Downloaded from http://sti.bmj.com/ on June 30, 2015 - Published by group.bmj.com
STI Online First, published on March 6, 2015 as 10.1136/sextrans-2014-051642 Epidemiology
ORIGINAL ARTICLE
Human papillomavirus genotype distribution among Cameroonian women with invasive cervical cancer: a retrospective study David Pirek,1 Patrick Petignat,1 Pierre Vassilakos,2 Jolanta Gourmaud,3 Jean-Claude Pache,3 Laura Rubbia-Brandt,3 Zacharie Sando,4 Thomas Alexander McKee,3 Liza Ho3 1
Department of Obstetrics and Gynecology, University Hospitals of Geneva, Geneva, Switzerland 2 Geneva Foundation for Medical Education and Research, Geneva, Switzerland 3 Department of Clinical Pathology, University Hospitals of Geneva, Geneva, Switzerland 4 Department of Clinical Pathology, Gyneco-Obstetric and Pediatric Hospital, Yaoundé, Cameroon Correspondence to David Pirek, Department of Obstetrics and Gynecology, University Hospitals of Geneva 1211, Switzerland; [email protected] Received 30 May 2014 Revised 27 January 2015 Accepted 14 February 2015
ABSTRACT Objectives We determined the human papillomavirus (HPV) types present in invasive cervical cancer (ICC) of women in Cameroon in order to estimate the potential efficacies of HPV prophylactic vaccines. Methods This is a retrospective study using 181 formalin-fixed paraffin-embedded cervical tissue samples of ICC collected from the Institute of Pathology, GynecoObstetric and Pediatric Hospital, Yaoundé, Cameroon. HPV was detected by PCR using modified GP5+/GP6+ (MGP) primers. Genotyping was performed by reverseblot hybridisation, which allowed the detection of 9 of the 14 high-risk HPV types. Results Of the 181 samples, 91.7% were squamous cell carcinomas and 6.6% were adenocarcinomas. Counting all the single and multiple infections, the three most common high-risk types in descending order were HPV16 (88%), HPV45 (32%) and HPV18 (14.8%). 54.9% of cases were infected with a single HPV type and 45.1% had two or more HPV infections. Conclusions The frequencies of HPV16, HPV45 and multiple infections are all higher than previously reported. These observations have significant implications on the consideration of vaccination strategies because each vaccine has different duration and efficacies in cross-protection of different HPV types. The method used proved to be sensitive and cost-efficient for retrospective studies where fresh materials are not available.
INTRODUCTION
To cite: Pirek D, Petignat P, Vassilakos P, et al. Sex Transm Infect Published Online First: [ please include Day Month Year] doi:10.1136/sextrans-2014051642
Invasive cervical cancer (ICC) is the second most common cancer among women worldwide accounting for 15% of cancers in women, after breast cancer. It is the leading cause of cancer mortality among women in developing countries with >80% of the cases arising in those regions. It is also replacing infectious diseases as a major cause of death. Every year, about 500 000 new cases of cervical cancers are diagnosed and 250 000 women die from the disease (585 278 incident cases and 327 899 cases of attributable deaths were predicted for the year 2010). Approximately one million women are living today with cervical cancer, and most of them do not have access to screening and treatment facilities.1–6 Infection with one of the few oncogenic human papillomavirus (HPV) types is a necessary cause of ICC. More than 118 different HPV types have
been isolated and sequenced, and about 40 of them are known to infect the genital tract and 14 have oncogenic potentials.2 Currently, the best way to prevent cervical cancer is through organised gynaecological screening programmes with associated treatment of the detected pre-cancerous lesions. In developed countries, introduction of those programmes has resulted in a significant reduction in death rates, burden of disease and cost of treatment. However, they have not been effectively implemented in some countries where they are most needed due to barriers of cost, trained personnel and infrastructure.1 6 While the type distribution in cervical cancer does vary somewhat worldwide, HPV16 and HPV18 are the most prevalent types, being found in >70% of samples from cervical cancer around the world and being the two types targeted by current vaccine formulations.1 6 Data on HPV-type distribution in women with ICC and its precursor lesions are essential to predict the potential impact of new prophylactic vaccine against HPV16 and HPV18, as well as to determine priorities for inclusion of HPV types in future HPV vaccines and HPV-based screening tests. It is known that a majority of worldwide ICC cases are associated with HPV16 and/or HPV18, but no reliable information exists about the type-specific contribution of HPV in ICC in many, mostly developing, countries, and data are limited or missing from many regions in Africa, such as the region of West–Central Africa where Cameroon is situated. In Africa, about 21% of women in the general population are estimated to harbour cervical HPV infections, and 72.9% of ICC is attributed to HPV16 and/or HPV18.4 7 Cameroon has a population of 5.24 million women aged 15 years and older who are at risk of developing cervical cancer. Current estimates indicate that every year 1474 women are diagnosed with cervical cancer and 995 die from the disease.1 6 There is no national cervical cancer screening programme, but sporadic screening limited to some main cities are conducted, in spite of 42% of the population of Cameroon being rural. This condition probably contributes to the high incidence and mortality rates of cervical cancer in the country. Based on hospital registry data, women most at risk for
Pirek D, et al. Sex Transm Infect 2015;0:1–5. doi:10.1136/sextrans-2014-051642
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
1
Downloaded from http://sti.bmj.com/ on June 30, 2015 - Published by group.bmj.com
Epidemiology cervical cancer are those >35 years with a median age at diagnosis of 49 years; most of them having an advanced and incurable disease at presentation.7 The present study was conducted in the framework of the Swiss–Cameroon cooperation. This study aims to provide information about HPV genotype distribution in samples of ICC from Cameroon, where the incidence of ICC is one of the highest in the world. It also offers a comparison with data of other countries worldwide in order to predict and evaluate how HPV vaccination and HPV-based screening might influence cervical cancer prevention.
METHODS Sample collection Cases were recruited between January 2006 and December 2010 from Yaoundé and Douala areas. All cases were referred for diagnostic purposes to the Pathology Department (ZS) of the Gyneco-Obstetric and Pediatric Hospital, Yaoundé, Cameroon. The biopsies were retrieved from the archives of the anatomopathology services of the two capitals (Douala and Yaoundé), which hold all specimens from Cameroon for research and complementary diagnostics purposes. Some samples were from preoperative biopsies and others from tissue fragments of surgical specimens. In total, 181 formalin-fixed paraffin-embedded samples were selected, following initial diagnosis of ICC. An independent histological examination of all the samples was carried out by a pathologist of the Clinical Pathology Department at the University Hospitals of Geneva, Switzerland. DNA extraction and HPV genotyping were carried out in the molecular biology laboratory of the same department.
DNA extraction and reverse-blot hybridisation Five to ten sections of 10 μm from each sample were used to extract DNA. The sections were incubated in xylene to remove paraffin. Xylene was subsequently removed by rinsing the tissue pellet in 100% ethanol, followed by rehydration in 70% ethanol. The pellet was air-dried and lysed in 600 μL lysis solution (50 mM Tris, pH 8.3, 1 mM NaEDTA, 0.5% Tween-20) and digested overnight at 56°C in 350 μg/mL proteinase K. Phenol chloroform extraction was carried out twice on the lysed material and DNA was precipitated, resuspended in water and quantified on NanoDrop ND-1000 (NanoDrop Technologies, USA). DNA was successfully obtained from 181 samples. A total of 100 ng DNA was used by PCR using modified general primers GP5+/GP6+ (MGP) and genotyping was performed by reverse-blot hybridisation using the probes from Dr R. Sahli ( personal communication), which allows the detection of 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 56, 58, 59, 66, 73 and 82) and 12 low-risk types (6, 11, 34, 40, 42, 43, 44, 53, 54, 57, 70 and 84).8–11 Note that the oligonucleotides used for 26, 52, and 68 lie outside the PCR fragment using MGP primer pairs, and therefore would not be detected in this study. In order to determine whether HPV negativity was due to poor quality materials from which amplifiable DNA may not be extracted, beta-globin of 200 bp was amplified by PCR to test the integrity of DNA extracted. For histopathology for cervical intraepithelial neoplasia grade 3 (CIN3), the sensitivity and specificity of GP5+/GP6+ primers are 100% and 23.5%, respectively.12 MGP has the advantage of being more sensitive in detecting multiple infections.9 2
RESULTS Patient characteristics The average age of the patients at the time of diagnostics was 51.8 years, bearing in mind that the ages of 59 patients (32.6%) were not known. The patients for whom we do not have the age were those who did not know their age and did not have identification cards. Further, initial clinical information from remote health services in rural areas is incomplete. Such were common situations in Cameroon. Between the initial diagnostics and the independent histological examination of sections before DNA extraction, there was a concordance of 98.3% (178 of 181 cases). HPV DNA positivity was detected in 175 (96.7%) cases. The result was that all six HPV-negative cases were negative for beta-globin (results not shown), confirming that HPV negativity was due to the poor qualities of these DNAs. The majority of samples (n=166; 91.7%) were squamous cell carcinomas (SCC) and 12 (6.6%) were adenocarcinomas (ADC). In three cases, there was a discrepancy between the initial diagnosis and the review diagnosis of tissue sections. In the 175 cases that were positive for HPV DNA, 9 of the 14 high-risk types were detected, of which 96 cases (54.9%) were infected with a single HPV type and 79 cases (45.1%) had two or more HPV infections (table 1).
Prevalence of HPV types in ICC samples The most common HPV types, taking into account single and multiple infections, in descending order, were HPV16, HPV45, HPV18, HPV82, HPV35, HPV56, HPV39, HPV51 and HPV33. HPV16 was identified in 154 HPV-positive cases (88%), which consisted of almost equal proportions of single and co-infections with other types (44.6% and 43.4%, respectively; table 2). HPV45 was the second most prevalent high-risk HPV type in Cameroonian women with ICC, and the majority of HPV45 was found in co-infections with HPV16 or HPV18 (figure 1). HPV82 was detected in 6.9% of the cases but only in co-infections with HPV16, HPV45, or both. Fifteen cases (8.6%) of ICC were found to have one of the other established high-risk HPV types as single or multiple infections. In both SCC and ADC, the prevalence of HPV16 was similar (86% and 83%). There were also seven cases with low-risk HPV types: HPV6, HPV11 and HPV83, all of which are present in co-infections with high-risk types (table 2). A graphical
Table 1
Characteristics of participants
HPV status HPV positive HPV negative Cancer type SCC ADC Other Tissue of non-cervical origin Infection type Single infections Multiple infections Age Median age (range) Total analysed
n=181
Per cent
175 6
96.7 3.3
166 12 2 1
91.7 6.6 1.1 0.6
96 79
54.9 45.1
52 (29–87) 181
ADC, adenocarcinomas; HPV, human papillomavirus; SCC, squamous cell carcinomas.
Pirek D, et al. Sex Transm Infect 2015;0:1–5. doi:10.1136/sextrans-2014-051642
Downloaded from http://sti.bmj.com/ on June 30, 2015 - Published by group.bmj.com
Epidemiology Table 2 Human papillomavirus (HPV)-positive cases with single and multiple infections HPV
n (%)
Single infections n (%)
Multiple infections n (%)
16 18 35 45 56 59 82 6 11 33 39 44 51 83
154 (88) 26 (14.8) 9 (5.1) 56 (32) 5 (2.9) 1 (0.6) 12 (6.9) 2 (1.1) 4 (2.3) 1 (0.6) 2 (1.1) 1 (0.6) 2 (1.1) 1 (0.6)
78 (44.6) 6 (3.4) 2 (1.1) 4 (2.3) 1 (0.6) 0 0 0 0 0 0 0 1 (0.6) 0
76 20 7 52 4 1 12 2 4 1 2 1 1 1
(43.4) (11.4) (4) (29.7) (2.3) (0.6) (6.9) (1.1) (2.3) (0.6) (1.1) (0.6) (0.6) (0.6)
representation of the data including only HPV16 and HPV18 as single and multiple infections is shown in figure 2.
DISCUSSION This is one of the few published studies on the HPV genotype distribution among women with ICC in the West–Central African region and the first one in Cameroon, a country where no data on this specific subject are currently available.1 6 Among the 175 HPV-positive cases analysed, ADC constituted 6.6% of all ICCs. This is in agreement with the study of Guan and colleagues, which showed that ADCs represent
STI Online First, published on March 6, 2015 as 10.1136/sextrans-2014-051642 Epidemiology
ORIGINAL ARTICLE
Human papillomavirus genotype distribution among Cameroonian women with invasive cervical cancer: a retrospective study David Pirek,1 Patrick Petignat,1 Pierre Vassilakos,2 Jolanta Gourmaud,3 Jean-Claude Pache,3 Laura Rubbia-Brandt,3 Zacharie Sando,4 Thomas Alexander McKee,3 Liza Ho3 1
Department of Obstetrics and Gynecology, University Hospitals of Geneva, Geneva, Switzerland 2 Geneva Foundation for Medical Education and Research, Geneva, Switzerland 3 Department of Clinical Pathology, University Hospitals of Geneva, Geneva, Switzerland 4 Department of Clinical Pathology, Gyneco-Obstetric and Pediatric Hospital, Yaoundé, Cameroon Correspondence to David Pirek, Department of Obstetrics and Gynecology, University Hospitals of Geneva 1211, Switzerland; [email protected] Received 30 May 2014 Revised 27 January 2015 Accepted 14 February 2015
ABSTRACT Objectives We determined the human papillomavirus (HPV) types present in invasive cervical cancer (ICC) of women in Cameroon in order to estimate the potential efficacies of HPV prophylactic vaccines. Methods This is a retrospective study using 181 formalin-fixed paraffin-embedded cervical tissue samples of ICC collected from the Institute of Pathology, GynecoObstetric and Pediatric Hospital, Yaoundé, Cameroon. HPV was detected by PCR using modified GP5+/GP6+ (MGP) primers. Genotyping was performed by reverseblot hybridisation, which allowed the detection of 9 of the 14 high-risk HPV types. Results Of the 181 samples, 91.7% were squamous cell carcinomas and 6.6% were adenocarcinomas. Counting all the single and multiple infections, the three most common high-risk types in descending order were HPV16 (88%), HPV45 (32%) and HPV18 (14.8%). 54.9% of cases were infected with a single HPV type and 45.1% had two or more HPV infections. Conclusions The frequencies of HPV16, HPV45 and multiple infections are all higher than previously reported. These observations have significant implications on the consideration of vaccination strategies because each vaccine has different duration and efficacies in cross-protection of different HPV types. The method used proved to be sensitive and cost-efficient for retrospective studies where fresh materials are not available.
INTRODUCTION
To cite: Pirek D, Petignat P, Vassilakos P, et al. Sex Transm Infect Published Online First: [ please include Day Month Year] doi:10.1136/sextrans-2014051642
Invasive cervical cancer (ICC) is the second most common cancer among women worldwide accounting for 15% of cancers in women, after breast cancer. It is the leading cause of cancer mortality among women in developing countries with >80% of the cases arising in those regions. It is also replacing infectious diseases as a major cause of death. Every year, about 500 000 new cases of cervical cancers are diagnosed and 250 000 women die from the disease (585 278 incident cases and 327 899 cases of attributable deaths were predicted for the year 2010). Approximately one million women are living today with cervical cancer, and most of them do not have access to screening and treatment facilities.1–6 Infection with one of the few oncogenic human papillomavirus (HPV) types is a necessary cause of ICC. More than 118 different HPV types have
been isolated and sequenced, and about 40 of them are known to infect the genital tract and 14 have oncogenic potentials.2 Currently, the best way to prevent cervical cancer is through organised gynaecological screening programmes with associated treatment of the detected pre-cancerous lesions. In developed countries, introduction of those programmes has resulted in a significant reduction in death rates, burden of disease and cost of treatment. However, they have not been effectively implemented in some countries where they are most needed due to barriers of cost, trained personnel and infrastructure.1 6 While the type distribution in cervical cancer does vary somewhat worldwide, HPV16 and HPV18 are the most prevalent types, being found in >70% of samples from cervical cancer around the world and being the two types targeted by current vaccine formulations.1 6 Data on HPV-type distribution in women with ICC and its precursor lesions are essential to predict the potential impact of new prophylactic vaccine against HPV16 and HPV18, as well as to determine priorities for inclusion of HPV types in future HPV vaccines and HPV-based screening tests. It is known that a majority of worldwide ICC cases are associated with HPV16 and/or HPV18, but no reliable information exists about the type-specific contribution of HPV in ICC in many, mostly developing, countries, and data are limited or missing from many regions in Africa, such as the region of West–Central Africa where Cameroon is situated. In Africa, about 21% of women in the general population are estimated to harbour cervical HPV infections, and 72.9% of ICC is attributed to HPV16 and/or HPV18.4 7 Cameroon has a population of 5.24 million women aged 15 years and older who are at risk of developing cervical cancer. Current estimates indicate that every year 1474 women are diagnosed with cervical cancer and 995 die from the disease.1 6 There is no national cervical cancer screening programme, but sporadic screening limited to some main cities are conducted, in spite of 42% of the population of Cameroon being rural. This condition probably contributes to the high incidence and mortality rates of cervical cancer in the country. Based on hospital registry data, women most at risk for
Pirek D, et al. Sex Transm Infect 2015;0:1–5. doi:10.1136/sextrans-2014-051642
Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.
1
Downloaded from http://sti.bmj.com/ on June 30, 2015 - Published by group.bmj.com
Epidemiology cervical cancer are those >35 years with a median age at diagnosis of 49 years; most of them having an advanced and incurable disease at presentation.7 The present study was conducted in the framework of the Swiss–Cameroon cooperation. This study aims to provide information about HPV genotype distribution in samples of ICC from Cameroon, where the incidence of ICC is one of the highest in the world. It also offers a comparison with data of other countries worldwide in order to predict and evaluate how HPV vaccination and HPV-based screening might influence cervical cancer prevention.
METHODS Sample collection Cases were recruited between January 2006 and December 2010 from Yaoundé and Douala areas. All cases were referred for diagnostic purposes to the Pathology Department (ZS) of the Gyneco-Obstetric and Pediatric Hospital, Yaoundé, Cameroon. The biopsies were retrieved from the archives of the anatomopathology services of the two capitals (Douala and Yaoundé), which hold all specimens from Cameroon for research and complementary diagnostics purposes. Some samples were from preoperative biopsies and others from tissue fragments of surgical specimens. In total, 181 formalin-fixed paraffin-embedded samples were selected, following initial diagnosis of ICC. An independent histological examination of all the samples was carried out by a pathologist of the Clinical Pathology Department at the University Hospitals of Geneva, Switzerland. DNA extraction and HPV genotyping were carried out in the molecular biology laboratory of the same department.
DNA extraction and reverse-blot hybridisation Five to ten sections of 10 μm from each sample were used to extract DNA. The sections were incubated in xylene to remove paraffin. Xylene was subsequently removed by rinsing the tissue pellet in 100% ethanol, followed by rehydration in 70% ethanol. The pellet was air-dried and lysed in 600 μL lysis solution (50 mM Tris, pH 8.3, 1 mM NaEDTA, 0.5% Tween-20) and digested overnight at 56°C in 350 μg/mL proteinase K. Phenol chloroform extraction was carried out twice on the lysed material and DNA was precipitated, resuspended in water and quantified on NanoDrop ND-1000 (NanoDrop Technologies, USA). DNA was successfully obtained from 181 samples. A total of 100 ng DNA was used by PCR using modified general primers GP5+/GP6+ (MGP) and genotyping was performed by reverse-blot hybridisation using the probes from Dr R. Sahli ( personal communication), which allows the detection of 14 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 56, 58, 59, 66, 73 and 82) and 12 low-risk types (6, 11, 34, 40, 42, 43, 44, 53, 54, 57, 70 and 84).8–11 Note that the oligonucleotides used for 26, 52, and 68 lie outside the PCR fragment using MGP primer pairs, and therefore would not be detected in this study. In order to determine whether HPV negativity was due to poor quality materials from which amplifiable DNA may not be extracted, beta-globin of 200 bp was amplified by PCR to test the integrity of DNA extracted. For histopathology for cervical intraepithelial neoplasia grade 3 (CIN3), the sensitivity and specificity of GP5+/GP6+ primers are 100% and 23.5%, respectively.12 MGP has the advantage of being more sensitive in detecting multiple infections.9 2
RESULTS Patient characteristics The average age of the patients at the time of diagnostics was 51.8 years, bearing in mind that the ages of 59 patients (32.6%) were not known. The patients for whom we do not have the age were those who did not know their age and did not have identification cards. Further, initial clinical information from remote health services in rural areas is incomplete. Such were common situations in Cameroon. Between the initial diagnostics and the independent histological examination of sections before DNA extraction, there was a concordance of 98.3% (178 of 181 cases). HPV DNA positivity was detected in 175 (96.7%) cases. The result was that all six HPV-negative cases were negative for beta-globin (results not shown), confirming that HPV negativity was due to the poor qualities of these DNAs. The majority of samples (n=166; 91.7%) were squamous cell carcinomas (SCC) and 12 (6.6%) were adenocarcinomas (ADC). In three cases, there was a discrepancy between the initial diagnosis and the review diagnosis of tissue sections. In the 175 cases that were positive for HPV DNA, 9 of the 14 high-risk types were detected, of which 96 cases (54.9%) were infected with a single HPV type and 79 cases (45.1%) had two or more HPV infections (table 1).
Prevalence of HPV types in ICC samples The most common HPV types, taking into account single and multiple infections, in descending order, were HPV16, HPV45, HPV18, HPV82, HPV35, HPV56, HPV39, HPV51 and HPV33. HPV16 was identified in 154 HPV-positive cases (88%), which consisted of almost equal proportions of single and co-infections with other types (44.6% and 43.4%, respectively; table 2). HPV45 was the second most prevalent high-risk HPV type in Cameroonian women with ICC, and the majority of HPV45 was found in co-infections with HPV16 or HPV18 (figure 1). HPV82 was detected in 6.9% of the cases but only in co-infections with HPV16, HPV45, or both. Fifteen cases (8.6%) of ICC were found to have one of the other established high-risk HPV types as single or multiple infections. In both SCC and ADC, the prevalence of HPV16 was similar (86% and 83%). There were also seven cases with low-risk HPV types: HPV6, HPV11 and HPV83, all of which are present in co-infections with high-risk types (table 2). A graphical
Table 1
Characteristics of participants
HPV status HPV positive HPV negative Cancer type SCC ADC Other Tissue of non-cervical origin Infection type Single infections Multiple infections Age Median age (range) Total analysed
n=181
Per cent
175 6
96.7 3.3
166 12 2 1
91.7 6.6 1.1 0.6
96 79
54.9 45.1
52 (29–87) 181
ADC, adenocarcinomas; HPV, human papillomavirus; SCC, squamous cell carcinomas.
Pirek D, et al. Sex Transm Infect 2015;0:1–5. doi:10.1136/sextrans-2014-051642
Downloaded from http://sti.bmj.com/ on June 30, 2015 - Published by group.bmj.com
Epidemiology Table 2 Human papillomavirus (HPV)-positive cases with single and multiple infections HPV
n (%)
Single infections n (%)
Multiple infections n (%)
16 18 35 45 56 59 82 6 11 33 39 44 51 83
154 (88) 26 (14.8) 9 (5.1) 56 (32) 5 (2.9) 1 (0.6) 12 (6.9) 2 (1.1) 4 (2.3) 1 (0.6) 2 (1.1) 1 (0.6) 2 (1.1) 1 (0.6)
78 (44.6) 6 (3.4) 2 (1.1) 4 (2.3) 1 (0.6) 0 0 0 0 0 0 0 1 (0.6) 0
76 20 7 52 4 1 12 2 4 1 2 1 1 1
(43.4) (11.4) (4) (29.7) (2.3) (0.6) (6.9) (1.1) (2.3) (0.6) (1.1) (0.6) (0.6) (0.6)
representation of the data including only HPV16 and HPV18 as single and multiple infections is shown in figure 2.
DISCUSSION This is one of the few published studies on the HPV genotype distribution among women with ICC in the West–Central African region and the first one in Cameroon, a country where no data on this specific subject are currently available.1 6 Among the 175 HPV-positive cases analysed, ADC constituted 6.6% of all ICCs. This is in agreement with the study of Guan and colleagues, which showed that ADCs represent
