Toshihito Suda

Copy number amplification of the PIK3CA gene is associated with poor prognosis in non-lymph node metastatic head and neck squamous cell carcinoma.

BackgroundDeregulation of the EGFR signaling pathway is one of the most frequently observed genetic abnormalities that drives cancer development. Although mutations in the downstream components of the EGFR signaling pathway, including KRAS, BRAF and PIK3CA, have been reported in numerous cancers, extensive mutation and copy number analysis of these genes in clinical samples has not been performed for head and neck squamous cell carcinoma (HNSCC).MethodsWe examined the mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients.We used DNA sequencing to detect mutations and the copy number changes were evaluated by qPCR and array comparative genomic hybridization (CGH) analysis.ResultsWe examined the mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients. We identified 3 mutations (2.6%) in K-RAS and 3 mutations (2.6%) in PIK3CA. Copy number amplification was found in 37 cases (32.2%) for PIK3CA, 10 cases (8.7%) for K-RAS and 2 cases (1.7%) for BRAF. Kaplan-Meier survival analysis revealed that copy-number amplification of PIK3CA was markedly associated with cancer relapse in patients without lymph node metastasis. (Log-rank test, p = 0.026)ConclusionsCopy number amplification of the PIK3CA gene is associated with poor prognosis in HNSCC patients without lymph node metastasis. The PIK3CA copy number status will serve as a marker of poor prognosis in patients with HNSCC.

Distinct effects of alcohol consumption and smoking on genetic alterations in head and neck carcinoma.

Background Tobacco and alcohol consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Recently, whole-exome sequencing clarified that smoking increased TP53 and other mutations in HNSCC; however, the effects of alcohol consumption on these genetic alterations remain unknown. We explored the association between alcohol consumption and somatic copy-number alterations (SCNAs) across the whole genome in human papillomavirus (HPV)-negative HNSCCs, and compared with the effects of smoking on genetic alterations. Methods SCNA and TP53 mutations in tumor samples were examined by high-resolution comparative genomic hybridization microarray 180K and by direct sequencing, respectively, and statistically analyzed for associations with alcohol consumption and smoking during the 20 years preceding diagnosis of HNSCC. Probes with a corrected p-value (=q-value) less than 0.05 and fold change greater than 1.2 or less than -1.2 were considered statistically significant. Results A total of 248 patients with HNSCC were enrolled. In the HPV-negative patients (n=221), heavy alcohol consumption was significantly associated with SCNAs of oncogenes/oncosuppressors that were previously reported to occur frequently in HNSCCs: CDKN2A (q=0.005), FHIT (q=0.005), 11q13 region including CCND1, FADD and CTTN (q=0.005), ERBB2 (HER2) (q=0.009), 3q25-qter including CCNL1, TP63, DCUN1D1 and PIK3CA (q=0.014), and CSMD1 (q=0.019). But, TP53 mutations were not affected. In contrast, smoking was associated with increased risk of TP53 mutations, but did not induce any significant SCNAs of oncogenes/oncosuppressors. Conclusion These results suggest that both alcohol consumption and smoking had distinct effects on genetic alterations in HNSCCs. Heavy alcohol consumption may trigger previously known and unknown SCNAs, but may not induce TP53 mutation. In contrast, smoking may induce TP53 mutation, but may not trigger any SCNAs.

Functional mutation analysis of EGFR family genes and corresponding lymph node metastases in head and neck squamous cell carcinoma

Tumors with certain mutations in the epidermal growth factor receptor (EGFR) family genes dramatically respond to EGFR inhibitors. Therefore, these mutations are important factors that influence disease progression and patient survival. We previously studied the mutation status of EGFR in patients with head and neck squamous cell carcinoma (HNSCC). However, the mutation status of lymph node metastases and the frequency of mutations in EGFR family genes have not been extensively studied. In this study, we sequenced the catalytic domains of the three other members of the EGFR family, HER2, HER3, and HER4 in 92 clinical samples of HNSCC. We identified a HER2 mutation (K716E) in one sample but no mutations were found in HER3 or HER4. Next to investigate the relationship between EGFR mutations and tumor metastasis, we compared the DNA sequences of the EGFR gene between the primary tumor and the lymph node metastasis in 31 clinical samples. Only one of the patients with an EGFR mutation in the primary HNSCC carried the same mutation (L858R) in the lymph node metastasis. Finally, we explored the tumorigenic potential of the EGFR mutations that we had previously identified and their sensitivity to two different EGFR tyrosine kinase inhibitors (CL-387785, OSI-420). Ba/F3 cells transformed with mutant EGFR genes were sensitive to treatment with lower concentrations of CL-387785 than of OSI-420. These results contribute to our understanding of the genetic basis of drug sensitivity and will help design drugs that specifically target different subtypes of HNSCC.

The Presence of HPV DNA in Neck Lymph Node Metastasis Correlates with Improved Overall Survival of Patients with Oropharyngeal Cancer Undergoing Surgical Treatment

Background: Few studies have addressed how human papilloma virus (HPV) infection in oropharyngeal squamous cell carcinoma (OPSCC) affects the outcome of surgical therapy; furthermore, the relationship between the presence of HPV DNA and neck lymph node (LN) metastasis has not been well established. Methods: A total of 65 patients who underwent surgery as a first-line therapy for OPSCC were enrolled in this study. In HPV-positive patients, the presence of HPV DNA in metastatic neck LN lesions was evaluated. Results: The HPV-positive patients had significantly better overall survival than the HPV-negative patients (log-rank test, p = 0.04), whereas HPV infection status did not significantly affect disease-free survival (log-rank test, p = 0.65). In all of the HPV-positive OPSCC patients who developed cervical LN metastasis, the same HPV DNA type was found in both the primary tumour and the metastases. Conclusions: The present results suggest that HPV infection is a determining factor for good prognosis in patients undergoing first-line surgical therapy for OPSCC.

Abstract 23: Prognostic significance of vitamin D receptor polymorphisms in head and neck squamous cell carcinoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Objective Many studies demonstrate associations between UVB rays and a lower risk of developing various cancers, implying that UVB rays induce vitamin D synthesis, which suppresses growth and induces differentiation/apoptosis of cancer. In patients with non-small-cell lung cancer, vitamin D receptor (VDR) polymorphisms and haplotypes are reported to be associated with survival. We hypothesized that a similar association would be observed in other type of cancers. In this study, we focused on head and neck squamous-cell carcinoma (HNSCC). Methods DNA was extracted from the frozen tumor and the VDR polymorphisms (Cdx2, FokI and BsmI) in 97 patients with HNSCC were genotyped by sequencing. Disease-free survival was compared between VDR polymorphisms using Kaplan-Meier survival with log-rank tests and using the Cox proportional hazard model adjusted for age, gender, primary tumor site, and postoperative stages. Adjusted hazard ratios (AHR) and 95% confidence intervals (95% CI) were determined. Results Tumor recurrence occurred in 41/97 (42%) patients during a median follow-up of 603 days. Genotype frequencies of the VDR polymorphisms were as follows: FokI: C/C, 39 (40%); C/T, 48 (50%); TT, 10 (10%); for Cdx2: G/G, 16 (17%); G/A, 41 (42%); A/A, 40 (41%) Bsm I: A/A, 5 (5.2%); A/G, 16 (16.5%); and G/G, 76 (78.3%). The FokI T/T genotype was associated with poor prognosis: median survival for T/T was 265 days compared with 959 days for C/C or C/T (log-rank: P=0.015; AHR, 2.65; 95% CI, 1.18 to 5.96; P=0.018). In contrast, Cdx2 and BsmI showed no significant association with survival. The G-T (Cdx2-FokI) haplotype was associated with worse disease-free survival compared with other haplotypes: median disease-free survival for G-T was 238 days compared with 959 days for other haplotypes (log-rank: P=0.0008; AHR, 6.67; 95% CI, 2.23 to 20.0; P=0.001). Conclusion These results suggest that VDR polymorphisms and haplotypes may be associated with prognosis of patients with HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 23. doi:10.1158/1538-7445.AM2011-23

A case of the hypopharyngeal cancer with laryngeal necrosis after chemoradiotherapy

Laryngeal necrosis is regarded as a disease due to disturbances of circulation in the larynx ; however, its incidence rate is quite low. We experienced a case in which laryngeal necrosis developed at a relatively early stage after chemoradiotherapy was performed for hypopharyngeal cancer. The patient was a 58-year-old man. A necrotic lesion of the mucous membrane in the primary lesion developed only three months after receiving chemoradiotherapy for double cancer of the hypopharynx and esophagus. It was unresponsive to conservative treatment and was diagnosed as no risk of cancer recurrence as a result of biopsy. In view of rapid exacerbation of the necrotic lesion, salvage operations (enucleations of the hypopharynx, larynx, and esophagus, and anterolateral thigh flap reconstruction) were performed in order to avoid fatal situations such as carotid collapse. We identified the causes of larynx necrosis at an early stage as the primary site of infection and wide range of radiation.

Homozygous deletions of UGT2B17 modifies effects of smoking on TP53-mutations and relapse of head and neck carcinoma

BackgroundSmoking induces oncogenic TP53-mutations in head and neck squamous cell carcinomas (HNSCCs). Disruptive mutations of TP53-gene and expression of p16 protein [p16 (+)] in tumor tissue associate with worse and better prognosis, respectively. UDP-glucuronosyltransferase 2 family, polypeptide B17 (UGT2B17) detoxifies smoking-related metabolites. Differences among ethnic groups in UGT2B17 are extremely high. Homozygous deletions of UGT2B17 gene (UGT2B17-deletion) are a common copy number variant (CNV) among Japanese, but not a common CNV among Africans and Europeans. Thus, we examined Japanese patients with HNSCC to explore if UGT2B17-deletion and/or p16 (+) modify effects of smoking on TP53-mutations and affect relapse.MethodsWe conducted a posthoc analysis of a prospective cohort. Polymerase chain reaction, immunohistochemistry, and direct sequencing were used to determine UGT2B17-deletion, p16 (+), and detailed TP53-mutations, respectively.ResultsUGT2B17-deletion was observed in 80% of this study population. For this 80%, TP53-mutations were significantly more common among smokers than non-smokers (P = 0.0016), but this difference between smokers and nonsmokers was not significant for the 20% with UGT2B17. In patients with UGT2B17-deletion and p16 (+), simultaneously, TP53-mutations were much more common among smokers than among non-smokers (81% versus 17%; P = 0.0050). Patients with both UGT2B17-deletion and disruptive TP53-mutations had higher relapse rates than other patients (hazard ratio, 2.22; 95% confidence interval, 1.30 to 3.80, P = 0.004) in a stepwise method.ConclusionsThese results suggest that UGT2B17-deletion interacting with p16 (+) may modify effects of smoking on TP53-mutations and may further interact with the disruptive TP53-mutations to raise relapse rates among Japanese patients with HNSCC.

Abstract 726: Copy number amplification of the PIK3CA gene is associated with poor prognosis in early-stage head and neck squamous cell carcinoma

Background: Deregulation of the EGFR signaling pathway is one of the most frequently observed genetic abnormalities that drive cancer development. Mutations in downstream components of the EGFR signaling pathway, including KRAS, BRAF and PIK3CA, have been reported in numerous cancers. However, extensive mutation analysis of these genes in clinical samples has not been performed for head and neck squamous cell carcinoma (HNSCC). Patient and methods: We examined mutations and copy number alterations of KRAS, BRAF and PIK3CA in 115 clinical specimens of HNSCC obtained from surgically treated patients. Results: We identified three mutations in K-RAS and two mutations in PIK3CA. Copy number amplification was found in 42 cases (36.4%) for PIK3CA, 12 cases (10.4%) for K-RAS and nine cases (7.8%) for BRAF. Tumor recurrence and death occurred in 49 (42.6%) and in 22 (19.3 %) patients during the median follow-up of 723 days, respectively. Kaplan-Meier survival analysis revealed that in patients with early-stage carcinoma (Stages I-III), copy-number amplification of PIK3CA was drastically associated with cancer relapse. Conclusion: Copy number amplification of the PIK3CA is associated with poor prognosis in early-stage HNSCC. The PIK3CA copy number status will serve as a marker of poor prognosis in patients with HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 726. doi:1538-7445.AM2012-726

Abstract 325: Mutation analysis of PIK3CA, KRAS, BRAF and Corresponding EGFR status in head and neck squamous cell carcinoma

Background: KRAS, BRAF, PIK3CA activate protein kinase signaling and an important pathway in cancer development. Recent reports show that these downstream signaling molecules are mutated in a number of cancers. However, in head and neck squamous cell carcinoma (HNSCC), the frequency and positions of these molecules mutations are not clear. In this study we detected mutations from the clinical specimens of HNSCC by High-Throughput sequencing KRAS, BRAF, PIK3CA. Next we also intended to discover whether these mutations in downstream signaling molecules of EGFR are corresponding with EGFR status. Patients and Method: The ninety-six tumors for this study were obtained from HNSCC patients surgically treated at the Department of head and neck surgery, Jikei University School of Medicine, Tokyo. The hot-spot regions in exons 1 and 2 of K-RAS, exon 15 of BRAF, exon 9 and 20 of PIK3CA were amplified by PCR and sequenced directly using DNA from samples. Next, we analyzed expression and phosphorylation of the EGFR protein by Western blotting as well as mutations encoding the hot-spot regions in exons 18 to 21. Results: We identified mutations in 6 of the 96 patients (6.2%). 3 mutations (G12A) were found in exon 1 of K-RAS. In PIK3CA, 2 mutations were found in exon 9 (E545K) and one was in exon 20(H1047R). No other mutation was found in BRAF. Overexpression of EGFR protein was observed in all clinical samples with these mutations. On the other hand, hyper-phospholyation of EGFR was confirmed in only one sample. Finally, we tried to elucidate associations between these molecular status and patient characteristics and disease-free survival to asses the prognostic values of these molecules factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 325. doi:10.1158/1538-7445.AM2011-325