Abstract: Background: In a prior single-institution trial, CLAG-M resulted in high rates of measurable residual disease (MRD)-negative remissions for adults with previously untreated AML or high-grade myeloid neoplasms (HG-MN) with ≥10% blasts in bone marrow and/or blood (Halpern et al. Leukemia 2018;32:2352-62). The CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO), reduces relapse and improves survival when added to other intensive chemotherapy regimens, prompting this phase 1/2 study (NCT03531918) combining GO with CLAG-M as initial therapy for fit adults with newly diagnosed AML or HG-MN. Patients and Methods: Adults ≥18 years were eligible if they had LVEF ≥45%, creatinine ≤2.0 mg/dL, bilirubin ≤2.5-times upper limit of normal, and treatment-related mortality (TRM) score of ≤13.1, previously corresponding to & lt;13.1% risk of 28-day mortality (Walter et al. J Clin Oncol 2011;29:4417-23). Patients with APL, CML in blast crisis, other illness with expected survival & lt;1 year, or uncontrolled infection were excluded. Phase 1 tested two dose levels of GO in combination with CLAG-M: "GO1": 3 mg/m 2 on day 1 and "GO3": 3 mg/m 2 on days 1, 4, and 7, with all GO doses capped at 4.5 mg. CLAG-M consisted of cladribine 5 mg/m 2/day (days 1-5), cytarabine 2 g/m 2/day (days 1-5), G-CSF 300 or 480 μg/day (for weight & lt;76 kg vs. ≥76 kg; days 0-5), and mitoxantrone (18 mg/m 2/day; days 1-3). A second course of CLAG-M (without GO) was given if MRD-negative (MRDneg) complete remission (CR) or MRDneg CR with incomplete count recovery (CRi) was not achieved. MRD was measured by multiparameter flow cytometry (MFC) and cytogenetics. One cycle of CLAG (mitoxantrone omitted) followed by 2 cycles of high-dose cytarabine was allowed as post-remission therapy. Dose escalation to GO3 was permitted if fewer than 2 dose-limiting toxicities (DLTs) were observed among 6 GO1 patients. DLT was defined as: 1) any grade 3 non-hematologic toxicity lasting & gt;48 hours that resulted in & gt;7-day delay of the subsequent treatment cycle, with the exception of febrile neutropenia/infection; 2) any grade ≥4 non-hematologic toxicity, with the exception of febrile neutropenia/infection or constitutional symptoms. The primary efficacy outcome was 6-month event-free survival (EFS). Results: Eighteen patients (median age 66 [range: 28-77] years, median TRM score 3.92 [range: 0.14-10.3] ) were treated in phase 1. One of 6 GO1 patients experienced a DLT of grade 3 left ventricular systolic dysfunction. Among 12 GO3 patients, there were 3 DLTs: 1 each of grade 4 aminotransferase level increase, grade 3 posterior reversible encephalopathy syndrome, and grade 3 intracranial hemorrhage. This established GO3 as the recommended phase 2 dose. A total of 60 patients (median age; 65 [range: 19-80] years) with either AML (n=48) or HG-MN (n=12) and a median TRM score of 3.4 (range: 0.02-11.8) were treated at GO3 in either phase 1 or phase 2. By ELN 2017 criteria, 20 had favorable-, 13 intermediate-, and 27 adverse-risk disease. Forty-six (77%) achieved CR and 6 (10%) achieved CRi for a CR/CRi rate of 87% (95% CI: 75-94%). Forty-five CR/CRi patients were negative for MRD by both MFC and cytogenetics for an overall MRDneg CR/CRi rate of 75% (95% CI: 62-85%). Two patients had morphologic leukemia free state (MFLS), 2 underwent hematopoietic stem cell transplant (HCT) in aplasia not meeting MLFS criteria, and 4 had resistant disease. With median follow-up of 15 months, 6-month EFS, 12-month EFS, 6-month overall survival (OS) and 12-month OS were 73% (95% CI: 61-85%), 59% (48-73%), 90% (83-98%) and 75% (65-89%), respectively. For patients achieving CR after cycle 1 (n=45), median time to neutrophil count of 1000/µL and platelet count of 100,000/µL was 32 days (range: 22-51) and 31 days (range: 21-48), respectively. Besides infections and neutropenic fever, hypertension and hemorrhage were the most common grade ≥3 adverse events. There were no deaths within 8 weeks of study start. Of note, 1 patient had reversible early liver injury not meeting sinusoidal obstructive syndrome (SOS) criteria, and 1/25 (4%) of patients who underwent allogeneic HCT after study therapy was diagnosed with nonfatal post-HCT SOS. Conclusion: CLAG-M with fractionated-dose GO resulted in an MRDneg CR/CRi rate of 75% and a 6-month EFS rate of 73% in adults with newly diagnosed AML/HG-MN. Formal comparison of outcomes with institutional patients given CLAG-M alone is ongoing. Disclosures Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Percival: Glycomimetics: Research Funding; Cardiff Oncology: Research Funding; BMS/Celgene: Research Funding; Biosight: Research Funding; Abbvie: Research Funding; Pfizer: Research Funding; Nohla Therapeutics: Research Funding; Oscotec: Research Funding; Trillium: Research Funding. Scott: Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Halpern: Pfizer: Research Funding; Nohla Therapeutics: Research Funding; Jazz Pharmaceuticals: Research Funding; Imago Pharmaceuticals: Research Funding; Novartis: Research Funding; Bayer: Research Funding; Tolero Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Research Funding; Abbvie: Consultancy; Gilead: Research Funding; Agios: Consultancy. Oehler: BMS: Consultancy; OncLive: Honoraria; Takeda: Consultancy; Pfizer: Research Funding. Orozco: Actinium Pharmaceuticals: Other: Principal Investigator, SIERRA Trial, Actinium, Research Funding. Cassaday: Pepromene Bio: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding; Kite/Gilead: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Research Funding; Vanda Pharmaceuticals: Research Funding; Servier: Research Funding; Pfizer: Consultancy, Research Funding. Walter: Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Amphivena: Consultancy, Other: ownership interests; Agios: Consultancy; Selvita: Research Funding; Astellas: Consultancy; BMS: Consultancy; Genentech: Consultancy; Janssen: Consultancy; Kite: Consultancy; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding. OffLabel Disclosure: The label for cladribine does not include acute myeloid leukemia, however cladribine is frequently used to treat AML and related conditions. In this study cladribine is used in induction chemotherapy for the treated of AML and related disorders.

Abstract: Heme is a component of cytochromes, catalases, glutathione peroxidase, hydroxylases, and nitric oxide synthase, as well as myoglobin and hemoglobin, and thus is critical to all aerobic cells. It is also a transcriptional and translational initiator of globin synthesis. However, free heme is toxic so that cells must balance synthesis with use. We recently determined that the cell surface receptor for Feline Leukemia Virus, subgroup C, (FLVCR), which causes pure red cell aplasia in cats, is a heme export protein (Cell; 118:6, 2004). To understand its physiologic significance, we generated constitutive (Flvcr+/−) and inducible (Flvcrflox/flox;Mx-cre) FLVCR null mice by targeted deletion of exon 3, and confirmed this produced a nonfunctional protein by zinc mesoporphyrin (ZnMP) and 55Fe-heme export studies. Intercrossing Flvcr+/− mice produced 0 null animals among 109 progeny genotyped. FLVCR null embryos die either before E8.5 or at E14.5, suggesting two separate mechanisms for their demise. In situ hybridization studies in normal mouse embryos show highest Flvcr transcript levels in the ectoplacental cone which forms placenta (E8.5), the placenta (E9.5), and high levels in the fetal liver (E12.5). These data plus studies with Flvcrflox/flox x Meox2-Cre mice (Cre inactivates Flvcr in all embryonic tissue, but not extraembryonic visceral endoderm and placenta) suggest that the early lethality of null mice is due to impaired maternal-embryo heme-iron transport. At E14.5, residual null mice were small with deformed limb buds and craniofacial abnormalities resembling the congenital anomalies of Diamond-Blackfan anemia. Flow cytometric analyses of fetal liver cells from deleted animals with Ter119 and CD71 demonstrated a block in erythroid differentiation at the CFU-E/proerythroblast stage, implying that their death results from a stage-specific failure in definitive erythropoiesis. Heterozygous null animals (Flvcr+/−) were viable with normal hematologic parameters. These mice had 30.9% of normal Flvcr mRNA by q RT-PCR, however, they demonstrated normal protein by western blot analysis and normal heme export function by ZnMP studies. Thus, heterozygous null mice compensate at the protein level, confirming FLVCR is critical in vivo. We next utilized our inducible mutant line (Flvcrflox/flox;Mx-cre) to generate a viable null mouse for study. Flvcr deleted mice developed pure red cell aplasia characterized by macrocytic anemia (deleted: HCT 13.8±1.2, MCV 68.35± 1.6; control: HCT 50.24 ± 2.0, MCV 51.3±1.5), reticulocytopenia, and reactive thrombocytosis. Marrow and spleen had excess cells with a proerythroblast morphology and lacked maturing red cells. Flow cytometry confirmed maturation arrest at the CFU-E/proerythroblast stage. CFU-E from FLVCR null mice were undetectable in culture. Deleted mice also demonstrated iron loading of hepatocytes. FLVCR is highly expressed in multiple human tissues with high heme-flux by western blot analyses, including liver, placenta, duodenum, and uterus, in addition to the bone marrow. Our studies prove that FLVCR is required for the survival or differentiation of proerythroblasts (that stage where heme synthesis intensifies). These results, plus additional data not shown, suggest that FLVCR may also protect nonerythroid tissues with high free heme exposure (uterus, duodenum), may mediate maternal-embryo heme-iron transport, and may mediate the transport of heme from hepatocyte to bile which would imply a novel mechanism for iron export from the body.

Abstract: The North American Shwachman-Diamond Syndrome Registry (SDSR) opened in 2008 to improve our understanding of the natural history of Shwachman-Diamond Syndrome (SDS), improve medical outcomes, and facilitate research. The diagnosis of SDS was defined by either biallelic SBDS gene mutations or by the clinical combination of exocrine pancreatic dysfunction with bone marrow failure. Median age of study subjects is 11.2 years (range, 0.6-52.8). SBDS genetic reports were available for 168 subjects. Eighty-one had biallelic SBDS mutations, while 48 individuals lacked SBDS mutations. Ongoing characterization of SBDS mutation-negative individuals has identified subgroups of SDS individuals meeting clinical diagnostic criteria as outlined above, as well as a more heterogenous subgroup with features of SDS but in whom a clinical diagnosis could not be confirmed by classic diagnostic criteria. Amongst those with biallelic SBDS mutations, cytopenias were noted in all but 1 subject. Intermittent neutropenia was noted in 97% (n=70/72) and was the most frequent hematologic abnormality. Anemia was noted in 53% (n=39/73) and thrombocytopenia in 64% (n=48/75). Congenital anomalies were seen in 50% (n=39/78). The mutational spectrum of SBDS was explored. In 80 of 81 patients harboring biallelic SBDS mutations, the c.258+2 T 〉 C intron 2 splice donor mutation was found in at least one of the two mutant SBDS alleles. In these cases, the mutation spectrum of the second mutant SBDS allele included missense mutations, splice site mutations, and truncating mutations. The one patient lacking the intron 2 splice donor mutation had a c.183_184delTAinsCT mutation together with a c.523 C 〉 T mutation in SBDS confirmed to be in trans. This results in the combination of a truncating p.Lys62X mutation with a p.R175W missense mutation at a conserved residue predicted to be deleterious. This patient presented with neonatal severe aplastic anemia requiring platelet and red cell transfusions with neutrophil counts of 0-200 unresponsive to G-CSF. The marrow showed irregular islands of cartilage surrounded by osteoid consistent with a disorder of bone formation. A low level of SBDS protein is expressed by the c.258+2T 〉 C variant, so the absence of this hypomorphic allele may have contributed to this exceptionally severe phenotype. Bone marrow reports were available for 67 subjects with biallelic SBDSmutations. Marrow hypocellularity was noted in 79% (n=49/62). Mild morphologic marrow dysplasia was observed in 58% (n=35/60). Clonal abnormalities developed in 36%. The most common clonal abnormality was del20q in 16% (n=10/64). Isochromosome 7 was noted in 2% (n=1/64). Three individuals developed AML at ages 19.5, 38, and 39 years. Eleven (14%) have undergone hematopoietic stem cell transplantation (HSCT), 10 for MDS or AML and 1 for severe aplastic anemia. Given the frequency of del20q clones in SDS, we studied clinical features of the 10 SDSR subjects with del20q clones. Median age of this group was 17 years (range, 10.8-29.3). Congenital anomalies were noted in 80% (n=8/10). Frequency of cytopenias was similar to that of non-del20q subjects, with neutropenia, anemia and thrombocytopenia seen in 90%, 50%, and 80% respectively. Bone marrow pathology reports were available for 9 subjects. All were noted to have hypocellular marrows as well as mild marrow dysplasia. In many patients, the clone was persistent or grew over time, with longest duration of 14.6 years. Progression to MDS was reported in 3 subjects who initially had an isolated del20q clone. Two developed an additional loss of chromosome 7. Progressive marrow dysplasia and falling blood counts were seen in two subjects. All three were treated with HSCT at 5.4, 5.7 and 7 years. Deletion of 20q in SDS has been hypothesized to result in a milder hematologic phenotype due to compensatory deletion of eIF6. eIF6 binds to the nascent 60S ribosomal subunit and sterically inhibits joining of the 60S to the 40S ribosomal subunits. SBDS functions to facilitate release of eIF6 thus promoting assembly of the mature 80S ribosome. These data from the SDSR suggest that SDS patients with del20q clones remain at risk for clonal evolution. Higher patient numbers are needed to quantitate risk of MDS in SDS patients with del20q. The SDSR continues to expand and mature as a resource for biological and clinical studies in this rare disorder advancing our understanding of marrow failure and clonal evolution. Disclosures Davies: Novartis: Honoraria. Dale:Amgen: Consultancy, Honoraria, Research Funding.

Abstract: Shwachman-Diamond syndrome (SDS) is an inherited marrow failure syndrome associated with exocrine pancreatic dysfunction and an increased risk of myelodysplasia and leukemia. The majority of individuals with SDS carry biallelic SBDS gene mutations, however a subset of patients remain genetically undefined. The objective of this study was to compare the clinical characteristics of patients with and without SBDS mutations. To address these questions, we conducted a retrospective study of patients enrolled on the North American Shwachman-Diamond Syndrome Registry (SDSR). Clinical data from the SDSR were available for 55 individuals with biallelic SBDS mutations and 16 individuals who fulfilled clinical diagnostic criteria for SDS but lacked biallelic mutations in the SBDS gene. Study subject ages for SBDS mutation positive and negative cohorts span 2-52.4 and 2.8-21.4 years with median ages of 12.4 and 10.9 years respectively. Cytopenias were present for both SBDS mutation positive and negative cohorts, with neutropenia the most common event in 94% and 81% respectively. Bone marrow hypocellularity was reported in 91% of those with SBDS mutations and 69% of those without. Marrow dysplasia was reported in 65% of those with SBDS mutations and none of those without. Clonal abnormalities were present in 44% and 25% of those with and without SBDS mutations with median age of initial appearance at 9 years (0.8-45.1) and 7 years (1.2-14) respectively. Abnormalities included del7q and del20q in both groups as well as iso7q, trisomy 8 and others in the SBDS mutation positive group. Clonal abnormalities were all transient in the SBDS mutation negative cohort. One SBDS mutation positive individual developed AML. None of the SBDS mutation negative individuals developed malignancy or progressed to require HSCT thus far. Pancreatic dysfunction determined by low serum trypsinogen or pancreatic isoamylase was similar in both cohorts 79% vs 80%. However, only 27% (15/55) of SBDS mutation positive individuals reported requiring enzyme therapy with 33% (18/55) documenting failure to thrive, in contrast to 75% (12/16) of SBDS mutation negative individuals with 73% (11/15) having failure to thrive. A broad spectrum of congenital anomalies were reported in 55% and 56% of SBDS mutation positive and negative individuals respectively, with skeletal anomalies being the most common in both groups. Medical comorbidities commonly reported in both groups included eczema and endocrinopathies. Elevated liver transaminases were seen in 27% of SBDS mutation positive individuals but this was not seen in the SBDS mutation negative cohort. Conclusion: Patients with genetically undefined (SBDS mutation negative) SDS share clinical characteristics with SBDS mutation positive patients; however, the risk of leukemia in the genetically undefined patients remains unclear due to low patient numbers with short follow-up. Further studies of this young cohort are required to inform medical management and to advance our understanding of genetic etiology, mechanism, disease pathophysiology and treatment of these marrow failure disorders. Disclosures Dale: Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract: Gemtuzumab ozogamicin (GO) improves outcomes when added to intensive AML chemotherapy. A meta-analysis suggested the greatest benefit when combining fractionated doses of GO (GO3) with 7 + 3. To test whether GO3 can be safely used with high intensity chemotherapy, we conducted a phase 1/2 study of cladribine, high-dose cytarabine, G-CSF, and dose-escalated mitoxantrone (CLAG-M) in adults with newly diagnosed AML or other high-grade myeloid neoplasm (NCT03531918). Sixty-six patients with a median age of 65 (range: 19–80) years were enrolled. Cohorts of six and twelve patients were treated in phase 1 with one dose of GO or three doses of GO (GO3) at 3 mg/m2 per dose. Since a maximum-tolerated dose was not reached, the recommended phase 2 dose (RP2D) was declared to be GO3. At RP2D, 52/60 (87%) patients achieved a complete remission (CR)/CR with incomplete hematologic recovery (CRi), 45/52 (87%) without flow cytometric measurable residual disease (MRD). Eight-week mortality was 0%. Six- and twelve-month event-free survival (EFS) were 73% and 58%; among favorable-risk patients, these estimates were 100% and 95%. Compared to 186 medically matched adults treated with CLAG-M alone, CLAG-M/GO3 was associated with better survival in patients with favorable-risk disease (EFS: p = 0.007; OS: p = 0.030). These data indicate that CLAG-M/GO3 is safe and leads to superior outcomes than CLAG-M alone in favorable-risk AML/high-grade myeloid neoplasm.

Abstract: The ClinGen MM-VCEP has specified RUNX1-specific curation rules to address gene function, gene-specific domains, and phenotypic criteria. RUNX1-specific criteria resulted in a reduction in CONF and VUS variants by 33%, emphasizing the need for expert variant curation.

Abstract: The NCCN Guidelines for Myelodysplastic Syndromes (MDS) provide recommendations for the evaluation, diagnosis, and management of patients with MDS based on a review of clinical evidence that has led to important advances in treatment or has yielded new information on biologic factors that may have prognostic significance in MDS. The multidisciplinary panel of MDS experts meets on an annual basis to update the recommendations. These NCCN Guidelines Insights focus on some of the updates for the 2022 version of the NCCN Guidelines, which include treatment recommendations both for lower-risk and higher-risk MDS, emerging therapies, supportive care recommendations, and genetic familial high-risk assessment for hereditary myeloid malignancy predisposition syndromes.