Thymic T-ALL Research Articles

Molecular subgroups of T-cell acute lymphoblastic leukemia in adults treated according to pediatric-based GMALL protocols

In contrast to B-cell precursor acute lymphoblastic leukemia (ALL), molecular subgroups are less well defined in T-lineage ALL. Comprehensive studies on molecular T-ALL subgroups have been predominantly performed in pediatric ALL patients. Currently, molecular characteristics are rarely considered for risk stratification. Herein, we present a homogenously treated cohort of 230 adult T-ALL patients characterized on transcriptome, and partly on DNA methylation and gene mutation level in correlation with clinical outcome. We identified nine molecular subgroups based on aberrant oncogene expression correlating to four distinct DNA methylation patterns. The subgroup distribution differed from reported pediatric T-ALL cohorts with higher frequencies of prognostic unfavorable subgroups like HOXA or LYL1/LMO2. A small subset (3%) of HOXA adult T-ALL patients revealed restricted expression of posterior HOX genes with aberrant activation of lncRNA HOTTIP. With respect to outcome, TLX1 (n = 44) and NKX2-1 (n = 4) had an exceptionally favorable 3-year overall survival (3y-OS) of 94%. Within thymic T-ALL, the non TLX1 patients had an inferior but still good prognosis. To our knowledge this is the largest cohort of adult T-ALL patients characterized by transcriptome sequencing with meaningful clinical follow-up. Risk classification based on molecular subgroups might emerge and contribute to improvements in outcome.

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

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

Development of constitutively synergistic nanoformulations to enhance chemosensitivity in T-cell leukemia

Advances in multiagent chemotherapy have led to recent improvements in survival for patients with acute lymphoblastic leukemia (ALL); however, a significant fraction do not respond to frontline chemotherapy or later relapse with recurrent disease, after which long-term survival rates remain low. To develop new, effective treatment options for these patients, we conducted a series of high-throughput combination drug screens to identify chemotherapies that synergize in a lineage-specific manner with MRX-2843, a small molecule dual MERTK and FLT3 kinase inhibitor currently in clinical testing for treatment of relapsed/refractory leukemias and solid tumors. Using experimental and computational approaches, we found that MRX-2843 synergized strongly—and in a ratio-dependent manner—with vincristine to inhibit both B-ALL and T-ALL cell line expansion. Based on these findings, we developed multiagent lipid nanoparticle formulations of these drugs that not only delivered defined drug ratios intracellularly in T-ALL, but also improved anti-leukemia activity following drug encapsulation. Synergistic and additive interactions were recapitulated in primary T-ALL patient samples treated with MRX-2843 and vincristine nanoparticle formulations, suggesting their clinical relevance. Moreover, the nanoparticle formulations reduced disease burden and prolonged survival in an orthotopic murine xenograft model of early thymic precursor T-ALL (ETP-ALL), with both agents contributing to therapeutic activity in a dose-dependent manner. In contrast, nanoparticles containing MRX-2843 alone were ineffective in this model. Thus, MRX-2843 increased the sensitivity of ETP-ALL cells to vincristine in vivo. In this context, the additive particles, containing a higher dose of MRX-2843, provided more effective disease control than the synergistic particles. In contrast, particles containing an even higher, antagonistic ratio of MRX-2843 and vincristine were less effective. Thus, both the drug dose and the ratio-dependent interaction between MRX-2843 and vincristine significantly impacted therapeutic activity in vivo. Together, these findings present a systematic approach to high-throughput combination drug screening and multiagent drug delivery that maximizes the therapeutic potential of combined MRX-2843 and vincristine in T-ALL and describe a novel translational agent that could be used to enhance therapeutic responses to vincristine in patients with T-ALL. This broadly generalizable approach could also be applied to develop other constitutively synergistic combination products for the treatment of cancer and other diseases.

Results of the Risk-Adapted, MRD-Stratified GMALL Trial 08/2013 in 281 T-ALL / T-Lbl Patients: Excellent Outcome of Standard Risk Thymic T-ALL

Results of the Risk-Adapted, MRD-Stratified GMALL Trial 08/2013 in 281 T-ALL / T-Lbl Patients: Excellent Outcome of Standard Risk Thymic T-ALL

Multi-Omics of Adult T Cell Acute Lymphoblastic Leukemia

Introduction:Risk stratification and minimal residual disease (MRD) monitoring improved outcome for adult T-ALL patients, but a relevant percentage of adult patients, particularly high-risk subtypes, still relapse with then very limited therapeutic options. For improved individualized treatment strategies, better understanding of molecular inter-leukemic heterogeneity would be important. Increasing amounts of unbiased next generation sequencing study data are available for pediatric T-ALL patients, but comprehensive data for adult T-ALL patients are scarce.Patients and methods:Eighty-four adult T-ALL patients (age: median 32, range 17-59 years) with different immunophenotypes were studied: 19 early, 14 mature and 51 thymic T-ALL. For all samples, deep targeted DNA sequencing with a gene panel consisting of 206 genes was performed (HighSeq 1500, 100 bp, average ~800 reads/bp). We sequenced mRNA with ~30 million reads/sample (HighSeq 2000, 125 bp). DNA methylation status of all samples were addressed by Infinium 450k methylation bead arrays.Results:Unsupervised clustering of 600 most variant expressed genes in our cohort identified five stable clusters with aberrant expression of oncogenes: TLX1 - (n=21), TLX3 - (n=10), HOXA - (n=12), TAL / LMO - (n=21) deregulated subgroups and cases with an immature gene expression signature (n=16). While the TLX1 - (thymic), the TAL / LMO - (thymic/mature), and the immature (early/mature) clusters are highly correlated with immunophenotype, HOX-A- and TLX3-cases occurred in any immunophenotypically defined subgroup. Different gene fusions were found in respective subgroups: TAL1 / 2 (n=7, fusion partners (FP): STIL1, TRDC, TCF7), LMO1/2 (n=2, FP: CAPRIN, RIC3), TXL1 (n=7, FP: TCRA, TCRB), and in the HOXA-cluster SET-NUP214-fusions (n=7). Differential expression (DE) analysis revealed an enrichment of stem cell genes like MEF2C, MN1, IGFBP7, FLT3 in the immature subgroup (2879 DE genes, DEG). TLX1 (2198 DEGs) and LMO/TAL (2165 DEGs) clusters were associated with mature T cell development stage (FDR<0.0001). Interestingly, HOXA-cluster (422 DEGs) showed a marked enrichment for ribosomal proteins (FDR<.00001).On genomic level, we found a slightly lower rate of mutations in the HOXA-cluster (median mutations/patient: TAL/LMO 8, TLX1 9, TLX3 8.5, HOXA 6, immature 9). Most frequently mutated genes included NOTCH1 (51%), PHF6 (32%), DNM2 (18%), PTEN (18%), FBXW7 (16%), and JAK3 (14%). Noteworthy, multiple NOTCH1 mutations were present in many patients and frequently on a subclonal level pointing to a late event in leukemogenesis. Mutations of epigenetic modifiers including KMT2D, DNMT3A, SUZ12, and EP300 were enriched in the TLX3 and immature cluster. JAK/STAT pathway was affected in all subgroups with frequently two hits in individual patients, e.g. 6/7 (86%) JAK1 mutations co-occurred with JAK3 mutations.On level of epigenetics, principal component analysis of the most variable CG-sites revealed five distinct subgroups. These are highly concordant with the subgroups based on gene expression profiling, with the exception of the HOXA-cluster. The increase of genomic altered epigenetic modifiers in TLX3 and immature cluster is reflected by a common DNA methylation signature (cluster M1).Conclusion:We characterized molecular defined subgroups of adult T-ALL on a genomic, transcriptional and epigenetic level finding high concordance for all subgroups across the platforms. We identified subgroup specific mutations and fusion genes for adult T-ALL patients reflecting inter-leukemic heterogeneity with association of molecularly defined subgroups to enriched pathways. These results might build the basis for the identification of patients eligible for targeted therapy like JAK/STAT inhibitors or epigenetic modulators. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular Subgroups of T Cell Acute Lymphoblastic Leukemia in Adults Treated According to GMALL Protocols

Molecular Subgroups of T Cell Acute Lymphoblastic Leukemia in Adults Treated According to GMALL Protocols

Post-Remission Treatment with Chemotherapy or Allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT) in Adult Patients with High-Risk (HR) Philadelphia Chromosome-Negative (Ph-neg) Acute Lymphoblastic Leukemia (ALL) According to Their Minimal Residual Disease (MRD). Final Results of the Pethema ALL-HR-11 Trial

Introduction: Recent studies have shown that young to middle-aged adults who receive a pediatric-inspired chemotherapy regimen for treatment of Ph-neg ALL do not appear to require an alloHSCT if they achieve good response on MRD testing after induction and/or consolidation therapy. Patients (pts) who are not good MRD responders achieve better outcomes with alloHSCT than their counterparts who do not receive alloHSCT. However, it is not clear if this approach can specifically apply to adult ALL pts with HR features at baseline. The aim of the prospective ALL-HR-11 trial (NCT01540812) from the Spanish PETHEMA Group was to evaluate response of HR Ph-neg adult ALL patients to a different post-induction therapy (chemotherapy or alloHSCT) according to MRD levels (centrally assessed by 8-color flow cytometry [FCM]) at the end of induction (week 5) and consolidation therapy (week 17).. Patients and methods: HR ALL included one or more of the following parameters at baseline: age 30-60y, WBC count &amp;gt;30x109/L for B-cell precursor ALL or &amp;gt;100x109/L for thymic T-ALL, pro-B, early or mature T-ALL, 11q23 or KMT2A rearrangements or complex karyotype. Induction therapy included vincristine, prednisone, daunorubicin and asparaginase (E coli native or PEG according to center availability) for 4 weeks (Induction-1). FLAG-Ida was administered as intensified induction (Induction-2) in pts not achieving CR or in those in CR with MRD≥0.1% at the end of induction. For pts in CR and MRD&amp;lt;0.1% early consolidation therapy included 3 cycles with rotating cytotoxic drugs with high-dose methotrexate, high-dose ARA-C and high-dose asparaginase (E coli native or PEG). These pts continued with delayed consolidation (identical to that of early consolidation) followed by maintenance therapy up to 2y in CR if MRD levels after consolidation were &amp;lt;0.01%; otherwise they were assigned to alloHSCT. Pts in CR after Induction-2 received one consolidation cycle and were assigned to alloHSCT. Main outcome measures were: complete response (CR), overall survival (OS) and cumulative incidence of relapse (CIR), assessed by competing risk analysis. Results: On April 2019, 307 HR ALL pts were evaluable. Median (range) age was 40 (15-60) y, 192 were males, 211 precursor B-ALL and 96 T-ALL, with a median WBC count of 12.9 (0.2-564) x109/L. Results of Induction-1 (n=304, 3 on induction): therapy-related death: 12(4%), resistance: 39(13%), CR: 253(83%). MRD&amp;lt;0.1% at the end of induction was observed in 77% of CR patients. Induction-2 was administered to 88 patients (due to no CR: 37, or to CR and MRD≥0.1%: 51). Overall response rate: 277 (91%). The 5y CIR and OS probabilities for the whole series were of 44%±8% and 48%±7% (median follow-up: 2.06y [range: 0-7.55y]). By intention-to treat after Induction-1, 94 pts were assigned to alloHSCT and 190 to delayed consolidation and maintenance. The 5y CIR and OS probabilities were of 37%±13% and 38%±11%, respectively, for pts assigned to alloHSCT, and of 48%±10% and 55%±10%, respectively, for those assigned to chemotherapy (P&amp;lt;0.001 for OS [Figure 1], and P=0.243 for CIR). Patients with MRD&amp;lt;0.1% at the end of induction and MRD&amp;lt;0.01% at the end of consolidation (n=137) showed a 5y CIR and OS of 42%±11% and 66%±11%, respectively (P&amp;lt;0.001 for both, Figure 2). Patients with MRD levels &amp;lt;0.01% on day14 of induction-1, end-induction-1 and end-consolidation (n=17) showed 5y CIR and OS probabilities of 17%±19% and 90%±19%, respectively. Conclusions: This trial, in which post-induction therapy was only based on MRD results assessed by FCM, suggests that avoiding alloHSCT does not hamper the outcome of HR Ph-neg adult ALL pts with adequate MRD response after induction and after consolidation. Better post-remission alternative therapies are specially needed for patients with poor MRD clearance. Supported by grants PI14/01971 FIS, Instituto Carlos III, and SGR225 (GRE), Spain. Disclosures Montesinos: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Teva: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Research support, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: Research support; Pfizer: Membership on an entity's Board of Directors or advisory committees, Other: Research support, Research Funding, Speakers Bureau. Esteve:Novartis: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy; Pfizer: Consultancy; Astellas: Consultancy, Speakers Bureau; Amgen: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy.

Comparison of Efficacy and Safety of Two Types of E.coli Asparaginase (Native or Pegylated) for Treatment of Adult Patients with High-Risk (HR), Philadelphia (Ph) Chromosome-Negative ALL Included in the Prospective MRD-Oriented Protocol ALL-HR-11 from the Spanish Pethema Group

Background and objective. Asparaginase (ASP) is an essential drug for ALL treatment. Two types of E.coli ASP (native and pegylated) are used in clinical trials for treatment of children and adults with ALL, but to our knowledge direct comparison between these two types of ASP in the same protocol has not been performed. This study aimed to compare the efficacy and safety of native vs. PEG-ASP in adult patients with HR, Ph-negative ALL.Patients and methods. HR ALL included one or more of the following parameters at baseline: age 30-60 yr., WBC count >30x109/L for B-cell precursor ALL or >100x109/L for thymic T-ALL, pro-B, early or mature T-ALL, 11q23 or MLL rearrangements or complex karyotype. Induction therapy consisted of vincristine, prednisone, daunorubicin and ASP (native 10,000 IU/m2, i.v., days 16-20 and 23-27 or PEG 2,000 IU/m2, iv, day 15 depending on center decision) for 4 weeks (Induction-1). FLAG-Ida was administered as intensified induction (Induction-2) in patients not achieving CR or in those in CR with MRD≥0.1% at the end of induction, and those patients proceeded to allogeneic HSCT if CR was attained. Patients in CR and MRD<0.1% proceeded to early consolidation therapy including 3 cycles with rotating cytotoxic drugs with high-dose methotrexate, high-dose ARA-C and ASP (native 20,000 IU/m2 on day 3 of each cycle or PEG 2,000 IU/m2 on day 3 of each cycle). Patients continued with delayed consolidation (identical to that of early consolidation) followed by maintenance therapy up to 2 yr. in continuous CR if MRD levels after consolidation were <0.01%, otherwise they were assigned to allogeneic HSCT. The cumulated doses of native ASP and PEG-ASP for patients who completed the induction and early+delayed consolidation were 220,000 IU/m2 and 14,000 IU/m2, respectively. No ASP levels were assessed.Results. Ninety-onepatients received native ASP and 35 PEG-ASP in Induction-1. The two groups of patients were comparable for the main clinical and hematologic ALL parameters. No differences were observed in the CR rate (86% vs. 86%), in the frequency of MRD level <0.1% after Induction-1 (63% vs. 70%), and that of MRD level <0.01% after early consolidation (74% vs. 92%, p=0.19), as well as in the proportion of patients submitted to allogeneic HSCT (20% vs. 14%). No differences were found in overall survival or disease-free survival probabilities at 3-years according to the type of ASP (OS: 60% [95%CI: 47% ; 73%] vs. 57%[95%CI: 36% ; 78%], p=0.872 and DFS: 40% [95%CI: 25% ; 55%] vs. 58% [95%CI: 36% ; 80%], p=0.302). Abnormalities in the coagulation parameters were significantly more frequent in patients receiving PEG-ASP in Induction-1 (18% vs. 35%, p=0.045). These abnormalities, together with hepatic toxicity were significantly more frequent in patients receiving PEG-ASP in early consolidation (1% vs. 13%, p=0.003, and 5% vs. 34, p<0.001, respectively). A trend for more allergic reactions was seen in patients receiving native ASP (18% vs. 5%, p=0.1) No differences in the frequency of ASP discontinuation rate were observed (6 % vs. 3%).Conclusions. In HR, Ph-negative adult ALL patients included in the PETHEMA ALL-HR 11 protocol the type of E.coli ASP (native vs. PEG) did not have impact on response and outcome. Allergic reactions were more frequently seen with native ASP and coagulation abnormalities and hepatic toxicity were most frequent with PEG-ASP. Most of the differences in toxicity can be explained by the schedule of ASP given in consolidation (single dose of native ASP vs. single dose of PEG-ASP in each cycle).Supported in part by grants RD12/0036/0029 (RTICC, FEDER), PI14/01971 FIS, Instituto Carlos III, and SGR225 (GRE), Spain DisclosuresNo relevant conflicts of interest to declare.

Acute Ph-negative lymphoblastic leukemias in adults: Risk factors in the use of the ALL-2009 protocol

to analyze well-known risk factors (RFs), such as age, immunophenotype, baseline leukocytosis, enhanced lactate dehydrogenase (LDH) activity, time to achieve complete remission, a risk group, and cytogenetic abnormalities) in patients with acute lymphoblastic leukemia (ALL) in the use of the ALL-2009 protocol. The protocol covered 298 patients (137 women (including 13 pregnant women) and 161 men) aged 15 to 55 years (median age 28 years) with Ph-negative ALL. The phenotype was unknown in 6 patients. Three (1%) were ascertained to have a biphenotypic variant. 182 (62.4%) patients were found to have B-cell ALL (early pre-B ALL (n=51); common ALL (n=92), and pre-B ALL (n=39); 107 (36.6%) patients had T-cell ALL (early T-ALL (n=56); thymic T-ALL (n=41), and mature T-ALL (n=10). According to the baseline clinical and laboratory parameters (leukocytosis of 30·109/l and more for B-ALL; and that of 100·109/l and more for T-ALL; phenotype В-I for B-ALL, phenotype Т-I-II-IV for T-ALL; LDH activity was more than twice the normal values; the presence of translocation t(4;11)), the high-risk group included most patients with B-ALL (n=110 (72.8%)) and T-ALL (n=76 (76%)). Thirty-five patients with T-ALL underwent autologous bone marrow transplantation (BMT). Allogeneic BMT was performed in 18 (7%) of the 258 patients who had undergone an induction phase. Five-year overall survival for all the patients included in the investigation was 59%; relapse-free survival was 65%, which was significantly different in the patients with B-ALL and in those with T-ALL: the overall survival rates were 53.3 and 67.5% (p=0.1); the relapse-free survival was 56 and 79% (p=0.005), respectively. Multivariate analysis including the well-known RFs demonstrated that the latter for T-ALL were of no independent prognostic value and only the patient's age was identified for B-ALL (p=0.013). A lower chemotherapeutic load and a small number of allogeneic BMTs did not affect total positive treatment results in adult patients with ALL, by complying with the principle achieving the continuity of cytostatic effects and by preserving the total cytostatic loading dose. The results of the Russian investigation casts some doubt on the necessity of using very intensive consolidation cycles and performing a large number of allogeneic BMTs in adult patients with ALL.

Post-Remission Treatment with Chemotherapy or Allogeneic Hematopoietic Stem Cell Transplantation (alloHSCT) of High-Risk (HR) Philadelphia Chromosome-Negative (Ph-neg) Adult Acute Lymphoblastic Leukemia (ALL) According to Minimal Residual Disease (MRD). Preliminary Results of the Pethema ALL-HR-11 Trial

▪Introduction: Recent studies have shown that young to middle-aged adults who receive a pediatric-inspired chemotherapy regimen for treatment of Ph-neg ALL do not appear to require an alloHSCT if they achieve good response on MRD testing after induction therapy. Patients (pts) who are not good MRD responders achieve better outcomes with alloHSCT than their counterparts who do not receive alloHSCT. However, it is not clear if this approach can be translated to adult ALL pts with HR features at baseline. The aim of the prospective ALL-HR-11 trial from the Spanish PETHEMA Group was to evaluate the response to a differentiated post-induction therapy (chemotherapy or alloHSCT) according to MRD levels (assessed by 8-color, centrally-performed flow cytometry at the end of induction-week 5- and consolidation therapy-week 17-) in HR Ph-neg adult ALL patients. Patients and methods: HR ALL included one or more of the following parameters at baseline: age 30-60 yr, WBC count >30x109/L for B-cell precursor ALL or >100x109/L for thymic T-ALL, pro-B, early or mature T-ALL, 11q23 or MLL rearrangements or complex karyotype. Induction therapy included vincristine, prednisone, daunorubicin and asparaginase (E coli native or PEG according to center availability) for 4 weeks (Induction-1). FLAG-Ida was administered as intensified induction (Induction-2) in pts not achieving CR or those in CR with MRD≥0.1% at the end of induction. For pts in CR and MRD<0.1% early consolidation therapy included 3 cycles with rotating cytotoxic drugs with high-dose methotrexate, high-dose ARA-C and high-dose asparaginase (E coli native or PEG). These pts continued with delayed consolidation (identical to that of early consolidation) followed by maintenance therapy up to 2 yr. in CR if MRD levels after consolidation were <0.01%, otherwise they were assigned to alloHSCT. Pts in CR after Induction-2 received one consolidation cycle and were assigned to alloHSCT. Results: On June 2015, 115 HR ALL pts were evaluable [mean (SD) age 38(13) yr, 67 males, 80/114 precursor B-ALL, 34/114 T-ALL, WBC count 56(96) x109/L]. Results of Induction-1: therapy-related death: 4(4%), resistance: 11 (10%), CR: 95(86%). MRD<0.1% at the end of induction was observed in 75% of CR patients. Induction-2 was administered to 33 patients (no CR: 11, CR and MRD≥0.1%: 22). No differences in the CR rate or in the rate of MRD clearance after induction were observed according to the type of asparaginase administered, although significantly increased hepatic toxicity in consolidation was observed in patients treated with PEG-asparaginase. The 2-yr DFS and OS probabilities for whole series were 51%±18% and 62%±13%. By intention-to treat after Induction-1 36 pts were assigned to alloHSCT and 68 to delayed consolidation and maintenance. The 2-yr DFS and OS probabilities were 54%±25% and 49%±20%, respectively, for pts assigned to alloHSCT, and 50%±22% and 73%±17%, respectively, for those assigned to chemotherapy (P =0.002 for OS comparison). Patients with MRD<0.1% at the end of induction and <0.01% at the end of consolidation (n=51) showed a 2-yr DFS and OS of 55%±25% and 81%±18%, respectively. Conclusions: The preliminary results of this trial, in which the post-induction therapy decision is only based on MRD results, suggest that in HR, Ph-neg adult ALL pts with adequate MRD response after induction and after consolidation the results of therapy are not hampered by avoiding alloHSCT. Supported by grants RD12/0036/0029 (RTICC, FEDER), PI14/01971 FIS, Instituto Carlos III, and SGR225 (GRE), Spain DisclosuresNo relevant conflicts of interest to declare.

Low Expression Of BCL11B Predicts Poor Overall Survival In Adult Standard Risk T-ALL

▪ IntroductionAlthough risk stratification, detection of minimal residual disease (MRD) and implementation of novel therapeutic agents have improved outcome in acute lymphoblastic leukemia (ALL), survival in adult T-ALL patients still remains unsatisfactory. Therefore, new prognostic markers and potential therapeutic targets are warranted. BCL11B, a key player in normal T-cell development, has recently gained interest due to its high mutation rate (9-16%) in T-ALL. We investigated the frequency of BCL11B mutations, expression levels and the prognostic value in a large uniformly treated cohort of adult T-ALL. Patients and methodsWe analyzed bone marrow (BM) samples of 201 adult T-ALL patients sent to the reference laboratory of the German Multicenter Study Group for Adult ALL (GMALL). BCL11B expression was determined in 195 patients by qRT-PCR, BCL11B mutations were assessed in 178 patients by Sanger sequencing of exon 4 (including all 6 zink finger [ZF] domains). Low expression of BCL11B was defined by expression levels in the lowest quartile (BCL11Blow), high expression by levels in the three remaining quartiles (BCL11Bhigh). Samples had previously been characterized for expression of BAALC, IGFBP7, MN1, WT1, GATA3, ERG as well as for the mutations status of NOTCH1, WT1, and TCR rearrangements. Clinical data were available for 169 patients enrolled on the GMALL trials. We generated BCL11B associated gene expression profiles (GEP) derived from an independent set of 86 T-ALL patients enrolled in the Microarrays Innovations in LEukemia multicenter study. ResultsBCL11B was aberrantly expressed in adult T-ALL with significantly higher expression levels in thymic compared to early T-ALL (0.6 vs. 0.3, P=0.01). Expression of genes associated with a prognostic impact (BAALC, IGFBP7, ERG) or/and T-cell stage dependent expression profile (GATA3, IGFBP7) showed that BCL11Blow (n=49) had higher MN1 (1.6 vs, 0.3, P=0.01), IGFBP7 (1.3 vs. 0.5, P=0.02), and lower GATA3 expression (2.1 vs. 5.7, P<0.01) compared to BCL11Bhigh patients (n=146). This maturation stage specific expression of BCL11B was stressed by a higher rate of TCR rearrangement in BCL11Bhigh patients (73% vs. 27%, P=0.005) and further underlined by the BCL11B derived GEP linking low BCL11B to an immature molecular signature characterized by high expression of BAALC, IGFBP7, FLT3, CD34. Regarding clinical characteristics, low BCL11B expression was associated with a poor prognosis (5-year overall survival (OS): low 35% (n=40) vs. high 53% (n=129), P=0.02) in the overall T-ALL cohort. In the standard risk group of thymic T-ALL (n=102), BCL11Blow identified patients with an unexpected poor outcome (5-year OS: 20%, n=18) compared to BCL11Bhigh (62%, n=84, P<0.001). In addition, BCL11Blow thymic T-ALL patients showed a lower remission rate (5 years: 38% vs. 72%, P=0.02).BCL11B mutations were found in 24 of the 178 (13.5 %) T-ALL patients. In 9 patients, mutations resulted in frame shifts, whereas the remaining, except for a single one, missense mutations were located in the ZF domains: three resulting in introduction of a stop codon. BCL11B mutations were enriched in the mature immunophenotype (thymic: 20%, mature: 8%, early: 3%, P=0.03). No differences were observed in mRNA levels for BAALC, IGFBP7, MN1, WT1, GATA3, ERG, but patients with BCL11B mutations were less frequently assigned to the BCL11Blow group compared to those with high expression (5% vs. 95%, P=0.02). Regarding the clinical characteristics, BCL11B mutations had no prognostic impact regarding OS, neither in the total T-ALL cohort (5-year OS: 56% vs. 48%, n.s.) nor in the thymic T-ALL subgroup (5-year OS: 65% vs. 51%, n.s). ConclusionOur data confirmingly show a high frequency of BCL11B mutations (13.5%) in the so far largest cohort of adult T-ALL patients. As loss of function mutations were restricted to functional ZF domains and recurrently occurred in thymic T-ALL, these data stress a potential pathogenetic role of BCL11B as T-cell specific transcription factor. Importantly, low expression was associated with poor prognosis; in particular in the standard risk group of thymic T-ALL, BCL11Blow is a novel marker that identifies patients with an unacceptable poor prognosis. These findings might help to improve risk stratification in a significant proportion of adult T-ALL patients, which fail to standard therapy despite the favourable immunophenotypic characteristics. Disclosures:No relevant conflicts of interest to declare.

FAT1 Expression and Mutation Status In Adult Acute Lymphoblastic Leukemia

IntroductionFAT1 belongs to the FAT protocadherin family, a drosophila homologous gene involved in development processes. Recently, FAT1 gained large interest as it is mutated in various cancers. Besides the known function of cell-cell interaction and polarity, FAT1 loss of function mutations have been linked to dysregulation of the WNT pathway in solid tumors. In acute lymphoblastic leukemia (ALL), aberrantly high expression of FAT1 was claimed to be associated with inferior outcome in pediatric B-lineage ALL. Herein, we investigated the yet unknown frequency and relevance of FAT1 expression and mutation in a large, homogenously treated cohort of adult ALL patients. Patients and MethodsWe investigated FAT1 expression in diagnostic bone marrow (BM) samples of 112 T-ALL, 122 B-lineage ALL, and additional 63 early T-cell precursor (ETP) ALL patients by real time (RT)-PCR. Patients were enrolled in trials of the German Multicenter Study Group for Adult ALL (GMALL) and outcome was investigated for patients into GMALL trials 06/99 and 07/03. Using the T-cell line BE13 as reference, we defined patients with FAT1 expression higher than BE13 as FAT1pos (0.01-38.5) and patients with a lower expression as FAT1neg (<0.01). We additionally examined peripheral blood (PB), BM, CD34+-, CD3+-cells from healthy donors. FAT1mutation status was investigated in 68 T-ALL patients. For mutation analyses customized biotinylated RNA oligo pools (SureSelect, Agilent) were used to hybridize the targeted regions, followed by 76-bp paired-end sequencing on an Illumina Genome Analyzer IIx platform. ResultsNormal hematopoietic cells including unselected BM cells, CD34+-progenitors, or CD3+ T-cells from healthy donors lacked FAT1 expression (<0.01) with the one exception of a CD34+-sample (0.03). In contrast, ALL samples aberrantly expressed FAT1: 32% of B-lineage ALL (0.01-38.5) and 54% of T-ALL cases (0.01-31.2) showed elevated FAT1 expression, with a lower frequency in the immature ETP-ALL subgroup (17%; 0.01-15.2). FAT1 expression was associated with a more mature immunophenotype (T-ALL: thymic 74%, mature 45%, early 4%, p<0.001; B-lineage ALL: pre B-ALL 57%, common ALL 26%, pro B-ALL 9%, p=0.04). No significant differences between FAT1pos and FAT1neg patients were observed regarding age and sex. FAT1pos T-ALL patients more frequently showed a white blood cell count (WBC) >30.000/µL at diagnosis compared to FAT1neg T-ALL patients (78% vs. 42%, p<0.01). Whereas no negative prognostic impact was observed for FAT1 expression in B-lineage ALL or T-ALL with respect to overall survival or remission duration, lack of FAT1 expression was associated with primary resistance to induction therapy in T-ALL (T-ALL: FAT1neg 12%, FAT1pos 0%, p=0.04). In addition, we found an unexpected high rate of FAT1 mutations (exclusively missense mutations) with 8 of 68 T-ALL patients (12%). The mutation spectrum, mainly located in the cadherin domains, was similar to the distribution of FAT1 mutations in solid tumors. No difference were observed between FAT1 mutated (FAT1mut) and FAT1 wild-type patients (FAT1wt) with regard to sex, age, WBC, and presentation of antigens associated with an early differentiation stage. FAT1 mutations were more frequent in early T-ALL (3/12, 25%) and in thymic T-ALL (5/41, 12%) than in T-ALL patients with a mature immunophenotype (0/15, 0%). FAT1 expression was more common in FAT1wt T-ALL compared to FAT1mut T-ALL patients (50% vs. 25%). ConclusionThis first comprehensive analysis on FAT1 in a large cohort of adult patients with ALL shows a high frequency of FAT1 expression. Higher FAT1 expression occurred in ALL patients with more mature immunophenotype linking FAT1 to cell-cell adhesion and polarity, thymic homing and interaction with the BM niche. This yet unreported high mutation rate of 12 % in adult T-ALL makes FAT1 to one of the most frequently mutated genes in T-ALL. The link of inactivating FAT1 mutations to aberrant activation of the WNT pathway, as reported in solid tumors, might allow the development of refined treatment options. In summary, these data make FAT1 a promising candidate for disease monitoring, risk stratification and development of targeted therapies. Disclosures:Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.

Mutational Spectrum Of Adult T-ALL

IntroductionT-cell acute lymphoblastic leukemia (T-ALL) in adults represents a disease with a rather unfavourable prognosis. Despite the fact that treatment stratification and minimal residual disease (MRD) monitoring have improved survival, there is still need to improve outcome by the development of novel targeted therapies. Therefore, molecular alterations are in the focus of on-going research. Until recently only few candidates were identified as recurringly mutated genes including NOTCH1, FBXW7, PTEN. The development of next generation sequencing (NGS) significantly enlarged this spectrum and identified alterations in additional genes (BCL11B, PHF6, DNM2, CNOT3, KRAS, NRAS, DNMT3A). Whereas a number of putative driver mutations have been characterized, the spectrum of recurring alterations in larger cohorts and their relevance in different leukemic subgroups remains unexplored. To unravel relevant recurring alterations in a large cohort of adult T-ALL and to explore potential target genes for novel therapeutic strategies, we performed targeted high throughput NGS of 88 candidates in 81 T-ALL samples. Patients and methodsWe investigated 67 adult T-ALL patients enrolled in the trial 07/2003 of the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL). In addition, 14 patients with early T-precursor ALL (ETP-ALL) from other GMALL trials were analysed. Customized biotinylated RNA oligo pools (SureSelect, Agilent) were used to select the targeted regions. We performed 76-bp paired-end sequencing on an Illumina Genome Analyzer IIx platform and reads were mapped to NCBI hg19 RefSeq. For a variant call we required at least a read depth of 20, an allele frequency of 20% and an average base calling quality of Q13. Polymorphisms annotated in dbSNP 135 were excluded. The targeted region comprised 88 genes known to be frequently mutated in ALL, acute myeloid leukemia, myelodysplastic syndrome as well as genes associated with epigenetic regulation, splicing, DNA mismatch repair, and the NOTCHpathway. ResultsWe obtained an average of 1.2 Mbp sequence for each sample, resulting in an average coverage of 120 reads for the target region. 79% of the targeted region was covered with a minimum of 20 reads. After exclusion of polymorphism annotated in dbSNP135, 473 single nucleotide variations (SNV) and small indels were identified, 294 of those resulted in changes on the protein level. On average three (3.1) genes per patient were mutated, and 66 (77%) of the 88 genes were mutated in at least one patient. As expected, the highest mutation rate with 53% was found for NOTCH1, with a higher frequency in thymic T-ALL (67.5%) than in early T-ALL (33.3%). Mutation frequencies of FBXW7 (10%), WT1 (10%), JAK3 (12%), and BCL11B (10%) were in the range of reported frequencies. Recently identified novel alterations in DNM2 (17%), PHF6 (11%), DNMT3A (5%) or RELN (5%) were confirmed in our cohort. Interestingly, genes that had not been described in T-ALL included recurring mutations in the histone methyl-transferase MLL2 (11%), frequently mutated in B-cell lymphomas. Like in lymphoma and in the Kabuki syndrome, MLL2 mutations were distributed over the entire gene without any obvious hot spot region. Also the protocadherins FAT1 (15%) and FAT3 (12%) were recurringly altered. FAT1and its inactivation by mutations were recently linked to activation of the WNT pathway in solid tumours.Affected pathways significantly differed in leukemic subgroups: whereas mutations involving the NOTCH pathway were predominately enriched in the thymic subgroup (75%) and less relevant in early T-ALL (33%, P=0.004), chromatin modifiying genes (17% vs. 5%, P=0.22) and signalling genes (42% vs. 15%, P=0.09) were more frequently mutated in early T-ALL. Spliceosome mutations described in myeloid and mature lymphoid malignancies were present only in a minority (7.4%) of T-ALL. ConclusionAdult T-ALL reveals a highly heterogeneous spectrum of candidate gene mutations. Here we provide an original and comprehensive overview of recurring mutations that unravel preferentially pathways altered in specific leukemic subgroups. In addition, we identified novel candidate genes with potential therapeutic implications (FAT1, EZH2, DNMT3A). These mutations have to be validated in a larger cohort with a focus on clinical implications accompanied by functional assays regarding their use as therapeutic targets. Disclosures:Krebs:Illumina: Honoraria. Greif:Illumina: Honoraria.

MicroRNA Profiling Reveals Aberrant MicroRNA Expression in Adult ETP-ALL and Functional Studies Demonstrate a Role of Mir-222 in Acute Leukemia,

Abstract 3458 Introduction:Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup in pediatric acute T-lymphoblastic leukemia (T-ALL). ETP-ALLs originate from early thymic progenitors that retain stem cell features and are characterized by a specific immunophenotype and by a specific gene expression profile. Recently, we have characterized ETP-ALL as subgroup of early T-ALL in adults. To unravel the underlying mechanisms for the aberrant and distinct gene expression profile in ETP-ALL we now explored the expression of microRNAs (miRNAs) as gene regulators in ETP-ALL and further investigated their functional role in acute leukemia. Patients and methods: We screened expression levels of 667 miRNAs in newly diagnosed T-ALL patients (n=14: ETP-ALL n=8, non-ETP early T-ALL n=3, thymic T-ALL n=3) and 2 healthy controls (CD3 selected T cells) using a Taqman Low Density Array (TLDA; Applied Biosystems). The expression levels of miR-221 and miR-222 were validated in a large cohort of adult T-ALL (n=178: ETP-ALL n=66 and typical T-ALL n=112 including early-, thymic-, and mature T-ALLs) and healthy controls (n=6) by real-time-PCR (Taqman microRNA assays; Applied Biosystems). These patients were enrolled on the GMALL study 07/03. Functional analysis was performed by AMAXA transfection of pSUPER vector containing the miR-221 and miR-222 DNA sequences in Jurkat (T-lineage ALL) and KG1a (AML) cells. Viability was determined by cell proliferation reagent WST-1, and BrdU ELISA. Apoptosis was measured by annexin-V/7AAD staining with subsequent flow cytometric analysis. Results: In our screen 229 (34%) of the 667 miRNAs represented by the TLDA were expressed at detectable levels in at least two-thirds of the T-ALL samples and the controls. Of these we identified 55 miRNAs to be differentially expressed in T-ALL samples compared to healthy controls: 49 miRNAs were upregulated and 6 miRNAs were downregulated in the patient samples. Of these, miR-221 and miR-222 were the only miRNAs significantly upregulated in the ETP-ALL compared to the combined early and thymic T-ALL subgroups (8.0-fold, P<0.01; 5.0-fold, P<0.01, respectively). Therefore, we further validated miR-221 and miR-222 expression in a larger set of 178 T-ALL samples and observed a 3.3-fold higher expression of miR-221 (P<0.0001) and 3.9-fold higher expression of miR-222 (P<0.0001) in ETP-ALL (n=66) compared to typical T-ALL (n=112). For the correlation of miR-221 and miR-222 expression with molecular features, samples were divided into groups (low and high) according to the median of the miRNA expression levels. High miR-222 expression was associated with an immature phenotype of early T-ALL (P<0.01) and cell surface expression of CD34 (mean: 32%) compared to low miR-222 expressers (mean: 8%; P<0.01). Moreover, high miR-222 expression was associated with expression of myeloid markers CD33 (mean: 23% compared to 4% in low miRNA expressers, P<0.01) and CD13 (mean: 18% vs. 4%, P<0.01). Similar results were obtained for miR-221. In vitro studies revealed that overexpression of miR-222 inhibited proliferation of Jurkat (58% reduction of proliferation compared to cells transfected with the empty vector, P<0.001) and KG1a cells (50% reduction of proliferation compared to control cells, P<0.01) determined by WST-1. A reduction of the DNA synthesis was detected by BrdU incorporation after miR-222 overexpression in Jurkat (37% reduction compared to control cells) and in KG1a cells (54% reduction compared to control cells). Overexpression of miR-222 induced a 1.3- (P=0.02) and 3.0-fold (P<0.01) increase in apoptosis in Jurkat and KG1a cells, respectively. No significant changes were observed for miR-221 transfected cells in these functional assays. Conclusion: In summary, our study revealed aberrant expression of miRNAs in ETP-ALL, with miR-221 and miR-222 as the most overexpressed miRNAs. Functional analysis demonstrated that miR-222 impaired the proliferation and induced apoptosis, indicating a potential role for miR-222 in acute leukemia. Importantly, miR-222 targets such as the proto-oncogene c-KIT and the ETS1oncogene are contained in the gene expression profile of ETP-ALL. Thus, aberrant expression of miR-222 may directly impact leukemogenesis by altering expression of key oncogenes in acute leukemia. Disclosures:Goekbuget:Micromet: Consultancy.

Successful Subtype Oriented Treatment Strategies in Adult T-All; Results of 744 Patients Treated in Three Consecutive GMALL Studies.

Abstract 324 Introduction and classification:This is the largest adult T-ALL cohort treated according to immunologic subtypes. All patients were immunophenotyped in one central lab (Berlin). T-ALL (cyCD3+, CD7+) were subclassified into early T-ALL (sCD3-, CD1a-), thymic T-ALL (sCD3-/+, CD1a+) and mature T-ALL (sCD3+, CD1a-). T-ALL constitutes in 3 consecutive GMALL-studies 24% of ALL patients. Patients and methods:A total of 744 T-ALL pts (15 to 55 yrs) were accrued in 102 hospitals in the GMALL studies 05/93, 06/99 and 07/2003. In GMALL 05/93 239 adult T-ALL patients, were treated according to a multi-agent chemoprotocol. Stem cell transplantation (SCT) was not recommended in CR1. In GMALL studies 06/99 and 07/03 505 T-ALL pts received intensified chemotherapy; particularly with introduction of PEG-asparaginase in induction as well as HDMTX/PEG-Asp consolidation cycles. Based on study 05/93 results, SCT from sibling (Sib) as well as matched unrelated (MUD) donor in CR1 was recommended for all patients with early T-ALL, mature T-ALL and for high-risk (HR) pts with thymic T-ALL (defined as late CR, complex karyotype or MRD positivity (MRD+)). Results:T-ALL subtype distribution in the total cohort of 744 adult T-ALL was early-T 23% (N=170), thymic-T 56% (N=420), mature-T 21% (N=154), without any differences between the studies. GMALL Study 05/93: The overall CR rate was 86% (early-T 72%, thymic-T 93%, mature-T 84%. The lower CR rate in early T-ALL was mainly due to early death (19%). The overall CCR rate was 47% (early-T 45%, thymic-T 54%, mature-T 30%). The overall survival rate at 10 yrs for all pts was 47% (early-T 47%, thymic-T 55%, mature-T 25%). GMALL Study 06/99 and 07/03:Of the 505 patients, 87% achieved CR (early-T 84%, thymic-T 92%, mature-T 77%). PR/Failure was higher in early-T (13%) and mature-T (17%) compared to thymic-T (5%). Early death was 4% and equally distributed. 267 pts (64%) received chemotherapy only and the majority were 229 pts (86%) with thymic T-ALL, not considered for SCT in CR1. The CCR rate was 61%. The few early (n = 15) and mature (n = 23) T-ALL pts, which could not have a transplant in CR1, are a negative selection (e.g. early relapse, comorbidity, no donor) and their CCR rate was 33% and 22% respectively. This was due to a high relapse rate in early T-ALL (60%) and mature-T (74%) compared to 33% in thymic-T. Overall survival rate at 8 yrs for thymic T-ALL with chemotherapy was 68%, for the 77 adolescent pts (15 to 25 yrs) even 76%. Stem cell transplantation:153 T-ALL pts in studies 06/99 and 07/03 received a SCT in first remission. SCT realisation rate in early T-ALL was 84%, in mature-T 68%. Overall CCR rate was 58% (early-T 47%, HR thymic-T 79%, mature-T 61%). Relapse rate after SCT was in early-T 33% and in mature-T 22%. The overall TRM rate was 18% despite more than half MUD SCT, without any TRM difference between the immunological subtypes. Overall survival rate after SCT in CR1 at 8 yrs was 53%, early-T 44%, thymic-T 67%, mature-T 59%. SCT modalit:49% received alloSib, 55% alloMUD and 20% auto-SCT. Overall CCR rate after alloSib for the total cohort was 65% (early-T 60%, thymic-T 73% and mature-T 69%); for alloMUD total 55% (early-T 45%, thymic-T 77%, mature-T 61%) and for the small cohort of 20 pts with auto-SCT CCR was 35%. Conclusion:The strategy in three consecutive GMALL studies to stratify and treat adult T-ALL pts according to the immunologic T-subtypes was successful. Overall survival at 5 yrs could be improved to 56% from 44%. There was a particular improvement for mature T-ALL (49% vs. 30%) and early-T (40% vs. 33%). This was mainly due to a high realisation rate of SCT in early T-ALL and mature T-ALL and the substantial better results of SCT. Results of alloMUD SCT were comparable to alloSib SCT. The small cohort of HR thymic T-ALL pts also had a benefit from SCT. The excellent outcome of SR thymic T-ALL (∼ 50% of all T-ALL) with the OS of 68% and 76% in adolescents due to intensified chemo, partic. PEG-Asp, does not suggest SCT in CR1. Several molecular markers, such as ERG, BAALC, WT1, had in a retrospective analysis some prognostic relevance in this pt cohort. The new GMALL study generation will however focus in thymic T-ALL on early evaluation of MRD to decide for SCT (MRD+) or not (MRD-) whereas early/mature T-ALL remain allocated to high risk groups with SCT in CR1.Supported by Deutsche Krebshilfe 702657Ho2 and BMBF 01GI9971/8 Disclosures:No relevant conflicts of interest to declare.

Early T-Cell Precursor-ALL in Adult T-ALL.

Abstract 911▪▪This icon denotes an abstract that is clinically relevant. Introduction:Recently, a small subgroup of pediatric acute T-lymphoblastic leukemia (T-ALL) was described, which is closely associated with the gene expression profile of early T-cell precursors (ETPs). This subtype, termed ETP-ALL, showed a highly unfavorable outcome compared to non-ETP(='typical')-ALL. Based on the results of Coustan-Smith et al. (Lancet Oncology, 2009), the Italian national study Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) and St-Jude Children's hospital modified their treatment in children with ETP-ALL to a more intensive regime including stem cell transplantation. ETP-ALL is characterized by a specific immunophenotype (CD1a-, CD8-, CD5weak with expression of stem cell or myeloid markers). Here we explored the existence of ETP-ALL in adults and further studied the molecular characteristics of this specific T-ALL subtype. Patients and methods:We examined the gene expression profiles of 86 adult T-ALL patients obtained from the Microarray Innovations in LEukemia (MILE) multicenter study (HG-U133 Plus 2.0, Affymetrix, Haferlach et al., JCO in press). In addition, bone marrow of 296 patients from the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL) were analyzed by flow cytometry and expression levels of BAALC, IGFBP7, MN1, and WT1 were determined by real-time-PCR. Results:Using the published list of differentially expressed genes in ETPs (Coustan-Smith et al. 2009) we performed unsupervised clustering analyses of the 86 T-ALL samples. A cluster of 17 samples (19.8%) displayed an ETP-associated gene expression profile and were defined as ETP-ALL. Comparing the gene expression profiles of ETP-ALL and typical T-ALL, 2065 probe sets were differentially expressed in ETP-ALL (FDR 0.05). In addition to genes used for classification, we also identified genes known to be involved in the pathogenesis of T-ALL (e.g. PROM1, BCL2, LMO2, LYL1). In particular, stem cell associated genes such as, BAALC (2.52-fold, p=0.003), IGFBP7 (2.76-fold, p=0.002) or MN1 (3.41-fold, p<0.001) were upregulated in ETP-ALL, whereas HOX11 (45-fold, p=0.004), a marker for thymic T-ALL, was downregulated. An independent cohort of 297 patient samples from the GMALL study group was examined by flow cytometry and real-time PCR. 19 (6.4%) samples revealed the ETP-ALL immunophenotype. As expected, all patient samples were found in the group of early T-ALL, representing 23.5% of all early T-ALLs. There was a significant correlation between a lower leukocyte count at first diagnosis and the classification of ETP-ALL (p=0.001). Gene expression measured by real-time-PCR was performed for genes associated with poor outcome in T-ALL: BAALC (2.11-fold, p<0.001) and IGFBP7 (3.59-fold, p=0.003) were significantly upregulated in the group of ETP-ALL. Similarly, the genes MN1 (4.52-fold, p<0.001) and WT1 (2.76-fold, p=0.036), described as poor prognostic markers in cytogenetically normal AML, were also upregulated in ETP-ALL. Conclusion:In adult T-ALL, a subset of patients shares the gene expression profil and immunophenotype of ETP-ALL, which is in line with recent findings in pediatric patients. The gene expression profile of this subset is significantly correlated to stem cell associated markers predictive for inferior outcome in T-ALL. Interestingly, adverse factors in CN-AML are also aberrantly expressed in ETP-ALL suggesting a myeloid origin of ETPs and indicating a closer relationship between ETP-ALL and AML. The prognostic impact and the determination of the most appropiate set of markers needs to be further investigated. These results support the GMALL strategy to regard early T-ALL patients as high risk with assignment to stem cell transplantation. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Wilms Tumor 1 Gene Mutations in Adult Acute T-Lymphoblastic Leukemia

The wilms tumor 1 gene (WT1) encodes a transcriptional regulator involved in normal hematopoietic development. The role of WT1 in acute leukemia has been underscored by the finding of WT1 overexpression in subsets of patients (pts) associated with an increased relapse risk. In addition mutations of WT1 have been found in about 10–15% of acute myeloid leukemia (AML) pts and have recently shown to predict inferior survival. Thus far, larger studies have not yet determined the frequency and impact of WT1 mutations in acute T-lymphoblastic leukemia (T-ALL). Herein, we have analyzed WT1 mutations and WT1 mRNA expression levels in a large cohort of T-ALL including 239 newly diagnosed adult pts treated on the GMALL protocols 0699 and 0703. Diagnostic bone marrow specimens were studied for WT1 mutations by DNA sequencing. In addition, samples were immunophenotyped, and mRNA expression of the molecular markers HOX11, HOX11L2, ERG, BAALC, as well as WT1 were determined by real-time RT-PCR. Twenty (8%) of the 239 analyzed T-ALL pts had WT1 mutations (WT1mut) [20 pts had mutations in exon 7 (WT1mut7), with 2 pts having coexisting mutations in exon 9 (WT1mut9)]. WT1mut7 were frameshift or nonsense mutations predicted to result in a truncated WT1 protein, whereas WT1mut9 were missense mutations leading to single amino-acid substitutions. WT1mut and WT1 wildtype (WTwt) pts did not significantly differ with respect to clinical parameters at diagnosis (e. g. age, leukocyte count, and sex). WT1mut cases were characterized by immature features such as an early immunophenotype (45% of WT1mut showed an early T-ALL immunophenotype as compared to only 25% of WT1wt), and WT1mut also showed higher levels of CD34 expression as determined by flow cytometry (WT1mut median: 46% vs. WT1wt median: 2 %; P=0.03). Moreover, WT1mut had significantly higher WT1 mRNA expression levels [WT1mut median: 0.05 (range: 0–0.395) vs. WT1wt median: 0 (range: 0–0.15); P<0.001]. Significant differences were not observed in the complete remission rate nor overall survival or relapse free-survival (RFS) between WT1mut and WT1wt pts. However, in the standard risk group of thymic T-ALL 80% (4/5) of WT1mut relapsed as compared to 28% (25/89) of WT1wt thymic pts [P=0.01; RFS at 18 months: 20% (SE: ±18) for thymic WT1mut vs. 82% (SE: ±4) for thymic WT1wt pts; P=0.008]. In conclusion, in adult T-ALL WT1 mutations are present in 8% of newly diagnosed pts and are located in the same region as reported in AML expected to impair the DNA binding ability of the WT1 protein. Similar to findings in AML, WT1mut cases are characterized by immature features pinpointing to a genetic hit in hematopoietic progenitors likely harboring bilineage potential. The prognostic implications of WT1 mutations in standard risk thymic T-ALL will have to be further validated in independent studies and may in future direct molecularly-based treatment stratification.

High Expression of the ETS Transcription Factor ERG Predicts Adverse Outcome in Acute T-Lymphoblastic Leukemia in Adults

In adult T-lymphoblastic leukemia (T-ALL) disease-free survival remains limited to 32% to 46%. The adverse prognosis in T-ALL has not been attributed to cytogenetic or molecular aberrations. We have determined the prognostic impact of the oncogenic transcription factor ERG in T-ALL. ERG expression was analyzed by real-time polymerase chain reaction (PCR) in 105 adults with newly diagnosed T-ALL treated on the German ALL protocols. Patients were dichotomized at ERG's median expression into low (n = 52) and high (n = 53) expressers. Homeobox (HOX) 11 and HOX11L2 expression was determined by real-time PCR. High ERG expressers compared with low ERG expressers had an inferior overall survival (OS, P = .02; 5-year OS: high ERG 26% v low ERG 58%) and relapse-free survival (RFS, P = .003; 5-year RFS: high ERG 34% v low ERG 72%). On multivariable analysis high ERG expression (P = .005), immunophenotypic subgroups (early v mature v thymic T-ALL; overall P = .04), HOX11L2 positivity (P = .055), and absence of HOX11 (P = .017) were independent adverse risk factors predicting RFS. Patients with high ERG expression had a hazard ratio (HR) for relapse of 3.2. Within the good prognostic subgroup of thymic T-ALL (n = 57), high ERG (HR, 4.1; P = .02) and presence of HOX11L2 (HR, 6.6; P = .008) were independent adverse factors for RFS. High expression of ERG is an adverse risk factor in adult T-ALL. Within thymic T-ALL, otherwise classified as standard-risk, high ERG expression-identified patients that were four times more likely to fail long-term RFS. The prognostic impact of ERG may assist treatment stratification and suggest the need of alternative regimens.

High expression of the transcription factor ERG predicts unfavorable outcome in acute T-lymphoblastic leukemia (T-ALL) in adults

6505 Background: In adult T-ALL long-term survival remains limited to 32–46%. Transcription factors are frequently targeted by chromosomal translocations resulting in disruption of hematopoietic proliferation and differentiation. The oncogenic ETS transcription factor ERG is expressed during early T-cell development and shut off once T-cell commitment is complete. We hypothesized that due to its specific involvement in T-cell maturation and oncogenic potential, ERG might contribute to leukemogenesis. Thus we have determined the prognostic impact of ERG expression in T-ALL. Patients and Methods: ERG mRNA expression was analyzed by real-time RT-PCR in pretreatment marrow samples of 105 adults with T-ALL treated on German ALL protocols. Patients (pts) were dichotomized at ERG’s median expression into low (n=52) and high (n=53) expressers. HOX11 and HOX11L2 expression was determined by real-time RT-PCR. Immunophenotyping was performed differentiating T-ALL into 3 subtypes: pre-T (CD2-), thymic (CD1a+), and mature (sCD3+). Results: High ERG expression was associated with a higher relapse rate (45%) compared to pts with low ERG expression (20%; P=0.01). High ERG expressers compared to low ERG expressers had an inferior overall survival (OS, P=0.02; 5-year OS: high ERG 26% vs low ERG 58%) and relapse-free survival (RFS, P=0.003; 5-year RFS: high ERG 34% vs low ERG 72%). On multivariable analysis high ERG expression (P=0.005), immunophenotypic subgroups (pre-T vs mature vs thymic; overall P=0.04), HOX11L2 positivity (P=0.055) and absence of HOX11 expression (P=0.017) were independent adverse risk factors predicting RFS. Patients with high ERG expression had a hazard ratio (HR) for relapse of 3.2. Within the good prognostic subgroup of thymic T-ALL (n=57) high ERG (HR 4.1; P=0.02) and presence of HOX11L2 (HR 6.6; P=0.008) were independent adverse factors for RFS. Conclusion: High expression of the oncogene ERG is an adverse factor in adult T-ALL. Within thymic T-ALL otherwise classified as standard risk, high ERG expression identified pts that were more than four times likely to fail long-term RFS. The prognostic impact of ERG may assist treatment stratification and suggest the need of more intensive regimens for these high risk thymic T-ALL pts. No significant financial relationships to disclose.

HOX11L2 Genotyping Identifies Subset of Adult Thymic T-ALL with Inferior Outcome.

Background: Studies evaluating the prognostic impact of the expression of the oncogenes HOX11 and HOX11L2 in adult T-ALL have been controversial so far.Methods: In the two consecutive German Multicenter ALL (GMALL) therapy trials 5/93 and 6/99, 503 T-ALL pts were recruited between 4/93 and 10/03. Of these, 286 patients with adult T-ALL have been retrospectively investigated for the expression of HOX11 and HOX11L2. This represents the largest cohort of adult T-ALL patients investigated so far. Expression was measured in pretreatment peripheral blood and bone marrow blasts by comparative real-time RT-PCR.Results: The median age was 30 (16–65)yrs, 76% were male, 69% had a mediastinal tumor, 24% WBC > 100.000 and 7% CNS involvement. 61 patients (21%) showed a high and 57 patients (20%) a low HOX11 expression, 165 patients (59%) did not express HOX11. In comparison, 29 patients (10%) expressed HOX11L2 whereas 256 (91%) did not show a HOX11L2 expression. The prevalence of HOX11L2 expression was higher in the youngest age group (median 20 yrs vs. 31 yrs in “negative” pts, p=0,025). CNS involvement was more frequently found in the high HOX11 expression group (p=0,025). High HOX11 expression and HOX11L2 expression were both significantly associated with the thymic immunophenotype (p=0.0077 and 0,031, respectively). There were no significant differences in CR rates between the HOX11- and HOX11L2-expression subgroups. In contrast, HOX11L2 positive patients showed a higher relapse rate (52% vs. 28%; P=0.011). The overall survival (OS) was higher for pts. with high HOX11 expression (OS at 5 years: high 69% vs low 36% vs negative 41%; p=0.007), and disease free survival (DFS) was significantly longer in the high HOX11 expression group (at 5 years high 73% vs low 41% vs negative 47%; p=0.007). For HOX11L2 expression the difference in OS did not reach statistical significance regarding all T-ALL patients (at 5 yrs: positive 13% vs negative 49%; p=0.06). Controlling for the immunophenotype, a significant difference in OS became evident (at 5 yrs: positive 19% vs negative 64%; p=0.01). DFS was significantly shorter in HOX11L2 positive patients (at 5 yrs: positive 24% vs negative 56%; p=0.03). In a multivariate analysis the expression of HOX11 could not be confirmed as independent prognostic factor for OS or DFS (p≥0,05). In contrast, HOX11L2 expression was found to be the only relevant prognostic factor besides the immunophenotype: The hazard ratio of death was 1,8 for HOX11L2 positive T-ALL patients (95% CI 1.1–3,0; P=0,021), and the hazard ratio for a relapse was 2,0 (95% CI 1.1–3,7; P=0.023).Conclusions: HOX11 expression is highly associated with the thymic immunophenoytpe and is not an independent prognostic factor. The expression of HOX11L2 is also predominantly found in thymic T-ALL. Whereas the majority of thymic T-All patients do not express HOX11L2 and have an improved outcome, HOX11L2 expression identifies a small subset of thymic T-ALL patients with a reduced OS and DFS, similarly to early and mature T-ALL. If this can be confirmed prospectively, thymic T-ALL patients need to be treated according to their HOX11L2 expression status as different entities: HOX11L2 positive thymic T-ALL patient requiring a rather aggressive therapeutical management.