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. 2013 May 29;5(5):49.
doi: 10.1186/gm453. eCollection 2013.

Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV+ and HPV- tumors

Matthias Lechner  1 , Garrett M Frampton  2 , Tim Fenton  3 , Andrew Feber  3 , Gary Palmer  2 , Amrita Jay  4 , Nischalan Pillay  3 , Martin Forster  1 , Maureen T Cronin  2 , Doron Lipson  2 , Vincent A Miller  2 , Timothy A Brennan  2 , Stephen Henderson  3 , Francis Vaz  5 , Paul O'Flynn  5 , Nicholas Kalavrezos  5 , Roman Yelensky  2 , Stephan Beck  3 , Philip J Stephens  2 , Chris Boshoff  3
Affiliations

Targeted next-generation sequencing of head and neck squamous cell carcinoma identifies novel genetic alterations in HPV+ and HPV- tumors

Matthias Lechner et al. Genome Med. .

Abstract

Background: Human papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) is an emerging disease, representing a distinct clinical and epidemiological entity. Understanding the genetic basis of this specific subtype of cancer could allow therapeutic targeting of affected pathways for a stratified medicine approach.

Methods: Twenty HPV+ and 20 HPV- laser-capture microdissected oropharyngeal carcinomas were used for paired-end sequencing of hybrid-captured DNA, targeting 3,230 exons in 182 genes often mutated in cancer. Copy number alteration (CNA) profiling, Sequenom MassArray sequencing and immunohistochemistry were used to further validate findings.

Results: HPV+ and HPV- oropharyngeal carcinomas cluster into two distinct subgroups. TP53 mutations are detected in 100% of HPV negative cases and abrogation of the G1/S checkpoint by CDKN2A/B deletion and/or CCND1 amplification occurs in the majority of HPV- tumors.

Conclusion: These findings strongly support a causal role for HPV, acting via p53 and RB pathway inhibition, in the pathogenesis of a subset of oropharyngeal cancers and suggest that studies of CDK inhibitors in HPV- disease may be warranted. Mutation and copy number alteration of PI3 kinase (PI3K) pathway components appears particularly prevalent in HPV+ tumors and assessment of these alterations may aid in the interpretation of current clinical trials of PI3K, AKT, and mTOR inhibitors in HNSCC.

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Figures

Figure 1
Figure 1
Workflow of FFPE sample preparation and selection. Eighty-two FFPE blocks [19] were stained for p16 of which eight samples were excluded from further analysis, showing mixed p16 staining. Eight samples were excluded after the LCM step, yielding insufficient amounts or quality of DNA and two further samples were excluded due to inconsistent or borderline results in repeat E6 qPCR measurements. In total, 22 confirmed HPV+ (p16+ and E6 qPCR+) and 34 HPV- (p16- and E6 qPCR-) samples were suitable for further analysis. Following age and gender matching, 20 HPV+ HNSCC samples (red) and 20 HPV- HNSCC samples (grey) were then selected for the final analysis (next-generation (NG) sequencing).
Figure 2
Figure 2
Illustration of somatic events in HPV+ and HPV- HNSCC revealed by NGS of cancer-related genes. Relevant demographic and histological data are described above the heatmap of genomic changes. The color coding of the observed changes and patient characteristics are explained in the key on the right.
Figure 3
Figure 3
Hierarchical clustering of HPV+ and HPV- HNSCC samples using all detected genetic changes. HPV+ and HPV- HNSCC samples clustered in 100% of cases.
Figure 4
Figure 4
Validation of copy number changes by Infinium CNA profiling across all samples. (A) Forty-eight of 50 (96%) copy number alterations detected by sequencing were confirmed (green: confirmed, pink: not confirmed, grey: no data); (B) Genetic changes in 'P17_neg' detected by NGS (extracted from Figure 2); (C) Illustration of copy number changes (obtained from Infinium CNA Profiling) in 'P17_neg'. Both the loss of the CDKN2A and CDKN2B genes (in a region of loss within chromosome 9) and the gain of the CCND1 gene (in an amplified region of chromosome 11) are shown. Y-axis: log fold change of copy number, X-axis: copy number changes across all chromosomes.
Figure 5
Figure 5
Validation of detected copy number alterations of Cyclin D1 (CCND1) by immunohistochemistry. Staining of HNSCC samples for Cyclin D1 confirmed strong expression in eight of nine CCND1 amplified cases (and intermediate expression in the remaining case) compared with samples harboring no copy number alteration; Representative samples shown: Low levels of CCND1 expression in the tumor tissue of sample 'P38_pos' (A) and sample 'P29_neg' (B); NGS: No CNA; High levels of Cyclin D1 expression in the tumor tissue of sample 'P12_neg' (C) and sample 'P17_neg' (D); NGS: CCND1 copy number gain.
Figure 6
Figure 6
Validation of detected PTEN copy number loss by immunohistochemistry. Staining of HNSCC samples for PTEN was negative in all cases in which deep sequencing revealed a homozygous deletion or mutation. Representative samples shown: Abundant PTEN expression in the tumor tissue of sample 'P26_pos' (A) and sample 'P70_neg' (B); Deep-sequencing: No CNA; Absence of PTEN protein in the tumor tissue of sample 'P60_pos' (C) and sample 'P13_pos' (D); Deep-sequencing: PTEN copy number loss.
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References

    1. Chaturvedi AK, Engels EA, Pfeiffer RM, Hernandez BY, Xiao W, Kim E, Jiang B, Goodman MT, Sibug-Saber M, Cozen W, Liu L, Lynch CF, Wentzensen N, Jordan RC, Altekruse S, Anderson WF, Rosenberg PS, Gillison ML. Human papillomavirus and rising oropharyngeal cancer incidence in the United States. J Clin Oncol. 2011;5:4294–4301. doi: 10.1200/JCO.2011.36.4596. - DOI - PMC - PubMed
    1. Mendenhall WM, Logan HL. Human papillomavirus and head and neck cancer. Am J Clin Oncol. 2009;5:535–539. doi: 10.1097/COC.0b013e31818b8fee. - DOI - PubMed
    1. Fakhry C, Westra WH, Li S, Cmelak A, Ridge JA, Pinto H, Forastiere A, Gillison ML. Improved survival of patients with human papillomavirus-positive head and neck squamous cell carcinoma in a prospective clinical trial. J Natl Cancer Inst. 2008;5:261–269. doi: 10.1093/jnci/djn011. - DOI - PubMed
    1. Agrawal N, Frederick MJ, Pickering CR, Bettegowda C, Chang K, Li RJ, Fakhry C, Xie TX, Zhang J, Wang J, Zhang N, El-Naggar AK, Jasser SA, Weinstein JN, Trevino L, Drummond JA, Muzny DM, Wu Y, Wood LD, Hruban RH, Westra WH, Koch WM, Califano JA, Gibbs RA, Sidransky D, Vogelstein B, Velculescu VE, Papadopoulos N, Wheeler DA, Kinzler KW. et al. Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science. 2011;5:1154–1157. doi: 10.1126/science.1206923. - DOI - PMC - PubMed
    1. Stransky N, Egloff AM, Tward AD, Kostic AD, Cibulskis K, Sivachenko A, Kryukov GV, Lawrence MS, Sougnez C, McKenna A, Shefler E, Ramos AH, Stojanov P, Carter SL, Voet D, Cortes ML, Auclair D, Berger MF, Saksena G, Guiducci C, Onofrio RC, Parkin M, Romkes M, Weissfeld JL, Seethala RR, Wang L, Rangel-Escareno C, Fernandez-Lopez JC, Hidalgo-Miranda A, Melendez-Zajgla J. et al. The mutational landscape of head and neck squamous cell carcinoma. Science. 2011;5:1157–1160. doi: 10.1126/science.1208130. - DOI - PMC - PubMed