Wayne M. Koch

Exome Sequencing of Head and Neck Squamous Cell Carcinoma Reveals Inactivating Mutations in NOTCH1

The mutational profile of head and neck cancer is complex and may pose challenges to the development of targeted therapies. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To explore the genetic origins of this cancer, we used whole-exome sequencing and gene copy number analyses to study 32 primary tumors. Tumors from patients with a history of tobacco use had more mutations than did tumors from patients who did not use tobacco, and tumors that were negative for human papillomavirus (HPV) had more mutations than did HPV-positive tumors. Six of the genes that were mutated in multiple tumors were assessed in up to 88 additional HNSCCs. In addition to previously described mutations in TP53, CDKN2A, PIK3CA, and HRAS, we identified mutations in FBXW7 and NOTCH1. Nearly 40% of the 28 mutations identified in NOTCH1 were predicted to truncate the gene product, suggesting that NOTCH1 may function as a tumor suppressor gene rather than an oncogene in this tumor type.

Molecular assessment of histopathological staging in squamous-cell carcinoma of the head and neck.

BACKGROUND Surgical oncologists rely heavily on the histopathological assessment of surgical margins to ensure total excision of the tumor in patients with head and neck cancer. However, current techniques may not detect small numbers of cancer cells at the margins of resection or in cervical lymph nodes. METHODS We used molecular techniques to determine whether clonal populations of infiltrating tumor cells harboring mutations of the p53 gene could be detected in histopathologically negative surgical margins and cervical lymph nodes of patients with squamous-cell carcinoma of the head and neck. RESULTS We identified 25 patients with primary squamous-cell carcinoma of the head and neck containing a p53 mutation who appeared to have had complete tumor resection on the basis of a negative histopathological assessment. In 13 of these 25 patients, molecular analysis was positive for a p53 mutation in at least one tumor margin. In 5 of 13 patients with positive margins by this method (38 percent), the carcinoma has recurred locally, as compared with none of 12 patients with negative margins (P = 0.02 by the log-rank test). Furthermore, molecular analysis identified neoplastic cells in 6 of 28 lymph nodes (21 percent) that were initially negative by histopathological assessment. CONCLUSIONS Among specimens initially believed to be negative by light microscopy, a substantial percentage of the surgical margins and lymph nodes from patients with squamous-cell carcinoma of the head and neck contained p53 mutations specific for the primary tumor. Patients with these positive margins appear to have a substantially increased risk of local recurrence. Molecular analysis of surgical margins and lymph nodes can augment standard histopathological assessment and may improve the prediction of local tumor recurrence.

Association between cigarette smoking and mutation of the p53 gene in squamous-cell carcinoma of the head and neck.

BACKGROUND Although epidemiologic studies have long associated tobacco and alcohol use with the development of squamous-cell carcinoma of the head and neck, the molecular targets of these carcinogens have yet to be identified. We performed a molecular analysis to determine the pattern of mutations in the p53 gene in neoplasms from patients with squamous-cell carcinoma of the head and neck and a history of tobacco or alcohol use. METHODS Sequence analysis of the conserved regions of the p53 gene was performed in tumor samples from 129 patients with primary squamous-cell carcinoma of the head and neck. We then used statistical analysis to identify any patient characteristics associated with mutation of the p53 gene. RESULTS We found p53 mutations in 42 percent of the patients (54 of 129). Fifty-eight percent of the patients who smoked cigarettes and used alcohol (37 of 64; 95 percent confidence interval, 45 to 70 percent), 33 percent of the patients who smoked but abstained from alcohol (13 of 39; 95 percent confidence interval, 19 to 50 percent), and 17 percent of the patients who neither smoked nor drank alcohol (4 of 24, 95 percent confidence interval, 5 to 37 percent) had p53 mutations (P = 0.001). (Two patients used alcohol but did not smoke, and neither had a p53 mutation.) Furthermore, 100 percent of the mutations in the patients who neither drank nor smoked occurred at sites containing cytidine phosphate guanosine dinucleotides (potentially representing endogenous mutations) within the p53 gene (5 of 5 mutations; 95 percent confidence interval, 48 to 100 percent), whereas only 23 percent of those in cigarette smokers consisted of such changes (12 of 53 mutations; 95 percent confidence interval, 12 to 36 percent; P = 0.001). CONCLUSIONS In our study, a history of tobacco and alcohol use was associated with a high frequency of p53 mutations in patients with squamous-cell carcinoma of the head and neck. Preliminary evidence linked cigarette smoking to p53 mutations at nonendogenous mutation sites. Our findings suggest a role for tobacco in the molecular progression of squamous-cell carcinoma of the head and neck and support the epidemiologic evidence that abstinence from smoking is important to prevent head and neck cancer.

Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloid-derived suppressor cell function

Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, tadalafil, and vardenafil) are agents currently in clinical use for nonmalignant conditions. We report the use of PDE5 inhibitors as modulators of the antitumor immune response. In several mouse tumor models, PDE5 inhibition reverses tumor-induced immunosuppressive mechanisms and enables a measurable antitumor immune response to be generated that substantially delays tumor progression. In particular, sildenafil, down-regulates arginase 1 and nitric oxide synthase–2 expression, thereby reducing the suppressive machinery of CD11b+/Gr-1+ myeloid-derived suppressor cells (MDSCs) recruited by growing tumors. By removing these tumor escape mechanisms, sildenafil enhances intratumoral T cell infiltration and activation, reduces tumor outgrowth, and improves the antitumor efficacy of adoptive T cell therapy. Sildenafil also restores in vitro T cell proliferation of peripheral blood mononuclear cells from multiple myeloma and head and neck cancer patients. In light of the recent data that enzymes mediating MDSC-dependent immunosuppression in mice are active also in humans, these findings demonstrate a potentially novel use of PDE5 inhibitors as adjuncts to tumor-specific immune therapy.

CYSTIC LYMPH NODE METASTASIS IN PATIENTS WITH HEAD AND NECK CANCER: AN HPV-ASSOCIATED PHENOMENON

Cystic lymph node metastases have been associated with tonsil cancer. A subset of oropharyngeal cancers contain human papillomavirus (HPV) DNA. The clinical and virologic associations of cystic nodal metastasis in head and neck cancer (HNSCC) were investigated.

Head and neck cancer in nonsmokers: A distinct clinical and molecular entity

Objectives/Hypothesis: Clinical and molecular patterns of head and neck squamous cell carcinoma (HNSCC) in nonsmokers and smokers may be different. Analysis of these patterns may improve understanding and management of this disease.

Inverse relationship between human papillomavirus-16 infection and disruptive p53 gene mutations in squamous cell carcinoma of the head and neck

Purpose: Squamous cell carcinomas of the head and neck (HNSCC) often harbor p53 mutations, but p53 protein degradation by the viral oncoprotein E6 may supercede p53 mutations in human papillomavirus 16 (HPV16)–positive tumors. The prevalence of p53 mutations in HPV-positive HNSCCs is indeed lower, but in some tumors these alterations coexist. The purpose of this study was to discern whether HNSCCs differ in the type of p53 mutations as a function of HPV16 status. Experimental Design: The study was nested within a prospective multicenter study (ECOGE 4393/RTOG R9614) of patients with HNSCC treated surgically with curative intent. Tumors from one study center were used to construct a tissue microarray. The tumors were well characterized with respect to p53 mutational status. The tissue microarray was evaluated by HPV16 in situ hybridization. HPV16 analysis was also done on a select group of tonsillar carcinomas known to harbor disruptive p53 mutations defined as stop mutations or nonconservative mutations within the DNA binding domain. Results: HPV16 was detected in 12 of 89 (13%) HNSCCs. By tumor site, HPV16 was detected in 12 of 21 (57%) tumors from the palatine/lingual tonsils, but in none of 68 tumors from nontonsillar sites (P < 0.00001). Both HPV16-positive and HPV16-negative HNSCCs harbored p53 mutations (25% versus 52%), but disruptive mutations were only encountered in HPV16-negative carcinomas. Of seven tonsillar carcinomas with disruptive p53 mutations, none were HPV16 positive, in contrast to HPV16-positive tonsillar carcinomas without disruptive p53 mutations (0% versus 57%; P = 0.008). Conclusions: Although HPV16 and mutated p53 may coexist in a subset of HNSCCs, HPV16 and disruptive p53 mutations seem to be nonoverlapping events. A less calamitous genetic profile, including the absence of disruptive p53 mutations, may underlie the emerging clinical profile of HPV16-positive HNSCC such as improved patient outcome.

Frequency and phenotypic implications of mitochondrial DNA mutations in human squamous cell cancers of the head and neck.

Mitochondrial genomic mutations are found in a variety of human cancers; however, the frequency of mitochondrial DNA (mtDNA) mutations in coding regions remains poorly defined, and the functional effects of mitochondrial mutations found in primary human cancers are not well described. Using MitoChip, we sequenced the whole mitochondrial genome in 83 head and neck squamous cell carcinomas. Forty-one of 83 (49%) tumors contained mtDNA mutations. Mutations occurred within noncoding (D-loop) and coding regions. A nonrandom distribution of mutations was found throughout the mitochondrial enzyme complex components. Sequencing of margins with dysplasia demonstrated an identical nonconservative mitochondrial mutation (A76T in ND4L) as the tumor, suggesting a role of mtDNA mutation in tumor progression. Analysis of p53 status showed that mtDNA mutations correlated positively with p53 mutations (P < 0.002). To characterize biological function of the mtDNA mutations, we cloned NADH dehydrogenase subunit 2 (ND2) mutants based on primary tumor mutations. Expression of the nuclear-transcribed, mitochondrial-targeted ND2 mutants resulted in increased anchorage-dependent and -independent growth, which was accompanied by increased reactive oxygen species production and an aerobic glycolytic metabolic phenotype with hypoxia-inducible factor (HIF)-1α induction that is reversible by ascorbate. Cancer-specific mitochondrial mutations may contribute to development of a malignant phenotype by direct genotoxic effects from increased reactive oxygen species production as well as induction of aerobic glycolysis and growth promotion.

Head and neck squamous cell carcinoma cell lines: established models and rationale for selection.

Head and neck squamous cell carcinoma (HNSCC) cell lines are important preclinical models in the search for novel and targeted therapies to treat head and neck cancer. Unlike many other cancer types, a wide variety of primary and metastatic HNSCC cell lines are available. An easily accessible guide that organizes important characteristics of HNSCC cell lines would be valuable for the selection of appropriate HNSCC cell lines for in vitro or in vivo studies.

Detection of transcriptionally active high risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method

Evidence for transcriptional activation of the viral oncoproteins E6 and E7 is regarded as the gold standard for the presence of clinically relevant human papillomavirus (HPV), but detection of E6/E7 mRNA requires RNA extraction and polymerase chain reaction amplification—a challenging technique that is restricted to the research laboratory. The development of RNA in situ hybridization (ISH) probes complementary to E6/E7 mRNA permits direct visualization of viral transcripts in routinely processed tissues and has opened the door for accurate HPV detection in the clinical care setting. Tissue microarrays containing 282 head and neck squamous cell carcinomas from various anatomic subsites were tested for the presence of HPV using p16 immunohistochemistry, HPV DNA ISH, and an RNA ISH assay (RNAscope) targeting high-risk HPV E6/E7 mRNA transcripts. The E6/E7 mRNA assay was also used to test an additional 25 oropharyngeal carcinomas in which the HPV status as recorded in the surgical pathology reports was equivocal due to conflicting detection results (ie, p16 positive, DNA ISH negative). By the E6/E7 mRNA method, HPV was detected in 49 of 282 (17%) HNSCCs including 43 of 77 (56%) carcinomas from the oropharynx, 2 of 3 (67%) metastatic HNSCCs of an unknown primary site, 2 of 7 (29%) carcinomas from the sinonasal tract, and 2 of 195 (1%) carcinomas from other head and neck sites. p16 expression was strongly associated with the presence of HPV E6/E7 mRNA: 46 of 49 HPV-positive tumors exhibited p16 expression, whereas only 22 of 233 HPV-negative tumors were p16 positive (94% vs. 9%, P<0.0001). There was also a high rate of concordance (99%) between the E6/E7 mRNA method and HPV DNA ISH. For the selected group of discordant HNSCCs (p16+/HPV DNA−), the presence of E6/E7 transcripts was detected in 21 of 25 (84%) cases. The E6/E7 mRNA method confirmed the presence of transcriptionally active HPV-related HNSCC that has a strong predilection for the oropharynx and is strongly associated with high levels of p16 expression. Testing for HPV E6/E7 transcripts by RNA ISH is ideal because it confirms the presence of integrated and transcriptionally active virus, permits visualization of viral transcripts in tissues, and is technically feasible for routine testing in the clinical laboratory.