Acute Undifferentiated Leukemia Research Articles

Genomic and global gene expression profiling in pediatric and young adult acute leukemia with PICALM::MLLT10 Fusion.

MLLT10 fusion is a rare but recurrent genetic driver in acute leukemias. To better understand the genomic landscape of PICALM::MLLT10 (PM) positive acute leukemia, we performed genomic profiling and gene expression profiling in twenty PM-positive patients, including AML (n = 10), T-ALL/LLy (n = 8), Mixed-phenotype acute leukemia (MPAL), T/B (n = 1) and acute undifferentiated leukemia (AUL) (n = 1). Besides confirming the known activation of HOXA, differential gene expression analysis compared to hematopoietic stem cells demonstrated the enrichment of genes associated with cell proliferation-related pathways and relatively high expression of XPO1 in PM-AML and PM-T-ALL/LLy. Our study also suggested PHF6 disruption as a key cooperating event in PICALM::MLLT10-positive leukemias. In addition, we demonstrated differences in gene expression profiles as well as remarkably different spectra of co-occurring mutations between PM-AML and PM-T-ALL/LLy. Alterations affecting TP53 and NF1, hallmarks of PM-AML, are strongly associated with disease progression and relapse, whereas EZH2 alterations are highly enriched in PM-T-ALL/LLy. This comprehensive genomic and transcriptomic profiling provides insights into the pathogenesis and development of PICALM::MLLT10 positive acute leukemia.

The ELN 2022 Risk Stratification Has No Impact on the Dismal Prognosis of Patients with Acute Undifferentiated Leukemia

Introduction: Acute undifferentiated leukemia (AUL) is a rare subtype of acute leukemia. It is characterized by lack of expression of myeloid or lymphoid lineage markers, frequently expressing CD56 or CD7. The dismal prognosis of AUL was reported in small studies; the largest previously reported group with this disorder included 24 patients (Weinberg, Mod Pathol, 2019). Furthermore, data are lacking regarding cytogenetic and molecular patterns of AUL. In this study, the cytogenetic and molecular changes of AUL patients are described, together with the clinical outcome. Methods: AUL was defined, in accordance with the WHO classification, as an acute leukemia that does not qualify for myeloid, B- or T-cell lineage. Immunophenotyping of BM cells at diagnosis was performed using 8-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. (Weinberg, Mod Pathol, 2019). Results: Three hundred and eighty-five patients with acute myeloid leukemia (AML) were diagnosed at Rambam Health Care Campus in Haifa, Israel between April 2015 and April 2021, of whom 30 (8%) were defined as AUL. The baseline characteristics are presented in Table 1. The median age was 62 years, none of the patients harbored NPM1 mutation, with 1 patient expressing FLT3-ITD mutation. Cytogenetic analysis was completed for all patients: 9 (30%) had adverse-risk cytogenetics by ELN 2022 criteria. A comprehensive molecular study using molecular inverted probe was performed on all patients, with final results available on 17. Nine of these 17 (53%) had high-risk mutations; RUNX1 was found in 4 patients. Notably, 4 other patients had JAK2 mutation, with or without previous myeloproliferative neoplasm. Only 3 patients had mutations characterizing secondary AML (Lindsley, Blood, 2015). Only 2 patients in this cohort had TP53 mutation or del17p. Fourteen patients were classified as adverse-risk AML by ELN 2022 using cytogenetic and molecular markers, while 15 were classified as intermediate risk. Only 4 patients in this adverse-risk cohort were not allogeneic hematopoietic stem cell transplant (HSCT) candidates due to age or comorbidity limitations. The outcome of the cohort is presented in Table 2. Twenty-two fit patients received 7+3 intensive chemotherapy, of whom 10 achieved complete remission (CR) after induction. Four of the 10 patients needed reinduction chemotherapy due to a high blast count on the bone marrow performed at day 14. Five patients were treated with low-intensity therapies such as hypomethylating agents ± novel agents; 2 achieved CR. One patient received FLAG-Ida chemotherapy and another patient was treated with ECOG1910 protocol due to clinical presentation more compatible with acute lymphoid leukemia; both achieved CR. Of the 14 patients achieving CR, 4 relapsed before HSCT and needed further salvage therapy. Eventually, 13 (50% of the transplant-eligible candidate cohort) patients proceeded to HSCT. Relapse occurred in 14 patients (10 post-HSCT and 4 before HSCT), none of them achieved long-term survival. Four of the 30 patients are alive, all in remission. The median overall survival of the entire cohort is 1 year (Fig 1). The outcome of patients with adverse or intermediate risk according to the ELN risk classification did not differ. The CR rate and relapse rate and median overall survival (OS) were similar in both groups (Table 2; Fig 1). Discussion: These data emphasize the dismal outcome of patients diagnosed with AUL, with the 5-year OS of 7.5%, far lower than the survival of other types of acute leukemia. In this cohort, 15 patients were classified as intermediate risk AML; yet, their CR rate and long-term survival were not different from the whole cohort. The CR rate achieved with the widely used 7+3 protocol, was inferior, raising the question whether this protocol should be the “standard of choice” in such patients. A limitation of this study is that not all patients were evaluated by the NGS panel, bringing about the issue whether more patients had high-risk mutations, who would then be classified as adverse risk. Conclusion: The unique subtype of AUL is associated with a particularly adverse risk genotype, irrespective of known prognostic determinants, as in the 2022 ELN classification.

Acute Leukemias of Ambiguous Lineage Have Durable Response to Hypomethylating Agents Plus Venetoclax in Older Patients

Background: The standard of care for frontline therapy of acute leukemias of ambiguous lineage (ALAL) remains controversial due to their rarity; however, outcomes are generally poor. Intensive chemotherapy (IC) regimens are standard, with evidence suggesting that acute lymphoblastic leukemia regimens are more effective than those used for acute myeloid leukemia (AML). Strategies for treating older or frail patients are limited. Although treatment with a hypomethylating agent plus venetoclax (HMA/Ven) is standard therapy for IC-ineligible AML patients, the role of HMA/Ven in ALAL remains poorly characterized. We performed a retrospective chart review of patients at our institution with Philadelphia-negative ALAL, comparing the outcomes of those treated with HMA/Ven and those who received IC. Method: Patients ≥ 18 years treated at Weill Cornell Medical Center with newly diagnosed, pathologically confirmed acute undifferentiated leukemia (AUL), T/myeloid, or Philadelphia chromosome-negative B- and B/T-myeloid leukemia were included. Frontline HMA/Ven was defined as Ven plus azacytydine, decitabine, or oral decitabine/cedazuridine (one patient had initial HMA monotherapy followed by HMA/Ven). Intensive chemotherapy included patients who received variations of standard ALL or AML IC regimens (hyper- or mini-hyper-CV[A]D, cytarabine plus daunorubicin or idarubicin, or cladribine plus idarubicin and cytarabine) with or without Ven. Tyrosine kinase inhibitors including midostaurin or gilteritinib were added for patients with fms-like tyrosine kinase 3 ( FLT3) mutations. Baseline and disease characteristics were collected as well as disease response, subsequent lines of therapy, whether the patient proceeded to allogeneic hematopoietic cell transplantation (HCT), rate of morphologic complete remission (CR), and overall survival (OS). Results: Of 16 patients, 7 received frontline HMA/Ven. B/myeloid was the most common disease type (n=8), followed by T/myeloid (n=4), AUL (n=3), and 1 patient with B/T/myeloid disease. The median age was 74 years (interquartile range [IQR] 62-80). Patients who received HMA/Ven were older than those who received IC, with median ages of 79 (IQR: 77-82) and 62 (IQR: 56-70; p = 0.01), respectively. Baseline white blood cell, hemoglobin, platelet counts, and lactate dehydrogenase, as well as blast percentages in both peripheral blood and bone marrow were similar between the groups. Although not validated in ALAL, markers of adverse myeloid disease as per the ELN 2022 schema were prevalent in both groups, including mutations in RUNX1, ASXL1, SRSF2, and TP53 (Figure A). One HMA/Ven patient and 3 IC patients underwent HCT. Of evaluable patients, 83% and 88% of HMA/Ven and IC patients achieved CRs, respectively. At 2 years, both groups had 50% overall survival (95% CI 22.5-100 in both). Among HMA/Ven recipients, overall survival ranged from 10-56 months, not including two patients with ongoing follow-up at 3 and 46 months (Figure B). Discussion: The treatment of ALAL remains a challenge, particularly in older patients who may be unfit for intensive treatment strategies. In our study, patients who received HMA/Ven had similarly durable disease control to younger patients who received IC in the frontline setting. Prolonged responses were seen in both AUL and MPAL. Multicenter registry and prospective analyses are needed; however, given the rarity of these diagnoses, our findings suggest that HMA/Ven should be considered as a frontline therapy for unfit patients with ALAL.

Treatment Outcomes of Childhood Picalm:MLLT10+ Acute Leukemias: An International Retrospective Study

Introduction: Acute leukemia with translocation t(10;11)(p12-13;q14-21) results in generation of the PICALM:MLLT10 fusion gene, and is distinct from t(10;11)(p11-13;q23) which gives rise to KMT2A (formerly MLL) gene rearrangement. Defective PICALM function leads to aberrant hematopoiesis, while defective MLLT10 function impairs chromatin-mediated gene regulation. The PICALM:MLLT10 gene has been reported in patients with Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) predominantly T-cell, Acute Undifferentiated Leukemia (AUL) and Mixed Phenotype Acute Leukemia (MPAL). MLLT10 interacts directly with DOT1L in a complex that regulates the methylation of H3K79. MLLT10 fusions are thought to misdirect DOT1L to the promoters of HOXA genes, leading to hypermethylation of H3K79. This hypermethylation causes constitutive activation of HOXA activity and prevents cell maturation and differentiation. The clinico-biological characteristics, response to conventional treatments, and survival outcomes of childhood PICALM:MLLT10+ leukemias are poorly understood. Methods: This was an international, multicenter, retrospective cohort study. Data were submitted by ten International-BFM (I-BFM) study Group-AML and ALL committees and by the Children`s Oncology Group (COG)-AML Study committee. For study inclusions, patients were required to be ≤ 21 years of age at diagnosis, diagnosed between January 1995 and December 2019 with de novo ALL, AML, AUL or MPAL and PICALM:MLLT10 fusion-positive. Patients with KMT2A-rearranged leukemia or acute promyelocytic leukemia were excluded. Kaplan Meier curves were employed for survival analysis with Cox proportional hazard modelling to compare effect of AML vs ALL therapy, and receipt of hematopoietic stem cell transplant (HSCT) vs no HSCT, on survival. Pearson's Chi Squared was employed as a non-parametric measure to determine the effect of end of induction-1 (EIO-1) MRD positive vs negative status on incidence of relapse. P value cut-off for data to reach statistical significance was <0.05. Results: Ninety-eight children with acute leukemia were included, 60 were males. Median age at diagnosis was 12 years (IQR:9-14.8 years) and median initial white cell count was 40.5 x 109/L (IQR:13.8-168.9). Fifty-five (55.6%) patients were diagnosed with T-ALL, 39 (39.4%) with AML, while precursor-B ALL, AUL and MPAL were each diagnosed in single patients; 1 subtype diagnosis was unknown. Ten (10.1%) patients presented with CNS disease and 13 (13.1%) presented with non-CNS extramedullary manifestations. Of 66 with cytogenetic/molecular data available, 33 (50%) had additional cytogenetic-molecular aberrations; NOTCH1, WT1 and RAS pathway mutations predominated. Most patients received an initial chemotherapy regimen guided by their disease immunophenotype: 58/98 (58.6%) received an ALL-like chemotherapy regimen upfront, 38 (38.4%) received an AML-like regimen, 1 (1.0%) a hybrid regimen, 1 initial chemotherapy regimen was unknown. Five-year event-free survival (EFS) was 50.4% (CI 39.1-64.9). For AML patients, 5-year EFS was 22.5% (CI 9.75-52.0) and for ALL 67.7% (CI 54.2-84.7), p=<0.001. Five-year overall survival (OS) was 56.3% (CI 46.3-68.5). For AML patients, 5-year OS was 26.7% (CI 13.3-53.6) and for ALL 75.7% (CI 64.6-88.8), p=<0.001. Thirty-eight patients died, 22 (57.9%) deaths were disease-related, 4 (10.5%) treatment-related. Patients who received HSCT in CR1, demonstrated a trend towards an inferior 5-year EFS (23.7% vs 56.5%) and OS (42.6% vs 64.5%) than those who did not; however, analysis was limited by small sample size. Four (22.2%) HSCT recipients had ALL but most, 14 (77.8%), had AML, which may account for the unfavourable outcomes associated with HSCT. There was no statistically significant difference in rate of relapse in the EOI-1 MRD-positive compared with MRD-negative patients (p=0.49). Of those who relapsed, 25 (75.8%) had an isolated bone marrow relapse, 3 (9.1%) isolated CNS, 3 (9.1%) other sites and, for 2 patients, site of relapse was unknown. Conclusions: Children with PICALM:MLLT10+ AML have inferior 5-year EFS and OS outcomes compared to their T-ALL counterparts. MRD status at EOI-1 was not predictive of relapse. Targeted therapies with DOT1L inhibitors or DNA demethylating agents should be explored, particularly for patients with AML.

A Novel Germline PAX5 Single Exon Deletion in a Paediatric Patient with B-Cell Leukaemia

Germline predisposition underlies leukaemia development in children in 5-10% of the cases. Over the past years, several somatic drivers for leukaemia, including IKZF1, RUNX1, ETV6 and PAX5, have also been identified as leukaemia predisposing genes. For example, two germline PAX5 missense variants (p.Arg38His and p.Gly183Ser) have been identified in five families with a high incidence of B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). The susceptibility to BCP-ALL in these patients is inherited in an autosomal dominant pattern with an incomplete but relatively high penetrance. The leukemic cells in the patients showed loss of the wild-type allele or a somatic mutation, suggesting a second hit is required for leukaemia development in this context. We present a boy who developed BCP-ALL at 5-years-old, with a father who was diagnosed with acute undifferentiated leukaemia at infancy. Because of his family history, germline testing of the patient and his parents was performed using SNP-based copy number analysis and whole exome sequencing (WES). This revealed a PAX5 exon 6 deletion in the child, which was absent in both parents. The PAX5 deletion causes a frameshift with premature stop in exon 7, resulting in a truncated protein with loss of the C-terminal transactivating and inhibitory domains. In contrast to what has been observed in patients with germline PAX5 missense variants, a second somatic hit in PAX5 was not observed. Genotype analysis, using heterozygous SNPs around the PAX5 deletion, revealed that the PAX5 deletion had occurred on the paternal allele. To test whether this deletion was present in (low) mosaic state in the father, we developed a sensitive breakpoint spanning PCR. Also with this method, the PAX5 deletion was not detected in DNA derived from blood and buccal cells of the father. Since we expected a causative germline variant to be inherited from the father, we re-analysed all sequence and copy number variants in the patient-parents' trio datasets for any other aberrations, but no plausible alternative candidates were identified. Since routine diagnostic analysis of PAX5 deletions is usually performed only in leukaemia samples, germline deletions could potentially be missed, particularly in isolated cases. To investigate the incidence of germline PAX5 deletions, we used a multiplex-ligated probe amplification (MLPA) dataset from 2041 ALL patients, from which we selected 303 cases with single-exon or partial (multiple exon) PAX5 deletion. Subsequently, we performed MLPA on 261 DNA samples from bone marrow or peripheral blood at time of remission. No additional case was found, suggesting that germline PAX5 deletions, if recurrent, are very rare. In conclusion, we present a boy with ALL carrying a de novo germline PAX5 exon 6 deletion leading to a truncated protein. Germline PAX5 deletions have not yet been described in ALL patients but since somatic PAX5 aberrations are common in ALL and germline PAX5 variants have been associated with ALL, we consider that this focal germline PAX5 deletion did contribute to leukaemia development in the boy.

Haploidentical Hematopoietic Cell Transplantation of Ex Vivo Tcrαβ-Depleted Grafts with CD45RA-Depleted Memory T Cell Add-Back Reduces Chronic Graft-Versus-Host Disease and Results in Favourable Gvhd-Relapse Free Survival: 5-Year Follow-up of 107 Patients Treated in a Multicenter Study in Singapore

Background: Haploidentical hematopoietic cell transplantation (HCT) provides an alternative option for patients without HLA-matched donors. Graft-versus-host disease (GVHD), engraftment failure, and infectious complications are main causes of non-relapse mortality (NRM). We hypothesized that selective depletion of TCRαβ+ and CD45RA+ naïve T cells will permit hematopoietic engraftment while effectively reducing GVHD and providing donor immune reconstitution through adoptive transfer of donor's mature NK cells, γδ T cells and CD45RO+ memory T cells. Methods: Mobilized PBSC products were divided into two fractions in 8-9 : 1-2 ratio and depleted using CliniMACS device after labelling with TCRαβ and CD45RA reagents (Miltenyi Biotec, Bergish-Gladbach, Germany) respectively. All except 13 patients received the standard conditioning regimen of fludarabine 160mg/m2 divided daily over 4 days, thiotepa 10mg/kg divided twice daily for 1 day, and melphalan 70 - 140 mg/m2 for 1 day, in combination with either total lymphoid irradiation 6 Gy (n=58) or 7.5 Gy (n=12) over 3 equal fractions, total body irradiation of 2 Gy (n=24), or thymoglobuline (n=2). Short term GVHD prophylaxis was given for 30 days to 1 patient using MMF, 95 using tacrolimus, and 2 using sirolimus. Results: Between January 2017 and July 2022, we transplanted 107 patients, including 100 adults (median age, 52 years; range 19- 70) and 7 children (median age, 13 years, range 7 - 17), with high risk AML (n=60), ALL (n=22), MDS (n=10), plasma cell neoplasm (n=3), mast cell leukemia (n=1), acute undifferentiated leukemia (n=1), CMMoL (n=2), CML (n=2), MPN (n=1) and lymphoma (n=5). Patients were infused with TCRαβ and CD45RA depleted grafts containing a median of 6.18 x 106 (range 2.13 - 20.78) CD34+ cells/kg, 0.00 x 104 (range 0 - 0.97) CD45RA+CD3+ cells/kg, and 1.21 x 106 (range 0.15 - 11.67) CD45RO+CD3+ cells/kg. TCRαβ depleted graft fraction contained a median of 0.37 x 104 (range 0 - 11.30) TCRαβ+ cells/kg, and 7.72 x 106 (range 0.62 - 42.00) TCRγδ+ cells/kg. Only 1 patient experienced primary graft failure with no secondary graft failures. All others had engraftment of ANC > 500 cells/µL at median of 12 days (range 8 - 25) and PLT > 20,000 cells/µL at median of 11 days (range 7 - 17). 10 patients with high donor-specific HLA antibodies (DSA) titres engrafted successfully after desensitisation with plasma exchange, rituximab, immunoglobulin, and irradiated donor buffy coat infusion (n=1). 38 patients (35%) developed acute GVHD of grade II - IV, with a cumulative incidence (CI) of grade II-IV and grade III-IV of 34%, and 12% respectively, at 180 days. Chronic GVHD was seen in only 4 patients at a 2-year CI of 5 % . 1-year CI of non-relapse mortality (NRM) and relapse were 18.4 % and 17.2%, respectively. 4 of the 22 NRM were attributed to aGVHD. Viral reactivation included CMV (n=39), HHV-6 (n=28), EBV (n=19), and adenovirus (n=8). 11 patients (11%) out of the 15 infection-related death (15%) occurred within 180 days. At a median follow up of 23 months (range 0.3- 67.1) in surviving patients, 2-year overall (OS), event-free (EFS), and GVHD-free/relapse-free (GRFS) survival were 69.9 %, 58.7 %, and 57.3 %, respectively (Figure 1). In multivariate analysis, HCT-comorbidity index (HCT-CI) has shown significant impact on OS (p=0.008), EFS (p=0.03), and NRM (p=0.002). Disease risk index (DRI) also showed significant impact on OS (p=0.042), EFS (p=0.02) and relapse (p=0.04). In contrast, NRM was only influenced by HCT-CI (p=0.002). In the subset analysis of 63 patients (adults, n= 58; children, n=5) with favorable HCT-CI score of 0/1 and low/intermediate risk DRI, the 2-year OS, EFS, and GRFS were 75.0 %, 68.6%, and 68.0 %, respectively (Figure 2). Conclusions: Our preliminary results suggest that RIC haplo-HCT with TCRαβ and CD45RA+ depleted grafts allow successful allograft in patients with high-risk haematological malignancies lacking suitable matched donors, including patients with high levels of DSA. Acute GVHD was generally abortive, leading to low incidence of chronic GVHD and favorable GRFS. Best outcomes are seen in patients with favourable HCT-CI and low/intermediate risk DRI. Further efforts are needed to reduce the risk of infection-related death in patients with high risk HCT-CI, and relapse risk in patients with high risk DRI, through optimization of conditioning regimen intensity, anti-microbial prophylaxis or prophylactic infusion of memory-cell DLI. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genomic Landscape of Mixed-Phenotype Acute Leukemia.

Mixed-phenotype leukemia (MPAL) is a type of acute leukemia in which the blast population shows mixed features of myeloid, T-lymphoid, and/or B-lymphoid differentiation. MPALs are rare and carry a poor prognosis, thus, often pose both a diagnostic and therapeutic challenge. Conventionally, the diagnosis of MPAL requires either a single blast population with a lineage-defining phenotypic expression of multiple lineages (myeloid, B-cell and/or T-cell) (biphenotypic) or two distinct blast populations that each independently satisfy criteria for designation as AML, B-ALL, and/or T-ALL (bilineage). Given the rarity of MPAL, minimal studies have been performed to describe the genomic landscape of these neoplasms. IRB approval was obtained. Central MCC database was searched for any patient with a diagnosis of acute undifferentiated leukemia (AUL), acute leukemia of ambiguous lineage (ALAL), and MPAL. All patient diagnoses were manually reviewed by a hematopathologist to confirm the diagnosis of MPAL. Genomic and molecular data were collated from the EMR and bioinformatically from MCC genomics repositories. Twenty-eight patients with MPAL were identified. Thirteen were female and 15 were male. Average age was 56 years old (range = 28–81). Ten cases were biclonal and 18 were biphenotypic. Diagnoses were as follows: B/myeloid (n = 18), T/myeloid (n = 9), and T/B (n = 1). Cytogenetic analysis (Karyotype +/− FISH) was available for 27 patients. The most frequent recurrent abnormalities were complex karyotype (n = 8), BCR/ABL1 translocation (n = 6), Del 5q/−5 (n = 4), Polysomy 21 (n = 4). Mutational analysis was available for 18 patients wherein mutations were detected in 45 unique genes. The most frequently mutated genes were TP53 (7), RUNX1 (6), WT1 (4), MLL2 (3), FLT3 (3), CBL (2), ASXL1 (2), TET2 (2), MAP3K6 (2), MLL (2), and MAP3K1 (2). Targetable or potentially targetable biomarkers were found in 56% of cases. Overall survival was 19.5 months (range = 0–70 m). Ten patients were treated with an allogeneic stem cell transplant and had superior outcome (p = 0.0013). In one the largest series of MPAL cases to date, we corroborate previous findings with enriched detection of RUNX1 and FLT3–ITD mutations along with discovery of unreported mutations (MAP3K) that may be amenable to therapeutic manipulation. We also report the frequent occurrence of AML with MDS-related changes (AML-MRC)-defining cytogenetic abnormalities (26%). Finally, we show that those patients that received stem cell transplant had a better overall survival. Our findings support the need to genomically profile MPAL cases to exploit opportunities for targeted therapies in this orphan disease with dismal prognosis.

Acute Leukemia Classification Using Transcriptional Profiles From Low-Cost Nanopore mRNA Sequencing.

Most cases of pediatric acute leukemia occur in low- and middle-income countries, where health centers lack the tools required for accurate diagnosis and disease classification. Recent research shows the robustness of using unbiased short-read RNA sequencing to classify genomic subtypes of acute leukemia. Compared with short-read sequencing, nanopore sequencing has low capital and consumable costs, making it suitable for use in locations with limited health infrastructure. We show the feasibility of nanopore mRNA sequencing on 134 cryopreserved acute leukemia specimens (26 acute myeloid leukemia [AML], 73 B-lineage acute lymphoblastic leukemia [B-ALL], 34 T-lineage acute lymphoblastic leukemia, and one acute undifferentiated leukemia). Using multiple library preparation approaches, we generated long-read transcripts for each sample. We developed a novel composite classification approach to predict acute leukemia lineage and major B-ALL and AML molecular subtypes directly from gene expression profiles. We demonstrate accurate classification of acute leukemia samples into AML, B-ALL, or T-lineage acute lymphoblastic leukemia (96.2% of cases are classifiable with a probability of > 0.8, with 100% accuracy) and further classification into clinically actionable genomic subtypes using shallow RNA nanopore sequencing, with 96.2% accuracy for major AML subtypes and 94.1% accuracy for major B-lineage acute lymphoblastic leukemia subtypes. Transcriptional profiling of acute leukemia samples using nanopore technology for diagnostic classification is feasible and accurate, which has the potential to improve the accuracy of cancer diagnosis in low-resource settings.

Multiple cells of origin in common with various types of mouse N-Myc acute leukemia

Little is known regarding whether the cell of origin differs among different leukemia types. To address this fundamental issue, we determined the cell of origin in five distinct types of acute leukemia induced by N-Myc overexpression in mice. CD150+CD48-CD41-CD34-c-Kit+Sca-1+Lin- (KSL) (HSC1) cells, CD150-CD48-CD41-CD34-KSL (HSC2) cells, CD150+CD41+CD34-KSL (HPC1) cells, CD150+CD41+CD34+KSL (HPC2) cells, and CD150-CD41-CD34+KSL (HPC3) cells were purified from the bone marrow of adult C57BL/6 mice, transduced with the N-Myc retrovirus vector, and transplanted into lethally irradiated mice. B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and mixed phenotype acute leukemia (MPAL) developed from five populations. RNA sequencing data supported the phenotypical diagnoses of leukemia, except that AUL appeared transcriptionally close to T-ALL. Whole-genome sequencing revealed that retroviral integration sites were irrelevant to the leukemia types and that T-ALL and AML of MPAL shared the same integration site and many gene mutations, suggesting their common origin. Additionally, leukemic stem cells were identified in the KSL cell population, suggesting that the phenotypes of leukemic stem cells are irrelevant to leukemia types. This study provides experimental evidence for the similar and multiple cells of origin in acute leukemia.

Early T-Cell Precursor ALL and Beyond: Immature and Ambiguous Lineage T-ALL Subsets.

Simple SummaryImmature T-cell acute lymphoblastic leukemias englobes a wide range of low prevalence subtypes, not well identified, that in some cases overlap with myeloid lineage subtypes. Globally, this “grey zone” of immature leukemias, are difficult to precisely diagnose using a classical immunophenotypic approach. Interesting, genomic data collected during last years has shown that these subtypes share several genomic alterations, raising the question of how their phenotypes reflect distinct AL entities. Here we provide a systematic overview of the genetic events associated with immature T-ALL and outline their relationship with treatment choices and outcomes. Our goal is to offer a basis for using the genetic information for new diagnostic algorithms. An immunogenetic classification of these immature subtypes will better stratify patients and improve their management with more efficient and personalized therapeutic options.A wide range of immature acute leukemias (AL), ranging from acute myeloid leukemias with minimal differentiation to acute leukemias with an ambiguous lineage, i.e., acute undifferentiated leukemias and mixed phenotype acute leukemia with T- or B-plus myeloid markers, cannot be definitely assigned to a single cell lineage. This somewhat “grey zone” of AL expresses partly overlapping features with the most immature forms of T-cell acute lymphoblastic leukemia (T-ALL), i.e., early T-cell precursor ALL (ETP-ALL), near-ETP-ALL, and pro-T ALL. These are troublesome cases in terms of precise diagnosis because of their similarities and overlapping phenotypic features. Moreover, it has become evident that they share several genomic alterations, raising the question of how their phenotypes reflect distinct AL entities. The aim of this review was to provide a systematic overview of the genetic events associated with immature T-ALL and outline their relationship with treatment choices and outcomes, especially looking at the most recent preclinical and clinical studies. We wish to offer a basis for using the genetic information for new diagnostic algorithms, in order to better stratify patients and improve their management with more efficient and personalized therapeutic options. Understanding the genetic profile of this high-risk T-ALL subset is a prerequisite for changing the current clinical scenario.

Morphological and Immunophenotypic Analysis in Diagnosis of Acute Leukaemia

Background: Leukaemias are neoplastic proliferations of haematopoietic stem cells and form a major proportion of haematopoietic neoplasms that are diagnosed worldwide.&#x0D; Objective: To differentiate between morphological and immunophenotypic analysis in the diagnosis of acute leukemia.&#x0D; Materials and method: This cross sectional study was conducted in the department of Haematology, Armed Forces Institute of Pathology (AFIP), Dhaka, Bangladesh from January 2008 to December 2008. Total 50 patients were included after fulfilling inclusion and exclusion criteria.&#x0D; Results: The total of 50 bone marrow samples from suspected cases of acute leukaemia were included in the study. Out of 50 samples, 48 cases were diagnosed as either acute myeloid leukaemia (19 or 38%) or acute lymphoblastic leukaemia (29 or 58%) and 02 (04%) cases were morphologically indistinguishable. All 50 cases were subjected to immunophenotypic study. Out of 50 cases immunophenotypically 14(28%) were acute myeloid leukaemia (AML), 32(64%) were acute lymphoblastic leukaemia (ALL), and bi-phenotypic leukaemia and acute undifferentiated leukaemia were 02(04%) each. In this study Male: Female ratio was 1.3:1. Out of 19(38%) cases of AML, 29(58%) cases of ALL and 02(04%) cases of indistinguishable diagnosed morphologically, 14(28%) were found to be AML, 32(64%) ALL, 02(04%) bi-phenotypic and 02(04%) were acute undifferentiated leukaemias on immunophenotyping respectively. Out of 29 cases identified as ALL on morphology 25(86.2%) were confirmed as ALL, 02(07%) turned out to be AML, 01(3.4%) was bi-phenotypic and 01(3.4%) was undifferentiated.&#x0D; Conclusion: In this study, acute lymphoblastic leukaemia was the commonest type of leukemia followed by acute myeloid leukaemia with male predominance seen in all types of leukaemia.&#x0D; Delta Med Col J. Jul 2020 8(1): 15-20

Prevalence And Diagnosis of Acute Leukaemia - Experience in 50 Cases in AFIP

Background: Haematological malignancies covers a wide range of diseases ranging from acute leukaemia to different type of lymphoproliferative disorders among which acute leukemia is a major concern all over the world. Objective: To evaluate the diagnosis of acute leukemia by morphological and immunophenotypic study. To find out the prevalence of acute leukemia among the 50 patients attending in the department Haematology, Armed forces Institute of Pathology (AFIP), Dhaka Cantonment. Materials and method: This cross-sectional study was conducted in the Department of Haematology, Armed forces Institute of Pathology (AFIP), Dhaka Bangladesh from January 2008 to December 2008. Total of 50 patients were included after fulfilling inclusion and exclusion criteria. Results: Out of 50 patients, 38 (76%) cases are in the age group of 06 months to 15 years followed by 12 (24%) patients belong to the age group of 16 to more than 75 years. Amongst the respondents 58% were male and 42% were female. Morphology of the bone marrow samples revealed that 19 (38%) cases were acute myeloid leukemia, 29 (58%) were found to be acute lymphoblastic leukemia and 02 (04%) were indistinguishable. Whether, from immunophenotyping of 50 cases, 14 (28%) were reported as acute myeloid leukemia, 32 (64%) were acute lymphoblastic leukemia and both bi-phenotypic and undifferentiated acute leukaemia were 02 (04%) of each. Conclusion: Acute lymphoid leukaemia was the commonest type of acute leukaemia. Male predominance was seen in all acute types of leukemia and age has a significant effect on the type of leuakemia.

Does hand mirror morphology a useful diagnostic tool for the treatment decision of acute undifferentiated leukemia at admission?

It is difficult how to manage acute undifferentiated leukemia in daily practice, but generally, hand mirror morphology provides ease to treat these patients. Thirty-nine years old male patient was admitted to with the complaints of echymosis, and pain at his left buttock due to an intramuscular injection for the treatment of previously diagnosed of the lower respiratory infection. Peripheral blood smear revealed >%50 blasts cells with a moderate nuclear: cytoplasmic ratio and one or more nucleoli. The blast cells showed a hand-mirror morphology and not harboring auer rods. According to the flow cytometric analysis the blastic cells do not represent to be originated from myeloid or lymphoid origin, because the cells harboring both of two cell lineages. AML-like therapy was commenced based on the positive myeloid markers including CD117 and CD135. Even though hand mirror morphology of the blasts usually demonstrates the lymphoid origin and the patients are treated as ALL like therapy, myeloid blasts rarely represents the same morphology, as was in our patient.

Patterns of Bone Marrow Confirmed Malignant and Non-Malignant Hematological Disorders in Patients with Abnormal Hematological Parameters in Northeast Ethiopia.

BackgroundHematological disorders are heterogeneous conditions ranging from malignant to non-malignant disorders. Hematological malignancies comprise a collection of heterogeneous conditions originating from cells of the bone marrow and the lymphatic system. Therefore, this study aimed to determine the pattern of bone marrow confirmed malignant and non-malignant hematological disorders in patients with abnormal hematological parameters.MethodsInstitutional-based cross-sectional study was conducted in Dessie town from April 2020 to June 2021. A total of 228 study participants who had abnormal hematological parameters and referred for bone marrow examination were included consecutively. About 1.5 mL of bone marrow sample and 3 mL of venous blood sample were collected for bone marrow examination, complete blood count analysis and peripheral blood morphology examination. Wright stain, Sudan black B, and Prussian blue stains were used for staining the bone marrow and peripheral blood smears. The result was expressed in mean and standard deviation and presented in texts and tables. Ratio, frequency, and percentage were used to express the magnitude of malignant and non-malignant hematological disorders.ResultsThe overall prevalence of hematological malignancies among the study participants was 11.4% with 8.8% in male patients. The prevalence of hematological malignancies were 3.5% CML, 2.6% AML, 1.8% CLL and MM, 0.9% ALL and undifferentiated acute leukemia. On the other hand, 57.0% of the study participants had non-malignant hematological disorders. Regarding non-malignant hematological cases, 24.6% were erythroid hyperplasia, 10.5% aplastic anemia, 8.8% concomitant IDA and MBA, 7.0% MBA, 3.5% leukemoid reaction, 1.8% IDA, and 0.9% visceral leishmaniasis. In patients with HM, 66.7% of AML, 100% of CML and CLL, and 75% of MM patients had increased total WBC count, whereas 66.7% of AML, 62.5% of CML, 75% of CLL, and 50% of MM patients had decreased hemoglobin level. On the other hand, 66.7% of AML, and 50% of CML, ALL, and CLL patients had decreased platelet count.ConclusionIn this study, 11.4% of the patients had hematological malignant cases, whereas 57% of the patients had non-malignant hematological cases. Therefore, in patients with hematological abnormalities and where conclusive diagnosis could not be made through clinical and other laboratory investigations, bone marrow examination should be done for definitive diagnosis, management and prognosis.

Acute undifferentiated/unclassifiable leukemia or disseminated non-hematologic neoplasm? When treatment is the compelling decision

The rapid collection of exact diagnostic and prognostic data is essential to guide therapy in patients diagnosed with acute leukemia. Basic diagnostic steps confirm the hematopoietic nature of the illness and allow distinction between Acute Lymphoid And Myeloid Leukemia (ALL and AML) throughout morphology, immunophenotype and cytogenetic/molecular analysis. However, because the complete results from the latter assays are not readily available, only morphology and Flow Cytometry (FC) permit to define quickly the cell lineage of the disease, thus orientating treatment. A small group of cases is referred to as Acute Undifferentiated Leukemia (AUL), because unclassifiable by current diagnostic markers [1]. AUL makes up 2-7% of Myeloperoxidase (MPO)-negative leukemias and poses a difficult treatment challenge [1,2]. The importance of a correct diagnostic input is further emphasized by the different results obtained with AML or ALL regimens in mixed lineage acute leukemia, another rare subset with debated diagnosis to treatment relationships [3]. Moreover, although rare in the modern era of improved diagnostic methodology, a pure leukemic presentation of an unrecognized metastatic carcinoma may need to be differentiated from AUL, AML or ALL, an issue which may be aggravated if FC results are inconclusive but treatment cannot be deferred until the availability of the other time-consuming diagnostic procedures.

The association of complex genetic background with the prognosis of acute leukemia with ambiguous lineage

Acute leukemia with ambiguous lineage (ALAL) is a rare and highly aggressive malignancy with limited molecular characterization and therapeutic recommendations. In this study, we retrospectively analyzed 1635 acute leukemia cases in our center from January 2012 to June 2018. The diagnose of ALAL was based on either EGIL or 2016 WHO criteria, a total of 39 patients were included. Four patients diagnosed as acute undifferentiated leukemia (AUL) by both classification systems. Among the patients underwent high-throughput sequencing, 89.5% were detected at least one mutation and the median number of gene mutation was 3 (0–8) per sample. The most frequently mutated genes were NRAS (4, 21%), CEBPA (4, 21%), JAK3 (3, 16%), RUNX1 (3, 16%). The mutations detected in mixed-phenotype acute leukemia (MPAL) enriched in genes related to genomic stability and transcriptional regulation; while AUL cases frequently mutated in genes involved in signaling pathway. The survival analysis strongly suggested that mutation burden may play important roles to predict the clinical outcomes of ALAL. In addition, the patients excluded by WHO criteria had even worse clinical outcome than those included. The association of the genetic complexity of blast cells with the clinical outcomes and rationality of the diagnostic criteria of WHO system need to be evaluated by more large-scale prospective clinical studies.

ACUTE UNDIFFERENTIATED LEUKEMIA WITH BCR-ABL TRANSLOCATION: CASE REPORT AND LITERATURE REVIEW

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 75.77×10 9 /L (77% blasts), platelets 175×10 9 /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.

ALL-144: Oncogenic Deregulation of BCL11B in Lineage Ambiguous Leukemia

<h3>Context:</h3> 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. <h3>Objective:</h3> 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. <h3>Results:</h3> 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 <i>BCL11B</i> in 100% of cases and <i>FLT3</i> mutations in 80% of cases. Most structural variants resulted in chromosomal translocations that placed the <i>BCL11B</i> gene in proximity to super-enhancers active in hematopoietic stem or early progenitor cells, a cell type where <i>BCL11B</i> is normally repressed. Additionally, 20% of cases harbored high-copy tandem amplification of a 2.5-kb non-coding element downstream of <i>BCL11B</i> that we term <i>BCL11B</i> enhancer tandem amplification or BETA. Analysis of 3D chromatin structure in primary leukemia samples demonstrated ectopic chromatin interactions between <i>BCL11B</i> and rearranged hematopoietic stem/progenitor cell super-enhancers, or between <i>BCL11B</i> and BETA, thereby demonstrating enhancer hijacking or <i>de novo</i> super-enhancer formation as the mechanism of aberrant <i>BCL11B</i> expression in this subtype. <i>In vitro</i> modeling in human CD34+ stem/progenitor cells demonstrated that ectopic <i>BCL11B</i> expression was sufficient to upregulate T-lineage gene expression programs and block myeloid differentiation, which was exacerbated by constitutive FLT3 signaling. <h3>Conclusions:</h3> <i>BCL11B</i> deregulation in primitive hematopoietic cells is an oncogenic driver of a subset of ALAL that transcends conventional immunophenotypic distinctions.

Comprehensive Immunophenotyping Panel in Japan Enables to Detect Rare Subtypes in Childhood Leukemia

[Background] Although the development of genome wide screening for risk stratification in childhood leukemia, immunophenotyping is still essential to decide initial treatment because of quick duration to make diagnosis. Immunophenotyping is also very useful to detect rare subtypes in leukemia. From 2011, Japanese pediatric leukemia/lymphoma study group (JPLSG) started nation-wide immunophenotyping with comprehensive panel including more than 50 antigens for childhood hematological malignancy. This panel led easier diagnosis not only for typical leukemia subtypes such as acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML), but for rare subtypes including mixed phenotype acute leukemia (MPAL), acute undifferentiated leukemia (AUL), and blastic plasmacytoid dendritic cell neoplasms (BPDCN). We report here the significance of comprehensive immunophenotyping panel, then clinical manifestations and immunophenotypic peculiarity of rare subtypes in childhood leukemia.[Methods] Immunophenotyping was done with comprehensive panel (Table 1) and diagnosis was done according to WHO 2008 classification. Briefly, determination of lineage was usually decided by major lineage specific markers such as myeloperoxidase (MPO), CD19, and cytoplasmic CD3. Then, more detailed categorization was done by analysis with other lineage, differentiation, or activation markers.[Result] In 4,033 case from January 2012 to December 2016, major immunophenotypes were as follows; B-precursor/pre-B (BCP-) ALL was 2,141, T cell (T-) ALL 348, AML 842, B-ALL 74, Lymphomas 171, transient myeloproliferative disorder (TAM) 244, chornic myelogenous leukemia (CML) 72. Out of other 141 cases, we also found 32 MPAL, 16 AUL, and 7 BPDCN. According to detailed immunophenotyping, MPAL cases could be divided into 3 distinct clusters, T-ALL/ AML-M1 biphenotypic subtype (8 cases), BCP-ALL/AML-M5 bilineal subtype (11), and BCP-ALL with MPO expression subtype (13). Furthermore, distinct cluster, ‘early T cell progenitor ALL (ETP)-like subtype’ was also distinguished from AUL.[Discussion] Acute leukemia of ambiguous lineage and BPDCN are both rare subtypes of acute leukemia. According to WHO 2008 classification, acute leukemia of ambiguous lineage is further classified as MPAL, AUL and natural killer lymphoblastic leukemia. In this study, we evaluate 32 MPAL, 16 AUL and 7 BPDCN. It is of note that we could not identify no natural killer lymphoblastic leukemia in this childhood leukemia cohort. We also evaluate 32 MPAL cases, which showed 3 distinct subgroups. T-ALL oriented cases indicate only one subtype ‘T-ALL/AML-M1 biphenotypic’, which indicated relatively older age, usually diagnosed as AML-M1 with morphologic FAB classification, and received mostly myeloid oriented therapy. BCP-ALL related cases were also divided into two groups, BCP-ALL/AML-M5 bilineal, and BCP-ALL with MPO expression. Bilineal leukemia often found in infantile ALL with MLL gene rearrangement (4 out of 11 cases), but still found 7 older cases without MLL gene rearrangemnt. Bilineal leukemia occasionally cause lineage-switch relapse, so diagnosis of bilineal leukemia seems to be very significant. Recent studies showed leukemia with ZNF384 fusion indicate CD10 negative or low BCP-ALL, and tended to express MPO. Our cases also tended to indicate negative or low CD10 expression. In addition, ETP-ALL like cases were positive for T cell antigens such as CD2 and/or CD7 and also some myeloid antigens but lacked cytoplasmic CD3 expression.In conclusion, we identified a spectrum of rare subtypes in childhood leukemia, including MPAL, AUL, and BPDCN by adoption of comprehensive immunophnotyping panel. In most of childhood MPAL cases were divided into 3 distinct clusters. It must be useful information for detection of genetic abnormality. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

First Results of a Prospective Multicenter Phase I/II Clinical Trial in Adult Patients Using Tcralpha/Beta and CD19 Depleted Haploidentical Stem Cell Transplantation Following Reduced Intensity Conditioning

We report the first prospective, multi-center, single-arm phase I/II study that assesses the safety and feasibility of haploidentical transplantation with TCRalpha/beta and CD19+ cells depleted peripheral blood stem cell grafts using the CliniMACS plus System (Miltenyi Biotec, Germany) in combination with a reduced-intensity conditioning (RIC) (www.clinicaltrialsregister.org; 2011-005562-38). The grafts were processed in 7 GMP laboratories and immune cell assays were performed in two core labs using standardized methods and the MACSQuant flow cytometry device (Miltenyi Biotec, Germany). The RIC regimen consisted of 15 or 30 mg ATG (Fresenius/Grafalon) or 7 Gy total nodal irradiation, 160 mg/m2 fludarabine, 10 mg/kg thiotepa, and 140 mg/m2 melphalan. MMF (40mg/kg/day) was used for GVHD prophylaxis until Day 30. The Day 100 data of the 30 pediatric patients have been presented at ASH2016; this report presents the data of the 30 adult patients with at least 100 days follow-up alive.Results: Seven hospitals enrolled 13 female and 17 male participants (median age 38.5 years, range 20-63 years), including 7 ALL, 17 AML, 3 MDS/MPS, 1 each with multiple myeloma, acute undifferentiated leukemia or sickle cell anemia. Malignant diseases were in complete remission (n=19), partial remission (n=6) or non-remission (n=4). Eight had one or more previous SCTs. The median follow-up is 360 days (range, 18 - 776). Of the 30 recipients, 9 already completed the 2-year follow-up.The median number of CD34+ cells, TCRab+ cells and CD20+ cells/kg was 8.1 x 106 (range, 4 - 18), 13.5 x 103 (range, 0.22 - 64.3) and 28 x 103 (range, 2 - 727), respectively. In addition, significant numbers of NK and TCRgd+ cells/kg were infused: median 39.3 x 106 (range, 9.4 - 102) and 8.9 x 106 (range, 1.1 - 30.7), respectively. All 30 patients tolerated the cell infusion well and 28 had primary engraftment of ANC >500 cells/µL at a median of 14 days (range, 9 - 41) and PLT >20,000 cells/µL at a median of 15 days (range, 12 - 38). Peripheral donor T-cell chimerism at the time of engraftment was complete in 27 patients, and mixed in one. On day 100, chimerism was completely donor in 24 patients and mixed in one. Two patients experienced primary graft failure after conditioning with ATG (one had sickle cell anemia, received 16 x 106 CD34 cells/kg; the other had high-risk AML in partial remission, received 18 x 106 CD34 cells). One patient had a secondary graft failure. All three patients were successfully re-transplanted.No patient developed severe acute GVHD grade III/IV. Five patients (16.7%) had grade II, 7 (23.3%) had grade I, and 18 (60%) had none despite minimal GVHD prophylaxis after transplantation.28 patients with primary engraftment were evaluable for immune reconstitution . The majority of lymphocytes on Day28 were NK cells (median 272 cells/µL; range, 10 - 1092); the median CD3+ cell count was 112 cells/µL (range, 9 - 911), with approximately two third of them being TCRgd+ (median 87 cells/µL; range, 7 - 891). Between day 28 and day 100, the median TCRab+ cells increased from 36 to 116 cells/µL (Figure 1).CMV (n=13) and HHV6 (n=10) reactivations were the most common infectious complications following transplantation. However, only one patient developed CMV colitis. Similarly, although ADV was detected in 4 patients in stool, no ADV disease occurred. Two patients were tested positive for BKV and both experienced hemorrhagic cystitis. Two patients had EBV reactivation and 3 had bacterial sepsis.At a median follow-up of 360 days 21 of 30 patients are alive; 9 patients died: 4 relapses, 5 organ failures (2 lung, 1 each liver, CNS, cardiac). After 100 days and 1 year the cumulative incidences of NRM and relapse were 7% and 8% and 20% and 13%, respectively.Kaplan-Meier estimated OS and DFS were 93% and 86% on day 100 and 73% and 70% at 1 year, respectively.Conclusion: The CliniMACSplus TCRalpha/beta and CD19-depletion System, when used in combination with a reduced-intensity conditioning regimen, resulted in favorable rates of primary engraftment (93%), no acute GVHD grade III/IV (0%), NRM (7%) and OS of 93% at day 100 and an estimated OS of 73% after 1 year. The immune reconstitution of innate immunity was rapid and there was no lethal viral and fungal infection. Longer follow up will provide more information on chronic GVHD and long term survival. [Display omitted] DisclosuresBethge:Neovii GmbH: Honoraria, Research Funding; Miltenyi Biotech GmbH: Consultancy, Honoraria, Research Funding. Mielke:ISCT: Other: Travel support; DGHO: Other: Travel support; Cellex GmbH: Other: Travel grants, Speakers Bureau; Gilead: Other: Travel grants; MSD: Consultancy, Other: Travel grants; Celgene: Other: Travel grants, Speakers Bureau; Novartis: Consultancy; Jazz Pharma: Speakers Bureau; KIADIS Pharma: Other: Travel grants. Kuball:Gadeta (www.gadeta.nl): Consultancy, Equity Ownership, Patents & Royalties: on gdT cells and receptors and isolation strategies , Research Funding; Miltenyi: Research Funding. Karitzky:Miltenyi Biotec GmbH: Employment. Holtkamp:Miltenyi Biotec GmbH: Employment. Siewert:Miltenyi Biotec GmbH: Employment. Pflitsch:Miltenyi Biotec GmbH: Employment. Handgretinger:Miltenyibiotec GmbH: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding.