Analysis of head and neck carcinoma progression reveals novel and relevant stage-specific changes associated with immortalisation and malignancy
Authors: Veeramachaneni Ratna , Walker Thomas , De Weck Antoine , Revil Timothée , Badescu Dunarel , O’Sullivan James , Higgins Catherine , Elliott Louise , Liloglou Triantafillos , Risk Janet M. , Shaw Richard , Hampson Lynne , Hampson Ian , Dearden Simon , Woodwards Robert , Prime Stephen , Hunter Keith , Parkinson Eric Kenneth , Ragoussis Ioannis , Thakker Nalin 
Published: July 2018

AbstractHead and neck squamous cell carcinoma (HNSCC) is a widely prevalent cancer globally with high mortality and morbidity. We report here changes in the genomic landscape in the development of HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Frequent likely pathological mutations are restricted to a relatively small set of genes includingTP53, CDKN2A,FBXW7,FAT1,NOTCH1andKMT2D; these arise early in tumour progression and are present in PPOLs withNOTCH1mutations restricted to cell lines from lesions that subsequently progressed to HNSCC. The most frequent genetic changes are of consistent somatic copy number alterations (SCNA). The earliest SCNAs involved deletions ofCSMD1(8p23.2),FHIT(3p14.2) andCDKN2A(9p21.3) together with gains of chromosome 20.CSMD1deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines (promoter hypermethylation ~63%, hemizygous deletions ~75%, homozygous deletions ~18%). Forced expression ofCSMD1in the HNSCC cell line H103 showed significant suppression of proliferation (p=0.0053) and invasioninvitro(p=5.98X10−5) supporting a role forCSMD1inactivation in early head and neck carcinogenesis. In addition, knockdown ofCSMD1in theCSMD1-expressing BICR16 cell line showed significant stimulation of invasionin vitro(p=1.82 x 10−5) but not cell proliferation (p=0.239). HNSCC with and without nodal metastases showed some clear differences including high copy number gains ofCCND1, hsa-miR-548k andTP63in the metastases group. GISTIC peak SCNA regions showed significant enrichment (adj P<0.01) of genes in multiple KEGG cancer pathways at all stages with disruption of an increasing number of these involved in the progression to lymph node metastases. Sixty-seven genes from regions with statistically significant differences in SCNA/LOH frequency between immortal PPOL and HNSCC cell lines showed correlation with expression including 5 known cancer drivers.Lay SummaryCancers affecting the head and neck region are relatively common. A large percentage of these are of one particular type; these are generally detected late and are associated with poor prognosis. Early detection and treatment dramatically improve survival and reduces the damage associated with the cancer and its treatment. Cancers arise and progress because of changes in the genetic material of the cells. This study focused on identifying such changes in these cancers particularly in the early stages of development, which are not fully known. Identification of these changes is important in developing new treatments as well as markers of behaviour of cancers and also the early or ‘premalignant’ lesions. We used a well-characterised panel of cell lines generated from premalignant lesions as well as cancers, to identify mutations in genes, and an increase or decrease in number of copies of genes. We mapped new and previously identified changes in these cancers to specific stages in the development of these cancers and their spread. We additionally report here for the first time, alterations inCSMD1gene in early premalignant lesions; we further show that this is likely to result in increased ability of the cells to spread and possibly, multiply faster as well.