ALBERT

Description: Background: The Revised International Prognostic Scoring System (R-IPSS) stratifies MDS patients better than the original IPSS scoring system. Although RBC-transfusion dependency (RBC-TD) is associated with poor prognosis, it is not included in the R-IPSS. Another limitation of R-IPSS is that it is designed to assess the prognosis of patients only at the time of diagnosis; it does not provide prognostic guidance during the disease course. We hypothesise that the use of RBC-transfusion dependency status as a time-varying covariate improves R-IPSS. Aim: To assess the impact of RBC-TD as a time-varying covariate in addition to R-IPSS in predicting survival outcome of MDS patients. Materials and Methods: To match the patient selection criteria as in R-IPSS, primary MDS patients, AML (blast 20-30%) and CMML (WBC≤12x109/L) not treated with disease modifying agents or stem cell transplantation were included. RBC-TD was defined as RBC transfusion of at least 1 unit/8 weeks for at least 4 months (Malcovati et al; JCO 2007). For the statistical analysis of overall survival (OS) measured in months since diagnosis, the Akaike Information Criterion (AIC) was used to assess the goodness-of-fit of a model. Landmark analyses at 6, 12 and 24 months after the diagnosis were also conducted; individuals who experienced the event (i.e. death) before the landmark time point were excluded. The remaining patients were then classified into two groups – RBC-TD noted at or before the landmark time point and transfusion independent at the landmark time point. Results: In our study, 295 patients met the inclusion criteria for analysis, their median age was 75 years (21-97 years) and 66% patients were male. The majority of patients were RCMD, RAEB1 and RAEB2. R-IPSS improved the risk stratification of MDS patients, predominantly for the IPSS-intermediate group (Table I). The median OS in R-IPSS Very Low, Low, Intermediate, High and Very High risk group was 87, 62, 28, 13 and 12 months respectively (p

Description: Introduction:Multiple myeloma (MM) disease progression is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow (BM), re-enter the peripheral blood (PB) and disseminate to other BM sites. Previous studies show that expression of CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome this retention signal enabling MM PC egress and dissemination via the PB are poorly understood. Previous studies in haematopoietic progenitor cells have demonstrated that CCL3 overcomes the CXCL12 retention signal to drive mobilisation to the PB (Lord et al. Blood 1995). Here, we examined the role of the CCL3 chemokine receptor CCR1 in driving MM PC dissemination. Methods and results: Initially, we assessed the expression of CCR1 protein on CD138+CD38++CD45loCD19- PC from 28 MM, 8 MGUS and 2 SMM patients by flow cytometry. Results show CCR1 expression is significantly increased in newly diagnosed MM compared with premalignant MGUS and SMM patients (p=0.03; CCR1 MFI mean±SEM, MGUS: 53.0±33.6; SMM: 37.6±8.9 MM: 250.9±71.6). Furthermore, CCR1 expression on PB MM PC positively correlated with PB MM PC numbers (p=0.03; n=11 patients). To identify mechanistically how CCR1 may promote dissemination, the effect of CCL3 on the response to CXCL12 in human myeloma cell lines (HMCL) was assessed in vitro. The migration of RPMI-8226 and OPM2 cells was induced by CCL3 or CXCL12 chemoattractant in a transwell assay. Notably, pre-treatment of RPMI-8226 or OPM2 with CCL3 abrogated migration towards CXCL12 and blocked F-actin remodelling in response to CXCL12 in vitro. These findings suggest that CCL3 can desensitise cells to exogenous CXCL12, providing a potential mechanism facilitating loss of the CXCL12 retention signal. To confirm whether CCR1 is required for driving MM PC dissemination, homozygous CCR1 knockout (KO) cells were generated using a lentiviral CRISPR/Cas9 system in OPM2 cells. CCR1-KO OPM2 cells were confirmed to have no detectable CCR1 expression by flow cytometry and could no longer migrate towards CCL3 in vitro. Empty vector (EV) or CCR1-KO OPM2 MM PC were injected into the tibia of immune-compromised NOD-scidgamma (NSG) mice. After 4 weeks, primary tumour within the injected tibia and disseminated tumour in the PB and the contralateral tibia and femur was assessed by flow cytometry. We found that mice bearing CCR1-KO cells have a 45.5% decrease in primary tumour growth (p=0.008; % GFP+ of total mononuclear cells, EV: 77.2±17.2; CCR1-KO: 42.1±24.4), a 97.8% reduction in PB MM PC (p

Description: BACKGROUND: We have previously shown that one target of hyper-methylation in AML is the promoter of the tumour suppressor and stress-response mediator Growth Arrest and DNA Damage inducible 45A (GADD45A) (GADD45AmeHI; 42% of AML). In mice Gadd45a has recently been shown to play a critical role in HSC stress responses. Gadd45a deficiency leads to enhanced HSC self-renewal, DNA damage accumulation in HSC, increased susceptibility to leukemogenesis, and impairment in HSC apoptosis after genotoxic exposure (Chen et al, Blood 2014). These findings suggest that hypermethylation of the GADD45A gene may play an important role in the altered properties of HSC, leukaemic initiation and progression. Promoter hypermethylation of this gene defines a patient group with poor survival on standard therapy (Perugini et al, Leukaemia 2012). To explore further the molecular basis of the GADD45AmeHI patient group weperformed genetic profiling of diagnosis samples using a Sequenom multiplex mutation panel, or using whole exome sequencing for broader coverage (n=95 patients).Sequenom MassARRAY was used for quantitative detection of GADD45A promoter methylation in patient samples. For a cohort of matched diagnosis and relapse samples we used CpG methylation data for GADD45A determined by ERRBS (Akalin et al, PLoSGenetics 2012). Response to cytotoxic drugs and assessment of drug combinations with 5-Aza-deoxycytidine (decitabine, DAC) and anthracycline (Daunorubicin, DNR) was performed in AML cell lines, and with primary leukemic cell populations. RESULTS: The association of the GADD45AmeHI patient group with poor outcome was validated in an independent AML patient cohort of 48 patients from the Alfred Hospital, Melbourne, Australia (p=0.003; HR3.35). Whole exome sequencing and Sequenom multiplex analysis of 95 AML patients revealed a striking co-occurrence of the GADD45AmeHI phenotype with mutations in IDH1, IDH2, and TET2 (p

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: Abstract 3059 Aim Despite novel agents, multiple myeloma (MM) remains incurable. The majority of patients with MM will relapse at some stage after induction therapy and high dose chemotherapy with autologous stem cell transplant (ASCT). Published studies have recommended salvage ASCT as an effective option in patients with relapsed MM. However, the patient cohort who will derive maximum benefit from salvage ASCT is still undefined and is likely to evolve with increasing therapeutic options. We aimed to evaluate the role of salvage ASCT in relapsed MM patients. Methods We performed a retrospective analysis of patients who underwent salvage ASCT for relapsed MM in South Australia between 1992 and 2011. Results During this period, autologous stem cell transplants were performed for 457 patients with newly diagnosed MM. Thirty-nine patients subsequently underwent salvage ASCT for relapsed MM. The median age of patients at salvage ASCT was 59 (34–73) years, and 85% were ISS I and II at diagnosis. The majority of patients (90%, n=35) had a progression free interval (PFI) of at least 12 months after initial ASCT, consistent with recommendations by current literature. Salvage ASCT was performed for four patients with PFI shorter than 12 months, due either to suboptimal response to novel agents (thalidomide, lenalidomide, bortezomib), or the lack of novel agent availability in the earlier years. The median progression-free survival (PFS) and median overall survival (OS) following salvage ASCT were 22 months (95% CI: 11–32) and 52 months (95% CI: 30–74) respectively. Non-relapsed mortality at 2 years was 7%. Following salvage ASCT, the median PFS (28 vs. 12 months, p = 0.006) and OS (83 vs. 26 months, p = 0.02) were significantly longer in patients whose progression free interval after the initial ASCT was 〉 24 months (Figures 1 and 2). Use of novel agents in salvage induction and maintenance therapy post salvage ASCT did not appear to influence outcome. Conclusion Our results suggest that there remains a role for salvage ASCT, even in the era of novel therapies. Salvage ASCT may result in durable responses, particularly in patients with longer progression free interval (〉 24 months) following initial ASCT. This interval could be included as a selection criterion for patients with relapsed myeloma who remain transplant eligible. Disclosures: No relevant conflicts of interest to declare.

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.

Description: While there have been extensive studies to define the roles of recurrent somatic mutations in AML, the contribution of germline variants to AML initiation and progression is less well established. DNA repair disorders often predispose patients to developing myeloid malignancies. In particular, biallelic mutations affecting FANC genes cause the recessive heritable bone marrow failure syndrome Fanconi Anemia (FA), which is associated with 〉800-fold increased risk of progression to AML. A recent explosion of cancer predisposition studies has also revealed the importance of germlineFANC variants in elevated cancer risk (Cancer Treat Rev 2012; 38:89). To investigate the role of FANC gene variants in AML we have performed a case-control study, analyzing rare, deleterious somatic and germline variants for the 19 FANC genes in adult AML and healthy controls cohorts. Whole exome sequencing was performed on diagnosis samples from 131 adult Caucasian AML patients from two major Australian centers, and a cohort of 329 healthy females. We identified rare Tier 1 variants using a minor allele frequency (MAF) 〈 0.001, as reported in common dbSNP137, 1000 Genome and NHLBI-ESP project databases. Combined Annotation Dependent Depletion algorithm (CADD, Nat Genet 2014; 46: 310) 〉10 was used to filter for FANC gene variants with high probability of pathogenicity. Sanger sequencing of matched tumour/non-tumour DNA showed the large majority of variants tested to be germline (90%), consistent with previous studies reporting that somatic FANC genes variants are extremely rare in AML (〈 1%). Overall, we identified 52 FANC gene variants in 44 cases with 34% of AML cases carrying one or more variant. For independent validation we determined the presence of somatic and germline FANC variants in the TCGA AML cohort using an identical pipeline and filtering analysis. In line with our results, we found that 36% of TCGA AML patients carry at least one germline FANC variant. We investigated known disease-causing (D-C) variants in these two AML cohorts using the FA (FAMutdb) and breast cancer (kConFab and BIC) mutation databases. We found 8 D-C FANC variants in the Australian AML cohort and 5 in the TCGA cohort, with 1 variant present in both cohorts. Moreover, the frequency of D-C variants in our cohort of females with AML (n=51) is 13.7%, while the frequency in the healthy female cohort is 4.5%, comparable to that reported in the ESP database for female European-Americans (2.1%, Hum Mol Genet 2014; 23: 6815). Accordingly, we determined that deleterious FANC germline variants confer a significant increased risk of AML (P=0.018, OR=3.3 for the Australian AML cohort). Finally, we performed mutational burden analysis to investigate enrichment of variants associated with particular FANC genes across the AML cohort. This revealed a significant enrichment of FANCL variants in AML vs healthy controls (P=0.008, Figure 1). FANCL is the enzymatic component of the FA core complex that monoubiquitinates the FANCD2/I heterodimer initiating DNA repair, and its down-regulation has been linked to AML (Oncogene 2016; doi:10.1038). Several FANCL variants, found in our AML cohort, affect the catalytic RING domain and are of particular interest. These include a D-C null variant present in 2 patients, a frame shift variant in 2 patients who presented with AML at a very early age (27 and 46 years old), and a variant affecting a critical conserved residue required for monoubiquitination of FANCD2/I. In conclusion, we show enrichment of rare potentially deleterious FANC gene mutations in AML, associated with a 3-fold increased risk of developing the disease. We hypothesise that, in hematopoietic stem/progenitor cells, these variants confer a subtle defect in interstrand cross-link repair leading to an increased accumulation of mutations and subsequent development of AML. Consistent with this there have been several reports of defective DNA damage repair and increased sensitivity to DNA damaging agents in cells from FANC carriers compared to normal controls (Nat Commun 2014; 5:5496; Mutagenesis 2009; 24:67). Importantly, it is possible to target defects in several DNA repair pathways, and our finding identifies a group of AML patients who may benefit from approaches that target defective FA and homologous recombination pathways. Figure 1. A significant increase mutational burden of FANCL was observed in our AML cohort (line represents P=0.05). Figure 1. A significant increase mutational burden of FANCL was observed in our AML cohort (line represents P=0.05). Disclosures Gill: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Imatinib has resulted in excellent and sustained clinical response in most patients with CML. For a few patients, a treatment limitation has been the development of imatinib resistance, frequently the result of kinase domain mutations. The cause of increased susceptibility to mutation development remains unknown. ABCB1 is a recognized efflux transporter of imatinib. Previous studies have demonstrated high ABCB1 expression in imatinib resistant cell lines. We hypothesize that ABCB1, by facilitating drug efflux and therefore limiting the intracellular concentration of imatinib, may contribute to resistance and mutation development. Using RQ-PCR for ABCB1 expression relative to the control gene BCR, flow cytometric analysis and radioactive drug intracellular uptake and retention studies (IUR) we have assessed 32 imatinib treated chronic phase CML patients pre therapy. 29/32(90%) patients had expression of ABCB1 mRNA less than 65% of control (median 49% range 21–65%). The three patients with higher expression of ABCB1 (73%, 130% and 105%) all subsequently developed kinase domain mutations and disease progression. Only 1/29 patients with low mRNA (

Description: Up to 90% of MDS patients require red blood cell (RBC) transfusions. Literature addressing incidence and impact of alloimmunization in MDS is limited. We previously reported that 11% RBC-transfused MDS patients develop alloantibodies and RBC-transfusion requirement increases following alloimmunization (Singhal et al Haematologica 2017). This study aims to assess mechanism of increased RBC transfusion requirement following alloimmunization in MDS patients by comparing RBC-transfusion requirement following single and multiple alloantibodies, and impact of autoantibody on transfusion requirement. Primary MDS (PMDS), oligoblastic acute myeloid leukemia (AML) and therapy-related myeloid neoplasm (t-MN) patients enrolled in the SA-MDS registry (n=1002) between Nov 1991-Jun 2017, followed up for 〉3 months, received at least 1 unit of RBC and did not develop alloantibodies before first RBC transfusion were selected for analysis. Cumulative incidence (CI) of RBC-alloimmunization and clinical impact of alloimmunization including autoantibody formation and change in RBC-transfusion requirements was assessed. We also assessed risk factors for alloimmunization using recursive partitioning and Cox-regression. Seven hundred and sixty-two patients (76%) were eligible for analysis; 584 (76.5%) PMDS, 56 (7.3%) oligoblastic AML and 123 (16%) were t-MN. The median age was 72 years (range 18-97) and 489 (64%) were males. According to the Revised International Prognostic Scoring System (IPSS-R), 44.9% and 54.9% patients were classified as IPSS-R Very low/Low risk and Intermediate/High/Very high risk, respectively. The CI of alloimmunization in RBC-transfused patients was 15% (Fig 1A) and alloantibodies were most commonly against K (32%), E (26%), C (18%), Jka (10%) & Duffy (3%) antigens. Interestingly, 53% (53/99) of alloimmunized patients had single alloantibody while 46% (46/99) had multiple alloantibodies detected simultaneously or subsequently. RBC requirement was significantly higher in alloimmunized compared to non-alloimmunized patients (80±95 vs 41±58, p