ORIGINAL ARTICLE Human papillomavirus integration pattern and demographic, clinical, and survival characteristics of patients with oropharyngeal squamous cell carcinoma Ming Yann Lim, MD, 1,3 Kristina R. Dahlstrom, PhD, 1 Erich M. Sturgis, MD, MPH, 1,2 Guojun Li, MD, PhD 1,2 * 1 Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas, 2 Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, Texas, 3 Department of Otolaryngology, Tan Tock Seng Hospital, Singapore. Accepted 30 December 2015 Published online 22 March 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.24429 ABSTRACT: Background. In human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (SCC), reported rates of HPV integration into the host genome vary. Among patients with oropharyngeal SCC, we determined differences in demographic, clinical, and survival characteristics between those with integrated and episomal HPV. Methods. The analysis included patients with newly diagnosed oropharyngeal SCC presenting to one institution during 1996 to 2011. HPVpositive samples were classified according to HPV physical state as integrated, episomal, or mixed. Overall, progression-free, and diseasespecific survival, distant metastasis rate, and second primary malignancy rate were calculated. Results. Among 307 patients, 179 had HPV-positive tumors. Of these, 22 (12%) had episomal, 42 (24%) had integrated, and 115 (64%) had mixed HPV. Among the 3 subgroups, there were no significant differences in demographic, clinical, or survival characteristics. Conclusion. The majority of HPV-positive oropharyngeal SCCs have either integrated or both episomal and integrated HPV with no significant differences in survival among the groups. VC 2016 Wiley Periodicals, Inc. Head Neck 38: 1139 1144, 2016 KEY WORDS: human papillomavirus, integration, episomal, viral physical state, oropharyngeal cancer INTRODUCTION Virtually all cervical cancers are attributable to human papillomavirus (HPV), 1,2 whereas population-based estimates suggest that approximately three quarters of oropharyngeal squamous cell carcinomas (SCCs) are HPVpositive. 3 Approximately 90% of HPV-positive oropharyngeal SCCs are attributable to HPV type-16 (HPV-16), compared with only 60% of cervical cancers. HPV is integrated into the host genome of the vast majority of cervical cancers; HPV-18 is integrated in 100% of HPV- 18-positive cervical cancers, and HPV-16 is integrated in approximately 80% of HPV-16-positive cervical cancers. 4 Only 3% of premalignant cervical lesions have integrated HPV, further supporting the importance of integration to carcinogenesis. 4 In contrast, epidemiological evidence shows that both episomal and integrated physical states exist in HPV-positive oropharyngeal SCC, 5 8 and HPV integration rates for HPV-positive oropharyngeal SCC in the current literature range from 15% to 100%. 5,9 13 However, many of the studies to date have had limited sample *Corresponding author: G. Li, Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 1445, Houston, TX 77030. E-mail: gli@mdanderson.org Ehab Y. Hanna, MD, Editor, was recused from consideration of this manuscript. Contract grant sponsor: National Institutes of Health through MD Anderson s Cancer Center Support; contract grant numbers: CA016672; CA135679 (to G.L.); CA133099 (to G.L.) sizes and/or have included head and neck tumors from nonoropharyngeal sites. Integration of HPV into the host genome disrupts or deletes the E2 viral gene, leading to increased expression of the E6 and E7 genes. The increased expression of E6 and E7, in turn, inactivates tumor suppressor protein p53 and the Rb pathway, resulting in increased proliferation and genomic instability. 14 It is well-established that HPV confers a survival advantage in oropharyngeal SCC, 15 19 but whether this survival advantage is associated with both the integrated and episomal physical states of HPV is unclear. This study had 3 purposes. The primary purpose was to determine, within a large sample of patients with HPV-positive oropharyngeal SCC, the relative proportions with integrated, episomal, and mixed HPV-16. The second purpose was to document any differences in the distribution of demographic and clinical characteristics between patients with oropharyngeal SCC with integrated, episomal, and mixed HPV-16. The third purpose was to document any differences in survival between patients with integrated versus episomal HPV-16. We hypothesize that the integrated physical state will demonstrate a superior survival advantage because the integrated physical state of HPV potentially allows a more direct action on the host genome. MATERIALS AND METHODS Patients with newly diagnosed, previously untreated oropharyngeal SCC who presented to The University of Texas MD Anderson Cancer Center between December HEAD & NECK DOI 10.1002/HED AUGUST 2016 1139
LIM ET AL. 1996 and August 2011 were included in this study. All subjects prospectively completed an institutional review board-approved informed consent form and an epidemiological questionnaire that included questions about demographic and exposure characteristics. Patients were considered smokers if they had smoked at least 100 cigarettes in their lifetime and were considered drinkers if they had consumed at least 1 alcoholic beverage per week for at least 1 year. Smokers or drinkers who had not smoked or consumed alcohol in the year before their cancer diagnosis were considered former smokers and former drinkers, respectively. Pack-years of smoking were calculated by multiplying the number of packs of cigarettes smoked per day by the number of years of smoking. At enrollment in this study, patients medical records and the institutional tumor registry were prospectively reviewed to obtain information about comorbidities, subsite, stage, T and N classification, histological grade, and treatment. The sixth edition of the American Joint Committee on Cancer TNM staging system was used to determine disease stage at the time of presentation for all patients. Patients were classified as having HPV-positive or HPV-negative oropharyngeal SCC on the basis of quantitative real-time polymerase chain reaction (PCR) for HPV-16 E6 and E7, in which positivity for one or both was considered HPV-positive. HPV integration was determined using the method of Peitsaro et al 20 with modifications described by Huang et al. 21 This quantitative realtime PCR method targets the HPV E2 open reading frame, which is most often deleted during HPV integration. The primer sequences for E2 and E6 for HPV-16 were previously reported by Ho et al. 22 The HPV-16 E2 and HPV-16 E6 amplimers are 101 and 107 base pairs, respectively. The specificities of the PCR products were monitored by analyzing the dissociation curves of each amplimer. Standard curves were established using a serial dilution (10 to 10,000,000 copies) of cloned partial-length HPV-16/18 plasmid DNA containing equivalent amounts of E2 and E6. Concentrations of HPV DNA were expressed as copies of HPV genome per 50 ng of cellular DNA. When the HPV is only episomal, the E2/E6 ratio should be 1.0 or greater. When the HPV exists in both integrated and episomal forms, the copy number of E2 should be less than that of E6 but not zero (ie, the E2/E6 ratio should be >0 but <1.0). When all HPV is integrated and no episomal HPV is present, the E2/E6 ratio should be zero. This assay has been shown to be highly effective for determining the integration status of the virus when various amounts of mixed episomal and integrated forms are present. 23 On the basis of results of this analysis, patients were classified as having episomal, integrated, or mixed episomal and integrated HPV physical pattern. Positive and negative controls were included and b-actin was used to ensure DNA integrity. Statistics Stata 12.0 (StataCorp, College Station, TX) was used for all statistical analyses. A p value of <.05 was considered statistically significant, and all tests were 2-sided. Standard descriptive statistical methods were used to describe patients demographic and clinical characteristics. TABLE 1. Demographic and exposure characteristics of patients with human papillomavirus-positive oropharyngeal squamous cell carcinoma by the pattern of human papillomavirus-16 integration into the host genome. Characteristics Episomal (n 5 22) No. of patients (%) Mixed (n 5 115) Integrated (n 5 42) p value Age, y.397 55 10 (46) 69 (60) 26 (62) >55 12 (54) 46 (40) 16 (38) Sex.401* Male 18 (82) 102 (89) 39 (93) Female 4 (18) 13 (11) 3 (7) Ethnicity.148* Non-Hispanic white 19 (86) 110 (96) 41 (98) Other 3 (14) 5 (4) 1 (2) Smoking status.764* Never smoker 13 (59) 61 (54) 17 (44) Former smoker 6 (27) 33 (29) 15 (38) Current smoker 3 (14) 20 (18) 7 (18) Missing 0 1 3 Pack-years of smoking.187* 10 4 (44) 19 (36) 4 (18) >10 5 (56) 33 (64) 18 (82) Missing 0 2 3 Alcohol use.047 Never 10 (46) 26 (23) 6 (15) Former 7 (32) 29 (25) 12 (30) Current 5 (23) 59 (52) 22 (55) Missing 0 1 2 * Fisher s exact test. Current and former smokers only. Overall survival was defined as the time from the first appointment to death from any cause, disease-specific survival was defined as the time from the first appointment to death from disease, and progression-free survival was defined as the time from the first appointment to the clinical detection of recurrent cancer (local, regional, or distant) or death from any cause. Patients lost to followup were considered censored. Patients alive at last followup were considered censored for overall survival, patients alive or dead from any cause other than disease were considered censored for disease-specific survival, and patients alive and progression-free were considered censored for progression-free survival. Kaplan Meier curves were created to compare differences in survival between the groups of patients, and the log-rank statistic was used to determine whether differences in survival between groups were statistically significant. RESULTS The study included 307 patients, 179 (58%) with HPV- 16-positive tumors and 128 (42%) with HPV-16-negative tumors. Of the 179 HPV-positive tumors, 22 (12%) had episomal, 42 (24%) had integrated, and 115 (64%) had mixed episomal and integrated HPV-16. The median follow-up time for patients alive at the end of follow-up was 46 months for patients with HPV-negative tumors and 52 months for patients with HPV-positive tumors. 1140 HEAD & NECK DOI 10.1002/HED AUGUST 2016
HPV INTEGRATION PATTERN OF PATIENTS WITH OROPHARYNGEAL SCC TABLE 2. Clinical characteristics of patients with human papillomavirus-positive oropharyngeal squamous cell carcinoma by the pattern of human papillomavirus-16 integration into the host genome. Characteristics Episomal (n 5 22) Mixed (n 5 115) Integrated (n 5 42) p value No. of patients (%) Comorbidity.220* None to mild 18 (82) 107 (93) 39 (93) Moderate to severe 4 (18) 8 (7) 3 (7) Subsite.249* Tonsil 12 (55) 62 (54) 16 (38) Base of tongue 9 (41) 51 (44) 24 (57) Other oropharynx site 1 (5) 2 (2) 2 (5) Stage 1.0* I or II 2 (9) 10 (9) 3 (7) III or IV 20 (91) 105 (91) 39 (93) T classification.188 0 2 13 (59) 89 (77) 32 (76) 3 or 4 9 (41) 26 (23) 10 (24) N classification.186 0 2a 10 (46) 41 (36) 10 (24) 2b 3 12 (55) 74 (64) 32 (76) Grade.144 Well to moderately differentiated keratinizing 10 (59) 37 (35) 12 (33) Moderately to poorly differentiated/nonkeratinizing 7 (41) 69 (65) 24 (67) Missing 5 9 6 Treatment.714 Radiation only 9 (41) 38 (33) 13 (31) Radiation 1 chemotherapy 6 surgery 13 (59) 77 (67) 29 (69) * Fisher s exact test. Patients with HPV-positive tumors were more likely to be men (89%) and never smokers (51%) than were those with HPV-negative tumors (81% men and 34% never smokers; p 5.062 and p 5.003, respectively; data not shown). Additionally, smokers with HPV-positive tumors had fewer pack-years of exposure than did smokers with HPV-negative tumors (p 5.002). The demographic and exposure characteristics of the patients with HPV-positive tumors by HPV-16 integration pattern are presented in Table 1. Compared with patients with episomal or mixed HPV, patients with integrated FIGURE 1. Progression-free survival in patients with human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinoma. HPV were more likely to be younger, men, and non- Hispanic white, but these differences were not statistically significant. Compared with patients with integrated or mixed HPV, patients with episomal HPV were more likely to be older, women, of an ethnicity other than non- Hispanic white, and never drinkers, but only the difference related to alcohol use was statistically significant. There were no statistically significant differences in the distribution of clinical variables between patients with episomal, mixed, and integrated HPV-16 (Table 2), although patients with integrated HPV were more likely than patients with episomal or mixed HPV to present with base of tongue primary tumors and higher N classification, and patients with episomal HPV were more likely than patients with integrated or mixed HPV to present with higher comorbidity, lower grade, and higher T classification. As expected, patients with HPV-positive oropharyngeal SCC had significantly better progression-free survival than patients with HPV-negative oropharyngeal SCC (p 5.021; see Figure 1). In addition, 3-year overall, disease-specific, and progression-free survival rates were better for patients with HPV-positive tumors than for patients with HPV-negative tumors (data not shown). Rates of second primary tumors and distant metastases were also lower for patients with HPV-positive tumors (data not shown). Although there was a trend toward patients with mixed HPV having better 3-year overall, disease-specific, and progression-free survival rates than patients with episomal or integrated HPV, there were no significant differences HEAD & NECK DOI 10.1002/HED AUGUST 2016 1141
LIM ET AL. TABLE 3. Rates of survival, distant metastasis, and second primary malignancies in patients with human papillomavirus-positive oropharyngeal squamous cell carcinoma by the pattern of human papillomavirus-16 integration into the host genome. Episomal (n 5 22) 3-year rate, % (95% CI) Mixed (n 5 115) Integrated (n 5 42) p value (hetero) Overall survival 90.5 (67.0 97.5) 95.1 (88.6 97.9) 87.6 (72.6 94.6).101 Disease-specific survival 95.2 (70.7 99.3) 97.0 (90.9 99.0) 90.2 (75.9 96.2).105 Progression-free survival 80.4 (55.6 92.2) 93.3 (86.5 96.8) 85.0 (69.5 93.0).233 Distant metastases 0 2.9 (1.0 8.8) 2.5 (0.3 16.5).756 Second primary malignancy 0 1.8 (0 6.8) 0 NC Abbreviations: CI, confidence interval; NC, not calculable because of zero cells. in survival among the 3 groups (Table 3). In particular, there was no significant difference in progression-free survival between the episomal and integrated groups (see Figure 2). DISCUSSION Our study demonstrated that the majority of HPVpositive oropharyngeal SCCs have either integrated HPV or mixed episomal and integrated HPV. Episomal HPV is less common. Several studies performed to date have examined HPV integration in HPV-positive oropharyngeal SCC (Table 4). 5,9 13 Various methods have been used in these studies to determine integration status, including E2/E6 ratio, the method used here; pattern of nuclear hybridization signals by in situ hybridization; mate pair gene sequencing; and restriction enzyme digestion and ligation with inverse PCR. These studies, together with other, smaller studies, 6 8 have provided epidemiological evidence that HPV exists in both episomal and integrated physical states in HPV-related oropharyngeal SCCs. In the present study, 179 patients had HPV-positive oropharyngeal SCC as determined by detection of HPV- 16 E6/E7 by quantitative real-time PCR. Of these patients, 12% had episomal HPV, 24% had integrated HPV, and 64% had mixed episomal and integrated HPV. These results suggest that most patients with HPV-related FIGURE 2. Progression-free survival in patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma by status of HPV-16 integration into the host genome (episomal, mixed, or integrated) and in patients with HPV-negative oropharyngeal squamous cell carcinoma. oropharyngeal SCC have integrated HPV either as the only state of HPV or in combination with episomal HPV. Our results are in agreement with both of the prior studies 10,13 that utilized E2/E6 ratio to determine HPV integration status, which found an HPV integration rate (integrated and mixed) of 60% and 78%, respectively. Both prior studies that used pattern of nuclear hybridization found a 100% HPV integration rate, whereas the study that used mate pair sequencing found only a 15% HPV integration rate. Although the chosen methodology likely affects reported integration rates, the impact of methodology can only be determined definitively if the various methodologies are used within the same cohort. The molecular mechanism by which integrated HPV results in a carcinogenic effect is well established, 14 and there is also molecular evidence to support a carcinogenic effect of episomal HPV. An in vitro study on cervical cell cultures demonstrated that cells with episomal HPV had viral mrna transcript levels and cell proliferation rate similar to those in cells with integrated HPV. In fact, the cells with episomal HPV developed a selective growth advantage and outcompeted the transcriptionally active integrant-containing cells. 24 In contrast, in a study by Jeon et al 25 on clonal populations of human cervical epithelial cells, it was demonstrated that clonal populations with integrated HPV DNA had E7 protein levels higher than those in the clonal populations with episomal HPV DNA, and cell populations with integrated HPV DNA outgrew cell populations with episomal HPV DNA. Jeon and Lambert 26 went on to demonstrate that integration of HPV-16 DNA leads to increased steady-state levels of mrnas encoding the viral oncogenes E6 and E7. This may explain the higher E7 levels seen in the cells with integrated HPV. Thus, integration seems to provide a selective advantage to cervical epithelial precursors of cervical carcinoma. In oropharyngeal SCC, both episomal and integrated HPV may lead to transcriptionally active HPV with high E6/E7 mrna levels and subsequently to cell proliferation and eventually to genomic instability. Olthof et al 27 examined 75 patients with oropharyngeal SCC, 29 of them (39%) with viral integration and the remaining 36 (61%) with episomal HPV only. There were no significant differences between the 2 groups in the mean RNA expression of viral genes E2, E6, and E7; in the number of viral copies per cell; or in the RNA expression of the HPV-disrupted genes. Consistent with these findings, our 1142 HEAD & NECK DOI 10.1002/HED AUGUST 2016
HPV INTEGRATION PATTERN OF PATIENTS WITH OROPHARYNGEAL SCC TABLE 4. Studies of human papillomavirus integration into the host genome in patients with oropharyngeal squamous cell carcinoma. Study, year No. of patients/ no. HPV-positive HPV type/detection method Method for determining integration HPV integration status Gao, 2014 9 20/13 HPV-16/DNA PCR E6/E7 Mate pair sequencing 1 integrated, 1 mixed, 11 episomal Deng, 2013 10 * 18/18 HPV-16/DNA PCR E2/E6 ratio 1 integrated, 13 mixed, 4 episomal Mooren, 2013 11 77/32 HPV-16/PCR and FISH Pattern of nuclear hybridization 31 integrated, 1 mixed signals Begum, 2005 12 8 tonsil (subset data) HPV-16/ PCR and FISH Pattern of nuclear hybridization 8 integrated signals Koskinen, 2003 13 5 tonsil/5 (subset data) HPV-16/DNA PCR E2/E6 ratio 3 integrated, 2 episomal Mellin, 2002 5 22/12 HPV-16 (11), HPV-33 (1) /DNA PCR Restriction enzyme digestion, ligation, and inverse PCR 11/11 HPV-16 positive episomal Abbreviations: HPV, human papillomavirus; PCR, polymerase chain reaction; FISH, fluorescence in situ hybridization. * Six patients with non HPV-16 not analyzed for integration. In this study, 37 of 45 oropharyngeal carcinomas were HPV-16 positive, but only 8 were analyzed for integration pattern. study showed no significant differences in survival at 3 years between the patients with episomal and those with integrated HPV, suggesting equivalent biologic activity of the 2 physical states. One possible explanation as to why the cases with integrated HPV did not demonstrate a significant difference in mean RNA expression of the key viral genes E2, E6, and E7, could be due to epigenetics. Epigenetics allows gene expression to be controlled by changes in DNA methylation and chromatin structure. DNA methylation typically occurs at cytosines (5-methylcytosine) that precede guanines in dinucleotide CpG sites and can impact the transcription of genes by impairing the binding of transcriptional proteins to the gene or by changing the chromatin structure to prevent transcription. Hypomethylation in head and neck cancers, in particular within the viral regulatory region, long control region, facilitates transcription of the E6 and E7 oncogenes and leads to increased levels of E6 and E7 expression. DNA hypermethylation may explain why integrated HPV does not necessarily lead to increased E6 and E7 levels. 28 Although our study included 307 patients with oropharyngeal SCC, the study was limited by the relatively small numbers of patients in the episomal (n 5 22) and integrated (n 5 42) groups, which limited our power to detect even modest differences in survival. Because of these small numbers, it was also not feasible to perform multivariable analysis to exclude confounding factors that may affect survival, such as smoking. However, there was no significant difference in the proportion of smokers or in other clinical characteristics between the episomal and the integrated groups. In a study of 138 patients with oropharyngeal SCC, Jung et al 8 showed that HPV-negative patients and patients with nontranscriptionally active HPV (HPV DNA positive, mrna E6/E7 expression negative) had similar 5-year survival rates, which were inferior to the 5-year survival rates of patients with transcriptionally active HPV (HPV DNA positive, mrna E6/E7 expression positive). Gene expression profiling analysis showed that CDKN2A gene expression was highest in the patients with transcriptionally active HPV. These findings suggest that survival may be influenced more by a transcriptionally active virus than by the viral physical state. Consistent with this interpretation is that the study by Olthof et al 27 showed no difference in E6 and E7 mrna expression between patients with episomal HPV and patients with integrated HPV. Several questions remain unanswered. These include why the episomal state is less common in cervical carcinoma than in oropharyngeal SCC, and how the episomal state generates a high E7 level without integration. Further studies are required to answer these questions. Notwithstanding the findings of Olthof et al 27 and the results of our study, emerging evidence suggests that integrated HPV may exert effects differently from episomal HPV. Parfenov et al 29 examined a series of head and neck cancers and scrutinized those that displayed evidence of high-risk HPV types 16, 33, and 35. Twentyfive of 35 such cases had integration of the viral genome into 1 or more locations in the human genome. Importantly, not all tumors with integrated HPV showed enhanced expression of viral E6 and E7 oncoproteins. In fact, at least 7 samples in which viral integration occurred showed an HPV expression profile similar to that of the integration-negative tumors. This was confirmed by Deng et al, 10 who demonstrated that, although 77% of HPV-16- positive samples had integrated or mixed episomal and integrated HPV-16, only 28% of HPV-16-positive cases had E6/E7 mrna transcripts detected. High HPV-16 load rather than physical state correlated significantly with E6 and E7 mrna expression. Hence, it is likely that integration drives tumorigenesis independent of the activities of E6 and E7. 29 Indeed, the study by Parfenov et al 29 went on to demonstrate that integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mrna transcript abundance and splicing, and both interchromosomal and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Therefore, in addition to inactivating p53 and Rb protein, integration led to amplification of host oncogenes and disruption of host tumor suppressor genes. Tumors that do and do not have HPV integrants display distinct gene expression profiles and DNA methylation patterns. 29 In conclusion, our study demonstrates that the majority of HPV-positive oropharyngeal SCCs have either integrated or mixed episomal and integrated HPV. Episomal HPV is uncommon. The significance of the episomal state HEAD & NECK DOI 10.1002/HED AUGUST 2016 1143
LIM ET AL. is unknown. Studies in the literature provide both epidemiological and molecular evidence for episomal states in the pathogenesis of HPV-related oropharyngeal SCC. The limited analyses in our study showed no significant difference in survival rates between the integrated and the episomal physical states, although our study was limited by the small sample size. Further studies with larger numbers of subjects will be required to evaluate this further. The current literature suggests that the integrated and episomal viral physical states are not associated with significantly different expression of key viral genes E2, E6, and E7. There is some evidence in the literature to suggest that survival outcomes may be related more to the presence of a transcriptionally active virus than to the physical state of the virus. Acknowledgments The authors thank Ms. Liliana Mugartegui, Ms. Kathryn Tipton, and Ms. Jenny Vo for their assistance with patient recruitment and Dr. Chong Zhao for laboratory support. We also thank Stephanie Deming for manuscript editing. REFERENCES 1. zur Hausen H. Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2002;2:342 350. 2. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999; 189:12 19. 3. Chaturvedi AK, Anderson WF, Lortet Tieulent J, et al. Worldwide trends in incidence rates for oral cavity and oropharyngeal cancers. J Clin Oncol 2013;31:4550 4559. 4. Cullen AP, Reid R, Campion M, L orincz AT. Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. J Virol 1991;65:606 612. 5. Mellin H, Dahlgren L, Munck Wikland E, et al. 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