ALBERT

Description: Antigen-directed immunotherapies for acute myeloid leukemia (AML), such as chimeric antigen receptor T cells (CAR-Ts) or antibody-drug conjugates (ADCs), are associated with severe toxicities due to the lack of unique targetable antigens that can distinguish leukemic cells from normal myeloid cells or myeloid progenitors. Here, we present an approach to treat AML by targeting the lineage-specific myeloid antigen CD33. Our approach combines CD33-targeted CAR-T cells, or the ADC Gemtuzumab Ozogamicin with the transplantation of hematopoietic stem cells that have been engineered to ablate CD33 expression using genomic engineering methods. We show highly efficient genetic ablation of CD33 antigen using CRISPR/Cas9 technology in human stem/progenitor cells (HSPC) and provide evidence that the deletion of CD33 in HSPC doesn’t impair their ability to engraft and to repopulate a functional multilineage hematopoietic system in vivo. Whole-genome sequencing and RNA sequencing analysis revealed no detectable off-target mutagenesis and no loss of functional p53 pathways. Using a human AML cell line (HL-60), we modeled a postremission marrow with minimal residual disease and showed that the transplantation of CD33-ablated HSPCs with CD33-targeted immunotherapy leads to leukemia clearance, without myelosuppression, as demonstrated by the engraftment and recovery of multilineage descendants of CD33-ablated HSPCs. Our study thus contributes to the advancement of targeted immunotherapy and could be replicated in other malignancies.

Description: Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (

Description: MDS is a disease of the elderly marked by dysplasia of hematopoetic cell lines resulting in cytopenias and AML progression. There is currently no treatment approved to alter the natural history of the disease. Aberrant methylation associated with cancer is a potential target for pharmacologic therapy, and DacogenTM (decitabine) for injection (SuperGen, Inc.), a cytosine analogue, indirectly depletes methylcytosine after incorporation into DNA with subsequent inactivation of DNA methyltransferases. We report the results of a Phase III trial of decitabine (DAC) vs. supportive care (Supp.Care) in adult MDS patients with IPSS Intermediate (Int)-1 (31%), Int-2 (44%) and high risk disease (26%). Secondary MDS (14%) and previously treated (27%) MDS patients were not excluded. Bone marrows were assessed by a blinded, independent pathologist. 170 patients (accrued from July 2001 through April 2003 at 23 centers) were randomized 1:1 to either Supp.Care or DAC (a 3 hr infusion of 15mg/m2/hr every 8 hrs on 3 consecutive days every 6 wks for up to 10 cycles. The groups were comparable for numerous risk factors, including time from diagnosis (median 29 weeks for DAC and 35 weeks for Supp.Care). Kaplan Meier (KM) curves for time to AML progression or death showed early and clinically meaningful separation (consistent with clinical benefit) in favor of DAC in all patients (intent to treat; ITT), Int-2/High Risk, and High Risk patients. Using the Cox proportional hazards model for the ITT population, the probability of progression to AML or death was 1.68-fold greater for Sup.Care than for DAC (p=0.023). Median Time to AML or Death (Days) MDS group (n) DAC Supp.Care p-value 1Protocol specified test. 2Preferred test for analysis of early separation of KM curves. n=89 n=81 Wilcoxon1,2 Log rank1 All Patients (170) 338 263 0.046 0.204 Int-2/High Risk (118) 334 189 0.005 0.040 High Risk (44) 260 79 0.001 0.006 Treatment naïve (124) 354 189 0.005 0.034 Figure Figure Investigator reported response rate by International Working Group criteria was 25% (10% CR, 15% PR) for DAC vs. 0% for Supp.Care (p〈 0.001), with responses equally distributed across baseline subgroups. Time to response was 100 days and median duration was estimated at 〉9 months. Responders (CR and PR) vs. non-responders had a median survival of 678 days vs. 406 days (p= 0.038 Wilcoxon). There were no treatment related deaths. As expected, grade 3–4 toxicity (including hematologic toxicity, febrile neutropenia) occurred in more DAC patients than in Supp.Care patients. Most patients tolerated treatment well. DAC appears a promising therapy for MDS with manageable toxicity. Final results of independently reviewed response rates and clinical benefit (transfusion independence; Quality of Life) will be presented.

Description: Background: Disease-specific measures of quality of life (QOL) can allow for improved assessment of disease-specific symptomatology and psychosocial factors. We recently reported on the development of the QUALMS, a 33-item QOL assessment tool for patients with myelodysplastic syndromes (MDS). We now report preliminary internal consistency and validity results from an international prospective study. Methods: From December of 2013 to July of 2014,an international cohort of MDS patients completed the QUALMS as well as the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) and the Functional Assessment of Cancer Therapy Anemia Scale (FACT-An). Eligible patients were 18 or older, and had to have biopsy-proven MDS; those who had undergone stem cell transplantation were excluded. Baseline medical record review was performed at the time of enrollment to document key clinical and laboratory data, including bone marrow pathology and treatments; a second QUALMS administration and medical record review is planned for each patient to assess responsiveness. Participants were recruited from MDS centers across the United States, Canada and Italy. Individual QUALMS items were scored on a 5-point scale ranging from “Never” to “Always”. Overall mean score was calculated by transforming the raw mean to a 100 point scale, with higher scores indicating better MDS-related QOL. Baseline QUALMS scores were compared to clinical factors such as hemoglobin (Hg) and transfusion dependence as well as with scores on the other QOL scales. Preliminary exploratory factor analysis was also undertaken to identify candidate subscales. Results: As of this analysis, 201 patients had enrolled. The mean age was 71.7 years; there were 55% men, and the IPSS distribution was 44% LO, 43% INT-1, 10% INT-2, 1% HI and 2% unclassifiable. The majority of patients (53%) were receiving an erythropoiesis-stimulating agent, hypomethylating agent or lenalidomide, and 29% of the overall cohort was transfusion-dependent. The geographical distribution was as follows: 20% from the Dana-Farber Cancer Institute (Boston, MA); 9% from Columbia University (New York, NY); 15% from the Moffitt Cancer Center (Tampa, FL); 25% from the Odette Cancer Center (Toronto, Canada); and 31% from two GIMEMA hospitals (Rome and Sardegna, Italy). Scores on the QUALMS ranged from 19 to 78; the mean and median scores were 49.6 and 50.0 respectively. The measure had excellent internal consistency (α=.91), and was moderately correlated with the EORTC QLQ-C30: correlations (r’s) with the global health status scale, functional scales (i.e., physical, role, emotional, cognitive and social) as well as fatigue, nausea and pain scales ranged from 0.33 to 0.63 (all p’s

Description: Background: Ring sideroblasts (RS) are erythroblasts with iron-loaded mitochondria, appears as blue granules when stained with Prussian blue, and are characteristic of myelodysplastic syndromes (MDS) subgroups refractory anemia with ring sideroblasts (RARS), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), and RARS with thrombocytosis (RARS-T) but are also infrequently found in other MDS groups. The etiology of RS remains unclear. Molecular genetic analysis of MDS patients with RS (MDS-RS) using exome and targeted sequencing identified mutations in several genes including SF3B1. While the RS phenotype was shown to be strongly associated with SF3B1 mutations, the association of SF3B1 mutations with better prognosis remained controversial. Also, there remained several patients who do not show any mutations in SF3B1 suggesting there are additional genes associated with RS phenotype. In addition to SF3B1 mutations, RARS-T patients show mutations in JAK2 (V617F and Exon 12), MPL and CALR genes, frequently mutated in Essential Thrombocytopenia (ET). But there remained a high number of patients who do not show any mutation in JAK2, MPL and CALR genes suggesting additional genes are associated with thrombocytosis. In order to gain further insight into the molecular genetics of MDS-RS patients and to elucidate their clinical significance we screened a large cohort of patients with RS (RARS/RCMD-RS) and a subgroup with thrombocytosis (RARS-T). Methods: This study is approved by the Institutional Review Board of Columbia University and informed consent was obtained from all the individuals participated in the study. Bone marrow mono nuclear cells were isolated from bone marrow aspirate and DNA was extracted using DNeasy Blood and Tissue kit. To screen for most frequent mutations in SF3B1 (exon 14 and 15), MPL (exon 10), JAK2 (exon 12), and CALR (exon 9) genes, primers were designed to amplify the exons and the exon-intron junctions using PCR and the amplified and purified PCR products were sequenced using Sanger sequencing on both strands. To screen for mutations V617F in JAK2, primers were designed to carry out allele specific PCR. Demographic and clinical data is collected from patient’s charts/reports. Statistical analysis including survival curve (Kaplan–Meier method) was performed using GraphPad Prism Software. Results: A total of 209 patients with RS phenotype were screened for SF3B1 mutations. Mutations in SF3B1 gene were detected in 62% (130/209) of RS patients, including 85% (23/27) of RARS-T and 59% (107/182) of RARS/RCMD-RS patients. Among all the SF3B1 mutations, K700E was the most frequent mutation (60%) followed by mutations at H662. Clinical significance of SF3B1 mutations on overall survival, using Kaplan-Meier method, showed SF3B1 mutations were associated with better prognosis (Fig 1). The median survival of patients with SF3B1 mutation is 110 months compared to those without mutations, 70 months (p value 〈 0.034). Studies to screen SF3B1 negative patients for additional mutations are being carried out using exome sequencing to identify genes associated with RS phenotype. In addition to SF3B1, RARS-T patients were also screened for JAK2 V617F and exon 12 mutations. JAK2 V617F mutations were detected in 11% of RARS-T patients and no mutations were found in JAK2 exon 12. Patients negative for JAK2 were screened for mutations in MPL and CALR genes. No mutations were found in MPL but 8% (2/23) of those negative for JAK2 were found to have CALR mutations. Both CALR mutations were frameshift mutations that alter the C-terminus thus abolishing the KDEL sequence required for CALR function. There are still 80% RARS-T patients in our cohort who do not show any mutations in JAK2, MPL, CALR genes. Conclusions: The association of SF3B1 mutation with prognosis is controversial with some studies suggesting a better prognosis while other reports no effect. Our study, using a large cohort of well-characterized RS patients provides an Independent verification of the observation that SF3B1 mutations are associated with better overall survival. There remains a large group of patients (40% in RARS/RCMD-RS groups) without SF3B1 mutation but with a RS phenotype suggesting yet other gene(s) is associated with RS phenotype. Similarly, there remain patients who do not show any mutations in JAK2, MPL and CALR suggesting other genes are involved in the etiology of thrombocytopenia. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.