Whole Exome Sequencing of Flow Purified Tumour Cells Reveals Recurrently Mutated Genes and Pathways in Adult T-Cell Lymphoma/Leukaemia (ATLL)

Introduction

ATLL is an aggressive lymphoid malignancy with a very poor prognosis arising in patients with HTLV-1 infection. Asymptomatic HTLV1+ individuals have a polyclonal expansion of CD4+ T-cells driven by the products of the HTLV-1 tax and HBZ genes.  In ATLL, integration site analysis shows a monoclonal outgrowth (frequently associated with downregulation of tax) which is presumed to be driven by acquisition of oncogenic somatic mutations.  Genome-wide studies identifying these mutations in patients of African or African Caribbean origin have not been reported; limited data are available on a Japanese population.

Methods

We obtained nine cryopreserved peripheral blood mononuclear cell samples from seven patients of African Carribean origin. ATLL cells (CD3+CD4+CD25+CCR4+) and healthy B- and myeloid cells (CD3-CD33+ and CD3-CD20+) were sorted to high purity (>99%) by flow cytometry. B- and myeloid populations were pooled and used as a germline control. DNA was extracted from the sorted populations and whole exome sequencing libraries were prepared using Sure Select XT v5 (Agilent) and sequenced using an Illumina HiSeq2500.  Mean exome coverage was 145.3x in tumour samples and 131x in control samples with 96.3% and 96% respectively covered at >20x. Variants were called using GATK, germline variants were filtered out using VarScan, and all remaining variants were manually inspected using IGV. Copy number changes were called using ExomeDepth.

Results

We identified somatic mutations in all cases, including short insertions or deletions (InDels, median 3 per exome, range 2-12), single nucleotide variations (SNV) affecting the amino acid sequence (median 40, range 4-132) and noncoding SNV (median 35, range 12-114). Median variant allele frequency (VAF) was >20% in 8/9 tumour samples (overall median 40%, range 24-47%) indicating reliable isolation of substantially pure clonal tumour cells.  We excluded one sample with a low VAF (18%) from further analysis.

We identified two recurrent somatic point mutations: CCR4 Y331I (2 patients; a known activating mutation present in ~25% of ATLL cases) and NRG1 T232M (2 patients; a novel mutation). Both patients with CCR4 Y331I had large duplications encompassing the mutant CCR4 allele along with CCR1-CCR9; one of the two patients had been treated with the anti-CCR4 antibody Mogamulizumab and had been in complete remission for 20 months. We identified a further six genes harbouring somatic mutations in more than one patient: ANKRD30A, CDH7, PRKCB, RYR2, SETD5 and STAT3(each mutated in two patients).

In addition to these recurrently mutated genes, pathway analysis using ConsensusPath identified three pathways which were mutated in most or all patients.  NOTCH pathway members were mutated in 6/6 cases (with 3/6 cases carrying two mutations) in one of the following genes: NOTCH1, NOTCH2, MAMLD1, DMXL2, PTCRA, NOTCH2NL, TBL1XR1, HDAC10.

T-cell activation and costimulation pathway members were mutated in 5/6 patients (range 1-5 mutations). The following genes were affected: CARD11, CCR4, CD40LG, CHUK, DPP8, HLA-A, HLA-B, IFNAR2, NLRP2, P2RX7, PLCG1, RASSF5, SH2B3, ZEB1.

Finally we observed multiple mutations affecting the FGFR/EGFR – PI3K signalling pathway (6/6 patients, 1-6 mutations per patient). The affected genes were ADCY1, APBB1B, FGFR2, HGF, LRRFIP1, NCAM1, NRG1, PDGFC, PI3KCD, PLCG1, PLCXD1, PRKCB, STAT3 and ZMYM2.

In one patient, all 261 somatic mutations present in the blood were detected in a lymph node biopsy taken at the same time; an additional mutation (a frameshift deletion in FIP1L1) was present in the node but not detected in the blood.  In another patient, a sample taken at the time of relapse five years after initial presentation was also analysed: all 97 mutations called at diagnosis were present at relapse, and 15 additional somatic mutations had been acquired.

Conclusions

Whole exome sequencing identified two recurrent somatic point mutations and six recurrently mutated genes which, given their co-occurrence in this small sample, are likely to be tumour drivers.  Genes encoding components of the NOTCH, T-cell activation and FGFR/EGFR-PI3K pathways were mutated in most or all ATLL samples analysed, identifying these pathways as potential therapeutic targets.  We now plan to validate these candidate genes in a large and longitudinal patient cohort and compare these with results from an ongoing study in the Japanese population.