Hypomethylating agents are frontline therapies for myelodysplastic neoplasms (MDS), yet clinical responses remain unpredictable. We conducted a phase 2 trial comparing injectable and oral azacitidine (AZA) administered over one or three weeks per four-week cycle, with the primary objective of investigating whether response is linked to in vivo drug incorporation or DNA hypomethylation. Our findings show that injection results in higher drug incorporation, but lower DNA demethylation per cycle, while global DNA methylation levels in mononuclear cells are comparable between responders and non-responders. However, hematopoietic stem and progenitor cells (HSPCs) from responders exhibit distinct baseline and early treatment-induced CpG methylation changes at regulatory regions linked to tissue patterning, cell migration, and myeloid differentiation. By cycle six-when clinical responses typically emerge-further differential hypomethylation in responder HSPCs suggests marrow adaptation as a driver of improved hematopoiesis. These findings indicate that intrinsic baseline and early drug-induced epigenetic differences in HSPCs may underlie the variable clinical response to AZA in MDS.

Hypomethylating agents are used as frontline therapy for myelodysplastic neoplasms (MDS), but clinical response is unpredictable. To determine whether response was associated with in vivo dynamics of DNA hypomethylation, we conducted a phase 2 trial for MDS using both injection and oral azacitidine (AZA). We established that global DNA methylation levels in peripheral blood and bone marrow mononuclear cells were comparable in AZA responders and non-responders during their course of treatment. However, there were distinct baseline and early drug induced differences in CpG methylation in haematopoietic stem and progenitor cells (HSPCs) in responders compared to non-responders that overlapped with regulatory regions of genes associated with tissue patterning, cell migration and myeloid differentiation. Following six cycles of therapy when clinical response typically manifests, differential hypomethylation in responder HSPCs pointed to marrow adaptation as a driver of enhanced haematopoiesis. Taken together, CpG methylation differences in HSPCs may explain variable response to AZA. ### Competing Interest Statement F.V. is affiliated with OmniOmics.AI Pty Ltd. C.F. is an advisory board member at Amgen, AbbVie, Adaptive Biotech, BeiGene, Pfizer, Otsuka, and Jazz, a consultant at Novotech, and received speaker fees from Amgen, Pfizer, Servier, BMS, and Astella. D.H. has consultancy agreements with GlaxoSmithKline and Pharming Corp. M.H. is a consultant/advisory board member at Roche, Gilead, Otsuka, Janssen, Beigene, and Takeda. M.N.P. received research funding and/or provision of drug for clinical trials (to institution) from AstraZeneca, BRII Biosciences, Celgene/BMS, CSL Behring, Eli Lilly, Emergent Biosciences, Gilead Pharmaceuticals, GlaxoSmithKline, Grifols, Janssen/Johnson and Johnson, Takeda, ViiV Pharmaceuticals and has advisory roles with Celgene/BMS, Gilead Pharmaceuticals, and ViiV Pharmaceuticals. J.E.P. received research funding and/or provision of drug for clinical trials (to institution) from Celgene/BMS, Astex, Verastem Oncology and received honoraria from Abbvie as an advisory board member. The remaining authors declare no competing financial interests. ### Clinical Trial NCT03493646 ### Funding Statement The investigator initiated clinical trial was funded in part by Celgene/BMS (RG172029) with research support from the National Health and Medical Research Council (RG170246, RG211412), Anthony Rothe Memorial Trust (RG182042, RG202657, RG213236), Leukaemia Foundation (RG231257). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The trial protocol received ethical approval from the South Eastern Sydney Local Health District Human Research Ethics Committee, and participating sites received Institution approval to conduct the trial prior to commencing recruitment. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Determinants of clinical response to hypomethylating agents (HMAs) in MDS/CMML are unclear. To address this knowledge gap, we enrolled 40 patients to NCT03493646 where they received 6 cycles of injection AZA (vidaza) or oral AZA (CC486) delivered for 7 or 21 days respectively in a 28-day cycle to study the kinetics of drug incorporation and DNA hypomethylation during treatment, and noted the following(i) Average DAC incorporation in DNA was higher with vidaza than CC486, but global DNA hypomethylation was greater with CC486. Although DAC incorporation was higher in responders (R) than in non-responders (NR) with vidaza, global DNA demethylation did not differ between response groups with either drug.(ii) DAC incorporation after vidaza correlated with the fraction of HPCs progressing through S/G2/M pre-treatment, but there were no pre-treatment differences between R and NR in the fraction of HSCs and HPCs progressing through S/G2/M. However, vidaza induced HSCs and HPCs to exit quiescence in R but not NR.(iii) Baseline CpG methylation was higher in CD34+ bone marrow cells in NR vs. R, and CpG sites that were associated with regulation of pattern specification (HOX cluster), epithelial cell migration/EMT and mesenchymal stromal cell differentiation were differentially hypomethylated in R vs. NR. Following vidaza, early drug-induced hypomethylation was seen at CpG sites in CD34+ that regulated genes associated with myeloid cell differentiation. By contrast, late drug-induced CpG hypomethylation in R was at sites associated with leukocyte development in response to marrow environmental changes.(iv) Variant alleles persist in R and NR following vidaza or CC486, and there was no association between change in variant allele frequencies and the degree of DNA hypomethylation.Site-specific differences in CpG hypomethylation in HSPCs may explain clinical response to HMAs.

Introduction: Anaemia is almost universal in MDNs with most patients eventually receiving blood products. Unfortunately, this can result in transfusion reactions. Apart from alloimmunisation and iron overload, there are limited studies of transfusion reactions for this cohort.1 We investigated the incidence and risk factors for transfusion reactions within a cohort of patients with MDNs.

Myelodysplastic neoplasms (MDS) are clonal myeloid neoplasms with an increased risk of progression to acute myeloid leukemia (AML). For patients unfit for hematopoietic cell transplantation (HCT), hypomethylating agents, such as azacitidine (AZA), represent the main therapeutic option for patients with higher-risk MDS, with an overall response rate (ORR) of 51% and median overall survival 25 months [1, 2]. Lenalidomide is an immunomodulatory drug that is effective in lower-risk MDS with isolated del(5q) [3], but may also have activity in non-del(5q) MDS [4]. Germline DDX41 mutations are found in approximately 5% of patients with MDS and AML, representing the most common myeloid neoplasms associated with germline predisposition [5-7]. To date, it is unclear if DDX41-mutated MDS should be treated differently than the standard treatment, such as AZA [8, 9]. While DDX41 (5q35.3) is outside the minimal deleted region (q31–q33) of most MDS cases with del(5q), it is variably deleted in 25% of patients [10]. Thus, there is considerable interest if DDX41-mutated MDS might also respond favorably to lenalidomide [10-13]. The Australasian Leukaemia and Lymphoma Group (ALLG) MDS4 phase II trial (ACTRN12610000271000) randomized 160 patients with higher-risk MDS, chronic myelomonocytic leukemia (CMML), and low blast AML to either AZA or combination AZA with lenalidomide (LEN, from cycle 3 onward) [14]. While the combination of LEN and AZA was tolerable, overall there was no improvement in clinical benefit, response rates, or overall survival in patients compared to treatment with AZA alone [14]. We performed a post-hoc genomic analysis to correlate genomic lesions in this trial cohort with clinical outcomes. This study was approved by the local ethics committee and conducted in accordance with the Declaration of Helsinki. A unique molecular index-based QIAseq targeted DNA panel (QIAGEN) was performed with a sensitivity of 0.5% variant allele frequency (VAF) [15]. HumanCytoSNP-12 BeadChip array (SNP-A) was previously reported [14]. The primary endpoint of the study was an ongoing clinical benefit (alive and progression-free) at 12 months. Kaplan−Meier overall survival was measured from the date of treatment commencement. R statistical software version 4.2 was used for analysis. A total of 66 patients had baseline DNA available for testing: 36 on AZA alone and 30 on AZA+LEN. Baseline characteristics are summarized in Figure 1. The median age was 71.6 years (range 52–87). IPSS-R risk groups were very low or low (n = 20), intermediate (n = 18), high or very high (n = 25), and not available (n = 3 due to missing/failed karyotype). Disease subtypes by WHO 2008 were MDS (n = 52), CMML (n = 9), and AML (n = 5). SNP-A was abnormal in 47% of patients, including 18% with complex (≥3) copy number changes. The most common mutations were ASXL1 (48.5%), TET2 (47%), and RUNX1 (30%). Median follow-up among survivors was 49 months and median overall survival was 33.2 months (95% CI, 21.7 to NE). DDX41 variants were found in six patients with all patients harboring ≥2 DDX41 variants (including the Arg525His variant in three). The germline versus somatic origin of these variants could not be proven; however, all six patients had one DDX41 variant at approximately 50% VAF with a second lower VAF DDX41 detected (Table S1). All patients with DDX41 variants had a normal karyotype and SNP-A (5/5, 100%) in comparison to 48% (26/55) DDX41 wildtype patients among evaluable patients. Additionally, five patients had monosomy 5 by G-banded karyotyping (expected to delete the DDX41 locus) but all were associated with complex karyotype and these five patients died after median 4.9 months (range 0.6−21.6). The most common co-mutation in DDX41 mutant group was ASXL1 (4/6 patients) (Table S2). One patient with a DDX41 mutation (low blast AML) died before receiving therapy due to disease progression. The remaining five patients, aged 64–76 years, received AZA (n = 4) or AZA+LEN (n = 1) and achieved complete remission (CR, n = 1), marrow CR (n = 1), hematologic improvement (HI, n = 1), and stable disease (n = 2), and remarkably all remained alive at last follow-up of median 53 months (range 47–58; Figure 2), despite having intermediate (n = 3) and high (n = 2) R-IPSS. No patients underwent HCT. Mutations in TP53, U2AF1, and EZH2, and abnormal SNP-A profile, including complex and abnormal 5q/7q/17p, were associated with inferior outcomes consistent with previous studies (Figures S1A and S2). Additional molecular subgroup analyses did not identify any significant interaction with the treatment arms (Figure S1B). Mutations were serially assessed on therapy in 45 patients after two (n = 38) to four cycles (n = 7) of therapy. The majority (67%) of the 166 mutations observed at baseline remained stable (< 10% VAF change). With the caveat of short interval between testing, no patient achieved complete molecular clearance. Fourteen (31%) patients had newly acquired mutations (n = 18 variants) or > 10% VAF increase (n = 2 variants). Twenty-six (58%) patients had mutation clearance of ≥1 variant (n = 20/166 [12%] variants) or > 10% VAF reduction (n = 33 [20%] variants). Of note, eight patients had mixed changes in VAF. Patients with mutation clearance/reduction had a trend toward better overall survival (Figure S3). Two patients with DDX41-mutated disease were assessed after two cycles of AZA with no significant change in the somatic mutations. One patient with DDX41-mutated MDS with increased blasts-1 achieved marrow CR after four cycles of AZA with approximately a 50% decrease in somatic DDX41, NRAS, and ASXL1 mutations (Figure S4). Our cohort demonstrated excellent outcomes among patients with DDX41-mutated myeloid neoplasms treated with AZA. Earlier retrospective studies suggested that patients with DDX41-mutated myeloid neoplasms might respond favorably to lenalidomide [10, 12, 13]. Sebert et al. studied 11 patients with DDX41-mutated MDS/AML who received AZA with 73% ORR and prolonged response duration (median 2.5 years); note seven patients underwent HCT [8]. A pooled analysis of additional patients (total 33 patients) showed similar ORR of 70% to AZA [9]. The small number (n = 5) in our trial cohort prevents any conclusion to be drawn regarding the impact of the addition of lenalidomide (n = 1) on DDX41-mutated MDS in comparison to AZA alone (n = 4). However, until further data become available, the management of DDX41-mutated MDS should follow the current standard of care. In summary, our correlative molecular analysis of the ALLG MDS4 trial of AZA versus AZA+LEN in MDS/AML has identified that patients with DDX41 mutations are associated with favorable outcomes with either AZA or AZA+LEN supporting the use of this therapy in these patients. This observation and subgroup of DDX41-mutated patients warrants further study in randomized trials. PB and WSS designed the study. IST, MW, YZY, and PB performed the molecular analyses. JFS and MK contributed essential clinical data. IST and PB wrote the manuscript. All authors reviewed and approved the submitted version. The authors gratefully acknowledge funding from the Wilson Centre for Blood Cancer Genomics and the Snowdome Foundation. The authors would also like to acknowledge all the participating patients in the Australasian Leukaemia and Lymphoma Group MDS4 study from the following sites (Principal Investigators): Austin Hospital (Daniela Zantomio); Barwon Health (Philip Campbell); Border Medical Oncology (Richard Eek); Cabrini Hospital (Melita Kenealy); Calvary Mater Newcastle (Sandra Deveridge); Canberra Hospital (James D'Rozario); Coffs Harbour Hospital (Martin Browne); Concord Hospital (Ilona Cunningham); Flinders Medical Centre (David Ross); Fremantle Hospital (Michael Leahy); Gosford Hospital (Campbell Tiley); Greenslopes Hospital (Anthony Mills); ICON (Kerry Taylor); Liverpool Hospital (Anne-marie Watson); Monash Medical Centre (Stephen Samuel Opat); Nepean Hospital (John Taper); Peter MacCallum Cancer Centre (John Seymour); Port Macquarie Hospital (Richard Stark); Princess Alexandra Hospital (Anthony Mills); Royal Adelaide Hospital (Devendra Hiwase); Royal Hobart Hospital (Rosemary Harrup); Royal Melbourne Hospital (Ashish Bajel); Royal North Shore Hospital (William Stevenson); Sir Charles Gardiner Hospital (Gavin Cull); St. George Hospital (Jing Hu) St. Vincent's Hospital Melbourne (Robin Filshie); St. Vincent's Hospital Sydney (Keith Fay); Tweed Hospital (Ehtesham Abdi); Western Health Hospital (Duncan Carradice); Westmead Hospital (Warwick Benson); Wollongong and Shoalhaven Hospital (Pauline Warburton). The authors declare no conflict of interest. Wilson Centre for Blood Cancer Genomics; Snowdome Foundation. This study was approved by the Northern Sydney Local Health District Human Research Ethics Committee (2019/ETH08385). All patients provided written informed consent to the ALLG MDS4 study and the storage and use of blood and tissue samples for research. anzctr.org.au (ACTRN12610000271000). The data that support the findings of this study are available from the corresponding author upon reasonable request. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Introduction: GB261 is a novel and highly differentiated CD20/CD3 bispecific T cell engager antibody computationally designed to maintain Fc effector function, i.e., antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) to broaden the mechanisms of action (MOA) for tumor cell killing. Furthermore, the “imbalanced” design of GB261 integrates de-tuned CD3 binding to reduce CRS incidence and improve safety features of the Fc effector function. Extensive pre-clinical studies have shown that GB261 has a highly advantageous safety/efficacy balance.Here, we present the preliminary clinical safety and efficacy results of an ongoing phase I/II study for GB261 (NCT04923048). Methods: This is an open-label, multicenter (China and Australia), dose-escalation/expansion phase 1/2 study of GB261 to evaluate the safety, tolerability and efficacy in patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma(B-NHL) and Chronic Lymphocytic Leukemia (CLL). Adult patients were eligible when they have CD20+ r/r B-NHL or CLL with no available standard of care treatments, adequate organ function, and no CNS involvement or other CNS disease, infections or other active/serious medical issues that may affect compliance or interpretation of results. GB261 was administered in 21-day cycles, weekly (QW) for the first 6 doses followed by every 3 weeks (Q3W) until disease progression, or other situation defined in protocol. Response assessment was based on Lugano 2014 and LYRIC 2016 criteria. Results: As of June 17, 2023, 47 r/r B-NHL patients (DLBCL:76.6%; FL:23.4%) were enrolled at flat or step-up doses of GB261 ranging from 1mg to 300 mg. Median age was 60.0 years (range: 28, 81), 55.3% of patients were male. Median prior lines of therapy were 3 (range: 1, 10). 78.7% of patients were refractory to any anti-CD20 therapy, 70.2% refractory to their last systemic therapy. Median time since last prior therapy to first study treatment was 1.9 months. In efficacy evaluable patients (n=22) from 3mg to 100mg, with at least 75% dose exposure before the first radiographic assessment, the median duration of study follow-up was 4.5 months (95%CI: 4.0, 7.4). The overall response rate (ORR) was 73% (16/22), and complete response rate (CRR) was 45.5% (10/22). ORR and CRR were 100% and 100% in 3mg, 56% and 22% in 10mg, 67% and 33% in 30mg. At 100mg dose, there were 5 evaluable patients, with ORR 100% (5/5), CRR 80% (4/5) and PR (20%, 1/5; mosunetuzumab-refractory rrDLBCL patient). Median time to response (TTR) was 1.3 months (95%CI: 1.2, 1.5), the same as median time to CR. Median duration of response (DOR) was not reached. In safety evaluable patients (n=47), the median duration of study follow-up was 4.1 months (95%CI: 2.9, 5.3). Treatment-emergent adverse events (TEAEs) were 95.7%, treatment-related adverse events were 85.1%. The most common TEAEs were COVID-19 infection (40.4%; grade 1 or 2: 27.6%; grade >=3: 12.8%) and neutropenia (31.9%; grade 1or 2: 14.9%; grade >=3: 17.0%). AE related treatment discontinuation and death were reported in 2 patients, which were all due to COVID-19 pneumonia. CRS occurred in 12.8% (6/47) patients, was mild and transient. CRS in 100mg were 14.3% (2/14). All cases of CRS were grade 1 (8.5%, 4/47) or 2 (4.3%, 2/47), no grade 3 (Lee et al., ASTCT criteria), no interruptions of treatment, and no administration of Tocilizumab. The median duration of CRS was 7 hours. No ICANS were reported. The PK profile of GB261 appeared to be linear across dose ranges tested (1mg-100mg). Effective half-life appeared to be 2-3 weeks which supports every 3-4 weeks dosing. Conclusion: GB261, a novel and highly differentiated CD20/CD3 bispecific antibody, is the first clinical stage Fc+ CD20/CD3 T cell engager. In heavily pretreated B-NHL patients, GB261 showed a highly advantageous safety/efficacy balance, consistent with the MOA. The safety profile is excellent especially for the CRS which is very mild, transient and less frequent compare with other CD20/CD3 bispecific antibodies. The response after GB261 treatment was early, deep and durable. At the 100mg dose level, 80% of patients achieved CR and with favorable safety. Additionally, clinical benefit seen in other CD20/CD3 bispecific antibody failed patients provides clinical support to the unique and differentiated MOA of GB261.

Myelodysplastic syndromes (MDS) have a major impact on quality of life (QoL). We performed a post hoc analysis of two multicenter trials of azacitidine-based disease-modifying therapy for patients with MDS and low blast count acute myeloid leukemia (AML), to identify factors associated with QoL. 231 patients were included (median age 70 years). At baseline, higher initial hemoglobin, but not neutrophil or platelet count, was associated with better global QoL and physical function (p < 0.001 and p = 0.001, respectively). During therapy, increase in hemoglobin was associated with improvement in QoL and physical function (p = 0.005 and p < 0.001, respectively). Lower initial hemoglobin was associated with higher dyspnea and fatigue scores (p < 0.001 and p = 0.001, respectively), and hemoglobin response was associated with improvement in dyspnea and fatigue (p < 0.001 for each). In patients with MDS and low blast count AML, hemoglobin level was strongly correlated with global QoL, physical functioning, dyspnea and fatigue, both before and during azacitidine-based therapy.

Introduction. The Australasian Leukaemia and Lymphoma Group (ALLG) MDS4 phase II trial randomized 160 patients (pts) with higher risk (by IPSS) myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML) and low blast acute myeloid leukemia (AML) to either azacitidine (AZA) or combination AZA with lenalidomide (LEN, from cycle 3 onwards) (Kenealy, Haematologica 2019; ANZCTR12610000271000). Whilst the combination of LEN and AZA was tolerable, there was no improvement in clinical benefit, response rates or overall survival in patients compared to treatment with AZA alone. We performed a post-hoc genomic analysis of pts on this trial to further understand the molecular changes in this cohort and any correlation with outcomes. Methods. A unique molecular index (UMI) based QIAseq targeted DNA panel (QIAGEN) was performed as previously described (Blombery, Blood 2020). Sensitivity for variant calling was 0.5% variant allele frequency (VAF). HumanCytoSNP-12 BeadChip array (SNP-A) was previously performed and reported (Kenealy, Haematologica 2019). Primary endpoint of the study was ongoing clinical benefit at 12 months, defined as being alive and progression-free. Logistic regression and Cox proportional hazards regression were used respectively to assess the differences in response rates and overall survival between two treatment groups according to the molecular subgroups. Results. A total of 66 pts, representative of the 160 enrolled, had baseline gDNA available for targeted panel testing; 36 and 30 pts on AZA alone and AZA+LEN therapy, respectively. Median age was 71.6 years. IPSS-R risk groups were very low or low (n=20), intermediate (n=18), high or very high (n=25), and not available (n=3 due to missing karyotype). Disease subtypes were MDS (n=52), CMML (n=9), and AML (n=5). 47% of pts had an abnormal SNP-A, including 18% with complex (≥3) copy number changes. The most common gene mutations observed were ASXL1 (48.5%), TET2 (47%) and RUNX1 (30%) (Figure 1). Overall clinical benefit at 12 months was achieved in 39 pts (59%). TET2 was the only gene mutation associated with significantly improved benefit when treated with AZA alone (OR 10 [95% CI, 2 to 81]). Median follow up among survivors was 49 months and the median overall survival was 33.2 months (95% CI, 21.7 - NE). Mutations in TP53, U2AF1, EZH2 and SRSF2 were associated with inferior overall survival. Molecular subgroup analyses did not identify any significant difference between the two treatment arms. Given the recent discovery of DDX41 mutations in MDS/AML and early data supporting a favorable response to AZA/LEN, we focused on 6/66 (9%) patients with detectable DDX41 mutations. All 6 pts had a second mutation detectable in DDX41 as well as no detectable abnormalities on SNP-A. The most commonly co-mutated gene in this group was ASXL1 (in 4/6). One patient with DDX41 mutation died before receiving any therapy. Best responses achieved in DDX41 mutated patients were complete remission (CR, n=1), marrow CR (n=1), hematologic improvement (HI, n=1), and stable disease (SD, n=2). Remarkably, of the 5 patients that received treatment (4 AZA, 1 AZA+LEN), the overall survival at 52 months (range 47-58) follow up was 100% (Figure 2), despite having intermediate (n=3) and high (n=2) R-IPSS. Mutations were also serially assessed on therapy in 45 pts: ≤2 cycles in 38 pts, and ≥4 cycles in 7 pts (2 AZA+LEN). Of 166 mutations observed at baseline, majority (67%) were stable with <10% change in VAF (median +2%). Overall, 27 (60%) pts had clearance or >10% VAF reduction in ≥1 variant, including clearance in 20 (12%) variants (most commonly TET2 [n=4] and RUNX1 [n=3]), and reduction in 33 (20%) variants (ASXL1 [n=6], SF3B1, SRSF2 and TET2 [n=5 each], but no pt had complete molecular clearance. In contrast, new mutations (n=18 variants) or >10% VAF increase (n=2 variants) were observed in 14 (31%) pts, most commonly TET2 (n=6), RUNX1 (n=4), and DNMT3A and BRAF (n=3 each). Conclusions. In summary, our correlative molecular analysis of the ALLG MDS4 trial of AZA vs AZA+LEN in MDS/AML has identified, in addition to established molecular risk factors for inferior outcomes, that pts with DDX41 mutations are associated with highly favorable outcomes with either AZA or AZA+LEN. This observation and subgroup of patients warrants further study in randomized trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Objectives Myelodysplastic syndromes (MDS) are characterised by ineffective haematopoiesis. Although hypomethylating agents (HMA) have improved survival in higher-risk MDS, most patients eventually succumb to progressive disease. Utilising samples collected prospectively from three MDS clinical trials, we analysed genetic and immunological biomarkers and correlated them with clinical outcomes. Methods A hundred and fifty four samples were analysed from 133 de novo MDS patients for T-cell and myeloid cell immunophenotyping and gene expression analysis. Treatments were with HMA or immunomodulatory drug (IMiD) alone or in combination. Results We observed differences in immune cell subsets between lower- and higher-risk IPSS groups with NKT cells, MDSCs, intermediate-proinflammatory and non-classical monocytes being higher in the latter group, while naive CD4+ T cells were reduced. Intermediate-proinflammatory monocytes were increased in non-responders and those failing to achieve at least a haematological improvement. Proinflammatory NKT cells were increased at diagnosis for patients failing to derive clinical benefit after 12 months of treatment. Gene expression analysis of paired bone marrow (BM) colony-forming units (CFUs) from diagnosis and 4 cycles post-treatment confirmed that genes involved in cytokine signalling were downregulated in C4 normal colonies. Conclusions These findings support the central roles of dysregulation in innate immunity and inflammatory signalling in the pathogenesis of MDS which correlated with clinical outcomes post-treatment.

Objectives Psychosocial interventions that mitigate psychosocial distress in cancer patients are important. The primary aim of this study was to examine the feasibility and acceptability of an adaptation of the Mindful Self-Compassion (MSC) program among adult cancer patients. A secondary aim was to examine pre-post-program changes in psychosocial wellbeing. Method The research design was a feasibility and acceptability study, with an examination of pre- to post-intervention changes in psychosocial measures. A study information pack was posted to 173 adult cancer patients 6 months-5 years post-diagnosis, with an invitation to attend an eight-week group-based adaptation of the MSC program. Results Thirty-two (19%) consented to the program, with 30 commencing. Twenty-seven completed the program (mean age: 62.93 years, SD 14.04; 17 [63%] female), attending a mean 6.93 (SD 1.11) group sessions. There were no significant differences in medico-demographic factors between program-completers and those who did not consent. However, there was a trend toward shorter time since diagnosis in the program-completers group. Program-completers rated the program highly regarding content, relevance to the concerns of cancer patients, and the likelihood of recommending the program to other cancer patients. Sixty-three percent perceived that their mental wellbeing had improved from pre- to post-program; none perceived a deterioration in mental wellbeing. Small-to-medium effects were observed for depressive symptoms, fear of cancer recurrence, stress, loneliness, body image satisfaction, mindfulness, and self-compassion. Significance of results. The MSC program appears feasible and acceptable to adults diagnosed with non-advanced cancer. The preliminary estimates of effect sizes in this sample suggest that participation in the program was associated with improvements in psychosocial wellbeing. Collectively, these findings suggest that there may be value in conducting an adequately powered randomized controlled trial to determine the efficacy of the MSC program in enhancing the psychosocial wellbeing of cancer patients.

is a bimonthly international journal of palliative medicine that focuses on the psychiatric, psychosocial, spiritual, existential, ethical, and philosophical aspects of palliative care.The journal's aim is to serve as an educational resource for practitioners from a wide array of disciplines engaged in the delivery of care to those with life threatening illnesses along the entire continuum of care from diagnosis to the end of life.

Introduction: 5'-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML). Response is associated with improved survival. However, only half of patients respond, and these responses are rarely durable. We recently reported that primary AZA resistance is associated with a molecular signature of cell cycle quiescence within bone marrow (BM) hematopoietic progenitor cells (Unnikrishnan et al, Cell Reports, 20:572-585 (2017)). As DNA incorporation of the deoxyribonucleic form of AZA (5-aza-2′-deoxycytidine, DAC) occurs during DNA replication, cell cycle quiescence is predicted to lead to less DAC in DNA and concomitantly less DNA demethylation. We recently developed a quantitative multi-parameter assay, AZA-MS (Unnikrishnan, Vo et al, Leukemia 32:900-910 (2018)), to measure the intracellular dynamics of AZA in patients. Using AZA-MS, we reported data supporting the predicted resistance model. CC486 is an oral formulation of AZA. A 28-day cycle of CC486 involves 21 continuous days (21/28) versus the standard 7/28 subcutaneous (SC) injection AZA scheme. Whether levels of in vivo DAC incorporation into DNA during a cycle of CC486 are comparable with that of SC AZA is unknown. AZA-MS provides us with a unique opportunity to empirically assess the in vivo intracellular dynamics of SC versus oral AZA. Study Design and Methods: To directly assess in vivo DAC incorporation and concomitant DNA demethylation with SC AZA and CC486 in the same patient, we initiated a phase II clinical trial (NCT03493646; Fig A). MDS (IPSS; intermediate-2 or high-risk), CMML (bone marrow [BM] blasts 10-29%) and AML (20-30%) patients were recruited for six cycles of SC AZA (75mg/m^2/day for 7/28 days) followed by six cycles of CC486 (100mg bid for 21/28 days in C7-C8 and 150mg bid for 21/28 in C9-C12). Clinical response was assessed at the end of C6 and C12 using International Working Group criteria. Clinical responders and non-responders to SC AZA at C6 received CC486 from C7 onwards. From each patient, 36 peripheral blood (PB) samples and five BM samples were collected over the study period. DNA, RNA and intracellular fractions were isolated from the PB MNCs, for intracellular DAC/AZA measurements by AZA-MS (primary endpoint; Fig A). BM MNCs were utilised for AZA-MS as well as flow cytometry-based cell cycle measurements (secondary endpoint). Results: 31 of 42 consented patients have commenced treatment since trial opening (Fig B-C). We applied the AZA-MS assay on the longitudinal PB and BM samples collected from the seven patients who had completed six months AZA and commenced CC486 as at 26th June 2019 (Fig D). DAC incorporation into DNA and DNA methylation levels were quantified within the same cells, in addition to measuring other parameters (Fig E). As represented by patient 61213-005 (Fig F) who had a complete response (CR) at cycle 6, after 7 days of injection AZA we observed robust incorporation of DAC within PB MNCs (left panel, Fig F) together with concomitant DNA demethylation (right panel, Fig F). DAC levels diminished upon cessation of AZA within a cycle, with corresponding increases in DNA methylation. There were quantitatively higher levels of DAC incorporated in DNA during SC AZA cycles versus CC486. The trend observed is also appreciated from 2.3x higher area under the curve (AUC) measurements in 61213-005 during the SC AZA cycle. DAC incorporation was higher at C9/10 (CC486 150mg bid 21/28) than at C7/8 (CC486 100mg bid 21/28) without appreciable changes in DNA demethylation. During SC AZA cycles, higher DAC levels (top panel, Fig G) and greater DNA methylation (lower panel, Fig G) were seen in the BM MNCs. In a non-responding patient at cycle 6 (61290-002, SD), we saw less DAC incorporation and DNA demethylation (Fig H). We also observed a positive correlation between baseline proportions of cycling BM cells (LIN-CD34+CD38+) and the amount of DAC incorporated in BM MNCs at C1 day 8 (Fig I). Conclusion: AZA-MS can be used to reliably measure in vivo DAC incorporation and concomitant DNA demethylation in PB MNCs and inform appropriate CC486 dosing. Figure Disclosures Unnikrishnan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fong:Astellas: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau. Roncolato:St. George Hospital: Employment. Enjeti:Roche: Honoraria, Speakers Bureau; Bayer and Sanofi: Honoraria, Speakers Bureau; Astellas: Consultancy; Novartis: Consultancy; Abbvie: Consultancy. Hertzberg:BMS: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Polizzotto:Janssen: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; ViiV: Research Funding. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Efficacy and safety of bortezomib-based consolidation following ASCT were investigated in newly diagnosed multiple myeloma patients from Australia, Korea, and China. Patients received three cycles of bortezomib-cyclophosphamide-dexamethasone induction followed by high-dose therapy/ASCT, then were randomized (1:1) to consolidation with TP (thalidomide 100 mg/d for <= 12 months/until disease progression; prednisolone 50 mg on alternate days indefinitely/until disease progression; n = 100) or VTP (subcutaneous bortezomib 1.3 mg/m(2) every 2 weeks for 32 weeks, plus TP; n = 103). The hypothesized difference in CR + VGPR rate (after <= 12 months consolidation therapy) was not met. The rate of CR + VGPR was numerically higher with VTP versus TP; however, this was not statistically significant (85.7% versus 77.1%; rate difference 8.6%; 95% confidence interval -2.3%-19.5%; p = .122). Secondary efficacy outcomes were similar between treatment arms. Addition of bortezomib to TP consolidation was associated with limited additional toxicity but did not significantly improve efficacy versus TP.

Abstract Background: Hypomethylating agents (HMA) used in higher-risk myelodysplastic syndromes (MDS) improve survival but HMA-failure has a poor prognosis. Abnormal bone marrow (BM) colony-forming units (CFUs) persist in treated MDS patients despite achievement of complete remission, suggesting persistent abnormal stem cell function. We aim to identify genetic biomarkers following treatment with Azacitidine ± Thalidomide or Lenalidomide that predict clinical outcomes in MDS. Methods: BM cells from patients enrolled in ALLG MDS3 and MDS4 clinical trials at baseline and after 4 cycles of treatment (C4) were grown in Methocult for 14 days. CFUs were pooled at baseline; C4 macroscopically normal and abnormal colonies were harvested separately. mRNA expression was quantified using the Nanostring nCounter PanCancer Pathways panel. Clinical outcomes analysed were: (1) clinical benefit at 12 months (haematological improvement or better as per IWG criteria) (2) best response achieved. Genes expressed above background level in ≥25% of samples were included for statistical analyses, resulting in 516 genes across 56 samples from 23 different patients. R limma package was used for differential expression analysis. Patients were weighted using limma's voomWithQualityWeights function. Moderated t-tests with empirical Bayes were done to identify differentially expressed genes. For testing between colonies, a log-fold-change cut-off of 0.5 was used with limma's treat function. P-values were adjusted for multiple hypothesis testing. Results: 98 genes exhibited significantly different expression (p <0.05) when comparing C4 normal to baseline colonies and 118 genes were differentially expressed between C4 normal to C4 abnormal colonies. Key results are summarised: (1) Within C4 normal colonies - RFC3 (p=0.04) and LTBP1 (p=0.04) were upregulated in patients with clinical benefit at 12 months (2) Within C4 abnormal colonies - FN1 (p=0.03) was upregulated in those failing to achieve at least a partial response (PR) (3) Comparing C4 normal colonies to baseline: a. The top 5 differentially expressed genes: MAPK12, PLAU, FGFR1, IL10 and FLNA (p<0.001) were downregulated in the C4 normal group; b. In addition, for patients with clinical benefit, MYD88 and PIK3R5 were downregulated (p<0.001) while; c. In patients who achieved at least a PR as best response, NFKB1, SYK and TGFBR2 were downregulated (p<0.001). d. Gene ontology analysis revealed upregulated genes involved in plasma membrane and cytokine production were over-represented at baseline while; e. KEGG pathway analysis showed upregulated genes involved in the cytokine receptor, Notch and NF-kB signalling pathways. Conclusion: We identified changes in gene expression following treatment in MDS that predict outcomes in response and clinical benefit. These genetic biomarkers require further validation and could define early markers of resistance for investigation of novel therapies. Citation Format: Lynette Chee, David Ritchie, Jessica Chung, Daniel Park, Mandy Ludford-Menting, Jane Ripley, Melita Kenealy, Rachel Koldej. Genetic biomarkers predict clinical response and survival in myelodysplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1548.

Standard treatment for higher risk myelodysplastic syndromes, chronic myelomonocytic leukemia and low blast acute myeloid leukemia is azacitidine. In single arm studies, adding lenalidomide had been suggested to improve outcomes. The ALLG MDS4 phase II trial randomized such patients to standard azacitidine or combination azacitidine (75mg/m2/d days 1 to 5) with lenalidomide (10mg days 1–21 of 28-day cycle from cycle 3) to assess clinical benefit (alive without progressive disease) at 12 months. A total of 160 patients were enrolled; median age 70.7 years (range 42.5-87.2), 31.3% female with 14% chronic myelomonocytic leukemia, 12% acute myeloid leukemia and 74% myelodysplastic syndromes. Adverse events were similar in both arms. There was excellent delivery of protocol therapy (median azacitidine cycles 11 both arms) with few dose reductions, delays or early cessations. At median follow up 33.1 months (range 0.7-59.5), the rate of clinical benefit at 12 months was 65% azacitidine arm and 54% lenalidomide+azacitidine arm (P=0.2). There was no difference in clinical benefit between each arm according to WHO diagnostic subgroup or IPSS-R. Overall response rate was 57% in azacitidine arm and 69% in lenalidomide+azacitidine (P=0.14). There was no difference in progression- free or overall survival between the arms (each P>0.12). Although the combination of lenalidomide and azacitidine was tolerable, there was no improvement in clinical benefit, response rates or overall survival in higher risk myelodysplastic syndrome, chronic myelomonocytic leukemia or low blast acute myeloid leukemia patients compared to treatment with azacitidine alone. This trial was registered at www.anzc-tr.org.au as ACTRN12610000271000.

Single agent azacitidine or immunomodulatory drugs are effective in myelodysplastic syndrome (MDS), with differing target mechanisms and toxicities. Objectives of this ALLG MDS3 study in clinically advanced MDS, AMML and low blast AML were to establish safety, response and quality of life of azacitidine and thalidomide. Patients received azacitidine (75mg/m2/d sc 7days every 28 days), and oral thalidomide up to 100mg/d for maximum 12months. Eighty patients registered; median age 68 years (range 42-82), 49% IPSS int2-high. With 36.5 months follow up, patients received median 9 cycles azacitidine, 6.1mths thalidomide. Nonhematologic toxicity grade 3+ in 85%, commonly infections. Overall response rate was 63%; 26% CR were unaffected by IPSS. Median response duration 26.3months; overall survival was 28.1months. This combination azacitidine and thalidomide in clinically advanced MDS, CMML and low-blast AML was tolerable without unexpected toxicity and encouraging responses support further investigation of combination approaches with hypomethylating agent and immunomodulatory drug.

We present a rare case of generalized crystal-storing histiocytosis (CSH) of IgM lambda (λ) subtype associated with Waldenstrom macroglobulinemia and responding to bortezomib-based combination chem...

13 THE ADDITION OF LENALIDOMIDE TO AZACITIDINE ACHIEVES HIGHER RESPONSES BUT NO IMPROVEMENT IN TWELVE MONTH CLINICAL BENEFIT OR PFS; MAIN ANALYSIS AUSTRALIAN ALLG MDS4 TRIAL M. Kenealy1, W. Benson2, W. Stevenson3, R. Eek4, D. Zantomio5, I. Cunningham6, D. Hiwase7, L. Cowan8, S. Vlachos8, D. Zannino8, J.F. Seymour9 1Haematology, Cabrini Hospital, Melbounre, Australia; 2Haematology, Westmead Hospital, Sydney, Australia; 3Haematology, Royal North Shore Hospital, Sydney, Australia; 4Haematology and Medical Oncology, Border Medical Oncology, Wodonga, Australia; 5Haematology, Austin Hospital, Melbourne, Australia; 6Haematology, Concord Hospital, Sydney, Australia; 7Haematology, Royal Adelaide Hospital, Adelaide, Australia; 8Biostatistics and Clinical Trials, Peter MacCallum Cancer Centre, Melbourne, Australia; 9Cancer Medicine, Peter MacCallum Cancer Centre, Melbourne, Australia