Aging and comprehensive molecular profiling in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aging-related and heterogeneous hematopoietic malignancy. In this study, a total of 1,474 newly diagnosed AML patients with RNA sequencing data were enrolled, and targeted or whole exome sequencing data were obtained in 94% cases. The correlation of aging-related factors including age and clonal hematopoiesis (CH), gender, and genomic/transcriptomic profiles (gene fusions, genetic mutations, and gene expression networks or pathways) was systematically analyzed. Overall, AML patients aged 60 y and older showed an apparently dismal prognosis. Alongside age, the frequency of gene fusions defined in the World Health Organization classification decreased, while the positive rate of gene mutations, especially CH-related ones, increased. Additionally, the number of genetic mutations was higher in gene fusion-negative (GF-) patients than those with GF. Based on the status of CH- and myelodysplastic syndromes (MDS)-related mutations, three mutant subgroups were identified among the GF- AML cohort, namely, CH-AML, CH-MDS-AML, and other GF- AML. Notably, CH-MDS-AML demonstrated a predominance of elderly and male cases, cytopenia, and significantly adverse clinical outcomes. Besides, gene expression networks including HOXA/B, platelet factors, and inflammatory responses were most striking features associated with aging and poor prognosis in AML. Our work has thus unraveled the intricate regulatory circuitry of interactions among different age, gender, and molecular groups of AML.

Transcriptome-based molecular subtypes and differentiation hierarchies improve the classification framework of acute myeloid leukemia.

The current classification of acute myeloid leukemia (AML) relies largely on genomic alterations. Robust identification of clinically and biologically relevant molecular subtypes from nongenomic high-throughput sequencing data remains challenging. We established the largest multicenter AML cohort (n = 655) in China, with all patients subjected to RNA sequencing (RNA-Seq) and 619 (94.5%) to targeted or whole-exome sequencing (TES/WES). Based on an enhanced consensus clustering, eight stable gene expression subgroups (G1-G8) with unique clinical and biological significance were identified, including two unreported (G5 and G8) and three redefined ones (G4, G6, and G7). Apart from four well-known low-risk subgroups including PML::RARA (G1), CBFB::MYH11 (G2), RUNX1::RUNX1T1 (G3), biallelic CEBPA mutations or -like (G4), four meta-subgroups with poor outcomes were recognized. The G5 (myelodysplasia-related/-like) subgroup enriched clinical, cytogenetic and genetic features mimicking secondary AML, and hotspot mutations of IKZF1 (p.N159S) (n = 7). In contrast, most NPM1 mutations and KMT2A and NUP98 fusions clustered into G6-G8, showing high expression of HOXA/B genes and diverse differentiation stages, from hematopoietic stem/progenitor cell down to monocyte, namely HOX-primitive (G7), HOX-mixed (G8), and HOX-committed (G6). Through constructing prediction models, the eight gene expression subgroups could be reproduced in the Cancer Genome Atlas (TCGA) and Beat AML cohorts. Each subgroup was associated with distinct prognosis and drug sensitivities, supporting the clinical applicability of this transcriptome-based classification of AML. These molecular subgroups illuminate the complex molecular network of AML, which may promote systematic studies of disease pathogenesis and foster the screening of targeted agents based on omics.

Transcriptome-wide subtyping of pediatric and adult T cell acute lymphoblastic leukemia in an international study of 707 cases.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy of T cell progenitors, known to be a heterogeneous disease in pediatric and adult patients. Here we attempted to better understand the disease at the molecular level based on the transcriptomic landscape of 707 T-ALL patients (510 pediatric, 190 adult patients, and 7 with unknown age; 599 from published cohorts and 108 newly investigated). Leveraging the information of gene expression enabled us to identify 10 subtypes (G1­G10), including the previously undescribed one characterized by GATA3 mutations, with GATA3R276Q capable of affecting lymphocyte development in zebrafish. Through associating with T cell differentiation stages, we found that high expression of LYL1/LMO2/SPI1/HOXA (G1­G6) might represent the early T cell progenitor, pro/precortical/cortical stage with a relatively high age of disease onset, and lymphoblasts with TLX3/TLX1 high expression (G7­G8) could be blocked at the cortical/postcortical stage, while those with high expression of NKX2-1/TAL1/LMO1 (G9­G10) might correspond to cortical/postcortical/mature stages of T cell development. Notably, adult patients harbored more cooperative mutations among epigenetic regulators, and genes involved in JAK-STAT and RAS signaling pathways, with 44% of patients aged 40 y or above in G1 bearing DNMT3A/IDH2 mutations usually seen in acute myeloid leukemia, suggesting the nature of mixed phenotype acute leukemia.

The novel TERF2::PDGFRB fusion gene enhances tumorigenesis via PDGFRB/STAT5 signalling pathways and sensitivity to TKI in ph-like ALL.

Patients with Philadelphia chromosome-like acute lymphoblastic leukaemia (Ph-like ALL) often face a grim prognosis, with PDGFRB gene fusions being commonly detected in this subgroup. Our study has unveiled a newfound fusion gene, TERF2::PDGFRB, and we have found that patients carrying this fusion gene exhibit sensitivity to dasatinib. Ba/F3 cells harbouring the TERF2::PDGFRB fusion display IL-3-independent cell proliferation through activation of the p-PDGFRB and p-STAT5 signalling pathways. These cells exhibit reduced apoptosis and demonstrate sensitivity to imatinib in vitro. When transfused into mice, Ba/F3 cells with the TERF2::PDGFRB fusion gene induce tumorigenesis and a shortened lifespan in cell-derived graft models, but this outcome can be improved with imatinib treatment. In summary, we have identified the novel TERF2::PDGFRB fusion gene, which exhibits oncogenic potential both in vitro and in vivo, making it a potential therapeutic target for tyrosine kinase inhibitors (TKIs).

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.

Ruxolitinib reduces severe CRS response by suspending CAR-T cell function instead of damaging CAR-T cells.

The therapeutic effect of CAR-T is often accompanied by sCRS, which is the main obstacle to the promotion of CAR-T therapy. The JAK1/2 inhibitor ruxolitinib has recently been confirmed as clinically effective in maintaining control over sCRS, however, its mechanism remains unclear. In this study, we firstly revealed that ruxolitinib significantly inhibited the proliferation of CAR-T cells without damaging viability, and induced an efficacy-favored differentiation phenotype. Second, ruxolitinib reduced the level of cytokine release not only from CAR-T cells, but also from other cells in the immune system. Third, the cytolytic activity of CAR-T cells was restored once the ruxolitinib was removed; however, the cytokines released from the CAR-T cells maintained an inhibited state to some degree. Finally, ruxolitinib significantly reduced the proliferation rate of CAR-T cells in vivo without affecting the therapeutic efficacy after withdrawal at the appropriate dose. We demonstrated pre-clinically that ruxolitinib interferes with both CAR-T cells and the other immune cells that play an important role in triggering sCRS reactions. This work provides useful and important scientific data for clinicians on the question of whether ruxolitinib has an effect on CAR-T cell function loss causing CAR-T treatment failure when applied in the treatment of sCRS, the answer to which is of great clinical significance.

Identification of fusion genes and characterization of transcriptome features in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignancy of immature T cells. Recently, the next-generation sequencing approach has allowed systematic identification of molecular features in pediatric T-ALL. Here, by performing RNA-sequencing and other genomewide analysis, we investigated the genomic landscape in 61 adult and 69 pediatric T-ALL cases. Thirty-six distinct gene fusion transcripts were identified, with SET-NUP214 being highly related to adult cases. Among 18 previously unknown fusions, ZBTB16-ABL1, TRA-SALL2, and involvement of NKX2-1 were recurrent events. ZBTB16-ABL1 functioned as a leukemogenic driver and responded to the effect of tyrosine kinase inhibitors. Among 48 genes with mutation rates >3%, 6 were newly found in T-ALL. An aberrantly overexpressed short mRNA transcript of the SLC17A9 gene was revealed in most cases with overexpressed TAL1, which predicted a poor prognosis in the adult group. Up-regulation of HOXA, MEF2C, and LYL1 was often present in adult cases, while TAL1 overexpression was detected mainly in the pediatric group. Although most gene fusions were mutually exclusive, they coexisted with gene mutations. These genetic abnormalities were correlated with deregulated gene expression markers in three subgroups. This study may further enrich the current knowledge of T-ALL molecular pathogenesis.

Identification of a Novel ß-Thalassemia Mutation at Codon 130 (+T) (HBB: c.391insT) in Han Chinese.

ß-Thalassemia (ß-thal) is an inherited blood disorder characterized by reduced or absent synthesis of the ß chains of hemoglobin (Hb). Although more than 900 ß-globin gene mutations around the world have been identified, here we report a novel mutation detected in a Chinese subject of Han ethnicity. This allele develops by insertion of one nucleotide (+T) at codon 130 (HBB: c.391insT) in the third exon of the ß-globin gene. The mutation causes a frameshift that leads to a termination codon at codon 139. The identification of the novel mutation will facilitate future diagnosis of ß-thal and will also be useful the genetic counseling and prenatal diagnosis.

Pattern and prognostic value of FLT3-ITD mutations in Chinese de novo adult acute myeloid leukemia.

FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in hematological malignancies. FLT3 internal tandem duplication (FLT3-ITD) mutations located in juxtamembrane domain (JMD) and tyrosine kinase domain 1 (TKD1) regions account for two-thirds of all FLT3 mutations. The outcome of patients remains unsatisfactory, with low survival rates. It is not yet known whether the different mutations within the FLT3 gene are all associated with patient outcome. In addition, the cause of FLT3-ITD in-frame duplication events remains unknown. Although there are some published studies investigating the FLT3-ITD mutation and its clinical implications in Chinese acute myeloid leukemia (AML) patients, sample sizes tend to be small and detailed molecular profiles of FLT3 mutations are lacking in these studies. In our study, 227 FLT3-ITD sequences were analyzed from 227 Chinese de novo AML patients. ITD were next classified into 3 types based on molecular profiles of insertion DNA sequences: DNA complete duplication (type I), DNA partial duplication (type II) and complete random sequence (type III). From the 154 patients, we confirmed that high ITD allelic ratio (≥.5) and allogeneic stem cell transplant treatment under CR1 are independent prognostic factors. We also presented evidence that ITD integration sites in the hinge region or beta1-sheet region are an unfavorable prognostic factor in adult AML patients with FLT3-ITD mutations. These findings may help to decipher the mechanisms of FLT3-ITD in-frame duplication events and stratify patients when considering different therapeutic combinations.

Application of liquid chromatographic/tandem mass spectrometric method to a urinary excretion study of subutinib and active metabolite in human urine.

A novel and selective liquid chromatographic-mass spectrometric method (LC-MS/MS) has been established and validated for simultaneous determination of subutinib and active metabolite in human urine. Urine samples were extracted by liquid-liquid extraction with ethyl acetate and separated on a Wondasil C18 (150 × 2.1 mm, 3.5 µm), with methanol-0.2% formic acid solution (73:27, v/v) as mobile phase at flow rate of 0.2 mL/min. The linear range was 0.5000-200.0 ng/mL for subutinib and active metabolite, with a lower limit of quantitation of 0.5000 ng/mL. Intra- and inter-run precisions were all <11.8 and 14.3%, and the accuracies were all <4.5 and 5.4%, with the extraction recoveries 88.8-97.5 and 93.8-99.4% for the two analytes, respectively. The carryover values were all <15% for the two anayltes. The method was successfully applied to study urinary excretion of subutinib and active metabolite in human after oral administration of subutinib maleate capsules in fed and fasting states.

[Effect of TP53 Allelic State on Clinical Performance and Prognosis of Patients with Myelodysplastic Syndrome].

OBJECTIVE: To investigate the clinical significance of TP53 allelic state in patients with myelodysplastic syndromes (MDS). METHODS: The clinical data of 858 MDS patients who underwent second-generation sequencing (NGS) testing in the First Affiliated Hospital of Soochow University from January 2019 to December 2021 were retrospectively analyzed. RESULTS: The median age of the patients was 52 years old, and median follow-up time was 23.8 (0.4-109.6) months. Four hundred and one patients (46.7%) had at least one chromosomal abnormality, including 106 complex karyotypes and 78 monosomal karyotypes. A total of 103 cases of TP53 mutations were identified, with a mutation rate of 12%. Compared with TP53 wild-type, various types of chromosomal abnormalities were significantly more common in patients with TP53 mutations (all P < 0.001). Patients with TP53 mutations had lower hemoglobin levels, lower platelet counts and higher percentage of bone marrow primitive cell compared with TP53 wild type (all P < 0.05), and significantly shorter overall survival (OS). Among 97 evaluable patients, 33 cases (34%) were mono-allelic TP53 mutation, while 64 cases were bi-allelic TP53 mutation. Patients in bi-allelic TP53 mutation subgroup had a higher proportion of chromosomal abnormalities and a lower number of co-mutations compared with mono-allelic TP53 mutation. The median OS was 33.6 months in patients with mono-allelic state and only 11.4 months in patients with bi-allelic state (HR=2.138, 95%CI : 1.053-4.343, P >0.05). Median OS was not reached in TP53 wild-type patients, and there was a significant difference in OS among TP53 wild-type, mono-allelic and bi-allelic TP53 mutation patients (P < 0.001). Multivariable Cox regression analysis showed that bi-allelic TP53 was an independent predictor of poor outcomes (HR=2.808, 95%CI : 1.487-5.003, P =0.001), while mono-allelic TP53 mutation and wild-type TP53 were not. CONCLUSION: Patients with TP53 mutations have a poor prognosis, and bi-allelic TP53 mutations have a worse prognosis compared with mono-allelic TP53 mutations and independently affect the prognosis of MDS patients.

Effective treatment with Gilteritinib-based regimens for FLT3-mutant extramedullary relapse in acute promyelocytic leukemia.

OBJECTIVE: Extramedullary relapse (EMR) is rare in acute promyelocytic leukemia (APL) and, there is a lack of information on its management. Current practices for EMR in APL are always to adopt strategies from other subtypes of Acute lymphoblastic leukemia (ALL) and Acute myeloid leukemia (AML). Gilteritinib, a highly selective FLT3 inhibitor, has demonstrated a remarkable effect on EMR in FLT3-mutant AML. Therefore, it is worthwhile exploring if FLT3 mutation can be a therapeutic target and assessing the efficacy of Gilteritinib on FLT3-mutant EMR in APL. METHODS: We described three cases of FLT3-mutant EMR in APL, comprising two isolated EMR cases and one systemic relapse. The patients underwent treatment with Gilteritinib-based regimens based on FLT3 mutation. RESULTS: All three patients achieved complete regression of EMR, and no signs of tumor lysis syndrome during Gilteritinib-based therapy, only patient 1 showed mild granulocytopenia. They all maintained molecular complete remission (mCR) during the follow-up period. CONCLUSIONS: The Gilteritinib-based regimen shows a high and sustained therapeutic effect with minimal adverse effects, and provides a valuable experience for further evaluation in EMR APL patients.

Effective Management of Polycythemia Vera With Ropeginterferon Alfa-2b Treatment.

Background: Polycythemia vera (PV) is a myeloproliferative neoplasm. Ropeginterferon alfa-2b is a new-generation polyethylene glycol-conjugated proline-interferon. It is approved for the treatment of PV at a starting dose of 100 µg (50 µg for patients receiving hydroxyurea (HU)) and dose titrations up to 500 µg by 50 µg increments. The study was aimed at assessing its efficacy and safety at a higher starting dose and simpler intra-patient dose escalation. Methods: Forty-nine patients with PV having HU intolerance from major hospitals in China were treated biweekly with an initial dose of 250 µg, followed by 350 µg and 500 µg thereafter if tolerated. Complete hematological response (CHR) was assessed every 12 weeks based on the European LeukemiaNet criteria. The primary endpoint was the CHR rate at week 24. The secondary endpoints included CHR rates at weeks 12, 36 and 52, changes of JAK2V617F allelic burden, time to first CHR, and safety assessments. Results: The CHR rates were 61.2%, 69.4% and 71.4% at weeks 24, 36, and 52, respectively. Mean allele burden of the driver mutation JAK2V617F declined from 58.5% at baseline to 30.1% at 52 weeks. Both CHR and JAK2V617F allele burden reduction showed consistent increases over the 52 weeks of the treatment. Twenty-nine patients (63.0%) achieved partial molecular response (PMR) and two achieved complete molecular response (CMR). The time to CHR was rapid and median time was 5.6 months according to central lab results. The CHRs were durable and median CHR duration time was not reached at week 52. Mean spleen index reduced from 55.6 cm2 at baseline to 50.2 cm2 at week 52. Adverse events (AEs) were mostly mild or moderate. Most common AEs were reversible alanine aminotransferase and aspartate aminotransferase increases, which were not associated with significant elevations in bilirubin levels or jaundice. There were no grade 4 or 5 AEs. Grade 3 AEs were reversible and manageable. Only one AE led to discontinuation. No incidence of thromboembolic events was observed. Conclusion: The 250-350-500 µg dosing regimen was well tolerated and effectively induced CHR and MR and managed spleen size increase. Our findings demonstrate that ropeginterferon alfa-2b at this dosing regimen can provide an effective management of PV and support using this dosing regimen as a treatment option.

Three times spontaneous remission of severe aplastic anemia following granulocyte transfusion from related donors: a case report and literature review.

Aplastic anemia (AA) is a bone marrow failure disease caused by abnormal activation of T lymphocytes, resulting in the apoptosis of hematopoietic cells and bone marrow failure. Currently, hematopoietic stem cell transplantation (HSCT), immunosuppressive - therapy (IST), and supportive care (e.g. transfusion adjuvant therapy, hematopoietic growth factors, and prevention of infection) are the main treatments of AA. Granulocyte transfusion has recently been accepted as an useful adjuvant therapy of HSCT and intensive IST. This article reported a severe AA patient who failed to respond to IST, but achieved spontaneous remission three times after granulocyte transfusions from related donors. Such cases have rarely been reported. Existence of human leukocyte antigen (HLA) cross between the patient and his relatives may influence the T cell-mediated immunity, which might explain this patient's recovery.

[Deletions and rearrangements of PAX5 gene in B-lineage acute lymphoblastic leukemia].

OBJECTIVE: To determine the frequency paired-box domain 5 (PAX5) gene alterations in B-lineage acute lymphoblastic leukemia (B-ALL) harboring 9p abnormalities and its implication for clinical prognosis. METHODS: Bacterial artificial chromosomes RP11-344B23 and RP11-652D9 encompassing the PAX5 gene were selected. DNA was extracted with conventional method and labeled with fluorescein by nicking transition. Fluorescence in situ hybridization (FISH) was used to determine the rearrangement or deletion of the PAX5 gene in B-ALL harboring chromosome 9p abnormalities. Clinical and laboratory features of patients were analyzed. RESULTS: Fifty cases were analyzed with FISH. Complete deletion was observed in 23 patients (46%), partial deletion was observed in 2 patients (4%), and rearrangement was detected only in 1 case. The total frequency of abnormalities was 52% (26/50). No significant difference was found in clinical features of patients with or without PAX5 rearrangement or deletion. CONCLUSION: The frequency of PAX5 gene alterations in B-ALL harboring 9p abnormalities was 52%. However, no significant difference was found between patients with and without PAX5 alterations.

[Clinical and cytogenetic study of 6 cases of hematological disorders associated with 20q- and t (20;21) (q11;q11) abnormalities].

OBJECTIVE: To analyze clinical and cytogenetic features of hematological disorders associated with 20q- and t (20;21) (q11;q11) abnormalities. METHODS: Following short-term culture of bone marrow cells, karyotypic analysis was carried out with R-banding. 20q- and t(20;21) (q11;q11) was detected by fluorescence in situ hybridization (FISH) using dual-color 20q11/12 probe, ST 20qter /ST 21qter probes, SE20(D20Z1)/SE 13/21 probes, and WC20/WC21 probes. RESULTS: Six (2.3%) of the 257 patients with 20q- detected by conventional karyotypic analysis were found to have t(20;21) (q11;q11) abnormality. Five cases had myelodysplastic syndrome, 1 had acute lymphoblastic leukemia. Above results were all confirmed by FISH. CONCLUSION: i (20q-), t(20;21) (q11;q11) seems to be a rare but recurrent chromosomal abnormality which is specifically associated with myeloid disease, late occurrence and poor prognosis. The translocation between chromosome 20q11 and 21q11 may form a novel fusion gene which has an important role in the pathogenesis of the disease.