BACKGROUND:TCF3::HLF-positive B-cell acute lymphoblastic leukemia (B-ALL) is a rare, highly aggressive subtype with historically poor outcomes. Despite its classification as a distinct entity, its clinical and molecular landscape remains poorly understood. METHODS:This study presents a single-center cohort of 34 TCF3::HLF-positive B-ALL patients, providing comprehensive clinical and molecular characterization by integrating clinical data, treatment responses, survival outcomes, whole-transcriptome sequencing (WTS), targeted sequencing, and flow cytometry. RESULTS:TCF3::HLF accounted for 1.59% of B-ALL cases. Three fusion isoforms were identified, with Isoform III likely arising from alternative splicing. No significant clinical or transcriptomic differences were observed between Isoform I and II. The 5-year overall survival (OS) was 35.2%. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) significantly improved OS and event-free survival (p < 0.0001), while chimeric antigen receptor T-cell (CAR-T) therapy facilitated allo-HSCT but lacked durable efficacy. RAS pathway mutations were prevalent (85.7%), and CD33 expression was frequent (79.4%), suggesting potential therapeutic targets. WTS analysis revealed dysregulation of epithelial-mesenchymal transition, coagulation, and immune pathways. CONCLUSIONS:TCF3::HLF-positive B-ALL represents an ultra-high-risk leukemia requiring allo-HSCT for long-term remission. CAR-T serves as a bridge to transplantation, while RAS and CD33-directed therapies warrant further investigation. These findings provide critical insights into disease biology and potential treatment.

Abstract: Accurate molecular classification is essential for diagnosis, risk stratification, and treatment selection in B-cell lymphoblastic leukemia (B-ALL). In this study, we performed a comprehensive, real-world reclassification of 1015 consecutively diagnosed B-ALL patients using the fifth edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5) and the International Consensus Classification (ICC). An integrative genomic strategy that combined whole transcriptome sequencing, fusion detection, mutational analysis, and cytogenetics enabled reclassification according to both the WHO-HAEM5 and ICC frameworks, thereby substantially reducing the proportion of unclassifiable B-ALL from 41.9% (2016 WHO revision [WHO-HAEM4R]) to 15.9% (WHO-HAEM5) and 11.9% (ICC). Distinct clinical and prognostic features were identified across newly defined subtypes. Multivariable analysis confirmed that this genomic classification is a robust, independent predictor of survival after adjusting for age, minimal residual disease status, and transplant intervention. Specifically, HLF-rearranged and MEF2D-rearranged B-ALL conferred a persistently poor prognosis across all age groups despite allogeneic hematopoietic stem cell transplantation, highlighting an urgent need for novel therapeutic strategies. Gene expression profiling resolved cryptic subtypes, including ETV6::RUNX1-like, ZNF384-rearranged-like, and BCR::ABL1-like B-ALL, and uncovered diagnostic ambiguity in patients with concurrent lesions. In addition, we report emerging high-risk groups, including IDH1/2- and ZEB2 Q1072-mutated B-ALL, that may warrant recognition as distinct molecular entities. Our findings demonstrate the clinical use of integrative transcriptomic profiling in refining B-ALL taxonomy in guiding risk-adapted therapies and informing future revisions of diagnostic standards. This study supports the incorporation of high-throughput molecular diagnostics into routine leukemia classification and precision treatment planning.

1. INTRODUCTION NUT midline carcinoma family member 1 (NUTM1) fusions are primarily known for their association with poorly differentiated and aggressive carcinomas, predominantly affecting midline structures in children and young adults. Recently, NUTM1 fusions have been identified various malignancies, including hematologic malignancies.1 Among these, NUTM1-rearranged B-cell lymphoblastic leukemia (B-ALL) has emerged as a distinct subtype that typically presents in children and is associated with positive clinical outcomes. This recognition is reflected in 2 major guidelines published in 2022 to refine the diagnosis and classification of B-ALL: the 5th edition of the World Health Organization Classification of Lymphoid Neoplasms and International Consensus Classification, both of which classify B-ALL with NUTM1 rearrangements as an independent subtype.2,3NUTM1 fusions are characterized by the overexpression of the normally silent NUTM1 gene and upregulation of the proto-oncogene BMI1.4,5 Although this subtype is very rare, with an incidence of 0.28% to 0.86% among pediatric B-ALL cases, it has garnered attention owing to its typically good prognosis.4,5 To date, 11 fusion partners of NUTM1 have been reported in B-ALL: ACIN1, BRD9, CUX1, ZNF618, AFF1, ATAD5, CHD4, RUNX1, SLC12A6, IKZF1, and KAT6A.4,6ZNF618::NUTM1 was observed in <20 patients, all of whom had successful outcomes.4,6–11 This case presents a unique case of ZNF618::NUTM1-positive B-ALL that exhibited a poor prognosis, challenging the current understanding that this subtype generally predicts favorable outcomes. 2. CASE PRESENTATION A 3-year-old boy was initially admitted to a local hospital due to an unexplained fever. Blood tests showed a hemoglobin level of 111 g/L, platelet count of 33 × 109/L, and white blood cell count of 7.17 × 109/L, and bone marrow (BM) smears revealed 70% lymphoblasts. Flow cytometry (FCM) showed that 29.78% of blast cells were positive for HLA-DR, TdT, CD10, CD19, CD22, CD38, CD24, and cCD79a. Additionally, multiplex-nested reverse transcription polymerase chain reaction (RT-PCR) screening for 41 common fusion genes in leukemia yielded negative results. Fluorescence in situ hybridization analysis for ABL1, ABL2, CSF1R, EPOR, CRLF2, P2RY8, PDGFRB, and JAK2 gene rearrangements was also negative, and chromosomal karyotyping showed normal results. He was diagnosed with B-ALL and immediately started on an induction chemotherapy regimen comprising vincristine, daunorubicin, L-asparaginase, and prednisone. The patient achieved complete remission (CR) after 1 course and received 4 additional consolidation courses. However, 11 months after the initial diagnosis, the patient experienced relapse and was transferred to our hospital for further treatment. At relapse, the BM smear showed 98.5% lymphoblasts, and FCM revealed 56.49% blast cells expressing B-lymphoid-related markers. Chromosomal karyotyping revealed 46, XY, del(6)(q13q23) in 20 analyzed metaphase cells; this deletion occurs in hematological malignancies and may involve the loss of tumor suppressor genes.12–14 Rescreening of the 41 fusion genes yielded negative results. To further investigate additional genetic abnormalities, a targeted next-generation sequencing analysis was performed on 300 frequently mutated genes in hematological malignancies. No pathogenic mutations were identified, suggesting that the aggressive clinical course was likely driven by the ZNF618::NUTM1 fusion, in conjunction with possible uncharacterized genetic or epigenetic changes. The patient underwent reinduction chemotherapy with vincristine, L-asparaginase, and prednisone. However, the BM smear and FCM revealed 60.5% and 25.33% of lymphoblasts, respectively. Then, the patient received lymphodepleting chemotherapy with fludarabine and cyclophosphamide, followed by a single-dose infusion of 1 × 106/kg autologous CD19 chimeric antigen receptor T (CAR-T) cells. However, 15 days post-infusion, BM examination indicated that CAR-T cell therapy was ineffective, with 89.5% blast cells in the BM smear and 16.07% in FCM. Subsequently, the patient underwent additional chemotherapy and autologous CD19/CD22 dual-targeted CAR-T therapy but still did not achieve CR. The patient was then treated with blinatumomab combined with cytarabine chemotherapy and allogeneic CD19/22-CAR T-cell infusion to achieve CR. Fifty days later, he underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) with his father as the 5/10 matched donor, however, the patient relapsed 7 months after the allo-HSCT. After this, the patient was treated with chemotherapy (mitoxantrone, cytarabine, and etoposide) and donor-specific infusion. Methotrexate was administered for graft-versus-host disease (GVHD) prophylaxis. The patient achieved CR again; however, he developed severe grade IV GVHD, characterized by widespread skin lesions and critical gastrointestinal symptoms. Quantitative PCR chimerism analysis was conducted with markers targeting chromosome 6p, revealing a loss of recipient-specific HLA genes post-allo-HSCT, rendering leukemic cells resistant to donor graft-versus-leukemia effects.15 Ultimately, the treatment was discontinued. To explore potential fusion genes in this patient, we performed whole transcriptome sequencing (WTS) on the relapsed BM sample and identified the in-frame ZNF618 (exon 10)::NUTM1 (exon 2) fusion (Fig. 1A). We obtained a cDNA sample from the diagnostic BM and confirmed the presence of the same fusion transcript using RT-PCR followed by Sanger sequencing. The predicted structure of the fusion protein contains 1456 amino acids and preserves 2 intact NUT domains of NUTM1 protein (Fig. 1B).Figure 1.: Transcriptomic sequencing results of the ZNF618::NUTM1-positive B-ALL patient. (A) Schematic representation of the ZNF618::NUTM1 fusion, where exon 10 of ZNF618 is fused with exon 2 of NUTM1. (B) Schematic diagrams of ZNF618, NUTM1, and the predicted ZNF618::NUTM1 fusion protein. Cleavage sites on the 2 proteins are indicated by red dashed lines. (C) Gene expression levels of NUTM1 as determined by transcriptomic sequencing. (D) Gene expression levels of BMI1 as determined by transcriptomic sequencing. Red asterisks indicate the expression levels of NUTM1 and BMI1 in the present case with the ZNF618::NUTM1 fusion. B-ALL = B-cell lymphoblastic leukemia.Herein, we also analyzed the expression levels of NUTM1 and BMI1, along with another 1013 B-ALL cases and 100 healthy individuals using WTS data. None of the control cases harbored NUTM1-related fusion genes. Notably, ZNF618::NUTM1 exhibited significantly elevated NUTM1 expression, whereas the other 2 groups showed minimal-to-no expression (Fig. 1C). To determine whether the upregulated NUTM1 expression originated from the ZNF618::NUTM1 fusion transcript or wild-type NUTM1, we examined the WTS read alignment. As shown in Figure 1A, most sequencing reads were mapped to the fusion junction and downstream region of NUTM1, whereas the 5' region of wild-type NUTM1 exhibited minimal coverage. This suggests that the increased NUTM1 expression was primarily derived from the fusion transcript rather than from the wild-type allele. The BMI1 gene was also overexpressed in this patient; however, this finding was not unique to this case (Fig. 1D). 3. DISCUSSION This case of ZNF618::NUTM1-positive B-ALL represents a rare instance that contradicts the generally favorable prognosis associated with NUTM1-rearranged B-ALL. Given the generally positive prognosis, the detection of NUTM1 fusion has led to considerations for less intensive chemotherapy regimens.4 Only 2 previously reported patients with B-ALL with NUTM1 rearrangements demonstrated poor outcomes: one case involved concurrent P2RY8::CRLF2 fusion and the other is the sole adult case documented to date.16This pediatric case did not present with any additional adverse molecular genetic abnormalities, however, it demonstrated a refractory and relapsed course despite aggressive treatment modalities, including CAR-T therapy and allo-HSCT. This highlights the complexity and variability of the clinical behavior of NUTM1-rearranged B-ALL, indicating that treatment strategies should be carefully considered and warrant further discussion. This clinical course can be explained using several mechanisms. First, the presence of del(6)(q13q23) may contribute to leukemogenesis by affecting tumor suppressor genes, as 6q deletions are recurrent in hematological malignancies and have been implicated in therapy resistance.12–14 Second, immune evasion mechanisms may contribute to this course, particularly in CAR-T cell therapy failure, where antigen escape or immune checkpoint upregulation causes relapse. FCM analysis of the relapsed BM revealed that malignant B lymphoblasts partially expressed PD-1 (CD279)—an uncommon finding in B-ALL. Although the functional significance of PD-1 expression in leukemic blasts remains unclear, recent studies have suggested that PD-1 expression in B-ALL may contribute to immune evasion and resistance to CAR-T therapy.17,18 However, PD-L1 (CD274) was not detected in the malignant blasts, indicating that PD-L1-mediated immune suppression might not have played a major role in this case. These findings highlight the complexity of immune interactions in NUTM1-rearranged B-ALL and suggest that additional immune profiling could provide deeper insights into treatment resistance mechanisms. Finally, epigenetic alterations may influence leukemic cell plasticity, immune regulation, and resistance to therapy. DNA methylation and histone modifications shape the transcriptional landscape of leukemia cells, potentially affecting key pathways involved in treatment response.19 For instance, epigenetic dysregulation is linked to the altered expression of immune checkpoint molecules and leukemic stem cell properties, which may contribute to resistance against CAR-T therapy and chemotherapy. Further studies, such as single-cell RNA sequencing and assays for transposase-accessible chromatin with high-throughput sequencing, are necessary to investigate the interplay between epigenetic mechanisms and immune escape in NUTM1-rearranged B-ALL. Currently, no direct experimental evidence exists that ZNF618::NUTM1 acts as an oncogenic driver in pediatric patients with B-ALL. However, previous studies have shown that NUTM1 fusion-positive B-ALL represents a distinct subtype with unique transcriptional activation and high BMI1 expression, which are features associated with leukemogenesis.4,5 Considering that other NUTM1 fusion partners (eg, ACIN1, BRD9, CUX1, AFF1, RUNX1) have well-characterized oncogenic functions,6ZNF618::NUTM1 may drive leukemogenesis through similar mechanisms. Further functional studies are required to elucidate their precise roles. Our case underscores the importance of continued vigilance in the clinical monitoring of ZNF618::NUTM1-positive B-ALL and suggests that the prognostic expectations for this fusion may require reassessment. In conclusion, our findings provide valuable insights into the complexities of NUTM1-rearranged B-ALL and underscore the need for further research to better understand the molecular mechanisms underlying variability in its clinical outcomes. ACKNOWLEDGMENTS We thank the patient and his legal guardians in this study. ETHICAL APPROVAL Samples were obtained in accordance with the principles of the Declaration of Helsinki and the Chinese legislation for the protection of personal data and research on human samples. The study was approved by the Institutional Review Board and Ethical Committee of the Hebei Yanda Lu Daopei Hospital. Informed consent was obtained from the guardian of the patient. AUTHOR CONTRIBUTIONS H.L. designed the research; X.C. designed molecular studies and wrote the paper; L.Y., X.M., J.W., F.W., Y.Z., and J.C. supervised clinical and experimental findings; P.C. performed bioinformatics analysis; J.Y. and R.S. were involved in the management of the patient and provided clinical data. X.Z. performed molecular studies. All authors reviewed the manuscript and contributed to the final draft.

Torque Teno Mini Virus (TTMV), a member of the Anelloviridae family, is a commensal component of the human virome. Since the initial identification of the TTMV::RARA fusion gene as a novel driver of acute promyelocytic leukemia (APL), 15 cases have been reported in retrospective studies. With advancements in diagnostic methods and increased awareness, the number of newly diagnosed cases has risen, and the clinical and molecular characteristics of TTMV::RARA-APL are becoming clearer. We systematically identified the clinical characteristics, fusion gene analysis, and treatment protocols of two pediatric APL patients harboring the TTMV::RARA fusion on this basis. While the detection of TTMV::RARA contributes to defining pathogenic fusion gene and MRD monitoring indicators in non-PML::RARA-APL cases, the precise pathogenic mechanisms of this ubiquitous symbiotic virus warrant further investigation.

ABSTRACT:Integration of torque teno mini virus (TTMV) generating the TTMV::RARA (retinoic acid receptor α) fusion represents a newly recognized subtype of acute promyelocytic leukemia (APL) that merits detailed investigation. We present, to our knowledge, the first comprehensive characterization of its epidemiologic profile, clinical presentation, virologic characteristics, and underlying molecular mechanisms. Our findings indicate that TTMV::RARA is more prevalent in pediatric patients and represents the second most common retinoic acid receptor fusion after PML::RARA. Affected patients exhibit a high incidence of extramedullary involvement, particularly myeloid sarcoma. Cytogenetic abnormalities involving i(17)(q10) or 7q22 were identified in 52.0% of cases, largely in a mutually exclusive manner. Co-occurring mutations in epigenetic regulators were present in 76.9% of patients. Although most patients achieved initial remission, relapse was common and associated with rapid acquisition of all-trans retinoic acid (ATRA)-resistant mutation and secondary chemoresistance. Venetoclax-containing regimens demonstrated encouraging clinical efficacy. Phylogenetic analysis indicated that patient-derived TTMV strains clustered into a distinct clade. TTMV integration consistently occurred within RARA intron 2, involving a consensus fragment of 510 to 610 base pairs encompassing the viral promoter and open reading frame 2 (ORF2) N terminus, likely mediated by microhomology-driven recombination. Tandem RUNX1-binding motifs within the integrated viral promoter may underlie the myelotropism of these TTMV strains and facilitate transcriptional activation of TTMV::RARA. The chimeric protein retains at least the first 56 N-terminal residues of ORF2 and remains transcriptionally responsive to pharmacological concentrations of ATRA. These findings establish TTMV::RARA-APL as a distinct leukemia entity, laying the foundation for future studies on virus-mediated leukemogenesis and therapeutic strategies.

Myeloid/lymphoid neoplasms with tyrosine kinase gene fusions (MLN-TK) are rare hematologic malignancies characterized by recurrent kinase rearrangements, including FGFR1, often associated with aggressive clinical behavior. We report the first case of acute myeloid leukemia (AML) harboring a novel TRAF3IP3::FGFR1 fusion, identified by whole transcriptome sequencing. The patient, a 35-year-old man, presented with monocytic AML and succumbed to disease within 40 days despite induction chemotherapy. Cytogenetic and molecular profiling revealed a complex monosomal karyotype and a pathogenic TP53 mutation, both known adverse prognostic markers. The in-frame fusion retained the coiled-coil domain of TRAF3IP3 and the full tyrosine kinase domain of FGFR1, suggesting preserved dimerization and oncogenic signaling. This case broadens the spectrum of FGFR1-rearranged neoplasms and highlights the importance of early genomic profiling in aggressive leukemia. It also underscores the potential therapeutic opportunities with FGFR1-targeted agents such as pemigatinib.

Myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (MLN-TK) are rare hematologic malignancies defined by recurrent kinase gene rearrangements. FGFR1 is a well-recognized partner in this category, but de novo B-lymphoblastic leukemia (B-ALL) as the initial presentation remains exceedingly rare. We report the first case of B-ALL with an LRRFIP1::FGFR1 fusion, identified by whole transcriptome sequencing in a 62-year-old male. The patient achieved sustained complete remission following intensive chemotherapy without hematopoietic stem cell transplantation. Only two prior cases of LRRFIP1::FGFR1 fusion have been reported, both presenting as acute myeloid leukemia. All three cases share an identical fusion structure. This case expands the clinical and molecular spectrum of FGFR1-rearranged neoplasms and underscores the importance of comprehensive molecular profiling for accurate classification, risk assessment, and individualized therapeutic planning in MLN-TK.

Background: TCF3::HLF-positive B-cell acute lymphoblastic leukemia (B-ALL) represents one of the most aggressive and treatment-refractory subtypes of ALL. It is characterized by the t(17;19)(q22;p13) translocation generating the TCF3::HLF fusion, leading to impaired B-cell differentiation, chemoresistance, and early relapse. Despite its classification as a distinct entity in the 2022 WHO and ICC frameworks, comprehensive clinical and molecular characterization remains limited due to its extreme rarity. Methods: We analyzed 34 consecutive TCF3::HLF-positive B-ALL patients diagnosed between 2012 and 2024 at a single leukemia center, representing the largest cohort to date. Multi-dimensional profiling included clinical data, immunophenotyping, karyotyping, gene mutation screening of 86 leukemia-related genes, and whole transcriptome sequencing (WTS). Comparisons were made with 129 TCF3::PBX1- and 25 TCF3::ZNF384-positive B-ALL cases to contextualize clinical outcomes and molecular features. Results: TCF3::HLF accounted for 1.6% of all B-ALL cases (n=2,136), with a pediatric predominance (88%, median age 11 years). Three fusion isoforms were identified, but Isoform III was always co-detected with Isoform II and showed significantly lower read support, suggesting an alternatively spliced variant rather than a distinct event. Immunophenotypically, CD33 and/or CD13 expression was present in 88% of cases, implying partial myeloid lineage priming. Karyotyping revealed a high frequency of complex abnormalities; only 59% had visible t(17;19). Mutational analysis showed a remarkably high frequency of isolated RAS pathway mutations (86%), distinguishing this subtype from other TCF3-fusion leukemias. No concurrent mutations outside the RAS pathway were observed. WTS analysis revealed 2,049 differentially expressed genes compared to other TCF3 fusions, including upregulation of EMT, interferon signaling, and coagulation-related genes (e.g., F3, NOX4), alongside downregulation of B-lineage and neuronal development programs. GSEA and KEGG/GO analyses supported enrichment of migration, adhesion, and immune dysregulation pathways, potentially contributing to leukemic aggressiveness and extramedullary behavior. Therapeutically, conventional chemotherapy was inadequate: all 12 patients not receiving allo-HSCT relapsed or died within 30 months (median time to relapse: 6.7 months). CAR-T therapy enabled MRD-negative remission in 13 patients, of whom 11 successfully proceeded to allo-HSCT in CR1. Two patients achieving MRD-negative remission after CAR-T without transplant relapsed and failed subsequent salvage, underscoring CAR-T's role as a bridge, not a cure. Allo-HSCT significantly improved overall and event-free survival (both p<0.0001), with 14 of 20 transplanted patients remaining in continuous CR, the longest exceeding 6 years. Post-HSCT relapse occurred in 4 patients, suggesting a need for better maintenance strategies. Use of blinatumomab showed mixed outcomes—effective in MRD-positive remission but ineffective in frank relapse. Venetoclax failed to induce remission in a multiply relapsed case. Notably, one patient received prophylactic blinatumomab post-HSCT without relapse, suggesting a possible preventive role. Conclusions: TCF3::HLF-positive B-ALL is an ultra-high-risk leukemia with distinct molecular and immunophenotypic features, a striking predominance of RAS-only mutations, and a unique transcriptional program marked by EMT and immune dysregulation. Allo-HSCT remains the only curative approach, ideally preceded by MRD-negative remission. CAR-T therapy is effective in bridging patients to transplant, but durable remissions remain elusive without allo-HSCT. Given consistent CD33 expression and RAS pathway dependency, this subtype may benefit from CD33-directed or MEK-targeted strategies. Our findings highlight the urgent need for international collaboration to design tailored treatment regimens and prospective trials in this challenging and lethal leukemia subtype.

Background Drug-resistant mutations in the ABL1 kinase domain (KD) of BCR::ABL1 are primary mechanisms of resistance to tyrosine kinase inhibitor (TKI) therapy in Ph-positive leukemia. These mutations alter the conformation of ABL1 within BCR::ABL1, impairing TKI and thus diminishing their anti-leukemia efficacy. Our previous studies, presented at the 61st and 62nd ASH Annual Meeting, demonstrated the superiority of next-generation sequencing (NGS) over Sanger sequencing for detecting BCR::ABL1-KD mutations. NGS offers higher sensitivity, accurate mutation frequency determination, and the ability to identify compound or polyclonal mutations within the same amplicon. However, NGS still needs to improve, including its short read length and the disadvantages in identifying in-cis compound mutations, lack of flexibility, and long turnaround time. Leveraging advancements in a novel third-generation sequencing (TGS) technology - characterized by single-molecule sequencing capability, long reads, real-time sequencing, and high accuracy - we have developed an approach for directly full-length in-cis BCR::ABL1-KD mutation screening. Methods The new approach allows for a comprehensive in-cis analysis of resistance mutations in the BCR::ABL1-KD using a novel TGS platform based on single-molecule side synthesis side nanopore sequencing (NSBS), distinct from PacBio or ONT sequencing. We retrospectively analyzed 30 specimens from 30 cases previously tested using NGS, including 15 mutation-positive and 15 mutation-negative specimens under previous investigation. Among them, there were ten single mutations, four double mutations, and one triple mutation, with the variant allele frequency (VAF) ranging from 7.8% to 99.6%. Results The TGS approach can directly analyze in-cis compound BCR::ABL1-KD mutations in a full-length BCR::ABL1-KD sequencing model with high single-base accuracy and superior to the NGS protocol. The novel TGS approach can detect the complete BCR::ABL1 fusion gene sequence (including p190 and p210 types, sequence range 1696~1717bp), which can more accurately obtain the ABL1 sequence in the BCR::ABL1 fusion gene, thus analyzing the veritable resistance mutations in the ABL1 KD of the BCR::ABL1 fusion gene; it has higher detection sensitivity (average sequencing coverage depth of 13086x, range 8226~24152x; Q30 can reach 99.39%; VAF as low as 1%). The TGS approach detected more mutations than NGS (27 vs. 21) and accurately distinguished more complex mutation patterns, including multiple compound or polyclonal mutations. It detected five mutations in the NGS mutation-negative group. The VAF obtained in mutation analysis was inconsistent compared to NGS, indicating that the mutations and mutation frequencies detected by TGS are more in line with the true situation and significant for continuous monitoring. Conclusion Through comparative analysis with the NGS project, the novel TGS approach shows the advantages of longer reads, higher sensitivity and accuracy, and the ability to complete the scheme from library construction to data output within 12 hours. It may provide the clinic with faster and more accurate results of in-cis BCR::ABL1-KD resistance mutation analysis with higher sensitivity and guide the clinic to quickly change treatment plans, thereby improving therapeutic outcomes.

Multiple myeloma (MM) is a malignant plasma cell disorder characterized by the clonal proliferation of malignant plasma cells in the bone marrow, leading to bone lesions, anemia, hypercalcemia, renal insufficiency, and immunodeficiency. Accurate diagnosis and minimal residual disease (MRD) monitoring are essential for personalized treatment strategies and long-term patient management. In this study, we aimed to evaluate the application of NGS-based immunoglobulin(IG) gene sequencing for the diagnosis and MRD monitoring of MM patients. We sequenced the IG gene heavy and light chain genes (IGH and IGK/IGL) using RNA-seq and targeted IGH-CDR3-DNA-NGS methods, and performed immunome analysis on the sequencing data using the MiXCR software. A total of 35 newly diagnosed or relapsed MM patients were included in this study, peripheral blood(PB) and bone marrow(BM) samples were collected at the time of diagnosis and during follow-up for NGS-based analysis, a total of 125 samples were analyzed. Our study results indicate that NGS-based IG gene sequencing offers a high level of sensitivity and specificity in identifying clonal IG gene rearrangements associated with MM. The RNA-seq method, which uses RNA as the substrate, detected clonal IG gene sequences in BM samples with a 100% detection rate (BM 33/33) and in PB samples with a 90.63% detection rate (29/32). In the 28 cases which clonal IGH gene sequences were detected using the RNA-seq method, the targeted IGH-NGS method using DNA as the substrate was able to detect clonal IGH gene sequences in 14/27 (53.85%) BM samples and in 10/25 (40%) PB samples, with sequence information consistent with the RNA-seq results. Next, we further compared whether there is a difference in the somatic hypermutation(SHM) rate among patients who can detect clonal sequences using the targeted IGH-CDR3-DNA-NGS method. By comparing the measured sequences with the IMGT human germline V gene using the IgBlast tool, we found that the average SHM rate of the IGH V region genes in patients with detectable clonal sequences by targeted IGH-CDR3-DNA-NGS was 7.15%, while the average SHM rate for the IGH V region in negative patients was 9.02%. Our results indicate that the RNA-seq sequencing method using RNA as the substrate has a much higher sensitivity in detecting clonal IG gene sequences than the targeted NGS method using DNA as the substrate. Additionally, we conducted further analysis of the clonal IG gene sequences expression in MM patients, B-ALL patients, as well as MM cell lines, B-ALL cell lines, and healthy controls. By conducting statistical analysis on the TPM of target genes among several groups, we found that the expression levels of clonal IG target genes in the BM and PB of patients with multiple myeloma were significantly elevated, much higher than those in B-ALL patients and healthy controls. Furthermore, there was a correlation between the expression levels of target sequences in the BM and PB of MM patients. The reasons for the superior use of the RNA-seq method to monitor the MRD status in MM may include: During the maturation of B cells into plasma cells, they undergo SHM and affinity maturation of antibodies. The IG gene sequences of mature plasma cells exhibit reduced homology with germline genes, leading to a decreased binding capacity for primers of targeted NGS. On the other hand, within plasma cells, the synthesis of IG are extremely active, with the quantity of IG mRNA greatly exceeding that of DNA. As demonstrated by our data, in MM patients, the TPM of clonal IG genes are approximately 10 times higher than those in B-ALL patients. In MM cell lines, the TPM of clonal IG genes are 3 times higher than those in B-ALL cell lines (P<0.05), indicates that the clonal IG RNA quantity of malignant plasma cells is significantly higher than malignant B lymphocytes. In summary, the use of NGS-based IG gene sequencing in MM patients can provide comprehensive genetic information, which aids in identifying the specific gene expression patterns of tumor cells, thereby facilitating more accurate disease diagnosis and MRD monitoring. The study also highlighted the potential of the RNA-seq method for monitoring the MRD status of MM, which is more sensitive than methods that use DNA as the substrate. The integration of NGS-based IG gene sequencing into routine clinical practice has the potential to transform the management of MM patients, leading to improved outcomes and better quality of life.

Acute myeloid leukemia (AML) is a hematopoietic malignancy with a high relapse rate and progressive drug resistance. Alternative polyadenylation (APA) contributes to post-transcriptional dysregulation, but little is known about the association between APA and AML. The APA quantitative trait locus (apaQTL) is a powerful method to investigate the relationship between APA and single nucleotide polymorphisms (SNPs). We quantified APA usage in 195 Chinese AML patients and identified 4,922 cis-apaQTLs related to 1,875 genes, most of which were newly reported. Cis-apaQTLs may modulate the APA selection of 115 genes through poly(A) signals. Colocalization analysis revealed that cis-apaQTLs colocalized with cis-eQTLs may regulate gene expression by affecting miRNA binding sites or RNA secondary structures. We discovered 207 cis-apaQTLs related to AML risk by comparing genotype frequency with the East Asian healthy controls from the 1000 Genomes Project. Genes with cis-apaQTLs were associated with hematological phenotypes and tumor incidence according to the PHARMGKB and MGI databases. Collectively, we profiled an atlas of cis-apaQTLs in Asian AML patients and found their association with APA selection, gene expression, AML risk, and complex traits. Cis-apaQTLs may provide insights into the regulatory mechanisms related to APA in AML occurrence, progression, and prognosis.

This manuscript reports a rare case of CUX1::NUTM1-positive B-cell lymphoblastic leukemia (B-ALL) in a 25-year-old female patient who experienced early relapse and a poor clinical outcome. Given the rarity of NUTM1 fusion-positive B-ALL in adults, this case contributes to the growing evidence that such cases may present with a more aggressive clinical course compared to the pediatric population. Our findings suggest the need for a re-evaluation of therapeutic strategies in adult patients with this subtype of B-ALL.

Background Fusion genes (FGs) are critical molecular abnormalities in acute myeloid leukemia (AML), serving as vital markers for diagnosis, classification, risk stratification, and targeted therapy. In 2021, we published the FG map of acute leukemia revealed by whole transcriptome sequencing (WTS) of 1000 cases, including 539 AML patients (Blood Cancer Journal. PMID: 34135310). Building upon this initial study, we have now expanded our study to include 1614 AML cases, aiming to further elucidate the FG landscape and explore additional insights into the clinical implications of these genetic alterations. Methods We enrolled 1614 AML cases diagnosed in our hospital from September 2018 to April 2024, with high-quality WTS data. Among them, 356 (22%) were children (≤18 years, median age 9 years, range 3 months to 18 years), and 1258 (78%) were adults (>18 years, median age 46 years, range 19 to 89 years). We used bone marrow samples from 100 healthy donors as controls. Written informed consent was obtained from all participants or their guardians in accordance with the Declaration of Helsinki. RNA quality assessment, sequencing library preparation, paired-end sequencing, and FGs detection were conducted as we previously reported. High-confidence in-frame FGs detected by WTS were classified into 4 tiers based on pathogenicity. (A) pathogenic: well-known FGs or new members of common FG families (FG-FMs) with definite pathogenicity in hematological malignancies. (B) likely pathogenic: FGs reported in hematologic malignancies without functional verification, or novel FGs involving genes associated with these malignancies. (C) uncertain significance: novel FGs involving genes without known associations with these malignancies. (D) non-pathogenic: FGs found in normal samples. Results A total of 269 different FGs were detected in 907 AML patients, including 90 tier A, 63 tier B, 108 tier C, and 8 tier D FGs. Tier D FGs were unlikely to be pathogenic and not analyzed further. 982 fusion events (771 tier A, 97 tier B, and 114 tier C, respectively) were detected in 889 (55%) cases. Most patients (n=800; 90%) carried only one FG. Additionally, 85 cases had 2 FGs, and 4 cases had 3 FGs. Only 12 patients had coexisting two tier A FGs, accounting for 0.7% of all cases enrolled in this study and 1.3% of all positive cases. We found 51 kinds of recurrent FGs that occurred at least twice, including 42 tier A, 4 tier B, and 5 tier C FGs, respectively. The positivity rate of FGs presented a typical long-tail distribution, with only 10 FGs having a positivity rate >1%: RUNX1::RUNX1T1 (12.2%), PML::RARA (6.9%), CBFB::MYH11 (3.7%), NUP98::NSD1 (3.5%), KMT2A::MLLT3 (3.1%), KMT2A::MLLT4 (1.9%), KMT2A::MLLT10 (1.7%), ZNF292::PNRC1(1.6%), DEK::NUP214 (1.1%), KMT2A::AFDN (1%). Notably, a considerable number of so-far unreported FGs were detected in this study. Totally, 181 kinds of novel FGs were discovered (15 tier A, 58 tier B, and 108 tier C), accounting for 69% of all FGs. However, only 7 of them were recurrent. We classified the 153 distinct tier A and tier B FGs according to FG-FMs, which referred to FGs that involve one protagonist gene and various fusion partners. More than half of them (99/153, 65%) could be classified into 25 FG-FMs, such as KMT2A-FM, NUP98-FM, and RUNX1-FM. When we focused on tier A FGs, 91% (82/90) could be clustered into FG-FMs, indicating the central involvement of key genes in AML pathogenesis. Conclusion This comprehensive analysis of 1614 AML cases using WTS provides an extensive and detailed landscape of FGs in AML. Our findings underscore the significant heterogeneity of FGs in AML, with a substantial proportion of patients harboring novel and previously unreported FGs. The identification of 269 distinct FGs, including 90 well-established pathogenic FGs, highlights the critical role of these genetic alterations in AML pathogenesis. Moreover, the classification into FG-FMs reveals the central involvement of key genes such as KMT2A, NUP98, and RUNX1 in a large proportion of pathogenic FGs. These insights not only enhance our understanding of the molecular complexity of AML but also have important implications for diagnosis, prognosis, and the development of targeted therapies. Future studies should focus on the functional validation of novel FGs and the exploration of their potential as therapeutic targets.

Background B-cell lymphoblastic leukemia (B-ALL) is a heterogeneous hematologic malignancy where fusion genes (FGs) play a crucial role in its pathogenesis. These genetic alterations drive oncogenesis and influence disease prognosis and treatment. In 2021, we published the FG map of acute leukemia revealed by whole transcriptome sequencing (WTS) of 1000 cases, including 365 B-ALL patients (Blood Cancer Journal. PMID: 34135310). Building upon this initial study, we have now expanded our research to include 1015 B-ALL cases, aiming to provide a comprehensive map of FGs in B-ALL, revealing novel fusions and assessing their clinical relevance, thereby enhancing our understanding of B-ALL's molecular landscape and informing targeted therapies. Methods We enrolled 1015 B-ALL cases diagnosed in our hospital from Sep. 2018 to Apr. 2024, with high-quality WTS data. Among them, 554 (55%) were children (≤18 years, median age 8 years), and 461 (45%) were adults (>18 years, median age 35 years). We used bone marrow samples from 100 healthy donors as controls. Written informed consent was obtained from all participants or guardians per the Declaration of Helsinki. RNA quality assessment, sequencing library preparation, paired-end sequencing, and FGs detection were conducted as we previously reported. High-confidence in-frame FGs detected by WTS were classified into 4 tiers based on pathogenicity. (A) pathogenic: well-known FGs or new members of common FG families (FG-FMs) with definite pathogenicity in hematological malignancies. (B) likely pathogenic: FGs reported in hematologic malignancies without functional verification, or novel FGs involving genes associated with these malignancies. (C) uncertain significance: novel FGs involving genes without known associations with these malignancies. (D) non-pathogenic: FGs found in normal samples. Results A total of 330 different FGs were detected in 717 cases, including 122 tier A, 77 tier B, 127 tier C, and 4 tier D FGs. Tier D FGs were excluded from further analysis. 881 fusion events (647 tier A, 95 tier B, and 139 tier C) were detected in 709 (70%) cases. Most patients (n=564; 80%) carried only one FG. Additionally, 125 cases had 2 FGs, 13 cases had 3 FGs, and 7 cases had 4 FGs. Only 26 patients had coexisting tier A FGs, accounting for 2.6% of all cases and 3.7% of all positive cases. We found 52 kinds of recurrent FGs that occurred at least twice, including 37 tier A, 9 tier B, and 6 tier C FGs, respectively. The positivity rate of FGs presented a typical long-tail distribution, with only 11 FGs having a positivity rate >1%: BCR::ABL1 (16.9%), ETV6::RUNX1 (6.1%), KMT2A::AFF1 (4.6%), DUX4::IGH (3.8%), TCF3::PBX1 (3.7%), EP300::ZNF384 (3.1%), MEF2D::BCL9 (2.5%), UBTF::ATXN7L3 (1.8%), TCF3::ZNF384 (1.4%), MEF2D::HNRNPUL1(1.0%), TCF3::HLF (1.0%). Notably, a considerable number of so-far unreported FGs were detected in this study. Totally, 236 kinds of novel FGs were discovered (40 tier A, 69 tier B, and 127 tier C), accounting for 72% of all FGs. However, only 13 of them were recurrent. We classified the 199 distinct tier A and tier B FGs into FG-FMs, which involve one protagonist gene and various fusion partners. More than half of them (130/199, 65%) could be classified into 25 FG-FMs, such as PAX5-FM, JAK2-FM, KMT2A-FM, and ZNF384-FM. When we focused on tier A FGs, 93% (114/122) could be clustered into FG-FMs, indicating the central involvement of key genes in B-ALL pathogenesis. Conclusion This analysis of 1015 B-ALL cases using WTS expands our understanding of the FG landscape in B-ALL. We identified FGs in 70% of cases, revealing a map that differed from previous expectations. Some FGs or FG-FMs were underestimated before due to their cryptic nature. For example, the positivity rate of ZNF384-FM is second only to BCR::ABL1, surpassing the well-known ETV6::RUNX1 and TCF3::PBX1 fusions in B-ALL. Classification of FGs into FG-FMs reveals the central involvement of key genes in pathogenic FGs. These findings highlight the role of FGs in B-ALL pathogenesis and their potential as biomarkers for diagnosis, prognosis, and targeted therapy. The long-tail distribution of FGs suggests that comprehensive genomic profiling is essential. This study enhances the FG map of B-ALL and provides a resource for future research and clinical applications, aiding in the development of precision medicine and improving patient outcomes.

Background : Emerging evidence suggests that viral infections play a crucial role in the pathogenesis of certain leukemias, with viral integration and its interaction with the human genome potentially contributing to disease onset and progression. Since Astolfi et al. identified the first case of TTMV::RARA-APL in a 6-year-old child, more than ten cases have been identified in the past two years. However, no other viruses have been reported to form fusion genes with human genes in leukemia. Our study leverages a vast repository of poly(A) RNA-seq data from AML patients to develop a sensitive bioinformatics workflow. This workflow aims to uncover the molecular footprints left by viruses in the leukemic transcriptome and discover novel viral-human fusion events that may have been previously overlooked. Methods : The cDNA library construction and sequencing were performed as previously described (Chen, Blood Cancer J 2021). Raw RNA sequencing data were initially processed using Fastp for adapter trimming, polyA/T trimming, and low-quality reads filtering. The high-quality reads were then mapped to the human reference genome (GRCH38) using the STAR aligner (version 2.7.10b). Samtools was used to sort the aligned BAM files by chromosome position and extract reads that were properly paired. A coustom Perl scripts was written to extract the reads without proper paired alignment. SPAdes was used to assemble the unmapped reads into scaffolds, which were then annotated by Blast against the NCBI Refseq Virus database, using an expected value (E-value) cutoff of less than 1e-10 and an alignment percent more than 80%. Due to the limited data available for TTV and TTMV in the NCBI RefSeq Virus database, we enhanced the accuracy of our annotations by incorporating sequences of TTV and TTMV from the NCBI Nucleotide database into our reference database. Reads with soft clipping were identified from the BAM alignment and grouped by the genome positions where soft clipping occurred. Cap3 was then used to assemble these soft clipping reads into consensus sequences (contigs) for each group. The soft-clipped contigs were aligned to the unmapped scaffolds using Blast to reconstruct the fusion transcripts. Results : A cohort of 712 AML cases was enrolled, including 154 children and 556 adults (divided by ≥18 years), with an age range of 0-89 years (median 42 years), and a male-to-female ratio of 375:337. Virus sequences were identified in 523 cases (73.46%), with an average of 4.25 viruses in one sample. Duplodnaviria was found in 414 cases (58.15%), Riboviria in 220 cases (30.90%), Varidnaviria in 38 (5.34%), Monodnaviria in 14 (1.97%) and other virus in 68 cases (9.55%). Alphatorquevirus (Torque Teno Viruses, TTVs) and betatorquevirus (Torque Teno mini Viruses, TTMVs) compose mostly of the Anelloviridae fraction, with TTV was found in 101 cases (14.19%) and TTMV in 30 cases (4.21%). Human herpesvirus 1 (HHV-1) was identified in 297 cases (41.71%), Cytomegalovirus (CMV) in 239 cases (33.57%), Epstein-Barr virus (EBV) in 25 cases (3.51%), and Human adenovirus C (HAdV-C) in 24 cases (3.37%), these were the most frequently identified pathogens in this cohort. Using this analytical approach, we detected one case with the TTMV::RARA fusion in this cohort. We re-analyzed the poly(A) transcriptome sequencing data from several cases of TTMV::RARA previously reported by our center using this analytical process. The re-analysis confirmed that the TTMV::RARA fusion could be successfully identified using this protocol. No other virus-human coding gene fusion was found in this cohort, underscoring the rarity of virus-human fusion genes. Conclusions : We present an analytical workflow for the detection of viral gene transcripts and viral-human gene fusions, providing a reference for future studies of tumor-associated viruses using poly(A) transcriptome sequencing data. This workflow facilitates the discovery of viral integration events and the identification of expressed viral transcripts in various diseases.

Atypical acute promyelocytic leukemia (aAPL) presents a complex landscape of retinoic acid receptor (RAR) fusion genes beyond the well-known PML::RARA fusion. Among these, 31 individually rare RARA and RARG fusion genes have been documented, often reported in the canonical X::RAR bipartite fusion form. Intriguingly, some artificially mimicked bipartite X::RAR fusions respond well to all-trans retinoic acid (ATRA) in vitro, contrasting with the ATRA resistance observed in patients. To unravel the underlying mechanisms, we conducted a comprehensive molecular investigation into the fusion transcripts in 27 RARA fusion gene-positive aAPL (RARA-aAPL) and 21 RARG-aAPL cases. Our analysis revealed an unexpected novel form of X::RAR::X or X::RAR::Y-type tripartite fusions in certain RARA- and all RARG-aAPL cases, with shared features and notable differences between these two disease subgroups. In RARA-aAPL cases, the occurrence of RARA 3' splices was associated with their 5' fusion partner genes, mapping across the coding region of helix 11_12 (H11_12) within the ligand-binding domain (LBD), resulting in LBD-H12 or H11_12 truncation. In RARG-aAPL cases, RARG 3' splices were consistently localized to the terminus of exon 9, leading to LBD-H11_12 truncation. Significant differences were also observed between RARA and RARG 5' splice patterns. Our analysis also revealed extensive involvement of transposable elements in constructing RARA and RARG 3' fusions, suggesting transposition mechanisms for fusion gene ontogeny. Both protein structural analysis and experimental results highlighted the pivotal role of LBD-H11_12/H12 truncation in driving ATRA unresponsiveness and leukemogenesis in tripartite fusion-positive aAPL, through a protein allosteric dysfunction mechanism.