Case report of pediatric TTMV-related acute promyelocytic leukemia with central nervous system infiltration and rapid accumulation of RARA-LBD mutations.

TTMV::RARA is a recently reported fusion gene associated with acute promyelocytic leukemia (APL), caused by the integration of torque teno mini virus (TTMV) genomic fragments into the second intron of the RARA gene. Currently, there have been only six documented cases, with clinical presentations showing significant variability. Although initial responses to all-trans retinoic acid (ATRA) treatment may be observed in patients with TTMV::RARA-APL, the overall prognosis remains unfavorable among infrequent reported cases. This article presents a pediatric case that manifested as PML::RARA-negative APL with central nervous system involvement at onset. The patient experienced both intramedullary and extramedullary relapse one year after undergoing allogeneic hematopoietic stem cell transplantation. Upon identification as TTMV::RARA-APL and subsequent administration of two rounds of ATRA-based treatment, the patient rapidly developed multiple RARA ligand-binding domain mutations and demonstrated extensive resistance to ATRA and various other therapeutic interventions. Additionally, the patient experienced ARID1A mutant clone expansion and progressed MYC-targeted gene activation. This case represents the first documentation of extramedullary involvement at both the initial diagnosis and relapse stages, emphasizing the intricate clinical features and challenges associated with the rapid accumulation of multiple ATRA-resistant mutations in TTMV::RARA-APL, characterizing it as a distinct and complex sub-entity of atypical APL.

Advances towards genome-based acute myeloid leukemia classification: A comparative analysis of WHO-HAEM4R, WHO-HAEM5, and International Consensus Classification.

Two recent guidelines, the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5) and the International Consensus Classification (ICC), were published to refine the diagnostic criteria of acute myeloid leukemia (AML). They both consider genomic features more extensively and expand molecularly defined AML subtypes. In this study, we compared the classifications of 1135 AML cases under both criteria. According to WHO-HAEM5 and ICC, the integration of whole transcriptome sequencing, targeted gene mutation screening, and conventional cytogenetic analysis identified defining genetic abnormalities in 89% and 90% of AML patients, respectively. The classifications displayed discrepancies in 16% of AML cases after being classified using the two guidelines, respectively. Both new criteria significantly reduce the number of cases defined by morphology and differentiation. However, their clinical implementation heavily relies on comprehensive and sophisticated genomic analysis, including genome and transcriptome levels, alongside the assessment of pathogenetic somatic and germline variations. Discrepancies between WHO-HAEM5 and ICC, such as the assignment of RUNX1 mutations, the rationality of designating AML with mutated TP53 as a unique entity, and the scope of rare genetic fusions, along with the priority of concurrent AML-defining genetic abnormalities, are still pending questions requiring further research for more elucidated insights.

Report of PRPF19 as a novel partner of RARG and the recurrence of interposition-type fusion in variant acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia (AML) which is characterized by specific clinical and biological features. Typical APL cases are caused by PML::RARA fusion gene and are exquisitely sensitive to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Rarely, APLs are caused by atypical fusions involving RARA or, in fewer cases still, fusions involving other members of the retinoic acid receptors (RARB or RARG). To date, seven partner genes of RARG have been reported in a total of 18 cases of variant APL. Patients with RARG fusions showed distinct clinical resistance to ATRA and had poor outcomes. Here, we report PRPF19 gene as a novel partner of RARG and identify a rare interposition-type gene fusion in a variant APL patient with a rapidly fatal clinical course. The incomplete ligand-binding domain of RARG in the fusion protein may account for the clinical ATRA resistance in this patient. These results broaden the spectrum of variant APL associated molecular aberrations. Accurately and timely identification of these rare gene fusions in variant APL is essential to guide therapeutic decisions.

DNTT activation, TdT-aided gene length mutation, and better prognosis in ATG-based regimen allo-HSCT in AML.

This study aimed to investigate the relationship between anomalous DNA nucleotidylexotransferase (DNTT) activation and the mutagenesis of gene length mutations (LMs) in acute myeloid leukemia (AML), and the relevance of their prognosis in antithymocyte globulin (ATG)-based regimen allogeneic hematopoietic stem cell transplantation (allo-HSCT). A cohort of 578 AML cases was enrolled. Next-generation sequencing was performed to screen mutations of 86 leukemia driver genes. RNA-seq was used to analyze gene expression. Prognostic analysis was investigated in 239 AML cases who underwent ATG-based regimen allo-HSCT. We report a refined subtyping algorithm of LMs (type I-IV) based on sequence anatomy considering the TdT-aided mutagenesis mechanism. GC content adjacent to LM junctions, inserted nontemplate nucleotide bases, and DNTT expression analysis supported the DNTT activation and TdT-aided mutagenesis in type II/III LMs in the total AML cohort. Both single-variate and multivariate analyses showed a better overall survival of FLT3 type III compared to type I in a subset of ATG-based regimen allo-HSCT cases. The novel LM subtyping algorithm not only deciphers the etiology of the mutagenesis of LMs but also helps to fine-tune prognosis differentiation in AML. The possible prognostic versatility of this novel LM subtyping algorithm in terms of chemotherapy, targeted therapy, and allo-HSCT merits further investigation.

Torque teno mini virus driven childhood acute promyelocytic leukemia: The third case report and sequence analysis.

In this manuscript, we report torque teno mini virus (TTMV) as a cause of acute promyelocytic leukemia (APL) lacking PML::RARA in a 3-year-old boy. Astolfi et al. firstly identified partial integration of the TTMV genome into RARA intron 2, which resulted in in-frame TTMV::RARA fusion in two APL-like pediatric cases without PML::RARA in November 2021. This fascinating report identified an unexpected exogenous genetic cause of APL and could be of great importance for diagnosing and managing APL. Here we report the third childhood APL-like case caused by TTMV integration and investigate the location and structure of the integrated TTMV sequence. These findings suggest TTMV::RARA is a recurrent cause of APL lacking PML::RARA. Considering the widespread prevalence of TTMV in the population, more TTMV::RARA positive APL-like cases might remain to be identified. Establishing a bioinformatic analysis strategy optimized for the highly variable TTMV genome sequence may facilitate the identification of TTMV::RARA by whole transcript sequencing. An effective PCR protocol to identify TTMV::RARA based on a profound analysis of the conservation of TTMV segments in the fusion transcript is also expected. Also, further investigation is needed to elucidate the oncogenic mechanisms of TTMV integration and the clinical features of TTMV::RARA positive patients.

Fanconi anemia gene-associated germline predisposition in aplastic anemia and hematologic malignancies.

Whether Fanconi anemia (FA) heterozygotes are predisposed to bone marrow failure and hematologic neoplasm is a crucial but unsettled issue in cancer prevention and family consulting. We retrospectively analyzed rare possibly significant variations (PSVs) in the five most obligated FA genes, BRCA2, FANCA, FANCC, FANCD2, and FANCG, in 788 patients with aplastic anemia (AA) and hematologic malignancy. Sixty-eight variants were identified in 66 patients (8.38%). FANCA was the most frequently mutated gene (n = 29), followed by BRCA2 (n = 20). Compared with that of the ExAC East Asian dataset, the overall frequency of rare PSVs was higher in our cohort (P = 0.016). BRCA2 PSVs showed higher frequency in acute lymphocytic leukemia (P = 0.038), and FANCA PSVs were significantly enriched in AA and AML subgroups (P = 0.020; P = 0.008). FA-PSV-positive MDS/AML patients had a higher tumor mutation burden, higher rate of cytogenetic abnormalities, less epigenetic regulation, and fewer spliceosome gene mutations than those of FA-PSV-negative MDS/AML patients (P = 0.024, P = 0.029, P = 0.024, and P = 0.013). The overall PSV enrichment in our cohort suggests that heterozygous mutations of FA genes contribute to hematopoietic failure and leukemogenesis.

Enriched clonal hematopoiesis in seniors with dietary vitamin C inadequacy.

BACKGROUND: The anti-cancer effect of vitamin C (VC) has long been speculated, but studies yielded inconsistency. Recent studies reported that supraphysiological concentration of VC have therapeutic or prevention effects for myeloid malignancies with certain mutation signatures. There was a notable proportion of DAT (i.e., DNMT3A, ASXL1, and TET2) and dozens of other genes that mutate in age-related clonal hematopoiesis (ARCH). METHODS AND RESULTS: Through analyzing the plasma VC concentration and mutations of 21 genes in 215 senior volunteers, we revealed that ARCH is significantly associated with dietary plasma VC concentrations, especially TET2 mutations and non-DAT mutations. CONCLUSION: This study firstly disclosed the significant association between VC inadequacy and ARCH in the senior population. It provides evidence that physiological VC concentration has ARCH prevention effect. It will illuminate future explorations on the oral VC supplement in maintaining sound hematopoiesis, reversal ARCH, adjuvant therapy for myeloid malignancies, and prevention of other ARCH related comorbidities.

Targeted minor histocompatibility antigen typing to estimate graft-versus-host disease after allogeneic haematopoietic stem cell transplantation.

Graft-versus-host disease (GVHD) is a critical complication after allogeneic haematopoietic stem cell transplantation induced by genetic differences in donor-recipient pairs. Rigorous HLA matching has reduced GVHD, but severe GVHD still occurs. Minor histocompatibility antigens (mHAs) are another source of GVHD inducers. We designed a multi-mHA panel with 35 valid mHA loci and retrospectively analyzed 391 donor-recipient pairs with the anticipation of implementing mHA typing into clinical practice to optimize donor selection. Results showed the total mismatching in mHA loci in this panel, as well as mismatching in the GVH direction in unmatched-related recipients (UMRs) were 1.8 times and 1.3 times as those in matched-sibling recipients (MSRs) (p = 4.1e-4, p = 0.012, respectively). There was no significant association between mHA loci mismatching and grades II-IV acute GVHD (aGVHD), III-IV aGVHD, extensive chronic GVHD (cGVHD), or relapse in neither group. UMRs had an increased cumulative incidence of II-IV aGVHD (p = 0.002), but there was no statistical difference of the incidences in severe aGVHD or cGVHD (p = 0.093; p = 0.930). This is a preliminary study to explore GVHD risks brought by mHA loci mismatching in both unmatched-related recipients and matched-full-sibling recipients. Our results confirmed that stringent HLA matching is the key to reduce the risks for GVHD.

Fusion gene map of acute leukemia revealed by transcriptome sequencing of a consecutive cohort of 1000 cases in a single center.

Fusion genes (FGs) are important genetic abnormalities in acute leukemias, but their variety and occurrence in acute leukemias remain to be systematically described. Whole transcriptome sequencing (WTS) provides a powerful tool for analyzing FGs. Here we report the FG map revealed by WTS in a consecutive cohort of 1000 acute leukemia cases in a single center, including 539 acute myeloid leukemia (AML), 437 acute lymphoblastic leukemia (ALL), and 24 mixed-phenotype acute leukemia (MPAL) patients. Bioinformatic analysis identified 792 high-confidence in-frame fusion events (296 distinct fusions) which were classified into four tiers. Tier A (pathogenic), B (likely pathogenic), and C (uncertain significance) FGs were identified in 61.8% cases of the total cohort (59.7% in AML, 64.5% in ALL, and 63.6% in MPAL). FGs involving protein kinase, transcription factor, and epigenetic genes were detected in 10.7%, 48.5%, and 15.1% cases, respectively. A considerable amount of novel FGs (82 in AML, 88 in B-ALL, 13 in T-ALL, and 9 in MPAL) was identified. This comprehensively described real map of FGs in acute leukemia revealed multiple FGs with clinical relevance that have not been previously recognized. WTS is a valuable tool and should be widely used in the routine diagnostic workup of acute leukemia.

[Identification of TCF3-ZNF384 fusion by transcriptome sequencing in B cell acute lymphoblastic leukemia and its laboratory and clinical characteristics].

OBJECTIVE: To detect fusion gene with pathological significance in a patient with refractory and relapsed acute B cell lymphoblastic leukemia (B-ALL) and to explore its laboratory and clinical characteristics. METHODS: Transcriptome sequencing was used to detect potential fusion transcripts. Other laboratory results and clinical data of the patient were also analyzed. RESULTS: The patient was found to harbor TCF3 exon 17-ZNF384 exon 7 in-frame fusion transcript. The minimal residual disease (MRD) has remained positive after multiple chemotherapy protocols including CD19-, CD22- targeted chimeric antigen receptor T cells immunotherapy. The patient eventually achieved complete remission and sustained MRD negativity after allogeneic hemopoietic stem cell transplantation (allo-HSCT). CONCLUSION: Transcriptome sequencing can effectively detect potential fusion genes with clinical significance in leukemia. TCF3-ZNF384 positive B-ALL has unique laboratory and clinical characteristics, may not well respond to chemotherapy and immunotherapy, and is more likely to relapse. Timely allo-HSCT treatment may help such patients to achieve long-term disease-free survival. TCF3-ZNF384 positive B-ALL is not uncommon in pediatric patients but has not been effectively identified.

Efficacy and Safety of CD28- or 4-1BB-Based CD19 CAR-T Cells in B Cell Acute Lymphoblastic Leukemia.

CD19-directed chimeric antigen receptor-T (CAR-T) cells with a 4-1BB or CD28 co-stimulatory domain have shown impressive antitumor activity against relapsed or refractory B cell acute lymphoblastic leukemia (r/r B-ALL). However, a parallel comparison of their performances in r/r B-ALL therapy has not been sufficiently reported. Here, we manufactured 4-1BB- and CD28-based CD19 CAR-T cells using the same process technology and evaluated their efficacy and safety in r/r B-ALL therapy based on pre-clinical and exploratory clinical investigations. In B-ALL-bearing mice, a similar antitumor effect and CAR-T kinetics in peripheral blood were observed at the CAR-T dose of 1 × 107/mouse. However, when the dose was decreased to 1 × 106/mouse, 4-1BB CAR-T cells were more potent in eradicating tumor cells and showed longer persistence than CD28 CAR-T cells. Retrospective analysis of an exploratory clinical study that used 4-1BB- or CD28-based CAR-T cells to treat r/r B-ALL was performed. Compared with CD28 CAR-T cells, 4-1BB CAR-T cells resulted in higher antitumor efficacy and less severe adverse events. This study demonstrated that the performance of 4-1BB CAR-T cells was superior to that of CD28 CAR-T cells in suppressing CD19+ B-ALL, at least under our manufacturing process.

Efficacy and safety of anti-CD19 CAR T-cell therapy in 110 patients with B-cell acute lymphoblastic leukemia with high-risk features.

Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is effective in patients with advanced B-cell acute lymphoblastic leukemia (B-ALL). However, efficacy data is sparse in subgroups of patients with high-risk features such as BCR-ABL+, TP53 mutation, extramedullary disease (including central nervous system leukemia) or posttransplant relapse. It is also uncertain whether there is an added benefit of transplantation after anti-CD19 CAR T-cell therapy. We conducted a phase 1/2 study of 115 enrolled patients with CD19+ B-ALL. A total of 110 patients were successfully infused with anti-CD19 CAR T cells. In all, 93% of patients achieved a morphologic complete remission, and 87% became negative for minimal residual disease. Efficacy was seen across all subgroups. One-year leukemia-free survival (LFS) was 58%, and 1-year overall survival (OS) was 64% for the 110 patients. Seventy-five nonrandomly selected patients (73.5%) subsequently received an allogeneic hematopoietic stem cell transplant (allo-HSCT). LFS (76.9% vs 11.6%; P < .0001; 95% confidence interval [CI], 11.6-108.4) and OS (79.1% vs 32.0%; P < .0001; 95% CI, 0.02-0.22) were significantly better among patients who subsequently received allo-HSCT compared with those receiving CAR T-cell therapy alone. This was confirmed in multivariable analyses (hazard ratio, 16.546; 95% CI, 5.499-49.786). Another variate that correlated with worse outcomes was TP53 mutation (hazard ratio, 0.235; 95% CI, 0.089-0.619). There were no differences in complete remission rate, OS, or LFS between groups of patients age 2 to 14 years or age older than 14 years. Most patients had only mild cytokine release syndrome and neurotoxicity. Our data indicate that anti-CD19 CAR T-cell therapy is safe and effective in all B-ALL subgroups that have high-risk features. The benefit of a subsequent allo-HSCT requires confirmation because of nonrandom allocation. This trial was registered at www.clinicaltrials.gov as #NCT03173417.

The incidence, genetic characteristics, and prognosis of leukemia with concurrent pathogenic fusion genes: a series of 25 cases from a large cohort of leukemia patients.

Recurrent fusion genes (FGs) with clinical significances in leukemias are mainly mutually exclusive, and the coexistence of different FGs has been rarely reported. In this study, we retrospectively analyzed the incidence, genetic characteristics, and prognosis of leukemias with concurrent pathogenic FGs, which commonly reported in hematological malignancies in 8226 leukemia patients. A total of 25 patients with coexistence of double FGs were identified, accounting for 0.30% of all cases enrolled. More than half of the cases (14/25, 56%) were diagnosed as chronic myeloid leukemia in accelerated or blast phase, another six and five cases were acute myeloid leukemia and acute lymphocytic leukemia, respectively. Most cases (20/25, 80%) carried constitutively activated tyrosine kinases FGs (BCR-ABL1 or ETV6-PDGFRB) and transcription factors associated FGs simultaneously. Of the 11 patients with contemporaneous karyotype, 5 (45%) showed visible chromosomal abnormalities corresponding to both FGs. The concurrency of FGs was often associated with disease progressions. The prognosis was pessimistic for patients with concurrent FGs, even with the combination of targeted therapy and chemotherapy. Performing allogeneic hematopoietic stem cell transplantation as soon as possible after complete remission can ameliorate the dismal prognosis.

Genome-Wide Transcriptome Analysis Reveals Extensive Alternative Splicing Events in the Protoscoleces of Echinococcus granulosus and Echinococcus multilocularis.

Alternative splicing (AS), as one of the most important topics in the post-genomic era, has been extensively studied in numerous organisms. However, little is known about the prevalence and characteristics of AS in Echinococcus species, which can cause significant health problems to humans and domestic animals. Based on high-throughput RNA-sequencing data, we performed a genome-wide survey of AS in two major pathogens of echinococcosis-Echinococcus granulosus and Echinococcus multilocularis. Our study revealed that the prevalence and characteristics of AS in protoscoleces of the two parasites were generally consistent with each other. A total of 6,826 AS events from 3,774 E. granulosus genes and 6,644 AS events from 3,611 E. multilocularis genes were identified in protoscolex transcriptomes, indicating that 33-36% of genes were subject to AS in the two parasites. Strikingly, intron retention instead of exon skipping was the predominant type of AS in Echinococcus species. Moreover, analysis of the Kyoto Encyclopedia of Genes and Genomes pathway indicated that genes that underwent AS events were significantly enriched in multiple pathways mainly related to metabolism (e.g., purine, fatty acid, galactose, and glycerolipid metabolism), signal transduction (e.g., Jak-STAT, VEGF, Notch, and GnRH signaling pathways), and genetic information processing (e.g., RNA transport and mRNA surveillance pathways). The landscape of AS obtained in this study will not only facilitate future investigations on transcriptome complexity and AS regulation during the life cycle of Echinococcus species, but also provide an invaluable resource for future functional and evolutionary studies of AS in platyhelminth parasites.