN012605000152628). One enrolled pt with non-CBF AML was excluded from analysis. Therapy consisted of induction with fludarabine 30 mg/m2/d followed by cytarabine 2 g/m2/d for 5 days (FA) and post-remission treatment with 3 cycles of HiDAC (2 g/m2 q 12 h Days 1, 3 and 5) followed by 3 cycles of FA for 4 days. No patient was transplanted in first complete remission (CR). Quantitative PCR (QPCR) for CBF-specific fusion transcripts was conducted at diagnosis, post induction, post consolidation cycles and 3 monthly during 2 years of post-treatment follow-up. The primary endpoints were failure free survival (FFS) at 2 and 4 years. Results Median age was 39 (17 – 62) years. 22 pts had t(8;21) and/or AML1-ETO and 30 had inv(16)/t(16;16) and/or CBFB-MYH11, of which 21 had Type A, 5 type D, 3 Type E and 1 Type G transcripts. CR was achieved in 48 of 52 pts (92%): 2 of 4 non-CR pts were responding (post-induction blast counts of 6 and 7%), 1 had residual extra-medullary disease and only 1 pt had refractory AML. 13 pts relapsed (3 AML1-ETO and 10 CBFB-MYH11 p=0.17; median time to relapse 12.4 (range 9.1 – 25.1) months) and 3 died in CR (sepsis). At a median follow-up of 4.6 (range 2.4 – 6.8) years, FFS was 62% (95% CI 50–77%) and 60% (47–75%) at 2 and 4 years respectively. Overall survival (OS) was 83% at 2 years (95% CI 73–94%) and 78% at 4 years (68–91%) reflecting the relative success of salvage (10 of 13 relapsed pts underwent allogeneic transplant). There was no statistical difference between the 2 molecular subtypes for either PFS (p=0.27) or OS (p=0.5). OS was similar to that achieved in our previous trial (Bradstock Blood 2005) utilizing ICE induction followed by either one further ICE or two IcE consolidation cycles (81% at 2 years). Time to neutrophil recovery (〉0.5 x 109/L on 2 consecutive measures) was longer for FA cycles compared to HiDAC (mean 10 vs 6 days; p = 0.001). The rate of grade 3,4 infection was high (88% of pts had at least 1 episode) with 17% coming off protocol for toxicity and only 29 pts (56%) completing all 6 cycles of post-remission therapy with no difference in cycles received between pts who relapsed and those who did not (median cycles delivered was 6 in both groups). Detectable QPCR levels in remission were significantly associated with risk of relapse (10 of 13 relapses were preceded by MRD positivity at a median of 13 weeks; p=0.001). All 10 patients with levels 〉250 copies in PB or BM at any post induction time point subsequently relapsed. LFS was significantly different for patients with MRD positivity (〉10 copies on 2 consecutive measurements or 〉250 at any time) during follow-up compared to those who remained negative (Fig 1). Multivariate analysis did not identify any significant associations between recognized clinical variables and either PFS or OS. LFS was not associated with c-kit mutational status for the subset of 33 pts with results available (p = 0.6). Conclusions The high CR rate and relatively favourable FFS and OS of pts with CBF AML treated with non-anthracycline containing fludarabine-modulated HiDAC based therapy, are similar to published results achieved with standard anthracycline combination treatments. Short-term infectious toxicity of this regimen, particularly during the prolonged post remission therapy, remained significant and the lack of association between relapse and number of cycles delivered suggests the duration may be shortened. QPCR monitoring was confirmed to be clinically useful in the prediction of relapse. Results will be updated after the planned final analysis. Disclosures: Off Label Use: Fludarabine in the treatment of AML.

Description: Hematopoietic stem cells (HSC) reside in specific peri-vascular niches in the bone marrow (BM). We have shown interactions with the inflammatory vascular adhesion molecule E-(endothelial)-selectin awakens HSC (Nat Med 2012). We now report that BM vascular cell-surface E-selectin expression is strongly upregulated during HSC mobilization regimens raising the question of a role for endothelial E-selectin in HSC transplant outcome. G-CSF was administered to cohorts of E gene-deleted or wildtype C57BL/6 mice together with E-selectin antagonist Uproleselan (Upro, GMI-1271). We found absence (Sele-/- mice) or therapeutic blockade (Upro) of E-selectin alone in steady-state hosts did not alter levels of circulating peripheral blood (PB) HSC. In contrast absence or therapeutic blockade of E-selectin strongly synergized with mobilizing regimens such as G-CSF or cyclophosphamide+G-CSF by boosting long-term engraftment and reconstitution potential of mobilized blood. The effect was pronounced boosting reconstitution potential over G-CSF-alone-mobilized blood 24-fold (

Description: Key Points Long-term remissions after FCR chemoimmunotherapy in previously untreated patients with CLL. Updated results on safety and efficacy of the CLL8 trial.

Description: Treatment Of Acute Promyelocytic Leukemia (APML) In The Jehovah’s Witness (JW) Population. Colm Keane, Peter Mollee, Paula Marlton, Devinder Gill. Background: Treatment of acute myeloid leukaemia in JW patients is challenging. The refusal to accept blood products is usually a contraindication to intensive chemotherapy and a potential cure. Methods: We review three cases of APML occurring in JW patients treated at our institution which demonstrate the benefit of newer targeted combination therapies that induce less marrow suppression. Suggested management principles are derived from these anecdotal rare cases. Case 1 was a 39 year old male who was initially treated with ATRA (25 mg/m2) and Darbopoietin 100μg twice weekly with arsenic added on day 10 (0.15mg/kg/day). He initially tolerated therapy well despite severe pancytopenia until (day 20) the hemoglobin fell to 52g/L when he developed chest pain. He was diagnosed to have had a myocardial infarct in the setting of fluid retention. His WCC was 10.4 × 109/L with platelet count of 20 × 109/L. He was managed conservatively with no anticoagulation. His hemoglobin nadir was 44 g/L before rising above 80 g/L by day 38 of therapy at which time he was in cytogenetic and molecular remission. He completed a further 28 weeks of consolidation with ATRA (45mg/m2 for 2 weeks every 4 weeks) and arsenic (0.15mg/kg/day Mon-Fri, d1-28 every 2 months for 4 cycles). He has completed 9 months of planned 2 yrs oral maintenance chemotherapy (ATRA, 6MP, MTX) without further incident. Case 2 was a 62 year old female who was treated with ATRA induction (initially at 25mg/m2 but increased to 45mg/m2 on day 18), plus Erythropoietin (Epo) 4,000U × 3 per week s/c. She was in complete cytogenetic remission on day 37 and complete molecular remission by day 87. She had a hemoglobin nadir of 60 g/L but remained asymptomatic. Consolidation therapy commenced on day 106 consisting 3 cycles of combination therapy with ATRA (45mg/m2/d po for 4 weeks) and arsenic (0.15mg/m2/d iv for 5d/week for 4 weeks). Between consolidation cycles there was a 3-week interval during which all therapy was ceased. This regimen was well tolerated. She remains in complete remission after 8 years. Case 3 was a 28-year-old male who was initially treated with ATRA, low dose cytarabine and Epo 4000U 3-times/week s/c. He developed symptoms of severe anemia when his hemoglobin dropped to 25g/L with syncopal episodes and hypotension. He was subsequently found to have t (11:17) mutation and the leukemia did not respond to ATRA. He died from severe anemia fourteen days after presentation. Case reports published subsequently have shown that ATRA resistance in t(11:17) APML may occasionally be overcome if combined with standard chemotherapy or GCSF. However to date Arsenic has not been useful in this variant form of APML. Recommendations: Therapy. Induction with ATRA 25mg/m2 followed on day 10 by arsenic 0.15mg/kg/day until morphologic remission. Consolidation with alternating cycles of ATRA and arsenic as per Case 1. Maintenance therapy with ATRA, 6MP and MTX for 2 years. Anemia. Our patients tolerated severe anemia reasonably well until hemoglobin dropped to approximately 50g/L (most deaths in the literature due to anemia in the JW population have occurred below this threshold). All JW patients receive maximal erythropoietin stimulation and sparing phlebotomy episodes using paediatric blood collection tubes. Thrombocytopenia and coagulopathy. Antifibrinolytics, DDAVP and newer agents such as FVIIa have been used successfully in JW patients undergoing high risk surgery and may be of benefit in the APML setting for patients with haemorrhagic complications. Differentiation syndrome (DS) Case 1 may have developed DS. The standard management of DS is to commence chemotherapy such as idarubicin, cytarabine or gemtuzumab ozogamicin but because of the potential prolonged myelosuppression such agents are not suitable options for JW patients. We would advocate corticosteroids for DS prophylaxis and if treatment of DS is required, hydroxyurea may be a safer option because of its limited and shorter duration of myelosuppression. Conclusion: Our series demonstrates the benefit of more targeted therapy in APML allowing patients who refuse transfusion of blood products a realistic chance of cure.

Description: Background: ET carries a significant risk of thrombotic complications which are difficult to predict, with a past history of thrombosis being the main predictor of subsequent events. The JAK2V617F mutation (JAK2) and spontaneous erythroid colony growth (SEC) have been inconsistently associated with increased thrombosis in these patients. We aimed to assess the predictive value of these assays for the development of thrombotic events post-diagnosis in patients with ET. Methods: A retrospective review of consecutive patients with ET diagnosed according to WHO criteria between January 1998 and August 2006 was performed. Data was abstracted from the medical record regarding baseline demographic characteristic, vascular risk factors, therapy, occurrence of thrombo-haemorrhagic events, and transformation to AML or myelofibrosis. Thrombotic events were defined as arterial or venous events occurring at or 12 months prior to diagnosis (prior thrombosis) and at any time post diagnosis of ET (subsequent thrombosis). Investigators were blinded to JAK2 and SEC results at the time of data collection. SECs were performed at the time of diagnosis in 53 patients. JAK2 analysis was performed by an allele specific PCR method on peripheral whole blood (n=32) or archived bone marrow samples (n=29). Results: 62 patients with ET were identified: median age 60yrs; 53% female; median platelet count at diagnosis 873 × 109/L; 10,8 and 44 being low, intermediate and high clinical risk, respectively. 67% (41/61) had a positive JAK2 test and 47% (25/53) had a positive SEC assay. 8 (13%) and 6 (10%) had a history of prior arterial and venous thrombotic complications. Median follow up was 39 months (range, 1 to 137 months). At diagnosis JAK2 positive patients had higher white cell counts (12.3 vs 8.9 ×109/L, p=0.01) and neutrophil counts (9.1 vs 5.7 ×109/L, p=0.01), and a trend to older age (62 vs 57yrs, p=0.3) and higher haemoglobin (139 vs 133 g/L, p=0.12), but not platelet count (870 vs 892 ×109/L, p=0.8). There was a trend for increased rates of prior thrombosis among JAK2 positive patients (27% vs 5%, p=0.08). Survival was not affected by JAK2 status (5yr OS 71% vs 72%, p=0.7). The presence of the JAK2 mutation predicted for the development of subsequent thrombosis (5yr event rate 32% vs 0%, p=0.01) which persisted when the analysis was stratified for a prior history of thrombosis (p=0.01). Further analysis demonstrated the JAK2 mutation to predict subsequent arterial thrombosis (5yr event rate 22% vs 0%, p=0.05) but not venous thromboembolism (5yr event rate 11% vs 0%, p=0.18). The increased risk of arterial thrombosis in JAK2 positive patients persisted when corrected for a prior history of vascular events (p=0.04). The SEC assay was not found to be predictive of any thrombotic events. Conclusions: Patients with ET who are JAK2 positive are at increased risk of thrombotic complications, particularly arterial thrombosis. This increased risk persists after correction for a history of prior thrombosis.

Description: Background: Majority of MDS cases appear to be sporadic in nature, but 10-15% have clear familial basis due to predisposing mutations in genes such as RUNX1, GATA2, CEBPA and DDX41. Contribution of germline variants in sporadic MDS is not studied. This study attempts to address the contribution of germline variants in MDS pathogenesis. Methods: We performed amplicon-based massively parallel sequencing (AmpliSeq custom panel adapted for Illumina HiSeq2500 sequencing) on all coding regions of 29 myeloid genes for 144 MDS samples. After identifying the variants in five genes (TET2, MET, GATA2, ASXL1, NOTCH1), we tested an additional 96 MDS samples including therapy-related myeloid neoplasm (T-MN) using a Sequenom assay. We also analyzed WES data for these variants in 178 AML samples and 758 normal controls and AmpliSeq data for ASXL1 and TET2 variants in 655 CML samples. Results: Collation of all coding variants in the 29 myeloid genes sequenced identified germline variants occurring in primary MDS at frequencies significantly higher than expected when compared to the normal population (ExAC and matched cohort were similar) (Table 1). These variants occurred in 5 genes (TET2, MET, GATA2, ASXL1 and NOTCH1) at increased frequencies of 1.5-16.6 fold. Numerous MDS samples had multiple variants (4 with 4 variants, 4 with 3 variants, 18 with 2 variants) while 70 had 1 variant. The 3 germline MET variants have been previously investigated in solid tumorigenesis and likely generate MET variant proteins that contribute to numerous cancer types including MDS. Interestingly, 7/17 (41%) MDS cases with germline MET variants also had other cancers including pancreatic, gastric and laryngeal cancers. Of the TET2 variants, Y867H and P1723S were concurrent in 5 MDS, 5 AML and 6 CML samples indicative of them being on the same allele (i.e. a haplotype). They were seen at higher than normal frequency in MDS and AML, but were not significantly enriched in CML. We are currently confirming their coexistence on the same allele and assaying for decreased TET2 activity to determine whether one or both variants contribute to the phenotype. Other variants identified in MDS include the rare GATA2 (P161A) variant which is present in 1% of the population and the nearby common GATA2 (A164T) allele (~20%). These were mutually exclusive in our cohort and were seen at 3.9 and 1.5-fold, respectively, above the expected population frequency. We generated the P161A variant using site-directed mutagenesis and assayed for GATA2 transactivation activity in HEK293 cells with a GATA2-responsive LYL1 promoter-Luciferase construct (Figure 1). We also included empty vector (EV), wildtype (WT) GATA2 and T354M which is the most common highly penetrant autosomal dominant mutation leading to familial MDS/AML. As expected, T354M displayed a marked decrease in transactivation ability when compared to WT. The P161A variant similarly displayed loss-of-function in this assay, but not to the same magnitude as T354M. This is consistent with the hypothesis that reduced GATA2 function predisposes to myeloid malignancy where decreasing GATA2 activity correlates with increasing risk of developing malignancy. In our study 10/36 (28%) cases harboring these variants were T-MN cases. Apart from MET (E168D) (11.4-fold), the 2 rare variants with highest frequency in MDS versus controls were ASXL1 (N986S) (16.6-fold) and NOTCH1 (R912W) (6.5-fold). ASXL1 is an epigenetic regulator often mutated in hematopoietic malignancy and aberrant NOTCH1 function has been associated with myeloid and lymphoid malignancies. Conclusions: We have identified common and rare germline variants in genes involved in myeloid malignancy that may contribute to MDS pathogenesis. It remains to be seen whether they contribute to initiation, maintenance and/or progression of MDS and other hematopoietic malignancies. This is the first study reporting higher frequency of germline variants in sporadic MDS cases. Table 1. Frequency of germline variants in MDS, AML and CML in comparison to ExAC. Table 1. Frequency of germline variants in MDS, AML and CML in comparison to ExAC. Disclosures Hiwase: Celgene Corporation: Research Funding.