Search

Patients with GATA2 deficiencyharbor de novo or inherited germline mutations in the GATA2 transcription factor gene, predisposing them to myeloid malignancies. There is considerable variation in disease progression, even among family members with the same mutation in GATA2. We investigated somatic mutations in 106 patients with GATA2 deficiency to identify acquired mutations that are associated with myeloid malignancies. Myelodysplastic syndrome (MDS) was the most common diagnosis (∼44%), followed by GATA2 bone marrow immunodeficiency disorder (G2BMID; ∼37%). Thirteen percent of the cohort had GATA2 mutations but displayed no disease manifestations. There were no correlations between age or sex with disease progression or survival. Cytogenetic analyses showed a high incidence of abnormalities (∼43%), notably trisomy 8 (∼23%) and monosomy 7 (∼12%), but the changes did not correlate with lower survival. Somatic mutations in ASXL1 and STAG2 were detected in ∼25% of patients, although the mutations were rarely concomitant. Mutations in DNMT3A were found in ∼10% of patients. These somatic mutations were found similarly in G2BMID and MDS, suggesting clonal hematopoiesis in early stages of disease, before the onset of MDS. ASXL1 mutations conferred a lower survival probability and were more prevalent in female patients. STAG2 mutations also conferred a lower survival probability, but did not show a statistically significant sex bias. There was a conspicuous absence of many commonly mutated genes associated with myeloid malignancies, including TET2, IDH1/2, and the splicing factor genes. Notably, somatic mutations in chromatin-related genes and cohesin genes characterized disease progression in GATA2 deficiency.

Objective Somatic mutations in UBA1 cause a newly defined syndrome known as VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic syndrome). More than 50% of patients currently identified as having VEXAS met diagnostic criteria for relapsing polychondritis (RP), but clinical features that characterize VEXAS within a cohort of patients with RP have not been defined. We undertook this study to define the prevalence of somatic mutations in UBA1 in patients with RP and to create an algorithm to identify patients with genetically confirmed VEXAS among those with RP. Methods Exome and targeted sequencing of UBA1 was performed in a prospective observational cohort of patients with RP. Clinical and immunologic characteristics of patients with RP were compared based on the presence or absence of UBA1 mutations. The random forest method was used to derive a clinical algorithm to identify patients with UBA1 mutations. Results Seven of 92 patients with RP (7.6%) had UBA1 mutations (referred to here as VEXAS-RP). Patients with VEXAS-RP were all male, were on average ≥45 years of age at disease onset, and commonly had fever, ear chondritis, skin involvement, deep vein thrombosis, and pulmonary infiltrates. No patient with VEXAS-RP had chondritis of the airways or costochondritis. Mortality was greater in VEXAS-RP than in RP (23% versus 4%; P = 0.029). Elevated acute-phase reactants and hematologic abnormalities (e.g., macrocytic anemia, thrombocytopenia, lymphopenia, multiple myeloma, myelodysplastic syndrome) were prevalent in VEXAS-RP. A decision tree algorithm based on male sex, a mean corpuscular volume >100 fl, and a platelet count

Somatic mutations in UBA1 involving hematopoietic stem and myeloid cells have been reported in patients with the newly defined VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. Here, we report clinical hematologic manifestations and unique bone marrow (BM) features in 16 patients with VEXAS. All patients were male and had a history of severe autoinflammatory and rheumatologic manifestations and a somatic UBA1 mutation (p.Met41). Ten patients had hematologic disorders: myelodysplastic syndrome (MDS; 6 of 16), multiple myeloma (2 of 16), monoclonal gammopathy of undetermined significance (2 of 16), and monoclonal B-cell lymphocytosis (2 of 16), and a few of those patients had 2 co-existing clonal processes. Although macrocytic anemia (100%) and lymphopenia (80%) were prevalent in all patients with VEXAS, thrombocytopenia and neutropenia were more common in patients with progression to MDS. All BMs in VEXAS patients had prominent cytoplasmic vacuoles in myeloid and erythroid precursors. In addition, most BMs were hypercellular with myeloid hyperplasia, erythroid hypoplasia, and varying degrees of dysplasia. All patients diagnosed with MDS were lower risk (low blast count, very good to intermediate cytogenetics) according to standard prognostic scoring with no known progression to leukemia. In addition, 10 of 16 patients had thrombotic events, including venous thromboembolism and arterial stroke. Although VEXAS presents symptomatically as a rheumatologic disease, morbidity and mortality are associated with progression to hematologic disease. Given the increased risk of developing MDS and multiple myeloma, surveillance for disease progression is important.

GATA2 is a transcription factor critical for hematopoiesis. Germline mutations in GATA binding protein 2 (GATA2) led to haploinsufficiency, severe cytopenias of multiple cell lineages, susceptibility to infections and strong propensity to develop myelodysplastic syndrome, and acute myeloid leukemia. Mechanisms of progressive cytopenias remain unclear. MicroRNA (miRNA) represents a unique mechanism of post-transcriptional gene regulation. In this study, miRNA profiles were evaluated and eight miRNAs were found to be differentially expressed (≥2-fold, P ≤ 0.05) in patient-derived cell lines (N = 13) in comparison to controls (N = 10). miR-9, miR-181a-2-3p, miR-181c, miR-181c-3p, miR-486-3p, and miR-582 showed increased expression, whereas miR-223 and miR-424-3p showed decreased expression. Cell death assays indicated that miR-181c potently induces cell death in lymphoid (Ly-8 and SP-53) and myeloid (HL-60) cell lines. miR-181c was predicted to target myeloid cell leukemia (MCL)1, which was confirmed by transfection assays, resulting in significantly reduced MCL1 mRNA and decreased live cell numbers. Bone marrow analysis of 34 GATA2 patients showed significantly decreased cellularity, CD34-positive cells, monocytes, dendritic cells, NK cells, B cells, and B cell precursors in comparison to healthy controls (N = 29; P

Genetic mutations associated with acute myeloid leukemia (AML) also occur in age-related clonal hematopoiesis, often in the same individual. This makes confident assignment of detected variants to malignancy challenging. The issue is particularly crucial for AML posttreatment measurable residual disease monitoring, where results can be discordant between genetic sequencing and flow cytometry. We show here that it is possible to distinguish AML from clonal hematopoiesis and to resolve the immunophenotypic identity of clonal architecture. To achieve this, we first design patient-specific DNA probes based on patient's whole-genome sequencing and then use them for patient-personalized single-cell DNA sequencing with simultaneous single-cell antibody–oligonucleotide sequencing. Examples illustrate AML arising from DNMT3A- and TET2-mutated clones as well as independently. The ability to personalize single-cell proteogenomic assessment for individual patients based on leukemia-specific genomic features has implications for ongoing AML precision medicine efforts. Significance: This study offers a proof of principle of patient-personalized customized single-cell proteogenomics in AML including whole-genome sequencing–defined structural variants, currently unmeasurable by commercial “off-the-shelf” panels. This approach allows for the definition of genetic and immunophenotype features for an individual patient that would be best suited for measurable residual disease tracking.

Purpose: To investigate the toxicity profile and establish an optimal dosing schedule of zotiraciclib with temozolomide in patients with recurrent high-grade astrocytoma. Patients and Methods: This two-stage phase I trial determined the MTD of zotiraciclib combined with either dose-dense (Arm1) or metronomic (Arm2) temozolomide using a Bayesian Optimal Interval design; then a randomized cohort expansion compared the progression-free survival rate at 4 months (PFS4) of the two arms for an efficient determination of a temozolomide schedule to combine with zotiraciclib at MTD. Pharmacokinetic and pharmacogenomic profiling were included. Patient-reported outcome was evaluated by longitudinal symptom burden. Results: Fifty-three patients were enrolled. Dose-limiting toxicities were neutropenia, diarrhea, elevated liver enzymes, and fatigue. MTD of zotiraciclib was 250 mg in both arms and thus selected for the cohort expansion. Dose-dense temozolomide plus zotiraciclib (PSF4 40%) compared favorably with metronomic temozolomide (PFS4 25%). Symptom burden worsened at cycle 2 but stabilized by cycle 4 in both arms. A significant decrease in absolute neutrophil count and neutrophil reactive oxygen species production occurred 12–24 hours after an oral dose of zotiraciclib but both recovered by 72 hours. Pharmacokinetic/pharmacogenomic analyses revealed that the CYP1A2_5347T>C (rs2470890) polymorphism was associated with higher AUCinf value. Conclusions: Zotiraciclib combined with temozolomide is safe in patients with recurrent high-grade astrocytomas. Zotiraciclib-induced neutropenia can be profound but mostly transient, warranting close monitoring rather than treatment discontinuation. Once validated, polymorphisms predicting drug metabolism may allow personalized dosing of zotiraciclib.

There is no standard or widely effective treatment of patients with moderate aplastic anemia (MAA) or hypo-productive uni-lineage cytopenias (UC). Eltrombopag (EPAG), a small molecule thrombopoietin mimetic, has previously been shown to result in durable multi-lineage hematologic responses with low toxicity in patients with refractory severe aplastic anemia (SAA). Its safety and efficacy in MAA are unknown. This prospective phase 2 study enrolled previously untreated and treated MAA and UC patients with clinically relevant cytopenias. EPAG was administered at doses escalating from 50 to 300 mg/d. Hematologic responses were assessed at 16 to 20 weeks. Responding patients were continued on EPAG until reaching defined robust or stable blood counts. EPAG was reinstituted for relapse. Thirty-four patients were enrolled between 2012 and 2017, including 31 with MAA and 3 with UC. Seventeen patients responded in at least 1 eligible lineage by the primary end point. A striking improvement in anemia was observed in a patient with Diamond-Blackfan anemia. EPAG was well tolerated, and it was discontinued for robust or stable blood counts in 12 of 17 patients after a median of 8 months. A majority required re-initiation of EPAG for declining counts, and all regained response. Two of 34 patients developed non–chromosome 7 bone marrow cytogenetic abnormalities while taking EPAG, without dysplasia or increased blasts. Somatic mutation allele frequencies in cancer genes did not increase overall on EPAG. EPAG is a well-tolerated oral treatment of cytopenias in patients with MAA/UC. This trial was registered at www.clinicaltrials.gov as #NCT01328587.

We previously reported an increased risk for monoclonal gammopathy of undetermined significance (MGUS), a precursor of multiple myeloma (MM), among Vietnam veterans exposed to Agent Orange and its contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dysregulated expression of certain microRNAs (miRNAs) has been demonstrated in MGUS and MM. Given the important role of miRNAs in cellular homeostasis, we sought to determine if there was an association between serum levels of selected miRNAs and TCDD in 47 MGUS cases identified in our previous study, using serum specimens and exposure data archived by the Air Force Health Study (AFHS). We measured 13 miRNA levels (let-7a, let-7i, miR-16, miR-20a, miR-21, miR-34a, miR-106b, miR-146a, miR-181a, miR-192, miR-205, miR-335, and miR-361) in serum stored during the 2002 AFHS follow-up and evaluated their relationship to lipid-adjusted serum TCDD levels in 1987 determined by the AFHS. miR-34a showed the strongest association with TCDD (coefficient 2.184, p-value 0.02); after age-adjustment, this positive association was more pronounced (coefficient 2.294, p-value 0.008). In contrast, the other 12 miRNAs had absolute values of age-adjusted coefficient estimates below 1.16 and p-values greater than 0.18. The observed strong positive relationship between high body burdens of TCDD and miR-34a, a tumor suppressor regulated by p53, in this MGUS population warrants clarification of the TCDD-miR-34a relationship and its role in the pathogenesis of MGUS and risk for MM.

Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3), also called SH3-containing lymphocyte protein (SLY1), is a putative adaptor protein that is postulated to play an important role in the organization of signaling complexes and propagation of signal transduction cascades in lymphocytes. The SASH3 gene is located on the X-chromosome. Here, we identified 3 novel SASH3 deleterious variants in 4 unrelated male patients with a history of combined immunodeficiency and immune dysregulation that manifested as recurrent sinopulmonary, cutaneous, and mucosal infections and refractory autoimmune cytopenias. Patients exhibited CD4+ T-cell lymphopenia, decreased T-cell proliferation, cell cycle progression, and increased T-cell apoptosis in response to mitogens. In vitro T-cell differentiation of CD34+ cells and molecular signatures of rearrangements at the T-cell receptor α (TRA) locus were indicative of impaired thymocyte survival. These patients also manifested neutropenia and B-cell and natural killer (NK)-cell lymphopenia. Lentivirus-mediated transfer of the SASH3 complementary DNA–corrected protein expression, in vitro proliferation, and signaling in SASH3-deficient Jurkat and patient-derived T cells. These findings define a new type of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in mice with Sly1–/– and Sly1Δ/Δ mutations, highlighting an important role of SASH3 in human lymphocyte function and survival.

Importance High-risk smoldering myeloma has a 5-year risk of progression to symptomatic multiple myeloma of approximately 75%. Treatment with lenalidomide decreases the risk of progression; however, novel triplet regimens are superior, and earlier disease may be more treatment sensitive. Objective To evaluate the use of carfilzomib, lenalidomide, and dexamethasone (KRd) with lenalidomide maintenance therapy as early intervention in high-risk smoldering myeloma and to determine the rates of minimal residual disease (MRD)–negative complete response (CR). Design, Setting, and Participants In this single-arm, single-center, phase 2 nonrandomized controlled trial, responses were evaluated at every cycle during KRd treatment and every 3 cycles subsequently. Bone marrow biopsies and imaging were performed by cycle 8 and then annually. The study enrolled patients from May 29, 2012, to July 23, 2020, at the National Institutes of Health Clinical Center, a highly specialized tertiary cancer center. Patient key eligibility criteria included a diagnosis of high-risk smoldering myeloma based on the Mayo Clinic, Spanish, and/or Rajkumar, Mateos, and Landgren criteria. Interventions Patients received eight 4-week cycles of intravenous carfilzomib 36 mg/m2(first 2 doses, 20 mg/m2), dexamethasone (20 mg, cycles 1-4; 10 mg, cycles 5-8 twice weekly), and lenalidomide 25 mg (days 1-21) followed by twenty-four 28-day cycles of maintenance lenalidomide 10 mg (days 1-21). Stem cell harvest and storage were optional. Main Outcomes and Measures The primary outcome was the MRD-negative CR rate. Key secondary outcomes included duration of MRD-negative CR and progression to multiple myeloma. Results A total of 54 patients (median age, 59 years [range, 40-79 years]; 30 men [55.6%]; and 2 Asian [3.7%], 15 Black [27.8%], 1 Hispanic [1.9%], and 36 White [66.7%] patients) were enrolled, with a median potential follow-up time of 31.9 months (range, 6.7-102.9 months). The MRD-negative CR rate was 70.4% (95% CI, 56.4%-82.0%), with a median sustained duration of 5.5 years (95% CI, 3.7 years to not estimable). The 8-year probability of being free from progression to multiple myeloma was 91.2% (95% CI, 67.4%-97.9%), and no deaths occurred. Nonhematologic grade 3 adverse events occurred in 21 patients (38.9%) and included thromboembolism, rash, and lung infection, with no grade 4 events. Conclusions and Relevance Results of this phase 2 nonrandomized controlled trial suggest that treatment of high-risk smoldering myeloma with novel triplet regimens, such as KRd and lenalidomide maintenance therapy, may alter the natural history of smoldering myeloma by significantly delaying development of end-organ disease. Randomized clinical trials are needed to confirm this favorable benefit-to-risk profile. Trial Registration ClinicalTrials.gov Identifier:NCT01572480

GATA2 deficiency was described in 2011, and shortly thereafter allogeneic hematopoietic stem cell transplantation (HSCT) was shown to reverse the hematologic disease phenotype. However, there remain major unanswered questions regarding the type of conditioning regimen, type of donors, and graft-versus-host disease (GVHD) prophylaxis. We report 59 patients with GATA2 mutations undergoing HSCT at National Institutes of Health between 2013 and 2020. Primary endpoints were engraftment, reverse of the clinical phenotype, secondary endpoints were overall survival (OS), event-free survival (EFS), and the incidence of acute and chronic GVHD. The OS and EFS at 4 years were 85·1% and 82·1% respectively. Ninety-six percent of surviving patients had reversal of the hematologic disease phenotype by one-year post-transplant. Incidence of grade III-IV aGVHD in matched related donor (MRD) and matched unrelated donor recipients (URD) patients receiving Tacrolimus/Methotrexate for GVHD prophylaxis was 32%. In contrast, in the MRD and URD who received post-transplant cyclophosphamide (PT/Cy), no patient developed grade III-IV aGVHD. Six percent of haploidentical related donor (HRD) recipients developed grade III-IV aGVHD. In summary, a busulfan-based HSCT regimen in GATA2 deficiency reverses the hematologic disease phenotype, and the use of PT/Cy reduced the risk of both aGVHD and cGVHD.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a newly characterized syndrome with underlying somatic UBA1 mutations in myeloid cells linking hematologic disease with autoinflammatory rheumatologic disorders. Hematologic abnormalities, particularly peripheral blood cytopenia(s) may prompt bone marrow evaluation in patients with known or unrecognized VEXAS syndrome. This review highlights key findings and diagnostic considerations encountered during bone marrow examination in patients with this disorder. Frequently reported hematologic changes include macrocytic anemia, cytoplasmic vacuoles in myeloid and erythroid precursors, marrow hypercellularity, and varying degrees of dysplasia. Myelodysplastic syndrome and plasma cell neoplasms have been diagnosed in patients with VEXAS syndrome. Macrophage activation syndrome and/or hemophagocytic lymphohistiocytosis and monoclonal B-cell lymphocytosis have also been reported. The bone marrow is a target organ in VEXAS syndrome. Heightened awareness of the bone marrow features and hematologic complications may aid in identifying individuals with VEXAS who may benefit from increased disease surveillance or alternative therapeutic strategies.

Background: HR-SMM is a plasma cell disorder with a 5-year risk of progression to symptomatic multiple myeloma (MM) of ~75% without therapy. Early treatment with novel therapies, may decrease the risk of progression and prolong survival as evidenced by studies (Quiredex and ECOG E3A06) comparing lenalidomide ± dexamethasone to observation. Randomized studies in MM have demonstrated that triplet are superior to doublet regimens and whole exome sequencing in HR-SMM suggests a more treatment-sensitive biology. Together, these support our initial pilot study (Korde et al, JAMA Onc 2015) in using effective therapy with KRd-R as early intervention. Given the favorable initial results of our pilot in terms of minimal residual disease (MRD) negativity rates, we designed a single-arm, phase 2 study with the primary objective of determining the rate of MRD negative complete remissions. Herein, we show that rates of MRD negativity are high and they are sustained with the use of KRd-R. Methods: Patients with HR-SMM (Mayo Clinic or PETHEMA models) received eight 28-day cycles of carfilzomib 20/36 mg/m2 IV days 1, 2, 8, 9, 15, 16; lenalidomide 25 mg PO days 1-21, and dexamethasone 20/10 mg days 1, 2, 8, 9, 15, 16, 22, 23. Transplant eligible patients underwent stem cell collection after 4 cycles of KRd and then resumed treatment without an intent for early high-dose melphalan with stem cell support (HDM-ASCT). After 8 cycles of KRd, patients transitioned to receive maintenance therapy with lenalidomide 10 mg PO days 1-21 for 24 additional cycles. Prophylactic antiviral and anticoagulation was mandated for all. MM laboratory evaluations were performed at the start of every cycle during KRd and every 3 cycles during -R and every 3 months during indefinite follow up. Bone marrow biopsies and PET/CTs were performed by the end of cycle 8 induction and then annually indefinitely. The primary objective was to determine the rate of MRD negative remissions by validated multi-color flow cytometry (≤10-5 sensitivity). Key secondary objectives included progression free survival (PFS) to symptomatic myeloma and biochemical progression per IMWG, duration of MRD negativity, overall response rate (ORR), and duration of response. Results: As of 7/15/2020, 52 patients meeting eligibility criteria were enrolled and their demographics and disease characteristics are shown in Table 1. With a median potential follow up of 27.3 months, the primary objective of MRD negative CR rate was 70.2% and the MRD negative ≥VGPR rate was 80.9%, Table 2. The median duration of MRD negativity was 5.5 years with 2 and 5 -year rates of 78% and 55%, respectively. The median time to progression to MM and time to biochemical progression was not reached with 90-month rates of 90% and 78%, respectively - 2 patients progressed to symptomatic MM and 4 patients biochemically. The ORR was 100% and 78% achieved a best response of stringent CR. No deaths occurred. All grade and Grade 3-4 treatment-related adverse events occurred in 90% and 33% of patients, respectively. Grade 3-4 toxicities occurring in >1 patient included neutropenia (19%), lymphopenia (13%), thromboembolism (12%), anemia (8%), rash (8%), leukopenia (6%), lung infection (6%), ALT increase (4%), diarrhea (4%), hyperglycemia (4%), hypophosphatemia (4%), and thrombocytopenia (4%). Other Grade 3-4 adverse events of interest occurring in ≤1 patient included atrial fibrillation, creatinine increase, dyspnea, febrile neutropenia, heart failure, hypertension, and neoplasm. Treatment discontinuation occurred in 4 patients; 3 due to toxicity and 1 due to patient withdrawal. Conclusions: Treatment of HR-SMM with KRd-R to prevent symptomatic MM resulted in an MRD negative CR rate of 70% with a median duration of 5.5 years. At the 5-year landmark, only 10% of patients developed MM which is favorable compared to historical rates with no treatment of ~75%. Alternative approaches using monotherapy lenalidomide (Lonial et al, JCO 2019) resulted in no CRs and a 5-year progression rate of 22% with a treatment discontinuation rate of 51% compared to 7% in our study. More aggressive approaches include GEM-CESAR (Mateos et al, ASH 2019) incorporating HDM-ASCT with KRd-R. Importantly, the rate of MRD negativity reported in GEM-CESAR was 56% compared to 70% in this study. Overall, the benefit compared to risk with KRd in SMM is very favorable. Future randomized trials will be needed to lock in this conclusion. Disclosures Korde: Amgen: Research Funding; Astra Zeneca: Other: Advisory Board. Mailankody:Physician Education Resource: Honoraria; PleXus Communications: Honoraria; Takeda Oncology: Research Funding; Janssen Oncology: Research Funding; Allogene Therapeutics: Research Funding; Juno Therapeutics, a Bristol-Myers Squibb Company: Research Funding. Manasanch:Quest Diagnostics: Research Funding; Sanofi: Research Funding; JW Pharma: Research Funding; Merck: Research Funding; Takeda: Honoraria; GSK: Honoraria; Sanofi: Honoraria; BMS: Honoraria; Adaptive Biotechnologies: Honoraria; Novartis: Research Funding. Bhutani:BMS: Other: Clinical trial funding to institute, Speakers Bureau; Amgen: Speakers Bureau; MedImmune: Other: Clinical Trial Funding to Institute; Janssen: Other: Clinical Trial Funding to Institute; Prothena: Other: Clinical Trial Funding to Institute; Sanofi Genzyme: Consultancy; Takeda: Other: Clinical trial funding to institute, Speakers Bureau. Landgren:Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Merck: Other; Karyopharma: Research Funding; Merck: Other; Pfizer: Consultancy, Honoraria; Juno: Consultancy, Honoraria; Cellectis: Consultancy, Honoraria; Glenmark: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Juno: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Karyopharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Glenmark: Consultancy, Honoraria, Research Funding; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Janssen: Consultancy, Honoraria, Other: Independent Data Monitoring Committees for clinical trials, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Binding Site: Consultancy, Honoraria; Takeda: Other: Independent Data Monitoring Committees for clinical trials, Research Funding; BMS: Consultancy, Honoraria; Cellectis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Binding Site: Consultancy, Honoraria; Adaptive: Consultancy, Honoraria; Seattle Genetics: Research Funding. OffLabel Disclosure: carfilzomib, lenalidomide, and dexamethasone are not approved for smoldering myeloma.

Distinguishing constitutional from immune bone marrow failure (BMF) has important clinical implications. However, the diagnosis is not always straightforward, and immune aplastic anemia, the commonest BMF, is a diagnosis of exclusion. In this review, we discuss a general approach to the evaluation of BMF, focusing on clinical presentations particular to immune and various constitutional disorders as well as the interpretation of bone marrow histology, flow cytometry, and karyotyping. Additionally, we examine the role of specialized testing in both immune and inherited BMF, and discuss genetic testing, both its role in patient evaluation and interpretation of results.

Key Points Leukemic blasts of a female carrier of an ATRX germline mutation have persistently skewed inactivation of the X chromosome. Germline mutation in leukemia needs to be interpreted with caution because it is not always pathologic.

ObjectivesThe 2017 Workshop of the Society for Hematopathology/European Association for Haematopathology aimed to review clinical cases with germline predisposition to hematolymphoid neoplasms.MethodsThe Workshop Panel reviewed 51 cases with germline mutations and rendered consensus diagnoses. Of these, six cases were presented at the meeting by the submitting pathologists.ResultsThe cases submitted to the session covering germline predisposition included 16 cases with germline GATA2 mutations, 10 cases with germline RUNX1 mutations, two cases with germline CEBPA mutations, two germline TP53 mutations, and one case of germline DDX41 mutation. The most common diagnoses were acute myeloid leukemia (15 cases) and myelodysplastic syndrome (MDS, 14 cases).ConclusionsThe majority of the submitted neoplasms occurring in patients with germline predisposition were myeloid neoplasms with germline mutations in GATA2 and RUNX1. The presence of a germline predisposition mutation is not sufficient for a diagnosis of a neoplasm until the appearance of standard diagnostic features of a hematolymphoid malignancy manifest: in general, the diagnostic criteria for neoplasms associated with germline predisposition disorders are the same as those for sporadic cases.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired condition in which, due to a mutation of the phosphatidylinositol glycan class A gene, hematopoietic cells lack proteins that are normally anchored to the cell surface by glycosylphosphatidylinositol (GPI). Thus, PNH cells show poor expression of surface proteins, such as CD55 and CD59, and dim or absent binding of fluorescently labeled modified aerolysin (FLAER). In clinical diagnostic laboratories, the detection and quantitation of PNH is currently performed by flow cytometry (FC) analysis of peripheral blood (PB) samples. Although PB remains the preferred source of cells for PNH detection, we and other authors have shown that a careful FC analysis of bone marrow (BM) aspirates can provide results for PNH detection and quantitation equivalent to those obtained with PB. Here, we review studies delineating the expression of GPI-anchored proteins and FLAER binding in normal BM cells, and summarize published findings demonstrating the feasibility of identifying and quantitating PNH cells in BM using FLAER as well as antibodies against GPI-anchored proteins. Detection of PNH cells in BM should be useful in patients with unsuspected PNH or with severe cytopenia in whom PB FC analysis may be challenging.

Bone marrow failure and related syndromes are rare disorders characterized by ineffective bone marrow hematopoiesis and peripheral cytopenias. Although many are associated with characteristic clinical features, recent advances have shown a more complicated picture with a spectrum of broad and overlapping phenotypes and imperfect genotype-phenotype correlations. Distinguishing acquired from inherited forms of marrow failure can be challenging, but is of crucial importance given differences in the risk of disease progression to myelodysplastic syndrome, acute myeloid leukemia, and other malignancies, as well as the potential to genetically screen relatives and select the appropriate donor if hematopoietic stem cell transplantation becomes necessary. Flow cytometry patterns in combination with morphology, cytogenetics, and history can help differentiate several diagnostic marrow failure and/or insufficiency entities and guide genetic testing. Herein we review several overlapping acquired marrow failure entities including aplastic anemia, hypoplastic myelodysplasia, and large granular lymphocyte disorders; and several bone marrow disorders with germline predisposition, including GATA2 deficiency, CTLA4 haploinsufficiency, dyskeratosis congenita and/or telomeropathies, Fanconi anemia, Shwachman-Diamond syndrome, congenital amegakaryocytic thrombocytopenia, severe congenital neutropenia, and Diamond-Blackfan anemia with a focus on advances related to pathophysiology, diagnosis, and management.

TO THE EDITOR: GATA2 encodes a zinc-finger transcription factor that is required for proliferation and survival of hematopoietic stem cells.[1][1] First described in 2011, germline heterozygous mutations in GATA2 lead to haploinsufficiency[2][2] and are associated with bone marrow failure

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome (MDS)/myeloproliferative disorder most commonly seen in the elderly. We describe an adolescent with monosomy 7 CMML presenting as central diabetes insipidus (DI), who was treated with venetoclax and decitabine as a bridge to hematopoietic stem cell transplantation (HSCT). Central DI is a rare manifestation of monosomy 7-associated MDS including CMML, itself a rare manifestation of GATA2 deficiency, particularly in children. Venetoclax/decitabine was effective for treatment of CMML as a bridge to HSCT.

Genetic mutations associated with acute myeloid leukemia can also be detected in age-related clonal hematopoiesis, making confident assignment of detected variants to malignancy challenging particularly in the post-treatment setting. This has implications for measurable residual disease monitoring, where the relationship between sequencing and flow cytometry is also imperfect. We show, using whole-genome-sequencing informed patient-personalized single-cell DNA and antibody-oligonucleotide sequencing, that it is possible to resolve immunophenotypic identity of clonal architecture.

Immunotherapeutic strategies targeting B-cell acute lymphoblastic leukemia (B-ALL) effectively induce remission; however, disease recurrence remains a challenge. Due to the potential for antigen loss, antigen diminution, lineage switch or development of a secondary or treatment-related malignancy, the phenotype and manifestation of subsequent leukemia may be elusive. We report on two patients with multiply relapsed/refractory B-ALL who, following chimeric antigen receptor T-cell therapy, developed myeloid malignancies. In the first case, a myeloid sarcoma developed in a patient with a history of myelodysplastic syndrome. In the second case, two distinct events occurred. The first event represented a donor-derived myelodysplastic syndrome with monosomy 7 in a patient with a prior hematopoietic stem cell transplantation. This patient went on to present with lineage switch of her original B-ALL to ambiguous lineage T/myeloid acute leukemia. With the rapidly evolving field of novel immunotherapeutic strategies, evaluation of relapse and/or subsequent neoplasms is becoming increasingly more complex. By virtue of these uniquely complex cases, we provide a framework for the evaluation of relapse or evolution of a subsequent malignancy following antigen-targeted immunotherapy.

The powerful “graft versus leukemia” effect thought partly responsible for the therapeutic effect of allogeneic hematopoietic cell transplantation in acute myeloid leukemia (AML) provides rationale for investigation of immune-based therapies in this high-risk blood cancer. There is considerable pre-clinical evidence for potential synergy between PD-1 immune checkpoint blockade and the hypomethylating agents already commonly used for this disease. We report here the results of 17-H-0026 (PD-AML, NCT02996474), an investigator sponsored, single-institution, single-arm open-label ten-subject pilot study to test the feasibility of the first-in-human combination of pembrolizumab and decitabine in adult patients with refractory or relapsed AML (R-AML). In this cohort of previously treated patients, this novel combination of anti-PD-1 and hypomethylating therapy was feasible and associated with a best response of stable disease or better in 6 of 10 patients. Considerable immunological changes were identified using TCRβ sequencing as well as single-cell immunophenotypic and RNA expression analyses on sorted CD3+ T cells in patients who developed immune-related adverse events (irAEs) during treatment. Clonal T cell expansions occurred at irAE onset; single-cell sequencing demonstrated that these expanded clones were predominately CD8+ effector memory T cells with high cell surface PD-1 expression and transcriptional profiles indicative of activation and cytotoxicity. In contrast, no such distinctive immune changes were detectable in those experiencing a measurable anti-leukemic response during treatment. Addition of pembrolizumab to ten-day decitabine therapy was clinically feasible in patients with R-AML, with immunological changes from PD-1 blockade observed in patients experiencing irAEs.One Sentence SummaryAML patients receiving a novel combination of a PD-1 immune checkpoint inhibitor with a hypomethylating agent demonstrated clear evidence of induced immunological responses in those developing autoimmune toxicity during treatment but not in those demonstrating an anti-leukemic response.

Simultaneous occurrence of hairy cell leukemia (HCL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (termed CLL) is very rare. Clinical characteristics, pathology and management of these cases have not been well described. We present six patients with CLL and HCL or HCL variant (HCL-v). Of six patients, three were initially diagnosed with CLL and later developed concurrent HCL. Two patients had concurrent HCL or HCL-v and CLL at initial diagnosis. One had HCL first, followed by concurrent CLL. Polymerase chain reaction analysis demonstrated B-cell clonality in all cases, with two distinct clonal populations in four cases, and three clonal populations in one case. Five patients were treated with a combination of a purine analog such as fludarabine, cladribine, and pentostastin with either rituximab or ibrutinib, while one received dabrefenib and trametinib. All patients achieved a durable response to either CLL or HCL-directed therapy with reduction or ablation of coexisting B-cell clones.

BACKGROUND. Undifferentiated systemic autoinflammatory diseases (USAIDs) present diagnostic and therapeutic challenges. Chronic interferon (IFN) signaling and cytokine dysregulation may identify diseases with available targeted treatments. METHODS. Sixty-six consecutively referred USAID patients underwent underwent screening for the presence of an interferon signature using a standardized type-I IFN-response-gene score (IRG-S), cytokine profiling, and genetic evaluation by next-generation sequencing. RESULTS. Thirty-six USAID patients (55%) had elevated IRG-S. Neutrophilic panniculitis (40% vs. 0%), basal ganglia calcifications (46% vs. 0%), interstitial lung disease (47% vs. 5%), and myositis (60% vs. 10%) were more prevalent in patients with elevated IRG-S. Moderate IRG-S elevation and highly elevated serum IL-18 distinguished 8 patients with pulmonary alveolar proteinosis (PAP) and recurrent macrophage activation syndrome (MAS). Among patients with panniculitis and progressive cytopenias, 2 patients were compound heterozygous for potentially novel LRBA mutations, 4 patients harbored potentially novel splice variants in IKBKG (which encodes NF-κB essential modulator [NEMO]), and 6 patients had de novo frameshift mutations in SAMD9L. Of additional 12 patients with elevated IRG-S and CANDLE-, SAVI- or Aicardi-Goutières syndrome-like (AGS-like) phenotypes, 5 patients carried mutations in either SAMHD1, TREX1, PSMB8, or PSMG2. Two patients had anti-MDA5 autoantibody-positive juvenile dermatomyositis, and 7 could not be classified. Patients with LRBA, IKBKG, and SAMD9L mutations showed a pattern of IRG elevation that suggests prominent NF-κB activation different from the canonical interferonopathies CANDLE, SAVI, and AGS. CONCLUSIONS. In patients with elevated IRG-S, we identified characteristic clinical features and 3 additional autoinflammatory diseases: IL-18-mediated PAP and recurrent MAS (IL-18PAP-MAS), NEMO deleted exon 5-autoinflammatory syndrome (NEMO-NDAS), and SAMD9L-associated autoinflammatory disease (SAMD9L-SAAD). The IRG-S expands the diagnostic armamentarium in evaluating USAIDs and points to different pathways regulating IRG expression. Fil: de Jesus, Adriana A.. National Institute Of Allergy And Infectious Diseases; Estados Unidos Fil: Hou, Yangfeng. Shandong University; China Fil: Brooks, Stephen. National Institute Of Arthritis And Musculoskeletal And Skin Diseases; Estados Unidos Fil: Malle, Louise. cahn School of Medicine at Mount Sinai; Estados Unidos Fil: Biancotto, Angelique. No especifíca; Fil: Huang, Yan. National Institute Of Allergy And Infectious Diseases; Estados Unidos Fil: Calvo, Katherine R.. National Institutes of Health; Estados Unidos Fil: Marrero, Bernadette. No especifíca; Fil: Moir, Susan. No especifíca; Fil: Oler, Andrew J.. National Institute Of Allergy And Infectious Diseases ; Estados Unidos Fil: Deng, Zuoming. National Institute Of Arthritis And Musculoskeletal And Skin Diseases; Estados Unidos Fil: Montealegre Sanchez, Gina A.. National Institute Of Allergy And Infectious Diseases ; Estados Unidos Fil: Ahmed, Amina. No especifíca; Fil: Allenspach, Eric. Washington State University; Estados Unidos Fil: Arabshahi, Bita. Virginia Commonwealth University; Estados Unidos Fil: Behrens, Edward. University of Pennsylvania; Estados Unidos Fil: Benseler, Susanne. University of Calgary; Canadá Fil: Bezrodnik, Liliana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bout Tabaku, Sharon. No especifíca; Fil: Brescia, AnneMarie C.. No especifíca; Fil: Brown, Diane. No especifíca; Fil: Burnham, Jon M.. University of Pennsylvania; Estados Unidos Fil: Caldirola, Maria Soledad. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Gobierno de la Ciudad de Buenos Aires. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas; Argentina Fil: Carrasco, Ruy. No especifíca; Fil: Chan, Alice Y.. University of California; Estados Unidos Fil: Cimaz, Rolando. Università degli Studi di Milano; Italia Fil: Dancey, Paul. Janeway Children's Hospital And Rehabilitation Centre; Canadá Fil: Dare, Jason. University of Arkansas for Medical Sciences; Estados Unidos Fil: DeGuzman, Marietta. Baylor College Of Medicine; Estados Unidos Fil: Dimitriades, Victoria. No especifíca; Fil: Ferguson, Ian. University of Yale. School of Medicine; Estados Unidos Fil: Ferguson, Polly. University of Iowa; Estados Unidos Fil: Finn, Laura. University of Washington; Estados Unidos Fil: Gattorno, Marco. No especifíca; Fil: Grom, Alexei A.. No especifíca; Fil: Hanson, Eric P.. No especifíca; Fil: Hashkes, Philip J.. No especifíca; Fil: Hedrich, Christian M.. University of Liverpool; Reino Unido Fil: Herzog, Ronit. University of New York; Estados Unidos Fil: Horneff, Gerd. Universitat zu Köln; Alemania Fil: Jerath, Rita. Augusta University; Estados Unidos Fil: Kessler, Elizabeth. University of Missouri; Estados Unidos Fil: Kim, Hanna. No especifíca; Fil: Kingsbury, Daniel J.. No especifíca; Fil: Laxer, Ronald M.. University Of Toronto. Hospital For Sick Children; Canadá Fil: Lee, Pui Y.. Children's Hospital Boston; Estados Unidos Fil: Lee Kirsch, Min Ae. Technische Universität Dresden; Alemania Fil: Lewandowski, Laura. No especifíca; Fil: Li, Suzanne. Hackensack University Medical Center; Estados Unidos Fil: Lilleby, Vibke. Oslo University Hospital; Noruega Fil: Mammadova, Vafa. Azerbaijan Medical University; Azerbaiyán Fil: Moorthy, Lakshmi N.. Robert Wood Johnson Medical School; Estados Unidos Fil: Nasrullayeva, Gulnara. Azerbaijan Medical University; Azerbaiyán Fil: O'Neil, Kathleen M.. Riley Hospital for Children; Estados Unidos Fil: Onel, Karen. Hospital for Special Surgery; Estados Unidos Fil: Ozen, Seza. Hacettepe University; Turquía Fil: Pan, Nancy. Hospital for Special Surgery; Estados Unidos Fil: Pillet, Pascal. Children Hospital Pellegrin-Enfants; Francia Fil: Piotto, Daniela G.P.. Universidade Federal de Sao Paulo; Brasil Fil: Punaro, Marilynn G.. University of Texas; Estados Unidos Fil: Reiff, Andreas. University of Nebraska; Estados Unidos Fil: Reinhardt, Adam. University of Nebraska; Estados Unidos Fil: Rider, Lisa G.. No especifíca; Fil: Rivas Chacon, Rafael. Nicklaus Children’s Hospital; Estados Unidos Fil: Ronis, Tova. Children’s National Health System; Estados Unidos Fil: Rösen Wolff, Angela. Technische Universität Dresden; Alemania Fil: Roth, Johannes. Children’s Hospital of Eastern Ontario; Canadá Fil: Mckerran Ruth, Natasha. Medical University of South Carolina; Estados Unidos Fil: Rygg, Marite. Norwegian University of Science and Technology; Noruega Fil: Schmeling, Heinrike. University of Calgary; Canadá Fil: Schulert, Grant. Children’s Hospital Medical Center; Estados Unidos Fil: Scott, Christiaan. University of Cape Town; Sudáfrica Fil: Seminario, Gisella. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina Fil: Shulman, Andrew. University of California at Irvine; Estados Unidos Fil: Sivaraman, Vidya. Nationwide Children’s Hospital; Estados Unidos Fil: Son, Mary Beth. Boston Children’s Hospital; Estados Unidos Fil: Stepanovskiy, Yuriy. Shupyk National Medical Academy for Postgraduate Education; Ucrania Fil: Stringer, Elizabeth. Dalhousie University Halifax; Canadá Fil: Taber, Sara. Hospital for Special Surgery; Estados Unidos Fil: Terreri, Maria Teresa. Universidade Federal de Sao Paulo; Brasil Fil: Tifft, Cynthia. No especifíca; Fil: Torgerson, Troy. University of Washington; Estados Unidos Fil: Tosi, Laura. Children’s National Health System; Estados Unidos Fil: van Royen Kerkhof, Annet. Wilhelmina Children’s Hospital Utrech; Países Bajos Fil: Wampler Muskardin, Theresa. New York University School of Medicine; Estados Unidos Fil: Canna, Scott W.. University of Pittsburgh; Estados Unidos Fil: Goldbach Mansky, Raphaela. National Institute Of Allergy And Infectious Diseases ; Estados Unidos

PURPOSE Single-agent purine analog, usually cladribine, has been the standard first-line therapy of hairy cell leukemia (HCL) for 30 years. High complete remission (CR) rates often include minimal residual disease (MRD), leading to relapse and repeated treatments. Rituximab can clear MRD, but long-term results are unknown and optimal timing of rituximab undefined. PATIENTS AND METHODS Patients were randomly assigned to first-line cladribine 0.15 mg/kg intravenously days 1-5 with 8 weekly doses of rituximab 375 mg/m2 begun either day 1 (concurrent, CDAR) or ≥ 6 months later (delayed) after detection of MRD in blood. MRD tests included blood and bone marrow (BM) flow cytometry, and BM immunohistochemistry. RESULTS Sixty-eight patients with purine analog-naïve classic HCL were randomly assigned 1:1 to concurrent versus delayed arms. At 6 months after CDAR versus cladribine monotherapy, CR rates were 100% versus 88% ( P = .11), MRD-free CR rates 97% versus 24% ( P < .0001, primary end point), and blood MRD-free rates 100% versus 50% ( P < .0001), respectively. At 96 months median follow-up, 94% versus 12% remained MRD free. Compared with CDAR, delayed rituximab after cladribine achieved lower rate (67% of 21 evaluable patients; P = .0034) and durability ( P = .0081, hazard radio favoring CDAR, 0.094) of MRD-free CR. Nevertheless, 12 patients in the delayed arm remained MRD free when restaged 6-104 (median, 78) months after last delayed rituximab treatment. Compared with cladribine monotherapy, CDAR led to brief grade 3/4 thrombocytopenia (59% v 9%; P < .0001) and platelet transfusions without bleeding (35% v 0%; P = .0002), but higher neutrophil ( P = .017) and platelet ( P = .0015) counts at 4 weeks. CONCLUSION Achieving MRD-free CR of HCL after first-line cladribine is greatly enhanced by concurrent rituximab and less so by delayed rituximab. Longer follow-up will determine if MRD-free survival leads to less need for additional therapy or cure of HCL.

RATIONALE Studies identified kininogen as a potential biomarker of preeclampsia, a major cause of adverse maternal outcomes. High-molecular-weight kininogen (HK) and its activated form participate in numerous pathways associated with establishing and maintaining pregnancy. However, dynamic changes in HK and naturally occurring HK-derived peptides during the natural course of pregnancy are largely unknown. METHODS Longitudinal serum samples during the course of normal pregnancy (trimesters T1, T2, T3) from 60 pregnant women were analyzed by western blot with an anti-HK antibody. Circulating peptides in longitudinal serum specimens derived from 50 participants were enriched using nanoporous silica thin films. Peptides were identified by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and database searching. Relative quantification was performed using MaxQuant and in-house scripts. Normality was evaluated by either ANOVA or Friedman tests with p

Secondary malignancies are a significant cause of non-relapse mortality in patients who undergo allogeneic HCT. However, secondary liver cancer is rare, and ICC following HCT has never been reported in the literature. Secondary solid cancers typically have a long latency period, and cholangiocarcinoma is classically a malignancy occurring in older individuals. Here, we report the first case of secondary ICC, which presented just 3 years after HCT in a young adult with a history of childhood ALL. A 26-year-old male with history of precursor B-cell ALL presented with asymptomatic elevated liver function tests 3 years after HCT. Laboratories were indicative of biliary obstruction. ERCP showed focal biliary stricturing of the common and left hepatic ducts. MRCP revealed left intrahepatic duct dilatation, suggestive of intrahepatic obstructing mass. Additional workup lead to a clinical diagnosis of ICC. The patient underwent left hepatectomy with extrahepatic bile duct resection and portal lymphadenectomy. Surgical pathology was consistent with moderately differentiated cholangiocarcinoma. Our case illustrates a rare SMN following HCT for ALL. It is the first case report of ICC occurring as a secondary cancer in this patient population. Although cholangiocarcinoma is characteristically diagnosed in the older population, it must remain on the differential for biliary obstruction in post-HCT patients.

Objectives To determine fluorescently labeled aerolysin (FLAER) binding and glycophosphatidylinositol–anchored protein expression in bone marrow (BM) cells of healthy volunteers and patients with paroxysmal nocturnal hemoglobinuria (PNH) detected in peripheral blood (PB); compare PNH clone size in BM and PB; and detect PNH in BM by commonly used antibodies. Methods Flow cytometry analysis of FLAER binding to leukocytes and expression of CD55/CD59 in erythrocytes. Analysis of CD16 in neutrophils and CD14 in monocytes in BM. Results FLAER binds to all normal BM leukocytes, and binding increases with cell maturation. In PNH, lymphocytic clones are consistently smaller than clones of other BM cells. PNH clones are detectable in mature BM leukocytes with high specificity and sensitivity using common antibodies. Conclusions PNH clone sizes measured in mature BM leukocytes and in PB are comparable, making BM suitable for PNH assessment. We further demonstrate that commonly used reagents (not FLAER or CD55/CD59) can reliably identify abnormalities of BM neutrophils and monocytes consistent with PNH cells.

Allogeneic hematopoietic stem cell transplantation (HSCT) reverses the bone marrow failure syndrome due to GATA2 deficiency. The intensity of conditioning required to achieve reliable engraftment and prevent relapse remains unclear. Here, we describe the results of a prospective study of HSCT in 22 patients with GATA2 deficiency using a busulfan-based conditioning regimen. The study included 2 matched related donor (MRD) recipients, 13 matched unrelated donor (URD) recipients, and 7 haploidentical related donor (HRD) recipients. MRD and URD recipients received 4 days of busulfan and 4 days of fludarabine. HRD recipients received low-dose cyclophosphamide for 2 days, fludarabine for 5 days, 2 to 3 days of busulfan depending on cytogenetics, and 200 cGy total body irradiation. MRD and URD recipients received tacrolimus and short-course methotrexate for graft-versus-host disease (GVHD) prophylaxis. HRD recipients received high-dose post-transplant cyclophosphamide (PTCy) followed by tacrolimus and mycophenolate mofetil. At a median follow-up of 24 months (range, 9 to 50), 19 of 22 patients were alive with reversal of the disease phenotype and correction of the myelodysplastic syndrome, including eradication of cytogenetic abnormalities. Three patients died: 1 from refractory acute myelogenous leukemia, 1 from GVHD, and 1 from sepsis. There was a 26% incidence of grades III to IV acute GVHD in the MRD and URD groups and no grades III to IV acute GVHD in the HRD cohort. Similarly, there was a 46% incidence of chronic GVHD in the MRD and URD cohorts, whereas only 28% of HRD recipients developed chronic GVHD. Despite excellent overall disease-free survival (86%), GVHD remains a limitation using standard prophylaxis for GVHD. We are currently extending the use of PTCy to the MRD and URD cohorts to reduce GVHD.

Fanconi anemia (FA) is an inherited disorder associated with loss of function mutations in gene members of the FA-BRCA pathway involved in interstrand cross-link DNA damage repair during cell division. Progressive bone marrow failure (BMF) is a primary cause of morbidity and mortality in patients with FA. Allogeneic hematopoietic stem and progenitor cell (HSPC) transplantation is the only curative treatment for FA-associated BMF. However, donor availability, graft failure, and FA-specific transplant toxicities remain significant hurdles. Attempts at genetic correction of FA are underway but collection of sufficient numbers of autologous HSPCs is challenging in subjects with advanced BMF. Androgens have been successfully used but side effects often prevent prolonged therapy. The orally bioavailable small molecule mimetic of thrombopoietin, eltrombopag (EPAG), was recently shown to stimulate multipotent long-term repopulating HSPCs in patients with acquired BMF, resulting in persistent trilineage hematopoiesis. EPAG promotes DNA repair (Guenther, Exp Hematol 2019) and maintains HSPCs under inflammatory conditions (Alvarado, Blood 2019), indicating potential relevance in FA. In this study, we investigated whether EPAG may offer a novel therapeutic modality for FA-associated BMF. We have enrolled 10 patients (FANCA, n=9 and FANCC, n=1) to date in a prospective phase I/II study of EPAG in Fanconi anemia (NCT03204188). All subjects received EPAG at an oral daily dose adjusted for age and ethnicity. The primary efficacy endpoint was the proportion of subjects with at least 2-fold increase in marrow cellularity or CD34+ HSPC frequency (marrow response), or clinically significant improvement in peripheral blood (PB) counts at 6 months (blood response). The primary safety endpoint was the global toxicity profile assessed at 6 months using CTCAE criteria. Responders were invited to continue on the extension phase of the study for an additional 3 years. Four of 10 patients have reached the 6-month assessment endpoint, and a pre-specified 3-month interim analysis is available on two additional subjects. A marrow response was observed in all 4 subjects at 6 months. Mean marrow cellularity increased by a factor of 4 (Fig. A/B), and CD34+ HSPC frequency improved 2.5-fold at 6 months relative to pre-treatment values (Fig. C/D). Marrow response criteria were also met in the other two subjects at the 3-month interim assessment. Primary PB response was observed in 2 of 4 patients in one (hemoglobin) or two lineages (hemoglobin and platelets) at 6 months of treatment. Responders continue to receive EPAG at 10 or 25 months with sustained improvements in blood counts and transfusion independence. The subject treated for 25 months recently met response criteria in a third lineage (neutrophils) (Fig. E). Both patients who had a marrow but no PB response are clinically well; one subject (FANCA) underwent an unrelated allogeneic HSPC transplantation one year ago, and the other subject (FANCC) recently entered the extension phase of this study. Unilineage PB response criteria were also met in one of two subjects at the 3-month interim assessment. No drug-related serious adverse events have occurred during EPAG treatment. All patients tolerated the maximum dose of EPAG and no interruption or dose reduction was required for adverse events attributable to EPAG. Patients without transfusional iron overload at study entry developed progressive depletion of iron stores attributable to the known potent iron chelating and mobilizing properties of EPAG. At a median of 3 months after initiating EPAG (range, 1 to 6 months), affected patients initiated daily oral iron supplementation with gradual amelioration of iron store levels. To date, there has been no occurrence of marrow fibrosis, solid malignancies or clonal evolution, defined as abnormalities on standard metaphase analysis of bone marrow or overt clinical transformation to MDS/AML. In sum, treatment with EPAG was associated with increased marrow HSPCs in all subjects and PB responses in a subset of patients with Fanconi anemia. The overall safety profile was favorable. Further follow-up and expansion of the ongoing patient cohort will confirm EPAG's potential as a safe and efficient long-term therapeutic modality for FA-associated BMF. Alternatively, EPAG could also be used safely to boost marrow CD34+ cell numbers prior to autologous gene therapy applications. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

BackgroundRelapsed or refractory Acute Myeloid Leukemia (R-AML) is a deadly disease with an inadequate response rate to current treatments. Recent advances in immunotherapy shed light on R-AML, and several clinical trials have shown promising potential for combining immune checkpoint inhibitors (ICIs) with hypomethylating agents. A deeper understanding of the tumor-immune microenvironment in R-AML during combination ICI treatment is urgently needed for developing better therapeutics and stratifying treatment strategies.MethodsTo dissect the tumor-immune interactions in the bone marrow microenvironment, we employed nanoString GeoMx Digital Spatial Profiler (DSP) and performed a spatial-transcriptomic analysis of patients with R-AML who received pembrolizumab and decitabine. We compared the transcriptomic profiles and TCR clonalities of tumor-interacting T cells, bystander T cells, and other cells at baseline, post-pembrolizumab treatment, and post-decitabine, which enable us to identify R-AML’s suppressive immune microenvironment and immune cells’ responses to ICI and hypomethylating agent.ResultsWe obtained the spatial-transcriptomic profiles of T cells, stromal cells, and leukemia cells in patients with R-AML at different treatment points. Our TCR-specific probes were able to track T cell clonal changes during treatments.ConclusionsR-AML harbored a complex tumor immune microenvironment and diverse T cell clonality.AcknowledgementsThis research was supported in part by the Intramural Research Program of the NCI (the Center for Cancer Research), NHLBI, and NIH Clinical Center.Ethics ApprovalThis study is approved by NHLBI IRB.

Introduction: Identifying the causes of adult-onset rheumatic diseases remains a challenge, and limits diagnosis, prognosis, and targeted treatment. We hypothesized that mutations in genes regulating the post-translational modification ubiquitin, previously implicated in two autoinflammatory diseases, may define new rheumatic disorders. Methods: We analyzed peripheral blood exome sequence data from 2,560 individuals with inflammation-related diagnoses for deleterious mutations in >800 ubiquitin-related genes. After discovering three patients with novel UBA1 mutations, we identified additional cases based on clinical similarities through screening multiple independent cohorts. Clinical evaluation of all patients combined with Sanger sequencing, digital droplet PCR, immunoblotting, immunohistochemistry, flow cytometry, and transcriptome/cytokine profiling were performed. CRISPR/Cas9 edited zebrafish provided an in vivo model to assess UBA1 gene function. Results: Twenty-eight adult males were identified with somatic mutations at methionine 41 in UBA1, an X-linked gene, encoding the major E1 enzyme that initiates ubiquitylation. Methionine 41 is highly conserved in UBA1, and these mutations were not observed in exome sequences from over 80,000 healthy controls. Among affected individuals, mutations were found in more than half of hematopoietic stem cells, exclusively in peripheral blood myeloid cells, and not in lymphocytes or fibroblasts. The variant allele fraction of UBA1 p.Met41 mutations in peripheral blood ranged from 20-95%. Patients developed an often fatal, treatment-refractory inflammatory syndrome in late adulthood, with fever, neutrophilic cutaneous and pulmonary inflammation, chondritis, and vasculitis and some individuals met clinical criteria for relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, or giant cell arteritis. In addition, the majority of subjects developed myelodysplastic bone marrow with cytopenias, characteristic vacuoles in myeloid and erythroid precursors cells, progressive bone marrow failure and thromboembolic disease, and some fulfilled clinical criteria for myelodysplastic syndrome or plasma cell dyscrasia. Transformation into MDS with excess blasts or acute myeloid leukemia did not occur in any case. Mutations at p.Met41, the initiation start site of the canonical cytoplasmic isoform, caused loss of this protein and expression of a novel, catalytically impaired isoform initiated at p.Met67. Mutant peripheral blood cells exhibited decreased ubiquitylation and activated innate immune pathways. Knockout of the zebrafish cytoplasmic UBA1 isoform homologue, but not the nuclear isoform, caused systemic inflammation. These results identify somatic mutations in UBA1 as the cause, and not the consequence, of this inflammatory disease. Conclusions: We have defined a novel disorder, VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic), which connects seemingly unrelated adult-onset inflammatory and hematologic diseases. Our work also reveals somatic mutations in a hematopoietic stem cell as a cause of adult-onset rheumatic syndromes that overlap with hematologic conditions. Identification of UBA1 mutations as a cause of these diseases has important implications for classification, prognosis, and treatment of patients, and for the role of somatic mutations of hematopoietic origin in multi-organ pathophysiology. Figure Disclosures Young: Novartis: Research Funding.

Eltrombopag (EPAG) is a non-peptide, thrombopoietin receptor agonist approved for several forms of thrombocytopenia, as well as severe aplastic anemia in first line (combined with immunosuppressive therapy) and refractory (single agent) settings. It is effective in moderate aplastic anemia and under investigation for myelodysplastic syndrome (MDS) and other marrow failure conditions. Although aplastic anemia (AA) patients often respond to EPAG and become drug-independent, this may take months to years, and some patients remain either continuously or intermittently EPAG-dependent. Previously, we and others have reported that EPAG chelates and mobilizes iron in a cohort of AA patients. We present a larger cohort of AA/MDS patients from 4 prospective clinical trials of EPAG, and report declining iron stores on extended EPAG therapy, including some patients developing frank iron deficiency anemia (IDA) responsive to oral or parenteral supplementation, and others normalizing prior severe iron overload during extended EPAG therapy. We report the kinetics of this effect and analyze its impact, if any, upon response and relapse. 317 patients were treated with EPAG for unilineage cytopenia, AA or MDS. 206 AA patients treated on non-EPAG protocols served as historical controls. Average follow-up was 42.9 months (2.2-206.7) for EPAG and 69.6 months (6.1-196) for controls. Average time on EPAG was 9.0 months (0.4-81.1); 73 patients (23.0%) were treated for ≥ 7 months. Average baseline serum ferritin (SF) was 1757 ng/mL (29-18977) for EPAG; 1628 ng/mL (14-8438) for controls (N.S.), with iron overload (SF ≥ 1000 ng/mL) in 56.5% of EPAG patients; 53.9% of controls (N.S.). On EPAG, serum iron (2x over baseline, P < 1.3 x 10-39) and transferrin saturation (85.1% of patients have 100% saturation) are elevated due to binding by EPAG, while serum ferritin (SF) does not correlate with iron or transferrin saturation. These effects resolve following EPAG therapy: iron is 15% lower than baseline (P = 5.1 x 10-5), median saturation is 40%, and SF and iron correlate. No such dissociation is noted for historical controls. This implies that SF is the most reliable measure of iron status while on EPAG due to its chelating properties. EPAG duration correlates with SF fold-reduction (R2 = 0.19, P < 4 x 10-14). SF levels during EPAG treatment follow first order (exponential) kinetics (R2 = 0.35, P < 2 x 10-20), with a clearance half-life of 15.3 months, independent of baseline SF. There is minimal correlation of treatment response with SF kinetics (P = 0.04). In comparison, historical responders demonstrate a significantly slower SF clearance (P < 8 x 10-10) with a half-life of 47.5 months. SF half-life on EPAG is comparable to that of chelators such as deferoxamine or deferasirox used for transfusion-related iron overload in AA/MDS. This similarity in kinetics and the fact that this effect is independent of treatment response (and thus transfusion burden) support a role for EPAG in actively depleting total body iron. Of 305 evaluable patients, 62 (9.8%) had iron-depletion as measured by ferritin. 30 (7.5%) were during EPAG treatment with a median time of 55.0 months. 11 (3.6%) of patients experienced falling hemoglobin or other signs of anemia. 9 started iron supplementation or discontinued EPAG, with improvement in the 5 patients who have had follow-up. Patients with EPAG-induced IDA follow the same kinetics (15.5-month half-life) as the general cohort. Logistic models do not predict response based upon either baseline ferritin or kinetics. Furthermore, neither logistic nor Kaplan-Meier models identify any timepoint ferritin, baseline iron overload, or kinetics as predictors of relapse risk. While iron and ferritin may be biomarkers of disease and transfusion burden, they do not appear to drive outcomes, and do not support the recent hypothesis that response of marrow failure to EPAG is based on iron chelation and reversal the toxic impact of intracellular iron on hematopoietic stem cells. Our data suggest a potential role for EPAG or its derivatives as iron chelators. More importantly, they demonstrate that EPAG can deplete iron stores, paradoxically requiring supplementation in previously overloaded patients. In addition to relapse, IDA should also be considered in responders on long term EPAG with declining hemoglobin. We recommend maintaining SF greater than 100 ng/mL so that these patients may continue this life-saving therapy. Figure 1 Disclosures Young: Novartis: Research Funding. Dunbar:Novartis: Research Funding.

Somatic mutations in UBA1 in hematopoietic stem cells and myeloid cells have recently been described and are associated with adult-onset severe autoinflammatory diseases including relapsing polychondritis, Sweet syndrome, polyarteritis nodosa, and giant cell arteritis. This newly defined syndrome is named VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic). We performed clinicopathologic, cytogenetic, flow cytometric and molecular bone marrow assessment of 15 patients diagnosed with VEXAS and peripheral cytopenias. All patients were male, with a median age of 64y (IQR 45-80) and all had somatic missense mutations affecting the p.Met41 residue in UBA1 with variant allele frequencies (VAF) over 20% from peripheral blood or bone marrow samples and with lineage restriction to mature myeloid cells. Germline mutations at this residue are not reported in any public databases. Peripheral blood findings included macrocytic anemia in all patients (100%), thrombocytopenia in 9/15, and neutropenia in 1/15. All bone marrow aspirates (15/15; 100%) showed prominent vacuolization of both myeloid and erythroid precursors with few vacuoles noted in mature cells. Of the 9 patients tested for serum copper levels all were normal or borderline low. Marrow was hypercellular with granulocytic and megakaryocytic hyperplasia in 12/15 (80%). Definitive WHO criteria for MDS was met in 5/15 (40%) with 3 cases of MDS-MLD, and 2 of MDS-SLD. The remaining cases were suspicious for MDS with low or borderline levels of dyspoiesis that did not exceed greater than 10% of cells in respective lineages meeting criteria for clonal cytopenia of undetermined significance (CCUS). Of the MDS cases, dysplasia was seen in megakaryocytes (5/5), myeloid (2/5) and erythroid (1/5) precursors. Cytogenetic abnormalities were detected in 2/5 MDS patients involving t(3;12)(q21;q13) in one case, and del (5q)/del (13q) in another case. Next generation sequencing analyses were performed in 9/15 patients and showed mutations in DNMT3A in 2/5 MDS cases (VAF 43% and 44%), CSF1R in 1/5 MDS (VAF 3.1%), GNA11 in 1/5 MDS (VAF3.3%) and EZH2 mutation (VAF 22%) in one of the patients without overt MDS. Clonal plasma cell or B-cell populations were diagnosed in 6/15 patients with 3 plasma cell dyscrasias, and 2 cases of monoclonal B-cell lymphocytosis. The most common abnormalities detected by bone marrow flow cytometry analysis were severely reduced or absent B-cell precursors, inverted CD4:CD8 ratios and abnormal expression of CD56 on monocytes. In summary, hematologic disorders were identified in 10/15 VEXAS patients, including both myeloid and lymphoid clonal disease comprising MDS, plasma cell dyscrasias, and monoclonal B cell lymphocytosis. Based on the presence of UBA1 somatic mutations in hematopoietic cells of all VEXAS patients, the patients without evidence of overt neoplasia in this study met criteria for CCUS. Importantly, all patients had characteristic vacuoles in myeloid and erythroid precursors in the marrow without evidence of copper deficiency. These findings suggest that vacuolization of hematopoietic precursors in marrow of cytopenic patients, particularly in male patients and in the setting of systemic inflammation and normal copper levels, should prompt evaluation for somatic UBA1 mutations. In addition to severe autoinflammatory disease manifestations, VEXAS patients appear to confer increased risk for development of both myeloid and lymphoid/plasma cell neoplasia and require surveillance for disease progression. Figure Disclosures Young: Novartis: Research Funding.

7036 Background: Anti-CD22 recombinant immunotoxin moxetumomab pasudotox (Moxe) is FDA-approved for hairy cell leukemia (HCL) patients who have received at least two prior systemic therapies including a purine nucleoside analog. In phase 3 testing the complete remission (CR) rate was 41%, and response was higher in patients with lower tumor burden and lower titers of antidrug antibodies (ADA). Phase 1 testing indicated that most CRs were without minimal residual disease (MRD) and eradication of MRD was associated with prolonged CR duration. Monoclonal antibody (Mab) rituximab binds to CD20 on HCL cells and induces apoptosis or immune-mediated killing, but as a single-agent achieved only 13% CRs in relapsed HCL requiring therapy. In a phase 1 trial to determine safety, rituximab was combined with Moxe, with the goal to help reduce tumor burden and to prevent or delay ADA by killing normal B-cells. Methods: To allow rituximab sufficient time to accomplish both goals, it was infused 3 days before day 1 of cycle 1 at 375 mg/m2, and Moxe was given by 30-minute infusion on days 1, 3 and 5. On repeat cycles of Moxe days 1, 3 and 5, rituximab was given on day 1. Cycles were generally spaced 4 weeks apart. Moxe was begun at a lower dose, 30 rather than the 40 mcg/kg dose used in phase 3 in case the rituximab would increase its toxicity. Bone marrow aspirate flow cytometry, which can detect 0.002% HCL cells, was the most sensitive test used for MRD detection, much more sensitive than BRAF V600E digital droplet PCR (ddPCR) or bone marrow biopsy immunohistochemistry (IHC). Patients could receive 4 cycles past MRD-free CR, but not more than 8 cycles. Results: Three patients received Moxe at 30 mcg/Kg/dose and 6 received 40 mcg/Kg/dose, all without dose limiting toxicity (DLT). There was no evidence of hemolytic uremic syndrome or capillary leak syndrome. To prevent intravascular hypovolemia due to expected third spacing, patients were encouraged to drink one cup per hour of water or other fluid from days 1 to 8 and take dexamethasone 4 mg orally if headache or nausea prevented good oral hydration. Of the 9 patients, 7 (78%) achieved CR after 2 (n = 6) or 3 (n = 1) cycles, and achieved MRD-free CR after 2 (n = 3), 4 (n = 3) or 6 (n = 1) cycles. No patients became infected with COVID-19. Conclusions: This phase 1 trial met its primary endpoint of determining whether rituximab could be safely combined with Moxe and will enroll 4 additional patients to further access clinical activity. Further testing will determine whether addition of a CD20 Mab to Moxe significantly improves clinical outcome compared to Moxe alone, particularly long-term MRD-free CR rate. Clinical trial information: NCT03805932.

GATA2 deficiency is a bone marrow failure syndrome effectively treated with hematopoietic cell transplantation (HCT), which also addresses the predisposition to many infections (prominently mycobacterial). However, many GATA2-deficient persons who come to HCT also have prevalent and refractory human papilloma virus disease (HPVD), which can be a precursor to cancer. We analyzed 75 HCT recipients for the presence of HPVD to identify patient characteristics and transplantation results that influence HPVD outcomes. We assessed the impact of cellular recovery and iatrogenic post-transplantation immunosuppression, as per protocol (PP) or intensified/prolonged (IP) graft-versus-host disease (GVHD) prophylaxis or treatment, on the persistence or resolution of HPVD. Our experience with 75 HCT recipients showed a prevalence of 49% with anogenital HPVD, which was either a contributing or primary factor in the decision to proceed to HCT. Of 24 recipients with sufficient follow-up, 13 had resolution of HPVD, including 8 with IP and 5 with PP. Eleven recipients had persistent HPVD, including 5 with IP and 6 with PP immunosuppression. No plausible cellular recovery group (natural killer cells or T cells) showed a significant difference in HPV outcomes. One recipient died of metastatic squamous cell carcinoma, presumably of anogenital origin, at 33 months post-transplantation after prolonged immunosuppression for chronic GVHD. Individual cases demonstrate the need for continued aggressive monitoring, especially in the context of disease prevalent at transplantation or prior malignancy. HCT proved curative in many cases in which HPVD was refractory and recurrent prior to transplantation, supporting a recommendation that HPVD should be considered an indication rather than contraindication to HCT, but post-transplantation monitoring should be prolonged with a high level of vigilance for new or recurrent HPVD.

GATA2 deficiency syndrome is caused by autosomal dominant, heterozygous germline mutations with widespread effects on immune, pulmonary and vascular systems. Patients commonly develop hematological abnormalities including bone marrow failure, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). We present a patient with GATA2 mutation and MDS who progressed to AML over four months. Whole exome and targeted deep sequencing identified a new p.Q61K NRAS mutation in the bone marrow at the time of AML development. Rapid development of AML is possible in the setting of germline GATA2 mutation despite stable MDS, supporting close monitoring and consideration of early allogeneic transplantation.

WHIM syndrome (warts, hypogammaglobulinemia, infections, and myelokathexis), a primary immunodeficiency disorder involving panleukopenia, is caused by autosomal dominant gain-of-function mutations in CXC chemokine receptor 4 (CXCR4). Myelokathexis is neutropenia caused by neutrophil retention in bone marrow. Patients with WHIM syndrome are often treated with granulocyte colony-stimulating factor (G-CSF), which can increase neutrophil counts but does not affect cytopenias other than neutropenia. In this investigator-initiated, open-label study, three severely affected patients with WHIM syndrome who could not receive G-CSF were treated with low-dose plerixafor, a CXCR4 antagonist, for 19 to 52 months. Myelofibrosis, panleukopenia, anemia, and thrombocytopenia were ameliorated, the wart burden and frequency of infection declined, human papillomavirus-associated oropharyngeal squamous-cell carcinoma stabilized, and quality of life improved markedly. Adverse events were mainly infections attributable to the underlying immunodeficiency. One patient died from complications of elective reconstructive surgery. (Funded by the National Institutes of Health.).

Germline mutation in GATA2 can lead to GATA2 deficiency characterized by a complex multi-system disorder that can present with many manifestations including variable cytopenias, bone marrow failure, myelodysplastic syndrome/acute myeloid leukemia (MDS/AML), and severe immunodeficiency. Penetrance and expressivity within families is often variable. There is a spectrum of bone marrow disease in symptomatic cytopenic patients ranging from hypocellular marrows without overt dysplasia to those with definitive MDS, AML, or chronic myelomonocytic leukemia. Relatives of probands with the same mutations may demonstrate minimal disease manifestations and normal marrows. A comprehensive clinical, hematological and genetic assessment of 25 patients with germline GATA2 mutation was performed. MDS-associated mutations were identified in symptomatic GATA2 patients both with overt MDS and in those with hypocellular/aplastic bone marrows without definitive dysplasia. Healthy relatives of probands harboring the same germline GATA2 mutations had essentially normal marrows that were overall devoid of MDS-associated mutations. The findings suggest that abnormal clonal hematopoiesis is a common event in symptomatic germline mutated GATA2 patients with MDS and also in those with hypocellular marrows without overt morphologic evidence of dysplasia, possibly indicating a pre-MDS stage warranting close monitoring for disease progression.

Eltrombopag (EPAG) received approval from the US Food and Drug Administration for the treatment of refractory severe aplastic anemia (rSAA) based on treatment of 43 patients with doses escalating from 50 to 150 mg daily for 12 weeks. Response kinetics suggested that more prolonged administration of EPAG at a dose of 150 mg could speed and improve response rates. We enrolled 40 patients with rSAA in a study of EPAG 150 mg daily, with a primary end point of response at 24 weeks. Twenty (50%) of 40 patients responded at 24 weeks; 5 (25%) of 20 would have been deemed nonresponders at 12 weeks, the end point of the previous study. Fifteen of the 19 responding patients continuing on EPAG had drug discontinued for robust response; 5 of the 15 required EPAG re-initiation for relapse, with all recovering response. To analyze risk of clonal progression, we combined long-term data from the 83 patients with rSAA enrolled in both studies. Evolution to an abnormal karyotype occurred in 16 (19%), most within 6 months of EPAG initiation. Targeted deep sequencing/whole-exome sequencing was performed pre-EPAG and at primary response end point and/or time of clonal evolution or longest follow-up. Cytogenetic evolution did not correlate with mutational status, and overall mutated allele fractions of myeloid cancer genes did not increase on EPAG. In summary, extended administration of EPAG at a dose of 150 mg for 24 weeks rescued responses in some patients with rSAA not responding at 12 weeks. The temporal relationship between clonal evolution and drug exposure suggests that EPAG may promote expansion of dormant preexisting clones with an aberrant karyotype. The studies were registered at www.clinicaltrials.gov as #NCT00922883 and #NCT01891994.

Lessons Learned Despite the initial optimism for using immune checkpoint inhibition in the treatment of multiple myeloma, subsequent clinical studies have been disappointing. Preclinical studies have suggested that priming the immune system with various modalities in addition to checkpoint inhibition may overcome the relative T-cell exhaustion or senescence; however, in this small data set, radiotherapy with checkpoint inhibition did not appear to activate the antitumor immune response. Background Extramedullary disease (EMD) is recognized as an aggressive subentity of multiple myeloma (MM) with a need for novel therapeutic approaches. We therefore designed a proof-of-principle pilot study to evaluate the synergy between the combination of the anti–PD-L1, avelumab, and concomitant hypofractionated radiotherapy. Methods This was a single-arm phase II Simon two-stage single center study that was prematurely terminated because of the COVID-19 pandemic after enrolling four patients. Key eligibility included patients with relapsed/refractory multiple myeloma (RRMM) who had exhausted or were not candidates for standard therapy and had at least one lesion amenable to radiotherapy. Patients received avelumab until progression or intolerable toxicity and hypofractionated radiotherapy to a focal lesion in cycle 2. Radiotherapy was delayed until cycle 2 to allow the avelumab to reach a study state, given the important observation from previous studies that concomitant therapy is needed for the abscopal effect. Results At a median potential follow-up of 10.5 months, there were no objective responses, one minimal response, and two stable disease as best response. The median progression-free survival (PFS) was 5.3 months (95% confidence interval [CI]: 2.5–7.1 months), and no deaths occurred. There were no grade ≥3 and five grade 1–2 treatment-related adverse events. Conclusion Avelumab in combination with radiotherapy for patients with RRMM and EMD was associated with very modest systemic clinical benefit; however, patients did benefit as usual from local radiotherapy. Furthermore, the combination was very well tolerated compared with historical RRMM treatment regimens.

Background Most patients infected with Epstein-Barr virus (EBV) are asymptomatic, have nonspecific symptoms, or have self-limiting infectious mononucleosis. EBV, however, may result in severe primary disease or cancer. Methods We report EBV diseases associated with GATA2 deficiency at one institution and describe the hematology, virology, and cytokine findings. Results Seven patients with GATA2 deficiency developed severe EBV disease. Three presented with EBV infectious mononucleosis requiring hospitalization, 1 had chronic active EBV disease (B-cell type), 1 had EBV-associated hydroa vacciniforme-like lymphoma with hemophagocytic lymphohistiocytosis, and 2 had EBV-positive smooth muscle tumors. Four of the 7 patients had severe warts and 3 had disseminated nontuberculous mycobacterial infections. All of the patients had low numbers of monocytes, B cells, CD4 T cells, and natural killer cells. All had elevated levels of EBV in the blood; 2 of 3 patients tested had expression of the EBV major immediate-early gene in the blood indicative of active EBV lytic infection. Mean plasma levels of tumor necrosis factor α, interferon γ, and interferon gamma-induced protein 10 were higher in patients with GATA2 deficiency than in controls. Conclusions GATA2 is the first gene associated with EBV hydroa vacciniforme-like lymphoma. GATA2 deficiency should be considered in patients with severe primary EBV infection or EBV-associated cancer, especially in those with disseminated nontuberculous mycobacterial disease and warts.

The incidence and importance of bone marrow involvement and/or early bone lesions in multiple myeloma (MM) precursor diseases is largely unknown. This study prospectively compared the sensitivity of several imaging modalities in monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM) and MM. Thirty patients (10 each with MGUS, SMM and MM) were evaluated with skeletal survey, [18F]FDG-PET/CT, [18F]NaF-PET/CT and morphologic dynamic contrast enhanced (DCE)-MRI. An additional 16 SMM patients had skeletal surveys and FDG-PET/CT. Among MGUS patients, DCE-MRI found only one focal marrow abnormality; other evaluations were negative. Among 26 SMM patients, five (19%) were re-classified as MM based on lytic bone lesions on CT and six had unifocal or diffuse marrow abnormality. Among MM, marrow abnormalities were observed on FDG-PET/CT in 8/10 patients and on DCE-MRI in nine evaluable patients. Abnormal NaF uptake was observed only in MM patients with lytic lesions on CT, providing no additional clinical information.

Purpose: Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B-cell receptor (BCR) signaling. Ibrutinib, a Bruton tyrosine kinase inhibitor, disrupts BCR signaling and leads to the egress of tumor cells from the microenvironment. Although the on-target effects on CLL cells are well defined, the impact on the microenvironment is less well studied. We therefore sought to characterize the in vivo effects of ibrutinib on the tumor microenvironment. Experimental Design: Patients received single-agent ibrutinib on an investigator-initiated phase II trial. Serial blood and tissue samples were collected pretreatment and during treatment. Changes in cytokine levels, cellular subsets, and microenvironmental interactions were assessed. Results: Serum levels of key chemokines and inflammatory cytokines decreased significantly in patients on ibrutinib. Furthermore, ibrutinib treatment decreased circulating tumor cells and overall T-cell numbers. Most notably, a reduced frequency of the Th17 subset of CD4+ T cells was observed concurrent with reduced expression of activation markers and PD-1 on T cells. Consistent with direct inhibition of T cells, ibrutinib inhibited Th17 differentiation of murine CD4+ T cells in vitro. Finally, in the bone marrow microenvironment, we found that ibrutinib disaggregated the interactions of macrophages and CLL cells, inhibited secretion of CXCL13, and decreased the chemoattraction of CLL cells. Conclusions: In conjunction with inhibition of BCR signaling, these changes in the tumor microenvironment likely contribute to the antitumor activity of ibrutinib and may impact the efficacy of immunotherapeutic strategies in patients with CLL. Clin Cancer Res; 22(7); 1572–82. ©2015 AACR. See related commentary by Bachireddy and Wu, p. 1547

Background Hairy cell leukemia (HCL) variant (HCLv) is considered a separate, more aggressive entity compared to classic HCL. HCLv responds poorly to single-agent purine analog with complete response (CR) rates below 10% and overall response rates under 50%. Rituximab combined with purine-analog can improve response rate and duration, but long-term data have not been reported for HCLv, particularly regarding minimal residual disease (MRD). We therefore update the results of a phase II trial with cladribine and concurrent rituximab in patients with HCLv, previously reported for 10 of the 20 patients enrolled. Methods Patients with HCLv with 0 to 1 prior courses of cladribine, and/or 0-1 prior courses of rituximab, received cladribine (0.15 mg/kg days 1-5), with 8 weekly doses of rituximab (375 mg/m2) beginning day 1. The primary endpoint was to determine CR rate and secondary endpoints included evaluating minimal residual disease (MRD) by blood and bone marrow aspirate flow cytometry, and bone marrow biopsy immunohistochemistry. Patients were able to receive a 2nd course of rituximab ≥ 6 months after the first, if and when MRD was detected in blood. Results Twenty patients were enrolled. Median age was 67 (range: 42-86) years. No patients had prior concurrent cladribine-rituximab. Eight were previously untreated, 1 had only splenectomy, 6 had prior cladribine, 1 had prior cladribine and splenectomy, 1 had prior rituximab, 1 had prior rituximab and splenectomy, 1 had cladribine, rituximab, and splenectomy, and 1 had combination rituximab-containing chemotherapy followed by cladribine. Out of 20 patients receiving concurrent cladribine-rituximab (CDAR), the CR rate was 95% (95% CI: 75-100%). This CR rate was superior to a historical control group of 3 of 39 HCLv patients who achieved CR to cladribine alone (p Conclusion Concurrent cladribine with rituximab is highly effective in HCLv irrespective of prior purine-analog treatment or IGHV4-34 status and should replace purine analog monotherapy as treatment. Patients with long-term MRD-free CR are being followed to determine whether concurrent cladribine-rituximab as 1st-3rd line systemic therapy can permanently eradicate HCLv. Patients who progress have limited OS. This provides a rationale for the testing of higher intensity approaches up front and the identification of additional treatment options for HCLv. Disclosures Kreitman: Genentech: Research Funding. OffLabel Disclosure: Rituximab for hairy cell leukemia

GATA2 deficiency is an immunodeficiency and bone marrow failure disorder caused by pathogenic variants in GATA2. It is inherited in an autosomal-dominant pattern or can be due to de novo sporadic germline mutation. Patients commonly have B-cell, dendritic cell, natural killer cell, and monocytopenias, and are predisposed to myelodysplastic syndrome, acute myeloid leukemia, and chronic myelomonocytic leukemia. Patients may suffer from disseminated human papilloma virus and mycobacterial infections, pulmonary alveolar proteinosis, and lymphedema. The bone marrow eventually takes on a characteristic hypocellular myelodysplasia with loss of monocytes and hematogones, megakaryocytes with separated nuclear lobes, micromegakaryocytes, and megakaryocytes with hypolobated nuclei.