Oliveira, Jennifer L.

Abstract Context.—The diagnosis and classification of leukemia is becoming increasingly complex. Current classification schemes incorporate morphologic features, immunophenotype, molecular genetics, and clinical data to specifically categorize leukemias into various subtypes. Although sophisticated methodologies are frequently used to detect characteristic features conferring diagnostic, prognostic, or therapeutic implications, a thorough microscopic examination remains essential to the pathologic evaluation. Detailed blast immunophenotyping can be performed with lineage- and maturation-specific markers. Although no one marker is pathognomonic for one malignancy, a well-chosen panel of antibodies can efficiently aid the diagnosis and classification of acute leukemias. Objective.—To review important developments from recent and historical literature. General immunohistochemical staining patterns of the most commonly encountered lymphoid and myeloid leukemias are emphasized. The goal is to discuss the immunostaining of acute leukemias when flow cytometry and genetic studies are not available. Data Sources.—A comprehensive review was performed of the relevant literature indexed in PubMed (National Library of Medicine) and referenced medical texts. Additional references were identified in the reviewed manuscripts. Conclusions.—Immunophenotyping of blasts using an immunohistochemical approach to lymphoid and myeloid malignancies is presented. Initial and subsequent additional antibody panels are suggested to confirm or exclude each possibility in the differential diagnosis and a general strategy for diagnostic evaluation is discussed. Although the use of immunohistochemistry alone is limited and evaluation by flow cytometry and genetic studies is highly recommended, unavoidable situations requiring analysis of formalin-fixed tissue specimens arise. When performed in an optimized laboratory and combined with a careful morphologic examination, the immunohistochemical approach represents a useful laboratory tool for classifying various leukemias.

Abstract Objectives SF3B1 mutations are the most common mutations in myelodysplastic syndromes (MDS). The International Working Group for the Prognosis of MDS (IWG-PM) recently proposed SF3B1-mutant MDS (SF3B1-mut-MDS) as a distinct disease subtype. We evaluated the spectrum and molecular landscape of SF3B1-mutated myeloid disorders and assessed the prognostication in MDS harboring SF3B1 mutations (MDS-SF3B1). Methods Cases were selected by retrospective review. Clinical course and laboratory and clinical findings were collected by chart review. SF3B1-mut-MDS was classified following IWG-PM criteria. Results SF3B1 mutations were identified in 75 of 955 patients, encompassing a full spectrum of myeloid disorders. In MDS-SF3B1, Revised International Prognostic Scoring System (IPSS-R) score greater than 3 and transcription factor (TF) comutations were adverse prognostic markers by both univariate and multivariate analyses. We confirmed the favorable outcome of IWG-PM-defined SF3B1-mut-MDS. Interestingly, it did not show sharp prognostic differentiation within MDS-SF3B1. Conclusions SF3B1 mutations occur in the full spectrum of myeloid disorders. We independently validated the favorable prognostication of IWG-PM-defined SF3B1-mut-MDS. However it may not provide sharp prognostication within MDS-SF3B1 where IPSS-R and TF comutations were prognostic-informative. Larger cohort studies are warranted to verify these findings and refine MDS-SF3B1 prognostication.

AbstractBackgroundHemolytic disease of the fetus and newborn (HDFN) attributable to anti‐M is rare, although case reports implicate anti‐M in varying severities of HDFN, including fetal hydrops and intrauterine death.Case DescriptionWe describe the case of a newborn with HDFN associated with an atypical immunoglobulin (Ig) G anti‐M that reacted best at cold temperatures. The maternal antibody detected in pregnancy was not reactive at 37°C, and a direct antiglobulin test (DAT) on red blood cells (RBCs) from the newborn was negative, suggesting an anti‐M that should not have been clinically relevant. However, the infant developed hyperbilirubinemia (bilirubin level, 17.6 mg/dL), hemolytic anemia (hemoglobin nadir, 5.5 g/dL), and reticulocytopenia. Laboratory testing demonstrated the presence of an IgG anti‐M in maternal and neonatal samples reacting best at 4°C. This passively acquired IgG anti‐M provoked hemolytic anemia in the infant and likely suppressed erythropoiesis, resulting in reticulocytopenia with prolonged anemia. He was treated for IgG anti‐M HDFN with 10 intravenous Ig infusions and 10 days of oral prednisone followed by a taper. He required seven transfusions with M− RBCs. His hemoglobin level normalized at 3 months of age. Follow‐up at 2 years revealed no hematologic or neuro‐developmental concerns.ConclusionTo our knowledge, this is the second report of HDFN attributable to an IgG anti‐M reacting preferentially at cold temperature with no 37°C reactivity. Clinically relevant IgG anti‐M may elude standard testing. Early recognition and testing for cold‐reacting IgG anti‐M should be considered for newborns with hemolysis, a negative DAT, and prolonged anemia.

Purpose: molecular testing is often indicated for recently transfused patients. However, there are no guidelines regarding the potential interference from donor DNA or whether it is necessary to wait for a period of time post-transfusion prior to genetic testing. While the majority of patients are transfused in the non-trauma setting using leukoreduced (LR) red blood cell products, the degree of leukoreduction varies among centers and is not universally practiced. Methods: whole blood units collected from anonymous donors were used in an in vitro transfusion model. One unit was split: half being leukoreduced simulating a leukopenic recipient and half left untreated. Donors were simulated by leukoreduced, partially leukoreduced (PLR), or non-leukoreduced units, transfused in 2, 5, or 16 unit equivalents. DNA from the combinations were subjected to short tandem repeat (STR) analysis for chimerism detection. Results: donor DNA was not detectable in any of the LR combinations, but detected in the PLR combinations, ranging from 0.1 to 1.5% donor DNA in the immunocompetent recipient and 6.3–27.8% in the leukopenic recipient. Non-LR donor DNA was also detected (13–95%). Conclusion: donor-derived DNA from leukoreduced blood products is unlikely to interfere with the interpretation of germline genetic testing in immunocompetent recipients but may interfere in immunocompromised recipients.

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

Background: Sickle cell disease is a homozygous hemoglobinopathy with vaso-occlusive complications secondary to abnormal sickling of red blood cells under stressful conditions such as hypoxia. Children with sickle cell trait have a heterozygous genetic state, typically without symptoms. Observation: An 8-year-old boy diagnosed with sickle cell trait was found to have multiple complications consistent with sickle cell disease, including pain crises, osteomyelitis, and priapism. Over a 6-year period, he underwent routine laboratory evaluations without a definitive diagnosis. The diagnosis of a compound heterozygous state of hemoglobin S/hemoglobin Quebec-Chori was eventually made on the basis of mass spectrometry and confirmed with hemoglobin subunit beta gene sequencing. Conclusion: Expanding diagnostic evaluation in patients with abnormal clinical presentations is vital to making the correct diagnosis and hence earlier institution of appropriate management of rare hemoglobinopathies.

Background In humans, maximal exercise capacity is reduced during hypoxic environmental conditions. In response to hypoxia, the hemoglobin binding affinity is decreased, promoting oxygen off‐loading at the tissue level and causing a right‐shift of the oxyhemoglobin dissociation curve (ODC). However, comparative biology demonstrates many species (e.g. Bar‐headed geese), who are adapted to hypoxia instead have high‐affinity hemoglobin (left‐shifted ODC). Therefore we studied unique human participants with hemoglobinopathies that result in high affinity hemoglobin (HAH) to investigate the role of oxygen affinity in exercise performance and tissue oxygenation during hypoxic conditions. Methods HAH participants (n=5, 1M/4F, Age= 34±11 yr, P 50 =16±1 mmHg) and matched control participants (n=12, 5M/7F, age=39±12 yr, and P 50 =26±1 mmHg) completed two incremental exercise tests to exhaustion on a cycle ergometer under two conditions: 1) room air (F i O 2 = 0.21, NORM), and 2) normobaric hypoxia gas mixture (F i O 2 = 0.15, HYP), separated by a 45 minute break. The vastus lateralis (VL) muscle was studied using frequency‐domain near infrared spectroscopy (NIRS) to measure concentrations of oxygenated ([OxyHb+Mb]), deoxygenated ([DeoxyHb+Mb]) hemoglobin and myoglobin, and tissue saturation (S t O 2 ). Data were analyzed using a three‐way (Inspirate [HYP, NORM], Hb type [Normal, HAH], exercise intensity [Rest, 40, 60, 80, 100% normoxic PPO]) univariate analysis of variance. Results HAH had an attenuated decline in exercise tolerance (peak power output at maximal exertion) in HYP compared to controls (HAH: 2.9±6.4% decline vs CTL: 11.7±5.4% decline, P<0.05). There was a significant effect of Hb type on [DeoxyHb+Mb] (P=0.048) suggesting less O 2 offloading in HAH compared to controls in the VL at rest and throughout graded exercise in both NORM and HYP. There were no significant effects of inspirate or exercise intensity on [DeoxyHb+Mb], and no significant differences in [OxyHb+Mb] between HAH and controls. There was a significant effect of Hb type on S t O 2 in NORM controls (P<0.01) However, during hypoxic exercise S t O 2 was the same in both HAH and controls except at end exercise (HAH 64.1±6.4% vs CTL: 62.1±4.2%, P<0.05). Conclusion HAH individuals have less oxygen off‐loading at rest and during exercise but higher tissue saturation during exercise in NORM compared to controls. Our results suggest that the similar levels of exercise capacity in hypoxia for HAH are due, at least in part, to maintenance of microvascular extraction similar to that during normoxic exercise. Future studies utilizing direct measurements of skeletal muscle blood flow during exercise are needed to understand integrative exercise in HAH patients. Support or Funding Information This work was funded by NIH R‐35‐HL139854 and the Mayo Foundation. Muscle oxygenation during graded exercise tests in normoxia and hypoxia. Data are presented as means ± standard error. Abbreviations: [OxyHb+Mb], concentration of oxygenated hemoglobin+myoglobin; [DeoxyHb+MB], concentration of deoxygenated hemoglobin+ myoglobin; PPO, peak power output. † denotes significant main effect of Hb types across all workloads. * denotes significant difference from CTL at same %PPO. Figure 1