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Neutrophils migrate into inflamed tissue, engage in phagocytosis, and clear pathogens or apoptotic cells. These processes require well-coordinated events involving the actin cytoskeleton. We describe a child with severe neutropenia and episodes of soft tissue infections and pneumonia. Bone marrow examination showed granulocytic hypoplasia with dysplasia. Whole-exome sequencing revealed a de novo heterozygous missense mutation in LCP1, which encodes the F-actin–binding protein Lymphocyte Cytosolic Protein 1. To determine its pathophysiological significance, we stably transduced cells with doxycycline-inducible wild-type LCP1 and LCP1 I232F lentiviral constructs. We observed dysplastic granulocytic 32D cells expressing LCP1 I232F cells. These cells showed decreased proliferation without a block in differentiation. In addition, expression of LCP1 I232F resulted in a cell cycle arrest at the G2/M phase, but it did not lead to increased levels of genes involved in apoptosis or the unfolded protein response. Both 32D and HeLa cells expressing mutant LCP1 displayed impaired cell motility and invasiveness. Flow cytometry showed increased F-actin. However, mutant LCP1-expressing 32D cells exhibited normal oxidative burst upon stimulation. Confocal imaging and subcellular fractionation revealed diffuse intracellular localization of LCP1, but only the mutant form was found in the nucleus. We conclude that LCP1 is a new gene involved in granulopoiesis, and the missense variant LCP1 I232F leads to neutropenia and granulocytic dysplasia with aberrant actin dynamics. Our work supports a model of neutropenia due to aberrant actin regulation.

Classification of myeloid neoplasms with isolated isochromosome i(17q) [17p deletion with inherent monoallelic TP53 loss plus 17q duplication] is controversial. Most cases fall within the WHO unclassifiable myelodysplastic/myeloproliferative neoplasms (MDS/MPN-U) category. The uniformly dismal outcomes warrant better understanding of this entity. We undertook a multi-institutional retrospective study of 92 adult MDS/MPN-U cases from eight institutions. Twenty-nine (32%) patients had isolated i(17q) [MDS/MPN-i(17q)]. Compared to MDS/MPN without i(17q), MDS/MPN-i(17q) patients were significantly younger, had lower platelet and absolute neutrophil counts, and higher frequency of splenomegaly and circulating blasts. MDS/MPN-i(17q) cases showed frequent bilobed neutrophils (75% vs. 23%; P = 0.03), hypolobated megakaryocytes (62% vs. 20%; P = 0.06), and a higher frequency of SETBP1 (69% vs. 5%; P = 0.002) and SRSF2 (63% vs. 5%; P = 0.006) mutations that were frequently co-existent (44% vs. 0%; P = 0.01). TP53 mutations were rare. The mutation profile of MDS/MPN-U-i(17q) was similar to other myeloid neoplasms with i(17q) including atypical chronic myeloid leukemia, chronic myelomonocytic leukemia, myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis, myelodysplastic syndrome and acute myeloid leukemia, with frequent concomitant SETBP1/SRSF2 mutations observed across all the diagnostic entities. Over a median follow-up of 52 months, patients with MDS/MPN-i(17q) showed a shorter median overall survival (11 vs. 28 months; P < 0.001). The presence of i(17q) retained independent poor prognostic value in multivariable Cox-regression analysis [HR 3.686 (1.17-11.6); P = 0.026] along with splenomegaly. We suggest that MDS/MPN-i(17q) warrants recognition as a distinct subtype within the MDS/MPN-U category based on its unique clinico-biologic features and uniformly poor prognosis.

PURPOSE Therapy-related acute myeloid leukemias (t-AML) are a heterogenous group of aggressive neoplasms that arise following exposure to cytotoxic chemotherapy and/or ionizing radiation. Many therapy-related myeloid neoplasms (t-MN) are associated with distinct chromosomal aberrations and/or TP53 alterations, but little is known about the clinicopathologic and molecular features of normal karyotype t-AML (NK-t-AML) and whether this t-MN subtype is distinctly different from NK de novo AML (NK-dn-AML). METHODS This multi-institutional study by the Bone Marrow Pathology Group retrospectively evaluated clinicopathologic and molecular characteristics of 335 patients with NK-AML, comprising 105 t-AML and 230 dn-AML cases. RESULTS Patients with t-AML compared with dn-AML exhibit significantly shorter overall survival (OS; median months: 17.6 v 44.2; P < .0001) and relapse-free survival (RFS; median months: 9.1 v 19.2; P = .0018). Frequency of NPM1, FLT3, KRAS, and GATA2 mutations were significantly different in NK-t-AML compared with NK-dn-AML ( NPM1 35% v 49%; P = .0493; FLT3 23% v 36%; P = 0494; KRAS 12% v 5%; P = .0465; GATA2 9% v 2% P = .0105), while TP53 mutations were rare. Patients with t-AML more often stratified into intermediate or adverse 2017 ELN genetic risk groups. Favorable ELN risk predicted favorable OS (hazard ratio [HR], 0.4056; 95% CI, 0 to 0.866; P = .020) and RFS (HR, 0.355; 95% CI, 0 to 0.746; P = .006). Among all patients with NK-AML, stem-cell transplant and favorable ELN risk both significantly affected RFS, while therapy-relatedness and age had a borderline significant impact on OS (HR, 1.355; 95% CI, 0.975 to 1.882; P = .070). CONCLUSION To our knowledge, this is the largest study to date to comprehensively evaluate NK-t-AML and provides a framework that may inform our understanding of NK-t-AML disease biology and could potentially help guide therapeutic management and improved disease classification in t-MNs that lack cytogenetic aberrations.

Myeloid/lymphoid neoplasms (M/LN) with 13q12/FLT3 rearrangement have been suggested as candidates for possible inclusion in the World Health Organization classification group of M/LN with eosinophilia (M/LN-eo). We report 12 patients with confirmed FLT3 rearrangement, six with t(12;13)/ETV6-FLT3; one with ins(13;22)/BCR-FLT3; and five with an unconfirmed partner gene located on chromosome bands 2p16, 3q27, 5q15, 5q35, and 7q36. Disease presentations were heterogeneous, including lymphoblastic leukemia/lymphoma, myeloid sarcoma, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, and myelodysplastic syndrome. However, some common features were observed, such as extramedullary involvement (n = 7, 58%), associated eosinophilia in blood, bone marrow, or tissue (n = 8, 67%), multilineage involvement, either as biphasic myeloid/lymphoid neoplasms (n = 2) or mixed phenotype acute leukemia (n = 2). Mutations were detected in 4/8 (50%) patients by next-generation sequencing. None (0/10) had FLT3 or KIT mutations. Eleven patients received disease-based chemotherapy or hypomethylating agents, three received FLT3 inhibitors, and five patients proceeded to hematopoietic stem cell transplant. Together with a review of 16 cases published in the literature, it is apparent that M/LNs with FLT3 rearrangement show disease features reminiscent of members in the category of M/LN-eo with PDGFRA, PDGFRB, FGFR1, and PCM1/JAK2 rearrangement, characterized by a specific gene rearrangement, frequent eosinophilia, multi-lineage involvement and therapeutic benefit from kinase inhibitors.

JAK2, CALR, and MPL are myeloproliferative neoplasm (MPN)-driver mutations, whereas SF3B1 is strongly associated with ring sideroblasts (RS) in myelodysplastic syndrome (MDS). Concomitant mutations of SF3B1 and MPN-driver mutations out of the context of MDS/MPN with RS and thrombocytosis (MDS/MPN-RS-T) are not well-studied. From the cases (

Here we investigate single codon SF3B1 mutations (K700E, K666, H662) to identify whether discrete amino acid substitutions may influence clinical phenotypes, survival and response to treatments in ...

Enumeration of blasts and promonocytes is essential for World Health Organization (WHO) classification of myelomonocytic neoplasms. The accuracy of distinguishing blasts, promonocytes and monocytes, including normal vs abnormal monocytes, remains controversial. The objective of this analysis is to assess concordances between experienced hematopathologists in classifying cells as blasts, promonocytes, and monocytes according to WHO criteria. Each of 11 hematopathologists assessed glass slides from 20 patients [12 with chronic myelomonocytic leukemia (CMML) and 8 with acute myeloid leukemia (AML)] including blood and BM aspirate smears, and limited nonspecific esterase (NSE) stains. All cases were blindly reviewed. Fleiss' extension of Cohen's kappa for multiple raters was used on these variables, separately for peripheral blood (PB) and bone marrow (BM). Spearman's rank correlation was used to assess correlations between each pair of hematopathologists for each measurement. For the classification based on the sum of blasts and promonocytes in the BM, Fleiss' kappa was estimated as 0.744. For PB, categorizing patients according to the sum of blasts and promonocytes, Fleiss' kappa was estimated as 0.949. Distinction of abnormal monocytes from normal monocytes in PB did not achieve a good concordance and showed strong evidence of differences between hematopathologists (P

Megaloblastic anemia causes macrocytic anemia from ineffective red blood cell production and intramedullary hemolysis. The most common causes are folate (vitamin B9) deficiency and cobalamin (vitamin B12) deficiency. Megaloblastic anemia can be diagnosed based on characteristic morphologic and laboratory findings. However, other benign and neoplastic diseases need to be considered, particularly in severe cases. Therapy involves treating the underlying cause-eg, with vitamin supplementation in cases of deficiency, or with discontinuation of a suspected medication.

Acute undifferentiated leukemia is a rare type of acute leukemia that shows no evidence of differentiation along any lineage. Clinical, immunophenotypic and genetic data is limited and it is uncertain if acute undifferentiated leukemia is biologically distinct from acute myeloid leukemia with minimal differentiation, which also shows limited myeloid marker expression and has been reported to have a poor prognosis. We identified 92 cases initially diagnosed as acute undifferentiated leukemia or acute myeloid leukemia with minimal differentiation from pathology databases of nine academic institutions with available diagnostic flow cytometric data, cytogenetic findings, mutational and clinical data. Outcome analysis was performed using Kaplan Meier test for the 53 patients who received induction chemotherapy. Based on cytogenetic abnormalities (N = 30) or history of myelodysplastic syndrome (N = 2), 32 cases were re-classified as acute myeloid leukemia with myelodysplasia related changes. The remaining 24 acute undifferentiated leukemia patients presented with similar age, blood counts, bone marrow cellularity, and blast percentage as the remaining 30 acute myeloid leukemia with minimal differentiation patients. Compared to acute myeloid leukemia with minimal differentiation, acute undifferentiated leukemia cases were characterized by more frequent mutations in PHF6 (5/15 vs 0/19, p = 0.016) and more frequent expression of TdT on blasts (p = 0.003) while acute myeloid leukemia with minimal differentiation cases had more frequent CD123 expression (p = 0.042). Outcome data showed no difference in overall survival, relapse free survival, or rates of complete remission between acute undifferentiated leukemia and acute myeloid leukemia with minimal differentiation groups (p > 0.05). Acute myeloid leukemia with myelodysplasia-related changes patients showed shorter survival when censoring for bone marrow transplant as compared to acute undifferentiated leukemia (p = 0.03) and acute myeloid leukemia with minimal differentiation (p = 0.002). In this largest series to date, the acute undifferentiated leukemia group shows distinct characteristics from acute myeloid leukemia with minimal differentiation, including more frequent PHF6 mutations and expression of TdT.

To describe the clinical, histologic, immunophenotypic, and genetic characteristics of myeloid sarcoma (MS) diagnosed in the testes of adults, 3 cases were identified, and information on their presentation, clinical features, treatment, and outcome was retrieved from the medical records. In addition, histologic, immunophenotypic, and molecular characteristics were reviewed. This showed that all patients had a previous history of acute myeloid leukemia (AML), in 2 cases diagnosed10 years before the testicular lesions. In 1 case, there was bilateral involvement, while in 2, involvement was unilateral. The neoplastic cells showed evidence of cytogenetic/molecular clonal evolution in all cases, 1 of which also had significant immunophenotypic changes. A mutational profile including NPM1 p.Trp288Cysfs*12, IDH1 p.Arg132His NRAS p.Gly12Asp was seen in 2 of the 3 cases. Concurrent bone marrow involvement by a myeloid neoplasm was diagnosed in 2 patients, in 1, there was AML in the second 8% blasts. These patients progressed rapidly after MS and had a dismal outcome. The patient with no concurrent bone marrow disease had a favorable outcome. In conclusion, MS involving the testes of adults is a rare event, and it may represent the clonal evolution of AML.

Myeloid/lymphoid neoplasms (M/LN) with 13q12/FLT3 rearrangement have been suggested as candidates for possible inclusion in the World Health Organization classification group of M/LN with eosinophilia (M/LN-eo). We report 12 patients with confirmed FLT3 rearrangement, six with t(12;13)/ETV6-FLT3; one with ins(13;22)/BCR-FLT3; and five with an unconfirmed partner gene located on chromosome bands 2p16, 3q27, 5q15, 5q35, and 7q36. Disease presentations were heterogeneous, including lymphoblastic leukemia/lymphoma, myeloid sarcoma, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, and myelodysplastic syndrome. However, some common features were observed, such as extramedullary involvement (n = 7, 58%), associated eosinophilia in blood, bone marrow, or tissue (n = 8, 67%), multilineage involvement, either as biphasic myeloid/lymphoid neoplasms (n = 2) or mixed phenotype acute leukemia (n = 2). Mutations were detected in 4/8 (50%) patients by next-generation sequencing. None (0/10) had FLT3 or KIT mutations. Eleven patients received disease-based chemotherapy or hypomethylating agents, three received FLT3 inhibitors, and five patients proceeded to hematopoietic stem cell transplant. Together with a review of 16 cases published in the literature, it is apparent that M/LNs with FLT3 rearrangement show disease features reminiscent of members in the category of M/LN-eo with PDGFRA, PDGFRB, FGFR1, and PCM1/JAK2 rearrangement, characterized by a specific gene rearrangement, frequent eosinophilia, multi-lineage involvement and therapeutic benefit from kinase inhibitors.

Thrombocytopenia 2 (THC2) is an autosomal dominant disorder characterized by ankyrin repeat domain 26 mutation and moderate thrombocytopenia. THC2 exposes patients to a low risk of bleeding and an increased likelihood of myelodysplastic syndrome/acute myeloid leukemia. Germline evaluation for a genetic disorder should be considered when a patient presents with isolated thrombocytopenia and associated dysmegakaryopoiesis. In this case report, we present a male patient who presented with isolated thrombocytopenia but was ultimately confirmed to have an inherited THC2 thrombocytopenia/myelodysplastic syndrome. Given the rarity of the disease, no clear guidelines on how to follow THC2 patients over the long term have been established. We recommend a monthly complete blood count and clinical visits every 3 months at a minimum.

JAK2, CALR, and MPL are myeloproliferative neoplasm (MPN)-driver mutations, whereas SF3B1 is strongly associated with ring sideroblasts (RS) in myelodysplastic syndrome (MDS). Concomitant mutations of SF3B1 and MPN-driver mutations out of the context of MDS/MPN with RS and thrombocytosis (MDS/MPN-RS-T) are not well-studied. From the cases (5% blasts) tested by NGS panels interrogating at least 42 myeloid neoplasm-related genes, we identified 18 MDS/MPN-RS-T, 42 MPN, 10 MDS, and 6 MDS/MPN-U cases with an SF3B1 and an MPN-driver mutation. Using a 10% VAF difference to define "SF3B1-dominant," "MPN-mutation dominant," and "no dominance," the majority of MDS/MPN-RS-T clustered in "SF3B1-dominant" and "no dominance" regions. Aside from parameters as thrombocytosis and ≥15% RS required for RS-T, MDS also differed in frequent neutropenia, multilineage dysplasia, and notably more cases with10% VAF of MPN-driver mutations (60%, p = 0.0346); MPN differed in more frequent splenomegaly, myelofibrosis, and higher VAF of "MPN-driver mutations." "Gray zone" cases with features overlapping MDS/MPN-RS-T were observed in over one-thirds of non-RS-T cases. This study shows that concomitant SF3B1 and MPN-driver mutations can be observed in MDS, MPN, and MDS/MPN-U, each showing overlapping but also distinctively different clinicopathological features. Clonal hierarchy, cytogenetic abnormalities, and additional somatic mutations may in part contribute to different disease phenotypes, which may help in the classification of "gray zone" cases.

The ongoing COVID-19 pandemic originated in Wuhan, Hubei Province, China, in December 2019. The etiologic agent is a novel coronavirus of presumed zoonotic origin with structural similarity to the viruses responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). Like SARS and MERS, COVID-19 infection manifests most frequently with lower respiratory symptoms. A minority of patients progress to acute respiratory distress syndrome/ diffuse alveolar damage. In addition to its central role in the diagnosis of COVID-19 infection, the clinical laboratory provides critical information to clinicians regarding prognosis, disease course, and response to therapy. The purpose of this review is to (a) provide background context about the origins and course of the pandemic, (b) discuss the laboratory's role in the diagnosis of COVID-19 infection, (c) summarize the current state of biomarker analysis in COVID-19 infection, with an emphasis on markers derived from the hematology laboratory, (d) comment on the impact of COVID-19 on hematology laboratory safety, and (e) describe the impact the pandemic has had on organized national and international educational activities worldwide.

This multi-institutional study retrospectively evaluated clinicopathologic and genetic characteristics in 351 patients with core-binding-factor acute myeloid leukemia (CBF-AML), comprising 69 therapy-related (t-CBF-AML) and 282 de novo cases. The T-CBF-AML patients were older, had lower WBC counts, and slightly higher hemoglobin than patients with de novo disease. Secondary cytogenetic abnormalities were more frequent in patients with de novo disease than t-CBF-AML (57.1% vs 41.1%, P = .026). Patients with secondary cytogenetic abnormalities had longer overall survival (OS) than those without abnormalities (median 190 vs 87 months, P = .021); trisomy 8, trisomy 22, and loss of the X or Y chromosome were associated with longer OS. In the 165 cases performed of targeted gene sequencing, pathogenic mutations were detected in 75.7% of cases, and were more frequent in de novo than in therapy-related disease (P = .013). Mutations were found in N/KRAS (37.0%), FLT3 (27.8%), KIT (17.2%), TET2 (4.9%), and ASXL1 (3.9%). The TET2 mutations were associated with shorter OS (P = .012) while N/KRAS mutation was associated with longer OS in t(8;21) AML patients (P = .001). The KIT mutation did not show prognostic significance in this cohort. Although they received similar therapy, t-CBF-AML patients had shorter OS than de novo patients (median 69 vs 190 months, P = .038). In multivariate analysis of all patients, older age and absence of any secondary cytogenetic abnormalities were significant predictors of shorter OS. Among the t-CBF-AML subset, age and hemoglobin were significant on multivariate analysis. This study demonstrated that although de novo and t-CBF-AML patients share many features, t-CBF-AML patients have worse clinical outcome than de novo patients.

Components of the pre-messenger RNA splicing machinery are frequently mutated in myeloid malignancies. Mutations in LUC7L2, PRPF8, SF3B1, SRSF2, U2AF1, and ZRSR2 genes occur at various frequencies ranging between 40% and 85% in different subtypes of myelodysplastic syndrome (MDS) and 5% and 10% of acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPNs). In some instances, splicing factor (SF) mutations have provided diagnostic utility and information on clinical outcomes as exemplified by SF3B1 mutations associated with increased ring sideroblasts (RS) in MDS-RS or MDS/MPN-RS with thrombocytosis. SF3B1 mutations are associated with better survival outcomes, while SRSF2 mutations are associated with a shorter survival time and increased AML progression, and U2AF1 mutations with a lower remission rate and shorter survival time. Beside the presence of mutations, transcriptomics technologies have shown that one third of genes in AML patients are differentially expressed, leading to altered transcript stability, interruption of protein function, and improper translation compared to those of healthy individuals. The detection of SF mutations demonstrates the importance of splicing abnormalities in the hematopoiesis of MDS and AML patients given the fact that abnormal splicing regulates the function of several transcriptional factors (PU.1, RUNX1, etc.) crucial in hematopoietic function. This review provides a summary of the significance of the most frequently mutated SF genes in myeloid malignancies and an update on novel targeted therapies in experimental and clinical trial stages.

The 2016 WHO update changed the diagnostic criteria for myeloid neoplasms with erythroid predominance, limiting the diagnosis of acute myeloid leukemia to cases with ≥20% blasts in the bone marrow or peripheral blood. Although acute myeloid leukemia with ≥50% erythroid cells has historically been presumed to represent acute myeloid leukemia with myelodysplasia-related changes, this hypothesis has never been systematically examined. We sought to investigate the clinicopathologic, cytogenetic, and molecular features of acute myeloid leukemia with erythroid predominance to subclassify cases as defined by the 2016 WHO. We retrospectively identified patients with ≥50% erythroid precursors and either ≥20% bone marrow blasts or ≥20% peripheral blood blasts at the time of initial diagnosis at seven major academic centers. Laboratory and clinical data were obtained. Patients were then reclassified according to 2016 WHO guidelines. A matched control group was also obtained. We identified 146 patients with acute myeloid leukemia with erythroid predominance (62% M, average age: 62 y, range: 5-93 y). Of these, 91 were acute myeloid leukemia with myelodysplasia-related changes, 20 (14%) were therapy-related myeloid neoplasm, 23 (16%) acute myeloid leukemia, not otherwise specified, and ten acute myeloid leukemia with recurrent cytogenetic/molecular abnormalities. The bone marrow blast count ranged from 9-41%. There was no difference in survival for patients with erythroid predominance compared to patients with acute myeloid leukemia without erythroid proliferations. In a multivariable analysis, cytogenetic risk was the only significant predictor of survival. We find a significantly lower rate of FLT3 and RAS pathway alterations in acute myeloid leukemia with erythroid predominance compared to controls. Our study is one of the first to apply the 2016 WHO guidelines for classification of acute myeloid leukemia. We find acute myeloid leukemia with erythroid predominance is a heterogeneous group and that erythroid richness has no impact on overall survival.

The JAK2 V617F mutation is characteristic of most Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) and occurs rarely in de novo acute myeloid leukaemia (AML). We sought to characterize AMLs that harbour this mutation and distinguish those that arise de novo (AML-DN) from those that reflect transformation of an underlying MPN (AML-MPN). Forty-five patients with JAK2 V617F-mutated AML were identified; 15 were AML-DN and 30 were AML-MPN. AML-MPN cases were more likely to have splenomegaly (P = 0·02), MPN-like megakaryocytes and higher mean JAK2 V617F VAF at diagnosis (P = 0·04). Mutations involving TET2 were exclusively identified in AML-DN patients. Mutations of genes affecting DNA methylation were more common in AML-DN (P < 0·01). A complex karyotype was more frequent in AML-MPN cases than in AML-DN (P < 0·01), with AML-DN more likely to display a normal karyotype (P = 0·02). Bone marrow histology after recovery from induction chemotherapy in AML-DN cases revealed no morphological evidence of any previously occult MPNs, while this was evident in most of the AML-MPN specimens (P < 0·01). These findings in this largest study of JAK2 V617F-mutated AMLs indicate that AML-DN is distinct from AML-MPN.

Objective T-cell large granular lymphocytic (T-LGL) leukemia is associated with B-cell lymphomas (BCLs), especially small BCLs. We aimed to explore and expand upon its association with BCLs. Methods We retrospectively studied clinicopathologic features of T-LGL leukemia patients with coexisting BCL from January 2001 to December 2016. Results Among 432 patients with T-LGL leukemia, 22 (5.1%) had an associated B-cell non-Hodgkin lymphoma. Thirteen (59%) patients had large and nine (41%) had small BCL. T-LGL leukemia occurred synchronously with BCL in five, preceded BCL in three, and followed BCL in 14 patients. Anemia was the most common cytopenia (68%). Only one patient had a history of rheumatoid arthritis. Conclusion To our knowledge, this is the first multicenter study looking at the spectrum and incidence of BCLs in patients with T-LGL leukemia and highlights its association with large BCLs (3% of T-LGL leukemias).

Extramedullary deposits of leukemic blasts, also often called myeloid sarcoma (MS), constitute an unusual presentation of AML, which can occur in isolation (IMS, often in the de novo setting) or in the context of a fully-blown leukemic process (LMS) either at diagnosis or relapse. This peculiar variant of an acute leukemia may be due to distinct phenotypic features promoting homing of blasts to non-hematopoietic locations or/and be a result of specific molecular lesions (perhaps related to a specific genotype). So far, systematic studies of these phenomenon have been scarce with the largest cohorts reporting on 70 pts and less over the last decade. By applying machine learning (ML) to perform latent class analysis of molecular features in AML patients (n=6,788), we have identified and validated 4 molecular clusters characterized by distinct clinical phenotypes (Awada et al Blood 2021). Genomic cluster-1 (GC-1) was characterized by predominantly normal karyotype and NPM1 MT that co-occurred with DNMT3A MT and FLT3 ITD, and IDH2 R140. In GC-2 mostly abnormal karyotype was present with CEBPA Bi, and IDH2 R172K, FLT3 ITD/TKD and lack of NPM1 MT. GC-3 had highest frequency of ASXL1 MT, BCORL1 MT, and DNMT3A MTalong with splicing factor mutations. Finally, GC-4 had the highest percentage of complex karyotype and TP53 MT. We observed a significant difference in survival [medians, months] across the molecularly-defined subgroups: GC-1 [34.1]; GC-2 [26.5]; GC-3 [15.8]; and GC-4 [9.2]. However, the genomic clustering (GCs 1-4) did not favor pAML over sAML (progressed from previous myeloid disorders). These unbiased molecular features were then compared to those found in 91 MS pts (median age 64 years) to discern their possible origins from specific genomic AML clusters vs. an as yet described unique MS genomic signature. Among the 91 pts, 19% of them were IMS while 81% were LMS (of which 43% were de novo vs. 57% refractory/relapsed). Soft tissue (32%), skin (22%), lymph nodes (13%), and gastrointestinal tract (13%) were the most common extramedullary sites involved. An involvement of more than two sites was observed in 15 (16.5%) cases. MS-specific phenotypic features were explored based on immunohistochemical (IHC) stains and flow cytometry studies. MS neoplastic cells showed reactivity with CD43 (54% of cases), muramidase/lysozyme (53%), CD117 (39%), myeloperoxidase (35%), CD33 (32%), CD45 (30%), and CD34 (30%). Flow cytometry of the LMS cases demonstrated that 90% of patients expressed surface CD13, CD33, CD45, and HLA-DR while positivity for CD38 was present in 83% of cases, and CD117 and CD34 in 60%. By comparison to our AML cohort, MS patients had a significant higher median age at the time of diagnosis (64 vs. 62 years p=0.041) and leukocyte counts (WBC 7k vs. 4k/uL, p=0.002), higher percentage of sAML (67% vs. 40% p Disclosures Balasubramanian: Servier Pharmaceuticals: Research Funding. Patel: Apellis: Consultancy, Other: educational talks, Speakers Bureau; Alexion: Consultancy, Other: educational talks, Speakers Bureau. Mukherjee: McGraw Hill: Honoraria, Other: Editor of Hematology Oncology Board Review (ongoing); Acceleron: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Membership on an entity's Board of Directors or advisory committees; AAMDS in Joint Partnership with Cleveland Clinic Taussig Cancer Institute: Honoraria; Genentech: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb Co.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Research/Independent Contractor, Research Funding; Eusa Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Teaching and Speaking; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research/Independent Contractor, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Partnership for Health Analytic Research: Honoraria; BioPharm: Consultancy; Jazz Pharmaceuticals: Research Funding. Advani: Macrogenics: Research Funding; Glycomimetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Research Funding; Kite Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Immunogen: Research Funding; OBI: Research Funding; Abbvie: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Carraway: Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Other: Independent review committee; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Astex: Other: Independent review committee; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy.

Introduction: As critical effectors of innate immune response, neutrophils migrate from the vasculature into inflamed tissue, where they engage in phagocytosis and clearance of pathogens and apoptotic cells. Migration, phagocytosis, and granule release require multiple well-coordinated events involving remodeling of the actin cytoskeleton. While mutations of some regulators of actin polymerization, such as Wiskott-Aldrich Syndrome gene (WAS) result in Severe Congenital Neutropenia (SCN), the role of actin cytoskeleton in regulating neutrophil ontogeny and functions remain poorly understood. Lymphocyte Cytosolic Protein 1 (LCP1) is an actin bundling protein encoded by LCP1 gene. A 7-year-old girl presented at age of 2 years with chronic severe neutropenia and frequent infections. Next generation sequencing did not reveal a variant in genes known to cause neutropenia. Whole exome sequencing identified a novel LCP1 p.I232F variant of unknown significance, which was not present in either parent. Bone marrow examination revealed a granulocytic hypoplasia with significant dysmorphic giant granulocytes with complex hypersegmentation and increased apoptosis. (Figure 1A) Based on these observations, we hypothesized that missense mutation in LCP1 (I232F) impairs granulocytic proliferation, and causes neutropenia. Aims: To investigate the role of LCP1 I232F, discovered in a child with severe symptomatic neutropenia, in granulopoiesis. Methods: in vitro experiments involving the expression of wild type and mutant LCP1 I232F in murine myeloblast cell (32D cells) and human cervical cancer HeLa cell. We deployed various biologic and functional studies to investigate the role of LCP1 I232F in granulocyte proliferation, differentiation, survival, and function. Results: To determine the pathophysiologic significance of LCP1 I232F, we stably transduced interleukin 3-dependent murine myeloblast 32D and human cervical cancer HeLa cell lines with a doxycycline-inducible lentiviral constructs containing the cDNA for either wild type LCP1 or LCP1 I232F. The mutant LCP1 I232F expressing 32D cells showed impaired proliferation with the appearance of dysplastic granulocytic cells. (Figure 1A, B) However, we did not observe block in differentiation. A cell cycle arrest at G2/M phase occurred only in the LCP1 I232F expressing cells (Figure 1B) and was associated with an upregulation of markers of cell cycle arrest (Cdkn1a and Tp53). LCP1 I232F overexpression did not lead to increased expression of genes involved in apoptosis or the unfolded protein response. The LCP1 I232F cells with dysplastic features were further analyzed by confocal microscopy, which demonstrated increased level of F-actin and enrichment of F-actin and LCP1 at the cell cortex. Flow cytometric evaluation of these cells further confirmed the presence of increased level of F actin. (Figure 1C) Expression of LCP1 I232F impaired cell motility and invasiveness in both 32D and HeLa cells. However, mutant LCP1 expressing 32D cells demonstrated normal oxidative burst upon treatment with N-formyl-Met-Leu-Phe (fMLP) and phorbol myristate acetate (PMA). Confocal imaging and subcellular fractionation revealed diffuse localization of LCP1, but only the mutant form was found in the nucleus. (Figure 1C) In LCP1, two tandem CH domains (CH1-CH2 and CH3-CH4) formed two actin-binding domains (ABD1 and ABD2) that bind to actin to participate in actin bundling. I232F is located at LCP1-CH1 that interacts with actin. To study how the mutation impacts LCP1-actin binding, we constructed a model of LCP1-ABD1 based on the cryo-EM structure of F-actin/LCP1-ABD2 and performed 40 ns of molecular dynamics (MD) simulations of LCP1-ABD1 and LCP1-ABD1 I232F alone to study their dynamical motions. We found that LCP1-ABD1 I232F adopted a more stable open conformation than LCP1-ABD1 to bind effectively with F-actin. The model implies a prolonged association of LCP1 I232F with actin than wild-type LCP1, and the consequence is the increased stabilization of actin bundles. Conclusions: Like the WAS gain-of-function mutations in SCN, LCP1 I232F resulted in severe neutropenia with dysplastic granulocytic precursors, cell cycle arrest, impaired motility, and increased F actin. We also found decreased proliferation of granulocytic progenitor and precursor cells. Our findings suggest that LCP1 is an important gene involved in granulopoiesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Inversion or translocation of the chromosome 3, specifically inv(3)(q21q26.2/ t(3;3)(q21;q26.2) are present in 1-2% of acute myeloid leukemia (AML) cases and are classified as a distinct entity in the 2016 WHO classification. Hallmark genetic alterations in this entity include mutations in GATA2 and MECOM. In fact, these rearrangements result in over activation of MECOM due to juxtaposition with a distal GATA2 enhancer. Cytomorphologic phenotypes include anemia, normal to elevated platelets and multilineage dysplasia in a hyperplastic bone marrow (BM). Studies have shown the frequent occurrence of NF1, NRAS and RUNX1 mutations. While proceeding towards our molecularly informed AML subtyping (Awada, Blood 2019;1406), we observed a high occurrence of somatic mutations in the splicing factor SF3B1 in inv(3)/t(3;3) AML. We were particularly intrigued by this observation considering several key aspects of SF3B1 mutations in the context of MDS. For instance, SF3B1 mutations are highly associated with clear phenotypic and morphologic features and carry favorable prognosis in MDS. These mutations are often found in patients carrying less deleterious abnormalities [e.g., del(5q)] and their founder clonal nature has been uncovered through experimental studies. Recent studies unveiled the occurrence of SF3B1 mutations in de novo AML and low complete remission rate when combined with other mutations (e.g., DNMT3A). To investigate whether SF3B1 mutations were unequivocally frequent in inv(3)/t(3;3) AML compared to other splicing factor mutations, we moved forward in dissecting the clinical, morphologic and molecular profiles of these cases. We analyzed results from whole exome sequencing and targeted deep sequencing from the Cleveland Clinic and publicly available data of AML with inv(3)/t(3;3) (de novo AML, n=32; secondary AML from antecedent myeloid neoplasms, n=11; t-AML, n=1). In our cohort, mutations in the most common components of the RNA splicing machinery (SF3B1, SRSF2, U2AF1, ZRSR2) were observed in 27% (n=12) of the patients. Among splicing factor mutations, SF3B1 was the most mutated gene (77%; 10/13 total mutations); 7 cases had inv(3) and 3 had t(3;3). Mutations were observed at canonical sites: K700E (70%) and K666N (30%) with no difference compared to the hotspots observed in MDS. Sixty% of patients were female. Median age was 61 years (range, 36-73). Anemia was present in 50%, leukopenia in 10% and thrombocytopenia in 50% of the patients. For 40% of the cases, BM smears for iron staining was available and showed absence of ringed sideroblasts. Complex karyotype (CK) was present in 20% of the patients; -7/del(7q) was present as the only cytogenetic abnormality in 30% or with CK in 10% pf the cases. Variant allele frequency (VAF) of SF3B1mutations in inv(3)/t(3;3) was not different than the those without inv(3)/t(3;3) (42% vs 40%). Survival analysis was performed in 3 subgroups: SF3B1MT AML (n=70), SF3B1MT AML + inv(3)/t(3;3) (n=10), AML + inv(3)/t(3;3) (n=34). SF3B1MT AML + inv(3)/t(3;3) and AML + inv(3)/t(3;3) had similar OS (11.7 vs 9.7 months) which was shorter than the that of SF3B1MT AML without any inv(3)/t(3;3) (19.4 months, P=0.002) suggesting that SF3B1MT in the context of inv(3)/t(3;3) might hold a different prognostic significance strongly due to the presence of inv(3)/t(3;3). Given this observation, we delved into the clonal diversity of SF3B1 mutations and its co-occurrence with other molecular mutations. Comparison of VAFs showed that SF3B1 mutations in relation to other mutations were dominant/founder in 30%, secondary/subclonal in 20% while co-dominant to another gene (VAF differences In sum, we describe that SF3B1 mutations occur in combination with inv(3)/t(3;3) in AML and might represent a subclass of this entity in which lesions in SF3B1 gene could potentially hide a cryptic association between splicing abnormalities and disease phenotypes. Figure 1 Disclosures Maciejewski: Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Objectives Isolated deletion of the long arm of chromosome 16 (del(16q)) is rare in myeloid neoplasms (MNs) and was historically considered a variant of inv(16)(p13.1q22) (inv(16)), a subtype of acute myeloid leukemia (AML) associated with CBFB-MYH11 rearrangement and favorable prognosis. This study aims to determine clinicopathologic characteristics of patients with isolated del(16q) in MNs in comparison to AMLs with isolated inv(16). Methods Clinicopathologic features were retrospectively reviewed in 18 MNs with del(16q) and 34 AMLs with inv(16) patients from seven institutions. Results MNs with del(16q) occurred in elderly patients, often as secondary MNs. Blood monocytes and marrow eosinophils were lower in del(16q) than inv(16). Deletion of CBFB but not CBFB-MYH11 rearrangement was confirmed by fluorescence in situ hybridization or reverse transcription polymerase chain reaction in 14 of 14 del(16q) patients. The median overall survival was shorter in del(16q) than in inv(16) patients (12 vs 94 months, log rank P = .0002). Conclusions Myeloid neoplasms with isolated del(16q) with deletion of the CBFB but lacking CBFB-MYH11 rearrangement should not be considered a variant of the AML-defining inv(16).

Objectives Single-nucleotide polymorphism (SNP) arrays have been shown to identify cytogenetic abnormalities in myeloid neoplasms that may be missed by metaphase cytogenetics alone at initial diagnosis. This study examines the utility of serial SNP arrays in follow-up testing of myeloid neoplasms. Methods We retrospectively reviewed results of SNP array testing in 44 patients with myeloid neoplasms and more than one SNP array study (n = 133 SNP arrays total; median, three per patient; range, two to eight per patient). Results Baseline abnormalities were identified by SNP array in 35 (79%) of 44 (79%) compared with 18 (50%) of 36 by metaphase karyotype. In follow-up studies, clonal evolution was found by both SNP array and karyotyping in seven (15.9%), by metaphase karyotyping alone in six (13.6%), and SNP arrays alone in two (4.5%). Overall survival was not significantly different between patients with or without clonal evolution detected by SNP array. Conclusions This study, the first systematic examination of serial SNP arrays in myeloid neoplasms, confirms the clinical utility of SNP arrays at initial diagnosis but shows that clonal evolution of the karyotype can be detected by metaphase cytogenetics alone in most patients. Follow-up SNP array testing is not required in routine clinical use in most cases.

Molecular lesions have diagnostic and prognostic impacts in myeloid neoplasia (MN). The beau idéal is the presence of SF3B1MT in MDS with ringed sideroblasts. Although most of these patients have a classic phenotype, little is known about the factors diverting it during the clonal evolution. Clinical trajectories of patients with SF3B1MT might depend on clonal hierarchy, dynamics and subclonal diversity in relation to other lesions. Herein, we studied the molecular architecture of patients with SF3B1MT to determine whether clonality and rank might infer distinct MDS features. We collected molecular and clinical information of 3561 patients with MN from Cleveland Clinic and publicly shared data. Results of targeted deep sequencing of 176 MDS/ AML genes were included. We applied an in-house variant allele frequency (VAF) based bioanalytic method to resolve clonal hierarchy. VAF (adjusted for copy number and zygosity) was used to classify the mutations into dominant (if a cutoff of at least 5% difference between VAFs existed), secondary (any subsequent subclonal hit) and codominant hits ( SF3B1 MT were detected in 9% of patients (n=335): 140 were SF3B1DOM (42%), 121 were SF3B1SEC (36%) and 74 were codominant (SF3B1COD, 22%). Comparison of SF3B1MT VAFs among the 3 groups showed no significant difference (40 vs 39 vs 44%, P= .6). Due to lack of distinct partition, we delved into the implications of SF3B1COD on mutational stability, disease phenotypes, molecular associations and overall survival (OS) as compared to the other 2 configurations. Serially-collected samples (n=21) showed clear scenarios of dominant clones remaining stable or switching to secondary due to impending acquisition of other hits or secondary clones remaining as such but denoted an ambivalent framework of codominant clones with "sweeping" features. Cases with SF3B1COD had a trend of OS similar to that of SF3B1SEC (median OS: 18 vs 15.9 mo.) and as such a poorer outcome compared to that of SF3B1DOM (39.8 mo.; P= .02). Moreover, the mutational profile of SF3B1CODvsSF3B1DOM was partially similar to that of SF3B1SECvs.SF3B1DOM with significantly higher odd ratios for DNMT3A (P< .0001), ASXL1, FLT3, RUNX1, TET2 (P≤ .05 for each). No variant morphologic features were observed when comparing SF3B1COD to the other 2 groups. By virtue, we strictly compared only cases with SF3B1DOM and SF3B1SEC, further supported by our single cell analysis(n=5), to showan unambiguous distinction between these two statii. Mutational burden analysis revealed that SF3B1DOM patients had a fewer number of mutations than those with SF3B1SEC (median 1.0 vs 2.6). SF3B1DOM was mostly detected in patients with a normocellular bone marrow as compared to SF3B1SEC (46 vs 29%, P= .02) and had a lower proportion of multidysplastic myeloid cells (29 vs 53%, P= .01). On the other side, SF3B1SEC were often present in the context of bilineage dysplasia (47vs. 26%, P= .02). Focused morphologic analysis revealed that SF3B1DOM cases were significantly associated with a higher RS% (median 37 vs 3%, P= .002; frequency of RS ≥15%: 77 vs 44%, P= .003) with higher VAF% significantly correlating with higher RS%. Patients with SF3B1SEC had shorter OS than those with SF3B1DOM (15.9 vs 39.7 mo., P< .0001). Even by dichotomizing according to VAF, the median OS of cases with VAF>40% was significantly shorter than those with VAF40%, the OS was shortened compared to SF3B1DOM (31 vs 11.6 mo., P= .001) and similarly when it laid between 20% and 40% (49.7 vs 25.6 mo., P= .01) suggesting a strong impact of associated hits. In SF3B1SEC, univariate analyses showed significantly higher odds of hits in RUNX1 (43 vs 19%, P< .0001), TET2 (29 vs 11%, P= .0005), FLT3 (22 vs 11%, P= .02), DNMT3A (20 vs 7%, P= .004), ASXL1 (16 vs 5%, P= .06), BCOR/L1 (17 vs 5%, P= .005), IDH1/2 (11 vs 2%, P= .008), CBL (8 vs 1%, P= .009) and CEBPA (7 vs 2%, P= .04) compared to SF3B1DOM. Interestingly, cases with co-existing TET2 mutations had a marked decrease in OS in SF3B1SECvsSF3B1DOM(10.1 vs 96.1 mo., P= .02) suggesting that the mutational ranking in a disease triggered by SF3B1MT can be skewed by stronger hits. In sum, our study suggests that molecular ranking in the context of SF3B1 clonal configuration is a key factor diverting the clinical and phenotypic trajectories of patients with MN and SF3B1MT. Disclosures Carraway: BMS: Consultancy, Other: Research support, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Abbvie: Other: Independent Advisory Committe (IRC); Novartis: Consultancy, Speakers Bureau. Sekeres:Pfizer: Consultancy; BMS: Consultancy; Takeda/Millenium: Consultancy. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Thrombotic thrombocytopenic purpura (TTP) is an uncommon, life-threatening disease requiring prompt diagnosis and initiation of therapeutic plasma exchange to improve patient survival. However, diagnosis is often difficult because of atypical presentations and signs and symptoms that resemble other conditions. Measurements of ADAMTS13 activity, ADAMTS13 inhibitor, and ADAMTS13 autoantibody are useful for diagnosing TTP, guiding therapy, and predicting relapse.

Summary Introduction Quantitation of ADAMTS13 activity, functional inhibitors, and autoantibodies is crucial in diagnosis and management of thrombotic thrombocytopenic purpura. We compared and optimized commercial assay kits and validated a testing panel. Methods Citrated plasma specimens from healthy volunteers and residual samples submitted for clinical testing were used in the study. Commercially available ADAMTS13 activity assays including ACTIFLUOR™ ADAMTS13 (Sekisui Diagnostics, Stamford, CT, USA), LIFECODES ATS-13 (Gen-Probe Inc., San Diego, CA, USA), and TECHNOZYM® ADAMTS-13 (Technoclone, Vienna, Austria) were evaluated. Functional inhibitor assays were performed using internally developed mixing protocols. Two autoantibody assays were also evaluated: IMUBIND® (Sekisui Diagnostics) and TECHNOZYM® ADAMTS-13 INH ELISA kits (Technoclone). Results A laboratory-developed assay using ACTIFLUOR™ reagents showed best agreement with the reference method, and full validation showed a reportable range of 5% (LLOQ) to 114% with a reference interval of ≥68%. Both intra- and interassay coefficients of variation were

Context: Myeloid neoplasia (MN) phenotype and outcome associates with the clonal architecture of SF3B1 mutations (SF3B1MT). Objective: To explore the implications of the clonal architecture of SF3B1MT in MN. Design/Setting: Clinical characteristics, mutational profile, and the clonal hierarchy of MN patients were reviewed at The Cleveland Clinic. Genomic studies were conducted by whole-exome and targeted deep sequencing. Patients: A cohort of 5102 patients was studied for detailed clinical and molecular annotation. Main outcome measures: To describe whether the clonal nature of SF3B1MT might alter the cellular trajectories in patients with MN. Results: The clonal hierarchy was resolved using our in-house designed VAF-based bioanalytic method and confirmed by the PyClone analysis. We first assigned clonal hierarchy to SF3B1MT by using VAFs (adjusted for copy number and zygosity) and classifying the mutations into dominant (if a cutoff of at least 5% difference between VAFs existed), secondary (any subsequent sub-clonal hit) and co-dominant hits (if the difference of VAFs between two mutations was Conclusions: Our study of the clonal architecture of SF3B1MT highlights that clonal progression of cases with MN harboring SF3B1MT might be inferred by the rank of additional genetic lesions cooperating with SF3B1.

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm, which is characterized by sustained peripheral leukocytosis with neutrophilia, hepatosplenomegaly, and hypercellularity of the bone marrow, with less than 5% myeloblasts along with normal neutrophil maturation and no dysplasia. In 2016, World Health Organization (WHO) included activating mutations in the gene for colony-stimulating factor 3 receptor (CSF3R) as one of the diagnostic criteria with CSF3RT618I being the most common mutation. We report a rare case of CNL (JAK2V617F negative, BCR-ABL1 negative, CSF3RT618I positive) in an elderly female who had an aggressive clinical course of the disease.

The purpose of this study is to examine hematopoietic neoplasms with 9p24/JAK2 rearrangement including neoplasms associated with t(8;9)(p22;p24)/PCM1-JAK2 fusion neoplasm as well as cases with translocations involving 9p24/JAK2 and other partner genes. From seven large medical centers, we identified ten patients with t(8;9)(p22;p24) /PCM1-JAK2 and 3 with t(9p24;v)/JAK2 at diagnosis. Majority of the cases showed myeloproliferative neoplasm (MPN) associated features (n = 7) characterized by variable degrees of eosinophilia, myelofibrosis, frequent proliferations of early erythroblasts in bone marrow and extramedullary sites, and infrequent/absent somatic mutations. Other less common presentations included myelodysplastic syndromes (MDS) or MDS/MPN (one each). Four patients presented with B-lymphoblastic leukemia (B-ALL), and of them, two patients with t(8;9)(p22;p24.1) were proven to be B-lymphoblastic crisis of MPN; and the other two cases with t(9p24;v) both were de novo B-ALL, BCR-ABL1-like (Ph-like). We show that the hematopoietic neoplasms with 9p24/JAK2 rearrangement are extremely rare, and most of them are associated with t(8;9)(p22;p24)/PCM1-JAK2, a recent provisional World Health Organization entity under “myeloid/lymphoid neoplasm with a specific gene rearrangement”. Cases of t(8;9)(p22;p24)/PCM1-JAK2, though heterogeneous, do exhibit some common clinicopathological characteristic features. Cases with t(9p24;v)/JAK2 are extremely rare; while such cases with a MPN presentation may resemble t(8;9)(p22;p24.1)/PCM1-JAK2, B-ALL cases presenting de novo B-ALL might belong to Ph-like B-ALL.

Chromosomal abnormalities can be founder lesions (e.g., t (8; 21), inv (16), inv (3)), initiate or advance disease progression (both founder and secondary hits e.g., ASXL1, TP53, RUNX1) or can be obligatory secondary hits (FLT3, NPM1). Hence, the rank of these mutations may determine the biological properties and clinical outcomes. However, while many mechanistic studies have been undertaken without identifying the key pathogenetic factors resulting from SF3B1 mutations, important biological clues can be derived from the consequences of SF3B1 alterations in the context of the clonal architecture of myeloid neoplasia (MN). SF3B1 mutant patients often have a homogeneous phenotype with isolated erythroid dysplasia, ring sideroblasts (RS) and favorable prognoses. Studies in primary MDS cells have suggested that SF3B1 mutations are initiating lesions and provide a marked clonal advantage to MDS-RS cells by propagating from rare lympho-myeloid hematopoietic stem cells. However, there is significant diversity of clinical phenotypes and outcomes including the observation that the disappearance of RS can be observed during the disease course of clonal MN and might suggest cellular shifts due to acquisition of additional hits. In such scenarios, the cell's fate in the context of SF3B1 mutations is pre-defined by the predominance of expanded hits. We took advantage of our detailed database of molecularly and clinical annotated cases with MN to study the SF3B1 mutatome and describe whether the clonal nature (ancestral vs. secondary) might change the clinical and phenotypic trajectories of MDS cells and whether the concatenation of mutations decreases the competitiveness of SF3B1 clones, leading to the dominance of other driver genes and subsequently to clonal evolution. The clonal hierarchy was resolved using our in-house designed VAF-based bioanalytic method and confirmed by the PyClone pipeline, which showed a high level of concordance. We first assigned clonal hierarchy to SF3B1 mutations by using VAFs (adjusted for copy number and zygosity) and classifying the mutations into dominant (if a cutoff of at least 5% difference between VAFs existed), secondary (any subsequent sub-clonal hit) and co-dominant hits (if the difference of VAFs between two mutations was In conclusion, our study of the clonal architecture of SF3B1 mutations highlights that clonal progression of cases with MN harboring SF3B1 mutations might be inferred by the rank of additional genetic lesions cooperating with SF3B1. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Advani:Abbvie: Research Funding; Macrogenics: Research Funding; Pfizer: Honoraria, Research Funding; Amgen: Research Funding; Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy. Nazha:Tolero, Karyopharma: Honoraria; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Abbvie: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.