Early T-cell Precursor Acute Lymphoblastic Leukemia Patients Research Articles

Homoharringtonine Synergizes Strongly with Venetoclax Against the Early T-Cell Progenitor Acute Lymphoblastic Leukemia：from Bench to Bed

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinct subtype of T-ALL with poor outcome and novel therapies are urgently needed. Venetoclax (VEN), a BCL2 inhibitor, has been approved by FDA for AML. Combination of VEN and demethylating drugs or targeted therapies such as MCL1 inhibitor showed promising effect in AML. Loucy, an ETP-ALL cell line, was also reported high expression of BCL2. But little is known about VEN combination treatment in ETP-ALL. Our data showed that homoharringtonine (HHT), a plant alkaloid, downregulated MCL1 in T-ALL. Herein, we examined the treatment effect of VEN combined with HHT on ETP-ALL using murine model and patient samples. First, we compared BCL2 levels and treatment effect of VEN alone on ETP-ALL (i.e., Loucy) and T-ALL (i.e., Jurkat, Molt4, and CCRF-CEM) cell lines. We observed higher levels of BCL2 and lower IC50 values (IC50 at 24h: 0.093 uM vs.>1uM) in Loucy vs T-ALL cells. Similar results were found in human samples, with IC50 of 0.539uM in ETP-ALL sample vs 3.165-23.25uM in T-ALL samples treated with VEN for 24h. Next, to determine whether the combination of VEN and HHT shows a synergistic effect on ETP-ALL, Loucy cells were treated with a serial concentration of VEN (range: 0.025-0.1uM) and HHT (range:10-40ng/mL) alone and in combination for 24h. VEN and HHT combination showed significant synergism in reducing cell viability, with a mean combination index (CI) of < 0.1. We observed similar results in patient samples, with mean CI lower than 0.01 indicating highly synergistic effect. To assess this in vivo, a cohort of ETP-ALL mice generated by transplanting Loucy cells into NSG mice were randomly divided into 4 groups and treated with vehicle, HHT (1 mg/kg, ip), VEN (25 mg/kg, oral gavage) or HHT+VEN combination. HHT and VEN combination significantly prolonged survival of the ETP-ALL mice compared to either drug alone or vehicle (median survival of combination vs VEN vs HHT vs control: 113.5 vs 95.5 vs 85.5 vs 69.0 days, p<0.01), suggesting a synergistic effect of HHT and VEN in vivo. To identify the mechanical basis for the synergy of VEN and HHT in ETP-ALL, we performed single-cell RNA-seq using primary patient ETP-ALL cells treated with vehicle, HHT, VEN or their combination for 24h. Based on the gene expression profile, we identified a cluster of ETP-ALL cells sensitive to HHT , and a cluster only sensitive to combination of VEN and HHT which showed higher levels of Bcl2 and Mcl1 and upregulated anti-apoptosis signal by gene set enrichment analysis (GSEA), suggesting intra-tumoral heterogeneity of ETP-ALL blasts. These results suggest that HHT or VEN alone is not sufficient to induce apoptosis in the resistant cells and combination of HHT and VEN could induce apoptosis by reducing MCL1 expression in these cells which was confirmed by western blot. Further, with the promising results from bench work and given that both HHT and VEN are approved anti-leukemia drugs, we translated these findings into clinic. The HAG (HHT, low dose Cytarabine, G-CSF priming) regimen has been widely used in China for the treatment of AML for 15 years and proved to be effective and safer than intensive chemotherapy. Based on above findings, with informed consent from patients, we applied venetoclax plus HAG(V-HAG) regimen to 3 relapsed/refractory(R/R) ETP-ALL patients and 4 newly diagnosed (ND) patients, including an 82-year-old refractory patient. CR/CRi rates after the first cycle treatment were 100%, which was significantly higher than the current induction therapy (CR rate ranging from 33.3-55.9% after the first induction cycle). Except for the 82-year-old patient who refused post-remission treatment, all the remaining 6 patients remained in remission until today. Of note, no clinical manifestations of tumor lysis syndrome were observed. The median time to absolute neutrophil count recovery of >500/μL and >1000/μL was 13 (range: 0-22) and 16 (range: 0-24) days, and to platelet count recovery of > 20,000/ μL was 16 (range: 0-21) days, indicating this regimen was relatively safe and well tolerated. In conclusion, our findings demonstrate that HHT and VEN showed synergistic effect on ETP-ALL and HHT/VEN based therapy is a promising and safe regimen in newly diagnosed and R/R ETP-ALL patients. With these promising results from bench to bedside, 2 multi-center phase Ib/II clinical trials using V-HAG in patients with R/R ETP-ALL and ND-ETP have been initiated at our institution.

Integrated clinical genotype-phenotype characteristics of early T-cell precursor acute lymphoblastic leukemia.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinct subtype of T-ALL with a unique immunophenotype and high treatment failure rate. The molecular genetic abnormalities and their prognostic impact in ETP-ALL patients are poorly understood. The authors performed systematic analyses of the clinicopathologic features with an emphasis on molecular genetic aspects of 32 patients with ETP-ALL. The median age was 43years (range, 16-71). The blasts were positive for cytoplasmic CD3 and CD7 and negative for CD1a and CD8. Other markers expressed included CD34 (88%), CD33 (72%), CD117 (68%), CD13 (58%), CD5 (partial, 56%), CD2 (38%), CD10 (25%), CD56 (partial, 19%), and CD4 (6%). Cytogenetic analyses revealed a diploid karyotype in 10 patients, simple (1-2) abnormalities in 10 patients, and complex karyotype in 10 patients. Next-generation sequencing for 21 patients demonstrated that all had gene mutations (median, four mutations per patient). The most frequently mutated genes were WT1 (38%), NOTCH1 (29%), NRAS (29%), PHF6 (25%), TP53 (24%), ASXL1 (19%), FLT3 (19%), and IKZF1 (19%). All patients except one received multi-agent chemotherapy, and 22 patients underwent allogeneic stem cell transplantation. Thrombocytopenia, an abnormal karyotype, and TP53 mutation were associated with markedly shortened overall survival. Stem cell transplantation significantly improved overall survival. Patients with ETP-ALL often have high mutation burden with increased genomic instability. TP53 mutation was the only molecular prognostic marker and was associated with complex karyotype and greater than or equal to five mutations. These patients may benefit from stem cell transplantation, and recurrent gene mutations may be novel therapeutic markers.

Computational Biology Model of ETP-ALL Used to Accurately Classify ETP-ALL Vs Non ETP-ALL Patients with Genomic Input

Background: Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive hematological malignancy with poor prognosis after chemotherapy compared to other ALLs. ETP-ALL cases typically display a high burden of chromosomal copy number variations (CNVs) and myeloid gene mutations. To enhance the understanding of this disease, a computational biological modeling approach was developed to classify ETP-ALL vs. non-ETP-ALL patient based on genomic abnormalities and protein biomarkers associated with ETP-ALL and non ETP-ALL profiles.Methods: Genomic profiling data for whole exome sequencing (WES), array CGH (Agilent) and karyotype data of 69 ETP- ALL and Non-ETP-ALL patient were used for this study. The genomic data for each patient were entered into a computational biology model (CBM) (Cellworks Group), which generates a disease-specific protein network map using PubMed and other online resources. CBM was used for a) Classification between ETP-ALL and Non-ETP ALL model, b) Identification of unique patient characteristics and c) Drug response.For the CBM approach, we divided the data into training and validation data sets. 40 patients were selected for training data and 39 patients selected for the validation data set. In a training dataset, genomic data from 22 ETP-ALL patients were used from a GEO database (GSE28703) (N=11) and a prior genomics report (PMID:22237106) (N=11). The training dataset also included non-ETP-ALL patients from a GEO database (GSE28703) (N=18). Each patient's data was analyzed by CBM, to translates genomic variants into protein networks.To validate these findings, genomic data from 15 ETP-ALL patients, different than training set patients, were used from a GEO database (GSE28703) (N=1), Moffitt Cancer Center (IRB201600284) (N=6), a prior genomics report (PMID: 22237106) (N=7), and an ETP-ALL LOUCY cell line with known genomic aberrations. The validation dataset also included Non-ETP-ALL patients different than training set patients, from a GEO database (GSE28703) (N=14).Results: Prediction accuracy for classification between ETP-ALL and Non ETP ALL patient was 89.65%. PPV value was 86.67% and NPV value was observed 92.86%. Sensitivity and Specificity of prediction outcome was observed as 92.86% and 86.67% respectively.The simulation model identified six protein biomarkers which predicted increased activity in ETP-ALL profiles compared to non-ETP-ALL cases: STAT3, RUNX1, HIF1A, FOXM1, CEBPA, mTORC1.Key genomic differences were also identified between ETP-ALL and non ETP-ALL patients (Table1).Conclusions: ETP-ALL is distinct from other T-ALL with respect to genomic and protein network abnormalities. Computational biology model can accurately classify between ETP-ALL and Non ETP-ALL patients. CBM also predicted unique biomarkers between ETP-ALL and Non-ETP-ALL patients. [Display omitted] DisclosuresKumar:Cellworks: Employment. K:Cellworks: Employment. Vasista:Cellworks Research India: Employment. Tyagi:Cellworks: Employment. Rajagopalan:Cellworks: Employment. Alam:Cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Cogle:Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

Personalized Therapy Design for Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) Using Computational Biology Modeling with in Vitro Validation

ETP-ALL is a high-risk subtype of T-ALL with poor prognosis and high risk of relapse in patients treated with standard of care (SOC). ETP-ALL patients have more complex cytogenetic profiles and a unique immunophenotype compared to other T-ALL cases. ETP-ALL is characterized by the expression of myeloid and hematopoietic stem cell (HSC) surface proteins, as well as gene expression profiles similar to myeloid progenitors and HSCs, including frequent mutations in myeloid oncogenes. Targeted therapies for ETP-ALL are limited because identified gene mutations are not actionable by currently available drugs. Recently we developed a predictive simulation modeling method that projects cancer patient-specific protein network maps based on the patient's unique cancer genomics (PMID 27855285). Here, we present a computational biology approach to analyze the genomic profile of ETP-ALL and identify novel treatment strategies using available FDA approved drugs. In this study, we used the LOUCY cell line as a model of ETP-ALL due to its high expression of characteristic ETP-ALL markers HHEX, LYL1, MEF2C and ZEB2.Aim: To identify and validate novel drug or drug combinations for ETP-ALL using an integrated genomics and computational biology modeling (CBM) approach.Methods: The LOUCY cell line was used as a model of ETP-ALL. Publicly available genomic profiling data (cBioPortal, Sanger) were collected. All available genomic data for the LOUCY cell line were entered into a CBM program (Cellworks Group) that generates a disease-specific protein network map using PubMed and other online resources. Disease-specific biomarkers unique to the LOUCY cell line were identified within the protein network maps. A digital drug library of FDA approved therapies and drugs in clinical development was developed and digitally simulated in the computational model at varying doses. Drug impact was quantitatively assessed by a cell growth score, which is a composite of cell proliferation, viability, apoptosis, along with the impact on disease-specific biomarkers. This CBM's predictions for drug response were compared with publicly available in vitro experimental data. Novel drug options and combinations to treat ETP-ALL were also identified by CBM. The novel drugs and drug combinations identified by CBM were then tested on the LOUCY cells at varying concentrations to calculate IC50 values. Drug activity was measured by XTT assay, using the Jurkat cell line as a non-ETP-ALL control and the KG-1 AML cell line as a non-ALL control.Results: CBM predicted extensive dysregulation of multiple protein signaling pathways in the LOUCY cell line: Cell cycle upregulation was predicted due to copy number loss of CDKN1B, CDKN2B and CDKN2A genes. BCL2 expression was predicted to be increased due to the presence of a NUP214 - SET fusion protein, activating the BCL2 transcription factor HOXA9. Knockdown of PTEN and TSC2 was predicted to activate the AKT-mTOR pathway, while knockdown of ETV6 and SOCS1 was predicted to activate the JAK-STAT pathway (Fig 1). The response predictions of 31 drugs were compared with data from in vitro experiments. LOUCY cells were predicted to respond to 9 of the drugs, and in vitro testing showed sensitivity to 8 out of 9 drugs (88.8%). LOUCY cells were predicted to show no response to 22 of the drugs, and in vitro testing showed resistance to all 22 drugs (100%) (Table 1). Additionally, CBM predicted everolimus, nelfinavir, and palbociclib as novel therapies for the ETP-ALL representative LOUCY cell line, and were validated (100%) by additional in vitro XTT assays (Fig 2). Key statistical correlations for drug efficacy were observed (Table 2).Conclusions: Predictive simulation modeling of the unique genomic abnormalities found in the ETP-ALL LOUCY cell line led to the creation of an ETP-ALL protein network map that shows multiple interacting and cooperative protein network disturbances including cell cycle, anti-apoptosis, AKT-mTOR and JAK-STAT pathways. Digital drug screening using this distinctive protein network map led to the discovery of new drugs with predicted efficacy. In vitro studies validated the computational method with high accuracy. Results from this work inform future clinical trials for patients with ETP-ALL. This CBM technology may be a useful tool for clinicians to personalize treatment for patients with relapsed/refractory ETP-ALL. [Display omitted] DisclosuresKumar:Cellworks: Employment. K:Cellworks: Employment. Kamath:cellworks: Employment. Nair:cellworks: Employment. Raju:cellworks: Employment. Abbasi:Cellworks Group Inc.: Employment. Vali:Cellworks Group Inc.: Employment. Cogle:Celgene: Other: Membership on Steering Committee for Connect MDS/AML Registry.

Early T-cell precursor acute lymphoblastic leukemia and other subtypes: a retrospective case report from a single pediatric center in China.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is rare in China and case reports are varied. We conducted an in-depth analysis of newly diagnosed children with T-ALL from January 1999 to April 2015 in our center, to show the biological differences between Chinese ETP-ALL children and other immune types of T-ALL. The newly diagnosed children with T-ALL were divided into four groups according to their immunophenotype: ETP-ALL, early non-ETP-ALL, cortical T-ALL and medullary T-ALL. Disease-free survival (DFS), event-free survival (EFS), and overall survival (OS) rates were estimated by the Kaplan-Meier method. The Cox regression model was used for multivariate analysis. A total of 117 newly diagnosed children with T-ALL were enrolled in this study. The 10-year EFS and OS rates for all patients were 59.0 ± 4.7% and 61.0 ± 4.7%, respectively, with a median follow-up of 64 (5-167) months. Univariate analysis showed that ETP-ALL patients had the lowest 10-year DFS rate of 32.1 ± 11.7%, while cortical T-ALL had the highest DFS rate of 81.3 ± 8.5% compared with early non-ETP-ALL (61.6 ± 7.0%) and medullary T-ALL (59.1 ± 10.6%). Multivariate analysis demonstrated that only ETP-ALL and involvement of the central nervous system were independent prognostic factors. Compared with other subtypes, pediatric ETP-ALL had a poor treatment response and high recurrence rate while cortical T-ALL appeared to have much better outcome. Our observations highlight the need for an individualized treatment regime for ETP-ALL.

A clinical study of allogeneic hematopoietic stem cell transplantation in 23 patients with early T-cell precursor acute lymphoblastic leukemia

目的探讨异基因造血干细胞移植（allo-HSCT）治疗急性早前T淋巴细胞白血病（ETP-ALL）的疗效及预后。方法回顾性分析2010-2018年在苏州大学附属第一医院接受allo-HSCT的23例ETP-ALL患者的疗效、相关并发症及预后。ETP-ALL根据流式细胞术免疫表型检测结果诊断。结果23例患者男18例，女5例，中位年龄26（12~47）岁，7例接受同胞全相合移植，4例接受无关供者全相合移植，12例接受单倍型移植，移植前19例患者为完全缓解（CR）状态，4例处于未缓解（NR）状态。ETP-ALL的临床特征：23例患者中，5例初诊时WBC>100×109/L，14例合并脾大，19例合并外周淋巴结肿大，5例合并胸腺受累，11例伴髓系突变，7例具有预后不良核型。所有患者确诊后接受诱导化疗，其中14例在初次化疗后即获得CR，5例经多次化疗后获得CR，4例患者在多次化疗后仍未获得CR。移植结果：22例患者完全植入，其中中性粒细胞和血小板植入中位时间分别为12（9~14）d及19（10~85）d，1例患者于+14 d死于移植相关性感染。中位随访11（1~33）个月，移植后18个月总生存（OS）率为（55.0±14.4）%，无复发生存（RFS）率为（48.1±14.7）%，移植相关死亡率为4.3%。移植前处于CR状态的患者中位OS时间为20个月，NR状态下的患者中位OS时间仅为13个月。19例移植前获得CR的患者中，单倍型移植和同胞全相合移植的OS及RFS差异无统计学意义（P=0.460，P=0.420）。结论allo-HSCT可以改善部分ETP-ALL患者的预后，挽救性移植的ETP-ALL患者仍然预后不佳，单倍型移植以及同胞全相合移植治疗ETP-ALL的疗效相仿。

The Functional Role of PRC2 in Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) - Mechanisms and Opportunities.

Early T-Cell precursor acute lymphoblastic leukemia (ETP-ALL) is a relatively newly identified subset of T-lineage ALL. There are conflicting results regarding prognosis, and the genetic basis of this condition is variable. Here, we summarize the current status of the field and discuss the role of mutations in the Polycomb Repressive Complex 2 frequently identified in ETP-ALL patients.

Co-Expression of NUP98-HOXD13 and Mutant IDH2 Triggers an Early T-Cell Precursor-like Leukemia in Mice

Background and hypothesis: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy, comprising of B lineage ALL and T-cell lineage ALL (about 85% and 15%, respectively). In most cases, treatment regimens for ALL patients have proved efficient and prognosis is usually encouraging. However, a recently described subgroup of T-cell ALL patients have been found to have a poor prognosis when treated with conventional therapy. These higher risk T-cell leukemias have a cell of origin thought to resemble an early T-cell precursor (ETP), which retains the capability to differentiate into both myeloid or T-lineage cells, and are therefore termed ETP ALL. ETP ALL is characterized by lack of or low expression of typical T-cell surface markers, aberrant expression of myeloid and hematopoietic stem cell (HSC) surface markers and a gene expression signature resembling that of murine ETP cells. Recently, ETP ALL patients were found to have a unique mutational landscape that includes acquired mutations in genes typically associated with myeloid differentiation. The ambiguous lineage attributes of ETP ALL may explain the ineffectiveness of current therapies, stressing the need for establishing pre-clinical animal models. Here we show that co-expression of Nup98-Hoxd13 (NHD13) fusion gene and a mutant IDH2 gene (IDH2 R140Q) triggers an ETP-like leukemia in mice. Expression of NHD13 has been reported in MDS and AML patients, and NHD13 mice have been previously shown to develop MDS, AML, and T-cell ALL. Mutations in IDH1/2 genes occur in several cancers, including AML; the most common IDH mutation in AML patients is the IDH2 R140Q. However, expression of mutant IDH1/2 in mice has been reported to induce an expansion of the HSC compartment but was not sufficient for establishment of leukemia. Targeted sequencing of NHD13 mice has identified recurrent mutations in IDH1, suggesting that these two aberrations may collaborate to cause leukemia.Study Design and Methods: IDH2 R140Q mice were crossed with NHD13 mice and the progeny were monitored for survival. Classification of leukemic samples was done using flow cytometry and PCR analysis of TCRβ gene rearrangements. Global expression and mutational patterns of IDH2/NHD13 leukemias were analyzed using gene chip arrays and whole exome sequencing (WES).Results and conclusions: Transgenic IDH2/NHD13 mice showed decreased survival compared to all control groups. Detailed analysis of surface marker expression revealed that most double Tg cases (10/11 tested) developed leukemias resembling ETP (cKit+CD44+CD25-) or double negative (DN) 2 cells (cKit+CD44+CD25+). PCR analysis of the TCRβ locus revealed that unlike NHD13 T-cell leukemias, IDH2/NHD13 leukemias do not have clonal VDJ rearrangements. However, most of IDH2/NHD13 leukemias have clonal or oligoclonal DJ rearrangements of the TCRβ locus, suggesting that these leukemias emerged during an early stage in T cell development. Expression analysis revealed that the IDH2/NHD13 leukemia expression signature is enriched in genes that are upregulated in ETP or DN2 cells. In addition, we found that the IDH2/NHD13 leukemia expression signature is also enriched in genes uniquely expressed in ETP ALL patients. Finally, WES showed correspondence between genes mutated in the mouse ETP-like leukemia and genes recurrently mutated in ETP ALL patients. These results indicate that IDH2/NHD13 leukemias resemble ETP ALL in terms of gene expression, immunophenotype, and cooperative mutations.Relevance and importance: The poor prognosis of ETP ALL may be related to incomplete effectiveness of treatment strategies, emphasizing the need for novel therapies. IDH2/NHD13 mice can serve as an excellent pre-clinical model to study ETP ALL and to test potential treatments. DisclosuresAplan:NIH Office of Technology Transfer: Patents & Royalties.

Outcome of Early T-Cell Precursor Acute Lymphoblastic Leukemia in AIEOP Patients Treated with the AIEOP-BFM ALL 2000 Study

Aim: To evaluate the outcome of the Italian Association of Pediatric Hematology and Oncology (AIEOP) patients diagnosed with Early T-cell Precursor (ETP) Acute Lymphoblastic Leukemia (ALL) in the period September 2000-December 2011 and treated in the context of the AIEOP-BFM ALL 2000 Study and the subsequent - very similar – AIEOP ALL R2006 Study.Patients and methods: ETP-ALL was defined by staining negative for CD1a and CD8, mildly positive or negative for CD5 and positive for at least one of CD34, CD117, HLADR, CD13, CD33, CD11b, or CD65 antigens. Treatment consisted of BFM protocol I either with dexamethasone or prednisone in phase IA, 4 HDMTX courses (5 g/sqm over 24 hrs) in non-high risk patients or 3 poly-chemotherapy blocks in high risk patients, delayed intensification based on protocol(s) II or III, protracted intrathecal therapy or cranial radiotherapy, maintenance therapy for a total of 2 years of treatment. Hematopoietic stem cell transplantation (HSCT) was indicated for patients with poor treatment response assessed either morphologically at day +8 (Prednisone Poor Response, PPR) or day +33 (no Complete Remission, CR) or by PCR at day +78 (high-level Minimal Residual Disease, HR-MRD; ≥10-3).Results: Of the 439 T-ALL eligible patients, 34 had ETP ALL. The incidence (7.7%) may be underestimated since the full set of the data needed was not available for all patients. Out of the 34 patients with ETP ALL 14 were at high risk due to PPR; of them, 3 were HR-MRD and 5 did not achieve CR after Phase IA of induction therapy (including one with HR-MRD). Of the remaining 20 patients (all prednisone good responders), 3 patients were at high risk due to resistance to Phase IA and HR-MRD (n=1) or HR-MRD only (n=2). 17/30 patients could not be monitored by MRD due to death in Induction (n=1), or absent (n=10) or inadequate PCR markers (n=6). Of the17 patients monitored by MRD, 13 had HR-MRD at day 33 and 6 of them also at day +78. One patient died during induction therapy. The remaining 33 achieved morphological CR: 27 after phase IA and 6 (those resistant to phase IA) after phase IB of protocol I. Of 12 patients who underwent HSCT, 3 died of HSCT-related complications and 3 relapsed. With a median follow-up of 6.6 years, the 5-year event-free survival (EFS) and Survival in the 34 ETP ALL patients were of 60.9%(SE 8.5) and 66.6%(8.3), versus 71.2%(2.3) and 77.1%(2.1) respectively in the non-ETP patients. The overall cumulative incidence of relapse was 27.3%(7.8) and 22.2%(2.1) in ETP and non-ETP T-ALL patients, respectively (p=0.52). EFS in ETP vs non-ETP patients was respectively 81.9%(9.5) vs 83.8%(2.4) in non high risk patients (p=0.87) and 41.2%(11.9) vs 53.2%(4.0) in high risk patients (p=0.24).Conclusions: The outcome of T-ALL patients treated with BFM-type therapy is comparable in ETP and non-ETP for those with good response to initial chemotherapy; it was slightly, but not significantly, inferior in ETP-ALL patients with poor initial response. Phase IB treatment element is very effective in ETP-ALL, suggesting that intensification with antimetabolite and alkylating agents may be beneficial, while the benefit of HSCT in first CR needs to be further investigated. DisclosuresNo relevant conflicts of interest to declare.

Abstract 997: Targeting the Jak/Stat signaling pathway is highly effective in xenograft models of early T cell precursor (ETP) acute lymphoblastic leukemia (ALL)

Abstract ETP ALL is a recently described subtype of T-ALL with poor prognosis and is defined by a unique immunophenotype, expressing T-lineage and myeloid/early progenitor markers. ETP ALL blasts have a diverse array of genetic alterations without a unifying mutated gene or pathway. A fraction have mutations in genes involved in Jak/Stat signaling, and we hypothesized targeting Jak/Stat may be effective for some ETP ALL patients. We evaluated Jak/Stat signaling by phosphoflow cytometry and immunoblotting of blasts collected from patients with ETP ALL with and without Jak/Stat pathway mutations and compared them to non-ETP T-ALL blasts. We xenografted blasts from 6 different ETP ALL patient samples with heterogeneous mutations (Table) into immunodeficient NSG mice and tested the in vivo efficacy of the Jak1/2 inhibitor ruxolitinib (rux). Mice were randomized to rux or vehicle after they developed &amp;gt;1% peripheral blood (pb) blasts (cytoCD3+/CD45+) by flow cytometry (FACS). Disease burden was assessed by FACS enumeration of pb blasts weekly and splenic blasts at sacrifice. We found that the Jak/Stat signaling pathway was dysregulated in ETP ALL samples, including in samples without identifiable mutations in Jak/Stat. ETP ALL blasts had marked increased expression of pSTAT1 (p = 0.01), pSTAT3 (p = 0.01) and pSTAT5 (p = 0.02) when compared to non ETP T-ALL by immunoblot, results confirmed by phosphoflow. All 6 ETP samples had a profound in vivo response to rux; circulating and splenic blasts were markedly reduced in all treated animals compared to controls (Table). The degree of response was comparable to that seen with TKIs in BCR-ABL ALL, using the same xenograft model. These results strongly suggest that ETP ALL blasts may be addicted to Jak/Stat signaling, irrespective of the presence of identifiable Jak/Stat mutations and that clinical trials testing Jak/Stat inhibitors are needed. Ruxolitinib is active in ETP ALLGenetic alterationAvg splenic blasts in millions. Control vs Rux Treated (p value by 2-tailed t-test)DNM2 m, JAK3 m157 vs 5 (p &amp;lt;0.01)PTPN11 SV124 vs 31 (p &amp;lt;0.01)GATA3 del, JAK1m, SH2B3 SV192 vs 18 (p &amp;lt;0.01)EZH2 m, RUNX1 m, NOTCH1 m, IKZF1m, SUZ12 m, SH2B3 SV452 vs 25 (p &amp;lt;0.01)EED m256 vs 1 (p &amp;lt;0.01)GATA3 m, JAK1 m, PTEN del, RB1 del, CDKN2A/B del332 vs 4 (p &amp;lt;0.01)m = mutation; del = deletion; SV = structural variationPK obtained from mice demonstrated serum levels that correlate with humans treated with drug and that would be expected to target JAK1 and JAK2 but not JAK3. Citation Format: Shannon L. Maude, Sibasish Dolai, Cristina Delagdo-Martin, Tiffaney Vincent, Alissa Robbins, Arthavan Selvanathan, Theresa Ryan, Stephen P. Hunger, Mignon L. Loh, Charles G. Mullighan, Brent L. Wood, Michelle L. Hermiston, Stephan A. Grupp, Richard B. Lock, David T. Teachey. Targeting the Jak/Stat signaling pathway is highly effective in xenograft models of early T cell precursor (ETP) acute lymphoblastic leukemia (ALL). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 997. doi:10.1158/1538-7445.AM2014-997

Immunophenotypic analysis of T‐acute lymphoblastic leukemia. A CD5‐based ETP‐ALL perspective of non‐ETP T‐ALL

T-cell antigens [CD5,CD1a,CD8] define early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). To understand immature T-ALL of which ETP-ALL is part, we used these antigens to subcategorize non-ETP T-ALL for examining expression of myeloid/stem cell antigens (M/S) and clinical features. Using CD5 (+/-) to start categorization, we studied 69 routinely immunophenotyped patients with T-ALL. CD5(-) was a homogenous (CD8,CD1a)(-) M/S(+) ETP-ALL group (n=9). CD5(+) cases were (CD8,CD1a)(-) pre-T-ALL (n=22) or (CD8,CD1a)(+) (n=38) thymic/cortical T-ALL; M/S(+) 20/22 (90.91%) in former and 22/38 (57.89%) in latter (P=0.007). ETP- and pre-T-ALL together (CD1a(-) ,CD5(-/+) immature T-ALL group) were nearly always M/S(+) (29/31; 93.55%). In multivariate analysis, only ETP-ALL predicted poor overall survival (P=0.02). We conclude (i) CD5 negativity in T-ALL almost always means ETP-ALL. CD1a and CD8 negativity, as much as CD5, marks immaturity in T-ALL, and the CD5(+/-) /CD1a(-) /CD8(-) immature T-ALL group needs further study to understand the biology of the T-ALL-myeloid interface. (ii) ETP-ALL patients may be pre-T-ALL if CD2(+) ; CD2(+) , conversely, CD5(-) /CD1a(-) /CD8(-) pre-T ALL patients are ETP-ALL. (iii) Immunophenotypic workup of T-ALL must not omit CD1a, CD5, CD8 and CD2, and positivity of antigens should preferably be defined as recommended for ETP-ALL, so that this entity can be better evaluated in future studies of immature T-ALL, a group to which ETP-ALL belongs. (iv) ETP-ALL has poor prognosis.

Mixed-phenotypic acute leukemia: cytochemically myeloid and phenotypically early T-cell precursor acute lymphoblastic leukemia.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is characterized by the specific immunophenotype of CD1a-, CD8-, and CD5weak/- with stem cell or myeloid marker expression [1]. This leukemia is associated with increased genomic instability, a high remission failure frequency, and hematological relapse. ETP-ALL patients also represent a distinct molecular T-ALL subgroup with a low frequency of NOTCH1 mutations and a high frequency of FLT3 mutations. ETP-ALL with FLT3 mutation has also been described as a new ETP-ALL subgroup with unique immunophenotypic features and high levels of CD2, myeloid antigen CD13, and CD117 expression, indicating a stem cell phenotype [2]. Mixed-phenotypic acute leukemia (MPAL), T/Myeloid subtype is rarely suspected from morphology, except in a small subset of cases, which have a distinct dual-blast population with both lymphoid and myeloid features. Therefore, final diagnoses of MPAL are based on flow cytometric immunophenotyping. Common cytogenetic abnormalities associated with MPAL include t(9;22) and MLL rearrangements, which may be restricted to B-Myeloid cases, whereas T-myeloid cases exhibit generally non-recurring chromosomal abnormalities [3]. Herein, we report a case of MPAL (T/Myeloid) with trisomy 4 and t(6;14)(q27:q22) in a 25-year-old man, the diagnosis of which was directed by the morphological presence of Auer rods and myeloperoxidase (MPO) staining, despite exhibiting a flow cytometric ETP-ALL phenotype.

FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.