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Human Papillomaviruses and Cervical Cancer in Bangkok. II. Risk Factors for in Situ and Invasive Squamous Cell Cervical Carcinomas

Human Papillomaviruses and Cervical Cancer in Bangkok. II. Risk Factors for in Situ and Invasive... Abstract To identify risk factors for progression of intraepithelial cervical lesions, 190 women with invasive cervical cancer were compared with 75 women with in situ disease diagnosed in Bangkok, Thailand, between September 1991 and September 1993. Polymerase chain reaction-based assays for type-specific human papillomavirus (HPV) DNA in cervical scrapings revealed oncogenic types in 79% of invasive and 57% of intraepithelial tumors. Types 16 and 18, but not types 31/33/35/39, were more common in invasive than intraepithelial tumors, and untyped HPV DNA was found more commonly in the in situ lesions, suggesting that in situ disease is four times more likely to become invasive if due to type 16 or 18 than to other causes, and that tumors with only untyped HPV are not at increased risk of progression. After controlling for HPV type, the risk of developing invasive diseases, compared with the risk of developing intraepithelial lesions, was not related to any of a large number of sexual and hormonal factors considered or to smoking, suggesting that any cofactors these variables represent act before the development of in situ carcinoma. Two indices of socioeconomic status were associated with a reduced risk of only invasive disease, suggesting the existence of unknown protective factors that operate after intraepithelial lesions develop. carcinoma, squamous cell, cervical intraepithelial neoplasia, cervix neoplasms, papillomavirus, human CIN-III, cervical intraepithelial neoplasia grade 3, HPV, human papillomavirus Some types of human papillomaviruses (HPVs) are carcinogenic for the cervix uteri (1). Although they are quite likely necessary causes of cervical carcinoma, they are not sufficient causes. Most infected women do not develop invasive cervical cancer, and cofactors must therefore play a role in allowing these infections to become persistent and carcinogenic. Cervical carcinoma in situ (cervical intra-epithelial neoplasia grade 3 (CIN-III)) is an intermediate morphologic lesion on the pathway from normal cervical epithelium to invasive disease, and cofactors for invasive cervical carcinoma could operate either prior to the development of carcinoma in situ or afterward. Those acting before the development of the intraepithelial lesion would be observed in relation to both in situ and invasive lesions, whereas those acting to enhance the probability that an in situ lesion becomes invasive would be observed only in relation to invasive disease. The identification of cofactors for preinvasive disease is best attempted in the context of prospective studies of HPV-infected women. Concurrent prospective studies of untreated women with carcinoma in situ to determine factors associated with progression to invasive diseases would be unethical, and studies of treated women would be uninformative because treatment removes or destroys the in situ lesion. A few historical cohort studies of HPV-infected subjects that included both in situ and invasive cervical carcinoma as endpoints (1–4) and case-control studies of invasive disease (1, 5–9) have identified a variety of possible cofactors with varying degrees of consistency that could act either before or after the development of in situ disease. These include the following: early age at first sexual intercourse, which may reflect particular vulnerability of the immature cervix to HPV-induced carcinogenesis; hormonal factors such as high parity and use of oral contraceptives; exposures to chemical agents in cigarette smoke; sexually transmitted agents other than HPV; deficiencies in certain micronutrients; an immunocompromised state resulting from human immunodeficiency virus (HIV) infection or immunosuppresion for prevention of organ transplant rejection; and various measures of socioeconomic status, suggesting the presence of additional unrecognized cofactors associated with social class. Most of these factors, however, have been observed in relation to both in situ and invasive disease (1) and, in studies in which the epidemiologic features of these two stages of cervical carcinoma can be compared, few consistent differences in risk factors have been observed (5, 10–15). These observations suggest that most putative cofactors are operative before the development of carcinoma in situ and that factors associated with progression to invasion have yet to be identified. This is a report of analysis performed to compare risk factors for in situ and invasive disease in order to identify variables associated only with the latter condition, after controlling for the presence of oncogenic HPV types in the tumor tissue. If women with in situ disease are considered the controls and if women with invasive disease are considered the cases in a standard case-control analysis, then elevated odds ratios in relation to a factor represent the risk of invasive disease relative to the risk of in situ disease in women with the factor (16). Because it is reasonable to assume that most or all invasive cervical carcinomas have passed through an in situ stage, an elevated odds ratio would imply that the factor enhances probability of progression from in situ to invasion. To our knowledge, results of direct comparisons of women with in situ and invasive disease have been reported only once previously (10). MATERIALS AND METHODS Women who were admitted to the public wards of Siriraj Hospital in Bangkok, Thailand, with a new histologically confirmed diagnosis of either CIN-III or invasive cervical carcinoma between September 1991 and September 1993, who were born after 1929, and who resided in Thailand for at least the past year were eligible as cases for this study. Although CIN-III includes both severe dysplasia and carcinoma in situ, the older terms, carcinoma in situ and in situ disease, are used interchangeably with CIN-III in this report. Two controls for each woman with invasive disease were selected from otolaryngology and general surgery wards of Siriraj Hospital, as previously described (17). The cases of carcinoma in situ were women who had had an abnormal cervical smear during a visit to either the family planning or gynecologic clinic associated with Siriraj Hospital, and an attempt was made to select two controls for each case from the same clinic from which the case came. Cervical smears are taken from many of the women who attend these clinics, and the women selected as controls were the next two women with appointments in the clinic after the appointment for the case, in the same 5-year age group and from the same region of the country as the case, who had returned to the clinic to learn the results of their cervical smears, and whose cervical smears revealed no suspicion of neoplastic change. If a woman refused to participate, the selection process continued until two controls per case were selected. All cases and hospitalized controls were interviewed while hospitalized; the controls selected for the cases of carcinoma in situ were interviewed in the clinics on the day they were selected. Information was obtained on sexual and reproductive history, prior cervical smears, use of tobacco and alcohol, and indices of prior use of medical resources and socioeconomic status. Dates of all prior cervical smears were obtained, and those taken within the past year were not considered prior screening cervical smears. The presenting symptom of each case was also ascertained and recorded as none (detected by routine cytology only), vaginal bleeding, abnormal vaginal discharge, or other. A 15-ml blood specimen was obtained from all interviewed women. Four aliquots of serum were stored at −70°C, two of which were retained in Bangkok and two of which were shipped to Seattle, Washington, on dry ice. Cervical scrapings for HPV DNA assays were obtained from cases by the patient's surgeon prior to treatment. Cervical smears and cervical scrapings were taken by these same surgeons from the hospital controls while they were hospitalized and from the clinic controls in the clinic on the same day they were selected and interviewed. The cervix, including the cervical os, was scraped with a Teflon (E. I. du Pont de Nemours and Company, Wilmington, Delaware)-coated swab, the end of which was then broken off into a vial containing 2 ml of specimen transport medium (Digene Diagnostics, Inc., Beltsville, Maryland). The specimens were stored at −70°C and periodically shipped on dry ice to Seattle. Histologic slides from the blocks that were used to make the diagnosis in the cases were read by a single collaborating pathologist in Bangkok. Information was recorded on source of specimen, tumor size, stage, and histologic diagnosis. A pathologist in Seattle reread the slides from 174 of the cases included in this report. Of 23 in situ cases, only one (4.3 percent) was considered invasive by the reference pathologist, and only eight (5.3 percent) of 151 cases considered invasive in Bangkok were read in Seattle as in situ with no evidence of microinvasion. The diagnosis made in Bangkok was thus used in the analysis in this report. As detailed elsewhere (17), serologic assays were performed for antibodies to human immunodeficiency virus, hepatitis B, Treponema pallidum, and herpes simplex virus types 1 and 2, as well as for hepatitis B surface antigen. Cervical scrapings were assayed for evidence of any HPV DNA and for type-specific DNA of types 6 and 11, type 16, type 18, types 31/33/35/39, and type 45 using polymerase chain reaction-based technology (17). Samples that hybridized with a generic probe but not with any of the type-specific probes were considered positive for untyped HPV. Cases and controls were compared, and odds ratios as estimates of relative risks were calculated using unconditional logistic regression (18) when comparing invasive cases with their unmatched controls and using conditional logistic regression (18) when comparing in situ cases with their matched controls. In addition, to directly compare risk factors for invasive and in situ disease, we performed unconditional logistic regression analyses using the women with invasive disease as “cases” and women with carcinoma in situ as “controls.” Because the purpose of these analyses was to identify any factors that distinguish invasive from in situ disease, and because no such factors were identified other than HPV status, no attempts were made to adjust odds ratios for factors other than age and (for the case-case comparisons) HPV status. RESULTS A total of 420 eligible cases were identified, of whom 338 (80.5 percent) were interviewed. Cervical scrapings were obtained from 337 (99.7 percent) of those interviewed, 216 and 75 of whom had invasive and in situ squamous cell carcinoma, respectively. Adequate amounts of DNA for HPV DNA assays were found in cervical scrapings from 191 of the women with invasive disease and all 75 of the women with carcinoma in situ. One woman with invasive disease who claimed to be a virgin was omitted, leaving 190 cases of invasive cervical carcinoma in the analyses. Controls were not found for 10 of the women with carcinoma in situ, leaving 65 of these cases for inclusion in the analyses in which cases and controls were compared; all 75 cases were included in the analyses in which women with invasive and in situ disease were compared. Of the 614 women selected as controls for the cases of invasive disease, 490 (79.8 percent) were interviewed. Cervical scrapings were obtained from 306 (62.4 percent) of these 490 women, 298 (97.4 percent) of which yielded adequate DNA samples. Three women with no history of sexual intercourse, one with a subtotal hysterectomy, and three with ineligible diagnoses were excluded, leaving 291 controls for analysis. Of the 161 women selected as controls for the in situ cases, 160 (99.4 percent) were interviewed, 144 of whom were subsequently confirmed as meeting the eligibility criteria for inclusion in the study. Cervical scrapings were obtained from 133 (92.4 percent) of these 144 women, 128 of which contained adequate amounts of DNA. Four of the 128 women from whom these samples were taken had been matched to excluded cases and were omitted, leaving 124 women as controls for the in situ cases in the analyses. As shown in table 1, women with invasive disease were slightly older than their controls. The women with carcinoma in situ and their controls were comparable in age and younger than the women with invasive carcinoma and their controls. The 10 additional cases of carcinoma in situ used in the comparisons of in situ and invasive disease are not appreciably different in age from the 65 cases shown in the table. All odds ratio estimates presented were adjusted for age using the age categories shown in the table. TABLE 1. Age distribution of invasive and in situ squamous cell cervical carcinomas and their corresponding controls, Bangkok, Thailand, 1991–1993* Age (years)  Invasive   In situ   Cases   Controls   Cases   Controls   No.  %  No.  %  No.  %  No.  %  <30  3  1.6  13  4.5  16  24.6  29  23.4  30–34  23  12.1  44  15.1  11  16.9  24  19.3  35–39  36  18.9  56  19.2  15  23.1  32  25.8  40–44  33  17.4  60  20.6  12  18.5  17  13.7  45–49  30  15.8  36  12.4  4  6.1  10  8.1  ≥50  65  34.2  82  28.2  7  10.8  12  9.7  Total  190  100.0  291  100.0  65  100.0  124  100.0  Age (years)  Invasive   In situ   Cases   Controls   Cases   Controls   No.  %  No.  %  No.  %  No.  %  <30  3  1.6  13  4.5  16  24.6  29  23.4  30–34  23  12.1  44  15.1  11  16.9  24  19.3  35–39  36  18.9  56  19.2  15  23.1  32  25.8  40–44  33  17.4  60  20.6  12  18.5  17  13.7  45–49  30  15.8  36  12.4  4  6.1  10  8.1  ≥50  65  34.2  82  28.2  7  10.8  12  9.7  Total  190  100.0  291  100.0  65  100.0  124  100.0  * The mean and median ages (years) are the following: invasive cases, 45.5 and 45.5; invasive controls, 43.9 and 43.0; in situ cases, 37.8 and 38.0; and in situ controls, 37.2 and 37.0, respectively. View Large Vaginal bleeding was the presenting symptom in 124 (65.3 percent) of the 190 women with invasive disease and in 47 (62.7 percent) of the 75 women with carcinoma in situ; 63 (33.2 percent) of the women with invasive carcinoma and 23 (30.7 percent) of those with carcinoma in situ reported no specific symptoms. Oncogenic types of HPV were strongly associated with both invasive and in situ disease (table 2). Type 16 DNA was the most frequently identified type in both invasive and in situ tumors. Both types 16 and 18 were found in a higher proportion of invasive than in situ carcinomas. Types 31/33/35/39 occurred with nearly equal frequency in both tumor types, and untyped HPV DNA was found in a higher proportion of the in situ than invasive lesions. Few women in either control group had any type of HPV in their cervical scrapings, although the frequency of a positive test was slightly higher in the controls for the in situ cases. The estimates of the odds ratio of both invasive and in situ carcinomas are large, with those in relation to types 16 and 18, any oncogenic type, and any HPV type being stronger for invasive disease; those in relation to types 31/33/35/39 being similar for both tumor types; but those in relation to untyped HPV being greater for in situ disease. As shown in table 3, after controlling for age and other HPV types, the odds ratio of invasive disease is four times greater than the risk of in situ disease in women with HPV types 16 and 18, twice as high in women with HPV types 31/33/35/39 (although possibly due to chance), and also significantly higher in relation to any oncogenic type and any type of HPV. The risk is not greater for invasive than in situ disease in women with untyped HPV DNA. TABLE 2. Age-adjusted odds ratios of invasive and in situ squamous cell cervical carcinoma in relation to specific types of human papillomaviruses, Bangkok, Thailand, 1991–1993 HPV* type†  Positive subjects           Invasive   In situ   Invasive  In situ  Cases (n = 190)   Controls (n = 291)   Cases (n = 65)   Controls (n = 124)           No.  %  No.  %  No.  %  No.  %  OR*  95% CI*  OR  95% CI  16  114  60.0  6  2.1  25  38.4  6  4.8  83  39, 232  11  3.9, 33  18  26  13.7  0  0  5  7.7  1  0.8  ∞  22, ∞  10  1.2, 86  31/33/35/39  25  13.2  3  1.0  9  13.8  1  0.8  14  4.9, 61  17  2.2, 135  Any oncogenic type‡  150  78.9  9  3.1  37  56.9  7  5.6  155  72, 384  16  5.9, 47  Untyped§  14  7.4  11  3.8  12  18.5  6  4.8  2.0  0.9, 4.7  4.9  1.6, 15  Any type§  164  86.3  20  6.9  50  76.9  13  10.4  97  52, 193  21  7.4, 57  HPV* type†  Positive subjects           Invasive   In situ   Invasive  In situ  Cases (n = 190)   Controls (n = 291)   Cases (n = 65)   Controls (n = 124)           No.  %  No.  %  No.  %  No.  %  OR*  95% CI*  OR  95% CI  16  114  60.0  6  2.1  25  38.4  6  4.8  83  39, 232  11  3.9, 33  18  26  13.7  0  0  5  7.7  1  0.8  ∞  22, ∞  10  1.2, 86  31/33/35/39  25  13.2  3  1.0  9  13.8  1  0.8  14  4.9, 61  17  2.2, 135  Any oncogenic type‡  150  78.9  9  3.1  37  56.9  7  5.6  155  72, 384  16  5.9, 47  Untyped§  14  7.4  11  3.8  12  18.5  6  4.8  2.0  0.9, 4.7  4.9  1.6, 15  Any type§  164  86.3  20  6.9  50  76.9  13  10.4  97  52, 193  21  7.4, 57  * HPV, human papillomavirus; OR, odds ratio in relation to women without the specific HPV type; CI, confidence interval. † One woman with carcinoma in situ had HPV type 6/11. No cases or controls had type 45. ‡ Includes types 16, 18, and 31/33/35/39. § Weakly reactive tests were considered negative. View Large TABLE 3. Risks of invasive squamous cell cervical carcinoma relative to risks of in situ squamous cell cervical carcinoma in relation to specific types of human papillomaviruses, Bangkok, Thailand, 1991–1993 HPV* type  No. of cases   OR*,†  95% CI*  Invasive  In situ  No HPV  26  18  1.0  Reference  16  114  29  4.2  1.8, 9.5  18  26  5  4.0  1.2, 13  31/33/35/39  25  11  2.1  0.8, 5.9  Any oncogenic type  150  42  3.5  1.6, 7.7  Untyped  14  14  0.7  0.3, 2.1  Any type  164  57  2.7  1.3, 5.2  Total cases  190  75      HPV* type  No. of cases   OR*,†  95% CI*  Invasive  In situ  No HPV  26  18  1.0  Reference  16  114  29  4.2  1.8, 9.5  18  26  5  4.0  1.2, 13  31/33/35/39  25  11  2.1  0.8, 5.9  Any oncogenic type  150  42  3.5  1.6, 7.7  Untyped  14  14  0.7  0.3, 2.1  Any type  164  57  2.7  1.3, 5.2  Total cases  190  75      * HPV, human papillomavirus; OR, odds ratio; CI, confidence interval. † Adjusted for age and all other types of HPV shown in the table. View Large In tables 4 through 6, estimated risks of invasive disease relative to risks of in situ disease are adjusted for age and the presence of oncogenic HPV, other HPV, or no HPV (three categories). These estimates did not differ appreciably from estimates adjusted only for age. No other factors were identified that appeared to confound the estimates presented. TABLE 4. Age-adjusted odds ratios of invasive and in situ squamous cell cervical carcinoma in relation to sexual factors and infectious agents other than human papillomaviruses, Bangkok, Thailand, 1991–1993 Factor/agent  Invasive   In situ   Invasive   In situ   Invasive/in situ†   Cases (no.)  Controls (no.)  Cases‡ (no.)  Controls (no.)  OR§  95% CI§  OR  95% CI  OR  95% CI  No. of sexual partners                       1  175  272  60/67  113  1.0  Reference  1.0  Reference  1.0  Reference   >1  15  19  5/8  11  1.2  0.6, 2.5  0.9  0.3, 2.9  0.8  0.3, 2.1  Age (years) at first intercourse                      ≤16  30  22  10/13  6  4.4  2.2, 9.1*  6.9  1.6, 30*  1.4  0.3, 3.9   17–18  45  47  19/21  21  2.8  1.6, 5.1  2.6  1.1, 6.3  1.0  0.4, 2.6   19–20  51  69  13/16  26  2.0  1.2, 3.5  1.5  0.6, 4.0  1.0  0.4, 2.6   21–23  33  66  11/12  35  1.4  0.8, 2.6  0.9  0.4, 2.4  1.0  0.3, 2.8  ≥24  31  87  12/13  76  1.0  Reference  1.0  Reference  1.0  Reference  HSV-2§ antibodies¶                       Negative  79  133  28/33  61  1.0  Reference  1.0  Reference  1.0  Reference   Positive  108  135  37/42  54  1.4  1.0, 2.0  1.5  0.8, 2.9  1.3  0.7, 2.4  Syphilis serology¶                       Negative  175  267  61/69  118  1.0  Reference  1.0  Reference  1.0  Reference   Positive  13  14  4/4  2  1.5  0.7, 3.2  3.6  0.7, 2.0  1.4  0.4, 5.1  HBsAg§,¶                       Negative  177  256  62/70  112  1.0  Reference  1.0  Reference  1.0  Reference   Positive  11  14  3/3  6  1.2  0.5, 2.8  0.9  0.2, 3.7  1.4  0.3, 6.1  Anti-HBs antibodies§,¶                       Negative  109  167  35/38  84  1.0  Reference  1.0  Reference  1.0  Reference   Positive  79  112  30/35  34  1.0  0.7, 1.5  2.3  1.2, 4.4  0.6  0.3, 1.1  Anti-HBc antibodies§,¶                       Negative  77  116  32/36  73  1.0  Reference  1.0  Reference  1.0  Reference   Positive  111  163  33/37  45  1.0  0.7, 1.4  1.9  1.0, 3.7  1.0  0.6, 1.9  Factor/agent  Invasive   In situ   Invasive   In situ   Invasive/in situ†   Cases (no.)  Controls (no.)  Cases‡ (no.)  Controls (no.)  OR§  95% CI§  OR  95% CI  OR  95% CI  No. of sexual partners                       1  175  272  60/67  113  1.0  Reference  1.0  Reference  1.0  Reference   >1  15  19  5/8  11  1.2  0.6, 2.5  0.9  0.3, 2.9  0.8  0.3, 2.1  Age (years) at first intercourse                      ≤16  30  22  10/13  6  4.4  2.2, 9.1*  6.9  1.6, 30*  1.4  0.3, 3.9   17–18  45  47  19/21  21  2.8  1.6, 5.1  2.6  1.1, 6.3  1.0  0.4, 2.6   19–20  51  69  13/16  26  2.0  1.2, 3.5  1.5  0.6, 4.0  1.0  0.4, 2.6   21–23  33  66  11/12  35  1.4  0.8, 2.6  0.9  0.4, 2.4  1.0  0.3, 2.8  ≥24  31  87  12/13  76  1.0  Reference  1.0  Reference  1.0  Reference  HSV-2§ antibodies¶                       Negative  79  133  28/33  61  1.0  Reference  1.0  Reference  1.0  Reference   Positive  108  135  37/42  54  1.4  1.0, 2.0  1.5  0.8, 2.9  1.3  0.7, 2.4  Syphilis serology¶                       Negative  175  267  61/69  118  1.0  Reference  1.0  Reference  1.0  Reference   Positive  13  14  4/4  2  1.5  0.7, 3.2  3.6  0.7, 2.0  1.4  0.4, 5.1  HBsAg§,¶                       Negative  177  256  62/70  112  1.0  Reference  1.0  Reference  1.0  Reference   Positive  11  14  3/3  6  1.2  0.5, 2.8  0.9  0.2, 3.7  1.4  0.3, 6.1  Anti-HBs antibodies§,¶                       Negative  109  167  35/38  84  1.0  Reference  1.0  Reference  1.0  Reference   Positive  79  112  30/35  34  1.0  0.7, 1.5  2.3  1.2, 4.4  0.6  0.3, 1.1  Anti-HBc antibodies§,¶                       Negative  77  116  32/36  73  1.0  Reference  1.0  Reference  1.0  Reference   Positive  111  163  33/37  45  1.0  0.7, 1.4  1.9  1.0, 3.7  1.0  0.6, 1.9  * p value of test for trend < 0.01. † Risks of invasive disease relative to risk of in situ disease, adjusted for age and presence or absence of oncogenic human papillomavirus (HPV) type or other and unknown HPV type or no HPV (three categories). ‡ In situ cases included in comparisons of cases versus controls/in comparisons of invasive versus in situ cases. § OR, odds ratio; CI, confidence interval; HSV-2, herpes simplex virus type 2; HBsAg, hepatitis B surface antigen; anti-HBs antibodies, anti-hepatitis B surface antibodies; anti-HBc antibodies, anti-hepatitis B core antibodies. ¶ Study subjects with no serologic tests were omitted from analyses. View Large TABLE 5. Age-adjusted odds ratios of invasive and in situ squamous cell cervical carcinoma in relation to hormonal factors, Bangkok, Thailand, 1991–1993 Factor  Invasive   In situ   Invasive   In situ   Invasive/in situ†   Cases (no.)  Controls (no.)  Cases‡ (no.)  Controls (no.)  OR§  95% CI§  OR  95% CI  OR  95% CI  No. of pregnancies                       0  2  9  4/6  17  0.6  0.1, 2.4*  0.5  0.1, 1.8*  0.3  0.1, 2.0   1–2  41  114  28/30  68  1.0  Reference  1.0  Reference  1.0  Reference   3–4  72  95  22/26  33  2.2  1.3, 3.6  1.9  0.9, 4.3  1.2  0.6, 2.5   5–8  67  66  11/13  6  3.0  1.0, 5.3  8.6  1.7, 4.2  1.9  0.8, 4.5   ≥9  8  7  0  0  3.4  1.7, 11          Ever had stillbirth¶                       No  180  272  59/66  106  1.0  Reference  1.0  Reference  1.0  Reference   Yes  2  10  2/3  1  1.1  0.4, 2.8  3.6  0.3, 4.0  0.6  0.1, 2.7  Ever had spontaneous abortion¶                       No  146  235  51/58  90  1.0  Reference  1.0  Reference  1.0  Reference   Yes  42  47  10/11  17  1.4  0.9, 2.8  1.0  0.4, 2.4  1.5  0.7, 3.3  Ever had induced abortion¶                       No  179  271  57/65  98  1.0  Reference  1.0  Reference  1.0  Reference   Yes  9  11  4/4  9  1.3  0.5, 3.3  0.7  0.2, 2.5  1.2  0.3, 4.5  Ever used oral contraceptives                       No  98  162  25/26  63  1.0  Reference  1.0  Reference  1.0  Reference   Yes  92  129  40/49  61  1.3  0.9, 2.0  1.7  0.9, 3.2  0.6  0.3, 1.2  Ever used DMPA§                       No  161  229  46/55  95  1.0  Reference  1.0  Reference  1.0  Reference   Yes  29  62  19/20  29  0.8  0.5, 1.3  1.4  1.3, 1.5  0.7  0.4, 1.6  Factor  Invasive   In situ   Invasive   In situ   Invasive/in situ†   Cases (no.)  Controls (no.)  Cases‡ (no.)  Controls (no.)  OR§  95% CI§  OR  95% CI  OR  95% CI  No. of pregnancies                       0  2  9  4/6  17  0.6  0.1, 2.4*  0.5  0.1, 1.8*  0.3  0.1, 2.0   1–2  41  114  28/30  68  1.0  Reference  1.0  Reference  1.0  Reference   3–4  72  95  22/26  33  2.2  1.3, 3.6  1.9  0.9, 4.3  1.2  0.6, 2.5   5–8  67  66  11/13  6  3.0  1.0, 5.3  8.6  1.7, 4.2  1.9  0.8, 4.5   ≥9  8  7  0  0  3.4  1.7, 11          Ever had stillbirth¶                       No  180  272  59/66  106  1.0  Reference  1.0  Reference  1.0  Reference   Yes  2  10  2/3  1  1.1  0.4, 2.8  3.6  0.3, 4.0  0.6  0.1, 2.7  Ever had spontaneous abortion¶                       No  146  235  51/58  90  1.0  Reference  1.0  Reference  1.0  Reference   Yes  42  47  10/11  17  1.4  0.9, 2.8  1.0  0.4, 2.4  1.5  0.7, 3.3  Ever had induced abortion¶                       No  179  271  57/65  98  1.0  Reference  1.0  Reference  1.0  Reference   Yes  9  11  4/4  9  1.3  0.5, 3.3  0.7  0.2, 2.5  1.2  0.3, 4.5  Ever used oral contraceptives                       No  98  162  25/26  63  1.0  Reference  1.0  Reference  1.0  Reference   Yes  92  129  40/49  61  1.3  0.9, 2.0  1.7  0.9, 3.2  0.6  0.3, 1.2  Ever used DMPA§                       No  161  229  46/55  95  1.0  Reference  1.0  Reference  1.0  Reference   Yes  29  62  19/20  29  0.8  0.5, 1.3  1.4  1.3, 1.5  0.7  0.4, 1.6  * p value of test for trend < 0.01. † Risks of invasive disease relative to risk of in situ disease, adjusted for age and presence or absence of oncogenic human papillomavirus (HPV) type or other and unknown HPV type or no HPV (three categories). ‡ In situ cases included in comparisons of cases versus controls/in comparisons of invasive versus in situ cases. § OR, odds ratio; CI, confidence interval; DMPA, depot-medroxyprogesterone acetate. ¶ Excluding women who had never been pregnant. View Large TABLE 6. Age-adjusted odds ratios of invasive and in situ squamous cell cervical carcinoma in relation to nonsexual, nonhormonal factors, Bangkok, Thailand, 1991–1993 Factor  Invasive   In situ   Invasive   In situ   Invasive/in situ*   Cases (no.)  Controls (no.)  Cases† (no.)  Controls (no.)  OR‡  95% CI‡  OR  95% CI  OR  95% CI  Ever used an IUD‡                       No  168  231  59/67  101  1.0  Reference  1.0  Reference  1.0  Reference   Yes  22  60  6/8  23  0.5  0.3, 0.9  0.4  0.2, 1.1  1.2  0.5, 3.1  Tubal ligation                       No  138  187  38/46  70  1.0  Reference  1.0  Reference  1.0  Reference   Yes  52  104  27/29  54  0.7  0.4, 1.0  0.9  0.5, 1.7  0.4  0.2, 0.8  Months since last cervical smear                       No cervical smear  165  192  50/56  75  1.0  Reference  1.0  Reference  1.0  Reference   1–12  7  1  11/11  30  0.2  0.1, 0.4  0.6  0.3, 1.3  0.3  0.1, 0.8   >12  5  2  4/7  14  0.3  0.1, 0.6  0.4  0.1, 1.3  0.3  0.1, 1.1   Unknown months  13  2  0  5  0.5  0.3, 1.0          Ever smoked ≥100 cigarettes§                       No  175  276  61/69  121  1.0  Reference  1.0  Reference  1.0  Reference   Yes  14  15  4/6  3  1.4  0.7, 2.0  2.2  0.5, 10  1.2  0.4, 3.7  Ever drank alcoholic beverages                       No  94  148  39/44  64  1.0  Reference  1.0  Reference  1.0  Reference   Yes  96  143  26/31  60  1.0  0.7, 1.5  0.7  0.4, 1.3  1.1  0.6, 2.0  Ever had a chest radiographic examination                       No  158  204  42/46  84  1.0  Reference  1.0  Reference  1.0  Reference   Yes  32  87  23/29  40  0.5  0.3, 0.7  1.2  0.6, 2.3  0.3  0.2, 0.6  Attended school                       No  31  27  3/4  10  1.0  Reference  1.0  Reference  1.0  Reference   Yes  159  264  62/73  114  0.6  0.3, 1.0  1.7  0.5, 6.6  0.5  0.2, 1.6  Factor  Invasive   In situ   Invasive   In situ   Invasive/in situ*   Cases (no.)  Controls (no.)  Cases† (no.)  Controls (no.)  OR‡  95% CI‡  OR  95% CI  OR  95% CI  Ever used an IUD‡                       No  168  231  59/67  101  1.0  Reference  1.0  Reference  1.0  Reference   Yes  22  60  6/8  23  0.5  0.3, 0.9  0.4  0.2, 1.1  1.2  0.5, 3.1  Tubal ligation                       No  138  187  38/46  70  1.0  Reference  1.0  Reference  1.0  Reference   Yes  52  104  27/29  54  0.7  0.4, 1.0  0.9  0.5, 1.7  0.4  0.2, 0.8  Months since last cervical smear                       No cervical smear  165  192  50/56  75  1.0  Reference  1.0  Reference  1.0  Reference   1–12  7  1  11/11  30  0.2  0.1, 0.4  0.6  0.3, 1.3  0.3  0.1, 0.8   >12  5  2  4/7  14  0.3  0.1, 0.6  0.4  0.1, 1.3  0.3  0.1, 1.1   Unknown months  13  2  0  5  0.5  0.3, 1.0          Ever smoked ≥100 cigarettes§                       No  175  276  61/69  121  1.0  Reference  1.0  Reference  1.0  Reference   Yes  14  15  4/6  3  1.4  0.7, 2.0  2.2  0.5, 10  1.2  0.4, 3.7  Ever drank alcoholic beverages                       No  94  148  39/44  64  1.0  Reference  1.0  Reference  1.0  Reference   Yes  96  143  26/31  60  1.0  0.7, 1.5  0.7  0.4, 1.3  1.1  0.6, 2.0  Ever had a chest radiographic examination                       No  158  204  42/46  84  1.0  Reference  1.0  Reference  1.0  Reference   Yes  32  87  23/29  40  0.5  0.3, 0.7  1.2  0.6, 2.3  0.3  0.2, 0.6  Attended school                       No  31  27  3/4  10  1.0  Reference  1.0  Reference  1.0  Reference   Yes  159  264  62/73  114  0.6  0.3, 1.0  1.7  0.5, 6.6  0.5  0.2, 1.6  * Risks of invasive disease relative to risk of in situ disease, adjusted for age and presence or absence of oncogenic human papillomavirus (HPV) type or other and unknown HPV type or no HPV (three categories). † In situ cases included in comparisons of cases versus controls/in comparisons of invasive versus in situ cases. ‡ OR, odds ratio; CI, confidence interval; IUD, intrauterine device. § One case with unknown smoking history was excluded. View Large As shown in table 4, the risk of both invasive and in situ disease tended to decline with increasing age at first intercourse. The risk of both tumor types was only weakly associated with positive serologic tests for herpes simplex virus type 2 antibodies, syphilis or hepatitis B antibodies, or surface antigen, and no associations with herpes simplex virus type 1 were found (not shown). Few women in this study admitted to having had more than one sexual partner, and no increase in risk of either tumor type was observed in relation to this variable. None of the sexual factors considered was significantly more strongly related to invasive than in situ disease. Only one woman with invasive disease and three with CIN-III were positive for the human immunodeficiency virus test (not shown). The risk of both in situ and invasive disease increased with increasing number of pregnancies (table 5). The risk of neither tumor type was significantly associated with the other hormonal factors considered, except for an increase in risk of in situ carcinoma in users of depot-medroxyprogesterone acetate for contraception. Use of oral contraceptives was weakly associated with both tumor types. None of the hormonal factors shown in table 5 was significantly more strongly associated with invasive than in situ disease. Ages at menarche and first livebirth were also not associated with either tumor type (not shown). Women who had used an intrauterine device and who had a history of prior cervical smears were at reduced risk of both invasive and in situ disease, and the risk of both types was slightly but not significantly increased in smokers (table 6). The risk of invasive but not of in situ carcinoma was lower in women with than without a history of a screening chest radiographic examination and formal schooling. The risk of invasive diseases was low relative to the risk of in situ disease in women with a tubal ligation, prior cervical smears, and a prior chest radiographic examination, and possibly in women who had attended school. When the analyses for the last columns in tables 4–6 were restricted to data on study subjects who had never had a cervical smear, to eliminate differences between women with invasive and in situ disease that may be due to prior screening behavior, the results were not appreciably different from those presented. Moreover, the same analyses were performed separately for invasive and in situ cases with an oncogenic HPV type (150 invasive and 42 in situ cases) and without an oncogenic HPV type (26 invasive and 18 in situ cases), and no significant differences (p > 0.05) were observed in the comparable odds ratio estimates from the two sets of analyses. DISCUSSION Oncogenic HPV DNA was found in a high proportion of invasive squamous cell cervical carcinomas, with type 16 being the predominant type. This is in accordance with prior observations (1, 6–8, 19) in which sensitive polymerase chain reaction-based methods were used to detect HPV DNA in cervical cancer cells. The oncogenic types that were assayed were demonstrated in a lower proportion of the in situ than invasive tumors, and untyped HPV DNA was found more frequently in the intraepithelial lesions. These observations are also in accordance with prior studies (1, 10, 20, 21) and are compatible with observations by others that the prevalence of oncogenic HPV types in tumors is directly related to the severity of the lesion (1, 22–26). There is little reason to suggest that the odds ratio estimates in this study in relation to HPV type are biased: cervical scrapings were obtained from a high proportion of both the invasive and in situ cases; both groups of women resided in the same areas of Thailand; and the prevalence of the HPV types that were assayed is similar to that observed by others (1). The odds ratio estimates shown in table 2 thus provide evidence that HPV types 16 and 18 are associated with a fourfold increase in the risk of progression to invasion, that types 31/33/35/39 are less strongly predictive of progression, and that the other (untyped) HPV types are not indicative of subsequent invasion. This interpretation is consistent with the observation (27) that squamous intraepithelial lesions with oncogenic HPV DNA tend to be monoclonal, and those with other HPV DNA types tend to be polyclonal. Although assays for types of HPV other than those that were investigated in this study are now available, and although some of these more recently identified types may be oncogenic, it is unlikely that their omission from this study influenced the results that pertain to types 16, 18, and 31/33/35/39, because only 14 women with invasive disease and 12 with in situ tumors had untyped HPV DNA in their cervical scrapings. The observation that untyped HPV types are more strongly associated with in situ than invasive disease would probably be strengthened if we had tested for the more recently recognized oncogenic types and removed them from the group with untyped DNA; our conclusion that there are HPV types associated with in situ disease that do not progress to invasion would not change. After controlling for age and HPV type, the epidemiologic features of in situ and invasive cervical carcinomas were found to be quite similar. The age at first intercourse was a strong risk factor for both tumor types in this largely monogamous population, suggesting either that early exposure to oncogenic types of HPV may enhance the risk of persistent infection and carcinogenesis, or that the sexual behavior of the husbands of women with early and late onset of sexual activity may differ (17). Serologic indices of exposure to sexually transmitted agents other than HPV were generally not strongly related to either invasive or in situ disease, and none was significantly more strongly related to one tumor type or the other. This is as expected, because these factors are more likely indicators of risk of initial infection with HPV rather than cofactors operative after neoplastic changes. Although quite possibly due to chance, the risk in relation to serologic evidence of hepatitis B surface antigen (table 4) was slightly greater for invasive than in situ disease. Because persistent hepatitis B virus antigenemia may suggest a deficient immune response, this observation is compatible with a role for immunodeficiency in the persistence of HPV infection that can lead to invasive carcinoma. An increase in the risk of both in situ and invasive disease with parity and use of oral contraceptives was observed, but the associations were not significantly stronger for invasive disease, suggesting that these hormonal factors operate prior to the development of in situ lesions. The risk of carcinoma in situ, but not of invasive disease, was elevated in users of depot-medroxyprogesterone acetate. This same observation was previously observed in a larger study conducted in Thailand and elsewhere (28, 29) and is compatible with the interpretation that any effect of depot-medroxyprogesterone acetate on the risk of cervical carcinoma in situ is reversible or that the lesions induced by this product have a low invasive potential. Smoking was weakly associated with the risk of both invasive and in situ lesions, but the prevalence of smoking among the study subjects was low and the odds ratio estimate had wide confidence limits. If smoking is a cofactor, it operates prior to the development of in situ disease and is not an important determinant of risk in Thailand. Having had a screening chest radiographic examination and having ever attended school were both associated with a reduced risk of invasive disease but not of in situ disease. These associations persisted after controlling for the frequency of prior cervical smear screening, suggesting that these associations are not due to more cervical cancer screening in the more educated women and in women who are screened for tuberculosis. Another possible explanation is that women of higher socioeconomic status may tend to seek care earlier than women of lower status and, hence, are more likely to have their cervical neoplasia diagnosed when still in a preinvasive stage. However, the observation that the presenting symptoms of women with in situ and invasive disease did not differ is not supportive of this interpretation. Other studies have also shown odds ratios in relation to school attendance to be less than unity for invasive but not in situ disease (10, 14, 15, 30), and the consistency of these results and ours suggests that factors associated with education or other measures of socioeconomic status, such as nutritional factors, may inhibit invasion. Prior cervical smears were shown to be related to a reduced risk of cervical carcinoma and, as expected, to be more strongly protective against invasive than in situ disease. As has also been observed elsewhere (31), reduced risks in relation to use of an intrauterine device and a tubal ligation most likely reflect screening for cervical cancer at the time of intrauterine device insertion or removal and at the time of tubal surgery. Although there has been only one prior report of studies in which direct comparisons of risk factors for in situ and invasive carcinomas were made after controlling for HPV type in the tumor (10), there have been other investigations in which women with both in situ and invasive disease have been studied using comparable methodology but without inclusion of HPV testing (13–15, 30). The results of all these studies are generally consistent with our findings in showing no sexual or hormonal factors, or smoking, to be more strongly associated with invasive than in situ carcinomas. Additional studies of the role of these factors in the genesis of cervical carcinoma should focus on the more proximal preinvasive stages of the carcinogenic process. Several possible sources of bias could have influenced the odds ratio estimates in this study. If women with carcinoma in situ were more likely than women with invasive carcinoma to have been screened for cervical cancer, then observed differences between women with these two tumor types could reflect factors associated with screening behavior rather than with development of invasive disease. This is an unlikely explanation for our results. There is no mass screening of asymptomatic women in Thailand, and cases of in situ disease in this study did not differ from women with invasive disease with respect to the presenting symptoms that lead to diagnosis. In addition, controlling odds ratio estimates for history of prior cervical smears had no appreciable effect on the results. Confounding by HPV status could have influenced the odds ratio estimates based on comparisons of cases and controls, although the consistency of our results with those of others suggests that this is not a likely explanation for the findings. More importantly, the direct comparisons of women with invasive and in situ carcinomas were controlled for HPV type, thus eliminating confounding by these viruses as an explanation for the few associations observed in those analyses. The comparison of invasive with in situ disease is an investigative approach that other investigators should consider in an attempt to identify factors that may enhance or prevent progression of intraepithelial lesions to invasive disease. Because HPV type was the only predictor of progression identified, other studies should focus on additional factors not adequately considered in this and other investigations. Correspondence to Dr. David B. Thomas, Program in Epidemlogy, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109 (e-mail: dbthomas@fhcrc.org). This study was supported by grant CA49044 from the US National Institutes of Health. The assistance of Drs. Anna Marie Beckmann and Larry Corey is gratefully acknowledged. REFERENCES 1. International Agency for Research on Cancer. Human papillomaviruses. IARC Monogr Eval Carcinog Risks Hum  1995; 64: 1–409. Google Scholar 2. Cavalcanti SM, Deus FC, Zardo LG, et al. Human papillomavirus infection and cervical cancer in Brazil: a retrospective study. Mem Inst Oswaldo Cruz  1996; 91: 433–40. Google Scholar 3. Shah KV, Viscidi RP, Alberg AJ, et al. Antibodies to human papillomavirus 16 and subsequent in situ or invasive cancer of the cervix. Cancer Epidemiol Biomarkers Prev  1997; 6: 233–7. Google Scholar 4. Lehtinen M, Dillner J, Knekt P, et al. Serologically diagnosed infection with human papillomavirus type 16 and risk for subsequent development of cervical carcinoma: nested case-control study. BMJ  1996; 312: 537–9. Google Scholar 5. Bosch FX, Muñoz N, de Sanjosé S, et al. Risk factors for cervical cancer in Colombia and Spain. Int J Cancer  1992; 52: 750–8. Google Scholar 6. Chaouk N, Bosch FX, Muñoz N, et al. The viral origin of cervical cancer in Rabat, Morocco. Int J Cancer  1998; 75: 546–54. Google Scholar 7. Chichareon S, Herrero R, Muñoz N, et al. Risk factors for cervical cancer in Thailand: a case-control study. J Natl Cancer Inst  1998; 90: 50–7. Google Scholar 8. Ngelangel C, Muñoz N, Bosch FX, et al. Causes of cervical cancer in the Philippines: a case-control study. J Natl Cancer Inst  1998; 90: 43–9. Google Scholar 9. Daling JR, Madeleine MM, McKnight B, et al. The relationship of human papillomavirus-related cervical tumors to cigarette smoking, oral contraceptive use, and prior herpes simplex virus type 2 infection. Cancer Epidemiol Biomarkers Prev  1996; 5: 541–8. Google Scholar 10. Moreno V, Muñoz N, Bosch FX, et al. Risk factors for progression of cervical intraepithelial neoplasms grade III to invasive cervical cancer. Cancer Epidemiol Biomarkers Prev  1995; 4: 459–67. Google Scholar 11. Muñoz N, Bosch FX, de Sanjosé S, et al. Risk factors for cervical intraepithelial neoplasia grade III/carcinoma in situ in Spain and Colombia. Cancer Epidemiol Biomarkers Prev  1993; 2: 423–31. Google Scholar 12. Ferrera A, Baay MF, Herbrink P, et al. A sero-epidemiological study of the relationship between sexually transmitted agents and cervical cancer in Honduras. Int J Cancer  1997; 73: 781–5. Google Scholar 13. Parazzini F, La Vecchia C, Negri E, et al. Reproductive factors and the risk of invasive and intraepithelial cervical neoplasia. 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Google Scholar 19. Bosch FX, Muñoz N, de Sanjosé S, et al. Human papillomavirus and cervical intraepithelial neoplasia grade III/carcinoma in situ: a case-control study in Spain and Colombia. Cancer Epidemiol Biomarkers Prev  1993; 2: 415–22. Google Scholar 20. Ho GY, Kadish AS, Burk RD, et al. HPV 16 and cigarette smoking as risk factors for high-grade cervical intraepithelial neoplasia. Int J Cancer  1998; 78: 281–5. Google Scholar 21. Kjaer SK, van den Brule AJ, Bock JE, et al. Human papillomavirus—the most significant risk determinant of cervical intraepithelial neoplasia. Int J Cancer  1996; 65: 601–6. Google Scholar 22. van den Brule AJ, Walboomers JM, du Maine M, et al. 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: 404–8. Google Scholar 23. Becker TM, Wheeler CM, McGough NS, et al. Cervical papillomavirus infection and cervical dysplasia in Hispanic, Native American, and non-Hispanic white women in New Mexico. Am J Public Health  1991; 81: 582–6. Google Scholar 24. Cuzick J, Terry G, Ho L, et al. Human papillomavirus type 16 DNA in cervical smears as predictor of high-grade cervical cancer. Lancet  1992; 339: 959–60. Google Scholar 25. Lungu O, Sun XW, Felix J, et al. Relationship of human papillomavirus type to grade of cervical intraepithelial neoplasia. JAMA  1992; 267: 2493–6. Google Scholar 26. Coker AL, Jenkins GR, Busnardo MS, et al. Human papillomaviruses and cervical neoplasia in South Carolina. Cancer Epidemiol Biomarkers Prev  1993; 2: 207–12. Google Scholar 27. Park TJ, Richart RM, Sun XW, et al. Association between human papillomavirus type and clonal status of cervical squamous intraepithelial lesions. J Natl Cancer Inst  1996; 88: 355–8. Google Scholar 28. The WHO Collaborative Study of Neoplasia and Steroid Contraceptives. Depot-medroxyprogesterone acetate (DMPA) and risk of invasive squamous cell cervical cancer. Contraception  1992; 45: 299–312. Google Scholar 29. Thomas DB, Ye Z, Ray RM, et al. Cervical carcinoma in situ and use of depot-medroxyprogesterone acetate (DMPA). Contraception  1995; 51: 25–31. Google Scholar 30. Kjaer SK, Dahl C, Engholm G, et al. Case-control study of risk factors for cervical neoplasia in Denmark. II. Role of sexual activity, reproductive factors, and venereal infections. Cancer Causes Control  1992; 3: 339–48. Google Scholar 31. Li HQ, Thomas DB, Jin SK, et al. Tubal sterilization and use of an IUD and risk of cervical cancer. J Womens Health Gend Based Med  2000; 9: 303–10. Google Scholar http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png American Journal of Epidemiology Oxford University Press

Human Papillomaviruses and Cervical Cancer in Bangkok. II. Risk Factors for in Situ and Invasive Squamous Cell Cervical Carcinomas

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Publisher
Oxford University Press
ISSN
0002-9262
eISSN
1476-6256
DOI
10.1093/aje/153.8.732
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Abstract

Abstract To identify risk factors for progression of intraepithelial cervical lesions, 190 women with invasive cervical cancer were compared with 75 women with in situ disease diagnosed in Bangkok, Thailand, between September 1991 and September 1993. Polymerase chain reaction-based assays for type-specific human papillomavirus (HPV) DNA in cervical scrapings revealed oncogenic types in 79% of invasive and 57% of intraepithelial tumors. Types 16 and 18, but not types 31/33/35/39, were more common in invasive than intraepithelial tumors, and untyped HPV DNA was found more commonly in the in situ lesions, suggesting that in situ disease is four times more likely to become invasive if due to type 16 or 18 than to other causes, and that tumors with only untyped HPV are not at increased risk of progression. After controlling for HPV type, the risk of developing invasive diseases, compared with the risk of developing intraepithelial lesions, was not related to any of a large number of sexual and hormonal factors considered or to smoking, suggesting that any cofactors these variables represent act before the development of in situ carcinoma. Two indices of socioeconomic status were associated with a reduced risk of only invasive disease, suggesting the existence of unknown protective factors that operate after intraepithelial lesions develop. carcinoma, squamous cell, cervical intraepithelial neoplasia, cervix neoplasms, papillomavirus, human CIN-III, cervical intraepithelial neoplasia grade 3, HPV, human papillomavirus Some types of human papillomaviruses (HPVs) are carcinogenic for the cervix uteri (1). Although they are quite likely necessary causes of cervical carcinoma, they are not sufficient causes. Most infected women do not develop invasive cervical cancer, and cofactors must therefore play a role in allowing these infections to become persistent and carcinogenic. Cervical carcinoma in situ (cervical intra-epithelial neoplasia grade 3 (CIN-III)) is an intermediate morphologic lesion on the pathway from normal cervical epithelium to invasive disease, and cofactors for invasive cervical carcinoma could operate either prior to the development of carcinoma in situ or afterward. Those acting before the development of the intraepithelial lesion would be observed in relation to both in situ and invasive lesions, whereas those acting to enhance the probability that an in situ lesion becomes invasive would be observed only in relation to invasive disease. The identification of cofactors for preinvasive disease is best attempted in the context of prospective studies of HPV-infected women. Concurrent prospective studies of untreated women with carcinoma in situ to determine factors associated with progression to invasive diseases would be unethical, and studies of treated women would be uninformative because treatment removes or destroys the in situ lesion. A few historical cohort studies of HPV-infected subjects that included both in situ and invasive cervical carcinoma as endpoints (1–4) and case-control studies of invasive disease (1, 5–9) have identified a variety of possible cofactors with varying degrees of consistency that could act either before or after the development of in situ disease. These include the following: early age at first sexual intercourse, which may reflect particular vulnerability of the immature cervix to HPV-induced carcinogenesis; hormonal factors such as high parity and use of oral contraceptives; exposures to chemical agents in cigarette smoke; sexually transmitted agents other than HPV; deficiencies in certain micronutrients; an immunocompromised state resulting from human immunodeficiency virus (HIV) infection or immunosuppresion for prevention of organ transplant rejection; and various measures of socioeconomic status, suggesting the presence of additional unrecognized cofactors associated with social class. Most of these factors, however, have been observed in relation to both in situ and invasive disease (1) and, in studies in which the epidemiologic features of these two stages of cervical carcinoma can be compared, few consistent differences in risk factors have been observed (5, 10–15). These observations suggest that most putative cofactors are operative before the development of carcinoma in situ and that factors associated with progression to invasion have yet to be identified. This is a report of analysis performed to compare risk factors for in situ and invasive disease in order to identify variables associated only with the latter condition, after controlling for the presence of oncogenic HPV types in the tumor tissue. If women with in situ disease are considered the controls and if women with invasive disease are considered the cases in a standard case-control analysis, then elevated odds ratios in relation to a factor represent the risk of invasive disease relative to the risk of in situ disease in women with the factor (16). Because it is reasonable to assume that most or all invasive cervical carcinomas have passed through an in situ stage, an elevated odds ratio would imply that the factor enhances probability of progression from in situ to invasion. To our knowledge, results of direct comparisons of women with in situ and invasive disease have been reported only once previously (10). MATERIALS AND METHODS Women who were admitted to the public wards of Siriraj Hospital in Bangkok, Thailand, with a new histologically confirmed diagnosis of either CIN-III or invasive cervical carcinoma between September 1991 and September 1993, who were born after 1929, and who resided in Thailand for at least the past year were eligible as cases for this study. Although CIN-III includes both severe dysplasia and carcinoma in situ, the older terms, carcinoma in situ and in situ disease, are used interchangeably with CIN-III in this report. Two controls for each woman with invasive disease were selected from otolaryngology and general surgery wards of Siriraj Hospital, as previously described (17). The cases of carcinoma in situ were women who had had an abnormal cervical smear during a visit to either the family planning or gynecologic clinic associated with Siriraj Hospital, and an attempt was made to select two controls for each case from the same clinic from which the case came. Cervical smears are taken from many of the women who attend these clinics, and the women selected as controls were the next two women with appointments in the clinic after the appointment for the case, in the same 5-year age group and from the same region of the country as the case, who had returned to the clinic to learn the results of their cervical smears, and whose cervical smears revealed no suspicion of neoplastic change. If a woman refused to participate, the selection process continued until two controls per case were selected. All cases and hospitalized controls were interviewed while hospitalized; the controls selected for the cases of carcinoma in situ were interviewed in the clinics on the day they were selected. Information was obtained on sexual and reproductive history, prior cervical smears, use of tobacco and alcohol, and indices of prior use of medical resources and socioeconomic status. Dates of all prior cervical smears were obtained, and those taken within the past year were not considered prior screening cervical smears. The presenting symptom of each case was also ascertained and recorded as none (detected by routine cytology only), vaginal bleeding, abnormal vaginal discharge, or other. A 15-ml blood specimen was obtained from all interviewed women. Four aliquots of serum were stored at −70°C, two of which were retained in Bangkok and two of which were shipped to Seattle, Washington, on dry ice. Cervical scrapings for HPV DNA assays were obtained from cases by the patient's surgeon prior to treatment. Cervical smears and cervical scrapings were taken by these same surgeons from the hospital controls while they were hospitalized and from the clinic controls in the clinic on the same day they were selected and interviewed. The cervix, including the cervical os, was scraped with a Teflon (E. I. du Pont de Nemours and Company, Wilmington, Delaware)-coated swab, the end of which was then broken off into a vial containing 2 ml of specimen transport medium (Digene Diagnostics, Inc., Beltsville, Maryland). The specimens were stored at −70°C and periodically shipped on dry ice to Seattle. Histologic slides from the blocks that were used to make the diagnosis in the cases were read by a single collaborating pathologist in Bangkok. Information was recorded on source of specimen, tumor size, stage, and histologic diagnosis. A pathologist in Seattle reread the slides from 174 of the cases included in this report. Of 23 in situ cases, only one (4.3 percent) was considered invasive by the reference pathologist, and only eight (5.3 percent) of 151 cases considered invasive in Bangkok were read in Seattle as in situ with no evidence of microinvasion. The diagnosis made in Bangkok was thus used in the analysis in this report. As detailed elsewhere (17), serologic assays were performed for antibodies to human immunodeficiency virus, hepatitis B, Treponema pallidum, and herpes simplex virus types 1 and 2, as well as for hepatitis B surface antigen. Cervical scrapings were assayed for evidence of any HPV DNA and for type-specific DNA of types 6 and 11, type 16, type 18, types 31/33/35/39, and type 45 using polymerase chain reaction-based technology (17). Samples that hybridized with a generic probe but not with any of the type-specific probes were considered positive for untyped HPV. Cases and controls were compared, and odds ratios as estimates of relative risks were calculated using unconditional logistic regression (18) when comparing invasive cases with their unmatched controls and using conditional logistic regression (18) when comparing in situ cases with their matched controls. In addition, to directly compare risk factors for invasive and in situ disease, we performed unconditional logistic regression analyses using the women with invasive disease as “cases” and women with carcinoma in situ as “controls.” Because the purpose of these analyses was to identify any factors that distinguish invasive from in situ disease, and because no such factors were identified other than HPV status, no attempts were made to adjust odds ratios for factors other than age and (for the case-case comparisons) HPV status. RESULTS A total of 420 eligible cases were identified, of whom 338 (80.5 percent) were interviewed. Cervical scrapings were obtained from 337 (99.7 percent) of those interviewed, 216 and 75 of whom had invasive and in situ squamous cell carcinoma, respectively. Adequate amounts of DNA for HPV DNA assays were found in cervical scrapings from 191 of the women with invasive disease and all 75 of the women with carcinoma in situ. One woman with invasive disease who claimed to be a virgin was omitted, leaving 190 cases of invasive cervical carcinoma in the analyses. Controls were not found for 10 of the women with carcinoma in situ, leaving 65 of these cases for inclusion in the analyses in which cases and controls were compared; all 75 cases were included in the analyses in which women with invasive and in situ disease were compared. Of the 614 women selected as controls for the cases of invasive disease, 490 (79.8 percent) were interviewed. Cervical scrapings were obtained from 306 (62.4 percent) of these 490 women, 298 (97.4 percent) of which yielded adequate DNA samples. Three women with no history of sexual intercourse, one with a subtotal hysterectomy, and three with ineligible diagnoses were excluded, leaving 291 controls for analysis. Of the 161 women selected as controls for the in situ cases, 160 (99.4 percent) were interviewed, 144 of whom were subsequently confirmed as meeting the eligibility criteria for inclusion in the study. Cervical scrapings were obtained from 133 (92.4 percent) of these 144 women, 128 of which contained adequate amounts of DNA. Four of the 128 women from whom these samples were taken had been matched to excluded cases and were omitted, leaving 124 women as controls for the in situ cases in the analyses. As shown in table 1, women with invasive disease were slightly older than their controls. The women with carcinoma in situ and their controls were comparable in age and younger than the women with invasive carcinoma and their controls. The 10 additional cases of carcinoma in situ used in the comparisons of in situ and invasive disease are not appreciably different in age from the 65 cases shown in the table. All odds ratio estimates presented were adjusted for age using the age categories shown in the table. TABLE 1. Age distribution of invasive and in situ squamous cell cervical carcinomas and their corresponding controls, Bangkok, Thailand, 1991–1993* Age (years)  Invasive   In situ   Cases   Controls   Cases   Controls   No.  %  No.  %  No.  %  No.  %  <30  3  1.6  13  4.5  16  24.6  29  23.4  30–34  23  12.1  44  15.1  11  16.9  24  19.3  35–39  36  18.9  56  19.2  15  23.1  32  25.8  40–44  33  17.4  60  20.6  12  18.5  17  13.7  45–49  30  15.8  36  12.4  4  6.1  10  8.1  ≥50  65  34.2  82  28.2  7  10.8  12  9.7  Total  190  100.0  291  100.0  65  100.0  124  100.0  Age (years)  Invasive   In situ   Cases   Controls   Cases   Controls   No.  %  No.  %  No.  %  No.  %  <30  3  1.6  13  4.5  16  24.6  29  23.4  30–34  23  12.1  44  15.1  11  16.9  24  19.3  35–39  36  18.9  56  19.2  15  23.1  32  25.8  40–44  33  17.4  60  20.6  12  18.5  17  13.7  45–49  30  15.8  36  12.4  4  6.1  10  8.1  ≥50  65  34.2  82  28.2  7  10.8  12  9.7  Total  190  100.0  291  100.0  65  100.0  124  100.0  * The mean and median ages (years) are the following: invasive cases, 45.5 and 45.5; invasive controls, 43.9 and 43.0; in situ cases, 37.8 and 38.0; and in situ controls, 37.2 and 37.0, respectively. View Large Vaginal bleeding was the presenting symptom in 124 (65.3 percent) of the 190 women with invasive disease and in 47 (62.7 percent) of the 75 women with carcinoma in situ; 63 (33.2 percent) of the women with invasive carcinoma and 23 (30.7 percent) of those with carcinoma in situ reported no specific symptoms. Oncogenic types of HPV were strongly associated with both invasive and in situ disease (table 2). Type 16 DNA was the most frequently identified type in both invasive and in situ tumors. Both types 16 and 18 were found in a higher proportion of invasive than in situ carcinomas. Types 31/33/35/39 occurred with nearly equal frequency in both tumor types, and untyped HPV DNA was found in a higher proportion of the in situ than invasive lesions. Few women in either control group had any type of HPV in their cervical scrapings, although the frequency of a positive test was slightly higher in the controls for the in situ cases. The estimates of the odds ratio of both invasive and in situ carcinomas are large, with those in relation to types 16 and 18, any oncogenic type, and any HPV type being stronger for invasive disease; those in relation to types 31/33/35/39 being similar for both tumor types; but those in relation to untyped HPV being greater for in situ disease. As shown in table 3, after controlling for age and other HPV types, the odds ratio of invasive disease is four times greater than the risk of in situ disease in women with HPV types 16 and 18, twice as high in women with HPV types 31/33/35/39 (although possibly due to chance), and also significantly higher in relation to any oncogenic type and any type of HPV. The risk is not greater for invasive than in situ disease in women with untyped HPV DNA. TABLE 2. Age-adjusted odds ratios of invasive and in situ squamous cell cervical carcinoma in relation to specific types of human papillomaviruses, Bangkok, Thailand, 1991–1993 HPV* type†  Positive subjects           Invasive   In situ   Invasive  In situ  Cases (n = 190)   Controls (n = 291)   Cases (n = 65)   Controls (n = 124)           No.  %  No.  %  No.  %  No.  %  OR*  95% CI*  OR  95% CI  16  114  60.0  6  2.1  25  38.4  6  4.8  83  39, 232  11  3.9, 33  18  26  13.7  0  0  5  7.7  1  0.8  ∞  22, ∞  10  1.2, 86  31/33/35/39  25  13.2  3  1.0  9  13.8  1  0.8  14  4.9, 61  17  2.2, 135  Any oncogenic type‡  150  78.9  9  3.1  37  56.9  7  5.6  155  72, 384  16  5.9, 47  Untyped§  14  7.4  11  3.8  12  18.5  6  4.8  2.0  0.9, 4.7  4.9  1.6, 15  Any type§  164  86.3  20  6.9  50  76.9  13  10.4  97  52, 193  21  7.4, 57  HPV* type†  Positive subjects           Invasive   In situ   Invasive  In situ  Cases (n = 190)   Controls (n = 291)   Cases (n = 65)   Controls (n = 124)           No.  %  No.  %  No.  %  No.  %  OR*  95% CI*  OR  95% CI  16  114  60.0  6  2.1  25  38.4  6  4.8  83  39, 232  11  3.9, 33  18  26  13.7  0  0  5  7.7  1  0.8  ∞  22, ∞  10  1.2, 86  31/33/35/39  25  13.2  3  1.0  9  13.8  1  0.8  14  4.9, 61  17  2.2, 135  Any oncogenic type‡  150  78.9  9  3.1  37  56.9  7  5.6  155  72, 384  16  5.9, 47  Untyped§  14  7.4  11  3.8  12  18.5  6  4.8  2.0  0.9, 4.7  4.9  1.6, 15  Any type§  164  86.3  20  6.9  50  76.9  13  10.4  97  52, 193  21  7.4, 57  * HPV, human papillomavirus; OR, odds ratio in relation to women without the specific HPV type; CI, confidence interval. † One woman with carcinoma in situ had HPV type 6/11. No cases or controls had type 45. ‡ Includes types 16, 18, and 31/33/35/39. § Weakly reactive tests were considered negative. View Large TABLE 3. Risks of invasive squamous cell cervical carcinoma relative to risks of in situ squamous cell cervical carcinoma in relation to specific types of human papillomaviruses, Bangkok, Thailand, 1991–1993 HPV* type  No. of cases   OR*,†  95% CI*  Invasive  In situ  No HPV  26  18  1.0  Reference  16  114  29  4.2  1.8, 9.5  18  26  5  4.0  1.2, 13  31/33/35/39  25  11  2.1  0.8, 5.9  Any oncogenic type  150  42  3.5  1.6, 7.7  Untyped  14  14  0.7  0.3, 2.1  Any type  164  57  2.7  1.3, 5.2  Total cases  190  75      HPV* type  No. of cases   OR*,†  95% CI*  Invasive  In situ  No HPV  26  18  1.0  Reference  16  114  29  4.2  1.8, 9.5  18  26  5  4.0  1.2, 13  31/33/35/39  25  11  2.1  0.8, 5.9  Any oncogenic type  150  42  3.5  1.6, 7.7  Untyped  14  14  0.7  0.3, 2.1  Any type  164  57  2.7  1.3, 5.2  Total cases  190  75      * HPV, human papillomavirus; OR, odds ratio; CI, confidence interval. † Adjusted for age and all other types of HPV shown in the table. View Large In tables 4 through 6, estimated risks of invasive disease relative to risks of in situ disease are adjusted for age and the presence of oncogenic HPV, other HPV, or no HPV (three categories). These estimates did not differ appreciably from estimates adjusted only for age. No other factors were identified that appeared to confound the estimates presented. TABLE 4. Age-adjusted odds ratios of invasive and in situ squamous cell cervical carcinoma in relation to sexual factors and infectious agents other than human papillomaviruses, Bangkok, Thailand, 1991–1993 Factor/agent  Invasive   In situ   Invasive   In situ   Invasive/in situ†   Cases (no.)  Controls (no.)  Cases‡ (no.)  Controls (no.)  OR§  95% CI§  OR  95% CI  OR  95% CI  No. of sexual partners                       1  175  272  60/67  113  1.0  Reference  1.0  Reference  1.0  Reference   >1  15  19  5/8  11  1.2  0.6, 2.5  0.9  0.3, 2.9  0.8  0.3, 2.1  Age (years) at first intercourse                      ≤16  30  22  10/13  6  4.4  2.2, 9.1*  6.9  1.6, 30*  1.4  0.3, 3.9   17–18  45  47  19/21  21  2.8  1.6, 5.1  2.6  1.1, 6.3  1.0  0.4, 2.6   19–20  51  69  13/16  26  2.0  1.2, 3.5  1.5  0.6, 4.0  1.0  0.4, 2.6   21–23  33  66  11/12  35  1.4  0.8, 2.6  0.9  0.4, 2.4  1.0  0.3, 2.8  ≥24  31  87  12/13  76  1.0  Reference  1.0  Reference  1.0  Reference  HSV-2§ antibodies¶                       Negative  79  133  28/33  61  1.0  Reference  1.0  Reference  1.0  Reference   Positive  108  135  37/42  54  1.4  1.0, 2.0  1.5  0.8, 2.9  1.3  0.7, 2.4  Syphilis serology¶                       Negative  175  267  61/69  118  1.0  Reference  1.0  Reference  1.0  Reference   Positive  13  14  4/4  2  1.5  0.7, 3.2  3.6  0.7, 2.0  1.4  0.4, 5.1  HBsAg§,¶                       Negative  177  256  62/70  112  1.0  Reference  1.0  Reference  1.0  Reference   Positive  11  14  3/3  6  1.2  0.5, 2.8  0.9  0.2, 3.7  1.4  0.3, 6.1  Anti-HBs antibodies§,¶                       Negative  109  167  35/38  84  1.0  Reference  1.0  Reference  1.0  Reference   Positive  79  112  30/35  34  1.0  0.7, 1.5  2.3  1.2, 4.4  0.6  0.3, 1.1  Anti-HBc antibodies§,¶                       Negative  77  116  32/36  73  1.0  Reference  1.0  Reference  1.0  Reference   Positive  111  163  33/37  45  1.0  0.7, 1.4  1.9  1.0, 3.7  1.0  0.6, 1.9  Factor/agent  Invasive   In situ   Invasive   In situ   Invasive/in situ†   Cases (no.)  Controls (no.)  Cases‡ (no.)  Controls (no.)  OR§  95% CI§  OR  95% CI  OR  95% CI  No. of sexual partners                       1  175  272  60/67  113  1.0  Reference  1.0  Reference  1.0  Reference   >1  15  19  5/8  11  1.2  0.6, 2.5  0.9  0.3, 2.9  0.8  0.3, 2.1  Age (years) at first intercourse                      ≤16  30  22  10/13  6  4.4  2.2, 9.1*  6.9  1.6, 30*  1.4  0.3, 3.9   17–18  45  47  19/21  21  2.8  1.6, 5.1  2.6  1.1, 6.3  1.0  0.4, 2.6   19–20  51  69  13/16  26  2.0  1.2, 3.5  1.5  0.6, 4.0  1.0  0.4, 2.6   21–23  33  66  11/12  35  1.4  0.8, 2.6  0.9  0.4, 2.4  1.0  0.3, 2.8  ≥24  31  87  12/13  76  1.0  Reference  1.0  Reference  1.0  Reference  HSV-2§ antibodies¶                       Negative  79  133  28/33  61  1.0  Reference  1.0  Reference  1.0  Reference   Positive  108  135  37/42  54  1.4  1.0, 2.0  1.5  0.8, 2.9  1.3  0.7, 2.4  Syphilis serology¶                       Negative  175  267  61/69  118  1.0  Reference  1.0  Reference  1.0  Reference   Positive  13  14  4/4  2  1.5  0.7, 3.2  3.6  0.7, 2.0  1.4  0.4, 5.1  HBsAg§,¶                       Negative  177  256  62/70  112  1.0  Reference  1.0  Reference  1.0  Reference   Positive  11  14  3/3  6  1.2  0.5, 2.8  0.9  0.2, 3.7  1.4  0.3, 6.1  Anti-HBs antibodies§,¶                       Negative  109  167  35/38  84  1.0  Reference  1.0  Reference  1.0  Reference   Positive  79  112  30/35  34  1.0  0.7, 1.5  2.3  1.2, 4.4  0.6  0.3, 1.1  Anti-HBc antibodies§,¶                       Negative  77  116  32/36  73  1.0  Reference  1.0  Reference  1.0  Reference   Positive  111  163  33/37  45  1.0  0.7, 1.4  1.9  1.0, 3.7  1.0  0.6, 1.9  * p value of test for trend < 0.01. † Risks of invasive disease relative to risk of in situ disease, adjusted for age and presence or absence of oncogenic human papillomavirus (HPV) type or other and unknown HPV type or no HPV (three categories). ‡ In situ cases included in comparisons of cases versus controls/in comparisons of invasive versus in situ cases. § OR, odds ratio; CI, confidence interval; HSV-2, herpes simplex virus type 2; HBsAg, hepatitis B surface antigen; anti-HBs antibodies, anti-hepatitis B surface antibodies; anti-HBc antibodies, anti-hepatitis B core antibodies. ¶ Study subjects with no serologic tests were omitted from analyses. View Large TABLE 5. Age-adjusted odds ratios of invasive and in situ squamous cell cervical carcinoma in relation to hormonal factors, Bangkok, Thailand, 1991–1993 Factor  Invasive   In situ   Invasive   In situ   Invasive/in situ†   Cases (no.)  Controls (no.)  Cases‡ (no.)  Controls (no.)  OR§  95% CI§  OR  95% CI  OR  95% CI  No. of pregnancies                       0  2  9  4/6  17  0.6  0.1, 2.4*  0.5  0.1, 1.8*  0.3  0.1, 2.0   1–2  41  114  28/30  68  1.0  Reference  1.0  Reference  1.0  Reference   3–4  72  95  22/26  33  2.2  1.3, 3.6  1.9  0.9, 4.3  1.2  0.6, 2.5   5–8  67  66  11/13  6  3.0  1.0, 5.3  8.6  1.7, 4.2  1.9  0.8, 4.5   ≥9  8  7  0  0  3.4  1.7, 11          Ever had stillbirth¶                       No  180  272  59/66  106  1.0  Reference  1.0  Reference  1.0  Reference   Yes  2  10  2/3  1  1.1  0.4, 2.8  3.6  0.3, 4.0  0.6  0.1, 2.7  Ever had spontaneous abortion¶                       No  146  235  51/58  90  1.0  Reference  1.0  Reference  1.0  Reference   Yes  42  47  10/11  17  1.4  0.9, 2.8  1.0  0.4, 2.4  1.5  0.7, 3.3  Ever had induced abortion¶                       No  179  271  57/65  98  1.0  Reference  1.0  Reference  1.0  Reference   Yes  9  11  4/4  9  1.3  0.5, 3.3  0.7  0.2, 2.5  1.2  0.3, 4.5  Ever used oral contraceptives                       No  98  162  25/26  63  1.0  Reference  1.0  Reference  1.0  Reference   Yes  92  129  40/49  61  1.3  0.9, 2.0  1.7  0.9, 3.2  0.6  0.3, 1.2  Ever used DMPA§                       No  161  229  46/55  95  1.0  Reference  1.0  Reference  1.0  Reference   Yes  29  62  19/20  29  0.8  0.5, 1.3  1.4  1.3, 1.5  0.7  0.4, 1.6  Factor  Invasive   In situ   Invasive   In situ   Invasive/in situ†   Cases (no.)  Controls (no.)  Cases‡ (no.)  Controls (no.)  OR§  95% CI§  OR  95% CI  OR  95% CI  No. of pregnancies                       0  2  9  4/6  17  0.6  0.1, 2.4*  0.5  0.1, 1.8*  0.3  0.1, 2.0   1–2  41  114  28/30  68  1.0  Reference  1.0  Reference  1.0  Reference   3–4  72  95  22/26  33  2.2  1.3, 3.6  1.9  0.9, 4.3  1.2  0.6, 2.5   5–8  67  66  11/13  6  3.0  1.0, 5.3  8.6  1.7, 4.2  1.9  0.8, 4.5   ≥9  8  7  0  0  3.4  1.7, 11          Ever had stillbirth¶                       No  180  272  59/66  106  1.0  Reference  1.0  Reference  1.0  Reference   Yes  2  10  2/3  1  1.1  0.4, 2.8  3.6  0.3, 4.0  0.6  0.1, 2.7  Ever had spontaneous abortion¶                       No  146  235  51/58  90  1.0  Reference  1.0  Reference  1.0  Reference   Yes  42  47  10/11  17  1.4  0.9, 2.8  1.0  0.4, 2.4  1.5  0.7, 3.3  Ever had induced abortion¶                       No  179  271  57/65  98  1.0  Reference  1.0  Reference  1.0  Reference   Yes  9  11  4/4  9  1.3  0.5, 3.3  0.7  0.2, 2.5  1.2  0.3, 4.5  Ever used oral contraceptives                       No  98  162  25/26  63  1.0  Reference  1.0  Reference  1.0  Reference   Yes  92  129  40/49  61  1.3  0.9, 2.0  1.7  0.9, 3.2  0.6  0.3, 1.2  Ever used DMPA§                       No  161  229  46/55  95  1.0  Reference  1.0  Reference  1.0  Reference   Yes  29  62  19/20  29  0.8  0.5, 1.3  1.4  1.3, 1.5  0.7  0.4, 1.6  * p value of test for trend < 0.01. † Risks of invasive disease relative to risk of in situ disease, adjusted for age and presence or absence of oncogenic human papillomavirus (HPV) type or other and unknown HPV type or no HPV (three categories). ‡ In situ cases included in comparisons of cases versus controls/in comparisons of invasive versus in situ cases. § OR, odds ratio; CI, confidence interval; DMPA, depot-medroxyprogesterone acetate. ¶ Excluding women who had never been pregnant. View Large TABLE 6. Age-adjusted odds ratios of invasive and in situ squamous cell cervical carcinoma in relation to nonsexual, nonhormonal factors, Bangkok, Thailand, 1991–1993 Factor  Invasive   In situ   Invasive   In situ   Invasive/in situ*   Cases (no.)  Controls (no.)  Cases† (no.)  Controls (no.)  OR‡  95% CI‡  OR  95% CI  OR  95% CI  Ever used an IUD‡                       No  168  231  59/67  101  1.0  Reference  1.0  Reference  1.0  Reference   Yes  22  60  6/8  23  0.5  0.3, 0.9  0.4  0.2, 1.1  1.2  0.5, 3.1  Tubal ligation                       No  138  187  38/46  70  1.0  Reference  1.0  Reference  1.0  Reference   Yes  52  104  27/29  54  0.7  0.4, 1.0  0.9  0.5, 1.7  0.4  0.2, 0.8  Months since last cervical smear                       No cervical smear  165  192  50/56  75  1.0  Reference  1.0  Reference  1.0  Reference   1–12  7  1  11/11  30  0.2  0.1, 0.4  0.6  0.3, 1.3  0.3  0.1, 0.8   >12  5  2  4/7  14  0.3  0.1, 0.6  0.4  0.1, 1.3  0.3  0.1, 1.1   Unknown months  13  2  0  5  0.5  0.3, 1.0          Ever smoked ≥100 cigarettes§                       No  175  276  61/69  121  1.0  Reference  1.0  Reference  1.0  Reference   Yes  14  15  4/6  3  1.4  0.7, 2.0  2.2  0.5, 10  1.2  0.4, 3.7  Ever drank alcoholic beverages                       No  94  148  39/44  64  1.0  Reference  1.0  Reference  1.0  Reference   Yes  96  143  26/31  60  1.0  0.7, 1.5  0.7  0.4, 1.3  1.1  0.6, 2.0  Ever had a chest radiographic examination                       No  158  204  42/46  84  1.0  Reference  1.0  Reference  1.0  Reference   Yes  32  87  23/29  40  0.5  0.3, 0.7  1.2  0.6, 2.3  0.3  0.2, 0.6  Attended school                       No  31  27  3/4  10  1.0  Reference  1.0  Reference  1.0  Reference   Yes  159  264  62/73  114  0.6  0.3, 1.0  1.7  0.5, 6.6  0.5  0.2, 1.6  Factor  Invasive   In situ   Invasive   In situ   Invasive/in situ*   Cases (no.)  Controls (no.)  Cases† (no.)  Controls (no.)  OR‡  95% CI‡  OR  95% CI  OR  95% CI  Ever used an IUD‡                       No  168  231  59/67  101  1.0  Reference  1.0  Reference  1.0  Reference   Yes  22  60  6/8  23  0.5  0.3, 0.9  0.4  0.2, 1.1  1.2  0.5, 3.1  Tubal ligation                       No  138  187  38/46  70  1.0  Reference  1.0  Reference  1.0  Reference   Yes  52  104  27/29  54  0.7  0.4, 1.0  0.9  0.5, 1.7  0.4  0.2, 0.8  Months since last cervical smear                       No cervical smear  165  192  50/56  75  1.0  Reference  1.0  Reference  1.0  Reference   1–12  7  1  11/11  30  0.2  0.1, 0.4  0.6  0.3, 1.3  0.3  0.1, 0.8   >12  5  2  4/7  14  0.3  0.1, 0.6  0.4  0.1, 1.3  0.3  0.1, 1.1   Unknown months  13  2  0  5  0.5  0.3, 1.0          Ever smoked ≥100 cigarettes§                       No  175  276  61/69  121  1.0  Reference  1.0  Reference  1.0  Reference   Yes  14  15  4/6  3  1.4  0.7, 2.0  2.2  0.5, 10  1.2  0.4, 3.7  Ever drank alcoholic beverages                       No  94  148  39/44  64  1.0  Reference  1.0  Reference  1.0  Reference   Yes  96  143  26/31  60  1.0  0.7, 1.5  0.7  0.4, 1.3  1.1  0.6, 2.0  Ever had a chest radiographic examination                       No  158  204  42/46  84  1.0  Reference  1.0  Reference  1.0  Reference   Yes  32  87  23/29  40  0.5  0.3, 0.7  1.2  0.6, 2.3  0.3  0.2, 0.6  Attended school                       No  31  27  3/4  10  1.0  Reference  1.0  Reference  1.0  Reference   Yes  159  264  62/73  114  0.6  0.3, 1.0  1.7  0.5, 6.6  0.5  0.2, 1.6  * Risks of invasive disease relative to risk of in situ disease, adjusted for age and presence or absence of oncogenic human papillomavirus (HPV) type or other and unknown HPV type or no HPV (three categories). † In situ cases included in comparisons of cases versus controls/in comparisons of invasive versus in situ cases. ‡ OR, odds ratio; CI, confidence interval; IUD, intrauterine device. § One case with unknown smoking history was excluded. View Large As shown in table 4, the risk of both invasive and in situ disease tended to decline with increasing age at first intercourse. The risk of both tumor types was only weakly associated with positive serologic tests for herpes simplex virus type 2 antibodies, syphilis or hepatitis B antibodies, or surface antigen, and no associations with herpes simplex virus type 1 were found (not shown). Few women in this study admitted to having had more than one sexual partner, and no increase in risk of either tumor type was observed in relation to this variable. None of the sexual factors considered was significantly more strongly related to invasive than in situ disease. Only one woman with invasive disease and three with CIN-III were positive for the human immunodeficiency virus test (not shown). The risk of both in situ and invasive disease increased with increasing number of pregnancies (table 5). The risk of neither tumor type was significantly associated with the other hormonal factors considered, except for an increase in risk of in situ carcinoma in users of depot-medroxyprogesterone acetate for contraception. Use of oral contraceptives was weakly associated with both tumor types. None of the hormonal factors shown in table 5 was significantly more strongly associated with invasive than in situ disease. Ages at menarche and first livebirth were also not associated with either tumor type (not shown). Women who had used an intrauterine device and who had a history of prior cervical smears were at reduced risk of both invasive and in situ disease, and the risk of both types was slightly but not significantly increased in smokers (table 6). The risk of invasive but not of in situ carcinoma was lower in women with than without a history of a screening chest radiographic examination and formal schooling. The risk of invasive diseases was low relative to the risk of in situ disease in women with a tubal ligation, prior cervical smears, and a prior chest radiographic examination, and possibly in women who had attended school. When the analyses for the last columns in tables 4–6 were restricted to data on study subjects who had never had a cervical smear, to eliminate differences between women with invasive and in situ disease that may be due to prior screening behavior, the results were not appreciably different from those presented. Moreover, the same analyses were performed separately for invasive and in situ cases with an oncogenic HPV type (150 invasive and 42 in situ cases) and without an oncogenic HPV type (26 invasive and 18 in situ cases), and no significant differences (p > 0.05) were observed in the comparable odds ratio estimates from the two sets of analyses. DISCUSSION Oncogenic HPV DNA was found in a high proportion of invasive squamous cell cervical carcinomas, with type 16 being the predominant type. This is in accordance with prior observations (1, 6–8, 19) in which sensitive polymerase chain reaction-based methods were used to detect HPV DNA in cervical cancer cells. The oncogenic types that were assayed were demonstrated in a lower proportion of the in situ than invasive tumors, and untyped HPV DNA was found more frequently in the intraepithelial lesions. These observations are also in accordance with prior studies (1, 10, 20, 21) and are compatible with observations by others that the prevalence of oncogenic HPV types in tumors is directly related to the severity of the lesion (1, 22–26). There is little reason to suggest that the odds ratio estimates in this study in relation to HPV type are biased: cervical scrapings were obtained from a high proportion of both the invasive and in situ cases; both groups of women resided in the same areas of Thailand; and the prevalence of the HPV types that were assayed is similar to that observed by others (1). The odds ratio estimates shown in table 2 thus provide evidence that HPV types 16 and 18 are associated with a fourfold increase in the risk of progression to invasion, that types 31/33/35/39 are less strongly predictive of progression, and that the other (untyped) HPV types are not indicative of subsequent invasion. This interpretation is consistent with the observation (27) that squamous intraepithelial lesions with oncogenic HPV DNA tend to be monoclonal, and those with other HPV DNA types tend to be polyclonal. Although assays for types of HPV other than those that were investigated in this study are now available, and although some of these more recently identified types may be oncogenic, it is unlikely that their omission from this study influenced the results that pertain to types 16, 18, and 31/33/35/39, because only 14 women with invasive disease and 12 with in situ tumors had untyped HPV DNA in their cervical scrapings. The observation that untyped HPV types are more strongly associated with in situ than invasive disease would probably be strengthened if we had tested for the more recently recognized oncogenic types and removed them from the group with untyped DNA; our conclusion that there are HPV types associated with in situ disease that do not progress to invasion would not change. After controlling for age and HPV type, the epidemiologic features of in situ and invasive cervical carcinomas were found to be quite similar. The age at first intercourse was a strong risk factor for both tumor types in this largely monogamous population, suggesting either that early exposure to oncogenic types of HPV may enhance the risk of persistent infection and carcinogenesis, or that the sexual behavior of the husbands of women with early and late onset of sexual activity may differ (17). Serologic indices of exposure to sexually transmitted agents other than HPV were generally not strongly related to either invasive or in situ disease, and none was significantly more strongly related to one tumor type or the other. This is as expected, because these factors are more likely indicators of risk of initial infection with HPV rather than cofactors operative after neoplastic changes. Although quite possibly due to chance, the risk in relation to serologic evidence of hepatitis B surface antigen (table 4) was slightly greater for invasive than in situ disease. Because persistent hepatitis B virus antigenemia may suggest a deficient immune response, this observation is compatible with a role for immunodeficiency in the persistence of HPV infection that can lead to invasive carcinoma. An increase in the risk of both in situ and invasive disease with parity and use of oral contraceptives was observed, but the associations were not significantly stronger for invasive disease, suggesting that these hormonal factors operate prior to the development of in situ lesions. The risk of carcinoma in situ, but not of invasive disease, was elevated in users of depot-medroxyprogesterone acetate. This same observation was previously observed in a larger study conducted in Thailand and elsewhere (28, 29) and is compatible with the interpretation that any effect of depot-medroxyprogesterone acetate on the risk of cervical carcinoma in situ is reversible or that the lesions induced by this product have a low invasive potential. Smoking was weakly associated with the risk of both invasive and in situ lesions, but the prevalence of smoking among the study subjects was low and the odds ratio estimate had wide confidence limits. If smoking is a cofactor, it operates prior to the development of in situ disease and is not an important determinant of risk in Thailand. Having had a screening chest radiographic examination and having ever attended school were both associated with a reduced risk of invasive disease but not of in situ disease. These associations persisted after controlling for the frequency of prior cervical smear screening, suggesting that these associations are not due to more cervical cancer screening in the more educated women and in women who are screened for tuberculosis. Another possible explanation is that women of higher socioeconomic status may tend to seek care earlier than women of lower status and, hence, are more likely to have their cervical neoplasia diagnosed when still in a preinvasive stage. However, the observation that the presenting symptoms of women with in situ and invasive disease did not differ is not supportive of this interpretation. Other studies have also shown odds ratios in relation to school attendance to be less than unity for invasive but not in situ disease (10, 14, 15, 30), and the consistency of these results and ours suggests that factors associated with education or other measures of socioeconomic status, such as nutritional factors, may inhibit invasion. Prior cervical smears were shown to be related to a reduced risk of cervical carcinoma and, as expected, to be more strongly protective against invasive than in situ disease. As has also been observed elsewhere (31), reduced risks in relation to use of an intrauterine device and a tubal ligation most likely reflect screening for cervical cancer at the time of intrauterine device insertion or removal and at the time of tubal surgery. Although there has been only one prior report of studies in which direct comparisons of risk factors for in situ and invasive carcinomas were made after controlling for HPV type in the tumor (10), there have been other investigations in which women with both in situ and invasive disease have been studied using comparable methodology but without inclusion of HPV testing (13–15, 30). The results of all these studies are generally consistent with our findings in showing no sexual or hormonal factors, or smoking, to be more strongly associated with invasive than in situ carcinomas. Additional studies of the role of these factors in the genesis of cervical carcinoma should focus on the more proximal preinvasive stages of the carcinogenic process. Several possible sources of bias could have influenced the odds ratio estimates in this study. If women with carcinoma in situ were more likely than women with invasive carcinoma to have been screened for cervical cancer, then observed differences between women with these two tumor types could reflect factors associated with screening behavior rather than with development of invasive disease. This is an unlikely explanation for our results. There is no mass screening of asymptomatic women in Thailand, and cases of in situ disease in this study did not differ from women with invasive disease with respect to the presenting symptoms that lead to diagnosis. In addition, controlling odds ratio estimates for history of prior cervical smears had no appreciable effect on the results. Confounding by HPV status could have influenced the odds ratio estimates based on comparisons of cases and controls, although the consistency of our results with those of others suggests that this is not a likely explanation for the findings. More importantly, the direct comparisons of women with invasive and in situ carcinomas were controlled for HPV type, thus eliminating confounding by these viruses as an explanation for the few associations observed in those analyses. The comparison of invasive with in situ disease is an investigative approach that other investigators should consider in an attempt to identify factors that may enhance or prevent progression of intraepithelial lesions to invasive disease. Because HPV type was the only predictor of progression identified, other studies should focus on additional factors not adequately considered in this and other investigations. Correspondence to Dr. David B. 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Journal

American Journal of EpidemiologyOxford University Press

Published: Apr 15, 2001

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