Fine-mapping loss of gene architecture at the CDKN2B (p15INK4b), CDKN2A (p14ARF, p16INK4a) and MTAP genes in head and neck sduamous cell carcinoma

Objective: To identify the extent and the smallest region of loss for CDKN2B(INK4nb), CDKN2A(ARF,INK4a), and MTAP. Homozygous deletions of human chromosome 9p21 occur frequently in malignant cell lines and are common in squamous cell carcinoma of the head and neck (HNSCC). This complex region encodes the tumor suppressor genes cyclin-dependent kinase 2B (CDKN2B) (p15(INK4b)) and CDKN2A (p14(ARF), p16(INK4a)) and the housekeeping gene methylthioadenosine phosphorylase (MTAP). Design: A targeted probe panel designed to finely map the region of 9p21 loss comprised 3 probes for CDKN2B(INK4b), 7 for CDKN2A(ARF,INK4a) and 3 for MTAP and was interrogated using the multiplex ligation-dependent probe amplification assay (MLPA). The MLPA genomic copy number alterations for CDKN2A were validated using real-time polymerase chain reaction. Subjects: Six HNSCC primary (A) and recurrent or metastatic (B) cell lines were examined: UMSCC-11A/11B, UMSCC-17A/17B, and UMSCC-81A/81B. Results: Cell line UMSCC-11B retained all 9p loci tested in the region. Cell lines UMSCC-17A/B indicated homozygous deletion of CDKN2A(ARF,INK4a) starting at p16(INK4) exon 1 alpha to include exons 2 and 3. Homozygous loss was indicated for CDKN2B(INK4b) and CDKN2A(ARF,INK4a) in UMSCC-11A, and UMSCC-81A. Cell line UMSCC-81B indicated retention of all 9p loci except for exon la (p16(INK4a)). Selective loss of the 3' end of MTAP was observed in UMSCC-11A. Genomic alterations by fine-mapping MLPA were validated at the DNA level for CDKN2A. Conclusions: We identified exon la (p16(INK4a)) as the smallest region of loss in the CDKN2A(ARF,INK4a) gene. The frequency and precise loss of CDKN2B(INK4b) CDKN2A(ARF,INK4a) and MTAP in the prognosis of 9p21-deleted HNSCC may provide impetus for use of these targets as therapeutic biomarkers in head and neck cancer.