Proposal of a Genetic Classifier for Risk Group Stratification in Pediatric T-Cell Lymphoblastic Lymphoma Reveals Significant Differences to T-Cell Lymphoblastic Leukemia

Introduction

T-cell lymphoblastic lymphoma (T-LBL) represent the second most common subtype of Non-Hodgkin lymphoma (NHL) in children and adolescents. In contrast to other pediatric NHL-subtypes and acute lymphoblastic leukemia (ALL) criteria for the stratification of treatment intensity are lacking in T-LBL. Consequently all patients receive identical treatment intensity resulting in over- and under-treatment of a relevant but not yet characterized subgroup of patients. Recently a genetic classifier for adult T-ALL patients was reported. Whether T-ALL and T-LBL represent one or two diseases remains an ongoing discussion. Whole exome sequencing data of pediatric T-LBL cases now support the hypothesis that T-ALL and T-LBL, despite pathogenic similarities are biologically different. Here we used our large dataset of well defined and uniformly treated pediatric patients with T-LBL to define molecular risk factors of the disease.

Methods

All pediatric T-LBL patients of the NHL-BFM group with sufficient material available were sequenced for abnormalities in the genes: NOTCH1, FBXW7, NRAS, KRAS, PTEN, PIK3R1, PIK3CA using Sanger sequencing of known mutational hotspots and loss of heterozygosity of chromosome 6q using fragment length analyses. Patients were treated uniformly according to NHL-BFM protocols for T-LBL. Clinical data were available from the data base of the NHL-BFM study center. Accompanying molecular research for the trials NHL-BFM 95 and EURO-LB 02, in which these patients were recruited, has been approved by the ethical committees of the Hannover Medical School and Justus-Liebig University Giessen, Germany.

Results

The observed frequencies of somatic mutations with 95% confidence intervals were: NOTCH1 61% (51-70%), FBXW7 18% (12-27%), PTEN 15% (9-23%) in 114 analyzed patients, N-RAS + K-RAS 10% (5-18%) in 99 analyzed patients, PIK3R1+PIK3CA in 8% (4-15%) in 107 analyzed patients, and LOH6q 12% (8-17%) in 217 analyzed patients. Detailed evaluation of potential associations of distinct mutation status and clinical characteristics revealed a statistically significant association of NOTCH1 mutations (p=0.006) and FBXW7 mutations (p=0.034) with age below 10 years compared to patients with germline status. All other analyses evaluated for each gene separately taking into account age, gender, stage of disease, CNS disease, bone marrow involvement, mediastinal tumor, and general condition at diagnosis did not identify any statistically significant association.

Concerning the concurrence or exclusion of the analyzed alterations, NOTCH1 mutations were significantly associated with FBXW7 mutations (p=0.03). LOH6q positive patients presented significantly more often in cases with NOTCH1 wildtype status (p=0.03) and PTEN mutations and FBXW7 mutations turned out to present mutually exclusive (p=0.03).

The analyses concerning patients outcome allowed the proposal of an new genetic classifier defining three risk groups:

1) Good risk group (GR) comprising 39% (35/91) of patients defined by NOTCH1 mutation and no RAS or PIK3 mutation with a cumulative incidence of relapse of 11+5%.

2) Intermediate risk group (IR) with all non-GR and non-HR patients including 46% (42/91) of patients with a cumulative incidence of relapse of 20+6%.

3) High-risk group (HR) of 15% (14/91) of patients defined by NOTCH1 wildtype in combination with PTEN mutation and/or LOH6q positivity associated with a cumulative incidence of relapse of 64+14%.

Except for an overrepresentation of patients 10 to 15 years of age in the HR arm, none of tested patients’ characteristic parameters of was associated with risk group.

Conclusion

Detailed analyses of genetic alterations in pediatric T-LBL revealed relevant somatic mutation frequencies for gene loci of the PTEN/PI3K pathway and the RAS pathway. Together with earlier published results on the prognostic relevance of NOTCH1 mutations and chromosome 6q alterations the analyzed cohort of about 100 uniformly treated pediatric T-LBL patients allowed the definition of a genetic classifier for risk group stratification. This proposed classifier requires prospective validation. The here proposed genetic classifier for T-LBL might be worth to be analyzed in pediatric T-ALL. Interestingly our proposed T-LBL classifier includes some aspects in parallel, but overall differed significantly from the earlier published classifier for adult T-ALL.