Blast phase of chronic myeloid leukemia with concurrent BCR::ABL1 and SET::NUP214: A report of two cases.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm defined by the presence of t(9;22)(q34;q11.2)/BCR::ABL1. Additional chromosomal abnormalities play an important role in the progression to CML. However, the additional fusion gene was rarely reported such as CBFB::MYH11. In this report, we described two cases of the co-occurrence of BCR::ABL1 and SET::NUP214 in CML-BP for the first time, which is associated with poor outcomes during tyrosine kinase inhibitor (TKI) treatment. Meanwhile, we retrospectively analyzed SET::NUP214 fusion transcript of the two cases at initial diagnosis of the CML chronic phase by quantitative RT-PCR, and detected at a ratio of 1.63% and 1.50%, respectively. SET::NUP214 may promote disease progression during the transformation of CML. This study highlights the importance of extended molecular testing at the initial diagnosis of CML-CP at TKI resistance and/or disease transformation.

Identification of variants in 94 Chinese patients with hereditary spherocytosis by next-generation sequencing.

Hereditary spherocytosis (HS) is the most common type of hereditary erythrocyte membrane disease and has varied phenotypic features and genetic patterns. We herein performed a retrospective study of 94 patients with HS and aimed to investigate the genetic variations and genotype-phenotype correlations using targeted next-generation sequencing. In 79/94 (84%) patients, 83 HS variants including 67 novel variants were identified. Pathogenic variants of SPTB, ANK1, SLC4A1, SPTA1, and EPB42 were found in 32/79(41%), 22/79(28%), 15/79 (19%), 8/79 (9%), and 3/79 (4%) of the patients respectively, revealing that SPTB is the most frequently mutated HS gene in Eastern China. Most SPTB and ANK1 gene variations were nonsense and frameshift variations. Missense variants were the main variant type of SLC4A1, SPTA1, and EPB42 genes. Interestingly, one SPTA1 variant (p. Arg1757Cys) showed an autosomal dominant inheritance pattern and one EPB42 variant (p. Gln377His) was apparent as a hotspot variation. Furthermore, genotype-phenotype analysis was performed among the five mutated gene groups. Besides the finding that patients with the SLC4A1 variant had the highest mean corpuscular hemoglobin levels, no clear correlations between genotype and phenotype were observed.

Integrative genomic and transcriptomic profiling reveals distinct molecular subsets in adult mixed phenotype acute leukemia.

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.

Transcriptome sequencing reveals CHD1 as a novel fusion partner of RUNX1 in acute myeloid leukemia with t(5;21)(q21;q22).

BACKGROUND: RUNX1/AML1, which is a Runt family transcription factor critical for normal hematopoiesis, is frequently mutated or translocated in a broad spectrum of hematopoietic malignancies. FINDINGS: We describe here the case of a 54-year-old female developed acute myeloid leukemia with a t(5;21)(q21;q22). Transcriptome sequencing identified the chromodomain-helicase-DNA-binding protein 1 gene, CHD1, as a novel partner gene of RUNX1. Furthermore, the patient was found to harbor FLT3-ITD mutation, which might collaborated with CHD1-RUNX1 in the development of acute myeloid leukemia. CONCLUSIONS: We have identified CHD1 as the RUNX1 fusion partner in acute myeloid leukemia with t(5;21)(q21;q22).

Insights from a rare myeloproliferative neoplasm with coexisting BCR-ABL1 fusion gene, CALR, and TET2 mutations treated with nilotinib and ruxolitinib.

Myeloproliferative neoplasms (MPNs) with concurrent BCR-ABL1 fusion gene and CALR mutation are especially rare. We report a patient with coexisting BCR-ABL1 fusion gene, CALR, and TET2 mutations who was treated with the combination of the second-generation TKI nilotinib and JAK1/JAK2 inhibitor ruxolitinib.

Identification of a venetoclax-resistance prognostic signature base on 6-senescence genes and its clinical significance for acute myeloid leukemia.

Background: Satisfactory responses can be obtained for acute myeloid leukemia (AML) treated by Venetoclax (VEN)-based therapy. However, there are still quite a few AML patients (AMLs) resistant to VEN, and it is critical to understand whether VEN-resistance is regulated by senescence. Methods: Here, we established and validated a signature for predicting AML prognosis based on VEN resistance-related senescence genes (VRSGs). In this study, 51 senescence genes were identified with VEN-resistance in AML. Using LASSO algorithms and multiple AML cohorts, a VEN-resistance senescence prognostic model (VRSP-M) was developed and validated based on 6-senescence genes. Results: According to the median score of the signature, AMLs were classified into two subtypes. A worse prognosis and more adverse features occurred in the high-risk subtype, including older patients, non-de novo AML, poor cytogenetics, adverse risk of European LeukemiaNet (ELN) 2017 recommendation, and TP53 mutation. Patients in the high-risk subtype were mainly involved in monocyte differentiation, senescence, NADPH oxidases, and PD1 signaling pathway. The model's risk score was significantly associated with VEN-resistance, immune features, and immunotherapy response in AML. In vitro, the IC50 values of ABT-199 (VEN) rose progressively with increasing expression of G6PD and BAG3 in AML cell lines. Conclusions: The 6-senescence genes prognostic model has significant meaning for the prediction of VEN-resistance, guiding personalized molecularly targeted therapies, and improving AML prognosis.

RUNX1 together with DAT mutations predicted poor outcome in acute myeloid leukemia.

We retrospectively explored the prognostic impact of DAT mutations at diagnosis in 122 RUNX1mut AML patients. RUNX1 missense mutation was dominant in the RUNT domain, and frameshift mutation was dominant in the TAD domain. DAT mutations occurred in 38.5% of RUNX1mut AML. After propensity score matching, DATpos patients had worse two-year relapse-free survival (RFS) than DATneg patients (p = .041). Moreover, RUNX1high (VAF ≥ 37.6%) patients showed poorer two-year overall survival (OS) and RFS than RUNX1low (VAF < 37.6%) patients (OS, p = .033; RFS, p = .027), especially in the RUNX1highDATpos group. Additionally, multivariate analysis confirmed that DAT mutations at diagnosis were an independent adverse factor for RFS. There were no significant differences in two-year OS and RFS between DATpos and DATneg patients or between RUNX1high and RUNX1low patients who undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Collectively, DAT mutations at diagnosis were adverse factors for RFS, and allo-HSCT could likely improve the poor outcomes of these patients.

[Application of Bionano-DNA Full-Length Optical Mapping Technology in Genetic Diagnosis of Hemophilia A Carriers].

OBJECTIVE: To apply Bionano Saphyr visual full-length DNA optical mapping technology to the precise genetic diagnosis of hemophilia A carriers. METHODS: For 2 suspected F8 gene deficiency female carriers who could not be diagnosed by conventional next-generation sequencing technology, the full-length DNA optical mapping technology was used to detect and scan the sample X chromosome full-length visual haplotype characteristic map, which was compared with the normal haplotype. The gene structure variation information of the samples was obtained by compare with DNA atlas library. RESULTS: The average fluorescent marker length of the X chromosome DNA molecular where the F8 gene was located in the two samples was greater than 2.5 Mbp, and the average copy number was greater than 20×. After comparative analysis, one of the samples was a proximal inversion of intron 22 of the F8 gene, and another was an inversion of intron 22 accompanied by multiple deletions of large fragments. CONCLUSIONS: Bionano technology has a good detection rate for gene defects with large length and complex variation. In the absence of a proband or accurate genetic diagnosis results of the proband, the application of this technology to detect the heterozygous complex variant of the F8 gene is of great significance for the prenatal diagnosis and pre-pregnancy diagnosis of hemophilia carriers.

Clinical and molecular characteristics of acute myeloid leukemia with MPL mutation.

OBJECTIVES: The current study aimed to explore the incidence of MPL mutations and the clinical and molecular characteristics of AML with MPL mutation. METHODS: In total, 1509 patients with newly diagnosed AML were retrospectively analyzed between January 2017 and December 2020. MPL mutations were detected via next-generation sequencing. During the same period, we also enrolled 30 patients with other myeloid neoplasms (MNs) with MPL mutation, which included myelodysplastic syndrome (n = 15), myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) (n = 6), and MPN (n = 9). The clinical characteristics of MPL-mutated AML and other types of MNs or MPL-wide type (MPL-wt) AML were compared, and the spectrum of co-mutations and MPL mutation profiles in MPL-mutated AML were analyzed. RESULTS: MPL mutations were identified in 19 (1.26%) of 1509 patients with AML. The waterfall diagram showed that the co-mutations were mainly epigenetic modifications (TET2, IDH1, and EZH2), spliceosomes (SRSF2), and transcription factors (RUNX1). The platelet count of the AML group was significantly lower than that of the MPN group (p = 0.001). MPL mutations were commonly observed in the intracellular region in AML but the transmembrane region in MPN (p = 0.013). The MPL-mutated AML group had a lower white blood cell count and a lower rate of complete remission than the MPL wild-type AML group (p = 0.037). CONCLUSION: MPL mutations are clinically relevant in patients with AML, and they may be a novel subtype characterized by lower white blood cell counts and poor complete remission rates. However, further studies must be conducted to identify its correlated mechanism.

Treatments of Ph-like acute lymphoblastic leukemia: a real-world retrospective analysis from a single large center in China.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of ALL. We retrospectively studied 70 cases with Ph-like ALL and here present the largest study of CAR-T cell treatment and haplo-HSCT for this leukemia. Median age was 26 years and median leukocyte count was 31.44 × 109/L. The proportion of patients receiving chemotherapy, KIs, CAR-T cells, and allo-HSCT was 19%, 30%, 46%, and 61%, respectively. The overall response rate was 62%, 73%, and 100% after one month of KI treatment combined with chemotherapy, CAR-T cell therapy, and allo-HSCT, respectively. Five-year DFS and OS were 35% and 51%, respectively. The five-year cumulative incidence of relapse and non-relapse mortality was 63% and 11%, respectively. Allo-HSCT was associated with a better DFS (p = 0.010) and OS (p = 0.000) by univariate analysis. In conclusion, allo-HSCT after KIs together with chemotherapy or CAR-T cell therapy is a safe and feasible treatment modality for Ph-like ALL.

Case Report: The Formation of a Truncated PAX5 Transcript in a Case of Ph-Positive Mixed Phenotype Acute Leukemia With dic(7;9)(p11-p13;p13).

PAX5 plays a critical role in B-cell precursor development and is involved in various chromosomal translocations that involve the fusion of a portion of PAX5 to at least 49 different partners reported to date. Here, we identified a novel PAX5 fusion transcript in a Ph-positive mixed phenotype acute leukemia case with dic(7;9)(q13;q13), in which a translocation juxtaposes the 5' region of PAX5 and the ubiquitin-conjugating enzyme E2D4 (UBE2D4) to generate a PAX5-UBE2D4 fusion gene. To further explore the general characteristics and function of PAX5-UBE2D4, we cloned the full-length cDNA, which was amplified from the bone marrow of the patient. Interestingly, the fusion was located in the nucleus and negatively affected PAX5 transcription activity. Importantly, the fusion promoted tumor growth in nude mice and the proliferation of NIH3T3 cells in vitro. In conclusion, the fusion resulted in partial oncogenic activity, in contrast to the tumor suppressor activity of wild-type PAX5.

A Recurrent Cryptic MED14-HOXA9 Rearrangement in an Adult Patient With Mixed-Phenotype Acute Leukemia, T/myeloid, NOS.

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.

Efficacy of ruxolitinib in a patient with myelodysplastic/myeloproliferative neoplasm unclassifiable and co-mutated JAK2, SF3B1 and TP53.

Myelodysplastic/myeloproliferative neoplasm, unclassifiable (MDS/MPN-U) is a rare but heterogeneous subtype of MDS/MPN, with no specific genetic alterations and standard treatments. ASXL1, SRSF2, TET2, JAK2 and NRAS are commonly mutated in MDS/MPN-U. Double gene mutations could be detected in MDS/MPN-U, however, co-mutations of 3 and more genes in this disease entity are very rare. Here, we present a case of MDS/MPN-U with triple mutations involving JAK2, SF3B1, and TP53. After failure of traditional therapy including hydroxyurea and interferon-α, the patient received ruxolitinib monotherapy and achieved hematological response quickly. Though mutations in TP53 implied a poor prognosis in myeloid malignancies, this patient has maintained no AML transformation for 26 months since diagnosis. Further research on complex mutations in the pathogenesis and prognosis of MDS/MPN-U is warranted.

[ARMS-PCR combined with capillary electrophoresis can be a sensitive and quantitative method for detection of MYD88-L265P mutation in lymphoma].

OBJECTIVE: To investigate the feasibility of sensitive and quantitative detection of MYD88 gene L265P mutation in lymphoma patients by using ARMS-PCR combined with capillary electrophoresis. METHODS: ARMS-PCR amplified MYD88 gene was analyzed by capillary electrophoresis in ABI 3730 sequencer; Exon 5 of the same gene was sequenced bi-directionally as reported. RESULTS: The sensitivity of detection L265P mutations by the ARMS-PCR combined with capillary electrophoresis and direct sequencing was 0.2% and 5%, respectively, according to the detection of the gradient-diluted plasmid standards. The detection rate of 184 patients was 13.59% and 8.28%, respectively (p<0.001). Moreover, the former method can successfully detect the mutation ratio(R2=0.979), and the repeatabilities (CV=2.86%, 1.94%, 5.49%) are acceptable. CONCLUSION: ARMS-PCR combined with capillary electrophoresis can quantitatively detect the MYD88 gene L265P mutation, and the detection sensitivity is significantly higher than sanger sequencing. As a supplement to the latter, it can effectively lead to the earlier diagnose and monitoring of minimal residual disease.

Application of High-Throughput Sequencing in the Diagnosis of Inherited Thrombocytopenia.

Inherited thrombocytopenia is a group of hereditary diseases with a reduction in platelet count as the main clinical manifestation. Clinically, there is an urgent need for a convenient and rapid diagnosis method. We introduced a high-throughput, next-generation sequencing (NGS) platform into the routine diagnosis of patients with unexplained thrombocytopenia and analyzed the gene sequencing results to evaluate the value of NGS technology in the screening and diagnosis of inherited thrombocytopenia. From a cohort of 112 patients with thrombocytopenia, we screened 43 patients with hereditary features. For the blood samples of these 43 patients, a gene sequencing platform for hemorrhagic and thrombotic diseases comprising 89 genes was used to perform gene detection using NGS technology. When we combined the screening results with clinical features and other findings, 15 (34.9%) of 43patients were diagnosed with inherited thrombocytopenia. In addition, 19 pathogenic variants, including 8 previously unreported variants, were identified in these patients. Through the use of this detection platform, we expect to establish a more effective diagnostic approach to such disorders.