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Natural killer (NK) cells are lymphocytes of the native immune system that play a pivotal role in host defense and immune surveillance. While the conceptual view of NK-neoplasms is evolving, little is known about the rare NK lymphoblastic leukemia (NK-LL), which remains as a provisional entity in the 2016 WHO Classification. The goal of this study is to characterize NK-LL cases and compare with other CD56 co-expressing acute leukemias. We identified 105 cases, diagnosed as NK-LL (6), CD56+ acute undifferentiated leukemia (AUL) (6), CD56+ T-lymphoblastic leukemia (T-LL) (51), and CD56+ acute myeloid leukemia (AML) (42). Compared to AUL patients, NK-LL patients were significantly younger (p = 0.021) and presented with higher white blood cell (WBC) (p = 0.037) and platelet counts (p = 0.041). Flow cytometry showed more frequent expression of cytoplasmic CD3 (cCD3, p = 0.064) and CD33, (p = 0.065), while HLA-DR was significantly absent from NK-LL (p = 0.035) compared to AUL. Compared to T-ALL, NK-LL cases showed less frequent cCD3 (p = 0.002), CD4 (p = 0.051), and CD10 expression (p = 0.06). The frequency of abnormal karyotypes was similar between NK-LL, AUL, and T-ALL. The mutational profile differed in four leukemia groups, with a significance enrichment of NOTCH1 (p = 0.002), ETV6 (p = 0.002) and JAK3 (p = 0.02) mutations in NK-LL as compared to AML. As compared to T-ALL, NK-LL cases showed a higher number of total mutations (p = 0.04) and significantly more frequent ETV6 mutations (p = 0.004). Clinical outcome data showed differences in overall survival between all four groups (p = 0.0175), but no difference in event free survival (p = 0.246). In this largest study to date, we find that that NK-LL shows clinical presentation, immunophenotypic and molecular characteristics distinct from AUL, T-ALL, and AML. Our findings suggest NK-LL is a distinct acute leukemia entity and should be considered in the clinical diagnosis of acute leukemias of ambiguous lineage.

Introduction: Leukemia is a hematological disorder which needs timely diagnosis and early treatment because at initially leukemic cell burden is low. So, the chances of achieving complete remission will be highest. Aims: The present study was undertaken for correlation of patient’s signs and symptoms with their laboratory findings like CBC, peripheral smear findings, immunophenotyping to know the lineage specificity and cytogenetics to know molecular abnormality. Methods and Material: The blood samples of suspected patients were received and examined at Hematology and Surgical pathology sections of Central Diagnostic Laboratory from November 2018 to June 2020. The clinical history was obtained, CBC was performed, peripheral smear and bone marrow samples were received for morphological examination and special investigations like Immunophenotyping, Cytogenetics and fish. Results: Out of the 110 cases, 17 were ALL, 33 were AML, 16 were CLL, 29 were CML. other 15 cases were distributed in acute leukemia, lymphoma, myelofibrosis and acute undifferentiated leukemia. Conclusions: Present study revealed that acute leukemia was more prevalent than chronic leukemia. The most common type of leukemia was AML followed by CML, ALL and CLL. ALL was mostly observed in children whereas CML and CLL were mostly observed in adults. AML mostly observed in adults.

Acute undifferentiated leukemia (AUL) is a subtype of acute leukemias of ambiguous lineage. There is no standard treatment approach for AUL, although acute lymphoblastic leukemia–like regimens for induction therapy have been used. Additional data suggest that AUL may be better treated as acute myeloid leukemia (AML), given their similarities in genetic, cytogenetic, and gene expression patterns. Somatic mutations of IDH1 are found in 7% to 14% of patients with AML; however, the patient in this study was the first patient with IDH1-mutated AUL treated with ivosidenib. In this case, a woman aged 39 years was found to have anemia and thrombocytopenia after presenting to her primary care physician with fatigue, weight loss, and persistent infections. During further workup of the cytopenia, she was diagnosed with AUL and received 7+3 (daunorubicin, 60 mg/m2/d intravenously on days 1–3, and cytarabine, 100 mg/m2 24-hour continuous intravenous infusion on days 1–7) due to the presence of the IDH1 mutation. Bone marrow biopsy performed on day 14 of 7+3 showed persistent disease, and ivosidenib was initiated due to severe HLA alloimmunization (panel-reactive antibody, 100%) and significant bleeding complications. The patient achieved a complete morphologic and molecular remission on ivosidenib monotherapy despite critical bleeding complications during induction. Targeted therapy using ivosidenib may represent an encouraging therapeutic option in patients with AUL and IDH1 mutations. Additional evaluation of ivosidenib in this subgroup of patients with AUL is needed.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) with associated prior or concurrent hematologic malignancy (BPDCN-PCHM) is an emerging phenomenon, increasingly seen in clinical practice in the...

Acute leukemias of ambiguous lineage (ALAL) include acute undifferentiated leukemia and mixed-phenotype acute leukemia (MPAL). This article provides an overview of the diagnosis of ALAL and focuses on the data accounting for the current lineage-assignment criteria for blasts harboring more than one lineage-associated marker. In addition, the currently known molecular data are reviewed, which show that MPAL-associated gene mutations, methylation signatures, and expression profiles are a mixture of those seen in both acute myeloid leukemia and acute lymphoblastic leukemia. Finally, the prognosis and current treatments of MPAL are briefly discussed.

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.

Background Acute leukemia (AL), not clearly assigned to myeloid, B-lymphoid, or T-lymphoid lineage, is classified as either biphenotypic acute leukemia (BAL) based on the European Group for Immunological Classification of Leukemias (EGIL) or acute leukemia of ambiguous lineage (ALAL) encompassing acute undifferentiated leukemia (AUL) and mixed-phenotype acute leukemia (MPAL) based on the World Health Organization (WHO) criteria. Methods Medical records of children newly diagnosed with BAL or ALAL, based on the EGIL or the 2008/2016 WHO criteria, respectively, admitted at Chonnam National University Hospital in 2001-2017 were retrospectively reviewed. Results Twelve (3.2%) of 377 AL patients satisfied the BAL or ALAL definitions based on the EGIL or the WHO criteria, respectively. Among 12 patients including 11 with BAL and another with undefined case based on the EGIL criteria, 7 (1.9%) had ALAL based on more stringent 2016 WHO criteria (AUL, 2; MPAL, 5). One patient had MPAL with t(9;22)(q34;q11.2), BCR-ABL+, and two had MLL gene abnormality. ALL-directed regimen was associated with better complete remission rate compared with AML-directed regimen (100.0% vs. 16.7%; P=0.015). The 5-year overall survival (OS) and event-free survival (EFS) were 51.1±15.8% and 51.9±15.7%, respectively. AUL was associated with poor OS and EFS compared with MPAL (0.0% vs. 75.0±21.7%; P=0.008). Conclusion Due to the rarity of the cases, future multicenter, prospective studies incorporating large number of cases are urgently warranted to identify the clinical, biologic, and molecular markers for the prediction of prognosis and determine the best tailored therapy for each patient.

Context: Acute leukemias of ambiguous lineage (ALAL) pose significant diagnostic and therapeutic challenges due to lack of clarity surrounding their cellular origins and driving genomic alterations, as well as poor long-term response to conventional chemotherapy. Most ALAL cases are diagnosed as mixed phenotype acute leukemia (MPAL) or acute undifferentiated leukemia (AUL); however, early T-cell precursor acute lymphoblastic leukemias (ETP-ALL, a subset of T-ALL), often express myeloid markers, suggesting a degree of lineage ambiguity. Biological distinctions between these diseases remain vague, as T/myeloid MPAL and ETP-ALL exhibit similar mutational and immunophenotypic profiles, highlighting the need for an improved understanding of ALAL etiology. Objective: To resolve biological heterogeneity of ALAL, we performed a large-scale genomic analysis, including whole-transcriptome and whole-exome/genome sequencing, of 1,114 primary leukemia samples spanning the stem, myeloid, and T lineages. Results: Transcriptional profiling identified a group of cases (N=61) with a distinct gene expression profile that included one-third of all T/myeloid MPAL and ETP-ALL cases examined, as well as subsets of acute myeloid leukemia (AML) and AUL. Whole-genome sequencing identified structural variants targeting the T-cell transcription factor gene BCL11B in 100% of cases and FLT3 mutations in 80% of cases. Most structural variants resulted in chromosomal translocations that placed the BCL11B gene in proximity to super-enhancers active in hematopoietic stem or early progenitor cells, a cell type where BCL11B is normally repressed. Additionally, 20% of cases harbored high-copy tandem amplification of a 2.5-kb non-coding element downstream of BCL11B that we term BCL11B enhancer tandem amplification or BETA. Analysis of 3D chromatin structure in primary leukemia samples demonstrated ectopic chromatin interactions between BCL11B and rearranged hematopoietic stem/progenitor cell super-enhancers, or between BCL11B and BETA, thereby demonstrating enhancer hijacking or de novo super-enhancer formation as the mechanism of aberrant BCL11B expression in this subtype. In vitro modeling in human CD34+ stem/progenitor cells demonstrated that ectopic BCL11B expression was sufficient to upregulate T-lineage gene expression programs and block myeloid differentiation, which was exacerbated by constitutive FLT3 signaling. Conclusions: BCL11B deregulation in primitive hematopoietic cells is an oncogenic driver of a subset of ALAL that transcends conventional immunophenotypic distinctions.

Acute undifferentiated leukemia is a rare hematologic malignancy with a poor prognosis. Venetoclax was highly active in two patients with relapsed acute undifferentiated leukemia and should be studied further.

Acute undifferentiated leukemia (AUL) is a very rare hematologic neoplasm that expresses no markers specific for either myeloid or lymphoid lineages. While commonly observed in several acute leukemias, KMT2A rearrangements in AUL have been rarely reported in the literature. We report the third case to our knowledge of AUL harboring a KMT2A rearrangement. Furthermore, the KMT2A/GIMAP8 gene fusion identified in this case represents a novel KMT2A rearrangement.

Acute leukemias of ambiguous lineage (ALAL) are rare hematologic malignancies with poor outcomes. Retrospective studies have suggested that acute lymphoblastic leukemia (ALL) regimens are more effective than acute myeloid leukemia (AML) regimens. We retrospectively examined the effectiveness of the widely-used adult ALL regimen hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyperCVAD) as initial therapy in patients with ALAL at five academic institutions. Twenty-five patients were identified, including 23 with mixed phenotype acute leukemia (MPAL) and two with acute undifferentiated leukemia. Five of 8 tested (63%) had FLT3-ITD and 3 of 25 (12%) were Philadelphia chromosome-positive. The complete remission (CR) rate was 76%, with CR with incomplete count recovery (CRi) in an additional 8%, for an overall response rate of 84%. Median number of cycles to CR/CRi was 1. There were no deaths in the first 30 days. Of the 21 patients achieving CR or CRi, 14 (66%) proceeded to allogeneic hematopoietic stem cell transplantation. With a median follow-up time of 31.6 months, median overall survival for the entire cohort was not reached, and the estimated 2-year survival was 63%. HyperCVAD can be considered an effective and tolerable front-line regimen for patients with ALAL, and warrants further prospective study.

Ambiguous lineage acute leukemia (ALAL) is a rare subtype of acute leukemia and is defined immunologically. ALAL consists of mixed phenotype acute leukemia (MPAL) and acute undifferentiated leukemia (AUL). MPAL is further divided into subtypes such as B/M MPAL, T/M MPAL, and B/T MPAL. Recently, the genetic basis of MPAL has been revealed as an acquisition of mutations in immature hematopoietic progenitors. There are shared genetic features between B/M MPAL with ZNF384 and B-cell acute lymphoblastic leukemia (ALL) with ZNF384 as well as T/M MPAL and early T-cell precursor ALL. Treatment for ALAL has not been established. However, treatment of ALL-type is significantly more effective than treatment of acute myelogenous leukemia. Generally, the prognosis of pediatric ALAL is worse than that of pediatric ALL. Treatment selection based on the genetic background is recommended. ALAL with genetic features of ALL, such as BCR-ABL or KMT2A alterations, and CD19 positive ALAL should be treated using ALL treatment. Treatment switching, either ALL-type to AML-type or vice versa, is beneficial for few ALAL cases. Hematopoietic stem cell transplantation is indicated for patients with poor efficacy of induction treatment.

Acute undifferentiated leukemia (AUL) is rare and defined by the absence of bona fide myeloid and lymphoid markers. Little is known about its incidence, survival and optimal management in the recent time period. Based on a case observed in our clinic, we queried the Surveillance, Epidemiology, and End Results database between 2000 and 2016. A total of 1,888 cases of AUL were diagnosed (1.34 per million person-years). The incidence of AUL has significantly decreased over time. Compared to other acute leukemias, patients with AUL have the highest median age (74 years); in contrast to acute myeloid leukemia (AML, 65) and acute lymphoblastic leukemia (ALL, 12). Excluding patients with preexisting malignancies, 1,444 patients with AUL were analyzed for survival. Only 35% of AUL patients had received chemotherapy. Comparatively, 94% of ALL and 71% of AML cases received chemotherapy. Among AUL patients who received chemotherapy, the median survival was 12 months as opposed to 1 month in the group who did not receive chemotherapy (or unknown status). Among adults, AUL patients had the worst prognosis, with a median overall survival (OS) of 9 months, compared to 27 months in ALL and 13 months in AML. Among children, the median OS was superior for all three groups of leukemias, the OS of AUL patients being better than in AML and very similar to ALL. On multivariate analysis, older age and time period were associated with worse outcome. We describe here the largest series of cases with AUL published to date.

The World Health Organization (WHO) has categorized acute undifferentiated leukemia (AUL) as a rare subtype of acute leukemias of ambiguous lineage (ALAL). The prognosis of AUL is considered poor a...

The 2016 WHO classification defines acute undifferentiated leukemia (AUL) as a rare type of acute leukemia (AL) without defining markers for either lymphoid or myeloid lineage commitment. Literature suggests AUL does not harbour the genetic mutations commonly seen in lymphoblastic leukemia or acute myeloid leukemia (AML) including FLT3, WT1, rearrangements of KMT2A or BCR-ABL1 and it highly expresses genes associated with poor prognosis in AML (BAALC, ERG, and MN1); therefore, AUL is often associated with dismal outcome (Arber et al. 2016). This case report depicts an unusual genetic presentation of an AUL with BCR-ABL fusion gene. A 54-year-old female patient presented to our hospital in July 2021 with fatigue and adynamia one month before. She was also found with deep vein thrombosis in the left leg and segmental pulmonary thromboembolism. Laboratory exams showed Hb 9,7 g/dL, WBC 75.77×109/L (77% blasts), platelets 175×109/L. Coagulogram and fibrinogen were normal. Bone marrow (BM) aspiration was hypercellular, showing 86% of agranular blasts. Flow cytometry (FC) from BM blasts exhibited expression of CD34, CD45, HLA-DR, CD33, CD38, and partial expression of CD10 and CD123. The blasts were negative for MPO, CD79a, CD19, CD7, CD3, CD13, C117, CD16, CD11b, CD64, CD15, CD14, CD300e, CD61, NG2, CD71, CD4, CD36, CD22, CD42b. Conventional cytogenetics revealed a Ph+ disease (46,XX,t(9;22)(q34q11.2)[7]/46, idem, add(4)(q35)[7]/46,XX[6]). Real-time quantitative PCR showed positive BCR-ABL with p190 transcripts. FLT3 and NPM1 were found negative. Diagnosis of AUL Ph+ was made. AML chemotherapy was started with 3+7 standard doses regimen plus imatinib 600 mg/day. On day 14th BM evaluation found 89% of blasts with the same immunophenotype as in the diagnosis. The patient presented neutropenic colitis with the need to delay rescue therapy. We searched articles in the Pubmed database using the following strategy: “Bcr-Abl Tyrosine Kinase”AND “undifferentiated leukemia”, “Philadelphia chromosome”AND “undifferentiated leukemia”between 1976 and 2021. We found two case reports that described patients with AUL and positive BCR-ABL gene. In 1983, Smadja, N et al. reported a patient with AL considered at diagnosis to be undifferentiated. Neither the diagnosis cytogenetic nor FC data was provided by the authors, and the AL classification was based only on morphological characteristics. After 17 months, the patient developed Ph+ chronic myelogenous leukemia with a complex translocation (2; 9; 22). Hattori, M et al., in 1995, described a patient that following treatment to non-Hodgkin lymphoma developed myelodysplastic syndrome (MDS) which progressed to AUL, both found with the presence of chimeric BCR-ABL transcripts. The FC showed blasts that expressed CD7 and HLA-DR, and were negative for lineage markers: CD14,CD33,CD2,CD3,CD4,CD5,CD8,CDl0,CD19,CD20 and CD41. Qasrawi, A. et al., in 2019 and 2020 performed two studies analyzing data from 24 and 1444 cases of AUL, respectively. The most common pathogenic mutations were PHF6, SRSF2, RUNX1, ASXL1, and BCOR. No BCR-ABL mutation was described. We believe that with the advent of target therapies, the prognosis of AUL can improve. The patient described has the BCR-ABL gene as a potential target to a tyrosine kinase inhibitor. Considering this is not a usual finding, we hope this report can further help treatment decisions.

Introduction MPAL (Multi Phenotypic Acute Leukemia), BAL (Biphenotypic Acute Leukemia), AUL (Acute Undifferentiated Leukemia), ALAL (Acute Leukemia of Ambiguous Lineage), HAL (Hybrid Acute Leukemia), HAL (Hybrid Acute Leukemia), MLL (Mixed Lineage Leukemia) and aBLL (acute BiLineal Leukemia) represent different names of the same pathology or do these entities represent completely separate disease processes? These rather uncommon manifestations of acute leukemia complicate strict taxonomical sub grouping as well as their management. Rapid identification & swift management may restrict further neurological damage, while achieving hematological remission. Case description A 16 year female presented with gradual onset paraplegia for 7 months, a history of multiple blood transfusions in the past 2 years, pancytopenia, atypical cells in peripheral smear; the hematological and bone marrow work up revealed a diagnosis of: MPAL, B/Myeloid , NOS [Multi Phenotypic Acute Leukemia, B cell {lymphoid}/Myeloid, not otherwise specified]. An ALL type induction regimen was started. The management strategy, it's rationale and the clinical outcome are discussed. Conclusion For routine neurosurgical practice, these entities are extremely rare; and hence a working knowledge is very essential for appropriate & timely management notwithstanding the neurosurgical desire to rule out the compressive lesions first. Neurological status deterioration may be halted with timely institution of appropriate chemotherapy. In the extensive literature review in pubmed, this may be only the 1st case of MPAL with extramedullary neurological manifestation, at the first clinical presentation.

Acute undifferentiated leukemias (AUL) are the leukemia which lack differentiating features of T-cell–specific, myeloid-specific, and B-lineage–specific markers. It is categorized under acute leukemia of ambiguous lineage in 2008 WHO classification of tumors of hematopoietic and lymphoid tissues. AUL is the diagnosis of exclusion and is very rare, the morphology of leukemic cells is not specific.1 Here we report a rare case of AUL with myelofibrosis with an unusual morphology. A 40 year female presented with anaemia and hepatospleenomegaly with pancytopenia and leucoerythroblastic blood picture on peripheral blood smear underwent bone marrow studies. Bone marrow biopsy showed infiltration of blasts with scant cytoplasm, round to irregular nucleus, dispersed chromation with grade 3 fibrosis by Reticulin stain and negative Masson trichrome stain. Immunohistochemistry (IHC) on bone marrow biopsy showed CD 34 positivity and negative for Tdt, MPO, CD3, CD20, CD61 markers. Thus diagnosed as Acute undifferentiated leukemia with myelofibrosis. AUL is the diagnosis of exclusion and before categorising leukemia as undifferentiated, it is necessary to perform immunophenotyping with a comprehensive panel of monoclonal antibodies. Key words: Pancytopenia, hepatospleenomegaly, Immunohistochemistry

ObjectivesOnly a small number of studies have reported on the association of parental occupational exposure to benzene and risk of childhood and adolescent leukaemias. We examined associations with acute lymphoblastic leukaemia (ALL) in this population-based study in Denmark.MethodsBenzene was largely banned from Danish workplaces after 1975, thus this case-control study focused on the immediately prior years. Paediatric cancer cases (ResultsWe identified 217 employed case fathers and 169 employed case mothers, of which 22 (10.1%) and 11 (6.5%), respectively, were exposed to benzene (vs 6.7% and 2.9% of control fathers and mothers). Most exposed parents worked as machine or engine mechanics, or in the shoe industry. Maternal occupational exposure to benzene in pregnancy was related to increased risk of ALL in offspring (adjusted OR=2.28, 95% CI 1.17 to 4.41), while paternal preconceptional benzene exposure was not as strongly associated (adjusted OR=1.40, 95% CI 0.88 to 2.22).ConclusionsOur study supports an increased risk for ALL with parental occupational benzene exposure.

Background For diagnosis, sub-categorization and follow up of Acute Leukemia (AL), phenotypic analysis using flow cytometry is mandatory. Material and methods We retrospectively analyzed immunophenotypic data along with cytogenetics/molecular genetics data (wherever available) from 631 consecutive cases of AL diagnosed at our flow cytometry laboratory from January 2014 to August 2017. Results Of the total 631 cases, 52.9% (n=334) were acute lymphoblastic leukemia (ALL), 43.9% (n=277) acute myeloid leukemia (AML), 2.2% (n=14) mixed phenotypic acute leukemia (MPAL), 0.5% (n=3) acute undifferentiated leukemia (AUL) and 0.5% (n=3) chronic myeloid leukemia in blast crisis (CML-BC). ALL cases comprised of 81.7% (n=273/334) B-cell ALLs (95.2%, n=260/273 common B-ALLs and 4.8%, n=13/273 Pro B-ALLs). CD13 was the commonest cross lineage antigen, expressed in B-ALL (25.6%, n=70/273), followed by CD33 (17.9%, n=49) and combined CD13/CD33 (11.3%, n=31/273) expression. T-ALLs constituted 18.3% (n=61/334) of total ALLs and included 27.9% (n=17/61) cortical T- ALLs. CD13 was commonest (32.7%, n=20/61) aberrantly expressed antigen in T-ALLs, followed by CD117 (19.1%, n=9/47). AML cases included 32.1% (n=89/277) AML with recurrent genetic abnormalities, 9.0% (n=25/277) with FLT3/NPM1c mutation and 58.9% (n=163/277) AML NOS including 14.7% (n=24/163) AML M4/M5, 1.8% (n=3/163) AML M6 and 3.7% (n=6/163) AML M7. In AMLs, CD19 aberrancy was the most common (20.2%, n=56/277) followed by CD56 (15.8%, n=42/265). Conclusions In this study, we document the spectrum, correlate the immunophenotype with genetic data of all leukemias, especially concerning T-ALL where the data from India is scarce.

Plasmablastic lymphoma (PBL) is an aggressive malignancy that usually occurs in the setting of immunosuppression. The immunohistochemical profile of PBL is that of terminally differentiated B lymphocytes. CD138, CD38, and MUM1 are usually immunopositive. However, pan B-cell markers such as CD20 and PAX-5 are usually negative.MYCrearrangement is the most commonly encountered genetic alteration, with immunoglobulin (IG), especially immunoglobulin heavy (IGH) chain, being the most frequent partner. We report a case of PBL in a 48-year-old human immunodeficiency virus- (HIV-) positive male who was admitted to the hospital with signs and symptoms suspicious for tumor lysis syndrome. Bone marrow examination revealed hypercellular marrow with trilineage hypoplasia and sheets of intermediate to large neoplastic cells with basophilic vacuolated cytoplasm comprising the majority of cellular elements of the bone marrow. The neoplastic cells were negative for conventional B-cell, T-cell, plasma cell, and myeloid markers, while flow cytometric analysis revealed an abnormal CD45-dim population that was partially weakly positive for CD71 and CD79b. The diagnosis was initially thought to be a high-grade primitive hematopoietic neoplasm, possibly an acute undifferentiated leukemia. BOB-1, however, was immunopositive in the neoplastic cells, confirming its B-cell origin. MYC was positive by immunohistochemistry and break-apart FISH, as were CD45, MUM-1, and EMA immunostains. There was immunoglobulin kappa (IGK) light chain gene rearrangement by polymerase chain reaction (PCR). Additionally, Epstein–Barr virus- (EBV–) encoded small RNAs (EBER) were positive by in situ hybridization (ISH). The tumor proliferation index by Ki-67 immunostaining approached 95%. Although the tumor cells were negative for CD38 and CD138, the diagnosis of PBL was still rendered. We recommend using a broad spectrum of B-cell markers, including BOB-1 and OCT-2, in such challenging cases of B-cell lymphomas with no expression of conventional B-cell markers. We also emphasize that the negative CD38 and CD138 should not exclude PBL from the differential diagnosis.

Acute undifferentiated leukemia (AUL) is a rare subset of acute leukemias, being included in the 2008 edition of the World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues as a special category called ʻacute leukemias of ambiguous lineageʼ, in which the leukemic cells express no more than one membrane marker of any given lineage. We report a rare case of therapy-related acute myeloid leukemia (AML), in which a strict myeloid marker myeloperoxidase (MPO) expression was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis but not immunophenotyping. Although the diagnosis of AUL was initially considered, the lineage of leukemic cells was demonstrated by qRT-PCR analysis. Our observations suggest that molecular analysis of lineage markers may help to establish the diagnosis of acute leukemias.

Introduction Acute undifferentiated leukemia (AUL) is rare and by definition has neither lymphoid nor myeloid lineage specific markers. Given its rarity, little is known about its incidence, survival, and optimal management. We queried the SEER (Surveillance, Epidemiology, and End Results) registry database to obtain information about the incidence and survival of AUL in comparison to acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Methods We identified patients with microscopically confirmed AUL from the SEER database between 2000 and 2016. Incidence rates of AUL were calculated and age-adjusted according to the 2000 US Standard Population. Annual percentage change was estimated using the weighted least square method to measure trends of AUL over this time period. Survival analysis compared AUL vs AML vs ALL patients who were diagnosed in the same period (patients with a preceding malignancy were excluded). Age, sex, race, ethnicity, year of diagnosis, and use of chemotherapy were compared across the three groups. Overall survival (OS) was estimated using the Kaplan-Meier method and comparisons were made using the log-rank test. All factors with p-values Results Between 2000 and 2016, a total of 1888 cases of AUL were diagnosed corresponding to an age-adjusted incidence rate of 1.34 per million person-years. The incidence of AUL has decreased over time, with an estimated annual percentage change of -6.5% (CI: -8.1% to -4.8%), p < 0.0001 (Fig. 1). The incidence increases dramatically in the older age groups. After excluding patients with a previous malignancy, 1444 patients with AUL were analyzed for survival. Table 1 summarizes patient baseline characteristics. Patients with AUL had the highest median age among the three groups (74 years) in comparison to 65 in AML and 12 in ALL. Only 35% of AUL patients were coded as having received chemotherapy (vs no/unknown). On the other hand, 94% of ALL and 71% of AML cases received chemotherapy. Among AUL patients who received chemotherapy, the median OS was 13 months (CI: 11-15), compared to 1 month in patients whose chemotherapy status was labelled as "No/Unknown," HR=3.45 (CI: 3.01-3.95). On multivariate analysis of AUL patients who received chemotherapy, older age was the strongest factor associated with worse survival followed by earlier year of diagnosis (Table 2). We compared the survival of AUL patients who were treated with chemotherapy with survival of ALL and AML patients. Adults (age 18+ years) and children (age On multivariate analysis (Table 3), in both the adult and pediatric subgroups, older age, non-White race, Hispanic ethnicity, and earlier year of diagnosis were independently associated with worse OS. Male sex was predictive of worse outcomes, but only in the adult subgroup. When AUL was compared to ALL and AML in the multivariate model in the adult subgroup, it had worse outcomes, with 47% and 35% increased risk of death, respectively. By comparison, pediatric AUL had similar outcomes to ALL but better survival than AML. Adjusted survival curves for the three types of leukemias in both adult and pediatric subgroups are shown in Fig. 2. Conclusions We describe the largest series of patients with acute leukemia without lineage defining markers. The incidence of AUL has decreased over time, likely due to better diagnostic tools assigning patients either to the myeloid or lymphoid lineage. The prognosis of AUL is generally poor but has improved over time. We also show that the prognosis of AUL relative to other acute leukemias is different between children and adults. This is probably related to distinct molecular and cytogenetic features. With the introduction of targeted therapies, we foresee further improvements in the prognosis of AUL. Disclosures No relevant conflicts of interest to declare.

International audience; Reproductive function is under the control of the neurohormone GnRH, which activates a G-protein-coupled receptor (GnRHR) expressed in pituitary gonadotrope cells. GnRHR activates a complex signaling network to regulate synthesis and secretion of the two gonadotropin hormones, luteinizing hormone and follicle-stimulating hormone, both regulating gametogenesis and steroidogenesis in gonads. Recently, in an attempt to identify the mechanisms underlying GnRHR signaling plasticity, we identified the first interacting partner of GnRHR, the proto-oncogene SET. We showed that SET binds to intracellular domains of GnRHR to enhance its coupling to cAMP pathway in alpha T3-1 gonadotrope cells. Here, we demonstrate that SET protein is rapidly regulated by GnRH, which increases SET phosphorylation state and decreases dose-dependently SET protein level. Our results highlight a post-translational regulation of SET protein involving the proteasome pathway. We determined that SET phosphorylation upon GnRH stimulation is mediated by PKC and that PKC mediates GnRH-induced SET down-regulation. Phosphorylation on serine 9 targets SET for degradation into the proteasome. Furthermore, a non-phosphorylatable SET mutant on serine 9 is resistant to GnRH-induced down-regulation. Altogether, these data suggest that GnRH-induced SET phosphorylation on serine 9 mediates SET protein down-regulation through the proteasome pathway. Noteworthy, SET down-regulation was also observed in response to pulsatile GnRH stimulation in L beta T2 gonadotrope cells as well as in vivo in prepubertal female mice supporting its physiological relevance. In conclusion, this study highlights a regulation of SET protein by the neurohormone GnRH and identifies some of the mechanisms involved.

Acute leukemias of ambiguous lineage are defined by the World Health Organization (WHO) classification as leukemias that either lack a specific lineage association or show features of concurrent multiple lineage associations [1, 2]. Ambiguous-lineage leukemias are more rare and more diagnostically challenging than conventional acute myeloid leukemias and lymphoblastic leukemias [1–11]. Given that the cytologic features of these leukemias are generally nonspecific, the diagnosis rests upon extensive immunophenotypic analysis. Multiparametric flow cytometric immunophenotyping is the preferred method for immunophenotyping, but in some cases, immunohistochemistry may be performed and may facilitate the recognition of two distinct blast populations in tissue sections. In addition to standard immunophenotypic studies, further evaluation should include cytogenetic studies to identify cases with t(9;22)(q34.1;q11.2); BCR-ABL1 or t(v;11q23.3); KMT2A rearranged. Table 13.1 highlights the diagnostic subcategories of acute leukemias of ambiguous lineage, Table 13.2 lists the diagnostic criteria for lineage determination, and Table 13.3 compares lineage-specific and lineage-associated markers in acute leukemia. This chapter provides pathologic examples of various ambiguous-lineage leukemias and illustrates the key diagnostic challenges, considerations, and caveats in establishing these rare diagnoses (Figs. 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 13.10, 13.11, 13.12, 13.13, 13.14, 13.15, 13.16, and 13.17).

Acute leukemias of ambiguous lineage may be divided into acute undifferentiated leukemia (AUL) and mixed-phenotype acute leukemias (MPALs). AULs are leukemias with very primitive phenotypes that lack definitive lineage-specific antigens. MPAL encompasses rare blastic hematopoietic cell neoplasms that express a mixture of myeloid and lymphoid (B- or T-lineage) antigens. The 2008 World Health Organization (WHO) classification defines several categories of MPALs based on the presence of recurrent cytogenetic lesions or in their absence, on the lineage of the leukemic blasts, defined as B-myeloid, T-myeloid, B-T, or B-T-myeloid. Any of these leukemias can develop as biphenotypic or bilineal (bilineage) acute leukemias. The WHO 2016 update will maintain WHO 2008 criteria for MPAL with additional clarifications. This chapter summarizes the clinical and pathologic features of this rare leukemic subentity.

N-Myc plays a role in the regulation of proliferation, differentiation, and survival of hematopoietic stem cells (HSCs) and leukemogenesis. We studied the cell of origin in the mouse acute leukemia model by N-Myc-overexpression. Our single cell RNA-seq data showed that N-Myc is restrictively expressed in HSCs and hematopoietic progenitor cells (HPCs) whereas c-Myc, but not L-Myc, is widely expressed in a variety of hematopoietic cells. HSC1, HSC2, HPC1, HPC2, and HPC3 with different self-renewal and differentiation potentials were isolated from the bone marrow of adult B6 mice. These cells were retrovirally overexpressed with N-Myc and transplanted into lethally irradiated mice with competitor cells. Acute leukemia developed from all these populations. Notably, not only B cell acute lymphoid leukemia (B-ALL), but also T cell ALL (T-ALL), acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and mixed phenotype acute leukemia (MPAL) developed. Metascape analysis of RNA-seq data showed that RNA profiles each from B-ALL, T-ALL, AML and AUL samples were enriched in special cellular signaling pathways. Collectively, the multiple cells of origin suggested that the self-renewal and differentiation potentials of cells were not related to leukemogenesis induced by N-Myc-overexpression. In addition, whole genome sequencing data suggested that retroviral integration sites play a role in the development of different types of leukemia.

C-kit receptor (CD117) and its ligand, stem cell factor, play a key role in normal hematopoiesis. It has been demonstrated that its expression extremely increases in leukemias with myeloid commitment. We analyzed findings on CD117 expression together with other myeloid related markers in 203 de novo acute leukemias, referred to Iranian immunophenotyping centers: Iranian Blood Transfusion Organization (IBTO) and Baghiatallah Hospital (BH). All cases were characterized based on the French American British cooperative group (FAB) and European Group for Immunological Classification of Leukemias (EGIL). The cases comprised of 111 acute myeloblastic leukemia (AML), 86 acute lymphoblastic leukemia (ALL), and 6 acute undifferentiated leukemia (AUL). CD117 was positive in 75 % of AML and 50 % of AUL, whereas none of the ALL cases was positive for this marker. Although CD117 was positive in 100 % of M5a cases, no M5b positive was found (p = 0.036). The calculated specificity for myeloid involvement was 100 % for CD117 and CD33, and 98 % for CD13 and CD15 (p < 0.001). The calculated sensitivity for myeloid involvement was 83, 76, 64, and 41 % for CD13, CD117, CD33, and CD15, respectively (p < 0.001). We concluded that CD117 expression is a specific and rather sensitive marker for differential diagnosis between AML and ALL, and except for M5 subtypes, it fails to determine FAB subtypes; lack of expression in M5 can identify M5b. Therefore, it should be included in the routine primary panel for diagnosis of acute leukemias.

Introduction: Acute undifferentiated leukemia (AUL) is a rare type of acute leukemia that shows no evidence of differentiation along any lineage. Clinical, immunophenotypic and genetic data is limited: the largest study to date reported 16 AUL cases but did not use the current WHO classification and included limited genetic data on 5 cases (Ann Hematol 2013 92:747-758). Moreover, it is uncertain if AUL is biologically distinct from acute myeloid leukemia with minimal differentiation (AML MD), which also shows limited myeloid marker expression and has been reported to have a poor prognosis. Methods: A total of 95 cases (36 AUL cases 59 AML MD) were identified from pathology databases of eight academic institutions with available diagnostic flow cytometric data, cytogenetic findings, and clinical data by searching for diagnoses of "AUL" or "AML MD". Diagnosis of AUL required absence of any lineage-defining markers including MPO, CD19 and CD3. Using the WHO classification, diagnosis of AML with MD required expression of at least 2 myeloid markers (CD117, CD13 or CD33), absence of myeloid maturation (CD15) or monocytic markers (CD64, CD11b, lysozyme or non-specific esterase). Next generation sequencing with extensive mutational panel data was available in 78 cases. Outcome analysis for overall survival (OS) and relapse-free survival (RFS) and were performed using Kaplan Meier and log rank test for patients who received induction chemotherapy. Results: Based on cytogenetic abnormalities (N=27) or history of MDS (N=2), according to the 2016 WHO Classification, 28 cases (6 AUL and 22 AML MD) were re-classified as AML with myelodysplasia related changes (AML MRC). The remaining 30 AUL patients presented with similar age, blood counts, bone marrow cellularity, and blast percentage as the 37 AML MD patients (all p > 0.05). Comparison of immunophenotype in the two groups showed that AUL blasts had more frequent expression of TdT (p=0.0003) and lacked myeloid markers (CD117, CD13 or CD33 p0.05) were seen between these two groups. The frequency of abnormal karyotype was similar between AUL and AML MD (16/30 [53%] vs 15/37 [41%], respectively). The most common mutations identified in AUL were PHF6 (7/18), SRSF2 (7/18), RUNX1 (7/23), ASXL1 (6/23) and BCOR (5/16). Compared to AML MD, AUL cases were characterized by frequent mutations in PHF6 (7/18 vs 1/23, p=0.013) and SRSF2 (7/18 vs 2/22 p=0.028). Limiting AUL cases to only those with 1 myeloid marker or less also showed similar findings with more frequent mutations in PHF6 (7/16 vs 1/25, p=0.0031), SRSF2 (6/15 vs 3/26 p= 0.018) and trend towards higher BCOR frequency (5/15 vs 2/26, p=0.078) in AUL patients as compared to AML MD. RUNX1 mutation was seen in 7/23 AUL and 8/29 AML MD (p>0.05). 19/30 AUL patients received induction chemotherapy (AML-type regimen in 18 cases and an ALL-type regimen in 1 case) and 15/30 achieved complete remission. In 10 AUL patients who relapsed, 9 showed identical immunophenotype and one case showed expression of CD13 and CD33. Outcome data in the subset of patients who received induction showed no difference in OS, RFS, or rates of complete remission between AUL and AML MD groups (p>0.05). The 28 AML MRC cases that were originally classified as AUL or AML MD presented with lower WBC (p=0.026), more frequent abnormal karyotype (27/28) specifically complex karyotype (20/28 p=0.002), frequent TP53 mutations (p=0.0002) when compared to the AUL group. AML MRC patients showed worse overall OS (p=0.029) as compared with AUL patients and a trend toward worse outcome as compared with AML MD (p=0.068). Conclusions: In this largest series to date, AUL group shows distinct characteristics from AML MD, including more frequent PHF6 and SRSF2 mutations and expression of TdT. However, clinical outcome is similar between the two groups in patients treated with induction chemotherapy. Cases reclassified as AML-MRC had shorter survival compared to de novo AUL and trend towards worse outcome when compared to AML MD patients. These results suggest a genetic rationale for the separation of AUL as a distinct entity from AML MD and also support the WHO classification of cases with history of prior MDS and/or MDS-type karyotype findings as AML-MRC. Disclosures Garcia: Celgene: Consultancy.

Introduction: Acute undifferentiated leukemia (AUL), also referred to as ambiguous lineage leukemia, has been recognized as a unique entity by the WHO classification of myeloid neoplasms since 2008, and is considered a rare subtype. It is characterized by lacking expression of markers diagnostic of myeloid or lymphoid lineages, and encompasses the provisional entity of myeloid/natural killer (NK) leukemia, which also expresses CD56 and other immature T-cell markers. Small studies have reported that AUL is associated with a poor prognosis; however, cytogenetic and molecular characterization of this subgroup of patients is lacking, as are the long-term outcomes of induction therapy and stem cell transplantation (SCT). The goal of this study was to assess clinical and laboratory data of patients diagnosed with AUL at our institution, focusing on its unique characteristics and response to initial therapy. Methods: Parameters assessed at diagnosis included: initial blood counts, bone marrow (BM) blast cell percentage by morphology, specific phenotypic characteristics of BM blasts, molecular mutations and translocations, as well as cytogenetic abnormalities. Immunophenotyping of BM cells at diagnosis was performed using eight-color flow cytometry. Multi-parameter Cytognos acute orientation tube (ALOT) and the combination of the following antigens were used: CD34/CD117/CD14/CD56/CD36/CD123/HLA-DR/CD45/CD64/CD11c. Data were acquired with FACSCanto II flow cytometer and analyzed with Infinicyt software. Myeloperoxidase staining by fluorescence-activated cell sorting (FACS) was considered negative if Results: One hundred and sixteen cases of acute myeloid leukemia were diagnosed at the Rambam Health Care Campus between 11/2016 and 5/2018; 16 (1.4%) of them were defined as AUL, since they did not qualify for myeloid, B- or T-cell lineage according to the WHO classification. CD56 positivity was demonstrated in 8 (50%) cases, 5 of which were also CD7 positive. Patient median age was 63 years, 56% were men. None of the patients was found positive for either NPM1 or FLT3 mutations. Likewise, no core binding leukemias were diagnosed in this cohort. Sixty six percent (10/15) of the BM samples demonstrated cytogenetic and/or FISH abnormalities, with 26% complex karyotype cases, and 20% chromosome 7 aberrancies. All but two patients were treated with AML-oriented therapies. None of the patients achieved remission with induction therapy regardless of whether it included a chemotherapy backbone (82% of patients) or a hypomethylating agent. Seven patients have undergone allogeneic SCT, 4 of whom are in remission to date. Two of the remaining transplanted patients died of progressive disease and one developed extramedullary relapse 6 months post-transplant. While 8/16 (50%) patients are alive at 18-month follow-up, only 4 of them (25%) are in remission. Notably, all of the latter patients are SCT recipients. Six out of 8 CD56-positive patients have died of progressive disease, and the remaining two have not achieved remission. Conclusions: The present study, while confirming the poor prognosis of AUL patients, also suggests that a certain subset of such individuals could benefit from an allogeneic SCT. The high percentage of complex karyotype abnormalities, the lack of NPM1 mutations, and the expression of T/NK cell immaturity markers could partly account for the established poor outcome. However, since other molecular abnormalities described in the AML mutational landscape may be involved in AUL initiation/progression, extensive molecular sequencing of this patient population is required. A better understanding of the unique characteristics of this leukemia subtype is also crucial for the design of potentially improved treatment strategies for AUL patients. Disclosures No relevant conflicts of interest to declare.

Background: Acute leukemias of ambiguous lineage (ALAL), including acute undifferentiated leukemia and mixed phenotype acute leukemia (MPAL), are rare hematologic malignancies, accounting for only 2-5% of cases of acute leukemia. Outcomes are generally poor, and retrospective studies have suggested that patients have better outcomes when treated with regimens used for acute lymphoblastic leukemia (ALL) rather than acute myeloid leukemia (AML). Due to heterogeneous definitions and rarity of these leukemias, no single ALL regimen has been studied extensively. Hyperfractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone (hyperCVAD) is one of the most widely used adult ALL regimens in the United States. We retrospectively examined the effectiveness of this regimen as initial therapy in patients with ALAL. Methods: We retrospectively reviewed records from adult patients treated initially with hyperCVAD-based chemotherapy for ALAL as defined by the World Health Organization at five academic institutions. Only patients whose diagnosis was verified at a participating institution were examined. Philadelphia (Ph) chromosome-positive ALAL patients treated concurrently with tyrosine kinase inhibitors were included. The primary objective of the study was to determine the overall response rate (ORR) and median overall survival (OS). Descriptive statistics were used for baseline characteristics and responses, and Kaplan-Meier estimates were used to calculate all time-to-event outcomes. Results: Twenty-two patients were identified, and pre-treatment characteristics are shown in the Table. Median age was 50 years (range, 22-69), with an even distribution of male and female patients (50% each). The majority (77%) were Caucasian. Two patients (9%) had acute undifferentiated leukemia, 11 (50%) T/myeloid MPAL, 8 (36%) B/myeloid MPAL, and 1 (5%) B/T MPAL. Seven (32%) had a complex karyotype (at least 3 abnormalities), 6 (27%) had a normal karyotype, 3 (14%) were Ph-positive and were treated concurrently with dasatinib, and 1 (5%) had an 11q23 rearrangement. FLT3-ITD was detected in 5 of 8 patients (63%) with an available mutation panel. Three (15%) had central nervous system (CNS) involvement at presentation, and one additional patient developed CNS disease during the course of therapy. There were no deaths within the first 30 days, and the median number of cycles given was 4 (range, 1-8). The CR rate was 73% (n=16) and an additional 14% (n=3) had CR with incomplete count recovery (CRi), for an ORR rate of 86%. One patient had a partial response, and 2 patients had no response. Of the 19 patients achieving CR or CRi, median number of cycles to CR was 1.5, and 12 (63%) proceeded to allogeneic hematopoietic stem cell transplantation (alloHSCT). Of the 7 responders who did not receive alloHSCT, 3 relapsed shortly after initial CR and 4 are alive and in remission 77, 39, 37 and 10 months from treatment initiation. All 3 patients with Ph-positive disease achieved CR. Only 1 proceeded with alloHSCT and all 3 patients are alive and in remission at 51, 39, and 10 months. With a median follow up of 37 months, 14 (64%) patients were alive and in remission, including 11 who had undergone alloHSCT. Four of the 6 patients who died had a complex karyotype. Median overall survival for the entire cohort was not reached (Figure). Conclusions: HyperCVAD results in high remission rates in patients with ALAL, with low incidence of early mortality and high ability to proceed to allogeneic transplantation following response. HyperCVAD can be considered an effective front-line therapy for patients with ALAL. Additionally, hyperCVAD may be a backbone for incorporation of novel immunotherapies and targeted therapies, which may improve results in patients with appropriate targets. Disclosures Sweet: Astellas: Consultancy; Agios: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Jazz: Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Agios: Consultancy; Celgene: Honoraria, Speakers Bureau; BMS: Honoraria; Phizer: Consultancy; Jazz: Speakers Bureau; Astellas: Consultancy; Phizer: Consultancy; BMS: Honoraria. Wang:Novartis: Speakers Bureau; Amgen: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Consultancy. Al-Kali:Novartis: Research Funding.

Introduction: Biphenotypic leukemia (BAL) or acute leukemias of ambiguous lineage (ALAL) is an uncommon manifestation of childhood acute leukemia. The aim of this study is to further assess clinicopathological characteristics and genetic mutation of de novo BAL or ALAL. Materials and Methods: Among 377 patients ≤ 18 years old who were diagnosed with de novo Acute leukemia from 2001 to 2017, 12 patients (3.2%) satisfied the definition of BAL by the European Group for the Immunological Classification of Leukaemias (EGIL) criteria or ALAL by World Health Organization (WHO) criteria. By the EGIL criteria, 11 patients were diagnosed as BAL: B-cell/myeloid (B/My) phenotype, 5; T-cell/myeloid (T/My) phenotype, 5. By the 2008 WHO criteria, 7 patients (1.9%) remained as ALAL: acute undifferentiated leukemia (AUL), 2; MPAL, 5. The remaining 5 BAL patients were redirected as ALL (n=3) and AML (n=2). Except one case with ALL by WHO criteria, 11 cases were examined by next generation sequencing using 60 gene panels. Results: In 5 patients with MPAL by the WHO criteria, a total of 31 distinct mutations were identified in 26 different genes. One case had BCR-ABL gene rearrangement. PTPN11 and NDS1 were recurrent mutation in 2 patients. In 2 patients with AUL, a total of 8 distinct mutations were identified in 8 different genes. In 2 cass of AML, 19 distinct mutations were identified in 15 differnet genes. FLT3-ITD was recurrent mutation. In 2 case of ALL, 17 distinct mutations were identified in 14 differrent genes. The median age of the patients at diagnosis was 8.6 years (range, 0 months-17.8 years). Six patients initially received AML-directed induction therapy, whereas 6 patients received ALL-directed induction therapy. Overall, 7 of the 12 (58.3%) patients achieved a complete remission (CR) after their initial induction therapy. ALL-directed induction chemotherapy was associated with higher chances of achieving a CR as compared to AML-directed regimens (100.0% vs. 16.7%; P =0.015). Nine patients except three cases (1, progression of disease; 1, intracranial hemorrhage during chemotherapy; 1, in continuous CR over 7 years chemotherpay only) underwent an allogeneic hematopoietic stem cell transplantation. With a median follow-up of 5 years, 5 year overall survival (OS) rate was 51.1±15.8% and event-free survival (EFS) rate was 51.9±15.7%. MPAL by WHO criteria seemed to have a better OS and EFS than AUL (both for 75.0 ± 21.7% vs. 0.0%; P=0.008). Conclusions: BAL or ALAL contains heterogeneous genetic mutation. ALL-directed induction chemotherapy was much better in achieving CR than AML-directed induction chemotherapy. And, patients with MPAL as defined by 2008 WHO definition have better clinical contcome compared with patients with AUL. Disclosures No relevant conflicts of interest to declare.

The tyrosine-kinase receptor c-KIT (c-KIT) plays an important role in proliferation, survival and differentiation of progenitor cells in normal hematopoietic cells. In human hematological malignancies, c-KIT is mostly expressed by progenitor cell neoplasia and seldom by those involving mature cells. Tyrosine kinase inhibitors (TKIs) are actually licensed for the first- and second-line treatment of human hematologic disorders. Aim of the present study was to evaluate c-KIT mRNA and protein expression and complementary DNA (cDNA) mutations in canine leukemia. Eleven acute lymphoblastic leukemia (ALL) and acute undifferentiated leukemia (AUL) and 12 chronic lymphocytic leukemia (CLL) were enrolled in this study. The amounts of c-KIT mRNA and protein were determined, in peripheral blood samples, by using quantitative real time RT-PCR, flow cytometry and immunocytochemistry, respectively. The presence of mutations on c-KIT exons 8-11 and 17 were investigated by cDNA sequencing. Higher amounts of c-KIT mRNA were found in ALL/AUL compared to CLL, and this latter showed a lower pattern of gene expression. Transcriptional data were confirmed at the protein level. No significant gain-of-function mutations were ever observed in both ALL/AUL and CLL. Among canine hematological malignancies, ALL/AUL typically show a very aggressive biological behavior, partly being attributable to the lack of efficacious therapeutic options. The high level of c-KIT expression found in canine ALL/AUL might represent the rationale for using TKIs in future clinical trials.

Cryptic deletions are occasionally reported in hematologic malignancies. The SET-NUP214 fusion gene has been rarely reported in acute myeloid leukemia, acute undifferentiated leukemia, and recurrently in T-cell acute lymphoblastic leukemia. The fusion product is generated by a submicroscopic deletion in the vicinity of 9q34. Herein we present a novel case of acute undifferentiated leukemia with SET-NUP214 rearrangement due to the cryptic deletion of the 9q34 region producing two different types of fusion transcripts by alternative splicing and molecular characterization of the fusion transcripts by fluorescence in situ hybridization, reverse transcriptase–polymerase chain reaction, and array comparative genomic hybridization analyses.