Wang, Vivian

Abstract Human papillomavirus (HPV) appears to be responsible for dramatic increases in the incidence of oropharyngeal squamous cell carcinoma (OPSCC) in the United States in the last two decades, in spite of decreases in the incidence of smoking. It is generally assumed that the role of HPV in the development of OPSCC is identical to the one that it plays in cervical cancer where HPV infection followed by integration into the host genome and the subsequent increased expression of the E6 and E7 oncoproteins leads to increased genomic instability. The common fragile sites (CFSs) are highly unstable chromosomal regions that will be particularly sensitive to increased genomic instability and they are also hot-spots for viral integrations in cervical cancer. The CFS regions span many megabases and many of them also span extremely large genes, a number of which have been demonstrated to function as important tumor suppressors. We therefore examined the expression of very large genes in OPSCC. An analysis of RNAseq data generated on 11 OPSCCs and matched normal oropharyngeal tissue from the same patients revealed that some of the tumors had greatly decreased expression for many of the largest genes. The two genes with the greatest decreases in expression in the tumors as compared to matched normal were the known tumor suppressor PARK2 and the putative tumor suppressor DLG2, but three other large CFS genes: CTNNA3, LRP1B and DMD, and one large gene not yet precisely localized within a CFS region: PDE4D, also had much more decreased expression than any of the other large genes. Real-time RT-PCR was used to measure the expression of these six genes in a much larger number of OPSCC tumor-normal pairs. The expression of these six genes was concordant in most OPSCCs. An individual tumor generally had either decreased expression of these six genes, no changes in the expression of these genes or increased expression of all six genes. When the tumors were grouped with respect to which ones were HPV-positive or HPV-negative we observed that 40% of the HPV-positive OPSCCs had greatly decreased expression (more than 100-fold) for all six genes. Half of the HPV-negative tumors had decreased expression of these six genes, but the amount of decreased expression was much more modest (4-16 fold). Most exciting, however was that 7 of the 12 HPV-positive tumors, and three of the four of the HPV-negative patients that had decreased expression of these genes were from patients who had tumor recurrence. In contrast only one of 18 patients whose tumors did not have decreased expression of the large genes had tumor recurrence. The loss of expression of multiple large genes, many of which are derived from CFS regions, could thus be a powerful predictor for which patients will have tumor recurrence. These results also suggest a linkage between HPV-induced chromosomal instability and dramatic decreases in the expression of multiple CFS genes in some OPSCCs. Citation Format: David I. Smith, Ge Gao, Nicole Tombers, Jan Kasperbauer, Vivian Wang. Human papillomavirus and the inactivation of expression of multiple large common fragile site genes in oropharyngeal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 582. doi:10.1158/1538-7445.AM2014-582

The common fragile sites (CFSs) are large regions of profound genomic instability found in all individuals. The frequent deletions and other alterations in these regions in multiple cancers has led to the discovery of a number of extremely large genes contained within these regions and several of the large CFS genes have already been demonstrated to function as tumor suppressors involved in the formation of many different cancers. To study the large CFS genes in oropharyngeal squamous cell carcinoma (OPSCC), we did RNA seq analysis from 11 head and neck cancer patients. This revealed that there are six large CFS genes which consistently had decreased expression in the tumor samples compared to their matched normal tissues. These six genes are PARK2, DLG2, NBEA, CTNNA3, DMD, and FHIT. PARK2 and FHIT are located within the two most frequently expressed CFSs and both have been demonstrated to function as tumor suppressors, while the other large genes are found to have frequent alterations in multiple cancers. Validation experiments using real time PCR indicated that over 60% of OPSCC tumors showed decreased expression for all six genes. Both HPV‐positive and HPV‐negative OPSCCs had similar proportions with loss of expression of these genes. Our results suggest that this selected group of large genes might serve as potential tumor suppressors involved in the development of OPSCCs. Further studies are needed to investigate whether the decreased expression observed is due to genomic instability within the CFS regions or the selection for alterations of specific large CFS genes. © 2014 Wiley Periodicals, Inc.

Abstract There are a number of human genes which span very large chromosomal regions. Forty genes span greater than 1 megabase and 200 are 500 Kb or larger. We and others have demonstrated that many of the largest genes are derived from within regions of profound genomic instability known as common fragile sites (CFSs). We have been studying oropharyngeal cancers which are derived from the base of tongue, tonsil and larynx. While the traditional etiologic factors underlying the development of this aggressive cancer is a history of smoking and drinking there are an increasing number of younger patients developing this cancer without these risk factors. The most likely cause of these cancers is the presence of human papillomavirus which we detect in over 90% of oropharyngeal cancers derived from the Mayo Clinic. In an attempt to better understand the molecular alterations that underlie oropharyngeal cancers arising from different risk factors we utilized the power of Next Generation sequencing on the Illumina GA IIx to compare the transcriptomes of 12 oropharyngeal cancers to patient matched normal tissue. We first observe a division of patients into two groups of oropharyngeal cancers: those caused by mutations and other alterations induced by a history of smoking and drinking and those associated with HPV infection caused by viral-induced genomic instability. We then examined the expression of the 200 largest human genes in the oropharyngeal tumors and find that each tumor has inactivated the expression of at least 20 different very large genes. There is a distinct pattern of inactivation of these genes in different oropharyngeal cancers depending upon the underlying etiology. Some of the genes most frequently inactivated include Dlg2 (the human homolog of the large discs tumor suppressor gene from drosophila and a mutation target in a number of other cancers); ERBB4 (Her4 and a member of the EGFR family), BAI3 (a brain specific angiogenesis inhibitor that is also mutated in multiple cancers), and ANK2 (mutated in long QT patients). Each of these genes may be functioning as tumor suppressors as has already been demonstrated for the two large CFS genes, FHIT and WWOX. The inactivation of so many of these genes in each oropharyngeal tumor may have a profound effect upon the phenotype of the resulting tumor. When we examine the expression of the large genes most frequently inactivated in each of the two groups of oropharyngeal cancers in panels of breast or lung cancer samples we find that we do not detect significant loss of expression of these genes. We believe that genomic instability promotes alterations in the unstable CFS regions but that there is selective inactivation of different large CFS genes in different cancers. The signature of which specific genes are inactivated may have phenotypic and clinical significance which we are just beginning to investigate. 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 3885. doi:10.1158/1538-7445.AM2011-3885

Abstract There is considerable clinical interest in the role that human papillomavirus (HPV) plays in the development of oropharyngeal cancers. A variety of assays are currently available to stratify patients based on HPV status prior to treatment. The most frequently utilized are PCR and In Situ hybridization (ISH) to detect the presence of HPV DNA sequences. The increasing clinical interest in determining HPV patient status relates to compelling evidence that a subset of patients with HPV positive tumors have better clinical outcomes. Evidence also suggests that patients with HPV positive tumors should be further divided into groupings based on levels of viral activity and that this may represent additional clinical information necessary to develop appropriate treatment plans. The most frequently applied assay to identify HPV activity in a patient sample is to detect the over-expression of the surrogate protein marker p16 by Immunohistochemisty (IHC), which has been shown to increase expression in response to HPV active infections in oropharyngeal cancer. An alternative technique to quantitatively detect the presence and level of transcriptional activity of HPV in tissue samples is to measure the expression of oncogenes E6 and E7. We have analyzed the fresh frozen tissue samples corresponding to matched tumor, normal and metastatic patient tissues using a quantitative PCR (qPCR) approach to determine the expression of HPV oncogenes and p16 gene expression. We observed that there are three groups of oropharyngeal tumors when grouped by HPV16 E6 and E7 gene expression. The first are the most rare and are tumors that contain no HPV sequences or expression of the E6 and E7 ocogenes. The second are tumors that contain HPV DNA sequences but very low expression of E6 or E7 and the third are tumors that contain HPV DNA sequence and robust expression of E6 and E7. Additionally, we found variable correlation between HPV16 E6 and E7 expression and p16 gene expression, suggesting that factors other than HPV oncogene expression may influence the expression of p16. Our results would indicate that the best assays for quantifying the activity of an HPV infection in oropharyngeal cancers would be those optimized to accurately measure either E6 and E7 transcripts or their protein products directly out of paraffin-embedded tissues. 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 3859. doi:10.1158/1538-7445.AM2011-3859