T-cell Precursor Acute Lymphoblastic Leukemia Research Articles

Early T-cell precursor acute lymphoblastic leukemia: diagnostic pitfalls, genomic alteration, novel therapeutics, and minimal residual disease monitoring

Early precursor T-cell acute lymphoblastic leukemia (ETP-ALL) is a rare type of T-cell ALL (T-ALL) that was initially described in 2009. Since its initial description, it has been formally recognized as a distinct entity, according to the 2016 World Health Organization classification. ETP-ALL is characterized by unique immunophenotypic and genomic profiles. The diagnosis and management of ETP-ALL remain challenging. Poorer outcomes, high incidence of induction failure and relapsed/refractory disease demand novel therapies. This review emphasizes the challenges of initial diagnosis, the molecular mechanisms underlying leukemogenesis, and the available therapeutic options. Additionally, it discusses the importance of induction failure, the prognostic value of detecting minimal residual disease, and the implications of hematopoietic stem cell transplantation, along with emerging novel therapies.

Single-cell sequencing technology to characterize stem T-cell subpopulations in acute T-lymphoblastic leukemia and the role of stem T-cells in the disease process.

Precursor T-cell acute lymphoblastic leukemia (Pre-T ALL) is a malignant neoplastic disease in which T-cells proliferate in the bone marrow. Single-cell sequencing technology could identify characteristic cell types, facilitating the study of the therapeutic mechanisms in Pre-T ALL. The single-cell sequencing data (scRNA-seq) of Pre-T ALL were obtained from public databases. Key immune cell subpopulations involved in the progression of Pre-T ALL were identified by clustering and annotating the cellular data using AUCell. Next, pseudo-temporal analysis was performed to identify the differentiation trajectories of immune cell subpopulations using Monocle. Copy number mutation landscape of cell subpopulations was characterized by inferCNV. Finally, cellphoneDB was used to analyze intercellular communication relationships. A total of 10 cellular subpopulations were classified, with Pre-T ALL showing a higher proportion of NK/T cells. NK/T cells were further clustered into two subpopulations. Stem T cells showed a high expression of marker genes related to hematopoietic stem cells, Naive T cells had a high expression of CCR7, CCR7, RCAN3, and NK cells high-expressed KLRD1, TRDC. The cell proliferation was reduced and the activation of T cell was increased during the differentiation of stem T cells to Naive T cells. We observed interaction between stem T cells with dendritic cells such as CD74-COPA, CD74-MIF as well as co-inhibition-related interactions such as LGALS9-HAVCR2, TGFB1-TGFBR3. Stem T cells were involved in the development of Pre-T-ALL through the regulatory effects of transcription factors (TFs) KLF2 and FOS and multiple ligand-receptor pairs.

Long-term exposure and response to azacitidine for post-hematopoietic stem cell transplantation relapse of early T-cell precursor acute lymphoblastic leukemia: a case report and review of the literature

Adult T-cell acute lymphoblastic leukemia has a poor outcome after relapse. Because the subtype of early T-cell precursor displays characteristics close of those of acute myeloid leukemia, such as epigenetic dysregulation, hypomethylating agents might prove of interest. We describe the case of a patient relapsing 3 months only after allogeneic stem cell transplantation who achieved complete remission on azacitidine, and is still on therapy 9 years later. We discuss the biological background of this very long-term response, underlining the immunological effects of hypomethylating agents, and the perspectives opened by combination of hypomethylating agents with other drugs such as venetoclax.

Homoharringtonine synergizes with venetoclax in early Tcell progenitor acute lymphoblastic leukemia: Bench and bed.

Early Tcell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinct subtype of T-ALL with a poor prognosis. To find a cure, we examined the synergistic effect of homoharringtonine (HHT) in combination with the BCL-2 inhibitor venetoclax (VEN) in ETP-ALL. Using invitro cellular assays and ETP-ALL xenograft models, we first investigated the synergistic activity of HHT and VEN in ETP-ALL. Next, to explore the underlying mechanism, we employed single-cell RNA sequencing of primary ETP-ALL cells treated with HHT or VEN alone or in combination and validated the results with western blot assays. Based on the promising preclinical results and given that both drugs have been approved for clinical use, we then assessed this combination in clinical practice. Our results showed that HHT synergizes strongly with VEN both invitro and invivo in ETP-ALL. Mechanistic studies demonstrated that the HHT/VEN combination concurrently downregulated key anti-apoptotic proteins, i.e., MCL1, leading to enhanced apoptosis. Importantly, the clinical results were very promising. Six patients with ETP-ALL with either refractory/relapsed (R/R) or newly diagnosed disease were treated with an HHT/VEN-based regimen. All patients achieved complete remission (CR) after only one cycle of treatment. Our findings demonstrate that a combination of HHT/VEN is effective on ETP-ALL and represents the "backbone" of a promising and safe regimen for newly diagnosed and R/R patients with ETP-ALL. This work was funded by the National Cancer Institute, Gehr Family Foundation, George Hoag Family Foundation, National Natural Science Foundation of China, and Key Research and Development Program of Zhejiang Province of China.

Epigenetic alterations affecting hematopoietic regulatory networks as drivers of mixed myeloid/lymphoid leukemia

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.

Flow Cytometry-Based Detection of Minimal/Measurable Residual Disease Predicts Survival Outcomes in Pediatrics, Adolescents, and Young Adults With T-acute Lymphoblastic Leukemia.

Measurable/minimal residual disease (MRD) is considered the single most powerful high-risk factor in acute leukemia, including T-cell acute lymphoblastic leukemia (T-ALL). In this study, we evaluated the impact of flow cytometry (FC)-based detection of MRD on survival outcomes in pediatrics, adolescents, and young adults (AYA) with T-ALL. We included 139 patients, 88 pediatric patients between the ages of one and 14 years, and 51 AYA patients between 15 and 39 years of age, over a period of three years and were treated with the Indian Collaborative Childhood Leukemia Group (ICiCLe) protocol. MRD assessment was performed on post-induction (PI) bone marrow aspirate samples using a 10-color 11-antibody MRD panel on a Gallios instrument (Beckman Coulter, Miami, FL, USA). MRD value > 0.01% was considered positive. PI-MRD status was available in 131 patients. The five-year event-free survival (5-year EFS) in PI-MRD positive patients was inferior to those of negative patients (13.56% vs 79.06%), which was statistically significant (P < 0.001). However, the five-year overall survival (5-year OS) did not show any statistically significant difference between PI-MRD positive and negative T-ALL patients (92.93% vs 94.28%). The hazard ratio (HR) for 5-year EFS and MRD positivity was 8.03 (p-value < 0.0001). HR for 5-year EFS and early T-cell precursor ALL (ETP-ALL) was 2.63 (p = -0.02). PI-MRD detected using FC is a strong predictive factor of inferior survival outcomes in pediatrics, AYA patients with T-ALL. PI-MRD positivity can be used to modify the treatment of T-ALL patients, especially in resource-constrained developing countries where molecular tests are not widely available.

Immunophenotypic but Not Genetic Changes Reclassify the Majority of Relapsed/Refractory Pediatric Cases of Early T-Cell Precursor Acute Lymphoblastic Leukemia.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) develops from very early cells with the potential for both T-cell and myeloid differentiation. The ambiguous nature of leukemic blasts in ETP-ALL may lead to immunophenotypic alterations at relapse. Here, we address immunophenotypic alterations and related classification issues, as well as genetic features of relapsed pediatric ETP-ALL. Between 2017 and 2022, 7518 patients were diagnosed with acute leukemia (AL). In addition to conventional immunophenotyping, karyotyping, and FISH studies, we performed next-generation sequencing of the T-cell receptor clonal repertoire and reverse transcription PCR and RNA sequencing for patients with ETP-ALL at both initial diagnosis and relapse. Among a total of 534 patients diagnosed with T-cell ALL (7.1%), 60 had ETP-ALL (11.2%). Ten patients with ETP-ALL experienced relapse or progression on therapy (16.7%), with a median time to event of 5 months (ranging from two weeks to 5 years). Most relapses were classified as AL of ambiguous lineage (n = 5) and acute myeloid leukemia (AML) (n = 4). Major genetic markers of leukemic cells remained unchanged at relapse. Of the patients with relapse, four had polyclonal leukemic populations and a relapse with AML or bilineal mixed-phenotype AL (MPAL). Three patients had clonal TRD rearrangements and relapse with AML, undifferentiated AL, or retention of the ETP-ALL phenotype. ETP-ALL relapse requires careful clinical and laboratory diagnosis. Treatment decisions should rely mainly on initial examination data, taking into account both immunophenotypic and molecular/genetic characteristics.

Clinical features of immature leukemias in children.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL), mixed phenotypic acute leukemia (MPAL), and acute myeloid leukemia with minimal differentiation (AML-M0) all originate from immature hematopoietic progenitor cells and have a poor prognosis. We investigated the clinical characteristics of these immature leukemias in 17 children (ETP-ALL: 8, MPAL: 5, AML-M0: 4) at seven institutions. Clinical and laboratory findings were comparable across disease types. Eleven and six patients received ALL- and AML-oriented induction chemotherapy, with six and four achieving complete remission (CR), respectively. Five additional patients achieved CR after salvage with the other type of chemotherapy. Eight patients received hematopoietic cell transplantation (HCT) in first CR, and six survived without relapse. However, six of seven patients who did not receive HCT during first CR relapsed; all underwent HCT later, and only three survived. The 5-year event-free survival (EFS) and overall survival (OS) rate were 37% and 69%, respectively. Patients who achieved CR after induction chemotherapy and received HCT in first CR had favorable EFS and OS. Notably, all patients who received HCT in first CR survived 5years after diagnosis. Appropriate induction chemotherapy and HCT in first CR could improve the outcome of immature leukemias.

The experience of relapsed Burkitt lymphoma treatment with targeted drugs and autologous/allogeneic stem cell transplantation

The use of high-effective, multicomponent, risk-adopted chemoimmunotherapy schemes in children with Burkitt lymphoma reached advanced long-term progression-free survival over 90 % even for high risk patients. Unfortunately, conventional therapeutic strategy for relapsed/refractory disease is not accepted, and the effectiveness of carboplat‑in- and gemcitabine-containing regimens is unsatisfactory. Clinical experience of rituximab, ibrutinib and nivolumab in combination with polychemotherapy and own clinical case of successful relapsed Burkitt lymphoma treatment with targeted therapy and following autologous and allogeneic hematopoietic stem cell transplantation are presented. Proposed program could achieve a complete remission of Burkitt lymphoma, but short-term after allogeneic hematopoietic stem cell transplantation diagnosed T-cell precursor acute lymphoblastic leukemia became fatal for the patient.

Synergistic Effect of Venetoclax and Bendamustine in Early T-cell Precursor Acute Lymphoblastic Leukemia.

To date, therapeutic options for T-cell acute lymphoblastic leukemia (T-ALL) remain very limited. This study evaluated the efficacy of monotherapies and combination therapies including a selective BCL-2 inhibitor for T-ALL cell lines, namely Jurkat, CCRF-CEM, and Loucy. Loucy is an early T-precursor ALL (ETP-ALL) cell line characterized by an immature phenotype, whereas Jurkat and CCRF-CEM are late T-cell progenitor ALL (LTP-ALL) cell lines. Monotherapy was conducted with venetoclax, cytarabine, bendamustine, or azacytidine, whereas combination therapy was performed with venetoclax plus cytarabine, venetoclax plus bendamustine, or venetoclax plus azacytidine. Cell viability assay was conducted after 48 h using Trypan blue and the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Statistical analysis for evaluating synergistic interactions between anticancer drugs was performed by using the SynergyFinder Plus and drc R package. Adding venetoclax to cytarabine, bendamustine, or azacitidine achieved an additive effect, with Loewe synergic scores ranging from -10 to 10 in Jurkat and CCRF-CEM. Conversely, the combination of venetoclax and cytarabine displayed an additive effect (Loewe synergic score: 8.45 and 5.82 with MTS and Trypan blue assays, respectively), whereas venetoclax plus bendamustine or azacitidine exhibited a synergistic effect (Loewe synergic score >10 with MTS assay) in Loucy. Remarkably, the Bliss/Loewe score revealed that the combination of venetoclax and bendamustine was the most synergistic, yielding a score of 13.832±0.55. The combination of venetoclax and bendamustine demonstrated the greatest synergistic effect in suppressing ETP-ALL cell proliferation. Further studies are warranted to determine the mechanisms for the synergism between venetoclax and bendamustine in high-risk T-ALL.

Rescue Therapy in Gastric Mucosal Bleed using Haemospray™

Abstract Hemostatic agents are used extensively in endoscopic management of gastrointestinal (GI) bleed. Conventional endoscopic techniques such as thermal, mechanical (clipping), and injection treatment fail in up to 5%–10%. These agents are used particularly in anatomically challenging areas, lesions with poor visibility, diffuse bleeding, and friable tissues. We present an endoscopic management of gastric bleed in a 17-year-old male with leukemia (early T-cell precursor acute lymphoblastic leukemia), who underwent haplotransplant. Posttransplant patient developed pneumonia with acute respiratory distress syndrome requiring extracorporeal membrane oxygenation support with multiorgan dysfunction syndrome, graft versus host disease. During his hospital stay, he developed persistent GI bleed from multiple large excavating gastric ulcers. Multiple sessions of conventional hemostatic endoscopic procedures such as hemoclips, glue injection, and sclerosants failed to achieve hemostasis. Mucosal surface bleeds are difficult to manage hence we used Hemospray for achieving good hemostasis.

Characterizing Morphometric and Nanomechanical Malignant Cell Features in a Rare Paediatric γδ T-acute Lymphoblastic Leukaemia: Insights from a Single Case Study Using Atomic Force Microscopy

The most common cancer in paediatric age is acute lymphoblastic leukaemia (ALL) which accounts for nearly a quarter of all cases of paediatric cancer. ALL cases are classified as B-cell or T-cell precursor ALL, based on their immunophenotypical features. Patients with T-cell ALL (T-ALL) comprise 10–15% of all newly diagnosed cases and are more prevalent in boys and in older age compared to the overall incidence peak age of ALL. In this case report, we present the morphometric and nanomechanical features of T-lymphoblasts derived from an 11-month-old infant with a rare subtype of γδ T-ALL with aggressive biological behavior. To investigate these features, we employed atomic force microscopy (AFM) and compared the blast cell deviations found in their nanostructure and elasticity to those of normal lymphocytes from a healthy child. The malignant T-lymphoblasts exhibited reduced roughness and Young’s modulus values. This single case analysis demonstrates the potential of the AFM method to provide additional information regarding the characteristics of malignant cells and suggests its potential as a complementary approach for distinguishing neoplastic cells from normal cells. The application of AFM could potentially facilitate the introduction of in vitro tests to determine the efficacy of anti-leukemic treatments.

Inflammatory Signatures Define a New High-Risk T-Lineage ALL Subtype

T-lineage acute lymphoblastic leukemia (ALL) is an aggressive cancer comprising of diverse subtypes that are challenging to stratify using conventional immunophenotyping and have historically exhibited poor treatment outcomes in response to cytotoxic chemotherapy ( Nguyen, K et al. Leukemia 2008). Early T-cell Precursor ALL (ETP-ALL) represent a distinct subtype that displays a developmental block at the earliest stages of T-cell commitment, accompanied by the aberrant expression of myeloid and stem cell markers ( Coustan-Smith, E. et al. Lancet Oncol. 2009). The classification can be further complicated by T/Myeloid Mixed phenotype acute leukemia (T/My-MPAL), a rare and aggressive malignancy characterized by blasts with both T-cell lymphoid and myeloid markers ( George, B. S. et al. Biomedicines 2022). Accurate clinical diagnosis of T-lineage ALL is hindered by phenotypic variations among its subsets. Nevertheless, the precise diagnosis holds critical importance as drug sensitivity in preclinical models of T-lineage ALL is closely linked to the differentiation state, such as the sensitivity of ETP-ALL to the BCL-2 inhibitor venetoclax ( Chonghaile, T. N. et al. Nat Cancer 2021). This highlights the critical need to identify consistent phenotypic traits associated with unique therapeutic vulnerabilities to effectively tailor therapy to individual patients. To gain insights into subset-specific therapeutic vulnerabilities, we performed an integrative multiomic analysis of bone marrow (BM) samples (n=21) from newly diagnosed T-lineage ALL patients, including T-cell ALL (T-ALL), ETP-ALL, and T/My-MPAL. Leveraging cellular indexing of transcriptomes and epitopes in conjunction with T-cell receptor sequencing, we identified a distinct subset of patients spanning all three subtypes characterized by the high frequency of leukemic cells that most closely resemble healthy hematopoietic stem and progenitor cells (HSPC) by Symphony ( Kang, J. B. et al. Nat Commun 2021) query mapping, indicative of an earlier stage of differentiation arrest ( Figure 1A bottom). Pathway analysis revealed an inflammatory signature in patients with high frequencies of stem-like cells suggestive of an inflammatory microenvironment akin to high-risk AML ( Lasry, A. et al. Nat Cancer 2023), which we refer to as inflammatory T-lineage ALL. Next, we mined our multiomic data to generate a comprehensive geneset for inflammatory T-lineage ALL scoring in single cells and bulk samples ( Figure 1A top). Interestingly, Olink cytokine array analysis uncovered upregulation of production of cytokines IL-1β, IL-18, and/or OSM in inflammatory T-lineage ALL samples. Thus, this comprehensive multiomic analysis identified a subset of T-lineage ALL patients characterized by early differentiation arrest and inflammatory signatures. To explore the relationship between inflammatory T-lineage status and clinical response, we utilized the ssGSEA algorithm to score activity of our inflammatory signature in bulk RNA-sequencing data from the TARGET T-lineage ALL dataset (n=265, 74/265 inflammatory) ( Liu, Y. et al. Nat Genet 2017) and the Tran et al. study (n=27, 17/27 inflammatory) ( Tran, T. H. et al. Blood Adv 2022). We found a significant association between high inflammatory ssGSEA score and elevated MRD. To uncover the inflammatory T-lineage ALL mutational landscape, we performed whole exome sequencing on our study cohort, and compiled the reported mutations in the TARGET dataset. Interestingly, inflammatory T-lineage ALL patients, as predicted by inflammatory signature activity, had a significantly higher prevalence of mutations affecting cytokine signaling ( NRAS, JAK3), chromatin remodeling genes ( EZH2), and the WT1 oncogene. These analyses demonstrate a significant relationship between inflammatory status, genomic lesions, and MRD. Finally, we demonstrate that inflammatory, but not non-inflammatory, T-lineage ALL cells are significantly more sensitive to BCL-2 inhibitor venetoclax ex vivo (Figure 1B).Overall, our study has identified a new subtype of T-lineage ALL defined by an early arrest in T-cell development, inflammatory signaling, a unique mutational landscape, sensitivity to venetoclax, and an association with adverse clinical parameters.

Picalm::MLLT10 can Identify a New Subgroup of Acute Leukemias of Ambiguous Lineage with Unique Extramedullary Disease and Treatment Response

Background: Mixed-phenotype acute leukemias (MPAL), which is included in acute leukemias of ambiguous lineage (ALAL), can be classified as four different subtypes based on recurring genetic alterations including BCR::ABL, KMT2A and ZNF384 gene rearrangement as well as BCL11B activation. The 2022 WHO classification has already mentioned PICALM::MLLT10 fusions are also enriched in MPAL but need more data. In 2022 ELN classification, PICALM::MLLT10 was identified as acute myeloid leukemia (AML) with other rare recurring translocations but not ALAL. In addition, there is no separate classification for PICALM::MLLT10 in the new ICC classification. Therefore, it is still confused whether PICALM::MLLT10 corresponds to AML or acute lymphoblastic leukemia (ALL) or ALAL. Methods: Fourteen PICALM::MLLT10 positive patients of 390 AL patients (14/390, 3.6%) were identified by RNA sequencing (RNA-seq) in our center from 2020-2022, including 8 female and 6 male, with a median age of 33 years (16-50 years). These patients were newly diagnosed AL according to bone marrow morphology and immunology including 5 ALAL, 5 T-ALL [ 4 early T-cell precursor ALL (ETP-ALL), 1 cortical T-ALL], 2 AML, 1 B-ALL with aberrant expression of myeloid antigen, and 1 B/T MPAL. Results: The mean white blood cell counts of these patients was 8.25×10 9/L (2.34-51×10 9/L) and platelet counts was 168.5×10 12/L (39-429×10 12/L) individually. It should be mentioned that extramedullary disease (EMD) was found in 7 cases (7/14), including mediastinum, tonsil, and skin. In terms of immunotyping, CD7 was identified in all patients (14/14) and CD33 in 71.4% (10/14) patients. The major concurrence mutations were PHF6 mutation (8/14), JAK3 mutation (5/14), and SUZ12 mutation (4/14). Characteristic cytogenetic abnormality t(10;11)(p12.3;q14.2) was only found in three cases (3/14) (Table 1). The PICALM breakpoints are mainly concentrated in exon 17 (n=6) and exon 19 (n=8). Exon 4 (n=8), exon 6 (n=2), exon 9 (n=2) and exon 10 (n=2) are the most commonbreakpointsof MLLT10. To our knowledge, this is the first report about the breakpoints and fusion gene forms of PICALM::MLLT10. For initial treatment, these patients individually received standard ALL induction chemotherapy (VDPCP, vincristine, idarubicin, pegaspargase, cyclophosphamide, prednisone) and AML induction chemotherapy (3+7 IA regimen including idarubicin, cytarabine). The initial complete remission (CR) rate was only 35.7% (5/14), and 9 patients showing no remission (NR). These NR patients subsequently received salvage chemotherapy. It is worth mentioning that 6 of them received combined chemotherapy regimen including low doses of cytarabine (LDAC), granulocyte colony-stimulating factor (G-CSF) and anthracyclines such as aclarubicin or idarubicin or homoharringtonine (CAG or IAG or HAG), and 2 of 6 patients also further received combination therapy with venetoclax (CAG or IAG +VEN). Finally, 5 of them dramatically achieved CR (5/6). In the other 3 patients, 1 patient who was failure to initial Hyper-CVAD A/B regimen also dramatically achieved CR after the more Hyper-CVAD A regimen combined with venetoclax, and 1 patient achieved CR while another still NR with both receiving CLAG (cladribine, cytarabine, G-CSF) regimen. Subsequently 11 patients (9 CR, 2 with refractory disease) received allogenic hematopoietic stem cell transplantation (allo-HSCT), and all the CR patients survived well after HSCT (follow up 3-24 months, median 15 months) without relapse, while 2 NR patients died soon after transplantation because of severe complications (Table 2). Conclusions: Our data suggested PICALM::MLLT10 positive AL should be more appropriately recognized as an independent ALAL entity and may benefit from LDAC, G-CSF and anthracyclines combination chemotherapy as well as venetoclax. Sequential HSCT after chemotherapy combined with venetoclax may further improve long-term survival in AL patients with CR even MRD positive.

Jmjd3 Drives Immature T-Cell Lymphoblastic Leukemia

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that accounts for 15% of pediatric and 25% of adult ALL cases. Despite an improved survival rate due to intensified chemotherapy and bone marrow transplantation, still 20% of T-ALL patients are primary refractory to the standard treatment or relapse, and have a poor clinical outcome. T-ALL patients can be classified into molecular genetic subgroups based on the expression or genomic alterations of specific oncogenic transcription factors. One such subgroup the early T-cell precursor ALL (ETP-ALL) is a heterogeneous subgroup of immature leukemias that often display high expression of LYL1 and LMO2. It's important to improve our knowledge of T-ALL biology and developing novel targeted anti-TALL therapies. We previously showed that the H3K27 demethylase Jmjd3 plays an important role in T-ALL and is a good candidate for the treatment of T-cell leukemia. Aim: Identify the role of Jmjd3 in the initiation of immature T-ALL. Methods: We generated a conditional Rosa26- Jmjd3 overexpression mouse model (Figure 1A), and crossed it with a hematopoietic-specific Vav-icre mice, to obtain Jmjd3 Vav mice. To investigate the role of Jmjd3 in tumor initiation, we monitored survival of a Jmjd3 Vav cohort. Arising tumors were characterized by immunohistochemistry, flow cytometry, and were transplanted into secondary immunodeficient recipients. To characterize the pre-leukemic compartment, we performed single cell feature barcoding and RNA sequencing. Also bulk RNAseq and proteomics were performed on primary and transplanted Jmjd3-driven tumors. To investigate the role of Jmjd3 in immature T-cell biology in human and mice, we analyzed thymi from 8-weeks-old control and Jmjd3 Vavmice, and performed OP9-DL4 stromal co-cultures with transduced CD34+ human cord blood cells that overexpressed JMJD3, respectively. We used ETP-ALL patient-derived xenografts (PDX) models to validate markers identified in single cell analysis. Results: We found that hematopoietic Jmjd3 overexpression resulted in hematological malignancies with 38% penetrance (n= 8/21) and a median survival of 63 weeks (Figure 1B). Immunohistochemistry confirmed overexpression of Jmjd3, decreased levels of H3K27me3, and increased levels of H3K27ac in Jmjd3 Vav tumors. Immunophenotypic analysis revealed immature Thy1.1 +CD4 -CD8 - cells in all obtained Jmjd3 Vav tumors (Figure 1C), indicative of an immature the early T-cell precursor (ETP)-like ALL. These immature T-ALLs gave rise to primary (7 weeks) and secondary (3 weeks) transplants upon their transplantation in immunocompromised mice. Bulk RNAseq confirmed that the transcriptional feature of the primary tumor were maintained upon transplantation. The role of Jmjd3 in stimulating immature thymocyte development was confirmed in thymi from 8-weeks-old Jmjd3 Vavmice and could be validated as well in human T-cell development using stromal OP9-DL4 co-cultures. Finally, we used single cell sequencing technologies to characterize both transcriptional features and cell-surface markers within the tumor fraction (Figure 1D). We confirmed that the tumor cluster was ETP-like with absence of CD1, CD5 and mature T-cell markers, and expression of CD44, Lyl1, and myeloid markers. Furthermore, we could identify cell-surface in immature Jmjd3-driven tumors that were also present in human T-cell stromal culture and ETP-ALL PDXs. Conclusion: We identified Jmjd3 as a driver of immature thymocyte populations that give rise to ETP-ALL.

Identification of vulnerabilities for targeting BCL-2 family members in T-Cell Acute Lymphoblastic Leukemia

T-Cell acute lymphoblastic leukemia (T-ALL) is a disease caused by the malignant transformation of T-cell lineage progenitors. With the use of intensive chemotherapies survival rates have improved, but outcomes particularly of relapsed patients remain poor. In addition, currently used intensive chemotherapies are associated with high rates of treatment-related morbidity and mortality, emphasizing the need to develop new and improved therapies. The intrinsic apoptosis pathway, one of the key pathways controlling cell death, is dysregulated in many cancers and contributes to leukemogenesis and treatment failure. The main steps of this pathway are controlled by proteins of the B-cell lymphoma (BCL-2) family at the outer mitochondrial membrane. Hence, targeting this pathway by BH3-mimetics has emerged as an effective new treatment strategy in different cancers. Venetoclax is a selective BCL-2 inhibitor and is successfully used in CLL and AML, but heterogeneous sensitivity to venetoclax has been described in ALL and inhibitors of other BCL-2 family members including BCL-XL-selective A-1331852 and MCL-1 selective AZD5991 have been developed. Moreover, a dual inhibitor of the anti-apoptotic BCL-2 family members BCL-2 and BCL-XL (AZD4320) with its dendrimer conjugate (AZD0466) has recently shown anti-tumor activity in hematologic cancer models with manageable toxicity. The aims of this study are to evaluate susceptibilities of T-ALL to inhibitors of BCL-2 family proteins, to identify markers of response, to elucidate mechanisms of action and to assess combination effects. First, we analyzed the anti-leukemia activities of inhibitors targeting BCL-2 (venetoclax), BCL-XL (A-1331852) and MCL-1 (AZD5991) and of the dual BCL-2/BCL-XL inhibitor AZD4320 in T-ALL cell lines (N=6) and a series of patient-derived xenograft (PDX) samples (N=8). Inhibition of MCL-1 alone was not effective in the cell lines tested (EC50 &amp;gt;1 µM), but heterogeneous sensitivity was found in PDX samples (EC50 &amp;lt;1µM in 3/8 samples). In cell lines, sensitivity for BCL-2 inhibition was exclusively found in a cell line with an early T-cell precursor ALL phenotype (Loucy). Similar to cell lines, we found insensitivity for BCL-2 inhibition in all PDX samples tested (N=8). For inhibition of BCL-XL, we found heterogeneous sensitivities with 4/6 cell lines and 5/8 PDX samples showing an EC 50 &amp;lt;1 µM. Interestingly, BH3-profiling confirmed BCL-2 dependence in Loucy and BCL-XL dependence in the other cell lines. Importantly, dual inhibition of BCL-2 and BCL-XL showed clear responses in cell lines and PDX samples (median EC 50s 796.1 nM and 721.6 nM). Notably, sensitivity was associated with priming of the BH3-peptide BAD (r s=0.89, p=0.03), providing a marker of response. We next sought to elucidate the molecular mechanisms by which different BH3-mimetics induce cell death in T-ALL. Analyzing protein interactions of BCL-2 family members by co-immunoprecipitation studies, we found that AZD4230 acts on-target by releasing BIM from BCL-2 and BCL-XL, but with compensatory increased protein complexes of BIM and MCL-1. Accordingly, combined inhibition of BCL-2 and BCL-XL with inhibition of MCL-1 leads to release of BIM from all three anti-apoptotic proteins enabling downstream apoptosis signaling. These findings suggest that combined inhibition of BCL-2 and BCL-XL together with MCL-1 may provide synergistic benefits. To further validate these data, we applied dynamic BH3-profiling and determined dependencies of T-ALL samples on BCL-2 family members in response to BCL-2/BCL-XL inhibition. Here, we found that the MCL-1 dependence strongly increases upon BCL-2/BCL-XL inhibition, suggesting MCL-1 as a target for combined inhibition. Based on these results, we performed dose-response matrix analyses to test the effects of combined BCL-2/BCL-XL and MCL-1 inhibition. Most importantly, combined inhibition showed synergism in all cell lines and PDX samples tested (Bliss scores &amp;gt;10), including leukemias being insensitive to single compounds. Taken together, we found vulnerabilities of T-ALL to BCL-2 family inhibition, particularly to dual BCL-2/BCL-XL inhibition by AZD4320, and we demonstrated on-target activities. Using BH3-profiling, we identified BAD-priming as a marker of response for AZD4320 and MCL-1 dependence as a resistance mechanism that can be targeted by combination treatment, suggesting further clinical evaluation.

Venetoclax Combined with Hag Regimen in Newly Diagnosed ETP-ALL: Interim Analysis of a Prospective, Multicenter, Phase 2 Trial

Background: Early T-cell precursor (ETP) acute lymphoblastic leukemia (ALL) is identified as a subset of T-lineage ALL with a unique immunophenotype and genome profile in 2009. Patients with ETP-ALL have a relatively poor outcome with standard chemotherapy and studies showed that therapies utilized for myeloid neoplasms may be effective in ETP-ALL given the relatively high frequency of molecular myeloid features. In recent years, targeting of the antiapoptotic protein BCL-2 has emerged as an effective approach in acute myeloid leukemia (AML). Preclinical studies reported that ETP-ALL cells also express high level of BCL-2 and are sensitive to the BCL-2 inhibitor venetoclax (VEN). However, the clinical efficacy of venetoclax combination therapies in ETP-ALL patients is still limited. Our data showed that homoharringtonine (HHT) showed strong synergistic effect with VEN in ETP-ALL (2022 ASH abstract #170604). We therefore conducted a study of combination of VEN and HHT-based HAG regimen (V-HAG regimen) in newly diagnosed ETP-ALL patients. The aim of this study was to investigate the activity and tolerability of V-HAG regimen for newly diagnosed ETP-ALL. Methods: In this prospective, multicenter, phase 2 clinical trial conducted in 7 different hematological centers in China, eligible patients were newly diagnosed ETP-ALL(aged≥16 years old) based on the 2016 WHO classification with an Eastern Cooperative Oncology Group performance status of 0-2. Patients received venetoclax (100mg on day1, 200mg on day 2 and 400mg on day 3-14, if the blast cells in bone marrow was more than 5% on day 14, the patient continued to receive venetoclax 400mg until day 28), homoharringtonine (1.4 mg/m 2,2mg maximum daily, intravenously daily from on d1-10), Low-dose cytarabine (10 mg/m2 subcutaneously every 12h on d1-14), and G-CSF (100ug/m 2 daily on d1-14). Venetoclax dose was reduced by at least 50% in the patients receiving concomitant moderate or strong CYP3A4 inhibitors (such as azole antifungals et al). The primary endpoint was the rate of composite complete remission (CRc) after one cycle of induction treatment, including CR and CR with incomplete count recovery (CRi). Secondary endpoints included bone marrow (BM) minimal residual disease (MRD) by flow cytometry after one cycle of induction treatment, overall survival (OS), event-free survival (EFS), and adverse events (AEs). Here, we report results of the interim analysis. Results: Between July 2022 and July 2023, we enrolled 18 patients with newly diagnosed ETP-ALL in 7 different hematological centers. 14 of 18 patients (77.8%) were male and 22.2% were female, with a median age of 51 years (range, 29 to 73 years). As of July 31, 2023, 15 patients were available for the assessment of treatment results. Among the 15 patients, 14 patients were given standard V-HAG regimen as showed above and one 73-year-old patient, who had interstitial pneumonia at the onset of ETP-ALL, was given dose-reduced V-HAG regimen. To our surprise, after the first cycle of treatment with V-HAG, all the 15 patients achieved CRc (100% CRc rate), including the 73-year-old patient with dose-reduced regimen. Then, after one to three cycles of consolidation treatment with V-HAG regimen, 7 of 15 patients have proceeded to allo-HSCT so far. With a median follow-up of 7.7 months, both the median OS and EFS were not achieved. In fact, to date, except for the 73-year-old patient who eventually relapsed (6% blasts in BM after receiving a second cycle of dose-reduced V-HAG regimen as consolidation) and died of respiratory failure, all the other 14 patients are remaining alive and event-free. For the adverse events, the most common toxicities were grade 4 neutropenia (93.33%), grade 4 thrombocytopenia (73.33%) and grade 3 febrile neutropenia (26.67%). No early death occurred. Conclusions: Venetoclax Combined with HAG regimen achieved an 100% CRc rate in newly diagnosed ETP-ALL to date. Enrollment is ongoing to assess its efficacy and safety in treating newly diagnosed ETP-ALL. This therapy is a promising and well-tolerated regimen in newly diagnosed ETP-ALL patients. Trial registration: The trial was registered in the Chinese Clinical Trial Register with the registration number ChiCTR2200061708.

Prognostic Value of MRD By Flow Cytometry in Adolescent and Adult Patients with Philadelphia Chromosome-Negative Acute Lymphoblastic Leukemia Treated with Pediatric-Inspired Regimen: A Post-Hoc Analysis from a Prospective Study of China

Background Minimal residual disease (MRD) is currently considered as one of the most powerful predictors in patients with Philadelphia chromosome-negative acute lymphoblastic leukemia (Ph - ALL).The time points and thresholds for MRD monitoring in pediatric Ph - ALL have been well established. However, surveillance strategy in adult ALL has not yet been standardized. In this study, we explored the prognostic value of MRD levels and monitoring time points by flow cytometry (FCM) in adolescent and adult patients with Ph - ALL treated with pediatric-inspired regimen. Methods This analysis includes adolescent and adult patients who were treated according to a pediatric-inspired regimen revised based on Children's Oncology Group protocol (CCG-1961). This prospective study was registered at Chinese Clinical Trial Registry Website with the registration number of ChiCTR-OOC-15006328. MRD was measured using bone marrow aspirates with eight-color multiparametric FCM on day 14 of induction, at end of induction (EOI, days 29-42) , post the first consolidation (post-C1), and throughout the treatment period. MRD was defined as the percentage of lymphoblasts present in the total nucleated cells counted. MRD levels of &amp;lt; 0.01% and ≥ 0.01% was considered as negative and positive, respectively.Patients who were MRD-positive were further stratified into three groups according to MRD level: MRD low positive (MRD-lp), ≥ 0.01% to &amp;lt; 0.1%; MRD high positive (MRD-hp), ≥ 0.1% to &amp;lt; 1%; and MRD very high positive (MRD-vhp), ≥ 1%. Results From 2014 to 2021, a total of 415 patients with a median age of 27 (range: 14-69) years were treated. Three hundred and twelve patients presented with B-precursor ALL and 103 with T-cell ALL. With a median follow-up time of 40.8 months, the median overall survival (OS) of the cohort has not yet been reached. There were 386, 389, and 346 BM samples available for MRD evaluation on day 14 of induction, at EOI, and post-C1, respectively. Figure 1A presents the distribution of MRD status over time. Figure 1B depicts the compositions of MRD levels at EOI stratified according to disease types. Notably, Ph-like ALL and early T-cell precursor (ETP) ALL were associated with extremely lower achievements of MRD negativity at EOI compared with other types. The 5-year OS rates of patients with MRD negativity, MRD-lp, MRD-hp, and MRD-vhp at EOI were 68.0% (95% CI: 60.2%-75.8%), 44.8% (95% CI: 28.9%-60.7%), 52.4% (95% CI: 34.2%-70.6%), and 36.7% (95% CI: 24.2%-49.2%), respectively ( P &amp;lt; 0.001) (Figure 2A). If patients were censored at the time of hematopoietic stem cell transplantation, the 5-year OS rates of these patients were 74.9% (95% CI: 65.1%-84.7%), 39.7% (95% CI: 18.7%-60.7%), 42.3% (95% CI: 11.7%-72.9%) and 0%, respectively ( P &amp;lt; 0.001) (Figure 2B). Patients with a negative MRD at EOI had a more favorable OS than those with a positive MRD at EOI. There was no statistically significant difference in terms of survival time between patients with MRD-lp and MRD-hp. Further, patients with MRD-vhp had the worst survival. The relapse probabilities had a similar pattern. Patients with MRD negativity at EOI had a lower 5-year CIR than those with MRD positivity at any levels. Compared to MRD-lp and MRD-hp patients, patients with MRD-vhp experienced a significantly higher rate of early relapse. However, with a longer follow-up, the 5-year cumulative incidence of relapse (CIR) did not differ significantly among patients with a positive MRD in any of the three groups, with a rate of 47.4%, 43.8%, and 49.1% in patients with MRD-lp, MRD-hp, and MRD-vhp, respectively (Figure 2C). Multivariate analysis showed that positive post-induction MRD was one of independent predictors of both OS and Disease-free survival. Conclusion Our study confirmed that MRD negativity at EOI is an independent prognostic factor of OS in adolescent and adult patients with Ph - ALL treated with pediatric-inspired regimen. Patients with MRD-lp and MRD-hp at EOI had a lower OS and a higher relapse incidence than those with MRD negativity. Therefore, an early and deep MRD clearance is important, and even extremely low MRD levels can predict poor prognosis.

Nelarabine (NEL), Pegylated Asparginase (PEG) and Venetoclax (VEN) Incorporated to HCVAD Chemotherapy in the Frontline Treatment of Adult Patients with T-Cell Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma (T-ALL/T-LBL)

Background: The incorporation of novel agents such as NEL and PEG to frontline therapy has been associated with improved outcomes in pediatric patients (pts) with T-ALL/LBL, but has not been extensively studied in the adult pts. BCL-2 upregulation has been demonstrated in T-lymphoblasts, especially of the early T-cell precursor (ETP) ALL and addition of VEN (BCL-2 inhibitor) may be beneficial. Methods: Pts with previously untreated or minimally pre-treated T-ALL/LBL were eligible if they had an ECOG PS ≤3, creatinine ≤ 2 mg/dL, total bilirubin ≤ 2 mg/dL and ALT/AST ≤ 4 x ULN. Pts received 8 cycles (C) of HCVAD (C 1,3, 5, 7) alternating with high dose Ara-C and methotrexate (MTX) (C 2, 4, 6, 8) at approximately 3-week intervals. Two cycles of NEL (650 mg/m 2 daily x 5) were initially administered after C8 (cohort 1). Later, after a protocol amendment, they were administered after C4 and C5 (cohort 2). Subsequently, the protocol was amended to add PEG (1500 IU/m 2 capped at 3750 IU; for pts age 60 or older, dose was 1000 IU/m 2, capped at 2000 IU) on day 5 of the NEL cycles (cohort 3) and more recently, VEN 400 mg daily was added on the first 7 days of each of 8 cycles of therapy (cohort 4); this was later modified to be given for 7 days during the induction cycle to all pts and reduced to 3 days per post-induction cycle only in pts with ETP-ALL or those with persistent measurable residual disease (MRD) and all other pts did not receive VEN after C1 (cohort 5). Pts with ETP-ALL were referred for allogeneic stem cell transplant (allo-SCT) in first complete remission (CR). After the completion of the intensive phase; pts in all cohorts received 30 cycles of maintenance therapy with monthly POMP (prednisone, vincristine, MTX, prednisone) and early intensification with NEL/PEG on C6 and 7 and late intensification with MTX/PEG in C18 and HCVAD on C19. All pts received 8 intrathecal chemotherapy with MTX alternating with Ara-C and mediastinal radiation was considered in pts with bulky mediastinal disease. Results: Between 7/2007 and 12/2022, 133 pts were enrolled in the 5 study cohorts sequentially (cohort 1= 30, 2= 49, 3=17, 4=16, 5=21) (Table 1). Eighty pts (60%) had T-ALL, 52 T-LBL (39%) and 1 pt had ETP/myeloid bi-phenotypic leukemia. The median age for the entire cohort was 35 years (yrs) (range 18-78), 102 pts were male (77%), 83 pts (62%) were white and 18 pts (14%) had PS ≥2. Overall, 24 pts (18%) had an ETP phenotype and another 19 pts (14%) had a near-ETP phenotype. Six pts (5%) had central nervous system disease at diagnosis, 72 pts (54%) had mediastinal disease, 49 of 80 (61%) T-ALL pts had extramedullary disease and 20 pts (15%) were in complete remission (CR) at trial enrollment after minimal pre-enrollment therapy. Overall response (CR+CRp+CRi+PR) on trial was attained in 109/113 (95%) pts [CR= 101 (89%), CRi/CRp= 3 (3%), PR=5 (4%)]. CR/CRi/CRp was attained in 36/41 (88%) pts with ETP/near-ETP ALL and 60/64 (94%) of confirmed near-ETP ALL pts ( p=0.31). At a median follow-up of 62 months (mos), the median overall survival (OS) was not reached (NR), 3-yr OS was 71% and 5-yr OS was 64% . The 3-yr OS for pts treated with HCVAD+NEL (cohort 1+2), HCVAD+NEL+PEG (Cohort 3) and HCVAD+NEL+PEG+VEN (Cohort 4+5) were 66%, 88% and 76% respectively (Figure 1). Median OS was not reached for both VEN cohorts, while 3-yr OS was 72% in cohort 4 compared to 88% in cohort 5 ( p=0.51), with significantly shorter follow-up for the latter (33 mos vs. 15 mos). Amongst the 124 pts who had CR/CRp/CRi, the median RFS was NR and 5-yr RFS was 68%. 24 (18%) pts underwent SCT in first remission, including 20 (83%) with ETP or near-ETP ALL. The median OS was shorter in the 44 ETP/near-ETP pts compared to the 77 pts with non-ETP phenotype (71 mos vs. NR, p=0.08). 30 and 60-day mortality was 0% and 1%. 10 pts (7.5%) died in remission (3, 5 and 2 pts in cohorts 1, 2 and 4 respectively) including 1 after SCT, 4 after developing therapy-related acute myeloid leukemia and 5 from infectious complications. Conclusion: The ongoing phase 2 trial of NEL, ASP, VEN added to the HCVAD regimen shows promising long-term survival in adult pts with T-ALL/LBL with 3-yr OS of 76%-88% in pts treated with HCVAD+NEL+PEG +/- VEN. Larger prospective trials and longer follow-up are needed to demonstrate further benefit.

Newly Dignosed Early T-Cell Precursor Acute Lymphoblastic Leukemia Successfully Treated with Venetoclax Combined with DA and IA Regime

Abstract Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinctive and extremely aggressive subtype of T-ALL. It is characterized by the aberrant expression of myeloid/stem cell antigens and the lack of Tlymphoid antigens including CD1a, CD4, CD5, and CD8. Currently, standard combination chemotherapy is still the mainstay of ETP-ALL treatment. However, the optimal management remains to be determined. ETP-ALL has low response rates to conventional chemotherapy and high relapse rates with a grim prognosis. Although some ETPALL patients achieved morphologic complete remission (CR), a more signifcant proportion maintained MRD positive. Under the traditional induction and intensifcation treatment strategy, the ETP-ALL group had worse long-term outcomes than the non-ETP-ALL group [1]. A recent preclinical study suggested that inhibition of B-cell lymphoma/leukemia-2 (BCL-2) might be a potential treatment option for patients with ETP-ALL [2]. Here, we present two cases of ETP-ALL clinical reactivity to venetoclax in combination with the IA regimen, providing a better bridge to allogeneic hematopoietic stem cell transplantation (allo-HSCT). Case 1 A 47-year-old female was diagnosed with ETPALL in October 2021. Flow cytometry analysis showed CD7,CD117,CD34,CD13,CD2,CD200,cyCD3 positive,suggestive of ETP ALL. Cytogenetic analysis was normal and molecular panel identified aberrations in TET, EP300 and EZH2. She then recived venetoclax plus DA regime（venetoclax, 100 mg d1, 200 mg d2, 400 mg qd d3-14；daunorubicin 40mg d1-3；cytarabine 100mg d1-5). A bone marrow biopsy after the 1st cycle revealed CR with minimum residual disease (MRD) 1.91% by flow cytometry. She then received 2nd cycle of venetoclax plus CAG treatment and got MRD negative. She received 2cycles of the same combination and remained in MRD-negative CR prior to proceeding with a reduced intensity haploidentical allo-transplantation. She is currently 1 years and 4 months post-transplant and in remission. Case 2 A 53-year-old man was diagnosed with ETP-ALL following bone marrow aspiration in August 2022. He presented with Leukopenia and anemia (WBC 2.53× 109 /L, HB 73 g/L, PLT 148 × 109 /L). Bone marrow biopsy revealed a hypercellular bone marrow with 80% lymphoid blasts, negative for MPO staining. The flow cytometry showed the immunophenotype of the lymphoid blasts (CD7+CD34+CD33+CD13+CD200+cyCD3+nTdT+), which were consistent with the immunophenotype of the ETP-leukemic cells. Cytogenetics revealed der(4)(q35).The molecular panel identified aberrations in CSF3R,DNMT3A,CCND3,CSF3R and WT1. Therefore, ETP-ALL was diagnosed. Initially, he received the first cycles of venetoclax combined with IA regimen (venetoclax, 100 mg d1, 200 mg d2, 400 mg qd d3-14;idarubicin 10mg d1-3,cytarabine 100mg d1-5), and CR was achieved this time. Then, he received a 3rd CAG therapy. Subsequently, he underwent hematopoietic stem cell transplantation (HSCT) successfully in December, and CR was still maintained at now 7 months after HSCT. Conclusion Combination therapy of venetoclax with IA regimen is an effective regimen for treating patients with ETP ALL.