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. 2013 Jul;3(7):770-81.
doi: 10.1158/2159-8290.CD-12-0537. Epub 2013 Apr 25.

Integrative genomic characterization of oral squamous cell carcinoma identifies frequent somatic drivers

Curtis R Pickering  1 , Jiexin Zhang, Suk Young Yoo, Linnea Bengtsson, Shhyam Moorthy, David M Neskey, Mei Zhao, Marcus V Ortega Alves, Kyle Chang, Jennifer Drummond, Elsa Cortez, Tong-Xin Xie, Di Zhang, Woonbok Chung, Jean-Pierre J Issa, Patrick A Zweidler-McKay, Xifeng Wu, Adel K El-Naggar, John N Weinstein, Jing Wang, Donna M Muzny, Richard A Gibbs, David A Wheeler, Jeffrey N Myers, Mitchell J Frederick
Affiliations

Integrative genomic characterization of oral squamous cell carcinoma identifies frequent somatic drivers

Curtis R Pickering et al. Cancer Discov. 2013 Jul.

Abstract

The survival of patients with oral squamous cell carcinoma (OSCC) has not changed significantly in several decades, leading clinicians and investigators to search for promising molecular targets. To this end, we conducted comprehensive genomic analysis of gene expression, copy number, methylation, and point mutations in OSCC. Integrated analysis revealed more somatic events than previously reported, identifying four major driver pathways (mitogenic signaling, Notch, cell cycle, and TP53) and two additional key genes (FAT1, CASP8). The Notch pathway was defective in 66% of patients, and in follow-up studies of mechanism, functional NOTCH1 signaling inhibited proliferation of OSCC cell lines. Frequent mutation of caspase-8 (CASP8) defines a new molecular subtype of OSCC with few copy number changes. Although genomic alterations are dominated by loss of tumor suppressor genes, 80% of patients harbored at least one genomic alteration in a targetable gene, suggesting that novel approaches to treatment may be possible for this debilitating subset of head and neck cancers.

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Conflict of interest statement

Conflicts: The authors disclose no potential conflicts of interest.

Figures

Fig. 1
Fig. 1
Integrated analysis of gene expression and copy number alterations. A) Copy number and outlier gene expression plot. Frequency indicates how often a gene with the indicated relative CN shows low, normal or high transcript expression. Frequencies were calculated from all genes and all samples with the indicated relative CN. High or low expression identifies samples outside of the 95% confidence interval for expression as described in B below. B) Gene expression vs. copy number for CCND1. Each dot represents one sample. Black horizontal bars designate gene expression mean. Red bars indicate the 95% confidence interval of gene expression for samples with relative CN=0. C) Modified IGV plot for the region around CCND1. Chromosome location is shown at the top. Each row is a sample, and the relative CN is indicated by color. Black boxes around a gene indicate it as an outlier with respect to expression.
Fig. 2
Fig. 2
Integrated mRNA and microRNA expression and DNA methylation clusters. A) Clustered heatmap of highly variable mRNAs and microRNAs. Sample IDs are shown at the bottom. B) Plots showing total number of mutations (top) and average ploidy (bottom) per sample for expression clusters 1 and 2. Median or mean values are shown by bars. P-values are indicated in the upper left and calculated by Mann Whitney test or Student’s t-test. C) Clustered heatmap of bimodally methylated probes. Methylated probes are shown in red. D) Plot showing the total number of CNAs per sample for methylation clusters 1 and 2.
Fig. 3
Fig. 3
NOTCH1 is detrimental to HNSCC cell lines. A) NOTCH1 genomic alterations identified in HNSCC cell lines. Mutations of unknown significance are indicated with an *. B) Western blot for total NOTCH1 protein. WT=wild-type, MS=missense, NS=non-sense. FL indicates full length NOTCH1, and TM indicates the transmembrane form. Actin is shown as a loading control. C) Flow cytometry for GFP in HN31 cells infected with retrovirus to express activated NOTCH1 (ICN1). The frequency of events is shown inside each gate. D) Relative cell fractions for the “horse-race” experiment. GFP-positive fraction was normalized to the frequency at day 4. Vector controls are shown with solid lines and ICN1-expressing cells are shown with dashed lines. E) Xenograft tumor growth in an orthotopic mouse model of tongue cancer. HN31 or UM-SCC-47 cell lines were infected with virus to express the full-length NOTCH1 (NFL), the activated form of NOTCH1 (ICN1), or the empty vector. F) Cell cycle analysis of HN31 cells expressing ICN1 or empty vector. Samples were analyzed at day 3 post infection. G) Western blot for p21 (CDKN1A) protein in cells expressing ICN1 or vector. Samples were collected that the indicated time post-infection. Actin is shown as a loading control. H) Senescence-associated beta-galactosidase staining in cells expressing ICN1 or vector. Cells with positive staining appear blue.
Fig. 4
Fig. 4
Driver pathways and key genes in OSCC. A) Pathway alteration in each sample is indicated by a black box. Sample IDs are shown at the top and pathway names at the left. Selected clinical variables and clusters from Fig. 3 are indicated with colored boxes. B) Notch pathway. C) Mitogenic signaling pathway. D) Cell cycle pathway. E) TP53 pathway. Pathway diagrams indicate the genes included in each pathway. Copy gains are indicated with red, copy losses with blue, mutations with black and methylation with purple. Percentages shown indicate the frequency of that event.
Fig. 5
Fig. 5
CASP8 mutations are associated with fewer CNAs and RAS-family mutations. A) Number of CNAs by CASP8 mutation status in OSCC. B) Focal CNAs by CASP8 status in TCGA HNSCC samples. C) Focal CNAs by CASP8 status in TCGA CRC samples. Median values are shown by the bar. P-values were calculated by Mann Whitney test. D) Number of samples with various CASP8 and Ras-family genotypes. P-values were calculated with Fisher’s exact test. E) CASP8 genomic alterations identified in HNSCC cell lines. Mutations of unknown significance are indicated with an *. F) Frequency of tumor formation based on CASP8 mutation status. Mean values are shown by the bar. P-value was calculated by Mann Whitney test. G) Difference in final tumor volume by CASP8 mutation status. P-value was calculated by t-test. Sample numbers are the same as in H. H) Overall mouse survival by CASP8 mutation status. P-value was calculated by log-rank test. Mouse tumor data are from Sano et.al.(14)
Fig. 6
Fig. 6
Targetable genomic alterations. Genes and frequencies are shown on the left. Approved therapeutics targeting each listed gene are shown on the right. Sample names are along the top. Only copy number events that also demonstrated high expression are shown.
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