Clinical Cancer Research Vol. 11, 2919-2923, April 15, 2005
© 2005 American Association for Cancer Research
Imaging, Diagnosis, Prognosis |
Detection of Human Papillomavirus Infection of the Cervix in Very Elderly Women Using PCR
Kaiyo Takubo1,
Naotaka Shimomura-Izumiyama1,
Hidemi Koiwai2,
Naoko Honma1,
Yukiyoshi Esaki3,
Tomomi Yoshida4,
Takashi Nakajima5,
Motoji Sawabe2 and
Tomio Arai2
Authors' Affiliations: 1 Human Tissue Research Group, Tokyo Metropolitan Institute of Gerontology; 2 Department of Pathology, Tokyo Metropolitan Geriatric Medical Center, Tokyo, Japan; 3 Department of Pathology, Sekishinkai Sayama Hospital, Saitama-ken, Japan; and 4 Department of Histopathology and Cytopathology, School of Health Science, Faculty of Medicine, Gunma University; 5 Department of Tumor Pathology, Gunma University Graduate School of Medicine, Maebashi, Japan
Requests for reprints: Kaiyo Takubo, Human Tissue Research Group, Tokyo Metropolitan Institute of Gerontology, Sakaecho 35-2, Itabashi-ku, Tokyo 173-0015, Japan. Phone: 81-3-3964-3241; Fax: 81-3-3579-4776; E-mail: takubo{at}tmig.or.jp.
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Abstract
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Purpose: To determine the incidence of human papillomavirus (HPV) infection and the morphologic lesions induced by HPV infection of the uterine cervix in elderly Japanese women (including very elderly women) and to clarify the natural history and outcome of HPV infection.
Experimental Design: We detected squamous intraepithelial lesions (SIL) by cytology and the presence of HPV infection by PCR on cervical smears obtained at autopsy from 335 women ages 60 to 105 years (mean, 82.7 years). Two primers were used for PCR, one for low-risk HPV subtypes and one for high-risk HPV subtypes.
Results: SILs were observed in 20 (6.0%) of the women autopsied; 18 (5.4%) had low-grade SIL and 2 (0.6%) had high-grade SIL. HPV-DNA was not detected in any of the women with normal cervical cytology but was found in 9 (45.0%) of the 20 with SIL (2.7% of all subjects). Of these 9 women, 2 (22%) were positive for low-risk types of HPV and 7 (78%) were positive for high-risk types. All 9 had spent their last days in hospitals or nursing homes, the duration of institutional care ranging from 17 days to 10 years 2 months.
Conclusions: Our results suggest that HPV infection of the cervix may occasionally persist for long periods of time (the maximum duration noted in this study being 10 years 2 months). It is also possible that HPV infection in young women with normal cervical cytology will not persist into advanced age.
Key Words: cervical carcinoma • natural history • intraepithelial squamous neoplasia • advanced age
Human papillomavirus (HPV) infection and cervical squamous cell carcinoma are very closely related; sexually transmitted HPV infection can induce the development of benign papilloma or intraepithelial neoplasia of the external genitalia and uterine cervix (1). Invasive squamous cell carcinoma of the cervix is usually preceded by a noninvasive squamous lesion. HPV infection seems to be the major etiologic factor for carcinoma of the uterine cervix, but additional genetic changes also seem to be required for the development and progression of such lesions. To date, most studies that have examined the prevalence of cervical HPV infection have concentrated on young and middle-aged women (2–4). Only a few studies have been conducted on older women (5), and it has been suggested that the prevalence of HPV infection is lower in very elderly women than in younger women. However, regardless of age, a high proportion of those who test positive for HPV infection are infected with subtypes that are associated with a high risk of cancer. A review of the literature conducted by Schiffman (6) suggested that only a minority of women infected with HPV develop persistent infection and that only a proportion of those infected with high-risk types of HPV progress to high-grade squamous intraepithelial lesions (SIL) and invasive carcinoma. These conclusions are supported by statistical evidence; the incidence of HPV-related cervical intraepithelial neoplasia and invasive cancer in White women in the United States has been reported to increase with age and to plateau between ages 24 and 29 years for intraepithelial neoplasia and between ages 35 and 39 years for invasive cancer (6). However, there have been no reports on the incidence, natural history, and outcome of HPV in elderly women, including women of very advanced age. To clarify the natural history and outcome of HPV infection, we conducted a systematic study to determine the incidence of HPV infection and the morphologic lesions associated with such infection in elderly Japanese women (including very elderly women).
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Materials and Methods
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Subjects. Cervical smears were done on 612 consecutive female autopsy subjects, ages from 60 to 105 years at the time of death (mean, 83.2 years), who had no recorded clinical diagnosis of cervical carcinoma. The smears from 335 of these (mean age, 82.7 years) were usable; the other 277 showed marked autolysis and were unsuitable for cytologic examination. The autopsies were done at the Department of Anatomical Pathology of Tokyo Metropolitan Geriatric Medical Center (Tokyo, Japan) between 1997 and 2002 (6 years). Concerning the ages of the subjects, 25 were ages 60 to 69 years, 104 were ages 70 to 79 years, 133 were ages 80 to 89 years, 70 were ages 90 to 99 years, and 3 were centenarians. As a control group for the PCR study, 163 smears from women ages <50 years were analyzed; these included 125 smears from women (mean age, 38 years) without cytologic evidence of SILs and 38 smears from women (mean age, 35 years) with SIL.
Cytology. For the very elderly autopsy subjects, the vaginas and uteri were opened anteriorly at autopsy, and cervical cytology specimens were obtained using a cotton-tipped applicator. The uteri and vaginas were generally very small and had very narrow lumina due to the advanced ages of the subjects, and the cotton-tipped applicator had to be used several times to obtain sufficient cells and DNA for the cytologic examination and PCR. Smears were collected for routine cytologic examination first followed by specimens for HPV-DNA analysis. After preparation of the cytology slides, the cotton applicators were washed in 4 mL PBS and stored at –80°C until required for PCR. For the control groups, cotton-tipped applicators were used to obtain cells and DNA for cytologic examination and PCR study.
The smears were examined microscopically by four of the authors (K.T., M.S., T.A., and H.K.) who are all specialists in cytology, surgical, and anatomic pathology. Smears with marked autolysis were excluded from the study. SILs, including dysplasia and carcinoma in situ, were classified as low or high grade (LSIL or HSIL) based mainly on the Bethesda system (7).
DNA extraction. The cells in PBS were mixed with lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 25 mmol/L EDTA, 500 mmol/L NaCl, 3 mmol/L 2-mercaptoethanol] containing proteinase K (500 µg/mL) and 2% SDS and gently shaken overnight at 50°C. The lysate was mixed with an equal volume of phenol/chloroform/isoamylalcohol (25:24:1) and gently shaken overnight. The resulting supernatant was again treated with phenol/chloroform/isoamylalcohol for 30 minutes and with chloroform/isoamylalcohol (24:1) for a further 30 minutes. Thereafter, it was mixed with 0.1 volume of 3 mol/L sodium acetate and 2.5 volumes of ice-cold ethanol, kept at –80°C for 1 hour, and centrifuged at 10,000 x g for 15 minutes. The pellet was mixed with 70% ethanol and centrifuged at 10,000 x g for 15 minutes. The final pellet of DNA was dried and resuspended with 60 µg TE buffer [10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA].
Primers and PCR. For PCR, we used two different consensus primers. The primer HPVpU-31B (5'-TGCTAATTCGGTGCTACCTG-3', Takara Shuzo Co. Ltd., Ohtsu, Japan) was used to amplify the DNA of HPV 6 and 11 ("low-risk HPV types") and HPVpU-1M (5'-TGTCAAAAACCGTTGTGTCC-3', Takara Shuzo) was used to amplify the DNA of HPV 16, 18, 31, 33, 35, 52b, and 58 ("high-risk HPV types"; ref. 8).
DNA solution (10 µL) was diluted to 62 µL in a 1.25 mmol/L deoxynucleoside triphosphate mixture, and 1 µL of 25 pmol/µL sense primer, 1 µL of 25 pmol/µL antisense primer, and 0.5 µL of 5 units/mL Taq polymerase (GeneAmp DNA Amplification kit, Takara Co., Kyoto, Japan) were added. After adding 100 µL mineral oil, 30 cycles of PCR were conducted at 94°C for 30 seconds, 55°C for 2 minutes, and 72°C for 2 minutes. After PCR, the DNA was electrophoresed on 4% NuSieve GTG agarose gel (Takara), stained with ethidium bromide, and photographed under UV light. HPV-DNA was observed within the bands lying between 228 and 268 bp.
Duration of hospital and/or nursing home stay before death and other autopsy findings. For subjects who were positive for HPV-DNA, the time spent in hospitals and/or nursing homes just before death and the duration of widowhood were determined. Other autopsy findings were researched from the autopsy reports of each case.
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Results
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Cytologic findings. Surface cells that stained positive for Orange-G were rarely observed in the smears taken from our group of elderly and very elderly women (Fig. 1). SILs were observed in 20 (6.0%) of the 335 women (Table 1); 18 (5.4%) of these had LSIL and 2 (0.6%) had HSIL. No invasive squamous cell carcinomas or adenocarcinomas were observed in the cervical smears from any of the 335 women. The smears from the 38 clinical control subjects with SIL included 9 with LSIL and 29 with HSIL.
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Fig. 1. Cytologic smear from the cervix of a very elderly woman (age. 96 years; subject 20 in Tables 2 and 3). Low-grade atypia and koilocytosis can be seen in the squamous and fusiform cells of the uterine cervix. We diagnosed this as a low-grade SIL. Original magnification, x200.
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Human papillomavirus-DNA detection. HPV-DNA was detected in 9 (2.7%) of the 335 women (Table 1). None of the remaining 315 women with normal cytology exhibited HPV-DNA; all 9 HPV-DNA-positive cases showed positive cytology for LSIL or HSIL, representing 45.0% of the 20 women with these abnormalities. DNA for low-risk subtypes of HPV was detected in 2 (22%) of the 9 women positive for HPV-DNA (data not shown); the other 7 (78%) exhibited DNA for high-risk subtypes (Fig. 2). The oldest woman to show LSIL and koilocytosis on cytology and to be positive for HPV-DNA was 96 years old (Table 2). For the younger clinical control subjects, HPV-DNA was detected in 16 (12.8%) of the 125 with normal cytology and 35 (92.1%) of the 38 with LSIL or HSIL. DNA for high-risk subtypes of HPV was detected in 4 (25.0%) of the 16 with normal cytology but positive HPV-DNA and 22 (62.9%) of the 35 with abnormal cytology and positive HPV-DNA.
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Fig. 2. Amplification and detection of high-risk HPV-DNA. High-risk HPV DNA was detected between 228 and 268 bp in lane 2 (a 77-year-old subject, subject 4 in Table 2), lane 5 (an 83-year-old subject, subject 12), and lane 7 (a 96-year-old subject, subject 20) but not in lane 1 (a 61-year-old subject, subject 1), lane 3 (a 73-year-old subject, subject 2), lane 4 (an 81-year-old subject, subject 9), lane 6 (an 86-year-old subject, subject 15), or lane 8 (a 91-year-old subject, subject 18). M, size marker; TM, a control template consisting of malignant-type HPV-DNA (length of amplified DNA, 63 bp).
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Time spent in hospital and/or nursing home before death and other autopsy findings. The nine women who were positive for HPV-DNA had all spent their last days in a hospital or nursing home, the duration of stay ranging from 17 days to 10 years 2 months (Table 3). In such institutions, the patients would not have been able to have sexual intercourse and would have had no chance of becoming infected with HPV. All nine women had led life as single widowed women for 2 to 22 years before death.
Of the 20 elderly autopsy subjects with cytologic SIL, malignancies were found in 12 (60%), aspiration pneumonia in 8 (40%), acute or healed myocardial infarction in 5 (25%), cerebral infarction in 6 (30%), and Alzheimer's or Parkinson's disease in 4 (20%). Of the 12 subjects with malignancies, 3 had hematopoietic malignancies. Of the remaining 315 subjects without SIL, malignancies were found in 145 (46%) and aspiration pneumonia in 113 (36%). There were no significant differences in the incidences of malignancy or aspiration pneumonia between the groups with or without SIL (
2 test).
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Discussion
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Our morphologic and PCR studies led to four major findings. (a) Our cytologic examination of cervical smears detected SIL in 6.0% of very elderly Japanese women (mean age, 82.7 years). (b) Our PCR study showed the presence of HPV-DNA in 2.7% of these very elderly women; however, HPV-DNA was not detected in all patients with positive cytology. (c) All subjects positive for HPV-DNA were also positive for LSIL or HSIL on cytology, and HPV-DNA was not found in any subject with normal cervical cytology; in contrast, HPV-DNA was detected in 12.8% of the younger clinical control subjects with normal cytology. (d) According to the subjects' medical records, one woman who was positive for HPV-DNA had been resident in an institution for >10 years before death. These findings indicate that HPV infection may be persistent but does not always induce invasive squamous cell carcinoma even when it has been present for >10 years.
HPV infection of the uterine cervix is sexually transmitted, and the virus has been reported to preferentially infect metaplastic squamous epithelial cells. HPV is a family of DNA viruses, with >60 subtypes having been characterized. We did not attempt to identify DNA of all the different subtypes but used two consensus primers to amplify DNA for types associated with a low and a high risk of cancer (HPVpU-31B for the DNA of HPV 6 and 11 and HPVpU-31M for the DNA of HPV 16, 18, 31, 33, 35, 52b, and 58). These are the subtypes that are most commonly detected in the uterine cervix (9, 10).
We detected HPV-DNA in only 45.0% of the subjects who were positive for LSIL or HSIL on cytology. In contrast to these findings, other studies have detected HPV-DNA in 
90% of women with invasive squamous cell carcinoma of the cervix overall (9, 10) and 99% of those with invasive squamous cell carcinoma or adenocarcinoma with associated SIL (11). In addition, in our control group of younger women, HPV-DNA was detected in 92% of those who had SIL on cytology. We did not use consensus primers to amplify all types of HPV because of the small amount of DNA obtained from the cervical samples of these very elderly women, but we used primers for the HPV subtypes that are most commonly detected in the uterine cervix. It is possible that autolysis may have prevented the demonstration of HPV-DNA by PCR in some cases, but our observations may also indicate that LSIL and HSIL may sometimes be induced by factors other than HPV infection in very elderly women. We now intend to perform a further study to detect HPV subtypes in elderly and very elderly women with invasive carcinoma of the cervix.
Park et al. (12) found a strong association between various HPV subtypes and the grade of SIL in the uterine cervix and reported that HSILs were associated with infection with high-risk or intermediate-risk types of HPV, whereas LSILs were associated with heterogeneous HPV types. Both subjects who were found to have HSIL in the present study were positive for high-risk types of HPV-DNA, whereas the subjects with LSIL were positive for either high-risk or low-risk types.
In a previous study reported by Yokota et al., HPV-DNA was detected in 3.3% of 666 Japanese women with normal cervical cytology (13). In that series, the highest incidence of positive HPV-DNA (7.3%) was found in the 30- to 39-year age group (n = 108), with much lower incidences in the 60- to 69-year (n = 126) and >70-year (n = 18) age groups (0.8% and 0%, respectively). In the present study, no HPV-DNA was detected in cervical cells of any of the 315 very elderly women with normal cytologic specimens. This finding is in accordance with those of several previous studies. HPV-DNA has been detected previously in 8.3% of 2,189 Brazilian women (2), 0.9% of 1,033 Dutch women (ages 35-55 years; ref. 14), and 9% of 8,755 German women (5). These studies, however, did not deal primarily with very elderly women, as was the case in our series. Our data also suggest that HPV infection in young women with normal cervical cytology often does not persist into very advanced age. This is consistent with a review by Schiffman (6), which found that most HPV-infected women do not have persistent infection, defined as the continual detection of the same viral type or types for a prolonged period of time.
We detected HPV-DNA in a considerable number of very elderly women (i.e., age >70 years). One 96-year-old woman had LSIL as well as HPV-DNA in her cervical smear. Thus, some types of HPV infection that are strongly associated with invasive carcinoma and that generally occur in women ages <50 years may persist for several decades. Although intraepithelial neoplasia is detected in some HPV-DNA positive cases, our findings suggest that intraepithelial neoplasia of the uterine cervix does not occur solely in association with HPV infection.
In conclusion, we conducted this study in an attempt to resolve, at least in part, the natural history of HPV infection of the uterine cervix in humans. We found that some types of HPV infection can persist for a very long time even until death at a very advanced age. However, it seems probable that HPV infection in young women with normal cervical cytology often does not persist into very old age. The results of our study suggest that young women with normal cervical cytology who are positive for HPV-DNA may not need to have long-term clinical follow-up.
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Acknowledgments
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We thank the medical technologists of the Department of Anatomical Pathology of Tokyo Metropolitan Geriatric Medical Center, who prepared the smears from all the subjects, and Dr. Neil K. Lambie (Department of Pathology, Canterbury Health Laboratories, Christchurch Hospital, Christchurch, New Zealand) for his help in the preparation of this article in English.
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Footnotes
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Grant support: Grants-in-aid 12218106 and 12218239 for Scientific Research on Priority Areas of Cancer from the Ministry of Education, Science, Sports and Culture of Japan.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 11/19/04;
accepted 12/ 1/04.
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Abstract
Materials and Methods
