Joseph A. Califano

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.

Trends in incidence and prognosis for head and neck cancer in the United States: A site-specific analysis of the SEER database

Despite recent advances in the diagnosis and treatment of head and neck cancer, there has been little evidence of improvement in 5‐year survival rates over the last few decades. To determine more accurate trends in site‐specific outcomes as opposed to a more general overview of head and neck cancer patients, we analyzed the site‐specific data collected in the Surveillance, Epidemiology, and End Results—SEER Public‐Use Database 1973–1999. Based on the selection criteria, 96,232 cases were evaluated for trend analysis in incidence, clinical stage, treatment and 5‐year survival. During the period 1973–1999, site‐specific incidence rates for head and neck cancer changed significantly. Site‐specific analysis of survival from 1974–1997 showed significant improvements in 5‐year survival rates for cancers of the nasopharynx, oropharynx and hypopharynx (38.1% to 56.7% for nasopharynx, p < 0.001; 36.3% to 49.1% for oropharynx, p = 0.001 and 28.3% to 33.3% for hypopharynx, p = 0.015). The prognosis for early‐stage salivary gland cancer during 1983–1997 and late‐stage larynx cancer during 1974–1997 also demonstrated improvement (82.7% to 88.5%, p = 0.012 and 22.2% to 38.3%, p = 0.013, respectively). On the other hand, the prognosis for regional stage oral cavity cancer as well as early‐stage larynx cancer patients declined during 1983–1997 (49.2% to 43.8%, p = 0.032 and 82.3% to 74.3%, p = 0.002, respectively). Site‐specific changes in treatment and staging were also noted. Site‐specific analysis allows for a more accurate description of incidence, staging, treatment, and prognostic trends for 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.

Consensus Statement on the Classification and Terminology of Neck Dissection

OBJECTIVE To update the guidelines for neck dissection terminology, as previously recommended by the American Head and Neck Society. PARTICIPANTS Committee for Neck Dissection Classification, American Head and Neck Society; representation from the Committee for Head and Neck Surgery and Oncology, American Academy of Otolaryngology-Head and Neck Surgery (T.A.D.). EVIDENCE Review of current literature on neck dissection classification. CONSENSUS PROCESS Semiannual face-to-face meetings of the Committee for Neck Dissection Terminology and e-mail correspondence. CONCLUSIONS Standardization of terminology for neck dissection is important for communication among clinicians and researchers. New recommendations have been made regarding the following: boundaries between levels I and II and between levels III/IV and VI; terminology of the superior mediastinal nodes; and the method of submitting surgical specimens for pathologic analysis.

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.

MicroRNA alterations in head and neck squamous cell carcinoma

MicroRNAs (mirs) are small noncoding RNA molecules (∼22 nucleotides) that regulate posttranscriptional gene expression. Currently, there has not been a comprehensive study of their role in primary head and neck squamous cell carcinoma (HNSCC). To determine the role of mirs in HNSCC, we screened for altered microRNA expression in HNSCC primary tissue and cell lines. We then further tested the functional impact of alterations of specific mirs. An initial screening of 4 primary HNSCC, 4 normal mucosal controls and 4 HNSCC cell lines was analyzed for mature microRNA expression by microarray. Significance was determined using significance analysis of microarrays (SAM). Nine microRNAs were found by SAM to be upregulated or downregulated in tumor tissue including mir‐21, let‐7, 18, 29c, 142‐3p, 155, 146b (overexpressed) and 494 (underexpressed). Mir‐21 was validated by qRT‐PCR. Functional validation by growth assays was performed, further validating mir‐21. Transfection of mir‐21 into JHU‐011 and JHU‐012 cell lines showed a 39% increase in cell growth at 72 hr relative to controls (p < 0.05). Transfection of the inhibitor into JHU‐O12 cell lines showed a 92% decrease in cell growth relative to controls at 72 hr (p < 0.05). In addition, flow cytometry analysis of JHU‐012 cells 48 hr after mir‐21 inhibitor transfection showed a statistically significant increase in cytochrome c release and increased apoptosis. These differentially expressed microRNAs may be of interest as potential novel oncogenes and tumor suppressor genes in HNSCC. Mir‐21 is a putative oncogenic microRNA in head and neck cancer.

Quantitative detection of promoter hypermethylation of multiple genes in the tumor, urine, and serum DNA of patients with renal cancer.

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of human cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the urine and serum samples of renal cancer patients. We examined the tumor and the matched urine and serum DNA for aberrant methylation of nine gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-β2, and ARF) from 17 patients with primary kidney cancer by quantitative fluorogenic real-time PCR. An additional 9 urine samples (total, 26) and 1 serum sample (total, 18) also were tested from renal cancer patients. Urine from 91 patients without genitourinary cancer and serum from 30 age-matched noncancer individuals were used as controls. Promoter hypermethylation of at least two of the genes studied was detected in 16 (94%) of 17 primary tumors. Aberrant methylation in urine and serum DNA generally was accompanied by methylation in the matched tumor samples. Urine samples from 91 control subjects without evidence of genitourinary cancer revealed no methylation of the MGMT, GSTP1, p16, and ARF genes, whereas methylation of RAR-β2, RASSF1A, CDH1, APC, and TIMP3 was detected at low levels in a few control subjects. Overall, 23 (88%) of 26 urine samples and 12 (67%) of 18 serum samples from cancer patients were methylation positive for at least one of the genes tested. By combination of urine or serum analysis of renal cancer patients, hypermethylation was detected in 16 of 17 patients (94% sensitivity) with high specificity. Our findings suggest that promoter hypermethylation in urine or serum can be detected in the majority of renal cancer patients. This noninvasive high-throughput approach needs to be evaluated in large studies to assess its value in the early detection and surveillance of renal cancer.

Promoter methylation and inactivation of tumour-suppressor genes in oral squamous-cell carcinoma

Genetic alterations that lead to loss or changes in tumour-suppressor genes are known to contribute to oral carcinogenesis. Traditional molecular methods to detect such losses have relied on mutation analysis or deletion of the gene. However, epigenetic mechanisms could also contribute to silencing of tumour-suppressor genes. Methylation regions rich in CpG promoters prevent DNA transcription by changing the binding of histone complexes. The substantial contribution of methylation, specifically in oral squamous-cell carcinoma, is now being realised and investigated.

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.

ΔNp63 induces β-catenin nuclear accumulation and signaling

Abstract The P53 homolog p63 encodes multiple proteins with transactivating, apoptosis-inducing, and oncogenic activities. We showed that p63 is amplified and that ΔNp63 isotypes are overexpressed in squamous cell carcinoma (SCC) and enhance oncogenic growth in vitro and in vivo. Moreover, p53 associated with ΔNp63α and mediated its degradation. Here, we report that ΔNp63 associates with the B56α regulatory subunit of protein phosphatase 2A (PP2A) and glycogen synthase kinase 3β (GSK3β), leading to a dramatic inhibition of PP2A-mediated GSK3β reactivation. The inhibitory effect of ΔNp63 on GSK3β mediates a decrease in phosphorylation levels of β-catenin, which induces intranuclear accumulation of β-catenin and activates β-catenin-dependent transcription. Our results suggest that ΔNp63 isotypes act as positive regulators of the β-catenin signaling pathway, providing a basis for their oncogenic properties.