- Email: [email protected]
Polymerase chain reaction analysis of human papillomavirus in adenocarcinoma and adenosquamous carcinoma of the uterine cervix
Int J Gynecol Obstet, 1993, 41: 251-256 International Federation of Gynecology and Obstetrics
251
Polymerase chain reaction analysis of human papillomavirus in adenocarcinoma and adenosquamous carcinoma of the uterine cervix N. Matsuo, T. Iwasaka, Y. Hayashi, K. Hara, M. Mvula and H. Sugimori Department of Obstetrics and Gynecology, Saga Medical School, Nabeshima, Saga 849 (Japan)
(Received February 19th, 1992) (Revised and accepted December 6th, 1992)
Abstract Thirty-two cervical adenocarcinomas and adenosquamous carcinomas were examined to search for human papillomaviruses (HP Vs) using the polymerase chain reaction system. Human papillomavirus type 16 (HPV16) and type 18 (HPV18) deoxyribonucleic acid was detected in 22% and 16% of these carcinomas, respectively. HPV16 was the most common type in both adenocarcinoma and adenosquamous carcinoma, and the mean age of the HPV negative patients was significantly higher than that of HPVpositive patients. There may be an association between HPVs and the development of certain adenocarcinomas and adenosquamous carcinomas of the cervix.
Keywords: Human papillomavirus; Polymerase chain reaction; Adenocarcinoma. Introduction Adenocarcinoma and adenosquamous carcinoma represent only a small proportion of all primary carcinomas of the cervix, probably in the order of S-10% [2,5], although this percentage may be increasing [ 15,201. Several types of human papillomavirus (HPV), 0020-7293/93/$06.00 0 1993 International
Federation of Gynecology and Obstetrics Printed and Published in Ireland
especially HPV 16 and 18, were found to be closely associated with invasive squamous cell carcinoma of the cervix and cervical intraepithelial neoplasia [22]. Factors such as early age at the first intercourse, early age of first pregnancy, and multiple sexual partners increase the risk for development of cervical squamous cell carcinoma [ 131. In contrast, cervical adenocarcinoma has not been linked with sexual promiscuity [8,15], thereby suggesting that these carcinomas may have different etiologies. However, Southern blot hybridization [9,16,21] or in situ hybridization [11,17] studies have shown that HPV deoxyribonucleic acid (DNA) is present in some glandular malignancies. In some studies [17,21], but not in others [9,19], HPV 18 was the most common type identified in adenocarcinemas, while HPV 16 is the most predominant type in cases of squamous cell carcinomas [22]. It is not clear whether the high incidences of HPV 18 in adenocarcinemas were linked to geographic deviation, to different races, or to differences in the histologic types of carcinoma. The polymerase chain reaction (PCR) is a highly sensitive technique for HPV detection as small segments of DNA flanked by oligonucleotide primers can be amplified [3,18]. In addition, PCR can be used for the analysis of paraffin-embedded tissues, which allows retrospective studies on a large scale [7]. Article
252
Matsuo et al.
To examine the possible role of HPV in adenocarcinoma and adenosquamous carcinoma of the uterine cervix and to analyze the prognostic factors in relation to HPV positivity, we adopted the PCR analysis to examine the presence of HPV DNA in paraffinembedded tissues. Materials and methods Patients and sample preparation
The tissues were excised from 32 Japanese women with cervical adenocarcinoma or adenosquamous carcinoma. Three patients were International Federation of Gynecology and Obstetrics Stage 0, 21 were stage IB, 1 was Stage IIA, 3 were Stage IIB, 2 were Stage IIIB, and 2 were Stage IVA. They include 20 endocervical type adenocarcinomas in which 2 cases of adenoma malignum are included, 2 endometrioid adenocarcinomas, 2 clear cell adenocarcinomas and 8 adenosquamous carcinomas. All these women were treated at the Department of Obstetrics and Gynecology, Saga Medical School, Japan, between 1982 and 1991. The mean age of the patients was 52 years with a range of 18-85 years. Histologic slides were reviewed, and a representative block from each tumor was selected for HPV assay. Sections (5 pm) of formalin-fixed, paraffin-embedded tissues were deparaffinized with sequential washes of xylene and 100% ethanol in microfuge tubes, and pelleted by centrifugation. An adjacent section was also prepared to confirm the exact histology of the tissue analyzed. The sections were incubated with digestion buffer containing 100 pg/ml of Proteinase K and 100 mM Tris-HCl (pH 8.5) at 40°C for 48 h. The samples were centrifuged and an aliquot of the supernatant was used for PCR after incubation at 95°C for 8 min to inactivate the proteinase. Polymerase chain reaction (PCR)
Oligonucleotide primers were synthesized on a DNA synthesizer (Model 380, Applied Biosystems). Sequences of the primer pairs Int J Gynecol Obstet 41
were 5 ‘-GCAACCAGAGACAACTGATC3 ’ and 5 ‘-ATTGTAATGGGCTCTGTCCG3 ’ for HPV16, and 5 ‘-TCACGAGCAATTAAGCGACT-3 ’ and 5 ‘-CTGAGCTTTCTACTACTAGC-3 ’ for HPV 18, respectively. The primers were used to amplify the segments of HPV DNA position 606-720 (HPV 16) and 670-827 (HPV 18). The regions of both HPV 16 and 18 were within the E7 open reading frame in which the DNA remains after viral integration into host genomic DNA. The primers for HPV 6/11 were synthesized according to the sequences described elsewhere (position 115- 133 and 208-227) [3]. Furthermore, consensus primers which amplify DNA sequences within Ll region of HPV types 6, 11, 16, 18, 31, 33, 42, 52, 58, and some other unknown types [23] were used to detect HPV DNA other than HPV types 16 and 18. Amplification of the HPV target sequences was carried out in 100 ~1 of a reaction mixture containing 1.0 pg of genomic DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl*, 200 PM deoxynucleoside triphosphates, 100 pmol of each primer and 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus). The samples were overlaid with mineral oil and subjected to 40 cycles of amplification on an automated thermal cycler (Astec). For PCR using specific primers, a cycle represents DNA denaturation for 1 min at 94°C primer annealing for 2 min at 55”C, and primer extention for 2 min at 72”C, while for PCR using consensus primers, a cycle consists of DNA denaturation for 1.5 min at 95”C, primer annealing for 1.5 min at 48”C, and primer extension for 2 min at 70°C. After the last cycle, a 15-~1aliquot of the reaction mixture was analyzed by electrophoresis on 3% NuSieve agarose gel (FMC) and ethidium bromide staining, either untreated or after digestion with restriction endonucleases. DNA cleavage with restriction endonuclease Sau961 (Nippon Gene) or Mae111 (Boehringer Mannheim) was performed under conditions recommended by the suppliers to confirm whether the amplified band was HPV 16 or 18
HPV in cervical adenocarcinoma
253
specific. HPV typing was also performed on the band amplified with use of consensus primers by the multiple endonuclease cleavage patterns as described by Yoshikawa et al. ]231. HPV 6, 11, 16 and 18 were provided by Japanese Cancer Research Resources Bank. HPV DNA (1 ng) was used in PCR as a positive control. Negative controls included placental DNA and water. Preliminary analysis revealed that no cross-amplification occurred with different type of primers. Results Two HPV 16 DNA-positive cases are shown in Fig. 1, lanes 3-6. The fragment 115 base pairs (bp) in length was amplified by PCR and cleaved with Sau96 I into two subfragments (74 bp and 41 bp), as expected from the HPV 16 DNA sequence [ 141, thereby confirming that the authentic target DNA had been amplified. Similarly, two HPV 18 DNApositive cases are shown in Fig. 2, lanes 3-6. The 158 bp fragment was amplified by PCR and digested with Mae111 into two sub-
12345670
Fig. 2. Amplification of HPV 18 DNA by PCR. The PCR products were electrophoresed either untreated (lanes 1, 3, 5, 7) or after digestion with endonuclease Mae111 (lanes 2, 4, 6). Lanes 1, 2: HPV 18-positive control; lanes 3, 4 and 5, 6: two cases of HPV ll-positive adenosquamous carcinoma; lane 7: HPV 18-negative adenocarcinoma; lane 8: DNA size marker (HaeIII digest of X174RFI DNA). Molecular sizes are indicated in base pairs.
fragments (92 bp and 66 bp), as expected from the HPV 18 DNA sequence [l]. As shown in Table 1, HPV DNA was detected in 11 of 32 cases (34%) of cervical adenocarcinomas and adenosquamous carcinomas. Seven cases (4 adenocarcinomas and 3 adenosquamous carcinomas) were found to contain HPV 16 DNA. HPV 16 DNA-positive adenocarcinomas included 3 cases of endoTable 1. Detection of HPV DNA in adenocarcinomas and adenosquamous carcinomas of the cervix by polymerase chain reactiona. Histologic type
Fig. 1. Amplification of HPV 16 DNA by PCR. The PCR products were electrophoresed either untreated (lanes 1, 3, 5, 7) or after digestion with endonuclease Sau96 I (lanes 2, 4, 6). Lanes 1, 2: HPV 16-positive control; lanes 3, 4: HPV 16positive adenocarcinoma; lanes 5, 6: HPV 16-positive adenosquamous carcinoma; lane 7: HPV 16-negative adenocarcinoma; lane 8: DNA size marker (HaeIII digest of X174RFI DNA). Molecular sizes are indicated in base pairs.
No. of Pt. tested
Adenocarcinoma 24 Endocervical 20 Endometrioid 2 Clear cell 2 Adenosquamous ca. 8 Total 32
No. of HPV-positive Pt. HPV16 W)
HPVl8 (W
Total W)
4 (17) 3 (15) 1 (50) 0 (0) 3 (38) 7 (22)
3 (13) 3 (15) 0 (0) 0 (0) 2 (25) 5 (16)
6 (25)b 5 (25)b 1 (50) 0 (0) 5 (63) 11 (34)b
aAll cases were negative for‘HPV DNA other than HPV 16 and HPV 18. bDouble infection with HPV 16 and HPV 18 was found in one of the endocervical type adenocarcinomas. Article
254
Matsuo ef al.
cervical type adenocarcinoma and 1 case of endometrioid.adenocarcinoma. Two cases of clear cell adenocarcinoma and two cases of adenoma malignum contained no HPV DNA. HPV 18 DNA was detected in 3 adenocarcinemas and 2 adenosquamous carcinomas. No other type of HPVs including HPV 6/l 1 was detected in any tissue. The mean age of the HPV DNA-positive patients was 43 years (range, 26-69 years), and that of HPV DNA-negative patients was 57 years (range, 18-85 years). As shown in Table 2, t-test analysis revealed a statistically significant difference of age distribution between these two groups (P < 0.05). Correlation between presence of HPV DNA and clinical stages, tumor grade, lymph node metastasis, tumor size, and prognosis is shown in Table 3. HPV DNA detection rate was significantly higher in patients in earlier stages (P < 0.05) and in those with tumor less than 2 cm in size (P < 0.05). Moreover HPV DNA positivity was significantly higher in relapse-free patients than those with recurrent disease (P < 0.05). While no significant correlation was observed between HPV positivity and lymph node status or tumor grade. None of the patients had a history of diethylstilbestrol exposure in utero. Discussion While a large body of evidence implicates HPV in the pathogenesis of squamous cell carcinoma of the cervix [22], little is known about the etiology of cervical adenocarcinema and adenosquamous carcinoma. Unlike squamous cell carcinoma, adenocarTable 2. Comparison of age distribution between the groups of HPV-positive patients and HPV-negative patients. Group
No. of Patients
Range of Age
Mean Age zt S.D.
HPV(-) HPV(+)
21 I1
18-85 26-69
57.2 f 17.2 42.9 zk 12.7;
*t-test revealed a statistically significant difference (P Int J Gynecol Obstet 41
c
0.05).
Table 3. Correlation between presence of HPV and prognostic factors. Prognostic factors Clinical stage Stage 0 & I Stage II-IV Tumor size <2cm 2 cm s Lymph Node Metastasis Negative Positive Tumor grade Well Moderate Poor Prognosisb Relapse-free Relapsed
HPV positivity (%)
1l/24 (46)a O/8 (0) 6/10 (60)a 5122(23) lo/27 (37) l/5 (20) a/20 (40) 218 (25) l/4 (25) 9/18 (5o)a l/10 (IO)
“x2 test revealed a statistically significant difference (P < 0.05). bTwo patients died of the other carcinomas and another 2 patients were lost.
cinema has not been associated with venereal transmission [8,15]. Such a correlation may be difficult to determine because of the limited numbers. An etiological link between cervical adenocarcinoma and the use of oral contraceptives remains speculative [4]. Adenocarcinoma of the cervix was at first considered to be unrelated to HPV infection, but recent studies have shown that some adenocarcinomas do contain HPV DNA [9,11,16,17,19,21]. Using Southern blot hybridization, Smotkin et al. found HPV 16 DNA in 1 of 2 adenocarcinomas and HPV 18 DNA in 4 of 5 adenosquamous carcinomas [16]. Wilczynski et al. showed that among 11 adenocarcinomas 2 contained HPV 16 DNA and 5 contained HPV 18 DNA [21]. On the other hand, de Villiers et al. found HPV 16 DNA in 2 of 6 adenocarcinomas [ 191, and Lorincz et al. identified HPV 16 DNA in 4 cases and HPV 18 DNA in 2 cases of 9 adenocarcinomas [9]. Although certain cases of adenocarcinoma appear to be associated with HPV, the predominant HPV type differed with the study.
HPV in cervical adenocarcinoma
Our present data seem to be the first to demonstrate HPV DNA in a considerable number of adenocarcinomas of the cervix by the PCR system. PCR is a highly sensitive technique and it facilitates retrospective studies on paraffin-embedded tissue. The exact typing follows digestion of the amplified fragments with appropriate restriction endonucleases. We found HPV 16 DNA in 4 of 24 adenocarcinomas and 3 of 8 adenosquamous carcinomas. HPV 18 DNA was present in 3 of 24 adenocarcinomas and 2 of 8 adenosquamous carcinomas. In our study, HPV 16 was the most common HPV type in cervical adenocarcinoma and adenosquamous carcinoma. Some studies [17,21], but not others [9,19] including our data, suggested that HPV 18 was the most frequently identified HPV type in adenocarcinoma. The reason for this discrepancy is unknown, but may be due to geographic differences or to the different races studied. However, prevalence of HPV 18 among HPV-positive adenocarcinomas(l6% in 34”/0 in the present study was much higher than that in squamous cell carcinoma(8% in 95O/, [6]. Therefore, the possibility of a link between HPV 18 and pathogenesis of cervical adenocarcinoma can remain to be solved. Further studies are on going using larger databases and also using established cell lines. HPV detection rate (38%) as a result of PCR analysis seems to be lower than expected. This might be caused by the specimen used because extracted DNA from paraffinembedded tissue is usually fragmented or disrupted and moreover it is known that contamination of paraffin interferes the activity of Tuq polymerase. Therefore the sensitivity under this condition might decrease to the level of Southern. In addition, geographic or ethnic differences might be reflected on the result. The evidence that HPV 18 prevalence in squamous cell carcinomas is much higher in Europe and America than in Asia might explain this phenomenon. Wilczynski et al. suggested that one population of cervical adenocarcinoma in young
255
women is related to HPV while another predominant in older women is unrelated to HPV [21]. Their data showed 7 HPV DNApositive patients with a mean age of 37 years and 4 HPV DNA-negative patients with a mean age of 49 years. In the larger database, we demonstrated that mean age of HPVpositive patients was significantly younger than that of HPV-negative ones, which is consistent with the observation by Wilczynski et al. Riou et al. demonstrated that patients with HPV-negative cervical carcinoma showed poorer prognosis than those with HPV positive one [ 121.We also reported the similar observation, although the number of HPVnegative cases was too small to be analyzed [6]. If it is true that HPV-negative cervical carcinomas have poorer prognosis than HPVpositive ones, our present observation that HPV DNA was rarely detected in advanced cases and in cases with large tumor might be explained by aggressive nature of HPVnegative cancer cells which would grow fast and early spread beyond the uterine cervix. Our observation that HPV positive patients were more prevalent in relapse-free group than in recurrent group is also consistent with the above hypothesis. The risk of development of clear cell adenocarcinoma in females who were prenatally exposed to diethylstilbestrol was high compared to that in control women [9]. The median age at diagnosis of the clear cell adenocarcinoma in the diethylstilbestrolexposed women was 19 years [9]. The youngest woman in our series (virgin, 18 years old, clear cell adenocarcinoma) was negative for HPV DNA and there was no history of diethylstilbestrol exposure. Adenoma malignum is a very unique category in cervical adenocarcinoma. Two cases of adenoma malignum were contained in our series and both were negative for HPV DNA. A single etiologic factor probably cannot explain the pathogenesis of cervical adenocarcinema. On the basis of our observation, we Article
256
Matsuo et al.
are tempted to speculate that development of clear cell adenocarcinoma, adenoma malignum, and some adenocarcinoma predominant in older population is unrelated to HPV, while HPV is a closely linked factor for development of certain adenocarcinoma and adenosquamous carcinoma predominant in relatively younger population. Acknowledgment We thank Mariko Ohara for reading the manuscript. References I
2
3
4
5
6
7
8
9
10
Cole ST, Danos 0: Nucfeotide sequence and comparative analysis of the human papillomavirus type 18 genome. J Mel Biol 193: 599, 1987. Cramer DW, Cutler SJ: Inckience and histopathology of malignancies of the female genital organs in the United States. Am J Obstet Gynecol 118: 443, 1974. Dallas PB, Flanagan JL, Nightingale BN, Morris BJ: Polymerase chain reaction for fast, nonradioactive detection of high- and low-risk papillomavirus types in routine cervical specimens and in biopsies. J Med Virol 27: 105, 1989. Dallenbach-HeAhveg G: On the origin and histological structure of adenocarcinoma of the endocervix in women under 50 years of age. Pathology Research and Practice 179: 38, 1984. Hurt WG, Silverberg SG, Frabfe WJ, Belgrad R, Crooks DLJr: Adenocarcinoma of the cervix: Histopathologic and clinical features. Am J Obstet Gynecol129: 304, 1977. Iwasaka T, Yokoyama M, Oh-uchida M, Matsuo N, Hara K, Fukuyama K, Hachisuga T, Fukuda K, Sugimori H: Detection of human papillomavirus genome and analysis of expression of c-myc and Ha-rar oncogenes in invasive cervical carcinomas. Gynecol Oncol 46: 298 1992. Kiyabu MT, Shibata D, Amheim N, Martin WJ, Fitzgibbons PL: Detection of human papillomavirus in formalinfixed, invasive squamous carcinomas using the polymerase chain reaction. Am J Surg Path01 13: 221, 1989. Korhonen MO: Epidemiological differences between adenocarcinoma and squamous cell carcinoma of the uterine cervix. Gynecol Oncol IO: 312, 1980. Lorincz AT, Temple GF, Kurman RJ, Jenson AB, Lancaster WD: Oncogenic association of specific human papillomavirus types with cervical neoplasia. J Nat1 Cancer Inst 79: 671, 1987. Melnick S, Cole P, Anderson D, Herbst A: Rates and risks of diethylstilbestrol-related clear-cell adenocarcinema of the vagina and cervix. N Engl J Med 316: 514, 1987.
Int J Gynecol Obstet 41
11 Nielsen AL: Human papillomavirus type 16/18 in uterine cervical adenocarcinoma in situ and adenocarcinoma. Cancer 65: 2588, 1990. 12 Riou G, Favre M, Jeanne1 D, Bourhis J, Le Doussal V, Orth G: Association between poor prognosis in earlystage invasive cervical carcinomas and non-detection of HPV DNA. Lancet 335: 1171, 1990. 13 Rotkin ID A comparison review of key epidemiological studies in cervical cancer related to current searches for transmissible agents. Cancer Res 33: 1353, 1973. 14 Seedorf K, Krammer G, Durst M, Suhai S, Rowekamp WG: Human papillomavirus type 16 DNA sequence. Virology 145: 181,1985. 15 Shingleton HM, Gore H, Bradley DH, Soong SJ: Adenocarcinoma of the cervix. I. Clinical evaluation and pathologic features. Am J Obstet Gynecol139: 799, 1981. 16 Smotkin D, Berek JS, Fu YS, Hacker NF, Major FJ, Wettstein FO: Human papillomavirus deoxyribonucleic acid in adenocarcinoma and adenosquamous carcinoma of the uterine cervix. Obstet Gynecol 68: 241, 1986. 17 Tase T, Okagaki T, Clark BA, Manias DA, Ostrow RS, Twiggs LB, Faras AJ: Human papillomavirus types and localization in adenocarcinoma and adenosquamous carcinoma of the uterine cervix: A study by in situ DNA hybridization. Cancer Res 48: 993, 1988. 18 Tidy JA, Mason WP, Farrell PJ: A new and sensitive method of screening for human papillomavirus infection. Obstet Gynecol 74: 410, 1989. 19 de Villiers EM, Schneider A, Gross G, zur Hausen H: Analysis of benign and malignant urogenital tumors for human papillomavirus infection by labelling cellular DNA. Med Microbial Immunol 174: 281, 1986. 20 Weiss RJ, Lucas WE: Adenocarcinoma of the uterine cervix. Cancer 57: 1996, 1986. 21 Wilczynski SP, Walker J, Liao SY, Bergen S, Berman M: Adenocarcinoma of the cervix associated with human papillomavirus. Cancer 62: 1331, 1988. 22 Wright TC, Richart RM: Role of human papillomavirus in the pathogenesis of genital tract warts and cancer. Gynecol Oncol37: 151, 1990. 23 Yoshikawa H, Kawana T, Kitagawa K, Mizuno M, Yoshikura H, Iwamoto A: Detection and typing of muhiple genital human papillomaviruses by DNA amplitication with consensus primers. Jpn J Cancer Res 82: 524, 1991.
Address for repriots:
N. Matsuo
Department of Obstetrics and Gynecology Saga Medical Sehwl l-l, s-ehome Nabeshima, Saga 849
Japra
251
Polymerase chain reaction analysis of human papillomavirus in adenocarcinoma and adenosquamous carcinoma of the uterine cervix N. Matsuo, T. Iwasaka, Y. Hayashi, K. Hara, M. Mvula and H. Sugimori Department of Obstetrics and Gynecology, Saga Medical School, Nabeshima, Saga 849 (Japan)
(Received February 19th, 1992) (Revised and accepted December 6th, 1992)
Abstract Thirty-two cervical adenocarcinomas and adenosquamous carcinomas were examined to search for human papillomaviruses (HP Vs) using the polymerase chain reaction system. Human papillomavirus type 16 (HPV16) and type 18 (HPV18) deoxyribonucleic acid was detected in 22% and 16% of these carcinomas, respectively. HPV16 was the most common type in both adenocarcinoma and adenosquamous carcinoma, and the mean age of the HPV negative patients was significantly higher than that of HPVpositive patients. There may be an association between HPVs and the development of certain adenocarcinomas and adenosquamous carcinomas of the cervix.
Keywords: Human papillomavirus; Polymerase chain reaction; Adenocarcinoma. Introduction Adenocarcinoma and adenosquamous carcinoma represent only a small proportion of all primary carcinomas of the cervix, probably in the order of S-10% [2,5], although this percentage may be increasing [ 15,201. Several types of human papillomavirus (HPV), 0020-7293/93/$06.00 0 1993 International
Federation of Gynecology and Obstetrics Printed and Published in Ireland
especially HPV 16 and 18, were found to be closely associated with invasive squamous cell carcinoma of the cervix and cervical intraepithelial neoplasia [22]. Factors such as early age at the first intercourse, early age of first pregnancy, and multiple sexual partners increase the risk for development of cervical squamous cell carcinoma [ 131. In contrast, cervical adenocarcinoma has not been linked with sexual promiscuity [8,15], thereby suggesting that these carcinomas may have different etiologies. However, Southern blot hybridization [9,16,21] or in situ hybridization [11,17] studies have shown that HPV deoxyribonucleic acid (DNA) is present in some glandular malignancies. In some studies [17,21], but not in others [9,19], HPV 18 was the most common type identified in adenocarcinemas, while HPV 16 is the most predominant type in cases of squamous cell carcinomas [22]. It is not clear whether the high incidences of HPV 18 in adenocarcinemas were linked to geographic deviation, to different races, or to differences in the histologic types of carcinoma. The polymerase chain reaction (PCR) is a highly sensitive technique for HPV detection as small segments of DNA flanked by oligonucleotide primers can be amplified [3,18]. In addition, PCR can be used for the analysis of paraffin-embedded tissues, which allows retrospective studies on a large scale [7]. Article
252
Matsuo et al.
To examine the possible role of HPV in adenocarcinoma and adenosquamous carcinoma of the uterine cervix and to analyze the prognostic factors in relation to HPV positivity, we adopted the PCR analysis to examine the presence of HPV DNA in paraffinembedded tissues. Materials and methods Patients and sample preparation
The tissues were excised from 32 Japanese women with cervical adenocarcinoma or adenosquamous carcinoma. Three patients were International Federation of Gynecology and Obstetrics Stage 0, 21 were stage IB, 1 was Stage IIA, 3 were Stage IIB, 2 were Stage IIIB, and 2 were Stage IVA. They include 20 endocervical type adenocarcinomas in which 2 cases of adenoma malignum are included, 2 endometrioid adenocarcinomas, 2 clear cell adenocarcinomas and 8 adenosquamous carcinomas. All these women were treated at the Department of Obstetrics and Gynecology, Saga Medical School, Japan, between 1982 and 1991. The mean age of the patients was 52 years with a range of 18-85 years. Histologic slides were reviewed, and a representative block from each tumor was selected for HPV assay. Sections (5 pm) of formalin-fixed, paraffin-embedded tissues were deparaffinized with sequential washes of xylene and 100% ethanol in microfuge tubes, and pelleted by centrifugation. An adjacent section was also prepared to confirm the exact histology of the tissue analyzed. The sections were incubated with digestion buffer containing 100 pg/ml of Proteinase K and 100 mM Tris-HCl (pH 8.5) at 40°C for 48 h. The samples were centrifuged and an aliquot of the supernatant was used for PCR after incubation at 95°C for 8 min to inactivate the proteinase. Polymerase chain reaction (PCR)
Oligonucleotide primers were synthesized on a DNA synthesizer (Model 380, Applied Biosystems). Sequences of the primer pairs Int J Gynecol Obstet 41
were 5 ‘-GCAACCAGAGACAACTGATC3 ’ and 5 ‘-ATTGTAATGGGCTCTGTCCG3 ’ for HPV16, and 5 ‘-TCACGAGCAATTAAGCGACT-3 ’ and 5 ‘-CTGAGCTTTCTACTACTAGC-3 ’ for HPV 18, respectively. The primers were used to amplify the segments of HPV DNA position 606-720 (HPV 16) and 670-827 (HPV 18). The regions of both HPV 16 and 18 were within the E7 open reading frame in which the DNA remains after viral integration into host genomic DNA. The primers for HPV 6/11 were synthesized according to the sequences described elsewhere (position 115- 133 and 208-227) [3]. Furthermore, consensus primers which amplify DNA sequences within Ll region of HPV types 6, 11, 16, 18, 31, 33, 42, 52, 58, and some other unknown types [23] were used to detect HPV DNA other than HPV types 16 and 18. Amplification of the HPV target sequences was carried out in 100 ~1 of a reaction mixture containing 1.0 pg of genomic DNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl*, 200 PM deoxynucleoside triphosphates, 100 pmol of each primer and 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus). The samples were overlaid with mineral oil and subjected to 40 cycles of amplification on an automated thermal cycler (Astec). For PCR using specific primers, a cycle represents DNA denaturation for 1 min at 94°C primer annealing for 2 min at 55”C, and primer extention for 2 min at 72”C, while for PCR using consensus primers, a cycle consists of DNA denaturation for 1.5 min at 95”C, primer annealing for 1.5 min at 48”C, and primer extension for 2 min at 70°C. After the last cycle, a 15-~1aliquot of the reaction mixture was analyzed by electrophoresis on 3% NuSieve agarose gel (FMC) and ethidium bromide staining, either untreated or after digestion with restriction endonucleases. DNA cleavage with restriction endonuclease Sau961 (Nippon Gene) or Mae111 (Boehringer Mannheim) was performed under conditions recommended by the suppliers to confirm whether the amplified band was HPV 16 or 18
HPV in cervical adenocarcinoma
253
specific. HPV typing was also performed on the band amplified with use of consensus primers by the multiple endonuclease cleavage patterns as described by Yoshikawa et al. ]231. HPV 6, 11, 16 and 18 were provided by Japanese Cancer Research Resources Bank. HPV DNA (1 ng) was used in PCR as a positive control. Negative controls included placental DNA and water. Preliminary analysis revealed that no cross-amplification occurred with different type of primers. Results Two HPV 16 DNA-positive cases are shown in Fig. 1, lanes 3-6. The fragment 115 base pairs (bp) in length was amplified by PCR and cleaved with Sau96 I into two subfragments (74 bp and 41 bp), as expected from the HPV 16 DNA sequence [ 141, thereby confirming that the authentic target DNA had been amplified. Similarly, two HPV 18 DNApositive cases are shown in Fig. 2, lanes 3-6. The 158 bp fragment was amplified by PCR and digested with Mae111 into two sub-
12345670
Fig. 2. Amplification of HPV 18 DNA by PCR. The PCR products were electrophoresed either untreated (lanes 1, 3, 5, 7) or after digestion with endonuclease Mae111 (lanes 2, 4, 6). Lanes 1, 2: HPV 18-positive control; lanes 3, 4 and 5, 6: two cases of HPV ll-positive adenosquamous carcinoma; lane 7: HPV 18-negative adenocarcinoma; lane 8: DNA size marker (HaeIII digest of X174RFI DNA). Molecular sizes are indicated in base pairs.
fragments (92 bp and 66 bp), as expected from the HPV 18 DNA sequence [l]. As shown in Table 1, HPV DNA was detected in 11 of 32 cases (34%) of cervical adenocarcinomas and adenosquamous carcinomas. Seven cases (4 adenocarcinomas and 3 adenosquamous carcinomas) were found to contain HPV 16 DNA. HPV 16 DNA-positive adenocarcinomas included 3 cases of endoTable 1. Detection of HPV DNA in adenocarcinomas and adenosquamous carcinomas of the cervix by polymerase chain reactiona. Histologic type
Fig. 1. Amplification of HPV 16 DNA by PCR. The PCR products were electrophoresed either untreated (lanes 1, 3, 5, 7) or after digestion with endonuclease Sau96 I (lanes 2, 4, 6). Lanes 1, 2: HPV 16-positive control; lanes 3, 4: HPV 16positive adenocarcinoma; lanes 5, 6: HPV 16-positive adenosquamous carcinoma; lane 7: HPV 16-negative adenocarcinoma; lane 8: DNA size marker (HaeIII digest of X174RFI DNA). Molecular sizes are indicated in base pairs.
No. of Pt. tested
Adenocarcinoma 24 Endocervical 20 Endometrioid 2 Clear cell 2 Adenosquamous ca. 8 Total 32
No. of HPV-positive Pt. HPV16 W)
HPVl8 (W
Total W)
4 (17) 3 (15) 1 (50) 0 (0) 3 (38) 7 (22)
3 (13) 3 (15) 0 (0) 0 (0) 2 (25) 5 (16)
6 (25)b 5 (25)b 1 (50) 0 (0) 5 (63) 11 (34)b
aAll cases were negative for‘HPV DNA other than HPV 16 and HPV 18. bDouble infection with HPV 16 and HPV 18 was found in one of the endocervical type adenocarcinomas. Article
254
Matsuo ef al.
cervical type adenocarcinoma and 1 case of endometrioid.adenocarcinoma. Two cases of clear cell adenocarcinoma and two cases of adenoma malignum contained no HPV DNA. HPV 18 DNA was detected in 3 adenocarcinemas and 2 adenosquamous carcinomas. No other type of HPVs including HPV 6/l 1 was detected in any tissue. The mean age of the HPV DNA-positive patients was 43 years (range, 26-69 years), and that of HPV DNA-negative patients was 57 years (range, 18-85 years). As shown in Table 2, t-test analysis revealed a statistically significant difference of age distribution between these two groups (P < 0.05). Correlation between presence of HPV DNA and clinical stages, tumor grade, lymph node metastasis, tumor size, and prognosis is shown in Table 3. HPV DNA detection rate was significantly higher in patients in earlier stages (P < 0.05) and in those with tumor less than 2 cm in size (P < 0.05). Moreover HPV DNA positivity was significantly higher in relapse-free patients than those with recurrent disease (P < 0.05). While no significant correlation was observed between HPV positivity and lymph node status or tumor grade. None of the patients had a history of diethylstilbestrol exposure in utero. Discussion While a large body of evidence implicates HPV in the pathogenesis of squamous cell carcinoma of the cervix [22], little is known about the etiology of cervical adenocarcinema and adenosquamous carcinoma. Unlike squamous cell carcinoma, adenocarTable 2. Comparison of age distribution between the groups of HPV-positive patients and HPV-negative patients. Group
No. of Patients
Range of Age
Mean Age zt S.D.
HPV(-) HPV(+)
21 I1
18-85 26-69
57.2 f 17.2 42.9 zk 12.7;
*t-test revealed a statistically significant difference (P Int J Gynecol Obstet 41
c
0.05).
Table 3. Correlation between presence of HPV and prognostic factors. Prognostic factors Clinical stage Stage 0 & I Stage II-IV Tumor size <2cm 2 cm s Lymph Node Metastasis Negative Positive Tumor grade Well Moderate Poor Prognosisb Relapse-free Relapsed
HPV positivity (%)
1l/24 (46)a O/8 (0) 6/10 (60)a 5122(23) lo/27 (37) l/5 (20) a/20 (40) 218 (25) l/4 (25) 9/18 (5o)a l/10 (IO)
“x2 test revealed a statistically significant difference (P < 0.05). bTwo patients died of the other carcinomas and another 2 patients were lost.
cinema has not been associated with venereal transmission [8,15]. Such a correlation may be difficult to determine because of the limited numbers. An etiological link between cervical adenocarcinoma and the use of oral contraceptives remains speculative [4]. Adenocarcinoma of the cervix was at first considered to be unrelated to HPV infection, but recent studies have shown that some adenocarcinomas do contain HPV DNA [9,11,16,17,19,21]. Using Southern blot hybridization, Smotkin et al. found HPV 16 DNA in 1 of 2 adenocarcinomas and HPV 18 DNA in 4 of 5 adenosquamous carcinomas [16]. Wilczynski et al. showed that among 11 adenocarcinomas 2 contained HPV 16 DNA and 5 contained HPV 18 DNA [21]. On the other hand, de Villiers et al. found HPV 16 DNA in 2 of 6 adenocarcinomas [ 191, and Lorincz et al. identified HPV 16 DNA in 4 cases and HPV 18 DNA in 2 cases of 9 adenocarcinomas [9]. Although certain cases of adenocarcinoma appear to be associated with HPV, the predominant HPV type differed with the study.
HPV in cervical adenocarcinoma
Our present data seem to be the first to demonstrate HPV DNA in a considerable number of adenocarcinomas of the cervix by the PCR system. PCR is a highly sensitive technique and it facilitates retrospective studies on paraffin-embedded tissue. The exact typing follows digestion of the amplified fragments with appropriate restriction endonucleases. We found HPV 16 DNA in 4 of 24 adenocarcinomas and 3 of 8 adenosquamous carcinomas. HPV 18 DNA was present in 3 of 24 adenocarcinomas and 2 of 8 adenosquamous carcinomas. In our study, HPV 16 was the most common HPV type in cervical adenocarcinoma and adenosquamous carcinoma. Some studies [17,21], but not others [9,19] including our data, suggested that HPV 18 was the most frequently identified HPV type in adenocarcinoma. The reason for this discrepancy is unknown, but may be due to geographic differences or to the different races studied. However, prevalence of HPV 18 among HPV-positive adenocarcinomas(l6% in 34”/0 in the present study was much higher than that in squamous cell carcinoma(8% in 95O/, [6]. Therefore, the possibility of a link between HPV 18 and pathogenesis of cervical adenocarcinoma can remain to be solved. Further studies are on going using larger databases and also using established cell lines. HPV detection rate (38%) as a result of PCR analysis seems to be lower than expected. This might be caused by the specimen used because extracted DNA from paraffinembedded tissue is usually fragmented or disrupted and moreover it is known that contamination of paraffin interferes the activity of Tuq polymerase. Therefore the sensitivity under this condition might decrease to the level of Southern. In addition, geographic or ethnic differences might be reflected on the result. The evidence that HPV 18 prevalence in squamous cell carcinomas is much higher in Europe and America than in Asia might explain this phenomenon. Wilczynski et al. suggested that one population of cervical adenocarcinoma in young
255
women is related to HPV while another predominant in older women is unrelated to HPV [21]. Their data showed 7 HPV DNApositive patients with a mean age of 37 years and 4 HPV DNA-negative patients with a mean age of 49 years. In the larger database, we demonstrated that mean age of HPVpositive patients was significantly younger than that of HPV-negative ones, which is consistent with the observation by Wilczynski et al. Riou et al. demonstrated that patients with HPV-negative cervical carcinoma showed poorer prognosis than those with HPV positive one [ 121.We also reported the similar observation, although the number of HPVnegative cases was too small to be analyzed [6]. If it is true that HPV-negative cervical carcinomas have poorer prognosis than HPVpositive ones, our present observation that HPV DNA was rarely detected in advanced cases and in cases with large tumor might be explained by aggressive nature of HPVnegative cancer cells which would grow fast and early spread beyond the uterine cervix. Our observation that HPV positive patients were more prevalent in relapse-free group than in recurrent group is also consistent with the above hypothesis. The risk of development of clear cell adenocarcinoma in females who were prenatally exposed to diethylstilbestrol was high compared to that in control women [9]. The median age at diagnosis of the clear cell adenocarcinoma in the diethylstilbestrolexposed women was 19 years [9]. The youngest woman in our series (virgin, 18 years old, clear cell adenocarcinoma) was negative for HPV DNA and there was no history of diethylstilbestrol exposure. Adenoma malignum is a very unique category in cervical adenocarcinoma. Two cases of adenoma malignum were contained in our series and both were negative for HPV DNA. A single etiologic factor probably cannot explain the pathogenesis of cervical adenocarcinema. On the basis of our observation, we Article
256
Matsuo et al.
are tempted to speculate that development of clear cell adenocarcinoma, adenoma malignum, and some adenocarcinoma predominant in older population is unrelated to HPV, while HPV is a closely linked factor for development of certain adenocarcinoma and adenosquamous carcinoma predominant in relatively younger population. Acknowledgment We thank Mariko Ohara for reading the manuscript. References I
2
3
4
5
6
7
8
9
10
Cole ST, Danos 0: Nucfeotide sequence and comparative analysis of the human papillomavirus type 18 genome. J Mel Biol 193: 599, 1987. Cramer DW, Cutler SJ: Inckience and histopathology of malignancies of the female genital organs in the United States. Am J Obstet Gynecol 118: 443, 1974. Dallas PB, Flanagan JL, Nightingale BN, Morris BJ: Polymerase chain reaction for fast, nonradioactive detection of high- and low-risk papillomavirus types in routine cervical specimens and in biopsies. J Med Virol 27: 105, 1989. Dallenbach-HeAhveg G: On the origin and histological structure of adenocarcinoma of the endocervix in women under 50 years of age. Pathology Research and Practice 179: 38, 1984. Hurt WG, Silverberg SG, Frabfe WJ, Belgrad R, Crooks DLJr: Adenocarcinoma of the cervix: Histopathologic and clinical features. Am J Obstet Gynecol129: 304, 1977. Iwasaka T, Yokoyama M, Oh-uchida M, Matsuo N, Hara K, Fukuyama K, Hachisuga T, Fukuda K, Sugimori H: Detection of human papillomavirus genome and analysis of expression of c-myc and Ha-rar oncogenes in invasive cervical carcinomas. Gynecol Oncol 46: 298 1992. Kiyabu MT, Shibata D, Amheim N, Martin WJ, Fitzgibbons PL: Detection of human papillomavirus in formalinfixed, invasive squamous carcinomas using the polymerase chain reaction. Am J Surg Path01 13: 221, 1989. Korhonen MO: Epidemiological differences between adenocarcinoma and squamous cell carcinoma of the uterine cervix. Gynecol Oncol IO: 312, 1980. Lorincz AT, Temple GF, Kurman RJ, Jenson AB, Lancaster WD: Oncogenic association of specific human papillomavirus types with cervical neoplasia. J Nat1 Cancer Inst 79: 671, 1987. Melnick S, Cole P, Anderson D, Herbst A: Rates and risks of diethylstilbestrol-related clear-cell adenocarcinema of the vagina and cervix. N Engl J Med 316: 514, 1987.
Int J Gynecol Obstet 41
11 Nielsen AL: Human papillomavirus type 16/18 in uterine cervical adenocarcinoma in situ and adenocarcinoma. Cancer 65: 2588, 1990. 12 Riou G, Favre M, Jeanne1 D, Bourhis J, Le Doussal V, Orth G: Association between poor prognosis in earlystage invasive cervical carcinomas and non-detection of HPV DNA. Lancet 335: 1171, 1990. 13 Rotkin ID A comparison review of key epidemiological studies in cervical cancer related to current searches for transmissible agents. Cancer Res 33: 1353, 1973. 14 Seedorf K, Krammer G, Durst M, Suhai S, Rowekamp WG: Human papillomavirus type 16 DNA sequence. Virology 145: 181,1985. 15 Shingleton HM, Gore H, Bradley DH, Soong SJ: Adenocarcinoma of the cervix. I. Clinical evaluation and pathologic features. Am J Obstet Gynecol139: 799, 1981. 16 Smotkin D, Berek JS, Fu YS, Hacker NF, Major FJ, Wettstein FO: Human papillomavirus deoxyribonucleic acid in adenocarcinoma and adenosquamous carcinoma of the uterine cervix. Obstet Gynecol 68: 241, 1986. 17 Tase T, Okagaki T, Clark BA, Manias DA, Ostrow RS, Twiggs LB, Faras AJ: Human papillomavirus types and localization in adenocarcinoma and adenosquamous carcinoma of the uterine cervix: A study by in situ DNA hybridization. Cancer Res 48: 993, 1988. 18 Tidy JA, Mason WP, Farrell PJ: A new and sensitive method of screening for human papillomavirus infection. Obstet Gynecol 74: 410, 1989. 19 de Villiers EM, Schneider A, Gross G, zur Hausen H: Analysis of benign and malignant urogenital tumors for human papillomavirus infection by labelling cellular DNA. Med Microbial Immunol 174: 281, 1986. 20 Weiss RJ, Lucas WE: Adenocarcinoma of the uterine cervix. Cancer 57: 1996, 1986. 21 Wilczynski SP, Walker J, Liao SY, Bergen S, Berman M: Adenocarcinoma of the cervix associated with human papillomavirus. Cancer 62: 1331, 1988. 22 Wright TC, Richart RM: Role of human papillomavirus in the pathogenesis of genital tract warts and cancer. Gynecol Oncol37: 151, 1990. 23 Yoshikawa H, Kawana T, Kitagawa K, Mizuno M, Yoshikura H, Iwamoto A: Detection and typing of muhiple genital human papillomaviruses by DNA amplitication with consensus primers. Jpn J Cancer Res 82: 524, 1991.
Address for repriots:
N. Matsuo
Department of Obstetrics and Gynecology Saga Medical Sehwl l-l, s-ehome Nabeshima, Saga 849
Japra