ALBERT

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

Description: Introduction: Although infections are a leading cause of morbidity and mortality in MDS patients, there is limited evidence on causative organisms and resistance patterns in this patient population. This study, the largest series of infection in MDS patients, seeks to comprehensively analyse infection in this cohort. Method: CT scan, microbiology results and hospital admission ICD codes from January 1999 to April 2017 were analysed for 657 MDS patients enrolled in the South Australian MDS (SA-MDS) registry. Cox regression analysis was performed to ascertain risk factors for developing infection. Results: There were 531 primary and 126 therapy-related myeloid neoplasm (t-MN) patients with median age of 69.2 (18-97) years. According to Revised International Prognostic Scoring System (IPSS-R), 44.7% of patients were classified as Intermediate, High and Very High risk. The majority of the cohort (377/657; 57.3%) received best supportive care and 265 had disease modifying therapy including azacitidine (n=142), chemotherapy (n=74) and stem cell transplantation (SCT) (n=25). There were 2450 hospital admissions and 1312(53.5%) were infection-related, in 464/657 patients. The most common sites of infection were lower respiratory tract infections (LRTI) n=449(33.2%), fever with no known source n=356(26.3%) and skin and soft tissue (SSTI; 235, 17.3%). During the study period, 1613/16176 (10%) of microbiology tests were positive. The most common positive tests were blood cultures (494, 30.6%), respiratory viral PCR (227, 14.0%) and bacteriology (other sites; 216, 13.4%). The most common isolates were bacteria (67.3%) followed by viruses (28.1%) and fungi (4.6%). The most common bacteria were gram negative bacilli (GNB); E. coli n=81(12%), Pseudomonas spp. n=64(9%) and Klebsiella spp. n=56(8%), and the most common gram-positive isolates were Staphylococcus aureus n=114(20%) and Enterococcus spp. (37, 7%). There were 192 episodes of bacteraemia in 179 patients. The most common causative organisms were E. coli n=32(16.7%), Klebsiella spp. n=18(9.4%) Pseudomonas spp. n=25(13%), coagulase negative staphylococci n=26(14%), Streptococcus spp. n=22(11%) and Enterococcus spp. n=14(11%). The proportion of GNB bacteremias significantly increased over time (45.8% 1999-2005; 40.0% 2006-2011 and 59.4% 2012-2017 [p=.004]) without increased resistance to commonly used antibiotics. There were 21 enterococcal bacteremias with significant increase in proportion of vancomycin resistant Enterococcus (VRE) after 2011 (p

Description: Background: The management of older patients with Myelodysplastic syndrome (MDS) and Acute Myeloid Leukaemia (AML) is currently based on disease biology and performance status despite the heterogeneity of ageing and the increasingly complex care needs of frail and multi-morbid patients. Performance status scores fail to capture deficits across all the domains of ageing therefore patients that are pre-frail or vulnerable fail to be identified early in their journey. Geriatric assessments have been firmly established in the management of oncological patients but not as yet in haematological malignancies. Aims and Methods: This prospective interventional study was conducted at the Royal Adelaide Hospital. The aim was to determine if deficits in ageing were associated with therapy completion rates and survival. After the treatment decision had been made by the haematologists, consented patients had nurse-led geriatric assessments followed by Geriatrician review in participants with abnormal assessments subjective concerns with interventions implemented. Results: A total of 108 patients were enrolled into the study over a 4 year period. Although only 29 (27%) patients had an Eastern Cooperative Oncology Group score ≥2, 86 (79%) patients had deficits in at least one domain of ageing. Deficits were spread across all domains, including dependence for instrumental activity of daily living (iADL) (n=32, 29%). Patients who were iADL-dependent (3.2±5 vs. 10.8±15; p=0.004), were cognitively impaired (2.8±4 vs. 9.9±15; p=0.010) or had impaired mobility measured by the timed-up and got test (3.3±5 vs. 11.±15; p=0.002), completed significantly less cycles of azacitidine therapy than patients without deficits in these domains (Fig 1A-C). The patients who ceased therapy prematurely (less than 6 cycles) also had significantly poorer overall survival (OS) of patients compared to patients completing at least six cycles of azacitidine. (Fig 1D). Other domains predictive of poorer survival were iADL dependence (HR 4.91; p=0.003) and increased comorbidities (HR 4.41; p=0.004) independent of age and disease factors (n=108) (Fig 2A). Additionally iADL dependence was associated with shortened survival regardless of therapy choice reflecting the frailty of this group of patients - azacitidine (6 vs. 19 months, p=0.002) and supportive care cohorts (28 months vs. not reached, p=0.04; Fig 2B-C). Seventy patients were reviewed by the Geriatrician which led to the identification of a significant degree of multimorbidity (87%) and polypharmacy (73%), which have been shown to negatively impact on morbidity. Interventions and recommendations included changes in medications (57%), investigations for further evaluation of non-oncologic conditions (61%), social support referral including Aged Care Assessment (34%), nutritional support by counselling or referral to dietician (37%), formal cognitive evaluation and management (60%), physical therapy program referral or counselling (39%), referral or shared care with other specialists besides haematology and the primary care provider (26%), and psychologist referral (16%). Conclusion: This study demonstrates that geriatric assessment is feasible and instrumental in identifying geriatric related health issues in a significant proportion of patients compared to the use of performance scores. Deficits associated with ageing are associated with premature cessation of therapy and poorer survival. Geriatric assessments should form part of the assessment of older persons with the aim of reducing adverse outcomes and maintaining quality of life. Disclosures Ross: Celgene: Research Funding; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria. Hiwase:Novartis: Research Funding; Celgene: Research Funding.

Description: Therapy-related myeloid neoplasm (t-MN) is considered to be a direct stochastic complication of chemotherapy and/or radiotherapy for primary cancer or autoimmune diseases. However, genetic predisposition is reported in 8-12% of sporadic adult cancer patients [Lu et al Nature Communication 2015 and Huang et al Cell 2018]. Similarly, genetic predispositions to t-MN have also been reported in limited single institute studies of small numbers of patients [Churpek et al Cancer 2016]. In this study, we performed comprehensive germline and somatic mutation profiling in t-MN using next generation sequencing. Matched germline material was available for 62/194 (32%) patients. Mutation profiling was correlated with clinical features including family history in 194 patients enrolled in the South Australian MDS (SA-MDS) registry and Cleveland Clinic (CC). An in-house well established filtering pipeline was used for identification of somatic mutations. Only variants with Genome Aggregation Database (gnomAD) minor allele frequency (MAF) of ≤0.01% and variant allele frequency (VAF) of ≥35% were selected for further analysis of germline variants. Variants reported in in the Catalogue of Somatic Mutations in Cancer database and MDS/AML were excluded from further analysis. Variants reported pathogenic in Breast Cancer Information Core (BIC) database and Leiden Open Variation Database (LOVD) were retained. Other variants were included if truncating (nonsense, indels, splice alterations), CADD〉20, or predicted deleterious by 〉4/6 scoring algorithms (GERP〉4, PhyloP〉2, SIFT, PolyPhen2, MutationTaster and FATHMM). Forty-one (21%) t-MN patients harbored 45 rare (MAF

Description: Abstract 3473 Introduction/Background: Diamond Blackfan Anaemia (DBA) is a rare bone marrow failure disorder characterised predominantly by severe erythroid (red blood cell) failure. While the causative mutation remains unknown in approximately 50% of DBA patients, a mutation resulting in haploinsufficient expression of one of a number of ribosomal proteins (RPS or RPL) has been identified in the remaining 50% of cases, leading to DBA being classified as a ‘ribosomopathy’(Dianzani & Loreni Haematologica. 2008). Activation of the p53 pathway in response to ribosomal disruptions and nucleolar stress has also been implicated in both animal and cellular models of DBA (Danilova et al. Blood 2008; McGowan et al. Nat. Genet. 2008; Fumagalli et al. Nat. Cell. Bio. 2009). Haploinsufficiency of ribosomal proteins appears to be the major cause of DBA although a recent study (Sankaran VG, et al. J. Clin. Invest. 2012) reported the discovery of mutations in the transcription factor GATA-1, in absence of any known ribosomal protein mutation. While GATA-1 mutations have only been detected in 2 DBA pedigrees to date, this exception to the ‘ribosomopathy’ rule may help identify the link between ribosomal protein disruption and the tissue-specificity of the erythroid defect. Results: We have developed a Doxycycline-inducible shRNA RPS19 knockdown model of DBA in the human erythroleukaemic cell line TF1.8. Two independent shRNA triggers targeting RPS19 (the most common gene affected in DBA patients), and a vector-alone control were introduced into cells. RPS19 knockdown was validated by Q-RTPCR which showed a 5-fold decrease in RPS19 mRNA levels upon the addition of 0.25ug/ml Dox (Figure 1). Expression at the protein level was validated by western blotting. Other ribosomal proteins, for example RPL11, are unaffected. The recent discovery of GATA-1 mutations in 2 unrelated DBA pedigrees led us to investigate whether the expression of GATA-1, or other key erythroid transcription factors, may be affected in our RPS19 DBA cell line model. Q-RTPCR results show decreased expression of two key transcription factors (GATA-1 and Myb) in cells with RPS19 knockdown compared to the controls (Figure 1). Furthermore, the expression of the GATA-1 target gene, FOG-1 is decreased compared to controls. MYBBP1A, a Myb target gene involved in ribosomal stress pathways (Yamauchi et al. Genes. Cells 2008) is increased, while another Myb target, GFI-1b is decreased in this model. Discussion/Conclusions: We have generated an inducible RPS19-knockdown model of DBA in TF1.8 cells, and have showed that several key transcription factors important in erythropoiesis (including GATA-1) display down-regulation associated with RPS19-knockdown. This suggests that changes to GATA-1 levels may be important not only in the rare cases of DBA where GATA-1 is mutated, but also in classical DBA associated with ribosomal protein haploinsufficiency. Based on the observation that GATA-1 associates with and inhibits p53 during normal erythropoiesis (Trainor et al. Blood 2009) we propose that the decreased GATA-1 expression may contribute to activation of the p53 pathway during impaired erythropoiesis in Diamond Blackfan Anaemia. Further studies are being undertaken to test this model using assays for transcription factor activity, which will then be further characterised in a DBA model based on CD34+ cord blood cells infected with our validated RPS19 knockdown constructs. Disclosures: No relevant conflicts of interest to declare.

Description: The Australian Familial Haematological Cancer Study (AFHCS) was initiated in 2004 with the aim to define genes predisposing to hematological malignancy (HM) to offer better options for clinical decision making and genetic counselling, and to identify therapeutic targets. The study is a referral centre for Australia and New Zealand, and currently has 230 families with multiple cases of myeloid and/or lymphoid malignancies or early onset cases (Figure 1), and is growing as clinical awareness of a germline genetic basis for blood cancers increases. To date, we have identified families with causal germline variants in several predisposition genes (five GATA2, ten RUNX1, one CEBPA, ten DDX41, one SAMD9L) including novel single nucleotide variants, deletions and insertions in coding and intronic sequences using traditional Sanger sequencing and now genomic and transcriptomic technologies. Of these, one GATA2 and four DDX41 germline mutations were identified during the screening of "sporadic" MDS samples. All four DDX41 mutant samples also harbored a somatic DDX41 (R525H) variant on the other allele at a low variant allele frequency. A comprehensive clinical analysis of the RUNX1 families has uncovered segregating phenotypes, in addition to thrombocytopenia and myeloid and lymphoid malignancies, including skin disorders such as psoriasis. In an increasing number of individuals in these families, important clinical decisions have been made dependent on mutation carrier status. Recently, we have identified and characterized a unique myeloproliferative neoplasm (MPN)/acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS) family with a germline Chr14q duplication that overlaps with duplications in two other reported MPN/AML families. This appears to be a unique genotypic/phenotypic entity when compared to other myeloid predisposition genes and their associated phenotypes. Interestingly, we have identified several families carrying heterozygous pathogenic/likely pathogenic variants in genes representing autosomal recessive genomic instability syndromes segregating with HM. Here mutations in the genes NBN, RECQL4, DDX11 and RAD21 appear to act in an autosomal dominant manner. Further, we have found DNA damage repair gene predicted pathogenic variants in PALB2 and BARD1 in families with both solid cancers and HM, predominantly lymphomas, implicating an expansion of the major predisposition phenotype of these gene perturbations. Familial cases of chronic lymphocytic leukemia (CLL) have been well recognized, but it has been particularly difficult to identify predisposing variants. We have identified a number of strong candidate genes/variants in CLL families including PRPF8 (Y208C and N400S) and SAMHD1 (R371H) although more families are required to confirm these. An integral part of the AFHCS is the continued generation of cell and animal models to help define mechanisms of action of predicted or known pathogenic variants, and functional model systems for testing of variants of unknown significance. To facilitate the collection of patient samples, we have adopted the use of hair bulbs as the main germline sample as they are easy to collect, can be easily sent long distance by mail at room temperature, require no culture, are quickly and cheaply processed and provide good quality DNA using automated procedures. Overall, collaborative efforts within Australia and New Zealand and internationally have been highly fruitful in solving familial cases of hematopoietic malignancies over the last 15 years, and even more concerted international efforts will be required in the future to uncover the familial basis of unsolved cases, particularly in the lymphoid lineage, and to clarify best approaches for clinical decision making and treatment options. Figure 1. Summary of AFHCS families with associated hematological malignancies. Figure Disclosures Scott: Celgene: Honoraria.

Description: Objectives: Older people (≥65 years) living with myelodysplastic syndrome (MDS) have a poor prognosis, often compounded by comorbidities and polypharmacy. Polypharmacy is usually defined as the regular use of ≥5 medications and is common among older adults; however the appropriateness of therapy in older patients with malignancies requires a consideration of quality of life as well as prognosis rather than a discrete number. Such a patient-centred approach would identify potentially inappropriate medications (PIM) and also potentially omitted medications (POM). There is little data on the impact of PP, PIM and POM on patient reported outcome measures at baseline in older people with MDS. This study assesses the prevalence of PP, PIM and POM in older patients with MDS. Methods: Patients ≥65 years with MDS were enrolled from Jan 2014 to Nov 2018 at the Royal Adelaide Hospital. Patients were included in the study if they were seen by the Geriatrician for a Comprehensive Geriatric Assessment (CGA; Table 1), had geriatric screening assessments performed for frailty, completed patient-reported outcome measures using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) at enrolment, specifically the domains of physical condition of medical treatment interfering on family (q26), social (q27) and financial aspects (q28), overall health (q29) and overall quality of life (q30). Results: The average age of patients at the time of enrolment was 75 years (62-89 years). Other patient characteristics are in Table 1. Forty-one (64%) patients were on supportive care and 21 (33%) were treated with azacitidine. The prevalence of PP, PIM and POM was 39% (25/64), 47% (30/64), and 64% (41/64) respectively. These were evaluated by a Geriatrician/Clinical Pharmacologist using the STOPP/START criteria after reviewing the CGA reports for each patient. The most common PIM was aspirin without an evidence-based clinical indication. The number of PIM ranged from 1 to 7 for an individual patient. The most common POM were vaccinations - only 1/64 (2%) patient had a documented influenza and pneumococcal vaccination status as per national guidelines, followed by laxatives for concurrent opioid use. Twenty-three percent (15/64) reported an overall poorer quality of life (defined as q30

Description: Introduction: A subset of patients with MDS and related myeloid disorders present with concomitant autoimmune rheumatological diseases (AIRD); however the prevalence ranges from 10-48% based on limited literature. Further, use of immunosuppressive agents in AIRD patients could confound the secondary diagnosis of MDS and in some cases cause it (therapy-related myeloid neoplasm; t-MN). The prevalence of cytopenia in AIRD patients is unknown and the genetic characteristics of MDS patients with concomitant AIRD have not been described. Hence, we interrogated two large multi-institutional databases -Royal Adelaide Hospital Rheumatology Database (RAH-RD) and South-Australian MDS (SA-MDS) registry in this study. Methods: Demographic, clinical, laboratory and treatment data of 2663 AIRD and 1157 MDS patients were analysed. In AIRD patients (autoimmune inflammatory arthritis, spondyloarthritis, vasculitis and connective tissue diseases), cytopenia (persisting 〉6 months) were defined as follows: hemoglobin

Description: Introduction: Myeloid neoplasms occurring after exposure to chemo- and/or radiotherapy, termed Therapy-related myeloid neoplasms (T-MN), are considered poor prognosis. This perception creates an inherent bias in treatment decision, resulting in either over- (allo-HSCT) or under-treatment (palliation) and exclusion from most clinical trials. The optimal T-MN treatment paradigm is unknown, partly because its mutational architecture is not well defined. Few small studies show mutations in only 50-60% (Ok, Leuk Res 2015; Lindsley, Blood 2015) of T-MN (compared to 80-90% in PMDS). This study compares the mutational architecture of T-MN and PMDS from the South Australian Myelodysplastic Syndrome registry. Methods: Demographic, clinical and laboratory data including cytogenetic profiles of 129 T-MN (95 T-MDS; 34 T-AML [≥20% blasts]) and 108 PMDS patients were analysed. Targeted Massively Parallel Sequencing of a custom panel of 43 myeloid neoplasms associated genes (all coding regions) was performed on diagnosis bone marrow samples. Mutations with VAF ≥3% were selected for further assessment. Overall survival (OS) was calculated from date of diagnosis to date of last follow-up or death and adjusted using time varying covariate to account for disease modifying therapy (DMT) exposure. Results: Compared to PMDS, T-MN patients were younger at diagnosis (71.1 vs 75.3 years, p

Description: Introduction: Therapy-related myeloid neoplasm (t-MN) is a lethal second hematological malignancy following chemotherapy (CT) and radiotherapy (RT) for primary cancers. It accounts for 15-20% of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). AML and MDS are considered to be hematopoietic stem cell (HSC)-autonomous disorders, in which initiation and progression are mainly driven by HSC-intrinsic genetic events. However, emerging data suggest that bone marrow (BM) microenvironment plays critical role in initiation and evolution of MDS and AML (Raaijmakers et al., 2010). Malignant clones can also shape the BM-microenvironment conducive for its survival and proliferation (Medyouf et al., Cell Stem Cell 2014). Although CT/RT can damage BM-microenvironment, very limited studies assessed role of BM-microenvironment in t-MN pathogenesis. Aim: To assess BM-microenvironment changes induced by malignant HSC and changes induced by previous genotoxic stress on BM-microenvironment, we compared BM-mesenchymal stromal cells (MSC) from t-MN patients, with BM-MSC from 1) patients with two unrelated cancers, one of the cancer being MDS/AML, without prior exposure to CT/RT (Double cancers, DC), 2) primary MDS patients (pMDS) and 3) age matched healthy controls (HC). Methods: We characterized BM-MSC from t-MN (n=10), DC (n=8), pMDS (n=6) and age-matched healthy controls (n=10). Morphology (Fei et al., 2014), clonogenic potential (Geyh et al., 2013), proliferation (Prata et al., 2010) and cellular senescence was assessed using previously described methodology. Differentiation potential was assessed by the respective lineage cytochemical staining and quantification of positive cells (mineral quantification for osteogenic cells, and Nile red for adipocytes). DNA damage response was determined by assessing H2AX phosphorylation in the MSC. Results: Only 70% of BM-MSC from t-MN cohort could be expanded to passage 6 compared to 100% of MSC cultures from pMDS, DC and HC. Proliferation rate, assessed by population doubling time, and clonogenic potential was significantly reduced in t-MN patients compared to p-MDS, DC and HC (Fig 1Ai-ii). This was further substantiated by higher senescence rates, assessed by β-galactosidase positive cells at passage 3 (HC 7%±1.9%; pMDS 39%±6%; DC 27%±1%; t-MN 68%±4%) (Fig 1B). Together, it demonstrates that MSC from t-MN have significantly impaired proliferation capacity and higher senescence rate compared to HC, pMDS and DC patients. Interestingly, proliferation capacity and senescence rate was not significantly different between MSC from DC and pMDS patients. We also compared DNA damage repair, following sub-lethal dose of RT, in MSC from t-MN, pMDS and HC. DNA damage repair in t-MN MSC was significantly impaired compared to pMDS and HC (Fig. 1F). Impaired DNA repair could be due to pathogenic germline mutation in DNA repair pathways in some t-MN patients (Singhal et al., ASH 2018). Although MSC from pMDS and DC appeared disorganized, they maintain fibroblast-like morphology similar to HC-MSC. Whereas, most of the MSCs from t-MN cases lost spindle shape morphology and were significantly larger than DC, pMDS and HC (p