T-cell Acute Lymphoblastic Leukemia Research Articles

Frailty in Children and Adolescents with Lymphoblastic Leukemia or Lymphoma Receiving Maintenance Chemotherapy - a Pilot Study

Background: Increased survivorship of childhood cancer has revealed a number of long-term morbidities related to treatment. Evidence suggests that cancer and its treatment may accelerate normal loss of physiologic capacity that occurs during aging.The advanced physiologic aging and related morbidities often seen in childhood cancer survivors suggests that these patients may present with signs of early-onset frailty. Frailty is a measure of global infirmity and impaired physical function and has been correlated with adverse health outcomes and elevated incidence of mortality in the general population. Research into the incidence and applicability of the frailty phenotype in children being treated for cancer may aid in the identification of the most vulnerable patients and identify those who may require intervention. The purpose of this study is to explore the frailty phenotype in children and adolescents receiving maintenance chemotherapy for acute lymphoblastic leukemia or lymphoma. Methods: We prospectively recruited children and adolescents (aged 5-18 years-old) diagnosed with acute lymphoblastic leukemia or lymphoma in the first cycle of their maintenance chemotherapy. Frailty was assessed twice: at an initial visit in cycle 1 maintenance treatment (V1), and 6-months following the initial assessment (V2). The frailty phenotype consists of 5 domains, which were assessed using developmentally appropriate measures: 1) Slowness: 6-minute walk test; 2) Weakness: Handgrip strength dynamometry - calibrated to the size of the participant's hand; 3) Exhaustion/Fatigue: The PedsQL TM Multidimensional Fatigue Scale; 4) Body Composition: InBody 270 body composition analyzer; and 5) Physical activity/Energy expenditure: Self-administered physical activity questionnaire for older children (PAQ-C) and physical activity questionnaire for adolescents (PAQ-A).Each assessment was scored individually and “Frailty points” were assigned based on age and sex-based normative z-scores or survey scores, and a composite “Frailty Score” ranging from 0=least frail to 25=most frail. Wilcoxon signed-rank test was used to compare V1 and V2 frailty scores. Results:The analysis sample consisted of 19 participants (mean age 9.1 [SD=3.4] years old; 63% female) who completed V1 and 7 participants that completed V2. Diagnoses of participants who completed V1 include precursor B-Cell acute lymphoblastic leukemia (n=15), T-Cell acute lymphoblastic leukemia (n=3), and T-Cell lymphoblastic Lymphoma (n=1). Median V1 z-scores and survey scores per domain are as follows: Slowness: z-score -3.11 [-4.02, -2.48]; Weakness: z-score -1.48 [-2.06, -1.38]; Exhaustion/Fatigue: score 58.3 [46.5, 70.8]; Body composition: z-score (total body fat) 0.90 [0.47, 1.79]; Physical activity/Energy expenditure: score 2.26 [1.73, 2.54]. Domain specific frailty score for V1 are displayed in Figure 1. Of the 7 participants with a V2 frailty assessment, paired analysis demonstrated significant improvement in Exhaustion/Fatigue (median score V1=56.94, V2=70.83 [p=0.016]). However, improvements in weakness, body composition, and physical activity/energy expenditure did not reach statistical significance. Median slowness z-scores at V2 were similar to V1. Median composite frailty score improved from 12 to 11 in V2 but was not statistically significant (p=0.054). Comparison of V1 and V2 overall composite scores are displayed in Figure 2. Conclusion: Children and adolescents receiving maintenance chemotherapy for acute lymphoblastic leukemia or lymphoma scored poorly in all five domains of frailty. We observed some improvements in exhaustion/fatigue, and trending improvements in subdomain and composite frailty scores in the small sample of participants with V1 and V2 assessments. Given that frailty has been shown to persist into adulthood in survivors of childhood cancer, the frailty assessment may be an effective tool for identifying patients who may require additional intervention. Initial and follow-up frailty measurements for this study are currently ongoing.

CD7 CAR-T Therapy Followed By a Second Transplant for T-Cell Acute Lymphoblastic Leukemia or Lymphoblastic Lymphoma Patients Who Relapsed after a Prior Transplant

Background The therapeutic options for patients with T-cell acute lymphoblastic leukemia or lymphoblastic lymphoma (T-ALL/LBL) who relapse after hematopoietic stem cell transplantation (HSCT) are currently restricted. CD7-targeted chimeric antigen receptor (CAR) T-cell therapy provides a promising avenue towards remission, yet the long-term prognosis for these patients still needs to be improved. The advantages of progressing to a second transplant post-CAR-T treatment remain uncertain due to limited data. In this study, we analyzed the clinical characteristics and transplantation outcomes of 16 T-ALL/LBL patients who, having relapsed after their initial transplantation, achieved complete remission (CR) in bone marrow (BM) and CR/partial remission (PR) in extramedullary lesions via CD7 CAR-T therapy and then opted for a second allogeneic HSCT (allo-HSCT) at our center. Our aim is to explore strategies to enhance the survival rate of these patients. Methods From January 2021 to January 2023, we analyzed T-ALL/LBL patients who underwent second transplantation at Hebei Yanda Lu Daopei Hospital. The patients included in this study met the following criteria: they had relapsed following their first HSCT and had subsequently undergone CD7 CAR-T therapy prior to the second transplantation. The follow-up cut-off date was till June 1, 2023. The median follow-up duration after the second transplantation was 178 days, ranging from 9 to 500 days. Results A total of 16 patients including 13 with T-ALL and 3 with T-LBL were enrolled. There were 14 males and 2 females. The donor of first transplantation was autologous(n=3), sibling identical(n=2), unrelated donor(n=2), or haplo-identical (n=9). After the first transplantation, 8 patients relapsed in bone marrow (BM) and 8 relapsed both in BM and extramedullary sites. Of these, 13 patients received autologous CD7 CAR-T, one patient received donor-derived CD7 CAR-T, and one patient was treated with universal CAR-T cells. After the CD7 CAR-T therapy, all 16 patients (100%) achieved minimal residual disease (MRD)-negative CR in BM, and 6 out of 8 patients (75%) achieved CR in extramedullary lesions. Only two patients exhibited PR in the extramedullary lesions. The median age at the time of the second transplantation was 28 years (range 14-42 years). The median time from diagnosis to the second transplantation was 683 days (range 385-2672 days). The median interval between the first and second transplantation was 438 days (range 229-2540 days), and the median interval from CD7 CAR-T infusion to the second transplantation was 72 days (range 31-92 days). For the second transplantation, 13 cases had haplo-identical donors and 3 cases had unrelated donors. The conditioning regimens utilized were either total body irradiation (TBI)-based (n=9), busulfan-based (n=6), or melphalan-based (n=1). Following the 2 nd transplant, the 1-year overall survival(OS) and leukemia-free survival(LFS) were 58.5% (95% CI, 28.9-85.0%) and 51.4% (95% CI, 22.4-79.9%), respectively(Figure 1). The 1-year relapse incidence (RI) and non-relapse mortality (NRM) were 15.9% (95% CI, 4.4-57.8%) and 31.5% (95% CI, 13.2-74.9%), respectively. The cumulative incidence of CMV and EBV viremia at day 100 were 75.0% (95% CI, 56.5-99.5%) and 19.8% (95% CI, 7.2-54.6%), respectively. The cumulative incidence of all grades acute graft-versus-host disease (aGVHD) and 3-4 grade aGVHD at day 100 were 56.3% (95%CI, 36.5-86.7%) and 25% (95%CI, 10.7-58.4%), respectively. One-year chronic GVHD(cGVHD) was observed in 25.1% of the cases (95%CI, 9.3-67.4%), while moderate to severe cGVHD was not found in any patient. Following the 2 nd transplant, a total of 6 patients died, including 3 from infection, 2 from relapse and 1 from intracranial hemorrhage. Conclusions Our initial findings suggest that even in refractory/relapsed T-ALL/LBL patients who have experienced relapse following previous HSCT, MRD-negative complete remission can potentially be achieved utilizing CD7 CAR-T cell therapy. The subsequent implementation of a second allo-HSCT appears feasible. Combining CAR-T therapy with a follow-up second HSCT presents a strategy for these heavily pre-treated patients, offering promising prospects for survival. However, further evaluation of this treatment approach is required, necessitating long-term monitoring and a larger patient cohort.

A Parallel Comparison of the Safety and Efficacy of Allogeneic Hematopoietic Stem Cell Transplantation for Refractory and Relapsed T-ALL/Lbl Patients Who Achieved Complete Remission with CD7 CAR-T Versus Patients Who Achieved First Complete Remission with Chemotherapy before Transplantation

Introduction We have developed a fratricide-resistant CD7 CAR-T cells from bulk T cells transduced with CD7 CAR without gene editing or additional gene transfer, named naturally selected CD7 CAR-T (NS7CAR-T). NS7 CAR-T therapy has shown a robust complete remission (CR) rate in patients with refractory or relapsed (R/R) T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma (T-ALL/LBL). Nonetheless, the risk of relapse remains a challenge post-CD7 CAR-T therapy alone, suggesting the potential benefits of consolidation allogeneic hematopoietic stem cell transplantation (allo-HSCT). The question remains whether CD7 CAR-T therapy could increase the incidence of post-transplant complications. Here we conduct a parallel study to compare the safety and efficacy of allo-HSCT in R/R T-ALL/LBL patients who achieved CR with NS7 CAR-T therapy vs those who reached first CR (CR1) using chemotherapy prior to transplantation. Methods Between November 2020 and January 2023, a total of 223 consecutive patients with T-ALL/LBL who underwent allo-HSCT at our center were retrospectively analyzed. Patients who met the following criteria were included in this parallel study: 1) underwent the first allo-HSCT in our hospital, 2) achieved CR before transplantation either from NS7CAR-T therapy or CR1 from chemotherapy (Fig. 1). A total of 139 patients met the criteria for analysis and underwent allo-HSCT after achieving CR either from NS7CAR-T therapy (n=43) or chemotherapy (CR1=96). The median patient age at the time of transplantation was 15 years (range: 1-51 years). The median follow-up time was 370 days (2-941 days). CD7 CAR-T patients were from two clinical trials registered at www.clinicaltrials.gov NCT04572308, NCT04916860. Transplant donor types included sibling identical (n=12), haploidentical (n=110), and unrelated donors (n=17). We used total body irradiation (TBI)-based conditioning regimens (n=128) or Busulfan/Melphalan-based regimens (n=11). To prevent graft-versus-host disease (GVHD), we used ATG along with a short-term regimen combining methotrexate, cyclosporine/tacrolimus, and mycophenolate mofetil. Results The characteristics of patients from both groups are detailed in Table 1. The median interval between CD7 CAR-T treatment and transplantation was 59 (39-131) days. Within the CAR-T group, 16 patients had primary refractory disease, 18 had relapsed disease and 9 in minimal residual disease (MRD)-positive status in bone marrow before CD7 CAR-T therapy. Prior to CAR-T therapy, 55.8% (24 patients) had extramedullary lesions. In comparing the CAR-T and chemotherapy CR1 groups, we observed no significant differences in long-term efficacy post-allo-HSCT. The 2-year leukemia-free survival (LFS) rates were 64.9 (95% CI, 49.1-79.2)% and 69.8 (95% CI, 59.0-79.6)% (p=0.340); the 2-year overall survival (OS) rates were 64.1 (95%CI, 48.0-78.6)% and 71.8 (95%CI, 60.5-81.9)% (p=0.107); the 2-year relapse incidence (RI) rates were 9.3 (95%CI, 3.7-23.7)% and 11.5 (95%CI, 6.4-20.7)% (p=0.873); and the 2-year non-relapsed mortality (NRM) rates were 28.3 (95% CI, 16.9-47.4)% and 21.6 (95% CI, 13.9-33.4)% (p=0.244), for the CD7 CAR-T and non-CAR-T groups, respectively. The incidence of all grades of acute GVHD (aGVHD) 37.2 (95%CI, 25.2-54.9)% vs. 42.1 (95%CI, 33.3-53.3)% did not significantly differ between the CAR-T and non-CAR-T groups. A trend toward an increased rate but no P significance was observed in Grade 3-4 aGVHD incidence of 20.93 (95%CI, 11.7-37.4)% vs. 9.5 (95%CI, 5.1-17.6)% (p=0.139). One-year incidences of moderate to severe chronic GVHD (cGVHD) were 12.2 (95%CI, 5.4-28.0)% and 18.0 (95%CI, 11.5-28.0)% (p=0.357) in the CD7CAR-T and non-CAR-T groups, respectively (Table 2). No statistically significant differences were detected in the occurrences of transplant-associated thrombotic microangiopathy (TA-TMA), CMV, EBV, and HHV-6 infections between the two groups. Upon analyzing, we found that r/r patients who received allo-HSCT after achieving remission with NS7 CAR-T had a similar OS to CR1 patients (Fig. 2). Conclusions Our parallel study showed that T-ALL/LBL patients, who achieved CR via CD7 CAR-T therapy followed by a consolidation allo-HSCT, had similar favorable OS and LFS compared to patients who underwent allo-HSCT in the CR1 state induced by chemotherapy. This was achieved without increasing the incidence of GVHD, infections, or TA-TMA.

Extracellular Vesicles Secreted from Daratumumab Resistant Cells Promote Resistance and Proliferation of Daratumumab Sensitive Cells, Possibly through the Transfer of miRNA Cargo

Multiple Myeloma (MM) is the second most common hematologic malignancy and remains incurable. Daratumumab (dara) is a potent anti-CD38 monoclonal antibody used for MM treatment, but responses are heterogeneous, and resistance is inevitable. There is hence an urgent need for identification of biomarkers to predict responses and in-depth characterisation of mechanisms of resistance. We have previously demonstrated that the exosome biogenesis pathway plays a significant role in the survival of dara resistant (dara R) cell lines. Here, we highlight that the enriched expression of exosome pathway proteins reported in dara R cell lines is not detected in Lenalidomide or Bortezomib-resistant cell lines, or in other monoclonal antibody resistant cell lines such as anti-CD20 (Rituximab)-resistant cell lines, thus indicating that this mechanism is unique to daratumumab resistance. Coculture of dara sensitive (dara S) cell lines with dara R cell lines separated by a cell-impermeable transwell insert promoted cell proliferation and reduced response to dara treatment in dara S cell lines thereby suggesting a potential role for extracellular vesicles (EVs) secreted by dara R cells in mediating these responses. In this study, we seek to investigate the role of EVs secreted by dara R cells, in particular, that of EV-derived miRNAs in the tumour microenvironment which can help predict and mediate dara resistance. We have originally described the establishment of three dara R cell lines in Natural Killer T-cell Lymphoma (NKTL), T-cell acute lymphoblastic leukaemia (T-ALL) and MM via long-term exposure to increasing concentrations of dara. To assess the specific role of EVs in mediating resistance, we isolated and purified EVs from the media of dara R cell lines and added it to the culture of dara S cells. To confirm uptake of dara R EVs by sensitive cell lines, EVs isolated from resistant cell lines were stained with PKH26 and characterised by binding to CD81 and CD63 beads via flow cytometry. Uptake of dara R EVs by dara S cell lines was confirmed through flow cytometry and confocal microscopy. We observed that uptake of dara R EVs directly promote proliferation and reduced cytotoxic response to dara treatment in dara S cells. Next, we assessed the effect of inhibiting dara R EV secretion by applying Neticonazole and Ketoconazole, prior to coculture of resistant and sensitive cell lines. Interestingly, coculturing azole-treated resistant cell lines with sensitive cell lines mitigated the increase in proliferation and dara resistance. Exosomal miRNAs are known to play a pivotal role in regulating cellular pathways driving cancer cell resistance and survival. To evaluate the potential role of EV-derived miRNA cargo, we first extracted miRNAs from the EVs purified from dara R and dara S cell lines and then performed an miRNA profiling screen with the PanoramiR panel (MiRXES) which targets 376 miRNAs expertly curated from miRBase 22, HMDD 3.2 and The Cancer Genome Atlas. We discovered that EVs derived from resistant cell lines expressed higher levels of proliferative miRNAs, miR-155 and miR-181a. These findings were validated through qPCR, confirming the increased expression of these miRNAs in both resistant cell lines and EVs derived from resistant cell lines. We also observed a concomitant downregulation of mir-155 target genes such as BACH1, JARID2 and SMAD5 in the resistant cell lines. This positive observation led us to extend the study into the clinic. Extracellular vesicles were isolated from the plasma fraction of bone marrow samples obtained from 17 patients with MM prior to treatment with dara based combinations either in the upfront or relapsed setting. miRNA extraction was performed and samples evaluated by miRNA sequencing. We found that the EVs from patients with a progression free survival (PFS) of less than 2 years after dara based therapy (dara R patients) had an enrichment of mir-155 and mir-181a as compared to those with a PFS longer than 2 years. These data align with our findings in the cell lines and suggest that these miRNAs may be utilised as potential biomarkers for daratumumab resistance. We are currently elucidating the biological basis for dara resistance mediated by these miRNAs. A deeper understanding of the mechanisms involved may pave the way for clinical evaluation of EV- directed therapeutics to overcome dara resistance.

Signaling Pathway Mutations Cooperate with the PICALM/MLLT10 Fusion in a Knock-in AML Mouse Model

The PICALM/MLLT10 (or CALM/AF10; C/A) is a rare but recurring fusion gene detected in patients with T-cell acute lymphoblastic leukemia, malignant lymphoma and acute myeloid leukemia (AML). Our lab has previously established a C/A knock-in mouse model, where the fusion gene preceded by a loxP-flanked transcriptional stop cassette was inserted into the Rosa26 locus (the R26LSLCA strain). To achieve tissue-specific expression of C/A, R26LSLCA mice were crossed with three different Cre-inducer lines, namely the Vav-Cre, MB1-Cre and CD19-Cre. In the Vav-Cre strain, Cre recombinase is expressed in the entire hematopoietic system, while in the MB1-Cre and CD19-Cre lines, Cre is expressed in the B-cell compartment. Using this model, Dutta et al. showed that C/A leads to leukemia only when expressed in all hematopoietic cells, including hematopoietic stem cells and does not lead to induction of leukemia when expressed in B-cells alone ( Dutta et al., Leukemia 2016). To further characterise leukemias driven by the C/A fusion, we established new R26LSLCA and Vav-Cre crosses. Similar to what was found previously, the Vav-Cre/R26LSLCA mice developed leukemia with a long median latency of 1 year (366.5 days; N=24) and complete penetrance. On post-mortem analysis, the Vav-Cre/R26LSLCA mice were found to have splenomegaly and leucocytosis. Expression of the fusion gene was confirmed in these mice by RT-PCR on the cDNA from bone marrow (BM) cells. Immunophenotyping analysis of the BM cells and splenocytes of these mice revealed a predominantly myeloid phenotype. To assess the effect of expression of C/A on the transcriptome, we performed RNA-seq analysis using BM cells from 17 leukemic mice and 12 healthy controls. In accordance with reports on C/A patient samples, the Hoxa genes Hoxa7 and Hoxa9 as well as the Hox-cofactor Meis1 were significantly upregulated in our C/A expressing mice. In addition, genes involved in B-cell development, such as Cd19, Pax5 and Ikzf3, and Gata1, which is essential for the development of erythrocytes, were downregulated. To prove that the Vav-Cre/R26LSLCA mice were indeed leukemic and had the potential to propagate the disease, we serially transplanted BM cells from 4 Vav-Cre/R26LSLCA mice into congenic recipients. The secondary (N=20) and tertiary (N=19) transplanted mice developed leukemia with a significantly shorter median latency of 21 and 17 days, respectively. Our group and others have shown that a single genetic lesion is in most cases not sufficient to cause leukemia. Thus, to determine if additional somatic mutations were acquired by the C/A knock-in mice, we performed whole exome sequencing on the DNA from BM cells of the 4 knock-in mice, some of their daughter secondary and tertiary leukemias as well as the corresponding germline DNA samples (N=19). We identified between 2-32 mutations per leukemia exome, including mutations in genes and/or members of gene families that have been implicated in human leukemias. Mutations were detected in the transcription factors Sfpi1, Zeb1, and Sox9; Aff2, a member of the Af4/Fmr2 gene family; and Sf3a2, a component of the RNA-splicing machinery, among others. In addition, in two C/A sister secondary leukemias and the corresponding tertiary leukemias, we detected a missense mutation at amino acid 507 (G507A) of the Ptpn11 gene, which is homologous to G507 of human PTPN11, a known hotspot mutation site in AML and juvenile myelomonocytic leukemia. Interestingly, Dutta et al. also reported a G507 mutation (G507V) in Ptpn11 in a C/A knock-in mouse. PTPN11 regulates the RAS/MAPK signaling pathway. This finding is reminiscent of what we have previously reported in a C/A driven murine bone marrow transplantation model, where we detected somatic mutations in several AML-associated signal transduction genes, including Flt3, Cbl and Kras ( Desai et al., Leukemia 2020). Taken together, our results strongly suggest that mutations in genes involved in signaling cascades, particularly those in the RAS/MAPK pathway, cooperate with the C/A fusion and lead to complete malignant transformation. We aim to functionally validate these findings by establishing mouse models that harbor the C/A fusion as well as mutations in Ptpn11 and other signal transduction genes. These murine models will be immensely valuable for gaining a deeper insight into PICALM/MLLT10-mediated leukemogenesis, studying cooperating mutations and for testing new targeted therapies.

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.

Association of Age with Acuity and Severity of Illness at Initial Presentation in Children, Adolescents, and Young Adults with Leukemia

Background: Adolescent and Young Adult (AYA) patients, historically defined by the National Cancer Institute as patients between 15-39 years, diagnosed with cancer are at risk for increased mortality compared with younger patients. Age 10 has been an alternate threshold for higher risk of treatment failure in leukemia. Acuity of illness - as defined by intensive care unit (ICU) level resource use at admission - in children with acute myeloid leukemia (AML) was shown in prior data from the Pediatric Health Information System to mediate excess mortality in Black patients (Winestone et al, Am J Hematol., 2017). This study aims to evaluate the acuity and severity of illness in patients presenting with new diagnoses of leukemia and evaluate whether AYAs have a higher acuity and severity of illness compared to younger patients. Methods: We performed a retrospective analysis of 771 patients aged 1-21 years with initial presentation of newly diagnosed leukemia at Children's Healthcare of Atlanta (CHOA) between 2010-2018. Exclusion criteria included those who received any treatment at another facility or those for whom information about the first 72 hours following presentation to the hospital was not available. High acuity of illness was defined (yes/no) as any ICU admission or ICU-level resource use - regardless of physical location in the hospital - by organ system (cardiovascular, respiratory, renal, hematologic, and neurologic) on manual chart review. High severity of illness was defined as initial white blood cell (WBC) count ≥ 50,000 cells/microliter or central nervous system (CNS) disease. Both acuity and severity were assessed during the first 72 hours following the point of diagnosis. Multivariable logistic regression was used to examine the association of age at diagnosis with acuity or severity of illness, controlling for sex, race/ethnicity, insurance status, and type of leukemia. Results: Among 536 patients with complete data to date, the median age was 6 years (interquartile range 3-12 years, 65.5% aged 1-9 years, 34.5% aged 10-21 years), 53.4% were male, 45.7% were Non-Hispanic White, and 65.7% had private insurance. Diagnoses included B-cell acute lymphoblastic leukemia (ALL) (70.5%), AML (16.4%), T-cell ALL (9.3%), and other leukemias (3.7%) (juvenile myelomonocytic, chronic myeloid, and Burkitt's). High acuity of illness was seen in 24.2% of patients and was more likely in older patients (10-21 years, 33.5% versus 1-9 years, 19.4%, p <0.01). Figure 1 illustrates involved organ systems by age group. High severity of illness was seen in 31.3% of cases, and although not significant, was also more likely in older patients (10-21 years, 32.5% versus 1-9 years, 27.9%, p=0.06). In multivariable analysis, compared to patients aged 1-9 years, patients aged 10-21 years had an increased odds of high acuity of illness [odds ratio (OR) 1.69, 95% confidence interval (CI 1.09-2.63)]. Patients with T-ALL also had an increased odds of high acuity of illness [OR 3.40 (CI 1.76-6.54); referent B-ALL]. The odds of high severity of illness did not differ by age [10-21 years OR 1.23 (CI 0.81-1.86)], but was significantly higher by Non-Hispanic Black race [OR 1.83 (CI 1.14-2.94); referent Non-Hispanic White] and type of leukemia [T-ALL OR 3.90 (CI 2.03-7.48); other leukemia OR 3.26 (CI 1.28-8.32); referent B-ALL] (Table 1). Discussion and Conclusions: AYA patients had an increased likelihood of high acuity of illness requiring ICU-level care at presentation, indicating a risk for disparities of outcomes from the point of initial presentation. Future directions will examine the association of acuity of illness with mortality in this cohort, as well as the role of race/ethnicity on severity of illness and mortality. This research will ultimately help inform strategies toward narrowing age disparities in outcomes of AYA leukemia.

Patterns of Care Among Adolescents and Young Adults Treated for Acute Lymphoblastic Leukemia: A Retrospective Study across Diverse US Practices

Background. Studies have demonstrated superior outcomes in adolescents and young adults (AYAs, age 15-39 years) with T-cell and B-cell/Philadelphia chromosome negative (Ph-) acute lymphoblastic leukemia (ALL) who are treated using pediatric-inspired regimens (PIRs). However, some AYAs continue to be treated with non-PIR or adult regimens (ARs) that are associated with less favorable outcomes. The extent to which hospitals and physicians in the United States (US) have adopted PIRs to treat ALL in AYA patients is not known. Additionally, limited knowledge exists about factors associated with use of AR. The objective of this study was to describe patterns of care in AYAs with newly diagnosed ALL, and identify patient, physician and hospital-level characteristics associated with use of PIRs vs. ARs in these patients. Methods. This retrospective study analyzed data collected from medical charts of AYAs with ALL across varied cancer care settings in the US. Patients were identified by treating oncologists, who were board certified in the relevant specialty and had 3-40 years of post-residency practice experience. Physicians were not eligible to participate if they were associated with a pharmaceutical company or a healthcare/government agency in paid capacity. Patients were included based on the following criteria: newly diagnosed with ALL, 15-39 years at diagnosis, and treated with frontline PIR or AR between January 2016 and April 2022. Patients with a prior history of cancer were excluded. Standardized data collection forms were used by participating physicians to collect patient and clinical characteristics, treatment patterns, and site characteristics. Physicians were compensated for participation for each completed chart. Descriptive statistics (mean, standard deviation [SD], frequency, percentages) were used to describe these characteristics. Chi-square and multivariate logistic regression analyses were conducted to evaluate potential associations of these variables with AR use. The study received IRB (Advarra, Inc.; IRB#: 00000971) exempt status. Results. The analytic sample consisted of 378 eligible patients. Patients were treated by 178 physicians, of which 16.8% were pediatric oncologists, 59.3% were adult oncologists, and 23.9% treated patients of all ages. The majority of patients were treated at adult centers (82 [21.1%]) or centers that treat patients of all age groups (279 [71.7%]); 24 patients (6.2%) were treated at pediatric centers. Approximately three-quarters (73.8%) were treated at academic centers and one quarter (26.2%) at community centers. Of the 378 patients, 230 (61%) patients were treated with a PIR and 148 (39%) with an AR. The proportions were similar for both B-cell/Ph- patients (71.3% and 28.3%, respectively) and T-cell phenotypes (46.5% and 53.4%, respectively). Children's Oncology Group (COG)-based protocols were the most common PIR used (60.4%) followed by CALGB 10403 (23%). Among those receiving ARs, HyperCVAD-based regimens was most commonly used (n=125 [84.7%]). Of patients receiving HyperCVAD, 85.6% did not receive any immunotherapy. In univariate analyses, the use of an AR was significantly higher in subgroups of older AYAs (20-39y), males, and patients with public insurance. AYAs who were obese, had CNS involvement at diagnosis, T-cell immunophenotype, Ph+, Ph-like, or early T-cell precursor (ETP) were associated with AR use. Patients treated at non-pediatric centers, small-medium size hospitals (<500 beds), or those treated by non-pediatric oncologists (those who treat adult only or both adult and pediatric patients) were more likely to receive AR (Table 1). In multivariate analysis, patient factors independently associated with use of an AR included older age, obesity, Ph+, and Ph-like. Patients treated at small/medium size hospitals or non-pediatric centers were also significantly more likely to receive an AR vs. PIR (Table 2). Conclusions. The study provides “real-world” insights into treatment approaches used for AYAs with ALL across diverse cancer care settings. Nearly one-half of AYAs with T-cell ALL and one-third of AYAs with B-cell/Ph- ALL received AR. Additional studies are needed to understand the reasons behind the limited uptake of PIR, especially among older AYAs and in smaller and adult treatment settings.

Outcome Disparities Among Hispanic and Non-Hispanic Patients with Acute Lymphoblastic Leukemia

Introduction: Acute lymphoblastic leukemia (ALL) is the predominant type of leukemia diagnosed in children and accounts for approximately 20% of all cases of acute leukemia in adults. In pediatric cases, ALL generally exhibits a favorable prognosis and outcome. Unfortunately, this optimistic scenario does not extend to adults, as the majority of deaths related to ALL occur in this age group. The outcomes observed in children with ALL are linked to the favorable cytogenetic profile of their leukemia. However, as individuals age, the cytogenetic profile progressively shifts towards high-risk groups. Furthermore, several epidemiologic studies have identified ethnicity, specifically Hispanic and African American backgrounds, as independent factors associated with worse outcomes. The factors contributing to these outcomes are diverse and include limited healthcare access, insurance coverage, immigration status, treatment adherence, socioeconomic status, and timeliness of treatment initiation, among others. Additionally, individuals of Hispanic ethnicity are more prone to complex cytogenetics and Central Nervous System involvement, both of which are commonly associated with a poor prognosis. Our study aimed to assess the differences in treatment outcomes among adults diagnosed with ALL by conducting a specific comparison between non-Hispanic and Hispanic individuals. Methods: In this retrospective observational study, we collected data from the electronic medical records (EMR) of patients aged 18 years and older who received a diagnosis of Acute Lymphoblastic Leukemia between February 1, 2011, and February 28, 2020 at Ascension Seton Hospitals in Texas. The collected data included sociodemographic factors such as gender, ethnicity, and age at diagnosis, as well as clinical characteristics such as cytogenetics, immunophenotype, treatment, and overall survival. Bivariate and multivariate analyses were conducted using a chi-squared test. Statistical significance was defined as a P-value less than 0.05. Results: A total of 56 patients with ALL were identified, with 45% being non-Hispanic and 55% being Hispanic. The incidence of ALL did not show significant variation based on gender or age, but it exhibited a significant difference depending on insurance status. The analysis revealed that Hispanic patients had a significantly higher likelihood of lacking insurance coverage (75%) compared to non-Hispanic patients (25%), with a statistically significant p-value of 0.001. With the exception of one non-Hispanic patient diagnosed with T-cell ALL, almost all participants in the study had B-cell ALL. Among the patients who received chemotherapy, 44% were non-Hispanic, while 56% were Hispanic. Among patients with t(9;22)/BCR-ABL1 positive ALL, non-Hispanics represented 76% and Hispanics represented 23%. Other cytogenetic abnormalities, including CRLF2-IGH fusion, IgH rearrangement, CRLF2 rearrangement, PBX1/TCF3 fusion, t(1;19) with PBX1/TCF3 fusion, ETV6 deletion, near haploidy, and 9p deletion, were more prevalent in Hispanic patients (79%) compared to non-Hispanic patients (21%). The overall survival rates at 1 year were 64% for non-Hispanic patients and 67.7% for Hispanic patients. However, at 3 years, the overall survival rates decreased to 32% for non-Hispanic patients and 25.8% for Hispanic patients. Conclusions: Our study provides important insights into the demographic and clinical characteristics of patients with ALL and highlights notable disparities between non-Hispanic and Hispanic patients. A higher proportion of Hispanic patients lacked any form of health insurance coverage at the time of diagnosis. Interestingly, the incidence of Philadelphia-positive ALL was found to be higher among non-Hispanic patients, whereas other cytogenetic abnormalities were more prevalent among Hispanic patients. Additionally, Hispanic patients experienced lower rates of overall survival at 3 years, suggesting potential disparities in outcomes. The differences observed between Hispanic and non-Hispanic patients highlight the necessity for additional investigation and tailored interventions to address the specific challenges faced by Hispanic patients, including access to healthcare, insurance coverage, and the prevalence of unfavorable cytogenetic abnormalities.

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 < 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 < 0.1%; MRD high positive (MRD-hp), ≥ 0.1% to < 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 < 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 < 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.

Retrospective Immunophenotypic Profile of Acute Leukemias in the State of Rio Grande Do Norte, Northeast, Brazil

INTRODUCTION:The main acute leukemias (ALs) are lymphoid (B or T lineage ALL) and myeloid (AML), in addition to biphenotype AL (BAL) and undifferentiated ALL (IAL), which are rarer. This variation reflects on therapy, requiring methods to characterize these leukemias. Thus, methods such as immunophenotyping by flow cytometry (ICF), multiparametric and quantitative, help in the follow-up of these neoplasms. Objective: The aim of this study was to demonstrate the immunophenotypic diagnostic profiles in patients with AL in Rio Grande do Norte, state, Brazil by FC. Methodology: Bone marrow and peripheral blood samples from 371 patients clinically diagnosed with AL were investigated by CF, using a panel of monoclonal antibodies for the detection of lymphoid (B, T and NK) and myeloid antigens, as well as markers related to cell immaturity such as CD34, Terminal deoxynucleotidyl Transferase (TdT).At the same time, information related to clinical data, age group and sex of patients was also collected. Results: Of all LA investigates 71 were B cell-ALL, 55 T cell-ALL, 239 AML, 04 BAL and 02 IAL. Regarding the immunophenotypic pattern, the main subgroups in relation to age group and were evidenced through the FC. 06/71 were Pro-B ALL, 48/71 Common ALL, 06/71 cyt mü chain ALL and 11/71 mature sIgM+ B-ALL. In B-cell lineage ALL, 51 (71.8%) of the cases were children (less than or equal to 18 years old) and 20 (28.2%) adults (over 18 years old). In T-cell ALL, 9 (16.4%) were pre-T ALL, 12 (21.8%) intermediate ALL and 29 (52.7%) medullary T-type ALL, corresponding to a total of 36.4% children and 63.6% adults. In AML, there was a predominance of involvement in adult patients, corresponding to47 (80.7%) of cases and 192 (80.3%) in children. Biphenotypic and undifferentiated AL were detected in adult patients. DISCUSSION: The B cell-ALL were characterized by natural arrest of B-cells precursors based on the expression of antigens related to this lineage in: i) Pro-B ALL (CD19+, HLA-Dr+, CD34+, TdT+); ii) Common ALL (CD10+, CD19+, cCD79a+, cytCD22+, HLA-Dr+, TdT+/- and CD34+/-); iii) cyt mü chain+ ALL (cyt IgM+, CD19+, HLA-Dr+, cytCD79a+, cytCD22+, CD10-, CD34-) and iv) mature B-ALL (sIgM+, sCD22+, CD10+/-, CD20+ and kappa+ or lambda+). The T-ALL were classified into: i) pre-T-ALL (CD7+, cytCD3+, HLADr+, CD34+ and TdT+), ii) intermediate T-ALL (CD1a+, CD2+, CD7+, cytCD3+, TdT+ and HLADr+, CD4+/CD8+) and Medullary T-ALL (CD4+ or CD8+, sCD3+, CD1a-, TdT+, HLADr+/- and CD34-). It was also possible to observe the aberrant expression of myeloid antigens in B and T-ALL, such as CD13, CD33 and CD66b, mainly related to ALL in the context of KMT2A rearrangement with lineage switching. In addition to transferrin receptors (CD71) and IL-2 receptor (CD25), which have been reported in the context of ALL BCR+/ABL+, as well as the presence of the Philadelphia chromosome. In AMLs, the most observed subtypes were those with little differentiation and with a monocyte component. (CD64+/CD14+). CD15+/CD34+ expression was also observed, indicative of asynchrony of maturation, and phenotypic aberrancy of lymphoid antigens in some cases, predominantly CD7. In addition to other antigens such as CD2 and CD19, CD19 and CD56, In addition, ALL was more frequent in children and males, while AML in adults and females in general, with a predominant incidence in males among adult patient. Conclusion: When properly applied, ICF can have a great impact on the diagnosis, classification and prognostic analysis of neoplastic processes.

CHD4 Promotes Tumor Formation, Survival, and Resistance to Genotoxic Therapeutics in TP53 Mutant T-Cell Acute Lymphoblastic Leukemia Cells through the NuRD Chromatin Remodeling Complex

T-Cell acute leukemia(T-ALL) though generally responsive to current chemotherapy remains incurable in over 50% of adult patients. Importantly, the survival for patients with TP53 mutant T-ALL disease is less than 10%. We have previously shown the role of the Methyl Binding Domain protein 2-Nucleosome Remodeling and Deacetylase (MBD2-NURD) chromatin remodeling complex in triple negative breast cancer cell growth and the role of the NuRD component, Chromatin-helicase-binding protein 4(CHD4), in tumor colony formation and genotoxic resistance in acute myeloid leukemia. In the present study, we have examined the role of specific NuRD complexes and CHD4 in T-cell acute leukemia cells with mutant TP53. The prototypical NuRD complex possesses both a histone deacetylase (HDCC) sub-complex and an ATPase chromatin remodeling sub-complex containing CHD4 linked to MBD2 or MBD3 of the HDCC. CHD4 is linked to the MBD2/3 anchors of the NuRD complex, via GATAD2A or GATAD2B. Depletion of CHD4 was found to increase the rate of apoptosis in several TP53 mutant T-ALL cell lines including Jurkat, MOLT4, RPMI and CCRF by up to 5 to 10-fold. Likewise, depletion of both MBD2 and MBD3 also increased apoptosis and sensitization to the standard genotoxic chemotherapeutic agent, cytosine arabinoside (Ara-C) but had only minimal effects in normal human CD34+ hematopoietic progenitor cells. Depletion of GATAD2B and GATAD2A by shRNA also resulted in a significantly increased survival of 60 days compared to 20 days in Scramble shRNA controls, in a xenogeneic mouse model of T-ALL. Depletion of CHD4 resulted in a profound (10 to 100-fold) decrease in colony formation of the TP53 mutant T-ALL cell lines in methylcellulose. RNA seq analyses identified the apoptosis, ferroptosis and mitophagy pathways as potential mechanisms for the striking effect of NURD disruption on colony formation. In summary, CHD4, through the NuRD complex, promotes tumor cell formation, survival and therapeutic resistance of TP53 mutant T-ALL cells. Current efforts are aimed at validating these findings in primary T-ALL cells and structure-function studies to advance development of tool compounds for disrupting the NuRD complex, with the long-term goal of developing improved small molecule therapeutics for treatment resistant acute leukemia.

Long-Term Outcomes of T-Cell Acute Lymphoblastic Leukemia and Risk Factors for Survival in a Multicenter Registry Study from Brazil

Introduction T-cell acute lymphoblastic leukemia (T-ALL) constitutes around 25% of all newly diagnosed cases of acute lymphoblastic leukemia (ALL) in adults. While most pediatric treatment protocols categorize patients (pts) based on their phenotype, this approach is seldom applied to adults. Nevertheless, there is a scarcity of reported cohorts specifically focusing on adult T-ALL. Often, these cases are studied alongside B-cell cases, despite their notable biological differences, or grouped with T-cell lymphoblastic lymphoma, which exhibits a more favorable prognosis compared to T-ALL. The objective of this study is to present outcomes and identify prognostic factors for survival of adults newly diagnosed with T-ALL, utilizing data from a real-world multicenter registry study conducted in Latin America. Methods This registry encompassed four different academic centers in Brazil. The study included pts aged 15 years and above newly diagnosed with T-ALL between January 2009 and June 2022. Clinical data were collected from medical records after ethical approval. Overall (OS) and event-free (EFS) survivals were calculated by Kaplan-Meier method, with Cox regression multivariate analysis (MVA) for finding risk factors and competing risk analysis for relapse. Results A total of 97 pts were included, with a median age of 27 years (range, 15 - 82). Thymic T-ALL accounted for 51% of the cases. Mediastinal bulky was found in 48% and positivity of initial cerebrospinal fluid (CSF) evaluation was 12.5% (by morphology or flow cytometry). Baseline features of this cohort are summarized in table 1. Most pts (88%) were treated with pediatric-inspired regimens (asparaginase [ASP]-based), mostly adapted GMALL (43%) and adapted BFM (22%). Native E. coli ASP was used in 76% and pegylated ASP (PEG-ASP) was used in remaining cases. During presentation or treatment, 15% of the subjects experienced thrombosis of any site, with most cases being associated with the use of ASP (64%) and diagnosed during induction (64%). Following the frontline regimen, 77.3% of pts achieved complete response (CR). Overall, 11.3% of pts died during the induction phase, while 8% were primary refractory. Univariate analysis for CR showed correlation with age (p=0.002), bulky size (p=0.016), and use of ASP (p=0.009). Median follow-up period was 6 years. Overall, allogeneic stem cell transplant (alloHSCT) was performed in 18 pts, being 11/18 in first CR. Five-year OS and 5y EFS were 43.7% (95% confidence interval [CI]: 34.4-55.5) and 36.4% (95% CI 27.9-47.5), respectively. Over the follow-up, 5-year relapse rate and non-relapse mortality (NRM) were 35.6% and 27.9%, respectively. Among pts who underwent alloHSCT, 5y OS was 64%. In the univariate analysis, older age (HR 1.02 [1.01-1.04], p<0.01) was associated with lower OS. Conversely, thymic T-ALL (HR 0.54 [0.3-0.96], p=0.03) and use of ASP (HR 0.35 [0.17-0.716], p<0.01) were associated with higher OS. Initial white blood cell count was not statistically associated with OS. Subsequently, we performed a subgroup analysis focusing on the adult and young adult population (AYA, age between 15-45 years). In this subgroup (n=85), 89% were treated with pediatric-inspired regimens and 5y OS was 46.5%. In the univariate analysis, thrombosis (HR 2.21 [1.09-4.48], p=0.02) was associated with lower OS, while thymic T-ALL (HR 0.48 [0.24-0.93], p=0.03) and the use of ASP (HR 0.317 [0.13-0.75], p<0.01) were associated with higher OS. In the MVA for AYA, thymic T-ALL (HR 0.50 [0.25-0.98], p=0.04) and thrombosis (HR 2.23 [0.97-5.13], p=0.054) remained associated with OS, while older age was not (HR 1.004 [0.96 - 1.04], p=0.81) (Figure 1). Conclusions Reported outcomes of T-ALL in this registry from Latin America are comparable to older reports from large groups worldwide. Currently, European groups have reported OS rates surpassing 70% for adults with T-ALL.However, in our setting, even in the AYA population, OS was disappointing. This could be attributed to increased NRM due to treatment toxicity and limited access to HSCT, which may have contributed to these findings. As previously reported, thymic T-ALL remains a lower-risk subtype. Additionally, thrombosis has emerged as an independent risk factor for OS in the AYA population, which is a novel finding that warrants further research. To the best of our knowledge, this is the first study specifically focusing on adult T-ALL from Latin America.

Frontline Consolidation with Nelarabine for Adults with High-Risk T-Cell Acute Lymphoblastic Leukemia. Results of the Graall-2014/T Atriall Phase 2 Study

Rationale. Acute lymphoblastic leukemia (ALL) with unfavorable biology and/or high minimal residual disease (MRD) levels are at high-risk of disease recurrence. Based on the results of the GRAALL-2005/T study (Trinquand A et al, JCO 2013; Beldjord K et al, Blood 2014), T-ALL patients enrolled in the GRAALL-2014/T trial were classified at high-risk (HR) if presenting an unfavorable 4-gene classifier (absence of NOTCH1/FBXW7 mutations or presence of RAS/PTEN alterations) and/or a post-induction IG/TR MRD1 ≥ 10 -4 (including the need of a salvage course to reach a complete remission [CR]). In the phase 2 ATRIALL sub-study, HR patients were offered to receive nelarabine (NELA) during consolidation and maintenance or as bridge to allogeneic hematopoietic stem cell transplantation (HSCT). The aim of the present report is to compare their outcome to that of similar patients treated in the same GRAALL-2014/T trial but without frontline NELA. Method. Between November 2015 and May 2022, the GRAALL-2014/T included 325 patients with T-ALL. Among these patients, 199 presented with HR features. The ATRIALL sub-study was activated in April 2017. ATRIALL eligibility criteria were defined as HR features, no CNS involvement at baseline, and continuous CR before to start consolidation 2 (first NELA course) at week 12. Based on the results of the COG phase 3 study, the protocol was amended in January 2021 to include patients with CNS involvement at baseline. NELA was given at 1,500 mg/sqm/day at day 1,3 and 5 in combination with etoposide and cyclophosphamide for a maximum of 5 courses during consolidation (2 cycles) and maintenance (3 cycles). Patients with MRD1 ≥ 10 -3 and/or with post-consolidation 1 MRD2 ≥ 10 -4 were eligible for HSCT after one NELA course. A total of 121 patients were included. One patient was excluded due to overt relapse before to start NELA. Eight patients had CNS involvement at baseline. A total of 33 patients included in the GRAALL-2014/T met initial ATRIALL criteria but were not included in the sub-study either before ATRIALL activation (N=21), or during accrual (N=10), or after accrual completion (N=2) and received standard of care (SOC) without NELA. The ATRIALL/CNS- cohort (N=112) was compared to the control cohort (N=33) for MRD response, cumulative incidence of relapse (CIR), disease-free survival (DFS) and overall survival (OS). Results. The median follow-up in the ATRIALL study (n=120) was 2.9 years (95%CI, 2.5-3.5). At 3 years, the CIR, DFS, and OS were 28% (95%CI, 20-37), 67% (95%CI, 55-76), and 71% (95%CI, 61-79), respectively. Patients from the ATRIALL/CNS- (N=112) and control (N=33) cohorts exhibited similar characteristics, except for a longer median follow-up in the control cohort (Figure A). Patients included in the ATRIALL study reached lower post-consolidation MRD3 levels (p=0.05). Among patients with MRD1 ≥10 -4, undetectable MRD3 was achieved in 36/55 (65%) after NELA versus 5/16 (31%) after SOC (p=0.02). When censoring at HSCT time, NELA was associated with a significantly reduced CIR (SHR=0.48; 95%CI, 0.23-0.99; p=0.045) and a trend for a prolonged DFS (HR=0.52; 95%CI, 0.26-1.07; p=0.075) (Figure B,C). This difference did not reach significance when patients were not censored at HSCT time. Indeed, high-risk patients not eligible for HSCT benefited from NELA in terms of CIR (SHR=0.43; 95%CI, 0.20-0.98; p=0.045) and DFS (HR=0.46; 95%CI, 0.21-1.03; p=0.06). Patients with non-ETP ALL had a significantly improved CIR (SHR=0.03; 95%CI, 0.17-0.89; p=0.025) and DFS (HR=0.47; 95%CI, 0.22-0.99; p=0.048), whereas ETP-ALL patients did not benefit from NELA over SOC. There was no benefit of prior NELA in patients who received alloHSCT per protocol. Conclusion. While NELA did not yield an overall improved outcome in the study population, benefits were observed in favorable MRD responders and non-ETP patients. Additional prospective studies are needed to further delineate the specific patient subgroups that might benefit from NELA.

In Vivo Modeling of T-Cell Acute Lymphoblastic Leukemia Reveals Synergistic Oncogenic Pathways and Bcl11b Haploinsufficiency As a Potential Therapeutic Vulnerability

For decades, T-ALL models were based on retroviral overexpression of NOTCH1 in murine hematopoietic stem cells (HSCs). This induces aggressive T-ALL but does not reflect the genetic and clinical heterogeneity of the disease. Large-scale genomic analyses have mapped the detailed landscape of T-ALL driver events over recent years, but how these diverse oncogenic events interact in T-ALL pathogenesis and which vulnerabilities this creates remains incompletely explored. This is in part due to the lack of appropriate mouse models that recapitulate the complex disease biology and are suitable to identify and study potential therapeutic targets and interventions in a more realistic context. We modeled the genetic heterogeneity of T-ALL through in vivo multiplexing of gain-of-function and loss-of-function oncogenic events known to drive the disease. We developed a novel mouse strain that expresses Cre-recombinase and an inducible Cas9 only in the thymus ( LSL.Cas9 x Lck-cre) along with a lentiviral vector system into which T-ALL specific oncogenes are cloned in antisense flanked by LOX66/71 sites. Gain-of-function events, such as the overexpression of transcriptional regulators ( Tlx1, Tal1, or Lmo2) and signal transducers ( NRAS G12D, PIK3CD E1021K) were induced in developing thymocytes by Cre-mediated inversion together CRISPR-Cas9 editing of recurrent T-ALL loss-of-function drivers. This combinatorial approach allowed to probe more than 2000 possible oncogenic mutational combinations in vivo. Transplantation of HSCs from LSL.Cas9 x Lck-cre mice transduced with our vector system into sublethally irradiated mice led to T-ALL development with different morphology and disease phenotypes. Developing leukemia harbored reproducible phenotypes, covering very immature CD4-CD8-CD25+CD44+ T-ALL in the Tlx1_NRAS subgroup, in contrast to the classical cortical CD4+CD8+CD3- phenotype (Tlx1_PIK3CD subgroup), as well as mature single-positive CD4+CD3+ or CD8+CD3+ blasts (Tal1_Lmo2 subgroup). We identified Tlx1, Tal1, Lmo2, NRAS G12Dand PIK3CD E1021Kas the main driver of disease phenotype and gene expression profile, whereas the loss-of-function mutations contributed to and accelerated the onset of the disease. Tumor-derived cell lines could be easily established and kept their phenotypic characteristics and mutation patterns. The resulting T-ALL tumors contained up to eight different disease-relevant genetic alterations, thus recapitulating the genetic complexity in humans and overcoming the long latency for spontaneous mutations in less complex mouse models. Among these, co-evolution of insertions and deletions in Notch1, Cdkn2a, Bcl11b, and Pten were among the most frequently observed mutations in definitive T-ALL, whereas others, e.g., in Dnm2, Phf6, Etv6, and Lef1, occurred more randomly. While gene editing by CRISPR-Cas9 mostly resulted in frame-shift mutations in both alleles, Bcl11b exhibited a strong haploinsufficient phenotype with exactly one knock-out and one intact allele in each case examined. Following this observation, further suppression of Bcl11b by shRNAs in Bcl11b +/- cell lines derived from our tumor models demonstrated rapid induction of cell death, which was more intense in T-ALL cells compared to non-malignant thymocytes. In addition, re-analysis of CRISPR-based loss-of-function screens of human hematopoietic cell lines retrieved from the Cancer Dependency Map confirmed BCLL11B as a strong selective dependency in human T-ALL. In summary, we model subgroup-specific T-ALL transformation through conditional and multiplexed in vivo gene editing and oncogene overexpression, thus creating a resource of novel mouse models that recapitulate the genetic and biological heterogeneity of the disease. Using our resource, we identified Bcl11b as a strong and selective context-specific dependency in T-ALL. Although Bcl11b is a transcription factor, our results could serve as an starting point for novel therapies that interfere with Bcl11b function, such as molecular glue degraders or protein interaction inhibitors.

Efficacy and Safety Analysis of Prophylactic Brentuximab Vedotin Administration after Pediatric Acute Leukemia Haploidentical Hematopoietic Stem Cell Transplantation

Objective: Acute graft-versus-host disease (aGVHD) is a common complication following allogeneic hematopoietic stem cell transplantation. Currently, the reported incidence of moderate to severe aGVHD in domestic cases is 13%-47%, while the incidence of grade III/IV aGVHD after haploidentical transplantation ranges from 7.9% to 13.8%. Severe aGVHD is one of the leading causes of early post-transplant mortality. This study aims to analyze whether the administration of Brentuximab vedotin after transplantation can reduce the incidence of aGVHD without increasing disease relapse rates and viral infections. Methods: A retrospective analysis was conducted on 50 pediatric leukemia patients who underwent haploidentical hematopoietic stem cell transplantation (HSCT) at our hospital between June 2017 and July 2022. These patients received prophylactic treatment with Brentuximab vedotin (20mg) on day +1 and day +4 post-transplantation to prevent aGVHD. The median age of the patients was 4.1 years (range: 0.6-16.0 years), with a gender ratio of 25:25 (male:female). At the time of diagnosis, the median white blood cell (WBC) count was 19.9×10 9/L (range: 2.0-863×10 9/L) for 41 patients, while the WBC count at initial diagnosis was unknown for 9 patients. Among the cases, 22 were acute lymphoblastic leukemia (ALL), and 28 were acute myeloid leukemia (AML). Before transplantation, 28 patients were in CR1, 14 in CR2, 2 in CR3, and 6 in NR. All patients received myeloablative conditioning regimens, with 33 receiving Bu/Cy, 15 receiving TBI/Cy, 1 receiving Bu/Flu, and 1 receiving TBI/Flu. The median follow-up time was 29.1 months (range: 3.1-70.1 months). Detailed patient data are presented in Table 1. Results: All 50 patients achieved 100% engraftment of white blood cells, with a median time of 15 days (range: +10 to +21 days). Platelet engraftment occurred in a median time of 9 days (range: +5 to +78 days), except for one patient who did not achieve platelet engraftment. This particular patient was diagnosed with AML and experienced hematologic relapse and extramedullary relapse before transplantation. The patient died due to disease relapse 90 days after transplantation.The incidence of aGVHD after transplantation was 44%, with 28% experiencing grade I-II and 16% experiencing grade III-IV aGVHD. The incidence of CMV viremia was 40%, with a median time to diagnosis of 36 days (range: +19 to +84 days), while the incidence of EBV viremia was 8%, with a median time to diagnosis of 47 days (range: +14 to +87 days). During the follow-up period, five patients experienced disease relapse, with a median time to relapse of 92 days after transplantation (range: +67 to +181 days). Among the relapsed patients, 2 had ALL (both in CR2), and 3 had acute myeloid leukemia (all in NR). Seven patients died during the follow-up, including 4 with AML and 3 with ALL. The causes of death were disease relapse in 3 cases, infection in 2 cases, TMA in 1 case, and cGVHD in 1 case. The estimated 5-year overall survival (OS) and leukemia-free survival (LFS) rates were 86%, and the 5-year cumulative relapse rate was 10% (see Figures 1, 2, and 3). In comparison, a control group of 44 pediatric acute T-cell lymphoblastic leukemia patients who underwent haploidentical transplantation without prophylactic Brentuximab vedotin treatment between January 5, 2021, and June 30, 2022, were analyzed. In the control group, the incidence of aGVHD was 56.8% (P=0.007), with 36.4% experiencing grade I-II and 20.4% experiencing grade III-IV aGVHD. The incidence of CMV viremia was 68.2% (P=0.006), and the incidence of EBV viremia was 29.5% (P=0.007). The relapse rate was 15.9%, and the mortality rate was 27.3%. Conclusion: For pediatric acute leukemia haploidentical HSCT, the administration of Brentuximab vedotin (20mg) on days +1 and +4 after transplantation, in addition to conventional aGVHD prophylaxis (CsA+MMF+sMTX), significantly reduced the overall incidence of aGVHD and grade I-II aGVHD. Administering Brentuximab vedotin on days +1 and +4 did not increase the rates of CMV or EBV infection nor did it increase the risk of disease relapse.

Targeting hyperactive platelet-derived growth factor receptor-β signaling in T-cell acute lymphoblastic leukemia and lymphoma.

T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are rare aggressive hematologic malignancies. Current treatment consists of intensive chemotherapy leading to 80% overall survival but is associated with severe toxic side effects. Furthermore, 10-20% of patients still die from relapsed or refractory disease providing a strong rationale for more specific, targeted therapeutic strategies with less toxicities. Here, we report a novel MYH9::PDGFRB fusion in a T-LBL patient, and demonstrate that this fusion product is constitutively active and sufficient to drive oncogenic transformation in vitro and in vivo. Expanding our analysis more broadly across T-ALL, we found a T-ALL cell line and multiple patient-derived xenograft models with PDGFRB hyperactivation in the absence of a fusion, with high PDGFRB expression in TLX3 and HOXA T-ALL molecular subtypes. To target this PDGFRB hyperactivation, we evaluated the therapeutic effects of a selective PDGFRB inhibitor, CP-673451, both in vitro and in vivo and demonstrated sensitivity if the receptor is hyperactivated. Altogether, our work reveals that hyperactivation of PDGFRB is an oncogenic driver in T-ALL/T-LBL, and that screening T-ALL/T-LBL patients for phosphorylated PDGFRB levels can serve as a biomarker for PDGFRB inhibition as a novel targeted therapeutic strategy in their treatment regimen.

Canonical BAF complex regulates the oncogenic program in human T-cell acute lymphoblastic leukemia

AbstractAcute leukemia cells require bone marrow microenvironments, known as niches, which provide leukemic cells with niche factors that are essential for leukemic cell survival and/or proliferation. However, it remains unclear how the dynamics of the leukemic cell–niche interaction are regulated. Using a genome-wide CRISPR screen, we discovered that canonical BRG1/BRM-associated factor (cBAF), a variant of the switch/sucrose nonfermenting chromatin remodeling complex, regulates the migratory response of human T-cell acute lymphoblastic leukemia (T-ALL) cells to a niche factor CXCL12. Mechanistically, cBAF maintains chromatin accessibility and allows RUNX1 to bind to CXCR4 enhancer regions. cBAF inhibition evicts RUNX1 from the genome, resulting in CXCR4 downregulation and impaired migration activity. In addition, cBAF maintains chromatin accessibility preferentially at RUNX1 binding sites, ensuring RUNX1 binding at these sites, and is required for expression of RUNX1-regulated genes, such as CDK6; therefore, cBAF inhibition negatively impacts cell proliferation and profoundly induces apoptosis. This anticancer effect was also confirmed using T-ALL xenograft models, suggesting cBAF as a promising therapeutic target. Thus, we provide novel evidence that cBAF regulates the RUNX1-driven leukemic program and governs migration activity toward CXCL12 and cell-autonomous growth in human T-ALL.

Idasanutlin and Navitoclax Induce a Synergistic p53-Dependent Apoptotic Cell Death in T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age. Existing therapies for T-ALL, particularly in adults, are intensive and poorly tolerated, with high rates of of treatment-related morbidity and mortality and a high frequency of relapse. In T-ALL, activating Notch pathway mutations are common and thought to induce transformation in part through activation of c-Myc. Typically, increased expression of c-Myc induces apoptosis through the p19(ARF)-Mdm2-p53 tumor suppressor pathway. Unsurprisingly, over 70% of T-ALL cases have inactivating mutations in CDKN2A (p19Arf). However, greater than 95% of primary and 75% of relapsed T-ALL cases maintain wild type p53. We hypothesized that MDM2 inhibition would lead to T-ALL cell apoptosis by bypassing the loss of ARF. We reported in a previous ASH abstract initial studies evaluating the MDM2 inhibitor idasanutlin in the context of T-ALL. We observed that T-ALL patient-derived xenograft (PDX) lines treated in vitro showed modest induction of apoptosis. Additionally, we observed upregulation of pro-apoptotic p53 target genes including Bax and Puma1, leading us to hypothesize that inhibition of pro-survival BH3 domain proteins Bcl-2, Bcl-xL, and Bcl-W by navitoclax (ABT-263) would synergize to enhance apoptosis. In vitro treatment of a panel of PDX lines with combined therapy led to robust cell death with strong induction of pro-apoptotic p53 targets in all lines. In vivo, we assessed the response of four T-ALL xenografts to dual treatment. All showed a significant reduction of tumor burden compared to the most efficacious single therapy and evidence of synergy in all tested lines, as evaluated by a modified Bliss Independence test analyzing average daily change in tumor burden. Overall survival was significantly increased in the combination treatment group. We report here further mechanistic characterization of idasanutlin and navitoclax dual therapy in the context of T-ALL, as well as several therapeutically-oriented comparisons. While our initial studies used navitoclax due to prior work showing that T-ALL frequently expresses and is dependent on Bcl-xL ( Chonghaile et al., 2014), navitoclax can cause a dose-limiting thrombocytopenia due to on-target Bcl-xL inhibition in circulating platelets. Therefore, we treated our PDX panel with a dose matrix of idasanutlin with navitoclax or venetoclax, which lacks Bcl-xL activity. We observed that the navitoclax combination showed strong evidence of synergic activity, as measured by zero interaction potency (ZIP) score ( Yadav et al., 2015). Idasanutlin and navitoclax combination therapy had global ZIP scores suggesting an additive effect in 1 line and strongly synergic activity in the remaining 4 lines. Venetoclax showed an additive benefit in 3 lines, but was only synergistic in 1 of 5 lines tested. These results underscore the importance of Bcl-xL targeting in T-ALL and support further investigation of whether combination treatment with idasanutlin permits a more tolerable navitoclax dosing regimen. We also evaluated ruxolitinib in combination with idasanutlin, as recent work has shown overlapping responses to inhibitors of Jak/Stat or Bcl-2 ( Yuan et al. 2022). While we observed moderate activity of ruxolitinib monotherapy, combination with idasanutlin was additive against one and synergistic in none of the tested lines. To confirm the p53-dependence of idasanutlin dual therapy, we engineered p53 knockout T-ALL cell lines and observed strong induction of apoptosis and p53 target genes in wildtype cells, which was entirely abrogated in p53 LOF lines. Additionally, we performed RNA-seq on PDX cells treated with single or dual therapy to mechanistically characterize transcriptional responses to the drug combination. We observed minimal changes in navitoclax-only treated cells at the RNA level, consistent with its on-target post-transcriptional regulation of apoptotic induction. Idasanutlin and combination therapy samples clustered closely in PCA analysis, with p53 response a strongly-induced pathway, as expected. Our data suggest that the rational combination of idasanutlin and navitoclax is highly active against T-ALL, with greater synergic opportunity compared to other available idasanutlin combination treatments, through induction of a p53-dependent apoptotic cell death.

Clonal Evolution Dissection Reveals High MSI2 Level Promotes Chemo-Resistance in T-Cell Acute Lymphoblastic Leukemia

Occurrences of T-cell acute lymphoblastic leukemia (T-ALL) arise in approximately 10-15% of pediatric ALL cases and the outcomes for high-risk T-ALL are inferior. Currently, the precise dissection of clonal evolution with intra-tumoral heterogeneity and to delineate targetable molecular events driving relapse in T-ALL subtypes have become area of interest. We carried out high-throughput droplet-based 5′-single-cell RNA-sequencing (scRNA-seq) and paired T-cell receptor sequencing (scTCR-seq) of isolated CD7 + BMMCs from five patients' Dx_Rel paired samples. To investigate the gene signature variation underlying leukemic progression, we observed two distinct patterns of evolutionary trajectories in the four Dx_Rel nonETP-ALL pairs. From T956 and T723, a significant outgrowth in TCR initially negligible diagnostic subclusters at relapse, to which surrogate TCR repertoires were correspondingly linked and enumerated, suggestive robust dominant “clonal shifts” under continuous chemotherapeutic pressure. Whereas in Dx_Rel pairs T593 and T856, which contained subclusters with distinct gene signatures at diagnosis or relapse respectively, shared an identical TCR genotype (TCR Dx-Rel major clone). The clonal architecture clues gained from simultaneous scRNA-seq and scTCR-seq are strongly indicative of the leukemic “clonal drift” that commonly occurs under chemotherapeutic stress, during which somehow, hypothetically, molecular signatures within the same TCR subclones intrinsically progressed over time ( Fig. A-H). Next, we designed a targeted scDNA-seq library within a custom panel covering 127 amplicons and 102 genes via Tapastri platform to validate these two patterns of clonal evolution during leukemia progression unambiguously. Likewise, we captured that in persisted subclones in T593 and T856 leukemic cells attained reproductive fitness from maintaining a mutational static status with remarkable transcriptomic variation from diagnosis to relapse. To further interpret the transcriptomic signature drift in the persisted TCR-defined major clones from T593 and T856 pairs, differential expression analysis was employed with RNA-binding protein Musashi-2 (MSI2) showing the most prominent differential expression pattern across drifted subclones catching our attention as a potential indicator of intrinsic clonal progression related to chemo-resistance. Comprehensive analysis of RNA-seq data from the CCCG-ALL-2015 cohort (n=72) and TARGET T-ALL cohort (n = 264) revealed that higher levels of MSI2 mRNA expression in pediatric T-ALL were correlated with higher measurable residual disease (MRD) after induction, and significant correlation between elevated MIS2 mRNA and resistance to prednisone, daunorubicin, and cytarabine were observed from ex vivo drug response assay. RNA immunoprecipitation and high-throughput mRNA sequencing from T-ALL cell lines detected a consistent significant enrichment of MYC transcript peaks in three T-ALL cell lines, indicating the molecular mechanism of MSI2 upregulation on MYC in the progression of T-ALL. Finally, In vitro and in vivo pharmacotyping analysis further validated the MSI2 inhibition significantly sensitized T-ALL to the chemotherapeutic drugs. Collectively, our study leveraging single-cell multi-omics profiling has provided a snapshot of the clonal architecture evolution that occurs from diagnosis to relapse in T-ALL, proving the coexistence of “clonal shift” and “clonal drift” in leukemic progression. Systemic analysis of drifted T-ALL clones has provided compelling evidence of the posttranscriptional regulatory effect of MSI2 on MYC and its potential correlation with leukemic reoccurrence, pinpointing MSI2 as a promising drug target of T-ALL in the future practice.