The overall mutation frequency of KRAS, BRAF and PIK3CA in our HNSCC samples was lower than reported for other cancers. We identified mutations in 2.6% of our samples for the KRAS gene, 2.6% for PIK3CA and 0% for BRAF. KRAS mutations are observed in approximately 45-60% of pancreatic cancers [21, 22], 30-50% of colorectal cancers [23, 24] and 30% of non-small-cell lung carcinomas . BRAF mutations are observed in approximately 60-70% of malignant melanomas [26, 27], 40% of thyroid carcinomas  and 5-10% of colorectal cancers . PIK3CA mutations are observed in 32% of colorectal cancers , 31% of endometrial cancers  and 14% of breast cancers . The small sample size may have caused less frequency of these mutations in the study.
The KRAS mutations identified in three of our tumor samples (2.6%) were identical G12A substitutions, which is a well-characterized activating mutation. The low KRAS mutation frequency in the present study is in agreement with previous small-scale studies of HNSCC that reported a mutation frequency of 2.4% (1/42 samples) in oral squamous cell carcinoma , 4.5% (1/22 samples) in oropharyngeal cancer  and 0% (0/16 samples) in HNSCC . We also found that the frequency of copy number amplification was not high (8.7%), suggesting that approximately 90% of the patients have normal KRAS proteins and that the hyper activation of KRAS, per se, is not a common feature of HNSCC. A recent study showed that KRAS mutations result in resistance to cetuximab in colorectal cancer, limiting the utility of this drug . In lung cancer, although the frequency of KRAS mutations is similar to that in colorectal cancer, these mutations are not reported to be a predictive marker. Therefore, the low KRAS mutation frequency of HNSCC may cause difficulty in predicting the effect of cetuximab and other EGFR inhibitors.
We analyzed exon 15 of BRAF to search for a V600E substitution, the most common activating mutation of BRAF, which is observed at high frequencies in various cancers . However, we did not detect any V600E mutations in our samples, and the frequency of copy number alteration was also low, at 1.7% (2/115). Together with a previous study that reported a BRAF mutation frequency of 2.4% (1/42) in oral squamous cell carcinoma , our results suggest that the BRAF mutation frequency is much lower in HNSCC than in other cancers.
Mutations in exons 9 and 20 of PIK3CA were found in three specimens (2.6%), and these mutations are known to be hotspot mutations that generate a constitutively active kinase in other cancers . The mutation frequency of PIK3CA in our study was somewhat lower than that reported in previous small-scale studies. One study of surgical specimens from 30 Americans with HNSCC and eight HNSCC cell lines found mutations in four out of the 38 samples (10.5%) . Another study found mutations in two surgical specimens and five cell lines among 54 cases, including 17 cell lines and 18 surgical specimens from Vietnamese patients and 19 surgical specimens from Indian patients . The lower mutation frequency may reflect differences in the genetic backgrounds or may be due to the small population sizes. Although the frequency of point mutations in PIK3CA was low, we found copy number amplification in 37 samples (32.2%), suggesting that the hyper activation of the PI3K pathway occurs in one-third of HNSCC patients. PIK3CA mutations are associated with resistance to cetuximab in colorectal cancer in vitro . Furthermore, a phase I study of a recently developed mTOR inhibitor found that colorectal cancer patients with PIK3CA mutations had a higher response rate than those without mutations . Thus, checking the status of the PIK3CA gene will be important when using these molecularly targeted drugs in HNSCC patients. However, the low frequency of mutation in PIK3CA complicates its utility as a predictive marker for use in molecularly targeted medicine in clinical settings.
In our analysis, an amplification of the PIK3CA copy number was found in many of the samples (32.2%). With regard to lung cancer, such amplification is more frequent in squamous cell carcinomas (33.1%) than adenocarcinomas (6.2%) . Interestingly, Fendri et al. reported that the frequency of amplification was 21.6% and was associated with lymph node metastasis and with the overall survival in nasopharyngeal cancer . In HNSCC, metastatic lymph nodes are very strongly associated with disease progression and clinical staging: the cancer is considered stage III or IV when lymph node metastases are found. Therefore, we hypothesized that the copy number amplification of PIK3CA was associated with a poor prognosis. The frequency of the copy number alteration observed in the present study was the same as in other reports, yet we found no significant difference in the disease-free survival and overall survival between patients with PIK3CA amplification and those without amplification. However, when the patients with lymph node metastases were excluded from the population, a significant correlation was found between PIK3CA copy amplification and the time to relapse (log-rank test, p =0.026). As cancer relapse occurs in the lymph nodes in most cases, the amplification of the PIK3CA copy number is likely to promote the process of lymph node metastasis in early-stage patients. Our evidence further suggests that those patients with early-stage HNSCC may be divided into two subgroups of good and poor prognoses, as defined by the copy number status of PIK3CA.
The limitations of this study include the small number of patients especially about the prognosis study by the status of the copy number and may be include the selection bias in undergoing surgery.