959
SHORT REPORT
Human papillomavirus type 16 DNA in cervical smears as predictor of high-grade cervical cancer
women with mild to cervical smears would moderately dyskaryotic from a test that non-invasive benefit predicts which women have high-grade cervical intraepithelial neoplasia. Detection of human papillomavirus type 16 (HPV16) DNA in cervical smears may be such a test. With the polymerase chain reaction (PCR), we estimated the amount of HPV16 DNA in cervical smears from 85 women referred for colposcopy because of abnormal cytology. An intermediate or high amount of HPV16 DNA predicted the presence of high-grade cervical intraepithelial neoplasia in a subsequent biopsy in almost 90% of patients irrespective of the cytological grade of the referral smear. This technique may allow early identification of those women with low-grade cytological abnormalities who have high-grade underlying cervical disease
The management of
Around 5% of the more than 5 million cervical smears examined each year in the UK show evidence of abnormality. Severe dyskaryosis, which usually indicates underlying high-grade cervical intraepithelial neoplasia (CIN3), is found in about 10% of abnormal smears; it is a clear and sufficient indication for referral for colposcopy. However, most abnormal smears show mild or moderate dyskaryosis, where the underlying pathology is highly variable. Biopsy shows that about a third of such cases have high-grade disease (CIN3), a third have CIN1/2, and the remaining third have either normal cervices or lesions not amounting to CIN1.1 The management of women with mild or moderate dyskaryosis is controversial and would benefit from a non-invasive test that predicts accurately which women have underlying CIN3 disease. Preferrably, the test should be based on material taken at the time of the smear rather than on colposcopic observation. Human papillomavirus type 16 (HPV 16) DNA is found in 60-70% of cervical cancers or high-grade precancerous lesions by Southern blotting or in-situ hybridisation.2,3 However, these tests cannot be used for routine screening because they are labour intensive and not very sensitive, and smears do not always contain enough (106) cells for Southern blotting. The polymerase chain reaction (PCR) is a simpler and far more sensitive procedure (practical limit of sensitivity about one copy in 104 cells and requiring less than
2
x
105
cells), but a study of normal and cancerous cervical
specimens with PCR has indicated that low levels of HPV 16 DNA may be virtually ubiquitous.4 Van den Brule et al5 used carefully standardised procedures, and found that, depending on history of cervical pathology, prevalence rates for HPV16 DNA in cytologically normal women were 0 5-12 % . The rates for HPV 16/18 DNA in cases of mild or moderate dyskaryosis or severe dyskaryosis were 41 % and 58%, respectively. Similar prevalence rates for HPV16/18 based on dot-blot hybridisation of smears have been reported.6 However, these studies did not attempt to relate the HPV status of the smear to underlying histology for individual women. We present data relating PCR detection of HPV 16 DNA in cervical smears to underlying histology. TABLE I-HPV16 DNA AND HISTOLOGICAL DIAGNOSIS IN WOMEN REFERRED WITH A SEVERELY DYSKARYOTIC SMEAR
Figures are numbers of women in each group. *See text for definition tThls group Includes women judged to be normal on colposcopy and from whom no biopsy was taken, and women whose histological diagnosis was less severe than CIN1 +Includes 1 case of invasive cancer
Patients referred for colposcopy because of a dyskaryotic cervical studied. At the time of colposcopy, another smear was taken and sent for routine cytological assessment. The same spatula was used to collect additional cells, which were then stored at - 20°C in phosphate buffered saline. Cells were pelleted and washed before DNA extraction. Any areas of abnormal epithelium found on colposcopy were biopsied with a punch biopsy, loop diathermy, or laser cone, as appropriate, and sent for routine histological examination. Women with no colposcopically visible abnormality were not biopsied and were assumed to be histologically normal. PCR was used for estimation of HPV 16 DNA in DNA extracted from cervical cells.* PCR was done with primers that amplify a 228 bp segment of the regulatory region of the virus genome (primer 1, nucleotides 7763-7781; primer 2, complementary to nucleotides 78-61).’ The PCR product was located by ethidium bromide staining after electrophoresis on a 2% agarose gel. Reaction mixtures containing 04, 2, 10, and 100 fg of HPV 16 plasmid DNA from a standard preparation were included in every PCR run. Amounts of HPV16 DNA in individual patient specimens were estimated visually by comparison with the standards. HPV16 DNA content was designated "high" if the intensity of the 228 bp band was equal to or greater than that of the 100 fg standard, "intermediate" if the band intensity was between that of the 100 fg and 2 fg standards, "low" if the band intensity was equal or below that of the 2 fg standard, and "negative" if the band was not visible. The 50-100-fold difference in amount of HPV16 DNA between the "high" and the "low" categories could be read unambiguously and was completely reproducible. Less than 5% of specimens were of the "intermediate" category. Assays were scored without knowledge of cytology or histology results. smear were
*Details of DNA extraction and PCR methods authors.
are
available from the
960
TABLE il-HPV16 DNA LEVELS AND HISTOLOGICAL DIAGNOSIS FOR WOMEN REFERRED WITH A MILDLY OR MODERATELY
DYSKARYOTIC SMEAR
I
Figures are numbers of women
I
m
I
I
I
each group
*See text for definition.
The relation between amount of HPV16 DNA and underlying histology in 30 women referred because of severely dyskaryotic smears is shown in table 1. High amounts of HPV 16 DNA were detected in 11 women and 10 of these women had CIN3 or invasive cancer. The 1 false-positive patient was pregnant. In the 19 women in whom HPV 16 DNA was not detected or was present only at low levels, a further 10 cases of CIN3 were found. This suggests that another HPV type was involved, active HPV16 infection was no longer present, or that the lesion was unrelated to HPV. Table n shows the relation between amount of HPV16 DNA in cervical smears and the colposcopic or histological diagnosis in 55 women referred with a mildly (23) or moderately (32) dyskaryotic smear. An intermediate or high amount of HPV 16 DNA was found in 16 (29%) of these women, of whom 14 (87%) had CIN3. By contrast, CIN3 was found in only 4 of 39 women (10%) with low or no detectable HPV16 DNA. Of 10 women diagnosed as CIN1 or 2, only 1 (CIN2) had high HPV16 DNA. 26 of 27 women (96%) who were normal or had disease of lower grade than CIN1 by histology and/or colposcopy had undetectable or low amounts of HPV16 DNA (22 were in fact negative). Although HPV16 DNA was not found by PCR in cervical smears from all patients with CIN3 lesions, almost 90% of patients with intermediate or high amounts of viral DNA had an underlying CIN3 lesion. This relation was equally strong in women with mild, moderate, or severe dyskaryosis. Of the 30 women with severe dyskaryosis on referral, 20 had underlying CIN3 lesions detectable by routine histology. Only 10 of these women were identified by detection of an intermediate or high amount of HPV16 DNA. The false-negative results could have been caused by another type of HPV being involved in lesions or the HPV16 genome becoming integrated in the human genome and no longer producing detectable viral DNA in exfoliated cells; alternatively, some high-grade lesions may be unrelated to HPV16. However, for these women, colposcopic examination and biopsy is indicated by the severely dyskaryotic smear results; detection of HPV 16 DNA would not help management. 18 of the 55 women with mildly or moderately dyskaryotic smears had CIN3 lesions; this finding was predicted correctly by an intermediate or high amount of HPV16 DNA in cervical smears in 14 (78%) patients.
Identification of these women is important because clinical be directed to those at greatest risk-ie, patients with existing high-grade lesions which, in most cases, will not regress.8 Of the 2 "false positives" in the mildly or moderately dyskaryotic group, 1 had a CIN2 lesion. Detection of HPV, including HPV16, in apparently normal epithelium or normal epithelium adjacent to high-grade lesions or tumours has been reported,9 and shown to have clinical relevance even when no histological evidence of a lesion is present.1O Follow-up studies are needed to clarify whether these cases represent the earliest stages of CIN3 lesions. A test for intermediate or high amounts of HPV16 DNA in cervical smears could help improve management of women with mild to moderate dyskaryosis. 34 of 38 women (90%) with colposcopically visible lesions that turned out to be CIN3 were identified by presence of severe dyskaryosis and/or high or intermediate HPV16 DNA levels. These findings indicate that colposcopic investigation of women with mild or moderate dyskaryosis is appropriate when increased amounts of HPV 16 DNA are also present. The 3 women with intermediate or high HPV16 DNA but without CIN3 lesions may be at high risk of developing these lesions and may need increased surveillance. The women who had mild to moderate dyskaryosis and who were negative for HPV 16 DNA would be managed in the usual manner. resources can
We thank Prof H.
zur
Hausen for use of the HPV16 plasmid.
REFERENCES 1. Soutter WP, Wisdom S, Brough A, Monaghan JM. Should patients with mild atypia in a cervical smear be referred for colposcopy? Br J Obstet Gynaecol 1986; 93: 70-74. 2. Nuovo GJ, Richart RM. A comparison of slot blot, Southern blot, and in situ hybridization analyses for human papillomavirus DNA in genital tract lesions. Obstet Gynecol 1990; 74: 673-78. 3. Nuovo GJ, Blanco JS, Leipzig S, Smith D. Human papillomavirus detection in cervical lesions nondiagnostic for cervical intraepithelial
neoplasia: correlation with Papanicolaou smear, colposcopy, and occurence of cervical intraepithelial neoplasia. Obstet Gynecol 1990; 75: 1006-11. 4.
5.
Johnson MA, Blomfield PI, Bevan IS, Woodman CBJ, Young LS. Analysis of human papillomavirus type 16 E6-E7 transcription in cervical carcinomas and normal cervical epithelium using the polymerase chain reaction. J Gen Virol 1990; 71: 1473-79. Van den Brule AJC, Walboomers JMM, Maine MD, Kenemans P, Meijer CJLM. Difference in prevalence of human papillomavirus genotypes in cytomorphologically normal cervical smears is associated with a history of cervical intraepithelial neoplasia. Int J Cancer 1991; 48:
404-08. 6. Kochel HG, Teichmann A, Eckardt N, Arendt P, Kuhn W, Thomssen R. Occurence of human papillomavirus DNA types 16 and 18 (HPV-16/18) in cervical smears as compared to cytological findings. Int J Gynaecol Obstet 1990; 31: 145-52. 7. Seedorf K, Krammer G, Durst M, Suhai S, Rowekamp WG. Human papillomavirus type 16 DNA sequence. Virology 1985; 145: 181-85. 8. McIndoe WA, McLean MR, Jones RW, Mullins PR. The invasive potential of carcinoma in situ of the cervix. J Am Coll Obstet Gynecol 1984; 64: 451-58. 9. Fuchs PG, Girardi F, Pfister H. Human papillomavirus DNA in normal, metaplastic, preneoplastic and neoplastic epithelia of the cervix uteri. Int J Cancer 1988; 41: 41-45. 10. Nuovo GJ, Darfler MM, Impraim CC, Bromley SE. Occurence of multiple types of human papillomavirus in genital tract lesions. Analysis by in situ hybridization and the polymerase chain reaction. Am J Pathol 1991; 138: 53-58.
ADDRESSES: Imperial Cancer Research Fund, PO Box 123, London WC2A 3PX, UK (J. Cuzick, PhD); Departments of Chemical Pathology (G. Terry, PhD) and Medical Microbiology (L Ho, MD), University College London; and Departments of Obstetrics (T. Hollingworth, MB) and Pathology (M Anderson FRCPath), University Hospital, Nottingham. Correspondence to Dr J Cuzick.
SHORT REPORT
Human papillomavirus type 16 DNA in cervical smears as predictor of high-grade cervical cancer
women with mild to cervical smears would moderately dyskaryotic from a test that non-invasive benefit predicts which women have high-grade cervical intraepithelial neoplasia. Detection of human papillomavirus type 16 (HPV16) DNA in cervical smears may be such a test. With the polymerase chain reaction (PCR), we estimated the amount of HPV16 DNA in cervical smears from 85 women referred for colposcopy because of abnormal cytology. An intermediate or high amount of HPV16 DNA predicted the presence of high-grade cervical intraepithelial neoplasia in a subsequent biopsy in almost 90% of patients irrespective of the cytological grade of the referral smear. This technique may allow early identification of those women with low-grade cytological abnormalities who have high-grade underlying cervical disease
The management of
Around 5% of the more than 5 million cervical smears examined each year in the UK show evidence of abnormality. Severe dyskaryosis, which usually indicates underlying high-grade cervical intraepithelial neoplasia (CIN3), is found in about 10% of abnormal smears; it is a clear and sufficient indication for referral for colposcopy. However, most abnormal smears show mild or moderate dyskaryosis, where the underlying pathology is highly variable. Biopsy shows that about a third of such cases have high-grade disease (CIN3), a third have CIN1/2, and the remaining third have either normal cervices or lesions not amounting to CIN1.1 The management of women with mild or moderate dyskaryosis is controversial and would benefit from a non-invasive test that predicts accurately which women have underlying CIN3 disease. Preferrably, the test should be based on material taken at the time of the smear rather than on colposcopic observation. Human papillomavirus type 16 (HPV 16) DNA is found in 60-70% of cervical cancers or high-grade precancerous lesions by Southern blotting or in-situ hybridisation.2,3 However, these tests cannot be used for routine screening because they are labour intensive and not very sensitive, and smears do not always contain enough (106) cells for Southern blotting. The polymerase chain reaction (PCR) is a simpler and far more sensitive procedure (practical limit of sensitivity about one copy in 104 cells and requiring less than
2
x
105
cells), but a study of normal and cancerous cervical
specimens with PCR has indicated that low levels of HPV 16 DNA may be virtually ubiquitous.4 Van den Brule et al5 used carefully standardised procedures, and found that, depending on history of cervical pathology, prevalence rates for HPV16 DNA in cytologically normal women were 0 5-12 % . The rates for HPV 16/18 DNA in cases of mild or moderate dyskaryosis or severe dyskaryosis were 41 % and 58%, respectively. Similar prevalence rates for HPV16/18 based on dot-blot hybridisation of smears have been reported.6 However, these studies did not attempt to relate the HPV status of the smear to underlying histology for individual women. We present data relating PCR detection of HPV 16 DNA in cervical smears to underlying histology. TABLE I-HPV16 DNA AND HISTOLOGICAL DIAGNOSIS IN WOMEN REFERRED WITH A SEVERELY DYSKARYOTIC SMEAR
Figures are numbers of women in each group. *See text for definition tThls group Includes women judged to be normal on colposcopy and from whom no biopsy was taken, and women whose histological diagnosis was less severe than CIN1 +Includes 1 case of invasive cancer
Patients referred for colposcopy because of a dyskaryotic cervical studied. At the time of colposcopy, another smear was taken and sent for routine cytological assessment. The same spatula was used to collect additional cells, which were then stored at - 20°C in phosphate buffered saline. Cells were pelleted and washed before DNA extraction. Any areas of abnormal epithelium found on colposcopy were biopsied with a punch biopsy, loop diathermy, or laser cone, as appropriate, and sent for routine histological examination. Women with no colposcopically visible abnormality were not biopsied and were assumed to be histologically normal. PCR was used for estimation of HPV 16 DNA in DNA extracted from cervical cells.* PCR was done with primers that amplify a 228 bp segment of the regulatory region of the virus genome (primer 1, nucleotides 7763-7781; primer 2, complementary to nucleotides 78-61).’ The PCR product was located by ethidium bromide staining after electrophoresis on a 2% agarose gel. Reaction mixtures containing 04, 2, 10, and 100 fg of HPV 16 plasmid DNA from a standard preparation were included in every PCR run. Amounts of HPV16 DNA in individual patient specimens were estimated visually by comparison with the standards. HPV16 DNA content was designated "high" if the intensity of the 228 bp band was equal to or greater than that of the 100 fg standard, "intermediate" if the band intensity was between that of the 100 fg and 2 fg standards, "low" if the band intensity was equal or below that of the 2 fg standard, and "negative" if the band was not visible. The 50-100-fold difference in amount of HPV16 DNA between the "high" and the "low" categories could be read unambiguously and was completely reproducible. Less than 5% of specimens were of the "intermediate" category. Assays were scored without knowledge of cytology or histology results. smear were
*Details of DNA extraction and PCR methods authors.
are
available from the
960
TABLE il-HPV16 DNA LEVELS AND HISTOLOGICAL DIAGNOSIS FOR WOMEN REFERRED WITH A MILDLY OR MODERATELY
DYSKARYOTIC SMEAR
I
Figures are numbers of women
I
m
I
I
I
each group
*See text for definition.
The relation between amount of HPV16 DNA and underlying histology in 30 women referred because of severely dyskaryotic smears is shown in table 1. High amounts of HPV 16 DNA were detected in 11 women and 10 of these women had CIN3 or invasive cancer. The 1 false-positive patient was pregnant. In the 19 women in whom HPV 16 DNA was not detected or was present only at low levels, a further 10 cases of CIN3 were found. This suggests that another HPV type was involved, active HPV16 infection was no longer present, or that the lesion was unrelated to HPV. Table n shows the relation between amount of HPV16 DNA in cervical smears and the colposcopic or histological diagnosis in 55 women referred with a mildly (23) or moderately (32) dyskaryotic smear. An intermediate or high amount of HPV 16 DNA was found in 16 (29%) of these women, of whom 14 (87%) had CIN3. By contrast, CIN3 was found in only 4 of 39 women (10%) with low or no detectable HPV16 DNA. Of 10 women diagnosed as CIN1 or 2, only 1 (CIN2) had high HPV16 DNA. 26 of 27 women (96%) who were normal or had disease of lower grade than CIN1 by histology and/or colposcopy had undetectable or low amounts of HPV16 DNA (22 were in fact negative). Although HPV16 DNA was not found by PCR in cervical smears from all patients with CIN3 lesions, almost 90% of patients with intermediate or high amounts of viral DNA had an underlying CIN3 lesion. This relation was equally strong in women with mild, moderate, or severe dyskaryosis. Of the 30 women with severe dyskaryosis on referral, 20 had underlying CIN3 lesions detectable by routine histology. Only 10 of these women were identified by detection of an intermediate or high amount of HPV16 DNA. The false-negative results could have been caused by another type of HPV being involved in lesions or the HPV16 genome becoming integrated in the human genome and no longer producing detectable viral DNA in exfoliated cells; alternatively, some high-grade lesions may be unrelated to HPV16. However, for these women, colposcopic examination and biopsy is indicated by the severely dyskaryotic smear results; detection of HPV 16 DNA would not help management. 18 of the 55 women with mildly or moderately dyskaryotic smears had CIN3 lesions; this finding was predicted correctly by an intermediate or high amount of HPV16 DNA in cervical smears in 14 (78%) patients.
Identification of these women is important because clinical be directed to those at greatest risk-ie, patients with existing high-grade lesions which, in most cases, will not regress.8 Of the 2 "false positives" in the mildly or moderately dyskaryotic group, 1 had a CIN2 lesion. Detection of HPV, including HPV16, in apparently normal epithelium or normal epithelium adjacent to high-grade lesions or tumours has been reported,9 and shown to have clinical relevance even when no histological evidence of a lesion is present.1O Follow-up studies are needed to clarify whether these cases represent the earliest stages of CIN3 lesions. A test for intermediate or high amounts of HPV16 DNA in cervical smears could help improve management of women with mild to moderate dyskaryosis. 34 of 38 women (90%) with colposcopically visible lesions that turned out to be CIN3 were identified by presence of severe dyskaryosis and/or high or intermediate HPV16 DNA levels. These findings indicate that colposcopic investigation of women with mild or moderate dyskaryosis is appropriate when increased amounts of HPV 16 DNA are also present. The 3 women with intermediate or high HPV16 DNA but without CIN3 lesions may be at high risk of developing these lesions and may need increased surveillance. The women who had mild to moderate dyskaryosis and who were negative for HPV 16 DNA would be managed in the usual manner. resources can
We thank Prof H.
zur
Hausen for use of the HPV16 plasmid.
REFERENCES 1. Soutter WP, Wisdom S, Brough A, Monaghan JM. Should patients with mild atypia in a cervical smear be referred for colposcopy? Br J Obstet Gynaecol 1986; 93: 70-74. 2. Nuovo GJ, Richart RM. A comparison of slot blot, Southern blot, and in situ hybridization analyses for human papillomavirus DNA in genital tract lesions. Obstet Gynecol 1990; 74: 673-78. 3. Nuovo GJ, Blanco JS, Leipzig S, Smith D. Human papillomavirus detection in cervical lesions nondiagnostic for cervical intraepithelial
neoplasia: correlation with Papanicolaou smear, colposcopy, and occurence of cervical intraepithelial neoplasia. Obstet Gynecol 1990; 75: 1006-11. 4.
5.
Johnson MA, Blomfield PI, Bevan IS, Woodman CBJ, Young LS. Analysis of human papillomavirus type 16 E6-E7 transcription in cervical carcinomas and normal cervical epithelium using the polymerase chain reaction. J Gen Virol 1990; 71: 1473-79. Van den Brule AJC, Walboomers JMM, Maine MD, Kenemans P, Meijer CJLM. Difference in prevalence of human papillomavirus genotypes in cytomorphologically normal cervical smears is associated with a history of cervical intraepithelial neoplasia. Int J Cancer 1991; 48:
404-08. 6. Kochel HG, Teichmann A, Eckardt N, Arendt P, Kuhn W, Thomssen R. Occurence of human papillomavirus DNA types 16 and 18 (HPV-16/18) in cervical smears as compared to cytological findings. Int J Gynaecol Obstet 1990; 31: 145-52. 7. Seedorf K, Krammer G, Durst M, Suhai S, Rowekamp WG. Human papillomavirus type 16 DNA sequence. Virology 1985; 145: 181-85. 8. McIndoe WA, McLean MR, Jones RW, Mullins PR. The invasive potential of carcinoma in situ of the cervix. J Am Coll Obstet Gynecol 1984; 64: 451-58. 9. Fuchs PG, Girardi F, Pfister H. Human papillomavirus DNA in normal, metaplastic, preneoplastic and neoplastic epithelia of the cervix uteri. Int J Cancer 1988; 41: 41-45. 10. Nuovo GJ, Darfler MM, Impraim CC, Bromley SE. Occurence of multiple types of human papillomavirus in genital tract lesions. Analysis by in situ hybridization and the polymerase chain reaction. Am J Pathol 1991; 138: 53-58.
ADDRESSES: Imperial Cancer Research Fund, PO Box 123, London WC2A 3PX, UK (J. Cuzick, PhD); Departments of Chemical Pathology (G. Terry, PhD) and Medical Microbiology (L Ho, MD), University College London; and Departments of Obstetrics (T. Hollingworth, MB) and Pathology (M Anderson FRCPath), University Hospital, Nottingham. Correspondence to Dr J Cuzick.
