T-cell Acute Lymphoblastic Leukemia Patients Research Articles

A System-Level Study Reveals That a Defective Thymic Niche Accelerates the T-ALL Initiation

The ontogeny of T-cell acute lymphoblastic leukemia (T-ALL) is unknown. In humans and mice, T-ALL is diagnosed when malignant T cells are detectable in the peripheral blood, i.e., at a stage when disease development is complete. Therefore, the malignant processes in the thymus - where the T cells develop - are an unexplored "black box". We took an interdisciplinary approach combining in vivo experiments with computational modeling to investigate the role of the thymic niche in disease manifestation. We focused on the role of interleukin 7 (IL-7) in the thymus, as approximately 70-80% of T-ALL patients have either increased IL-7 receptor (IL-7R) expression or gain-of-function mutations in this gene. In addition, autocrine production of IL-7 by T-ALL blasts has been detected in some patients. Using our recently developed "virtual thymus" model (Aghaallaei et al., Science Advances, 2021), we first simulated more than 200 scenarios to find out the conditions that lead to massive expansion of a single clone in the thymus. We then genetically engineered thymocytes and thymic epithelial cells (TECs) in medaka fish based on in silico predictions. Consistent with our computational model, we found that manipulation of thymocytes or TECs alone resulted in only 25% (N=36) and 10% (N=29) T-ALL development, respectively, in the thymus of Medaka larvae after five days; however, this frequency was accelerated to 54% (N=45) when both cell types were manipulated simultaneously. Our results strongly suggest that defective thymic niche has a strong impact on T-ALL development. Taken together, we propose a previously unknown mechanism that may trigger T-ALL initiation in the thymus. In addition, we introduce the "virtual thymus" as a new versatile in silico model that can predict the outcome of conditions for the development or treatment of T-ALL.

The Expression of SREBF1 Inducing By Dexamethasone Promotes Bone Marrow Mesenchymal Stromal Cells Differentiate to Adipocytes to Attract and Protect the Residual T-Cell Acute Lymphoblastic Leukemia Cells

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood malignant disorder with high rate of relapse. The patients relapse as a result of minimal residual disease (MRD), which is originated from residual T-ALL cells in the bone marrow microenvironment (BMM). In the present study, we observed that adipocytes increased dramatically in BMM of T-ALL patients after being exposed to chemotherapeutic drugs. Then, we proved adipocytes attracted T-ALL cells by releasing CXCL13 and supported leukemia survival by activating Notch1 signaling pathway via DLL1 and Notch1 binding. Furthermore, we verified that DEX induced adipogenic differentiation by enhancing the expression of SREBF1 in BMSCs, and SREBF1 inhibitor significantly decreased adipogenic potential of BMSCs and the following ability of adipocytes to support T-ALL cells in vitro and in vivo. These findings confirmed that differentiation of BMSCs to adipocytes induced by DEX contributed to MRD of T-ALL and provided an auxiliary clinical treatment to reduce the recurrence rate of it.

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

We report here investigation of the combination of idasanutlin and navitoclax in the context of T-cell acute lymphoblastic leukemia (T-ALL), including detailed mechanistic characterization and in vivo patient derived xenograft results. T-ALL is an aggressive hematologic malignancy with a five-year event-free survival rate that declines with age, ranging from 70-75% for pediatric patients to 21.7% for patients aged 60-79. An unmet clinical need remains for the development of better therapies for T-ALL. Notch pathway activation mutations are present in the majority of T-ALL and are 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, and critical to our approach, greater than 95% of primary T-ALL cases and 75% of relapsed cases maintain wild type p53. Based on these genetic data, we hypothesized that treatment with MDM2 inhibitors, by bypassing the loss of ARF, would increase p53 expression and lead to T-ALL cell apoptosis. To test this hypothesis, we initially treated T-ALL patient derived xenograft (PDX) lines in vitro with single-agent idasanutlin (a second generation MDM2 inhibitor) and observed upregulation of pro-apoptotic p53 target genes Bax and Puma1. However, only modest T-ALL cell apoptosis was observed in response to MDM2 inhibition alone. Based on these observations, particularly the induction of pro-apoptotic p53 targets, we hypothesized that inhibition of pro-survival BH3 domain proteins would synergize with MDM2 inhibition to kill T-ALL cells. Of note, a prior study showed that most T-ALL (~62%) respond to treatment with navitoclax (ABT-263), an inhibitor of BCL2, BCLXL, and BCLW, although most responses were transient and dose-limiting thrombocytopenia was observed. Combination therapy with idasanutlin could permit on-target inhibition with tolerable dosing. In vitro treatment of a panel of 5 different PDX lines derived from pediatric and adult T-ALL with dual idasanutlin and navitoclax led to robust cell death in all lines. In addition, treatment of a T-ALL PDX line with a matrix of dose combinations showed strong evidence of synergic activity. Synergy was assessed by ZIP (zero interaction potency) score, a statistical method developed by B Yadav et al. (Comput. Struct. Biotechnol. J. 2015) that compares the change in potency of individual dose response curves in the presence of a second agent. Generally, a ZIP score greater than 5 is considered significant; Idasanutlin and navitoclax combination therapy had a global ZIP score=16-18, with the most synergic dose area ZIP score=40-52. Combined treatment resulted in marked apoptosis, with strong induction of pro-apoptotic p53 targets, including PUMA and BAX. Finally, we assessed the in vivo response of four different T-ALL xenografts to dual treatment. In each case, human T-ALL cells were injected into immunodeficient NSG mice and randomized into treatment groups. Monotherapy with idasanutlin or navitoclax resulted in a modest but significant decrease in T-ALL burden in all cases, with the exception of an ETP T-ALL, which was resistant to idasanutlin monotherapy. A marked response to combination treatment with idasanutlin and navitoclax was seen in all four T-ALL xenografts. Specifically, the combination was found to be synergistic in each case based on a modified Bliss Independence test, analyzing average daily change in tumor burden for synergy interaction. Furthermore, overall survival was significantly increased in the combination treatment group. These data suggest that the rational combination of idasanutlin and navitoclax is highly active, with induction of an apoptotic cell death in T-ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Influence of Genetic Ancestry on Disease Biology in Pediatric T-Cell Acute Lymphoblastic Leukemia

Introduction: Pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) is a biologically heterogenous disease that can be grouped into 16 subtypes based on integration of gene expression and somatic genomic alterations that have variable prevalence of activated targetable pathways (Pölönen P., et al ASH 2022). In both adult cancers and pediatric B-cell ALL, genetic ancestry has been associated with tumor biology and clinical outcomes; however, there is a paucity of data exploring the influence of genetic ancestry in T-ALL. Thus, we sought to determine the association of genetic ancestry with tumor biology in pediatric T-ALL. Methods: Whole genome sequencing (germline and tumor) and bulk RNA-sequencing was performed to characterize T-ALL subtype and pathway aberrations among 1312 newly diagnosed T-ALL patients treated on Children's Oncology Group trial AALL0434. Genetic ancestral composition was determined for each individual using the iAdmix program comparing allele frequencies between patients and reference genomes from the 1000 Genomes Project. Coefficients from the 5 reference populations (European (EUR), African (AFR), East Asian (EAS), South Asian (SAS), and American Admixture (AA)) were assumed to sum to 100%. Categorical genetic ancestral groups were classified from prior methods: "European" (EUR >90%), "African" (AFR >70%), "East Asian" (EAS >90%), "South Asian" (SAS >70%), and "Amerindian" (AA >10% and AA greater than AFR), with the rest defined as "Other." We evaluated associations of genetic ancestry with T-ALL molecular subtypes and pathways. Ancestry was first examined as a categorical variable using Fisher's exact test for its association with subtypes and pathways. Ancestry was also analyzed as a continuous variable using logistic regression for association with pathways and multinomial regression for association with subtype (TAL1-RB subtype as reference). The effect of ancestry was summarized as odds ratios (OR) for every 25% increase in a non-European ancestry, with a concurrent 25% decrease in European ancestry and remaining ancestries fixed. Results: Categorical ancestral composition in this cohort was 59% European, 11% African, 15% Amerindian, 3% East Asian, 2% South Asian, 10% Other. The prevalence of T-ALL subtypes differed by genetic ancestry (Figure 1). For example, HOXA9 and BCL11B, which are linked to ETP T-ALL, were significantly enriched in children of African genetic ancestry as compared to European ancestry (HOXA9: 11% AFR vs. 6% EUR; BCL11B: 3% AFR vs. 1.5% EUR). ETP phenotype was also more common in children of African descent as compared with European (11.9% vs. 7.5%, p =0.031). In analyses of ancestry as a continuous variable (with European as the reference), four subtypes and four pathways exhibited differences in odds of expression (Table 1). For every 25% increase in African ancestry, the odds of BCL11B subtype was 41% higher (p=0.034), odds of HOXA9 subtype was 26% higher (p=0.020), odds of MLLT10_TRA was 68% higher (p<0.001), and odds of tumor microenvironment (TME) aberrant subtype was 33% higher (p=0.027). For every 25% increase in African ancestry, patients were also 1.13-fold more likely to harbor JAK/STAT mutations (p=0.049) and 1.18-fold for MAPK/RAS pathway alterations (p=0.011), with decreasing odds of NOTCH mutations (OR=0.88, p=0.014) or ribosomal gene alterations (OR=0.69, p=0.003). For increasing East Asian ancestry, patients were also less likely to have NOTCH pathway alteration (OR=0.85, p=0.027), whereas there was no difference for increasing South Asian ancestry. Conclusion: Disease biology varies substantially among children of different genetic ancestries with T-ALL. Given that subtypes may inform future risk stratification, and pathways may guide novel treatment strategies, examining subtype and pathway expression for children of all ancestral backgrounds is critical. Important biological indicators of risk may be missed when studying patients of a single genetic ancestral group or when focusing on averages for the entire cohort without taking into consideration variation in genetic ancestry. Analyses of survival by genetic ancestry within genomic groups are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Clinico-Genomic Landscape of Induction Failure in Childhood and Young Adult T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy comprising 10-15% of childhood ALL. Previously, we found that Induction Failure (IF) occurs in approximately 10% of T-ALL cases, 3-fold more than in B-ALL, with only 50% achieving long-term survival (O'Connor et al, JCO 2017). Despite the preponderance of IF in T-ALL, little is known about the factors that predict treatment resistance and influence outcome, or how best to treat these patients. To address this, we present the largest cohort of T-ALL IF reported to date, comprising all IF cases on two large multi-national consecutive randomized trials, UKALL2003 and UKALL2011. Methods The UKALL2003 and UKALL2011 trials recruited 711 patients with T-ALL between 2004-2019; 688 of these patients were assessable at the end of induction (EOI). We supplemented existing trial data with new clinical data, and combined whole genome sequencing (WGS) and RNA-sequencing (RNAseq), to comprehensively characterize the clinical and genetic landscape of T-ALL IF. Results Of 688 T-ALL patients, 10.3% experienced IF. IF was significantly associated with increasing age occurring in 5.6% of patients below 10 years, 12.9% of those aged 10-16 years and 20% of patients over 16 years (p<0.001). Survival was significantly worse in IF (5yr Event-free survival (EFS) 47.9% (95%CI: 35.8 - 59.1) vs. 84.0% (95%CI: 80.7 - 86.8); HR:5.14 (95% CI: 2.82-6.07), p<0.001; 5yr Overall survival (OS) 52.1% (95%CI: 39.7 - 63.1) vs. 90.2% (95%CI: 87.4 - 92.5); HR: 6.38 (95% CI: 4.17-9.76), p<0.001). There was a clear trend towards increasing use of nelarabine over the study period; IF patients treated on UKALL2003 commonly remained on standard trial chemotherapy (74.2%; 26/35), while IF patients treated on the later UKALL2011 trial were more frequently treated with nelarabine and HSCT (68.6%; 24/35, p=0.0007). However, this did not translate into an improved outcome for IF patients, with no clear benefit of either HSCT or Nelarabine therapy. WGS revealed a picture of remarkable genetic heterogeneity, with 25 different initiating lesions converging on 10 subtype-defining T-ALL genes, rather than a single unifying driver of IF (Figure 1). Allocation of cases to conventional T-ALL subtypes found almost half were of the HOXA subtype, significantly more than in responsive T-ALL (p<0.001). This was consistent with immunophenotypic characterisation showing a significant enrichment of the Early T-cell precursor (ETP) subtype, present in 48% of IF cases vs. 10% of non-IF cases (p<0.001), indicating a less differentiated, more stem cell-like leukemia. Although the overall proportion of TAL1 cases was lower in IF, there was an unexpected dominance of non-coding TAL1 enhancer mutations, accounting for 87.5% of TAL1 cases. Importantly, TAL1 cases had a particularly dismal outcome with an OS of 12.5% (0.6-42.3). Notably, none of the 7 patients with a TAL1 non-coding driver lesion survived, and 2 never achieved remission, identifying this as a very high-risk genetic lesion in the context of IF. Mutational profiling found a reduction in mutations in several highly recurrent T-ALL genes (NOTCH1, FBXW7, CDKN2A, LEF1) coupled with an increase in mutations in WT1 and MED12, both of which have been associated with disease resistance in other leukemias. In addition, 2 genes not previously reported in T-ALL were recurrently mutated in IF with mutations in CHD4 in 5 cases and KAT6A in 4 cases, suggesting they may be novel drivers of refractory disease. Survival analyses also showed a significantly poorer survival in patients with mutations in MYCN and NRAS. WGS of samples collected at the end of induction showed no new lesions demonstrating that disease at presentation is representative of true refractory leukemia and that IF does not occur as a result of chemotherapy-induced mutagenesis. Conclusions Our results confirm the poor prognosis of IF in T-ALL and highlight a lack of progress in treatment of this high-risk leukemia, with no clear improvement in outcome despite increased use of nelarabine and HSCT. Genomic analyses identified a marked enrichment for non-coding TAL1 enhancer mutations, characterizing an ultra-high-risk group of patients in the context of IF, in which there were no survivors. The lack of a readily targetable pathway of primary chemoresistance supports alternative strategies, such as those employing immunotherapeutic agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Progenitor Sub-Populations in Treatment Resistant T-ALL

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid malignancy for which optimal therapeutic approaches for relapsed/refractory disease and biomarkers for upfront risk stratification are currently unknown. Comprehensive, large-cohort bulk-level profiling of T-ALL is currently underway with the promise to reveal novel drivers and associate genetics with treatment response. To synergize with these studies, we designed a single-cell (sc) genomics study powered to comprehensively characterize intra-tumoral arrest states in T-ALL. Using integrated scRNA and scATAC-seq analysis on 40 T-ALL cases alongside a novel, single cell atlas of pediatric T-cell development, we identified and characterized a bone-marrow progenitor-like (BMP-like) tumor sub-population associated with resistance to conventional therapy across distinct immunophenotypic subtypes of T-ALL. We first compared 10 treatment-refractory ETP-ALL patients (End of Induction MRD > 20%) to 10 treatment sensitive ETP-ALL patients (EOI MRD negative), where we identified a strong and consistent enrichment of ETP-ALL blasts that were transcriptomically and epigenetically analogous to multipotent BMP. BMP-like blasts had higher surface protein expression of myeloid and stem cell markers, such as CD33, CD123, HLA-DR, and CD34, and lowered expression of T-lineage surface protein markers, such as sCD3, CD4, CD2, and CD10. The top differentially expressed (DE) genes for BMP-like blasts included stem cell markers (C1QTNF4, CD44, LGALS1, HOPX), myeloid-lineage (S100A4, SPINK2), and B-lineage markers (IGLL1, IGKC, IGHM). The top DE transcription factors (TFs) in BMP-like blasts included TFs associated with self-renewal (MEF2C, HOXA3/4/5/6/9/10/11, MEIS1, HHEX, SPI1) and recovery from genotoxic stress (BCL11A). Several of the genes involved in self renewal (MEF2C, HOXA9, FLI1), or T-cell developmental block (SPI1) also had increased motif accessibility. We integrated these signatures with large-cohort bulk-genomic results of 1300+ T-ALL patients treated in the Children's Oncology Group (COG) AALL0434 clinical trial. The molecular signature of BMP-like blasts predicted poor outcome across multiple subtypes of T-ALL (ETP & Non-ETP) and were validated in two independent COG trials (AALL0434, full cohort sequenced; AALL1231, partially sequenced cohort) with corresponding bulk-RNAseq data (Panel A, ETP-ALL patient stratification). We also utilized single-cell derived transcriptional signatures in conjunction with bulk-sample-derived mutation calls to identify fusion drivers (eg, MLLT10, KMT2A, NUP98, NUP214) and gene mutations (eg, NOTCH1 WT, IL7R WT, ETV6 mut, and SAT1B mut) that are associated with the BMP-like state and poor outcome. Computational analysis and in vitro experiments indicated that BMP-like tumors would be highly resistant to conventional ALL-directed cytotoxics. To address the pressing need for targeted therapy in chemo-refractory T-ALL, we performed multi-database drug screening against the BMP-like signature derived from our single cell data, nominating a number of surface markers (CD44, ITGA4, LGALS1) and intracellular targets (BCL-2, MCL-1, BTK, NFKB) for experimental follow-up in patient derived xenografts (PDX) which we generated and characterized in our single cell study. Notably, experimental studies of PDX expanded blasts indicated consistent and selective vulnerability of BMP-like blasts to apoptosis-inducing agents (Panel B). Other agents with potent activity in this population included TEC-kinase inhibitors and proteasome inhibitors. Follow-up in-vivo studies in PDX are underway with the promise to bridge these therapies to the clinic. Our work comprehensively defines the developmental arrest states of T-ALL in reference to human early T-cell development. By doing so, we reveal a previously unappreciated sub-population level overlap between patients with distinct subtypes of T-ALL and identify one common BMP-like sub-population that associates with poor response to current therapy. As T-ALL thus far is solely defined by bulk-sample immuno-phenotype, we propose the prospective identification of BMP-like sub-populations and use of non-conventional cytotoxic agents for upfront-risk-stratification and targeted therapy to be realized in high-risk T-ALL. JX & CC, as well as DTT & KT, contributed equally to the work. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genomics improves risk stratification of adults with T-cell acute lymphoblastic leukemia enrolled in measurable residual disease-oriented trials.

Genetic information has been crucial to understand the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) at diagnosis and at relapse, but still nowadays has a limited value in a clinical context. Few genetic markers are associated with the outcome of T-ALL patients, independently of measurable residual disease (MRD) status after therapy. In addition, the prognostic relevance of genetic features may be modulated by the specific treatment used. We analyzed the genetic profile of 145 T-ALL patients by targeted deep sequencing. Genomic information was integrated with the clinicalbiological and survival data of a subset of 116 adult patients enrolled in two consecutive MRD-oriented trials of the Spanish PETHEMA (Programa Español de Tratamientos en Hematología) group. Genetic analysis revealed a mutational profile defined by DNMT3A/ N/KRAS/ MSH2/ U2AF1 gene mutations that identified refractory/resistant patients. Mutations in the DMNT3A gene were also found in the non-leukemic cell fraction of patients with T-ALL, revealing a possible mutational-driven clonal hematopoiesis event to prime T-ALL in elderly. The prognostic impact of this adverse genetic profile was independent of MRD status on day +35 of induction therapy. The combined worse-outcome genetic signature and MRD on day +35 allowed risk stratification of T-ALL into standard or high-risk groups with significantly different 5- year overall survival (OS) of 52% (95% confidence interval: 37-67) and 17% (95% confidence interval: 1-33), respectively. These results confirm the relevance of the tumor genetic profile in predicting patient outcome in adult T-ALL and highlight the need for novel gene-targeted chemotherapeutic schedules to improve the OS of poor-prognosis T-ALL patients.

Deep learning-based transcriptome model predicts survival of T-cell acute lymphoblastic leukemia

Identifying subgroups of T-cell acute lymphoblastic leukemia (T-ALL) with poor survival will significantly influence patient treatment options and improve patient survival expectations. Current efforts to predict T-ALL survival expectations in multiple patient cohorts are lacking. A deep learning (DL)-based model was developed to determine the prognostic staging of T-ALL patients. We used transcriptome sequencing data from TARGET to build a DL-based survival model using 265 T-ALL patients. We found that patients could be divided into two subgroups (K0 and K1) with significant difference (P< 0.0001) in survival rate. The more malignant subgroup was significantly associated with some tumor-related signaling pathways, such as PI3K-Akt, cGMP-PKG and TGF-beta signaling pathway. DL-based model showed good performance in a cohort of patients from our clinical center (P = 0.0248). T-ALL patients survival was successfully predicted using a DL-based model, and we hope to apply it to clinical practice in the future.

Mapping the genetic features of T-ALL cases through simplified NGS approach

BackgroundDespite the irruption of massive sequencing technologies in clinical routine, the genetic diagnosis of T-cell acute lymphoblastic leukemia (T-ALL) continues to be based on traditional techniques. The integration of old and new technologies with diagnostic and prognostic purposes represents a major challenge. MethodsA High-Throughput Sequencing (HTS) approach was applied to analyze the genetic landscape of two patients diagnosed with T-ALL and one early T cell precursor acute leukemia. Orthogonal standard techniques were used to confirm the findings of NGS analysis. ResultsBy using a single test, a complete genetic map including 2 previously unreported missense mutations in BCL11B gene are reported. Cooperating oncogenic lesions including CDKN2A/B deletions, SIL-TAL1 rearrangement and FLT3 amplification were also captured by using a single test. ConclusionsHTS is a useful approach that allows simultaneously analyzing mutations, CNVs and the clonal repertoire in T-ALL patients. This approach may simplify the genetic assessment of ALL.

ALL-017 Adult T-Cell Acute Lymphoblastic Leukemia With Transducin-Like Enhancer Gene Expression

Transducin-like enhancer of split 1 (TLE1) is a member of the TLE family of transcriptional co-repressors that control the transcription of a wide range of genes. The aim of this study was to evaluate the prognostic role of TLE1 gene expression in patients with T-cell acute lymphoblastic leukemia (T-ALL). This study was conducted on 97 newly diagnosed T-ALL patients admitted to the Mansoura University oncology center (59 males and 38 females) with median age 33 years, in addition to 102 apparently healthy individuals who served as a control group. TLE1 gene expression was measured in both patients and control groups by real time - PCR. The calculation of relative gene expression was done using the AACt method. TEL1 gene expression was significantly down regulated in T-ALL cases (median 2.83) as compared to controls (median 84.65) (p < 0.001). Low TEL1 gene expression was significantly associated with CNS infiltration, failure to achieve 1st complete remission and higher relapse rate (p< 0.001, 0.001 and 0.023, respectively). Early T-cell precursor (ETP)-ALL/LBL subtype was found to be significantly associated with low TEL1 gene expression (P 0.04), however there was no relation between TEL1 gene expression and TCR rearrangement or other available molecular abnormalities. Likewise, low TEL1 gene expression was significantly associated with shorter overall survival (OS) and disease-free survival (DFS) (P= 0.012 and 0.011, respectively). Furthermore, Low TEL1 gene expression was considered a risk predictor of relapse with OR 3.636(CI.1.422-9.295) (P =0.007); and OR 0.803(CI. 0.609-0.96) (P=0.021) and an independent predictor of T-ALL patients' outcomes with OR 0.619 (CI. 0.44-0.872) (P=0.006). TLE1 gene expression was significantly down regulated in T-ALL as compared with controls. Low TLE1 expression is an independent predictor of the T-ALL patient's outcome and could open the way for further risk stratification and targeted therapy in T-ALL.

ALL-312 Ig/T-Cell Receptor Clonality Testing as a Method for Minimal Residual Disease Monitoring in T-Cell Acute Lymphoblastic Leukemia Patients

Assessment of early response to therapy by minimal residual disease (MRD) monitoring in T-cell acute lymphoblastic leukemia (T-ALL) has been proven to be a fundamental tool for guiding further therapy. Polymerase chain reaction (PCR)-based immunoglobulin (Ig)-T-cell receptor (TCR) clonality testing is a standardized technique for routine diagnosis of lymphoproliferation. It is also widely applied for MRD detection in T-ALL and is most suitable for patients without additional rearrangements detected. To present the clinical significance and utility of a molecular-based approach for post-induction MRD detection in patients with T-ALL. Single-center, descriptive, retrospective study including 11 adult patients with T-ALL treated at the University Clinic for Hematology - Skopje from January 2018 to January 2022. TCR gene analyses were performed in 11 adults with T-ALL. TCR rearrangements in bone marrow samples were analyzed by multiplex fluorescent PCR amplifications of the TCRB-Vβ-Jβ, Dβ-Jβ, TCRG-Vγ-Jy, TCRD-Vδ-Dδ-Jδ, Dδ-Dδ, Vδ-Dδ, and Dδ-Jδ regions and subsequent capillary electrophoresis of PCR products by automatic DNA analyzer, following EuroClonality/BIOMED-2 guidelines. MRD status was assessed at the end of the induction cycle. Patients received a BFM-based protocol or Hyper-CVAD/high-dose methotrexate (HDM) and cytarabine (ARA-C) regimen. TCR rearrangements were detected in all 11 patients. All patients were negative for additional molecular prognostic markers. TCRB and TCRG gene rearrangements were slightly dominant over TCRD (64.3% vs. 50%). A total of 6 adolescents and young adults (mean age: 24 years) were treated with BFM. At the end of the induction regimen, MRD negativity was obtained in 5 patients (83.3%) and was sustained up to 3 months from stem cell transplantation (SCT) in 3 patients. The other 2 patients died from CNS infiltration. Hyper-CVAD/HDM-ARA-C was administered in 5 patients (mean age: 49 years). One patient died during the induction protocol due to intracerebral hemorrhage. Among the others, CR at the end of the first cycle was achieved in 3 patients, and a total of 4 cycles were applied. Two patients proceeded to SCT. The molecular technique for testing Ig/TCR clonality is a useful tool for post-induction MRD monitoring in patients with ALL, directing the further approach and clinical decisions.

Near-tetraploid T-cell acute lymphoblastic leukaemia in childhood: Results of the AIEOP-BFM ALL studies

BackgroundNear-tetraploidy–defined by DNA index 1.79–2.28 or 81–103 chromosomes–is a rare cytogenetic abnormality observed both in children and adults with T-cell acute lymphoblastic leukaemia (T-ALL) and its prognostic value is not yet determined. Patients and methodsWe report a retrospective study conducted in paediatric patients with newly diagnosed T-ALL treated in AIEOP-BFM ALL 2000 and 2009 studies. 31 near-tetraploid T-ALL patients (1.4%) are compared to T-ALL patients without near-tetraploidy. ResultsNear-tetraploid karyotype was associated with lower frequency of high-risk features: white blood cells count at diagnosis ≥100,000/μL (19.3% versus 41.0%, p-value < 0.001), PPR (13.3% versus 35.8%, p-value = 0.01) and minimal residual disease high-risk at the end of consolidation phase Induction B (4.03% versus 14.6%, p-value = 0.001). Complete remission was achieved at the end of induction phase (day 33) in 100% near-tetraploid T-ALL patients, compared to 93.2% T-ALL without near-tetraploidy. ConclusionOverall, we found that near-tetraploid T-ALL in newly diagnosed paediatric patients is associated with low-risk presenting features, with favourable treatment response and outcome.

TNFAIP3 mutation is an independent poor overall survival factor for patients with T-cell acute lymphoblastic leukemia.

It is imperative to explore potential biomarkers for predicting clinical outcome and developing targeted therapies for T-cell acute lymphoblastic leukemia (T-ALL). This study aimed to investigate the mutation patterns of tumor necrosis factor-alpha-inducing protein 3 (TNFAIP3, also known as A20) and its role in the prognosis of T-ALL patients. Polymerase chain reaction (PCR) and Sanger sequencing data from T-ALL (n= 49, JNU) and targeted sequencing data from T-ALL (n= 54, NFH) in our clinical center and a publicly available dataset (n= 121, PRJCA002270), were used to detect TNFAIP3 mutation. Three TNFAIP3 single nucleotide polymorphisms (SNPs; g.3033 C > T, g.3910 G > A, and g.3904 A > G) were detected in T-ALL in the JNU dataset, and g.3033 C > T accounted for the highest proportion, reaching 60% (6/10). Interestingly, TNFAIP3 mutation mainly occurred in adults but not pediatric patients in all three datasets (JNU, NFH, and PRJCA002270). T-ALL patients carrying a TNFAIP3 mutation were associated with a trend of poor overall survival (OS) (p= 0.092). Moreover, TNFAIP3 mutation was also an independent factor for OS for T-ALL patients (p= 0.008). Further subgroup analysis suggested that TNFAIP3 mutation predicted poor OS for T-ALL patients who underwent chemotherapy only (p< 0.001), and it was positively correlated with high risk and early T-cell precursor ALL (ETP-ALL) in two independent validation datasets (NFH and PRJCA002270). TNFAIP3 mutation mainly occurs in adult T-ALL patients, and it was associated with adverse clinical outcomes for T-ALL patients; thus, it might be a biomarker for prognostic stratification.

Effect of pediatric- versus adult-type chemotherapy regimens on outcomes of allogeneic hematopoietic stem cell transplants for adult T-cell acute lymphoblastic leukemia in first complete remission.

The optimal chemotherapy regimen pre-transplantation for adult T-cell acute lymphoblastic leukemia (T-ALL) patients remains unknown. Here, we compared the transplant outcomes in 127 subjects receiving pediatric- (N = 57) or adult-type (N = 70) regimens pre-transplant. The corresponding 3-year cumulative incidences of relapse (CIR) was 7% (95% CI: 3-11%) and 29% (95% CI: 23-35%; P = 0.02), leukemia-free survivals (LFS) was 86% (95% CI: 81-91%) and 57% (95% CI: 51-63%; P = 0.003), overall survivals (OS) was 88% (95% CI: 84-92%) and 58% (95% CI: 52-64%; P = 0.002), the 1-year NRM was 4% (95% CI: 1-7%) and 9% (95% CI: 4-14%; P = 0.40). Multivariate analysis showed that pediatric-type regimen was associated with lower CIR (Hazard Ratio [HR] = 0.31 [95% CI: 0.09-1.00]; P = 0.05), better LFS (HR = 0.34 [95% CI: 0.15-0.78]; P = 0.01) and OS (HR = 0.30 [95% CI: 0.13-0.72]; P = 0.01). Our results suggested that adult T-ALL patients undergoing allo-HSCT might benefit from pediatric-type chemotherapy.

Modern treatment approaches to adult acute T-lymphoblastic and myeloid/T-lymphoblastic leukemia: from current standards to precision medicine.

To review the most recent advancements in the management of adult T-cell acute lymphoblastic leukemia (T-ALL), we summarize insights into molecular diagnostics, immunotherapy, targeted therapy and new techniques of drug sensitivity profiling that may support further therapeutic progress in T-ALL subsets. With current induction/consolidation chemotherapy and/or risk-oriented allogeneic stem cell transplantation programs up to 95% adult T-ALL patients achieve a remission and >50% (up to 80% in adolescents and young adults) are cured. The group of patients who fail upfront therapy, between 25% and 40%, is enriched in high-risk characteristics (unfavorable genetics, persistent minimal residual disease) and represents the ideal setting for the study of molecular mechanisms of disease resistance, and consequently explore novel ways of restoration of drug sensitivity and assess patient/subset-specific patterns of drug vulnerability to targeting agents, immunotherapy and cell therapy. The emerging evidence supports the contention that precision medicine may soon allow valuable therapeutic chances to adult patients with high-risk T-ALL. The ongoing challenge is to identify the best way to integrate all these new data into the therapeutic path of newly diagnosed patients, with a view to optimize the individual treatment plan and increase the cure rate.

Low Expression of CD5 and CD6 Is Associated with Poor Overall Survival for Patients with T-Cell Malignancies.

Background T-cell malignancies (TCMs), including T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoma (TCL), are highly aggressive and have a poor prognosis. To further understand prognostic stratifications and to design targeted therapies, this study aims to explore novel, potential biomarkers based on alterations in immune costimulatory molecules (CMs) for TCMs. Methods Peripheral blood from 25 de novo T-ALL patients in our clinical center and transcriptome data from 131 to 162 patients with peripheral TCL (PTCL) from the GSE19069 and GSE58445 dataset, respectively, were obtained to assess the expression levels of CMs and their prognostic significance. Results Seven CMs were associated with overall survival (OS). Among these CMs, CD5 and CD6 had the highest pairwise positive correlation (R = 0.69). CD5 and CD6 were significantly down-regulated in TCM patients compared with healthy individuals (HIs), and lower CD5 and CD6 expression was associated with poor OS for both T-ALL and TCL patients, particularly for patients greater than 60 years old. Furthermore, CD5 was positively correlated with CD6 in TCM patients. Compared with patients who were CD5highCD6high, T-ALL and TCL patients who were CD5lowCD6low had poor OS. Importantly, CD5highCD6high was an independent prognostic predictor for OS in T-ALL (HR = 0.39, 95% CI: 0.23–0.65, P < 0.001) and TCL (HR = 0.35, 95% CI: 0.19–0.62, P < 0.001) patients. Conclusions Low expression of CD5 and CD6 was associated with poor OS for TCM patients, and this may be a potential immune biomarker panel for prognostic stratification of TCM patients.

Effect of Netrin-1 on VEGFA Expression in T-ALL Cells and Its Related Mechanism

ELISA assays were applied to detect the levels of Netrin-1 and VEGFA in the bone marrow (BM) samples from children in the T-ALL and control group. The level of Netrin-1 and VEGFA were compared between control children and patients, and the liner correlation between Netrin-1 and VEGFA was analyzed. The T-ALL cells Jurkat and Molt-4 were culture in vitro, and the cells were treated with different concentration of Netrin-1 (0, 25, 50, 100 ng/ml) for 24 h, quantitative RT-PCR (qRT-PCR) and Western blot were used to detect the VEGFA expression in Jurkat, Molt-4 cells. The expression of Netrin-1 receptors in T-ALL cells was detected by qRT-PCR and the interaction between Netrin-1 and receptor in each cells was detected by co-IP. Furthermore, Western blot was used to detect the phosphorylation level of key prateins of AKT signal transduction pathway including Akt and mTOR in T-ALL cells treated with Netrin-1 (100 ng/ml). The expression of VEGFA and phosphorylation of AKT pathway transducers were detected by Western blot, after T-ALL cells treated with Netrin-1 (100 ng/ml) combined with inhibitors specific to Akt or mTOR. The expression level of Netrin-1 and VEGFA in T-ALL patients BM samples were both signi-ficantly higher than that of control group. And the expression level of Netrin-1 was positively correlated with that of VEGFA（r2=0974). With the increase of Netrin-1 concentration, the expression level of VEGFA also increased(P<0.05）. Netrin-1 interacted with its receptor, integrin-β4 at the Netrin-1 concentration of 100 ng/ml. Further, the treatment of Netrin-1 could increase the phosphorylation of Akt and mTOR, which were the key transducers of AKT pathway. After treatment of T-ALL cells with Netrin-1 (100 ng/mL) and Akt inhibitor, the expression of VEGFA and phosphorylation of Akt or mTOR decreased. When the cells were treated with Netrin-1(100 ng/ml) and mTOR inbititor, the phosphorylation level of mTOR and the expression of VEGFA decreased, the phosphorylation level of Akt increased. The expression of Netrin-1 and VEGFA in bone marrow of childred with T-ALL were abnormal, and there was a linear relationship between them. Netrin-1 can interact with its receptor, integrin-β4 and activate AKT transduction pathway to elevate the expression of VEGFA in T-ALL cells.

Long non-coding RNA signatures and related signaling pathway in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy caused by clonal proliferation of T-cell pre-cursors arising from the thymus. Although the optimized chemotherapy regimen could improve the outcome of such patients, some challenges such as higher risk for induction failure, early relapse and isolated central nervous system (CNS) relapse occurring in T-ALL patients are of great significance, leading to increased mortality rates. Long non-coding RNA (lncRNA) is a key component involved in cell signaling through a variety of mechanisms in regulating gene expression. Oncogenes and tumor suppressors are no exception and their expression can be affected by lncRNAs. In addition, accumulating researches in samples from T-ALL patients as well as pre-clinical studies in mice suggest that the expression profile of lncRNAs in T-ALL could be aberrant, resulting in deregulation of target genes and downstream signaling pathways. In addition, accumulating researches in samples from T-ALL patients as well as pre-clinical studies in mice suggest that the expression profile of lncRNAs in T-ALL could be aberrant, resulting in deregulation of target genes and downstream signaling pathways. These lncRNAs may be determinants of proliferation, apoptosis, and drug resistance observed in T-ALL. Thus, lncRNAs can be a good tool to develop novel strategies against cancer cells in the treatment of relapsed and refractory T-ALL. They can also act as promoting biomarkers in assessing T-ALL and differentiating between patients with poor prognosis and good prognosis.

Multiomics to investigate the mechanisms contributing to repression of PTPRC and SOCS2 in pediatric T-ALL: Focus on miR-363-3p and promoter methylation.

T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous and aggressive malignancy arising from T-cell precursors. MiRNAs are implicated in negative regulation of gene expression and when aberrantly expressed contribute to various cancer types, including T-ALL. Previously we demonstrated the oncogenic potential of miR-363-3p overexpression in a subgroup of T-ALL patients. Here, using combined proteomic and transcriptomic approaches, we show that miR-363-3p enhances cell growth of T-ALL in vitro via inhibition of PTPRC and SOCS2, which are implicated in repression of the JAK-STAT pathway. We propose that overexpression of miR-363-3p is a novel mechanism potentially contributing to overactivation of JAK-STAT pathway. Additionally, by combining the transcriptomic and methylation data of T-ALL patients, we show that promoter methylation may also contribute to downregulation of SOCS2 expression and thus potentially to JAK-STAT activation. In conclusion, we highlight aberrant miRNA expression and aberrant promoter methylation as mechanisms, alternative to mutations of JAK-STAT-related genes, which might lead to the upregulation of JAK-dependent signaling in T-ALL.

Characteristics and prognostic effects of NOTCH1/FBXW7 gene mutations in T-cell acute lymphoblastic leukemia patients

Objective: To explore the characteristics, clinical features and prognostic effects of NOTCH1/FBXW7 gene mutations in T-cell acute lymphoblastic leukemia (T-ALL) patients. Methods: The clinical data of 61 T-ALL patients who underwent second-generation gene sequencing in Henan Provincial People's Hospital from March 2016 to March 2021 were retrospectively analyzed. There were 46 males and 15 females, with a median age [M (Q1, Q3)] of 18 (11, 30) years. The relationship between NOTCH1/FBXW7 gene mutation characteristics, clinical and laboratory parameters and their impact on event free survival (EFS) and overall survival (OS) were analyzed. Results: NOTCH1 gene mutations were found in 34 cases (55.7%, 34/61), including 22 cases of heterodimer domain (HD) mutations (64.7%), 7 cases of proline/glutamate/serine/threonine (PEST) mutations (20.6%), and 5 cases of both HD and PEST mutations (14.7%). FBXW7 gene mutations were detected in 9 cases (14.8%, 9/61), of which 5 cases had both NOTCH1 and FBXW7 gene mutations. Twenty-three (37.7%, 23/61) cases were wild type. The median white blood cell count of patients in NOTCH1/FBXW7 gene mutations group and wild-type group was 76.4×109/L (8.3×109/L, 149.2×109/L), 54.1×109/L (5.3×109/L, 156.6×109/L), respectively. Moreover, the hemoglobin was (89.1±27.1) g/L and (99.5±23.1) g/L, respectively, and the median proportion of bone marrow primordial cells was 84.5% (69.0%, 91.3%) and 60.0%(35.0%, 80.0%), respectively. The gene expression rate of SIL-TAL1, Hox11 and Hox11L2 was 7.9% (3/38) vs 17.4% (4/23), 18.4% (7/38) vs 4.3% (1/23), 5.3% (2/38) vs 13.0% (3/23), respectively (all P>0.05). However, the median platelet level in the NOTCH1/FBXW7 gene mutations group was 60.5×109/L (36.8×109/L, 100.3×109/L), which was lower than that in the wild-type group [116.0×109/L (63.0×109/L, 178.0×109/L)] (P=0.018). The median number of gene mutations in the group with NOTCH1/FBXW7 gene mutations group was 2.5 (1.8, 4.0), which was more than that in the group without NOTCH1/FBXW7 gene mutations group [0 (0, 1.0)] (P<0.001). The median EFS and OS of adult NOTCH1/FBXW7 gene mutations group were 28.0 (95%CI: 7.3-48.7) months and 30.0 (95%CI: 8.9-51.1) months, respectively, which were better than those of adult wild-type group [4.5 (95%CI: 0-11.6) months and 9.0 (95%CI: 0-19.1) months] (P=0.008 and 0.014).The median EFS and OS of children NOTCH1/FBXW7 gene mutations group were 12.0 (95%CI: 10.4-13.6) months and 19.0 (95%CI: 13.6-24.4) months, respectively, and those of wild-type group were 10.0 (95%CI: 8.9-11.1) months and 21.0 (95%CI: 0-51.4) months, respectively (P=0.673 and 0.434). Conclusions: The mutation rate of NOTCH1/FBXW7 gene is higher in T-ALL patients. Patients with NOTCH1/FBXW7 gene mutations group have lower platelet count and better EFS and OS. NOTCH1/FBXW7 gene mutation may be used as a hierarchical basis for individualized treatment of adult T-ALL patients.