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Mixed phenotype acute leukemia (MPAL) is a subtype of leukemia in which lymphoid and myeloid markers are co-expressed. Knowledge regarding the genetic features of MPAL is lacking due to its rarity and heterogeneity. Here, we applied an integrated genomic and transcriptomic approach to explore the molecular characteristics of 176 adult patients with MPAL, including 86 patients with T-lymphoid/myeloid MPAL (T/My MPAL-NOS), 42 with Ph+ MPAL, 36 with B-lymphoid/myeloid MPAL (B/My MPAL-NOS), 4 with t(v;11q23), and 8 with MPAL, NOS, rare types. Genetically, T/My MPAL-NOS was similar to B/T MPAL-NOS but differed from Ph+ MPAL and B/My MPAL-NOS. T/My MPAL-NOS exhibited higher CEBPA, DNMT3A, and NOTCH1 mutations. Ph+ MPAL demonstrated higher RUNX1 mutations. B/T MPAL-NOS showed higher NOTCH1 mutations. By integrating next-generation sequencing and RNA sequencing data of 89 MPAL patients, we defined eight molecular subgroups (G1-G8) with distinct mutational and gene expression characteristics. G1 was associated with CEBPA mutations, G2 and G3 with NOTCH1 mutations, G4 with BCL11B rearrangement and FLT3 mutations, G5 and G8 with BCR::ABL1 fusion, G6 with KMT2A rearrangement/KMT2A rearrangement-like features, and G7 with ZNF384 rearrangement/ZNF384 rearrangement-like characteristics. Subsequently, we analyzed single-cell RNA sequencing data from five patients. Groups G1, G2, G3, and G4 exhibited overexpression of hematopoietic stem cell disease-like and common myeloid progenitor disease-like signatures, G5 and G6 had high expression of granulocyte-monocyte progenitor disease-like and monocyte disease-like signatures, and G7 and G8 had common lymphoid progenitor disease-like signatures. Collectively, our findings indicate that integrative genomic and transcriptomic profiling may facilitate more precise diagnosis and develop better treatment options for MPAL.

To define the fusion genes in T/myeloid mixed-phenotype acute leukemia (T/M MPAL), we performed transcriptome sequencing of diagnostic bone marrow samples from 20 adult patients. Our analysis identified a second instance of a recurrent MED14-HOXA9 chimeric gene resulting from the in-frame fusion of exon 23 of MED14 and exon 1 of HOXA9, the first in an adult patient. The MED14-HOXA9 fusion gene was detected in both the diagnostic and relapsed blasts with reverse transcription-polymerase chain reaction and Sanger sequencing. The patient received combined conventional chemotherapy but suffered relapse at 11 months and died of disease progression one year after the initial diagnosis. Our data suggest that MED14-HOXA9 is a cryptic recurrent aberration in T/M MPAL, which might indicate an aggressive clinical course and inferior outcome after conventional chemotherapy. Further studies will be carried out to reveal the effects of the MED14-HOXA9 fusion on the differentiation and proliferation of leukemia stem cells, as well as suitable treatment strategies for this emerging entity.

Numerous acquired molecular and cytogenetic abnormalities are strongly associated with hematological malignancies. The breakpoint cluster region-ABL proto-oncogene 1 (BCR-ABL) rearrangement leads to a p210 chimeric protein in typical chronic myeloid leukemia (CML), whereas 17–25% of patients with acute lymphocytic leukemia and 0.9–3% patients with de novo acute myeloid leukemia (AML) carry a p190BCR-ABL fusion protein. Cases of patients with AML/CML carrying two specific primary molecular changes, BCR-ABL and core binding factor-β-myosin heavy chain 11 (CBFβ-MYH11) fusion genes have been rarely reported. The present study aimed to understand the nature and mechanism of this particular type of leukemia through case reports and literature review. A total of four patients who were diagnosed as AML/CML with BCR-ABL and CBFβ-MYH11 fusion genes in the First Affiliated Hospital of Soochow University (Suzhou, China) between January 2004 and December 2012 were examined. Morphological analysis of bone marrow cells, flow cytometry, quantitative polymerase chain reaction of p210BCR-ABL and CBFβ-MYH11 transcripts as well as cytogenetic and fluorescence in situ hybridization analyses were performed. A total of 4 patients who exhibited fusion of p210BCR-ABL and CBFβ-MYH11 were identified. A single patient (case 1) was first diagnosed CML-acute phase (AP), which progressed rapidly to CML-blast crisis (BC), and three patients (cases 2, 3 and 4) were diagnosed with AML with bone marrow eosinophilia at first presentation with no evidence of previous onset of CML. All cases achieved remission following conventional chemotherapy/hematological stem cell transplantation combined with the inhibitor of tyrosine kinase (TKI) maintenance therapy. The patients with CML carrying and expressing BCR-ABL and CBFβ-MYH11 fusion genes appeared more likely to rapidly progress to AP or BC. Therefore, the product of the CBFβ-MYH11 fusion gene may serve an important role in the transformation of CML. The co-expression of p210BCR-ABL and CBFβ-MYH11 fusion genes in myeloid leukemia may be a molecular event occurring not only during the development of CML, but also in AML.

To investigate the clinical and molecular cytogenetic features of myeloid diseases characterized by i(20q-).The clinical data of 7 patients with myeloid diseases, 6 with myelodysplastic syndrome (MDS) and one with acute myelocytic leukemia (AML), 4 males and 3 females, aged 51 - 74, were analyzed. Chromosome specimens were prepared by direct method and/or short-time culture of bone marrow cells. Karyotyping was performed by R banding technique. Two kinds of probes (20q subtelomere probe and 20q12 unique sequence probe) were used in dual-color fluorescence in situ hybridization (D-FISH) assay.Of the seven patients, 4 died and 3 survived by the end of this study. The patients survived for 6 months (case 1), 7 months (case 2), 17 days (case 4), and 28 days (case 5) respectively. Karyotype analysis showed that one of the normal chromosomes 20 was lost and substituted by one or two small metacentric isochromosomes smaller than the normal chromosome 20 in all these seven cases. It was proved to be ider(20)(q10) del(20)(q11q13), i(20q-) in six cases by D-FISH assay.i(20q-) is a novel and rare recurrent chromosome abnormality which may be specifically associated with myeloid diseases and poor prognosis.

Fms-Like tyrosine kinase 3 (FLT3) mutations are one of the most frequent genetic changes in acute myeloid leukemia (AML) and are related to poor prognosis. However, in acute promyelocytic leukemia (APL) the prognostic significance of this mutation is not firmly established. We investigated FLT3 internal tandem duplications (FLT3/ITD) or point mutation of the activation loop domain (FLT3/ALM) in initial marrow samples in 160 APL patients, also studied the impact of FLT3 mutations on disease characteristics and clinical outcome. Some of FLT3/ITD+ samples were further examined to determine the ITD allelic ratio(ITD-AR) using Genescan analysis. FLT3/ITD and FLT3/ALM were detected in 31 (19.4%) and 17(10.6%) of the patients, 2(1.25%) showed both ITD and ALM mutations. 19 of 31 FLT3-ITD+ patients were examined to determine ITD-AR, which varied from 0.11 to 1.79, with a median of 0.92. ITD-AR in 8 patients were greater than 1.0. Both mutations were associated with higher white blood cell(WBC) count at presentation(P0.05). Six originally FLT3-ITD+ patients were not detectable mutation after remission; so were six patients with originally FLT3-LAM+; however FLT3-LAM in one originally FLT3-LAM+ patient reappeared after relapse. In conclusion, FLT3 mutations (FLT3/ITD or FLT3/ALM) were frequently identified in patients with newly diagnosed APL, and both mutations were associated with higher WBC count at presentation. FLT3/ITD is more frequent than ALM mutation, and predicts a poorer prognosis because of the lower CR rates, while, FLT3/ALM mutation did not show the same unfavorable prognostic effect. In our study, ITD-AR were not found significantly difference between CR and no remission patients (P>0.05)

The most frequent chromosomal abnormalities in B-cell chronic lymphocytic leukaemia (B-CLL) are deletions on 13q14 and 17p13, trisomy 12 and 14q32 rearrangement. Conventional metaphase cytogenetic analysis underestimates the frequency of specific chromosome aberrations in B-CLL due to the low rate of spontaneous mitoses and poor response to mitogen stimulation. The aim of this study was to investigate the incidence of chromosomal changes in bone marrow or peripheral blood cells (or both) of B-CLL patients using a molecular cytogenetic method, interphase fluorescence in situ hybridization (I-FISH). Probes for 13q14 (D13S319), 17p13 (P53 gene), the centromere of chromosome 12 (D12Z3) and 14q32 (Ig10 and Y6) were applied to detect chromosomal aberrations on bone marrow and peripheral blood smears from 83 B-CLL patients (60 male, 23 female,). Molecular cytogenetic aberrations were found in 60 (72.3%) cases, and 8 (9.6%) patients showed two kinds of abnormalities. The most frequent abnormalities detected in our patients was deletions of 13q14 in 34 cases (41.0%), followed by trisomy of chromosome 12 in 16 patients (19.3%), deletions of 17p13 in 10 patients (12%) and 14q32 rearrangement in 8 patients (9.6%). Statistical analyses were performed to correlate the molecular cytogenetic findings with Binet stages. No apparent differences in distribution were noted for anomalies del(13q14), del(17p13), +12 or 14q32 rearrangement among patients with various Binet stages. FISH was found to be a more rapid, exact and sensitive technique for the analysis of chromosome aberrations in CLL. FISH could provide accurate information of molecular cytogenetics for CLL.

The NUP98 gene has been reported to be fused with at least 17 partner genes in leukemia with 11p15 translocation. An adult patient with de novo acute myeloid/T lymphocytic leukemia harboring t(3;11)(q29q13;p15) has been investigated to characterize the genes involved in that translocation. Through molecular cytogenetic analysis, we identified a fusion transcript between NUP98 gene and a novel partner gene named as NUP98 related gene (NRG) at 3q29. Further molecular analysis showed that exon 13 of NUP98 was fused in-frame to exon 10 of NRG. Moreover, the segment from 3q13 to 3q29 translocated at 11p15 had been inverted and accompanied by the deletion of the distal portion of breakpoint at chromosome 3q29. Interestingly, the NUP98-NRG protein showed nuclear and cytoplasmic distribution, a pattern different from that of wild type NUP98 or NRG. When assayed in a GAL 4 reporter system, the fusion gene showed an aberrant trans-regulatory activity. Transfection in HL-60 cells demonstrated that NUP98-NRG could promote cell proliferation, survival and arrest differentiation. Therefore, NUP98-NRG may exert transforming effects by interfering with the cellular mechanism of transcriptional regulation. Our data provide thus new evidence that NUP98-related molecular abnormality is a recurrent genetic event in leukemogenesis.