HPV-positive head and neck squamous cell carcinoma (HNSCC) has been described as molecularly distinct from traditional head and neck cancer [5
]. The human papillomavirus (HPV) oncoproteins E6 and E7 promote carcinogenesis by degrading the tumor suppressors p53 and retinoblastoma protein (Rb), respectively. In contrast, p53 is not degraded in HPV-negative HNSCC but is frequently mutated, and p16 is often lost through homozygous deletion, methylation, or, less frequently, point mutation [5
]. This might lead one to believe that carcinogens like tobacco and alcohol would promote HNSCC comprised of a large number of mutations in many different pathways. In contrast HPV-positive cancers, modulated by the activities of viral oncoproteins, might not accumulate a large number of cellular mutations. In our study, we provided quadruple confirmation of tumor HPV status with p16 immunohistochemistry, HPV in situ
hybridization, HPV detection by PCR, and detection of the HPV 16 genome sequences within patient 1's sequenced exome. We observed more mutations in the HPV-negative tumor when compared to the HPV-positive tumor, although the absolute difference was not dramatic (73 versus 58, resp.). Two large-scale exome sequencing efforts characterizing HNSCC have been reported recently [6
]. The study led by Stransky et al. reported twice as many mutations in the HPV-negative samples (4.83 mutations/Mb versus 2.28 mutations/Mb) [7
]. The second group examined a set of 32 patients, four of which were HPV positive and reported on a subset of mutations that were identified by exome sequencing and confirmed by PCR. In this subset of genes, there were four times as many mutations in the HPV-negative tumors (20.6 ± 16.7 versus 4.8 ± 3 mutations in the HPV-positive tumors) [6
]. Given the broad range of mutations seen in the HPV-negative cancers, our finding of slightly more mutations in the HPV-negative tumor is consistent with their results. As expected we did not identify TP53 or p16 mutations in the HPV-positive tumor; however these two genes appeared as wild type in the HPV-negative tumor as well. The lack of a p16 mutation in the setting of low expression levels as evidenced by immunohistochemistry may reflect that it has been inactivated by promoter methylation, the second most common cause of p16 loss [14
Only a single genetic mutation (Muc12) was shared by both HPV-positive and HPV-negative tumor samples. The cell surface associated Muc12 was the only mucin identified in the HPV-negative tumor. In contrast, the HPV-positive tumor had five mutations in four different mucin genes, including the secreted Muc6, and the transmembrane bound Muc4, Muc12 and Muc16. Stransky et al. reported mutations in all the above mucins except for Muc12 [7
]. Mucins are known to be involved in the development of epithelial cancer where they are often overexpressed, disrupting the epithelial cell polarity and promoting the epithelial to mesenchymal transition (EMT) phenotype [15
]. Multiple damaging mutations within the mucins of HPV-positive tumor may suggest another cellular difference between these two distinct tumor types.
We also found multiple mutations in the zinc finger (ZNF) family genes in both tumor types. The ZNF family represents a large group of molecules which are involved in various aspects of transcriptional regulation [16
]. There were almost twice as many ZNF mutated genes in the HPV-positive sample. Although there were a total of 11 ZNF mutations between the two tumor types, there were no shared ZNF members mutated in both cancers. Stransky et al. reported 50% of the same ZNF mutations that we found in both tumor samples suggesting that genetic changes to this family of transcription regulators may be important in the development of HNSCC [7
]. Not enough is known about the role of mucins and ZNF proteins in HNSCC. These molecules may warrant further study.
We confirmed that the sequence from the human pathogen HPV type 16 was identified within exome sequence of a HNSCC tumor. In order for HPV to be oncogenic, the viral E2 protein, which represses the expression of E6 and E7, must be lost [17
]. This only occurs during integration when the episomal HPV DNA breaks within the E2 gene. PCR detection of E6 and E7 can detect both episomal and integrated forms and thus cannot distinguish between a superficial HPV infection and integrated viral DNA causing the cancer [17
]. An additional benefit of whole exome sequencing is the detection of integrated HPV DNA. In Stransky's study, exome sequencing appeared even more sensitive than PCR for detection of HPV, as it identified the presence of HPV 16 in 14 of 73 cases versus 11 for PCR [7
]. Perhaps more interesting is the concept of screening human disease genomes against pathogen datasets. In fact, it was this exact strategy that led to the discovery of Merkel cell polyomavirus in 2008 [18
]. It may be that a subset of other cancers have a yet undiscovered viral etiology.
This study represents a pilot effort to gain experience with this exciting new technology, which was instructive as our group moves forward with large-scale projects. In addition to the small sample size, the quality of data generated limited by the ABI SOLiD platform with an average 30-fold coverage with 50 base pair paired-end reads yielded only 10-fold coverage over approximately two-thirds of the coding sequence. Thus, approximately a third of the exome was not adequately evaluated and important mutations could have been missed. We have recently completed characterizing a panel of head and neck cancer cell lines with 100-fold coverage with 100 base pair paired-end reads, and the results were vastly superior [19
]. An average of 99% of the targeted exome had at least 10 reads and 90% had fiftyfold coverage. Perhaps more importantly, an expert bioinformatics team is critical to properly analyze the data. Although there are standard steps involved with aligning the sequencing data to the reference genome, false positive results can be frequent without adequate quality control measures. A carefully validated pipeline is necessary to filter spurious results in order to generate valid data.
It should be noted that tremendous insights can be gained by exome sequencing; however, whole genome sequencing offers the advantage to identify other genetic changes that can lead to tumorigenesis including copy number variation and translocations, in addition to point mutations, insertions, and deletions. Alterations in noncoding regions that may be important, such as promoters and miRNAs, would also be identified. The study by Stransky et al. reported whole genome sequencing in two patients and revealed markedly more translocations in the HPV-negative versus the HPV-positive tumors [7
]. Ideally, future large-scale initiatives will be carried out using this more extensive but also more expensive technique to identify additional important genetic changes underlying HNSCC.