HPV genotype distribution in older Danish women undergoing surgery due to cervical cancer

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1 A C TA Obstetricia et Gynecologica AOGS ORIGINAL RESEARCH ARTICLE HPV genotype distribution in older Danish women undergoing surgery due to cervical cancer ANNE HAMMER 1,2, ELSE MEJLGAARD 3, PATTI GRAVITT 4, ESTRID HØGDALL 5, PERNILLE CHRISTIANSEN 5, TORBEN STEINICHE 2,3 & JAN BLAAKÆR 1,2 1 Department of Obstetrics and Gynecology, Aarhus University Hospital, Aarhus, Denmark, 2 Department of Clinical Medicine, Aarhus University, Aarhus, Denmark, 3 Department of Pathology, Aarhus University Hospital, Aarhus, Denmark, 4 Department of Pathology, University of New Mexico, Albuquerque, NM, USA, and 5 Department of Pathology, Copenhagen University Hospital, Herlev, Denmark Key words Cervical cancer, human papillomavirus, genotypes, human papillomavirus vaccine, older women Correspondence Anne Hammer, Department of Obstetrics and Gynecology, Aarhus University Hospital, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark. ahlauridsen@clin.au.dk Conflict of interest AH received a non-restricted grant from Sanofi Pasteur MSD, Denmark. The remaining authors have stated explicitly that there are no conflicts of interest in connection with this article. Please cite this article as: Hammer A, Mejlgaard E, Gravitt P, Høgdall E, Christiansen P, Steiniche T, et al. HPV genotype distribution in older Danish women undergoing surgery due to cervical cancer. Acta Obstet Gynecol Scand 2015; 94: Received: 8 April 2015 Accepted: 10 August 2015 Abstract Introduction. The prevalence of human papillomavirus (HPV)16/18 in cervical cancer may decrease with age. This study aimed to describe the HPV genotype distribution in Danish women aged 55 years or older with cervical cancer. Material and methods. In this cross-sectional study we identified 153 cases of cervical cancer diagnosed at Aarhus University Hospital, Denmark ( ) and Copenhagen University Hospital Herlev, Denmark ( ). All women had surgery to treat the disease. HPV genotyping was performed on cervical cancer tissue using the INNO LiPA HPV genotyping extra (Fujirebio, Belgium) at the Department of Pathology, Aarhus University Hospital, Denmark. The main outcome was to estimate the age-specific prevalence of high-risk HPV genotypes included in the bivalent, the quadrivalent, and the nonavalent vaccine. Results. Of 121 cases of cervical cancer included in this study, 113 were HPV-positive (93.4%). Although HPV16 and 18 were the most common genotypes overall, the prevalence of HPV16/18 decreased significantly from 78.1% in women aged years to 45.5% in women aged 75 or older (p < 0.001), whereas the prevalence of other HPV types and HPV-negative cases tended to increase with age (p = 0.1). The prevalence of HPV types included in the nonavalent vaccine was stable around 90% until the age of 75 years and then dropped to 63%. Conclusion. In the absence of waning immunity, the nonavalent HPV vaccine would be predicted to reduce cervical cancer burden in Denmark across a broader age-range compared with the reduced type-spectrum vaccines. Abbreviations: HE, hematoxylin and eosin staining; HPV, human papillomavirus; HR, high-risk; LiPA, Inno LiPA HPV genotyping extra. DOI: /aogs Introduction Human papillomavirus (HPV) has been described as a necessary though not a sufficient cause of cervical cancer and it has been estimated that approximately 78% of these cancers in Europe can be attributed to HPV16 and 18 (1). However, recent studies have indicated that the prevalence of HPV16 and 18 in cervical cancer decreases Key message HPV16/18 prevalence in cervical cancer decreases with age, whereas the prevalence of other types increases. Thus, the nonavalent HPV vaccine may offer better protection against cervical cancer across the life span compared with the bivalent and the quadrivalent vaccine. 1262

2 A. Hammer et al. HPV types in older women with cervical cancer with age, suggesting that the bivalent and the quadrivalent HPV vaccines will reduce cervical cancer incidence rate to a lesser extent in older women (>60 years) than younger women (<40 years) (2 4). However, research is lacking on which other genotypes occur in older women with cervical cancer. In Denmark, >30% of cervical cancers occur in women aged 60 years or older. In December 2014 the US Food and Drug Administration approved a new nonavalent HPV vaccine for use in girls aged 9 26 years. This vaccine has a very high efficacy against seven high-risk (HR) HPV genotypes (HPV16, 18, 31, 33, 45, 52 and 58) (5) that account for approximately 95% of all cervical cancers (6). It is of great clinical importance to investigate which genotypes can be detected in older Danish women with cervical cancer to better evaluate which vaccine may offer the best protection against cervical cancer across all ages. Hence, by analyzing cervical cancer specimens from women 55 years we aimed to describe the prevalence of HPV genotypes included in the different HPV vaccines (bivalent, quadrivalent, and nonavalent vaccine) categorized by age. Material and methods We searched the Danish Pathology Registry for women who had undergone surgery (i.e. hysterectomy or cone Identified through search in Aarhus n = 121 biopsy) due to cervical carcinoma at the Department of Obstetrics and Gynecology, Aarhus University Hospital, Denmark, in the period 1 January December 2013 and at the Department of Obstetrics and Gynecology, Copenhagen University Hospital Herlev, Denmark, in the period 1 January December 2013 (Figure 1). The archived samples were collected in the period from May 2013 to June Eligible cases were women aged 55 years or older at cervical carcinoma diagnosis, with no synchronic gynecological malignancy. All histological subtypes of cervical carcinoma were included. Two experienced gynecological pathologists (Aarhus: EM, Herlev: PC) reviewed slides from all cases to confirm the diagnosis and to verify the quality of the specimens (i.e. confirm the presence of tumor tissue). If several formalin-fixated and paraffin-embedded blocks were available from a woman, the most representative block was chosen. All blocks were sectioned at the Department of Pathology, Aarhus University Hospital. To avoid contamination, the microtome, tweezers, brush and knife were carefully cleaned with 1% sodium dodecyl sulfate and 99% ethanol before and after cutting every formalin-fixated and paraffin-embedded block. The sandwich technique was applied to enable histopathologic review of sections flanking the sections subjected to HPV testing as follows. First, a 3-lm section was cut for Identified through search in Herlev n = 32 Blocks could not be retrieved n = 14 Blocks could not be retrieved n = 5 Cases reviewed in Aarhus n = 107 Cases reviewed in Herlev n = 27 Diagnosis not confirmed, n = 5 Specimen of poor quality, n = 2 Disconcordance in diagnoses (HE before HE after), n = 3 Diagnosis not confirmed, n = 0 Specimen of poor quality, n = 0 Use of macroslices, n = 2 Disconcordance in diagnoses (HE before HE after), n = 1 Cases included n = 97 Cases included n = 24 Figure 1. Flowchart of included cases. Cases from Aarhus University Hospital to the left and cases from Copenhagen University Hospital Herlev on the right. HE, hematoxylin and eosin staining. 1263

3 HPV types in older women with cervical cancer A. Hammer et al. hematoxylin and eosin staining (HE). Then, four 10-lm sections were cut for HPV analysis. Finally, a 3-lm section was cut for HE staining. If presence of tumor tissue could not be confirmed in the flanking HE-stained sections, the case was excluded from further analysis. DNA extraction and HPV analysis took place at the Department of Pathology, Aarhus University Hospital. DNA extraction was performed using the QIAsymphony DSP DNA mini kit (Qiagen, Venlo, the Netherlands) following the manufacturer s instructions. For HPV analysis, the INNO-LiPA â HPV Genotyping Extra (LiPA) (Fujirebio Europe, Ghent, Belgium) was chosen. LiPA allows detection and genotyping of all high-risk or possible high-risk HPV genotypes (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82), a number of low-risk HPV genotypes (6, 11, 40, 43, 44, 54, 70) and a few additional types (69, 71, 74). All analyses were performed according to the manufacturer s recommendations and all samples were run in duplicate. If HPV was detected after amplification but the genotype could not be specified, the type was designated HPVX. We did not sequence samples designated HPVX, as our main goal was to report the prevalence of HPV genotypes included in the three HPV vaccines. Data on the cervical cancer incidence were retrieved from the Danish Cancer Registry. In the current study, case counts are a compilation of all cases of cervical cancer diagnosed among Danish women 55 years (ICD-10 codes: DC539 and DC53*) in the period and the census population encompasses all Danish women aged 55 years or older. This study was approved by the Central Denmark Region Committees on Health Research Ethics and by the Danish Data protection Agency. Informed consent was not obtained, which is in accordance with Danish regulations. We searched the Danish Tissue Use Registry to verify that we were allowed to use the specimens for medical research. Statistics We estimated the crude prevalence for each of 28 individual HPV genotypes and for a combination of genotypes. The prevalence of each genotype was calculated as the proportion of women testing positive for the genotype with or without co-infection by other LR or HR genotypes. For type combinations, for example HR HPV genotypes included in the nonavalent vaccine, the prevalence was estimated as the proportion of women testing positive for one or more HR HPV genotypes included in the vaccine with or without co-infection with other genotypes not included in the vaccine. In the case of multiinfections we applied a hierarchical classification of genotypes. For example; if a case was HPV16/18/53-positive, the case was classified as HPV16/18-positive and negative for other HPV genotypes (i.e. we assumed that HPV16 and/or 18 were the causal genotypes, which may be a reasonable assumption since these types have been shown to have a larger oncogenic potential). We used the total number of cervical cancer cases tested for HPV as the denominator. Spearman s correlation was performed on age-specific HPV prevalence rates using STATA 12 software (StataCorp, College Station, TX, USA). A p-value <0.05 was considered statistically significant. We also conducted a simulation to illustrate the possible age-specific impact of the different vaccine types under assumptions outlined below. We first calculated the age-specific cervical cancer incidence as the number of cervical cancers diagnosed in Danish women in a given age group (numerator) divided by the sum of the personyears of the at-risk female population in the same given age group (denominator). The anticipated age-specific cervical cancer incidence rate in a fully vaccinated cohort (i.e. all women vaccinated with the bivalent/quadrivalent vaccine or with the nonavalent vaccine) was estimated as follows: (1 proportion of cases positive for HR HPV types included in the HPV vaccines in a given age) * agespecific cervical cancer incidence rate in a given age. When calculating the anticipated incidence rates, we assumed that the vaccinated women will achieve 100% lifelong protection against the types included in the vaccines and that the HPV genotype distribution in cervical cancer will not change over time in Denmark. We also assume there is no cross protection. Results Through the Pathology Registry we identified 153 cases of cervical carcinoma, 121 of which were included in this study (Figure 1). Median age at cervical cancer diagnosis was 65 years (range years). The most common histological diagnosis was squamous carcinoma (76.0%) followed by adenocarcinoma (20.7%); 3.3% were classified as other carcinomas such as adenosquamous carcinoma and basaloid carcinoma. The distribution of HPV genotypes in cervical carcinoma is summarized in Table 1. Of 121 cases of cervical carcinoma, eight cases (6.6%) were negative for HPV and 11 cases (9.1%) were negative for HR HPV. The proportion of HPV-negative cases was higher in adenocarcinomas (24%) than squamous carcinoma (2.2%) and other carcinomas (0%). Overall, HPV16 was the most prevalent HR HPV genotype (50.4%) followed by HPV18 (16.5%) (i.e. including single and multiple infections). Stratified by histology, the prevalence of HPV16 and HPV18 single infections was 51.1% (HPV16) and 9.8% (HPV18) in squamous carcinoma, 32.0% (HPV16) and 32.0% (HPV18) in 1264

4 A. Hammer et al. HPV types in older women with cervical cancer adenocarcinoma, and 25% (HPV16) and 25.0% (HPV18) in other carcinomas. An additional five cases of squamous carcinoma and one case of other carcinoma had a multiinfection involving either HPV16 or HPV18. Table 1. HPV genotype prevalence in cervical cancer. Type-specific HPV prevalence is calculated by dividing the number of specimens positive for a given genotype with the number of specimens tested for HPV HPV genotype No. of cases Percentage of all cases Single infections HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPVX Multiple infections HPV HPV HPV HPV HPV HPV HPV HPV HPV-negative The prevalence of multiple HPV infections was 13.2% when encountering both HR and LR HPV genotypes and 5.0% when including HR HPV genotypes only. When stratifying by age, we found that the prevalence of HPV16 or 18 decreased significantly with age (p < 0.001) (Table 2). This decline is most likely due to a significant decline in HPV16 prevalence (p < 0.05) since we did not observe a difference in HPV18 prevalence with age. We also observed that the prevalence of other HPV genotypes increased with age (p < 0.05). The prevalence of HPV negative cases tended to increase with age; however, this finding was not statistically significant (p = 0.1; Table 2). The percentage positive for HR HPV genotypes included in the quadrivalent and the bivalent HPV vaccine (i.e. HPV16 and 18) decreased from 78.1% in women aged years to 45.4% in women 75 years, whereas the percentage positive for HR HPV genotypes included in the nonavalent vaccine (i.e. HPV16, 18, 31, 33, 45, 52 and 58) was more stable, at around 90% until age 75 years. In women older than 75 years, the percentage dropped to 68.2% (Table 2). Figure S1 illustrates the anticipated impact of the different vaccines on cervical cancer incidence among Danish women aged 55 years or older. All three vaccines will likely reduce the cervical cancer incidence, but the nonavalent vaccine might further reduce the cervical cancer incidence, especially in women younger than 75 years, since the vast majority of these women were infected with HPV types included in the nonavalent HPV vaccine. Women older than 75 years were more commonly HPV35-positive (9.1%) and HPV-negative (18.1%) compared with women younger than 75 years (Supporting Information Table S1). Table 2. Prevalence of vaccine-related genotypes, non16 or 18 HPV types, and HPV-negative cases by age. Numbers denote cases positive for a given genotype. In the case of multi-infections we applied a hierarchical classification of genotypes. For example; if a case was HPV16/18/53- positive, the case was classified as HPV16/18-positive and negative for other HPV genotypes (i.e. we assumed that HPV16 and/or 18 were the causal genotypes, which may be a reasonable assumption since these types have been shown to have a larger oncogenic potential). Type-specific HPV prevalence is calculated by dividing the number of specimens positive for one or more genotypes by the number of specimens tested for HPV Age (years) HPV type n % n % n % n % n % Spearman s rho (p-value) (p < 0.05) (p =0.62) 16 or (p < 0.001) 16, 18, 31, 33, 45, 52 or (p =0.39) Non 16 or 18 HPV types (p < 0.05) HPV neg (p =0.10) Total, n

5 HPV types in older women with cervical cancer A. Hammer et al. Discussion In the present study, we observed that HPV16 and 18 were by far the most common genotypes in cervical cancer overall (66.1%). This HPV16/18 prevalence is lower than the HPV16/18 prevalence reported in previous studies, where 74% of all cervical cancers in Denmark (7) and 78% of cervical cancers in Europe (1) could be attributed to HPV16 or 18. This difference in HPV16/18 prevalence between our study and previous studies is likely due to the fact that the previous studies included women of all ages with cervical cancer, whereas we only included women aged 55 years or older. In our study, we found that the prevalence of HPV16/18 decreased significantly with increasing age, which is consistent with findings in previous studies (3,4,8). Tjalma et al. found a significant difference in mean age at cervical cancer diagnosis between cancers positive for HPV16 or 18 compared with cancers positive for other HPV types, such as HPV31 and 33 (1). One explanation for the age-specific difference in HPV16/18 prevalence may be that pre-cancerous lesions attributed to HPV16/18 seem to progress to invasive cancer within a shorter time frame compared with pre-cancers caused by other HPV genotypes (1,9). This correlates well with the finding that the mean age of women diagnosed with HPV16/18-positive cervical cancer is lower than the mean age of women diagnosed with other HPV types such as HPV31 (1,10). This may be because HPV16 and 18 are more commonly integrated in the host genome, whereas other genotypes are more commonly episomal (10). Surprisingly, we also found a rather high prevalence of HPV44, which is categorized as a low-risk HPV genotype. In our study, HPV44 only occurred as a co-infection with HPV31. According to the manufacturer of the HPV assay used for HPV genotyping, these findings are likely due to cross-reactivity between the two types (i.e. HPV31 and HPV44) and thus reflect the limitations of the HPV assay. In Denmark, the quadrivalent and the bivalent vaccine might prevent almost 80% of cervical cancers in women aged years but only 45.4% in women 75 years. Compared with the bivalent or the quadrivalent vaccine, the nonavalent vaccine might offer better long-term protection assuming life-long immunity and no cross-protection. Hence, we estimated that the nonavalent vaccine might prevent around 90% of cervical cancers in women younger than 75 years and 68.2% in women >75 years. The low percentage in women older than 75 years is, especially, due to a high proportion of HPV-negative cases (18%) but also due to HPV35, which occurred in 9.1% of the cases. Thus, our results may indicate that the burden of cervical cancer in a fully vaccinated cohort, independent of vaccine type, is likely to shift to older ages assuming life-long immunity and assuming that the HPV genotype distribution in cervical cancer will not change over time. The latter assumption seems reasonable since the HPV genotype distribution in cervical cancers in a large multicenter study has not changed over a 70-year period (11). However, our results are based on a small sample size and should therefore be viewed with caution. The overall proportion of HPV-negative cases was 6.6%, which is consistent with previous studies. The proportion of HPV-negative cases was higher in adenocarcinomas (24%) than squamous carcinomas (2.2%). Previous studies have confirmed that adenocarcinomas tend to have a higher rate of HPV-negative cases compared with squamous carcinoma (4,12). This could be due to diagnostic difficulties of distinguishing between adenocarcinoma of cervical vs. endometrial origin. In our study we found that HPV negativity tended to increase with age, although not statistically significantly. Nevertheless, our findings are consistent with results from previous studies, including the results from the multicenter study by de Sanjose et al., where the proportion of HPVnegative cases increased from 12.3% in women <30 years to 27.5% in women aged 80 years or older (6). One explanation for the disparity in HPV negativity by age could be that the oldest women are more commonly diagnosed with adenocarcinomas compared with the youngest women; however, this was not the case in our study. Also, the disparities could be due to the fact that other factors, besides HPV, contribute to cervical cancer development in older women, including p53 mutations. Alternatively, they may be due to loss of HPV. However, a previous paper did not find an association between p53 mutations and age (13). We acknowledge that our results should be viewed with caution due to the small sample size. Nevertheless, the finding poses an important concern; that absence of HPV is not a completely valid predictor of disease. In fact, a recent study reported that almost 20% of women diagnosed with cervical cancer had had a negative HPV test 1 year prior to their diagnosis (14). Our study has some limitations that need to be addressed. First, we only included cervical cancer specimens from women who had undergone surgery due to the disease. Since women who have local disease are more likely to undergo surgery than women with advanced stage disease (>IIA) (15), we cannot rule out a bias. However, this is unlikely to have a major impact since type of HPV does not seem to be associated with disease stage (16). To avoid possible bias, a future study should include all cases of cervical cancers for HPV analysis. Second, we acknowledge that our sample size is relatively small and our results therefore require verification in a larger study. Nevertheless, our results are consistent with 1266

6 A. Hammer et al. HPV types in older women with cervical cancer previous studies (3,4). They are also consistent with the results of a previous Danish study in which the authors found that HPV16 positivity was significantly more common in women <40 years with precancerous lesions compared with women >40 years (17). Conclusion HPV16 and 18 may be the most prevalent HPV genotypes in cervical cancers overall; however, the proportion of HPV16/18-positive cases decreased significantly with age in Denmark. The bivalent and the quadrivalent vaccine may prevent almost 80% of cervical cancers in women aged years but only 45% in women aged 75 years or older. Given the type distribution found in cervical carcinomas of older women and in the absence of waning immunity, the nonavalent vaccine may offer protection over a broader age span compared with the other vaccines; however, more research in this area is needed. Acknowledgments The authors wish to thank Helle Knakkergaard for valuable assistance in the laboratory. Funding This study was supported by the Danish Cancer Society, Aase og Einar Danielsens Fond, Fabrikant Einar Willumsens Mindelegat, and Aarhus University. References 1. Tjalma WA, Fiander A, Reich O, Powell N, Nowakowski AM, Kirschner B, et al. Differences in human papillomavirus type distribution in high-grade cervical intraepithelial neoplasia and invasive cervical cancer in europe. Int J Cancer. 2013;132: Serrano B, Alemany L, Tous S, Bruni L, Clifford GM, Weiss T, et al. Potential impact of a nine-valent vaccine in human papillomavirus related cervical disease. Infect Agent Cancer. 2012;7: de Sanjose S, Wheeler CM, Quint WG, Hunt WC, Joste NE, Alemany L, et al. Age-specific occurrence of HPV16- and HPV18-related cervical cancer. Cancer Epidemiol Biomarkers Prev. 2013;22: Joste NE, Ronnett BM, Hunt WC, Pearse A, Langsfeld E, Leete T, et al. Human papillomavirus genotype-specific prevalence across the continuum of cervical neoplasia and cancer. Cancer Epidemiol Biomarkers Prev. 2015;24: Joura EA, Giuliano AR, Iversen OE, Bouchard C, Mao C, Mehlsen J, et al. A 9-valent HPV vaccine against infection and intraepithelial neoplasia in women. N Engl J Med. 2015;372: de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B, et al. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol. 2010;11: Kirschner B, Junge J, Holl K, Rosenlund M, Collas de Souza S, Quint W, et al. HPV genotypes in invasive cervical cancer in danish women. Acta Obstet Gynecol Scand. 2013;92: Wheeler CM, Hunt WC, Joste NE, Key CR, Quint WGV, Castle PE. Human papillomavirus genotype distributions: implications for vaccination and cancer screening in the United States. J Natl Cancer Inst. 2009;101: Castle PE, Shaber R, LaMere BJ, Kinney W, Fetterma B, Poitras N, et al. Human papillomavirus (HPV) genotypes in women with cervical precancer and cancer at Kaiser Permanente Northern California. Cancer Epidemiol Biomarkers Prev. 2011;20: Vinokurova S, Wentzensen N, Kraus I, Klaes R, Driesch C, Melsheimer P, et al. Type-dependent integration frequency of human papillomavirus genomes in cervical lesions. Cancer Res. 2008;68: Alemany L, de Sanjose S, Tous S, Quint W, Vallejos C, Shin HR, et al. Time trends of human papillomavirus types in invasive cervical cancer, from 1940 to Int J Cancer. 2014;135: Andersson S, Rylander E, Larson B, Sigurdardottir S, Backlund I, Sallstrom J, et al. Types of human papillomavirus revealed in cervical adenocarcinomas after DNA sequencing. Oncol Rep. 2003;10: Saito J, Hoshiai H, Noda K. Type of human papillomavirus and expression of p53 in elderly women with cervical cancer. Gynecol Obstet Invest. 2000;49: Blatt AJ, Kennedy R, Luff RD, Austin RM, Rabin DS. Comparison of cervical cancer screening results among 256,648 women in multiple clinical practices. Cancer Cytopathol. 2015;123: Erickson BK, Zhang B, Straughn JM Jr. Screening behaviors and cultural barriers in women with newly diagnosed cervical cancer. J Low Genit Tract Dis. 2013;17: Lombard I, Vincent-Salomon A, Validire P, Zafrani B, de la Rochefordiere A, Clough K, et al. Human papillomavirus genotype as a major determinant of the course of cervical cancer. J Clin Oncol. 1998;16: Baandrup L, Munk C, Andersen KK, Junge J, Iftner T, Kjaer SK. HPV16 is associated with younger age in women with cervical intraepithelial neoplasia grade 2 and 3. Gynecol Oncol. 2012;124:

7 HPV types in older women with cervical cancer A. Hammer et al. Supporting information Additional Supporting Information may be found in the online version of this article: Table S1. HPV genotype distribution by age. Figure S1. Impact of HPV vaccination on the cervical cancer incidence. Age-specific cervical cancer incidence in Denmark in women aged 55 years and older (black line); anticipated cervical cancer incidence following vaccination with the bivalent or quadrivalent vaccine (dashed line); anticipated cervical cancer incidence following vaccination with the nonavalent vaccine (dotted line). 1268