Reichard, Kaaren K.

Abstract Objectives Pure erythroid leukemia (PEL) is exceptionally rare in the pediatric setting. Four pediatric PEL cases with t(1;16)(p31;q24) NFIA-CBFA2T3 were reported previously. We present a case of an infant with PEL presenting with erythroblastic sarcoma and harboring a novel t(1;8)(p31.3;q21.3) NFIA-RUNX1T1 fusion detected by RNA sequencing and conventional karyotype. Methods Bone marrow (BM) and abdominal mass biopsies from the patient were evaluated with extensive immunohistochemical, flow cytometric, cytogenetic, and molecular studies. Results The patient was a female infant who presented between 2 and 5 months of age with cytopenias and an enlarging abdominal mass. Blasts in the BM and abdominal mass expressed CD71 and CD117 with focal expression of CD43, E-cadherin, epithelial membrane antigen, and hemoglobin A. They were negative for additional myeloid, lymphoid, and nonhematolymphoid markers. These findings were most consistent with PEL and erythroblastic sarcoma. RNA sequencing revealed the novel NFIA-RUNX1T1 fusion. Conclusions Along with the previously reported PELs with NFIA-CBFA2T3 fusions, we describe a subset of PELs that occur in children, that frequently display extramedullary disease, and that harbor rearrangements of NFIA with core binding factor genes. We hypothesize that, together, these cases represent a rare but distinct clinicopathologic group of pediatric PELs with recurrent genetic abnormality.

Abstract Objectives The 2019 Workshop of the Society for Hematopathology/European Association for Haematopathology received and reviewed cases covering the spectrum of mastocytosis and related diseases, including morphologic mimics, focusing on recent updates and relevant findings for pathologists. Methods The workshop panel reviewed 99 cases of cutaneous and systemic mastocytosis (SM) and SM and associated hematologic neoplasms (SM-AHN). Results Despite a common theme of KIT mutation (particularly D816V), mastocytosis is a heterogeneous neoplasm with a wide variety of presentations. This spectrum, including rare subtypes and extramedullary organ involvement, is discussed and illustrated by representative cases. Conclusions In the age of targeted treatment aimed at KIT, the accurate diagnosis and classification of mastocytosis has major implications for therapy and further interventions. Understanding the clinical, pathologic, and genetic findings of mastocytosis is crucial for selecting the proper tests to perform and subsequent arrival at a correct diagnosis in this rare disease.

AimsRearrangement of the platelet‐derived growth factor receptor B (PDGFRB) gene defines a unique group of myeloid/lymphoid neoplasms with frequent eosinophilia and high sensitivity to tyrosine kinase inhibitors. This genetic abnormality is also rarely reported in Philadelphia‐like B‐cell acute lymphoblastic leukaemia/lymphoma (B‐ALL). PDGFRB rearrangement was initially thought to only occur in cases with 5q31–33 rearrangement as determined with conventional cytogenetics; however, there are reported cases with cryptic rearrangements. We aim to develop a broader strategy for screening of PDGFRB rearrangements of patients with myeloid/lymphoid neoplasms.Methods and resultsWe performed fluorescence in‐situ hybridisation (FISH) for PDGFRB rearrangement in 197 patients, including 70 with B‐ALL, 10 with myeloid neoplasms with 5q31–33 rearrangements, and 117 with eosinophilia (≥0.5 × 109/l in peripheral blood or ≥5% in bone marrow), and identified PDGFRB rearrangement in four of 197 (2.0%) cases. In an attempt to identify clinicopathological and genetic features that may have a stronger association with PDGFRB rearrangement, we analysed 13 patients with confirmed PDGFRB rearrangements, including 10 with myeloid neoplasms and three with B‐ALL. Among the 10 patients with myeloid neoplasms, eosinophilia was present in eight, monocytosis in two, 5q31–33 rearrangement in seven, and abnormal bone marrow morphology in all. All patients with myeloid neoplasms showed an excellent response to imatinib, including a patient in blast crisis. The three B‐ALL patients presented de novo, showed no eosinophilia, had a complex karyotype including 5q31–33 rearrangement, and had clinically aggressive courses with ultimate patient demise.ConclusionsThese findings suggest that a higher yield for the identification of PDGFRB rearrangement may result from an index of suspicion in patients with eosinophilia, monocytosis, bone marrow features of a myeloid neoplasm, and 5q31–33 rearrangement, and patients with Philadelphia‐like B‐ALL.

Abstract Objectives Acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) is a heterogeneous category with a broad range of underlying genetic abnormalities. We investigated the significance of genetic factors in a large series of AML-MRC cases. Methods The morphologic findings, genetic data, and patient outcomes were assessed in 186 AML-MRC cases. Results The median overall survival (OS) was dismal in AML-MRC patients (median, 7.6 months; 95% confidence interval, 5-10.6 months). Karyotypically normal cases and cytogenetically abnormal cases without myelodysplastic syndrome (MDS)-related cytogenetic abnormalities showed similar OS, significantly better than cases carrying MDS-related cytogenetic abnormalities. MDS-related cytogenetic abnormalities, monosomal or complex karyotype, and history of MDS or myelodysplastic/myeloproliferative neoplasm were all associated with dismal outcome. Conclusions AML-MRC predicts a poor prognosis. Our study supports the finding that the genetic profile plays a key role in determining prognosis in AML-MRC as defined according to the World Health Organization revised fourth edition (2017) diagnostic criteria.

Abstract BACKGROUND: Thrombocytopenia is a common hematologic condition associated with multiple etiologies ranging from benign to malignant to potentially life-threatening disorders. Given the heterogeneity of clinical presentations, available clinical information, and pertinent clinical history, there are inter-physician variations in the approach to the workup of thrombocytopenia in the hematology clinic. While a limited test repertoire may be adequate for many cases, more extensive testing may be warranted in others. We were interested in analyzing the various tests performed and testing approaches in the initial workup of thrombocytopenia. METHODS: We reviewed the records of 69 patients who were referred to our center between 2010 and 2015 for an initial workup of thrombocytopenia. We collected epidemiologic data, laboratory testing results, and pathologic findings. Pathologic results were classified as "normal" or "abnormal" and further subcategorized on the basis of review by two clinicians. Quantitative data were analyzed using JMP Pro 10.0.2 software. RESULTS: At the time of thrombocytopenia diagnosis, the median age was 59 years (range: 17-90) and majority were males (65%). The median platelet count was 91,000/µL (range: 3,000-146,000). Isolated thrombocytopenia was present in 51 cases (74%). Forty-four patients (64%) had a peripheral blood smear review and 4 (9%) contained abnormalities including hypogranular neutrophils, rouleaux formation, and target cells. Autoimmune workup included anti-platelet antibody (APA) in 34 (49%), anti-nuclear antibody (ANA) in 21 (30%), lupus anticoagulant (LA) in 4 (6%) and rheumatoid factor (RF) in 13 (19%) of cases. Autoimmune testing was positive for APA in 2 (5.9%), ANA in 4 (19%), LA in 0 (0%), and RF in 1 (8%) of patients who underwent testing, respectively. Common infectious workups included human immunodeficiency virus in 23 (33%), hepatitis A virus in 2 (3%), hepatitis B virus in 11 (16%), hepatitis C virus in 22 (32%), Epstein-Barr virus in 5 (EBV, 7%), cytomegalovirus in 7 (10%) and Helicobacter pylori in 5 (7%) of patients, and were negative in all cases except for one patient with evidence of active EBV infection. Sixteen patients (23%) underwent bone marrow biopsy, and 2 (12.5%) were diagnosed with hematologic malignancies including myelodysplastic syndrome and hairy cell leukemia. Based on results of these tests, 28 (41%) patients were diagnosed with primary immune thrombocytopenia, 19 (27%) with thrombocytopenia secondary to another medical condition, and 22 (32%) with thrombocytopenia of undefined etiology. CONCLUSION: Thrombocytopenia is a common laboratory finding, and workup involves significant inter-clinician variation, often involving multiple laboratory tests and in some cases invasive tests such as bone marrow biopsy. We found that autoimmune causes of thrombocytopenia were moderately common and infectious and malignant causes were rare. Our findings were based on a small cohort of patients but are likely to be representative of the clinical practice in a large tertiary care center. Large scale studies may be warranted to devise a protocol for a thorough yet cost-effective and stepwise initial workup of thrombocytopenia and to minimize unwarranted inter-clinician variation in such investigations. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders characterized by dysplasia in one or more hematopoietic cell lines, peripheral cytopenia(s), and distinct cytogenetic abnormalities. The presence of certain cytogenetic abnormalities [e.g. 7/del(7q), -5/del(5q), 13/del(13q), i(17p)], in the setting of unexplained persistent cytopenia, has been considered presumptive evidence of MDS, despite definitive morphologic features of MDS. Conversely, chromosomal abnormalities such as isolated 20q- are not considered definitive evidence for MDS, in the absence of dysplasia. Based on our previous findings, patients with isolated 20q- without morphologic features of a myeloid neoplasm have varied clinical outcomes (Jawad MD et al. Am J Hematol.2016). In this study, we investigate the frequency of recurrent somatic gene mutations in these patients, and their relationship to progression to a myeloid neoplasm. Methods We retrospectively reviewed the bone marrow pathology reports of patients evaluated at our institution from 01/2005 to 09/2015 who had isolated 20q- in at least 2 of 20 metaphases. Patients with isolated 20q- and without myeloid neoplasms were included in the study, along with medical record review. A 35-gene next generation sequencing (NGS) panel (OncoHeme) targeting myeloid neoplasms was performed on available archived bone marrow aspirate pellets. Statistical analyses were performed using JMP Pro software version 13. Parametric (t-test) or nonparametric tests such as the Fisher-exact test, chi-square test, or Wilcoxon rank-sum test, were utilized as appropriate for test variables. Statistical significance was based on a 2-sided significance level of .05. Kaplan-Meier analysis was used for analyzing time to progression to myeloid neoplasms and progression-free survival (i.e. time to progression to myeloid neoplasms or death from any cause). Results Fifty-six (42 males, 14 females) patients with isolated 20q- were identified. The average percentage of 20q- was 38.5 % (±29.3). The median age was 68 years (range: 44-90) and the median hemoglobin (Hb), absolute neutrophil count (ANC) and platelet count (Plt) were 11.7 g/dL (range: 8.3-15.1), 2.7 x 10(9)/L (range: 0.6-9.4) and 138 x 10(9)/L (range: 16-392), respectively. The indications for bone marrow biopsies ranged from cytopenia (21), multiple myeloma (20), non-Hodgkin lymphoma (10), amyloidosis (2), CLL (2) and transplant donor evaluation (1). Twenty-three patients harbored pathogenic mutations (15 had one mutation, 1 had two mutations and 7 had three mutations). The identified pathogenic mutations included ASXL1 (8), TET2 (8), SRSF2(3),PHF6 (2), SF3B1 (2), CBL (2), U2AF1 (2), IDH1 (1), IDH2 (1), BCOR (1), JAK2 (1), PTPN11(1), TP53 (1) and RUNX1 (1). Nine patients (16.1%) progressed to myeloid neoplasms (5 MDS, 2 acute myeloid leukemia, 2 chronic myeloid neoplasm-not otherwise specified) at a median follow-up of 36.7 months (range: 1.9-57.4). Of these 9 patients, 5 had one mutation (BCOR, TET2, DNM3TA, CBL, IDH2), 1 had two mutations (SF3B1/CBL) and 3 had three mutations (ASXL1/IDH1/SRSF2,DNM3TA/PTPN11/TET2, PHF6/TET2/TET2). In contrast, only 29.8% of non-progressed cases showed at least 1 mutation. Kaplan-Meier analysis demonstrated the presence of genetic mutations in patients with isolated 20q- carried significantly higher chance of progression to myeloid neoplasms (p<.05, Figure 1A and 1B). However, the number of genetic mutations was not predictive of progression in our relatively small study cohort (p=.66). There was no significant difference between those who did and did not progress with respect to age, gender, Hb, ANC, Plt, 20q- or mutational allele burden. Conclusions Most of the patients with isolated 20q- and without morphologic features of a myeloid neoplasm did not harbor myeloid neoplasm associated gene mutations and further did not develop a myeloid neoplasm during the course of our study. Among patients with pathogenic mutations, nearly one-third showed progression to a myeloid neoplasm. Of the clinical and laboratory variables, the presence of genetic mutation is a significant risk factor for progression to a myeloid neoplasm. This study revealed the underlying molecular heterogeneity of isolated 20q- in the absence of morphologic features of a myeloid neoplasm, and underscored the prognostication value of genetic mutation analysis in these cases. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction: Chronic myelomonocytic leukemia (CMML) is a chronic myeloid malignancy associated with monocytosis, autoimmunity (~30%) & an inherent risk for leukemic transformation. Bone marrow (BM) dendritic cell (DC) populations occur in ~30% of patients, with a poorly defined biological & prognostic role. The malignant immune microenvironment is regulated by indoleamine 2,3-dioxygenase-1 (IDO-1) expressing DCs, which modulate regulatory T (Treg) cells & block their conversion into proinflammatory T helper (Th17)-like cells. IDO-1 is a known immune checkpoint & functions by catabolizing tryptophan, an amino acid essential for T cell function. We hypothesized that distinct IDO-1 expressing DC populations in CMML modulate Tregs & contribute towards immune tolerance & aggressive disease biology. Methods: Primary diagnostic CMML peripheral blood mononuclear cells (PBMC) & BM biopsy specimens were obtained after Mayo Clinic IRB approval. A DC population was defined on H&E stained biopsy sections as focal collections (>10) of cells with characteristic elongated nuclei & cytoplasmic extensions. Transcriptomic & protein expression studies assessing IDO-1 expression were done by previously described methods. In addition, IHC expression of PD-1, PD-L1 & CTLA-4 was also done. IDO-1 promoter methylation studies with DIP-seq were performed. The impact on immune tolerance was assessed using mass cytometry (CyTOF). Results: Cohort: Twenty eight patients with CMML were included in the study, median age 70 (range: 51-80) years; 71% males. Eleven (39%) patients had coexisting autoimmune conditions. Of these, 8 (73%) had detectable DC populations either at diagnosis, or during the course of their disease. At a median follow-up of 46 (95% CI 27, 84) months, there were 14 (50%) deaths & 9 (32%) leukemic transformations.IHC results: Nine (32%) patients were identified to have a DC population at CMML diagnosis. CD123 & TCL1 staining was performed in 5 (56%) patients, with 3 being positive for both, & 2 positive for CD123 only (additional IHC studies ongoing). IDO1 expression by IHC was documented in all 9 (100%) cases (Fig 1A & 1B), while rare populations of PD-1, PD-L1 & CTLA-4 lymphocytes were also seen in all cases. Due to the low DC burdens (median cellularity ≤ 5%) & uniform staining intensity, IHC-based grading was not done. Samples at serial time-points, post-HMA therapy & at the time of blast transformation, were available in 5 & 3 patients respectively. Among the patients who did not have DC populations at diagnosis, 5 (42%) developed them post-HMA therapy, while 3 (50%) developed them at the time of LT. The development of DC populations was associated with loss of response to HMA (50%) & disease progression (50%).Transcriptomic analysis: RNA expression data was available on 7 (25%) patients, of whom only 1 (14%) had DC populations at diagnosis. The IDO-1 RPKM value in the former was higher than the mean pooled value in the latter group (330 versus 74, p=0.05).Methylation studies: DIP-seq was performed on 12 (43%) cases from the primary IHC cohort. Qualitative analysis of IDO-1 promoter hypomethylation was conducted & confirmed in all 9 (100%) cases with 5-mC & 5-hmC marks compared to input as displayed in figure 1C.Immune profiling: CyTOF was performed on 4 CMML samples (3 with IDO-1 expressing DC populations at diagnosis) from the primary IHC cohort & compared to a normal PBMC control. Results confirmed an increase in DC populations (fig 1D& 1E), & reduced % of Th17-like T cells in CMML samples compared to control (1.1 versus 5.07, p=0.05, fig 1F).Clinical correlates & survival analysis: With the exception that CMML patients with DC populations had a higher frequency of NRAS (P=0.007) mutations, the two groups were comparable for cytogenetic & molecular abnormalities. The median OS for the cohort was 45 (95% CI 29, 84) months. CMML patients with IDO-1 expressing DC populations at diagnosis had a shorter median OS, in comparison to those without (median OS 30 vs 45, p=0.03, Kaplan-Meier analysis in fig 1G). Conclusions: In conclusion, we demonstrate that DC populations are seen in ~30% of patients with CMML with a uniform expression of IDO-1 & limited expression of PD-1, PD-L1 & CTLA-4. CMML patients with BM DC populations have a higher frequency of NRAS mutations & DC IDO-1 expression is associated with tumor induced immune tolerance. Additional IHC, genomic & preclinical studies with IDO-1 inhibitors are ongoing. Figure 1. Figure 1. Disclosures Al-Kali: Novartis: Research Funding.