Pediatric T-cell Research Articles

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

Phospho-Proteomic Profiling of T-Cell Acute Lymphoblastic Leukemia Identifies Targetable Kinase Activities and Novel Treatment Combination Strategies

Introduction Intensive multi-agent treatment has boosted survival up to 80% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Nevertheless, relapsed patients have a poor prognosis due to acquired therapy resistance while most survivors have detrimental chemotherapy-induced side effects. Therefore, novel targeted therapies are urgently needed since further intensification of the current standard treatment regimen is not feasible for refractory/relapsed cases. Protein kinase inhibitors (PKIs) are amongst the most successful cancer treatments. Targetable kinases activated by gene fusions are rare in T-ALL and include subclonal NUP214-ABL1 fusion in 6% of cases or other rare clonal ABL1 fusions. Nevertheless, leukemic blasts rely on enhanced kinase signaling to sustain their dysregulated proliferation. Protein kinases can be hyper-activated even in the absence of defects in their genes. Thus, together with the identification of genomic aberrations, phospho-proteomics can provide information on pathway activation, signaling networks and aberrant kinase activities that offer important opportunities for targeted therapies. Aim Here, we aimed to identify and quantify kinase activation in T-ALL cell lines that may yield differential sensitivity to PKIs in vitro. This approach could pinpoint targetable leukemia vulnerabilities and provide effective (combination) treatment strategies. Methods Protein extracts from 11 T-ALL cell lines were enriched for phospho-peptides by titanium dioxide enrichment and anti-phospho tyrosine immunoprecipitation followed by liquid chromatography - tandem mass spectrometry (MS). Subsequently, the Integrative Inferred Kinase Activity (INKA) pipeline was used to rank activated kinases in our panel (Beekhof et al., 2019). Based on these data, selected kinase inhibitors were tested in vitro as single treatment or in combinations. Eventually, drugs of interest were further tested ex vivo in a cohort of T-ALL patient-derived xenografts (PDXs). Results MS-based phospho-proteome profiling of 11 T-ALL cell lines identified about 3700 tyrosine phospho-sites and more than 13300 serine/threonine phosphorylation sites. We found SRC-family kinases including LCK, SRC, FYN, and YES1 as most activated kinases in many T-ALL cell lines while ABL1, ZAP70, LYN, and FGR were detected only in specific lines. Additionally, other kinases including CDK1/2 and PAK1/2 were found to be activated in all the cell lines while activation of the INSR/IGF-1R axis was detected only in a subset of lines. We then tested cellular response to multiple clinically relevant PKIs based on predicted kinase activities. The in vitro drug screening showed an effective response and G1-arrest following treatment with the CDK1/2 inhibitor milciclib in all the cell lines tested, with IC50 values between 10nM and 1uM. Despite a general SRC-family kinases activation profile, dual SRC/ABL inhibitors like dasatinib reduced cellular viability only in cell lines with ABL1 fusions or LCK translocations (IC50 < 10nM) while other lines lacking ABL or LCK rearrangements were affected at much higher drug concentration (IC50 > 3uM, which is beyond the clinical achievable plasma concentration). Thus, PKIs were tested in combination with other relevant inhibitors based on additional kinase activities detected. Interestingly, the concomitant inhibition of the SRC-family kinases by dasatinib and the INSR/IGF-1R axis by BMS-754807 led to a drastic reduction of cell viability at nanomolar concentrations even in cell lines that did not respond to dasatinib, identifying a novel possible effective combination strategy for T-ALL. Eventually, we tested clinically relevant PKIs in 50 PDXs ex vivo and identified various T-ALL samples with a high sensitivity to dasatinib single treatment (IC50 < 100nM) as previously reported by others (Frismantas et al., 2017). Moreover, 70% of our PDXs efficiently responded to the broad spectrum kinase inhibitor midostaurin (IC50 < 1uM), highlighting the importance of targeting multiple signaling nodes simultaneously to tackle T-ALL vulnerabilities. Conclusions Ranking kinase activities and signaling networks from phospho-proteomic data can guide the use of PKIs as treatment option for T-ALL patients. Moreover, kinase activity profiling can provide insights for efficient treatment combination strategies to develop personalized medicine. Disclosures No relevant conflicts of interest to declare.

Differences in Influenza-Specific CD4 T-Cell Mediated Immunity Following Acute Infection Versus Inactivated Vaccination in Children.

Early childhood influenza infections imprint influenza-specific immune memory, with most studies evaluating antibody specificity. In this study, we examined how infection versus inactivated influenza vaccination (IIV) establish pediatric CD4 T-cell mediated immunity to influenza and whether this poises the immune system to respond differently to IIV the following year. We tracked influenza-specific CD4 T-cell responses in 16 H3N2 infected and 28 IIV immunized children following both initial exposure and after cohorts were revaccinated with IIV the following fall. PBMCs were stimulated with peptide pools encompassing the translated regions of the H3 HA and NP proteins and were then stained to assess CD4 T-cell specificity and function. Compared to IIV, infection primed a greater magnitude CD4 T-cell response specific for the infecting HA and NP proteins, with more robust NP-specific immunity persisting through year 2. Post infection, CD4 T cells preferentially produced combinations of cytokines that included interferon-γ. Interestingly, age-specific patterns in CD4 T-cell reactivity demonstrated the impact of multiple influenza exposures over time. These data indicate that infection and vaccination differentially prime influenza-specific CD4 T-cell responses in early childhood, with these differences contributing to the lasting immunologic imprinting established following early influenza infection. NCT02559505.

Identification of prognostic factors in childhood T-cell acute lymphoblastic leukemia: Results from DFCI ALL Consortium Protocols 05-001 and 11-001.

While outcomes for pediatric T-cell acute lymphoblastic leukemia (T-ALL) are favorable, there are few widely accepted prognostic factors, limiting the ability to risk stratify therapy. Dana-Farber Cancer Institute (DFCI) Protocols 05-001 and 11-001 enrolled pediatric patients with newly diagnosed B- or T-ALL from 2005 to 2011 and from 2012 to 2015, respectively. Protocol therapy was nearly identical for patients with T-ALL (N=123), who were all initially assigned to the high-risk arm. End-induction minimal residual disease (MRD) was assessed by reverse transcription polymerase chain reaction (RT-PCR) or next-generation sequencing (NGS), but was not used to modify postinduction therapy. Early T-cell precursor (ETP) status was determined by flow cytometry. Cases with sufficient diagnostic DNA were retrospectively evaluated by targeted NGS of known genetic drivers of T-ALL, including Notch, PI3K, and Ras pathway genes. The 5-year event-free survival (EFS) and overall survival (OS) for patients with T-ALL was 81% (95% CI, 73-87%) and 90% (95% CI, 83-94%), respectively. ETP phenotype was associated with failure to achieve complete remission, but not with inferior OS. Low end-induction MRD (<10-4 ) was associated with superior disease-free survival (DFS). Pathogenic mutations of the PI3K pathway were mutually exclusive of ETP phenotype and were associated with inferior 5-year DFS and OS. Together, our findings demonstrate that ETP phenotype, end-induction MRD, and PI3K pathway mutation status are prognostically relevant in pediatric T-ALL and should be considered for risk classification in future trials. DFCI Protocols 05-001 and 11-001 are registered at www.clinicaltrials.gov as NCT00165087 and NCT01574274, respectively.

LncRNA CDKN2B-AS1 contributes to tumorigenesis and chemoresistance in pediatric T-cell acute lymphoblastic leukemia through miR-335-3p/TRAF5 axis.

T-cell acute lymphoblastic leukemia (T-ALL) is the most prevalent malignancy in children. Long non-coding RNAs are being found to have relevance to the pathogenesis of pediatric T-ALL. However, the function of cyclin-dependent kinase inhibitor 2B anti-sense RNA 1 (CDKN2B-AS1) in pediatric T-ALL progression and chemoresistance has not been illuminated. The levels of CDKN2B-AS1, miR-335-3p and tumor necrosis factor receptor-associated factor 5 (TRAF5) were assessed by quantitative real-time PCR. Cell proliferation and the 50% inhibitory concentration (IC50) value were detected using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetr-azolium assay. Cell cycle and apoptosis were evaluated by flow cytometry. Western blot analysis was performed to measure protein expression. Targeted interactions among CDKN2B-AS1, miR-335-3p and TRAF5 were determined by the dual-luciferase reporter and RNA immunoprecipitation assays. Animal studies were conducted to observe the function of CDKN2B-AS1 in vivo. Our data indicated that CDKN2B-AS1 was highly expressed in pediatric T-ALL peripheral blood mononuclear cells and cells, and high CDKN2B-AS1 level was associated with adriamycin (ADR) resistance. CDKN2B-AS1 depletion hindered T-ALL/ADR cell proliferation and cell cycle progression, and promoted cell apoptosis and ADR sensitivity in vitro. Moreover, CDKN2B-AS1 knockdown repressed tumor growth and enhanced ADR sensitivity in vivo. CDKN2B-AS1 sequestered miR-335-3p, and CDKN2B-AS1 depletion exerted regulatory effect in T-ALL/ADR cell progression by up-regulating miR-335-3p. TRAF5 was a direct target of miR-335-3p, and TRAF5 mediated the regulation of miR-335-3p in T-ALL cell behaviors. Furthermore, CDKN2B-AS1 positively modulated TRAF5 expression through sponging miR-335-3p. The current work suggested that CDKN2B-AS1 knockdown repressed the progression and enhanced ADR sensitivity of pediatric T-ALL at least partly through targeting miR-335-3p/TRAF5 axis, highlighting a promising therapeutic target for the treatment of pediatric T-ALL patients.

Long non‑coding RNA AWPPH interacts with ROCK2 and regulates the proliferation and apoptosis of cancer cells in pediatric T‑cell acute lymphoblastic leukemia

The long non-coding (lnc)RNA associated with poor prognosis of hepatocellular carcinoma (AWPPH) serves as an oncogene in several cancers, such as liver and bladder cancers, however, to the best of our knowledge, its function in T-cell acute lymphoblastic leukemia is unknown. The results of the present study revealed that the expression levels of lncRNA AWPPH and Rho-associated protein kinase 2 (ROCK2) were upregulated in the bone marrow of patients with pediatric T-cell acute lymphoblastic leukemia compared with healthy controls. Expression levels of lncRNA AWPPH and ROCK2 were positively correlated with each other. lncRNA AWPPH and ROCK2 overexpression promoted the proliferation and inhibited the apoptosis of Loucy cells, an acute lymphoblastic leukemia cell line. Overexpression of lncRNA AWPPH resulted in upregulation of ROCK2 expression in Loucy cells. Similarly, ROCK2 overexpression also resulted in upregulation of lncRNA AWPPH in Loucy cells, suggesting an element of reciprocity in the function of lncRNA AWPPH and ROCK2. It was concluded that lncRNA AWPPH promoted the proliferation and inhibited the apoptosis of cancer cells in pediatric T-cell acute lymphoblastic leukemia possibly through interactions with ROCK2.

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t(9;17)染色体異常を有した小児Tリンパ芽球性リンパ腫の1例

Advantages and Limitations of SNP Array in the Molecular Characterization of Pediatric T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous disease, and numerous genetic aberrations in the leukemic genome are responsible for the biological and clinical differences among particular ALL subtypes. However, there is limited knowledge regarding the association of whole-genome copy number abnormalities (CNAs) in childhood T-ALL with the course of leukemia and its outcome. The aim of this study was to identify the pattern of whole-genome CNAs in 86 newly diagnosed childhood T-ALL cases using a high-density single-nucleotide polymorphism array. We analyzed the presence of whole-genome CNAs with respect to immunophenotype, clinical features, and treatment outcomes. A total of 769 CNAs, including trisomies, duplications, deletions, and segmental loss of heterozygosity, were detected in 86 analyzed samples. Gain or loss of chromosomal regions exceeding 10 Mb occurred in 46 cases (53%), including six cases (7%) with complex chromosomal alterations. We observed that microdeletions in selected genes (e.g., FIP1L1 and PDGFRB) were related to the clinical features. Interestingly, 13% of samples have a duplication of the two loci (MYB and AIH1—6q23.3), which never occurred alone. Single-nucleotide polymorphism array significantly improved the molecular characterization of pediatric T-ALL. Further studies with larger cohorts of patients may contribute to the selection of prognostic CNAs in this group of patients.

Abstract B47: Overexpression of TLX3 or HOXA9 in association mutant IL7Rα are sufficient to generate T-ALL in vivo

Abstract Background: Mutations of the IL7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Rα is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Rα alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present, contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Rα plus TLX3 or HOXA9 overexpression indicate in vitro grow advantage, suggesting these two genes as potential collaborative candidates. Furthermore, patients with mutated IL7Rα were more likely to have TLX3 or HOXA subgroup leukemia. Objective: We sought to determine whether combination of mutant hIL7Rα plus TLX3 or HOXA9 overexpression is sufficient to generate T-cell leukemia in vivo. Methods: Double-negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7Rα plus TLX3 or HOXA9. The same combinations were tested for the hIL7Rα wild-type. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 7-13 days and accessed for driver oncogenes expression and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Results: Thymocytes transduced with muthIL7Rα-TLX3 showed overexpression of c-Myc and Pim-1. Nonetheless, there was no difference in the protein level expression for these two proteins in either thymocytes transduced with muthIL-7Rα-HOXA9 or in the control wthIL7Rα-TLX3. Mice injected with either muthIL7Ra-TLX3 or muthIL7Rα-HOXA9 cells, but not the controls (wthIL7Rα-TLX3 or wthIL7Rα-HOXA9), developed leukemia approximately 14 days post injection, characterized by GFP-expressing T-cells in blood, spleen, liver, lymph nodes, and bone marrow. Conclusion: Thymocytes transduced with muthIL7Rα-TLX3 showed c-Myc and Pim-1 upregulation. Cells expressing the combination muthIL7Rα-TLX3 or muthIL7Rα-HOXA9 were sufficient to trigger T-cell leukemia in vivo. Citation Format: Gisele O.L. Rodrigues, Julie Hixon, Hila Winer, Wenqing Li, Scott Durum. Overexpression of TLX3 or HOXA9 in association mutant IL7Rα are sufficient to generate T-ALL in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B47.

Subcutaneous panniculitis-like T-Cell lymphoma in a child presenting as an abdominal wall mass

Abstract Childhood panniculitides are not common. Histopathological diagnostic approach is vital to differentiate this group of pathologies that commonly present with inflammation of the subcutaneous adipose tissue. Pediatric subcutaneous panniculitis-like T-cell lymphomas are rare. Treatment modalities include observation, surgical excision, radiotherapy, immunosuppression, and chemotherapy. The case of an 18-month female who presented with a flank mass that was diagnosed as a subcutaneous panniculitis-like T-cell lymphoma and successfully managed with intensity-reduced, dual agent immunosuppression is described.

COTI-2 induces cell apoptosis in pediatric acute lymphoblastic leukemia via upregulation of miR-203

ABSTRACT COTI-2 is a third-generation thiosemicarbazone, which is effective against a diverse group of human cancer cell lines at nanomolar concentrations. COTI-2 also showed superior activity against tumor cells, in vitro and in vivo. As a high efficacy and low toxicity agent, it currently candidates in a phase I clinical study of gynecological malignancies and head and neck squamous cell carcinoma (HNSCC). However, its effect in pediatric T-cell acute lymphoblastic leukemia (T-ALL) is not clear. This study investigates the effect of COTI-2 on T-ALL Jurkat cells in vitro and in vivo. Jurkat cells were exposure to COTI-2 at different concentration and time. Cell apoptosis was detected by flow cytometry to examine the sensitivity of Jurkat cell lines treated with either COTI-2 alone or in combination with MiR-203 mimic or inhibitor in vitro. An orthotopic mouse model was used to examine the sensitivity of Jurkat cells treated with COTI-2 in vivo. Western blotting and RT-qPCR were performed to dissect molecular mechanisms. The results showed that COTI-2 promotes apoptosis of Jurkat cells in dose-and time-dependent way. Enforced expression of miR-203 promotes COTI-2-mediated cell apoptosis, whereas miR-203 silencing attenuates COTI-2-mediated cell apoptosis in Jurkat cells in vitro. COTI-2 is also effective against growth of Jurkat cells in vivo. Mechanistically, COTI-2 induced miR-203 upregulation and inhibited caspase-3/9 activaty leading to inhibition of cell apoptosis. Taken together, COTI-2 inhibits tumor growth in vitro and in vivo in Jurkat cells likely through miR-203-dependent mechanisms. COTI-2 may be a potential approach for T-ALL treatment.

PTEN and AKT1 Variations in Childhood T-Cell Acute Lymphoblastic Leukemia

Objective:PTEN/AKT pathway deregulations have been reported to be associated with treatment response in acute leukemia. This study examined pediatric T-cell acute lymphoblastic leukemia (T-ALL) samples for PTEN and AKT1 gene variations and evaluated the clinical findings.Materials and Methods:Fifty diagnostic bone marrow samples of childhood T-ALL cases were investigated for the hotspot regions of the PTEN and AKT1 genes by targeted next-generation sequencing.Results:A total of five PTEN variations were found in three of the 50 T-ALL cases (6%). Three of the PTEN variations were first reported in this study. Furthermore, one patient clearly had two different mutant clones for PTEN. Two intronic single-nucleotide variations were found in AKT1 and none of the patients carried pathogenic AKT1 variations.Conclusion:Targeted deep sequencing allowed us to detect both low-level variations and clonal diversity. Low-level PTEN/AKT1 variation frequency makes it harder to investigate the clinical associations of the variants. On the other hand, characterization of the PTEN/AKT signaling members is important for improving case-specific therapeutic strategies.

Phospho-Proteomic Profiling of T-Cell Acute Lymphoblastic Leukemia Identifies Specific Kinase Activation Signatures That Can Predict Response to Targeted Therapy

Introduction In the last decade, intensive multi-agent combination treatment has boosted survival and cure to approximately 80% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients. Nevertheless, patients that relapse have a very poor prognosis due to acquired therapy resistance. The current highly intensive chemotherapy is accompanied by severe toxicity, resulting in either death or frequent detrimental long-term side effects for survivors, with a relevant impairment of their quality of life. Therefore, there is an urgent need for new targeted therapies as well as reliable biomarkers that can predict treatment outcome and therapy resistance. Protein kinase inhibitors are amongst the most successful small-molecule drugs used for cancer treatment. However, for T-ALL patients the use of protein kinase inhibitors is still limited to clinical trials. Furthermore, targetable kinases activated by gene fusions are rare in T-ALL in contrast to other types of leukemia, and are often present at the subclonal level (e.g. episomal amplification of the NUP214-ABL1 fusion in 6% of patients at diagnosis). Nevertheless, leukemic blasts rely on enhanced kinase signaling to sustain dysregulated proliferation. Therefore, protein kinases can be activated even in the absence of genetic mutations in their coding sequences. In addition to screening for disease-associated genomic alterations in patient biopsies, profiling of aberrant protein kinase activity may offer opportunities for targeted therapeutic approaches. Aim High-throughput phospho-proteomics can provide direct information on pathway activation, kinase signaling networks and may therefore be used to identify novel therapy targets. Here, we aimed to identify kinase activation patterns in established T-ALL cell lines and to test their differential sensitivity to targeted kinase inhibitors in order to develop a strategy to predict leukemia dependencies. Methods We performed unbiased, mass spectrometry-based phospho-proteomic profiling of 11 established T-ALL cell lines. By combining titanium dioxide-based enrichment with selective phospho-tyrosine immunoprecipitation, we identified about 3700 tyrosine phospho-sites and more than 13300 serine/threonine phosphorylation sites. Multiple approaches have been developed to infer kinase activity from phospho-proteomic data. We applied the Inferred Kinase Activity (INKA) scoring to rank kinase activation in our cell lines. This ranking is not only based on the phosphorylation of kinases but also integrates information on phosphorylated substrates (Beekhof et al, Mol Syst Biol. 2019). Results We found SRC-family members as most activated kinases in T-ALL cell lines, with a major role for LCK, SRC, FYN, and YES1. Certain cell lines also revealed high activity of ABL1, ZAP70, LYN, and FGR. Additionally, other active kinases identified include INSR, CLK1, CDK1/2/7 and PAK1/2. To test the dependency of the cell lines to the predicted SRC-family kinases activation, cellular response levels were measured towards dasatinib, a SRC/ABL multi-kinase inhibitor. Surprisingly, only the cell lines with known genetic kinases aberrations-e.g. HSB-2 (TCRB-LCK translocated) and ALL-SIL (NUP214-ABL1 rearranged)-showed strong sensitivity to dasatinib. The remaining 9 lines were resistant to dasatinib treatment and seemed not to be solely dependent on the predicted kinase activities for their survival, indicating the need for therapeutic combinations that target additional parallel kinase activities. To unravel actual kinase dependencies rather than kinase activities, analysis of the overall phosphorylation profiles yielded specific 'phospho'-signatures that associate with dasatinib responsiveness. Therefore, individual T-ALL cell lines can be used as calibrator to predict and quantify signaling dependencies that associate with response to targeted kinase inhibitors in primary T-ALL biopsies. Conclusion The identification of oncogenic dependencies by ranking kinases activities and their signaling networks from phospho-proteomic profiling data can guide the assignment of T-ALL patients to specific kinase inhibitors treatment. Furthermore, these phospho-signatures may provide important drug response biomarkers as well as explain possible compensatory mechanisms for therapy resistance. Disclosures No relevant conflicts of interest to declare.

Novel KMT2A Partner Gene NUTM2A Revealed By Anchored Multiplex PCR in ALL

Objectives Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric and adult acute leukemias. Such rearrangements are more frequent in infants, in whom they are found in 60% - 80% of acute lymphoblastic leukemias (ALL). KMT2A-r group itself is genetically heterogeneous. KMT2A-r can occur with at least 94 partner genes previously described in «THE MLL RECOMBINOME &lt;…&gt; IN 2017». Detection of KMT2A-r is an essential part of AL initial diagnostics. Also, the accurate detection of all KMT2A-r types is crucial in order to perform minimal residual disease (MRD) monitoring, as it is clear now that MRD-based therapy adjustment has a very strong impact on outcome. Here we report a case of novel KMT2A partner in pediatric ALL - NUT family member 2A (NUTM2A). Methods The patient is 8 y.o. girl with T-cell acute lymphoblastic leukemia with pleural effusion. Bone marrow and pleural fluid aspirates were analyzed by G-banded karyotyping and FISH with KMT2A breakapart probe. Pleural fluid aspirates were also analyzed by real-time RT-PCR for 8 most common KMT2A rearrangements screening, long-distance inversed PCR and targeted RNA-seq with FusionPlex Myeloid kit (ArcherDX, CO, USA). Sanger sequencing was used for validation. Results Conventional cytogenetics and FISH showed 47,XX,t(10;11)(q22;q23),+mar[5] karyotype with 100% KMT2A-rearranged nuclei. Gene fusion analysis identified novel fusion KMT2A-NUTM2A with exon 11 - exon 1 breakpoint junction. NUTM2A is a gene at 10q23.2. This gene is not fully described in the literature. Rearrangements of this gene were identified in endometrial stromal sarcomas (ESS) (Cheng-Han Lee et al. 2012) and small round cell sarcoma (SRCS) (Sugita et al. 2017). In case of ESS, this rearrangement results in an in-frame fusion between YWHAE and NUTM2A (or highly homologous gene NUTM2B), and in case of SRCS it results in an in-frame fusion between NUTM2A and CIC. To our knowledge, KMT2A-NUTM2A fusion in our study is the first case demonstrating NUTM2A rearrangement in hematological malignancies. Conclusions Here we for the first time show the novel KMT2A-NUTM2A fusion transcript, which was found in pediatric T-cell acute lymphoblastic leukemia. Anchored multiplex PCR is one of the most sensitive way to detect rare variants of KMT2A rearrangements. It also allows selecting patient-specific primers for further PCR detection of MRD. Disclosures No relevant conflicts of interest to declare.

Next-generation sequencing of PTEN mutations for monitoring minimal residual disease in T-cell acute lymphoblastic leukemia.

Minimal residual disease (MRD) analysis has become a powerful indicator to refine therapy in acute lymphoblastic leukemia (ALL). Here, we present an MRD detection based on the next-generation sequencing of PTEN exon 7 mutations (NGS-PTEN) in 30 pediatric T-cell ALL patients. By comparing the NGS-PTEN results with current quantitative PCR of antigen receptor gene rearrangements (qPCR-Ig/TR), an overall concordance of 80% was found between the two methods. However, the NGS-PTEN qualified a lower number of high-risk patients than qPCR-Ig/TR. These findings suggest that NGS-PTEN is a promising tool that could potentially be used to support current MRD methodologies for T-ALL patients.

Clinical and biological features of PTPN2-deleted adult and pediatric T-cell acute lymphoblastic leukemia

AbstractProtein tyrosine phosphatase nonreceptor type 2 (PTPN2) is a phosphatase known to be a tumor suppressor gene in T-cell acute lymphoblastic leukemia (T-ALL). Because the full clinicobiologic characteristics of PTPN2 loss remain poorly reported, we aimed to provide a comprehensive analysis of PTPN2 deletions within a cohort of 430 patients, including 216 adults and 214 children treated according to the GRAALL03/05 (#NCT00222027 and #NCT00327678) and the FRALLE2000 protocols, respectively. We used multiplex ligation-dependent probe amplification to identify an 8% incidence of PTPN2 deletion, which was comparable in adult (9%) and pediatric (6%) populations. PTPN2 deletions were significantly associated with an αβ lineage and TLX1 deregulation. Analysis of the mutational genotype of adult T-ALL revealed a positive correlation between PTPN2 deletions and gain-of-function alterations in the IL7R/JAK-STAT signaling pathway as well as PHF6 and WT1 mutations. Of note, PTPN2 and PTEN (phosphatase and tensin homolog) deletions were mutually exclusive. Regarding treatment response, PTPN2-deleted T-ALLs were associated with a higher glucocorticoid response and a trend for improved survival in children, but not in adults, with a 5-year cumulative incidence of relapse of 8% for PTPN2-deleted pediatric cases vs 26% (P = .177).

Abstract 5233: MEF2C dysregulation is associated with immature T cell immunophenotype, absence of biallelic deletion of TCR-gamma and inferior survival

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy affecting both children &amp; adults. Unlike B-ALL, the prognostic markers are not clearly defined in T-ALL. In this study, we evaluated the correlation of MEF2C gene expression in T-ALL with immunophenotype &amp; absence of biallelic deletion of TCR-gamma (ABD) &amp; tested its potential significance as prognostic marker. A total of 106 T-ALL patients, including 75 children &amp; 31 adults (males 93; females 13) were included. Immunophenotyping was done in all cases at diagnosis on bone marrow/peripheral blood samples using CD3, CD7, CD4, CD2, CD45, CD5, CD8, CD1a, CD13, CD33, CD117, HLA-DR, CD34, CD65 &amp; CD11b antibodies. The patients were categorized into immature (pre &amp; pro T-ALL; n=48), cortical (n=47) &amp; mature (n=11) T-ALL based on immunophenotypic features. MEF2C gene expression was quantified by RQ-PCR. ABD was done in 90 patients in which DNA was available by quantitative DNA PCR (Q-PCR) for TCR-gamma rearrangements using the protocol described before1. Minimal residual disease in bone marrow samples at the end of induction therapy was also measured. Kruskal-Wallis test was performed to determine the correlation of MEF2C expression with immunophenotypes &amp; TCR-gamma chain status. Kaplan Meier survival analysis was done to evaluate the significance of MEF2C dysregulation on event free survival &amp; overall survival. We found MEF2C gene to be significantly overexpressed (p=0.013) in patients with immature T-ALL as compared to cortical &amp; mature T-ALL cases. It was significantly overexpressed in early T-cell precursor (ETP-ALL) cases (n=15) versus non ETP-ALL (p=0.03). By qPCR, 29 of 90 patients (32.22%) were classified as ABD, 44 (48.89%) as non-ABD &amp; 17 (18.89%) as indeterminate. MEF2C expression was higher in ABD patients (p=0.011). Minimal residual disease was found more frequently in MEF2C dysregulated patients (p=0.033). On Kaplan Meier survival analysis, event free survival (EFS) was not significantly different between two groups (p=0.33). Overall survival (OS) was worse in MEF2C dysregulated patients as compared to others (p=0.017). ETP-immunophenotype was associated with poorer EFS (p=0.013). ABD did not correlate with EFS &amp; OS. To conclude, MEF2C dysregulation was associated with immature T-immunophenotype, ABD &amp; inferior survival at diagnosis. MEF2C gene expression can be used as potential candidate to risk stratify T-ALL cases. Reference 1. Gutierrez A, Dahlberg SE et al. Absence of biallelic TCRgamma deletion predicts early treatment failure in pediatric T-cell acute lymphoblastic leukemia. J Clin Oncol. 2010;28:3816-23. Citation Format: Anita Chopra, Jay Singh, Deepak Verma, Rajive Kumar, Sameer Bakhshi, Jayanth Kumar, Rachna Seth, Atul Sharma. MEF2C dysregulation is associated with immature T cell immunophenotype, absence of biallelic deletion of TCR-gamma and inferior survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5233.

Abstract 1314: MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia

Abstract Background: Early T-precursor acute lymphoblastic leukemias (ETP-ALL) account for 15% of pediatric T-cell ALL (T-ALL) cases and are characterized by an immature phenotype, resistance to therapy, and high relapse rates. MERTK receptor tyrosine kinase is ectopically expressed in ~50% of T-ALLs, particularly those with an immature T cell phenotype, suggesting a role in ETP-ALL. The anti-apoptotic protein B-cell lymphoma-2 (BCL-2) is specifically expressed in immature T cell precursors, is preferentially expressed in ETP-ALL compared to other T-ALLs, is essential for ETP-ALL cell survival, and is regulated downstream of MERTK in acute leukemia cells. Thus, combination therapies targeting these two proteins may be particularly effective to treat ETP-ALL. Methods: MERTK and BCL-2 mRNA expression was assessed in T-ALL patient samples using publicly available data. Loucy and PEER ETP-ALL cell lines were cultured with vehicle or MRX-2843, a dual MERTK/FLT3 inhibitor, alone or in combination with the BCL-2 inhibitor venetoclax. Phosphorylated and total MERTK were assessed by immunoblot. Cells were stained with PoPro-1-iodide and propidium iodide dyes and analyzed by flow cytometry to assess cell death. Relative cell numbers were assessed using Presto Blue reagent. Orthotopic xenografts were established in NSG or NSGS mice using luciferase-expressing Loucy cells or an ETP-ALL patient sample and leukemia burden was monitored by bioluminescence imaging or flow cytometry. MRX-2843 or saline vehicle were administered orally once daily. Median survival was determined by Kaplan-Meier analysis. Results: MERTK and BCL-2 mRNAs were expressed at significantly higher levels in ETP-ALL patient samples relative to other T-ALLs. MRX-2843 mediated a dose-dependent decrease in phosphorylated MERTK and induced dose-dependent cell death (43.2% vs 16% in vehicle-treated cultures, p&amp;lt;0.01) in PEER cells. Moreover, in a patient-derived ETP-ALL xenograft model, treatment with MRX-2843 reduced peripheral blood disease burden by 83% (p&amp;lt;0.001) and decreased spleen weight by 64% (p&amp;lt;0.001) compared to vehicle-treated mice. Furthermore, treatment with MRX-2843 prolonged median survival by 11 days compared to control mice (n=8/group, p=0.0016). In a cell line-derived xenograft model, MRX-2843 reduced disease burden by 60% (n=10/group, p&amp;lt;0.0001) compared to vehicle. Treatment with a combination of MRX-2843 and venetoclax decreased cell density more effectively than either single agent in cultures of the Loucy and PEER cell lines (p&amp;lt;0.05). Conclusions: MERTK and BCL-2 are preferentially expressed in ETP-ALL relative to T-ALL and MRX-2843 has robust therapeutic activity in cell culture and xenograft models of ETP-ALL. These data validate MRX-2843 as a novel agent with potential for clinical application in patients with ETP-ALL. Combined targeting of MERTK and BCL-2 may be particularly effective. Citation Format: Ryan J. Summers, Katherine A. Minson, Eleana Vasileiadi, Xiaodong Wang, Steven V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1314.

Gene expression signature associated with in vitro dexamethasone resistance and post-induction minimal residual disease in pediatric T-cell acute lymphoblastic leukemia.

10033 Background: T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogeneous disease, which has largely precluded the use of genetic mutations for risk stratification. We hypothesized that despite this heterogeneity, diverse T-ALLs may have functional similarities that underlie patterns of chemotherapy sensitivity. Methods: We used flow cytometry to evaluate in vitro dexamethasone (DEX) sensitivity and baseline expression of signal transduction effectors and BCL2-family proteins in 68 fresh diagnostic T-ALL samples from patients enrolled on the Children’s Oncology Group (COG) trial AALL1231. We also performed RNA-sequencing (RNA-seq) on 40 AALL1231 samples and used hierarchical clustering and linear regression to analyze these and published T-ALL RNA-seq data from COG AALL0434. Comparisons between groups were made using t-tests and Fisher’s exact tests. Results: Of the proteins analyzed, only high BCL2 expression was significantly associated with increased in vitro DEX resistance (p = 0.002). Hierarchical clustering of the AALL1231 RNA-seq data identified two distinct clusters. Cluster 1 was associated with significantly higher BCL2 transcript expression (p = 0.0002) and in vitro DEX resistance (p = 0.04) relative to cluster 2. We defined a gene set consisting of the top 210 differentially expressed genes between these clusters and applied this gene set to the COG AALL0434 cohort. In this analysis, the early T-cell precursor (ETP) and near-ETP samples clustered together (p &lt; 0.0001) in cluster 1 along with 39 of 146 non-ETP samples. Not only did these cluster 1 non-ETP samples have significantly higher BCL2 transcript expression relative to the non-ETP samples in cluster 2 (p &lt; 0.0001), but 54% of these non-ETP samples were minimal residual disease (MRD) positive (≥0.01%) at the end of induction, as opposed to only 16% of the non-ETP samples in cluster 2 (p &lt; 0.0001). Conclusions: Gene expression profiling identifies non-ETP T-ALLs that cluster with ETP/near-ETP T-ALLs and have significantly higher BCL2 expression and increased rates of post-induction MRD.

The Profile of Immunophenotype and Genotype Aberrations in Subsets of Pediatric T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a biologically heterogeneous malignancy, which reflects distinctive stages of T-cell differentiation arrest. We have revisited a cohort of pediatric T-ALL, in order to test if immunophenotypes associated with molecular alterations would predict the patient's outcome. Genetic mutations, translocations and copy number alterations were identified through Sanger sequencing, RT-PCR, FISH and multiplex ligation-dependent probe amplification (MLPA). We defined 8 immunophenotypic T-ALL subtypes through multiparametric flow cytometry: early T-cell precursor (ETP, n = 27), immature (n = 38), early cortical (n = 15), cortical (n = 50), late cortical (n = 53), CD4/CD8 double negative mature (n = 31), double positive mature (n = 35) and simple positive mature (n = 31) T-ALL. Deletions (del) or amplifications (amp) in at least one gene were observed in 87% of cases. The most frequent gene alterations were CDKN2A/Bdel (71.4%), NOTCH1mut (47.6%) and FBXW7mut (17%). ETP-ALL had frequent FLT3mut (22.2%) and SUZ12del (16.7%) (p < 0.001), while CDKN2A/Bdel were rarely found in this subtype (p < 0.001). The early cortical T-ALL subtype had high frequencies of NOTCH1mut and IL7Rmut (71%, 28.6%, respectively), whereas, mature T-ALL with double positive CD4/CD8 had the highest frequencies of STIL-TAL1 (36.7%), LEF1del (27.3%) and CASP8AP2del (22.7%). The co-existence of two groups of T-ALL with NOTCH1mut/IL7Rmut, and with TLX3/SUZ12del/NF1del/IL7Rmut, were characterized with statistical significance (p < 0.05) but only STIL-TAL1 (pOS 47.5%) and NOTCH1WT/FBXW7WT (pOS 55.3%) are predictors of poor T-ALL outcomes. In conclusion, we have observed that 8 T-ALL subgroups are characterized by distinct molecular profiles. The mutations in NOTCH1/FBXW7 and STIL-TAL1 rearrangement had a prognostic impact, independent of immunophenotype.