Genetics of Teenage and Young Adult Acute Lymphoblastic T-Cell Leukaemia.

The outcome for Teenagers and Young Adults (TYA) with T-cell Acute Lymphoblastic Leukaemia (T-ALL) has improved significantly using paediatric based chemotherapy protocols. Event free survival figures from the recently closed UKALL2003 highlight this observation. It remains poorly understood however why their outlook remains inferior to children aged 1 – 15 years whilst receiving identical treatment. Possible explanations for this phenomenon are distinct tumour biology, host factors and treatment adherence. The primary aim of this study is to characterise the tumour associated genetic events leading to TYA T-ALL and establish whether T-ALL in Teenagers and Young Adults is a distinct disease entity. The secondary aims are to identify new prognostic markers and potential targets to optimise therapy.

DNA and RNA were extracted from 60 TYA T-ALL (15 – 25 years) historic samples. The DNA was derived from blasts at diagnosis or relapse, and matched with germ line DNA where available. The DNA was examined at high resolution for Copy Number Alterations (CNAs) and Loss of Heterozygosity (LOH) using the Affymetrix SNP6.0 platform and analysed by CNAG, dCHIP and Partek. The most frequent CNAs were confirmed by MLPA (P-383-T-ALL). In addition the DNA was screened for mutations in NOTCH1/FBXW7/PTEN using conventional Sanger sequencing and for mutations in NRAS, KRAS, CBL, FLT3 and SHP2 using denaturing high performance liquid chromatography. In cases without CDKN2A gene deletion we initially performed CDKN2A gene promoter methylation by SABiosciences EpiTect Methyl qPCR Arrays, followed up by Methylation Specific PCR (MSP). Where available, RNA was extracted from viable cells and the quality confirmed by Bioanalyser. Next the RNA was reverse transcribed to cDNA and subjected to real-time PCR gene expression analysis for CDKN2A and T-ALL related oncogenes.

The most frequent CNA was deletion of CDKN2A in 72.7% of patient samples (91.6% homozygous deletions). Other frequent CNAs were loss of MLLT3 (31%), STIL (27%), PTEN (19%), LEF1 (15%) and gain of MYB (12%). These CNAs were confirmed by MLPA. The STIL-TAL fusion occurred in 27% of patients. The only recurrent region of Copy Number Neutral LOH encompassed 9p24.3-p13.3, including the gene CDKN2A (27%). NOTCH1, FBXW7 and PTEN mutations occurred at expected frequencies. Rare mutations were identified in NRAS and CBL, and these mutations represented known variants. Bisulphite modified DNA was subjected to CDKN2A gene promoter methylation analysis. All samples examined were negative by Epitect Methyl Array, including the positive control cell line Raji. Promoter methylation was positive by MSP for the Raji cell line and some TYA T-ALL cases without CDKN2A gene deletion.

This data identifies the frequency of recurrent mutations in T-ALL of Teenagers and Young Adults but reveals no striking differences with those observed in T-ALL in younger children. We are in the process of analysing additional cases to complete a total of 75 patients, including whole exome sequencing of a subset of cases. This sample size will allow us to detect genomic aberrations with a 5% incidence within this cohort (>90% probability) and to determine any prognostic significance. The most frequently observed aberration is CDKN2A gene deletion. In some cases without CDKN2A deletion we identified CDKN2A gene promoter methylation, offering an alternative mechanism of CDKN2A inactivation. To be able to appreciate potential cooperation between genetic abnormalities, we are correlating aberrant expression of the oncogenes TAL1, LYL1, LMO1, LMO2, TLX1, TLX3, NKX2, ERG and MEF2C with the genomic abnormalities identified.

Gribben:Celgene: Honoraria; Roche: Honoraria; Merck: Honoraria; Mundipharma: Honoraria; Pharmacyclics: Honoraria.