Prevalence of cervical dysplasia and HPV infection according to sexual behavior.

A total of 165 uterine cervix smears from Venezuelan women were examined by cytological techniques to identify malignant and pre-malignant cervical changes, as well as to identify Human Papilloma Virus (HPV) DNA types 6, 11, 16, 18, 31, 33 and 35. Of these smears, 119 were from nonmonogamous women who participated in a cervical carcinoma screening program. In this group, HPV-DNA was detected by hybridization in 42 samples (35%) and cervical intraepithelial neoplasia (CIN) in 13 (11%). Forty-six monogamous aboriginal women were similarly studied and no evidence of abnormal cytology or HPV-DNA of the types studied here was found in any of them. In the non-monogamous group, age at first sexual intercourse and index of parity were not associated with cervical HPV infection and/or CIN. The rates of HPV infection, however, were significantly different between the two populations, confirming that sexual behavior involving multiple partners is associated with HPV infection.

In situ hybridization analysis of HPV DNA in cervical precancer and cervical cancers from China.

A series of 103 cervical biopsies derived from 103 women during July 1958 to September 1963 from Beijing, China were investigated with in situ hybridization for the presence of HPV6, 11, 16, 18, 31 and 33 DNA. The mean age of the patients was 46.1 + 10.6 years with a range of 24-74 years. Morphological features of HPV infection were found in 80 (77.7%) biopsies. Invasive cervical cancer was diagnosed in 43 biopsies and cervical intraepithelial neoplasia CIN I, CIN II and CIN III in 9, 9, and 27 cases, respectively. A total of 63.1% (65/103) of the lesions had morphological features of HPV infections associated with CIN or invasive carcinomas. Altogether, 31.1% (32/103) of the biopsies were shown to contain HPV DNA. Of the cases showing HPV morphology, 43.1% were HPV DNA positive. HPV16 (30/32) was the most frequent type, followed by HPV11 and 18, whereas no lesions with HPV6, 31 or 33 were found. A total of 19/43 (44.2%) of the invasive carcinomas contained HPV DNA. HPV DNA positivity and the grade of CIN showed a statistically significant correlation (P = 0.0011). Our study demonstrated the presence of HPV in cervical lesions among Chinese women in the late 1950's and early 1960's when a single sexual partner was the rule and also supports the concept that HPV has as an important etiological role in cervical cancer, the highest risk being associated with HPV type 16. The applicability of in situ hybridization in retrospective assessment is emphasized.

[Detection of HPV-DNA in the various uterocervical lesions by in situ hybridization].

We detected HPV (Human Papilloma Virus) -DNA in various uterocervical lesions by in situ hybridization using biotinylated DNA probes. In cases positive for HPV, the nuclei of the epithelial cells was purple to blackish purple. In 2 of 6 cases of chronic cervicitis, HPV-DNA was detected in the outer layer of the squamous epithelium. Eleven of 19 with mild dysplasia (57.9%) showed a positive reaction in the upper one-third of the epidermis in a mainly consisting of koilocytotic cells. All 6 patients with moderate dysplasia had positive cells among the koilocytotic cells and atypical cells in the middle layer. Five of 11 patients with severe dysplasia had scattered positive cells. Two of them had atypical condylomatoid lesions. Eight of 32 patients with squamous cell carcinoma were positive for HPV-DNA, but there was no consistent distribution pattern of the positive cells.

[Human papilloma virus (HPV) antigens and DNA in dysplasia and carcinoma of the uterine cervix].

Routinely paraffin-embedded sections of dysplasia, carcinoma in situ (CIS) and invasive (squamous) carcinoma of the cervix were studied to determine the participation of human papilloma virus (HPV) in these tissues. Morphological observation (1,059 cases) revealed condylomatous changes to reach 54% in dysplasia, 25% in CIS and 25% in invasive carcinoma. Condylomatous changes were also found to be 25 to 40% in the non-cancerous epithelia adjacent to in situ or invasive carcinomas. The immuno-peroxidase-PAP-method using anti-HPV serum was applied to 98 selected sections in which condylomatous changes were morphologically observed. HPV antigens were found to reach 56% in dysplasia, 42% in CIS and 35% in invasive carcinoma, and this result suggested that the morphologically observed condylomatous changes did not always coincide with virus maturation in the infected cells. By means of the in situ hybridization technique, HPV type-6, -11, -16 and -18 DNAs were all detected in dysplasia sections, whereas HPV type-16 DNA was demonstrated distinctively at a high rate among in situ and invasive carcinomas.

[Immunohistochemical studies on epidermal growth factor receptor in oncogenesis of uterine cervical cancer].

Recent studies have demonstrated that epidermal growth factor receptor (EGF-R) shows great homology with the v-erbB transforming protein and the amplified expression of EGF-R accompanies the malignant transformation of squamous epithelium of the uterine cervix. In this study, the tissue localization of EGF-R in the oncogenesis of uterine cervical cancer was examined by the avidin/biotin immunoperoxidase technique using anti-EGF-R monoclonal antibody. Normal squamous and columnar epithelium was almost negative for EGF-R. The positive rate of EGF-R increased in the precancerous lesions, whereas it decreased in invasive and metastatic cancer (mild dysplasia: 36%, moderate dysplasia: 57%, severe dysplasia: 77%, carcinoma in situ: 82%, microinvasive carcinoma: 80%, squamous cell carcinoma: 24%, glandular dysplasia: 67%, adenocarcinoma in situ: 75%, adenocarcinoma: 8%, adenosquamous carcinoma: 33%, metastatic carcinoma of the pelvic lymph node: 21%). The positive rate of dysplasia in follow up cases was high in the progressive group (regressive group: 0%, persistent group: 62%, progressive group: 80%). These results suggest that EGF-R may play an important role in the early stage of carcinogenesis of uterine cervical cancer, and it will be used as one of the markers in the prognosis of precancerous lesions of the uterine cervix.

Is it safe to prescribe hormonal contraception and replacement therapy to patients with premalignant and malignant uterine cervices?

The levels of estrogen and progesterone receptors in normal and abnormal uterine cervices were determined. The study group consisted of 14 patients with cervical intraepithelial neoplasia (CIN III) and 7 patients with invasive carcinoma of the cervix (stage IB-IIA). The control group included 23 patients who underwent total abdominal hysterectomy for menorrhagia, leiomyoma, etc. The concentration of total estrogen receptors in premalignant and malignant cervices did not differ from the patients with benign conditions of the cervix. The concentration of progesterone receptors was significantly higher in the nonaffected cervices than in the patients with preinvasive and invasive carcinoma of the cervix (P less than 0.05). We have shown that estrogen receptor concentrations do not differ between women with normal and abnormal uterine cervices. Therefore, we feel that the contraceptive pill is not contraindicated in women who have been treated for CIN III. We also maintain that hormone replacement therapy should be given, when indicated, to women who have been castrated following surgery and/or radiotherapy for invasive carcinoma of the cervix.

[In situ hybridization study on the localization of HPV DNA in the precancerous lesions of uterine cervix].

Human papillomavirus (HPV) types 16 and 18 have been found closely associated with cervical cancer. In order to investigate the relationship between HPV DNA and cervical precancerous lesions, we examined the formalin fixed specimens obtained from 22 cases of mild dysplasia, 33 cases of moderate dysplasia and 31 cases of severe dysplasia of the uterine cervix for the presence of HPV 6/11, 16 and 18 DNAs by in situ hybridization using the biotinylated HPV DNA probes. We also followed some HPV DNA positive cases of cervical dysplasia for more than 6 months prospectively. The results of in situ hybridization analysis revealed that HPV DNA was detected in the nuclei of koilocytosis, dysplastic cells and metaplastic cells. HPV 6/11 was positive in 27.3% (6/22) of mild dysplasia and 21.2% (7/33) of moderate dysplasia. On the other hand, HPV 16 positive rate increased with the grade of dysplasia and 36.4% (12/33) of moderate dysplasia, 61.3% (19/31) of severe dysplasia were positive for HPV 16 DNA. Some of the follow-up cases which were positive for HPV 16 DNA were later found to have carcinoma in situ. Our results suggest that HPV type 16 might play an important role in cervical carcinogenesis.

[Biometric research in the diagnosis of precancerous states and endocervical cancer]. / Biometricheskie issledovaniia v diagnostike predopukholevykh sostoianii i éndotservikal'nogo raka.

Smears of the endocervical channel taken from 58 patients with various endocervical conditions (reserve cell hyperplasia and dysplasia, reserve cell carcinoma in situ. Adenocarcinoma in situ, invasive glandular and poorly differentiated carcinoma) were stained by Feulgen method. Morphometrical (surface, perimeter, maximum and minimum diameter) and light microscopic (mean optical density of staining, its dispersion, DNA content) parameters of the epithelial cell nuclei were measured by means of telemetric image analyzer. The most informative biometrical indexes are found, the role of the DNA cytophotometry in the differential diagnosis is determined, the algorithm of the automated cytomorphological diagnosis of the dysplasia and endocervical carcinoma is developed.

A nuclear DNA study of uterine cervical dysplasia with reference to its prognostic significance.

To further define the nuclear DNA content of uterine cervical dysplasia and its relationship to prognosis and epidemiological features, a retrospective study using Papanicolaou stained cytological specimen and TICAS was undertaken. 1. Dysplasia patients was common among young females who had a background of low age first pregnancy, multiple Gravidity-Parity, the complication of inflammation and the use of hormonal contraceptives and progressed rapidly. It is recommended that a test should be repeated within 2 to 3 months regardless of the severity of the dysplasia and patients should be followed up for at least 2.5 to 3 years. 2. The DNA histograms were classified into 3 types (A,B and C): Type C, which had the stem line in an aneuploidy area, showed more severe dysplastic cases. This may be due to the proliferation rate and significant alternation in the chromosomes and mitoses. Nuclear DNA analysis using TICAS and Papanicolaou stained cytological material could discriminate between the progressive group and the persistent or regressive group. In addition, the mean nuclear area might be the best indicator of prognosis in uterine cervical dysplasia.

Identification of transferrin receptor in cervical and endometrial tissues.

The location of transferrin receptor (Tf.R) which is abundantly contained in proliferating cells and positional relationships of cells containing much Tf.R was observed on cervical and endometrial tissue sections by immunohistochemistry. In normal squamous epithelium, the parabasal cell layer was positive and the basal, intermediate, and superficial cell layers negative. This shows that the cells of the parabasal cell layer are proliferating most actively among those layers. Squamous carcinoma contained many positive cancer cells. But negative as well as positive cells were present, indicating that cancer cells proliferate under various conditions. Cancer cells infiltrating the stroma were made clear by Tf.R staining, and it became easier to identify them when only a few were present. Endometrial glandular cells in the basal layer stained only on the basal side of a cell regardless of the menstrual cycle, whereas in the functional layer glandular lumen side also stained positive in the secretory phase. Based on the present data, an immunohistochemical study of Tf.R seems useful to confirm the localization of proliferating cells and cancer cells and determine the conditions for cancer cell proliferation on the tissue sections.

Comparison of computerized analysis of nuclear DNA changes in uterine cervix dysplasia and in urothelial non-invasive papillary carcinoma.

The nuclear DNA content was measured in preneoplastic lesions of the uterine cervix and in papillary carcinomas of the bladder. Three groups of features were calculated from the raw data: basic DNA, DNA deviation and DNA distribution. The basic DNA features, concerning both the cervix and the bladder, showed a progressive increase in the mean DNA content, a decrease in the percentage of diploid nuclei and steadily increasing values of polyploid and aneuploid nuclei. Among the DNA deviation features, the malignancy grade value was zero in the normal cervical epithelium and in the normal urothelium. An increase in this value was evident in moderate dysplasia and in urothelial papillary carcinoma of grade 2, the highest value being in CIS and in grade 3. Concerning the DNA distribution features, the values of the 15th and 95th percentiles and their difference were progressively higher both in the cervix and in the bladder, expressing a continuous shift and spread of the DNA content measurements in the different diagnostic categories, with respect to normal epithelium and urothelium. The statistical analysis showed that the strongest correlation is between 2c D.I. and % of polyploid nuclei in the cervix and between M.I. and % of aneuploid nuclei greater than 4c in the bladder. In the cervix the most discriminating feature is the Malignancy Grade, whereas in the bladder it is the percentage of diploid nuclei. The comparison between the results of the three groups of features showed that: 1) Mild dysplasia of the cervix and urothelial papillary carcinoma of grade 1 showed similar changes in DNA features. Both were basically characterized by increased proliferative activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen receptor distribution in the normal and pathologically changed human cervix uteri: an immunohistochemical study with use of monoclonal anti-ER antibody.

Normal and pathologically changed structures of human cervix uteri were examined for estrogen receptor (ER) content using monoclonal antiestrophilin antibody (H 222 SP gamma) and the peroxidase-antiperoxidase method (PAP). The study was performed on fresh-frozen cervical specimens from 30 women; the staining was evaluated semiquantitatively. The ER expression in cervical mucosa was generally strong and comparable to the ER expression by proliferating endometria. The only exception was the endocervical stroma, which showed distinctly weaker specific staining than other cervical structures. This might account for ER concentration gradients observed in biochemical studies. The ER content in endocervical glands varied in different cases and in different areas of the same specimen, but no correlation to the functional activity of the genital tract or to the morphology of the glands could be found. The squamous epithelium of the portio vaginalis contained relatively large amounts of ER in the basal, parabasal, and intermediate layers. The superficial layer was ER-negative. The only factor we observed that strongly influenced the ER content in cervical mucosa was a local inflammatory process. Our results suggest that the synthesis of ER in cervical epithelium can be influenced by underlying stroma.

[Relation between human papillomavirus (HPV) and cancer of uterine cervix].

HPV-DNA fragments were detected in biopsy specimens (29 cases of cancer of uterine cervix, 2 cervical dysplasia and 9 normal cervix) using DNA hybridization technique. It was demonstrated that 52% of biopsy specimens of the cancer of uterine cervix was positive for HPV 16 DNA probe, while 9% was positive for HPV 18 DNA probe. 11% of non-cancerous biopsy specimens had a positive result for HPV 16 DNA probe. It was also demonstrated that the positive rate of HPV 16 DNA was 75% in grossly cauliflower and nodular type tumors but only 25% in erosion type. It seems that the positive rate of HPV 16 DNA is correlated to gross appearance of the tumor. The positive rates of HPV 16 DNA were different in 6 provinces in China. It was 64% in Shanxi Province, a high incidence area but 36% in Sichuan Province, a low incidence area. These results suggest that the carcinogenesis of cancer of the uterine cervix, be related to HPV infection.

Immunocytochemical localization of prolactin in carcinoma of the cervix.

Immunocytochemical methods were used to demonstrate prolactin in both normal and malignant human cervices. Four of five cervices with epidermoid carcinoma and three of four cervices with adenocarcinoma demonstrated prolactin. One of four normal cervices stained positive for prolactin in the endocervical glands. Four cervices from pregnant patients were positive for prolactin. The results of this study show prolactin in human cervical carcinomas. The role and source of cervical prolactin is unknown.

A new marker of maturation in the cervix: the estrogen-regulated 24K protein.

The presence of an estrogen-regulated protein (24K) in normal, dysplastic, metaplastic, and neoplastic cervical epithelium correlates with histologic criteria for squamous cell maturation. The 24K protein, originally discovered in the MCF-7 breast cancer cell line, was studied in 51 cases by the modified immunoperoxidase Avidin-Biotin Complex method, using an anti-24K mouse monoclonal antibody. Immunostained sections were compared to hematoxylin-and-eosin-stained sections cut from the same tissue block. The 24K protein was observed to be located primarily in the parabasal or "prickle cell layer" of normal cervical tissue (6 of 7 normal cervical tissue specimens tested were positive for 24K protein. Specimens were obtained from surgery for nonneoplastic causes) and in all cases (12 of 12) of dysplasia and carcinoma in situ. Intercellular bridges of these cells showed prominent immunostaining in normal cervix and dysplasia. 24K protein was observed as a granular cytoplasmic stain in all cases of squamous metaplasia (5 of 5) and keratinizing squamous cell carcinoma (9 of 9), and in 8 of 14 cases of nonkeratinizing squamous cell carcinoma. In this latter group, immunostaining was confined to only those cells showing cytoplasmic eosinophilia on H&E sections. In no case was the presence of the 24K protein associated with areas of mature keratin. 24K immunostaining was also observed in the reserve cells of morphologically normal endocervical glands adjacent to areas of dysplasia and carcinoma. We conclude that 24K protein is associated with squamous cell maturation and may be an important marker of reserve cell hyperplasia and squamous metaplasia.

Localization of an estrogen-responsive protein in the human cervix during menstrual cycle, pregnancy, and menopause and in abnormal cervical epithelia without atypia.

The presence and distribution of an estrogen-responsive protein with a molecular weight of 28,000 were investigated in the human cervix with use of a monoclonal antibody. This biochemical marker protein was localized with a light microscope and by immunocytochemical studies of the different cell types and cell layers of the cervix. The study involved 60 patients, 48 of whom were sexually active, eight pregnant, and four in menopause; strips of endometrial tissue were analyzed in 22 patients. In cycling women the estrogen-responsive protein was identified in the subcolumnar cells of the endocervix, whereas in the ectocervix the protein was detected mainly in the parabasal and intermediate cell layers but alternating with unstained areas. There were no significant variations in the presence of the protein in the ectocervical and endocervical epithelium during the different phases of the menstrual cycle. During pregnancy a more intense and homogeneous immunostaining of the protein was seen in the ectocervix, endocervical areas with squamous metaplasia showed strong estrogen-responsive immunostaining, and predecidual and decidual cells were positive for the protein. There were no prominent changes in the presence and distribution of the protein in the abnormal ectocervical samples without atypia. However, in the endocervix the protein detection was useful to follow the evolution of the subcolumnar cells to simple squamous metaplasia. These samples displayed intense estrogen-responsive immunostaining. No immunoreaction was observed in the cervix of menopausal women. The results of the present study have shown that the response of the normal uterine cervix to estrogenic influence is heterogeneous in different cervical cell types and in different sites within the same cell layer; during the normal menstrual cycle the capability of response of the cervical cells to variations of estrogen levels is limited when compared with the endometrium; and during pregnancy and in the process of indirect squamous metaplasia some of the cervical cells seem to be very reactive to estrogenic stimulation. This study defines the normal baseline for further analysis of the estrogen-regulated protein in the uterine cervix during abnormal growth.

Correlation of cellular atypia and human papillomavirus deoxyribonucleic acid sequences in exfoliated cells of the uterine cervix.

Restriction enzyme digestion and Southern blot hybridization were used to analyze deoxyribonucleic acid (DNA) extracted from exfoliated cervical cells for the presence of human papillomavirus sequences and these results were correlated with cytologic findings on Papanicolaou smears. Specimens (N = 204) were obtained from a nonselected population of women undergoing routine cytologic screening and human papillomavirus DNA sequences were detected in 33 (16%) women. Thirteen smears contained atypical squamous cells, ranging from very mild dysplasia to moderate dysplasia; all showed associated morphologic evidence of human papillomavirus infection characterized by koilocytosis, nuclear enlargement, wrinkling, and hyperchromasia, and human papillomavirus DNA was demonstrable in 12 (92%) smears. Of the remaining 191 samples with normal cytology, 21 (11%) also contained human papillomavirus DNA sequences. Reevaluation of the smears from these women resulted in a revision of the cytologic diagnosis to very mild dysplasia in four cases. These data suggest that human papillomavirus infection occurs more frequently than predicted by cytologic screening.

Proliferative activity in dysplasia, carcinoma in situ and microinvasive carcinoma of the uterine cervix.

Proliferative activity of various human uterine cervical lesions obtained by conization was examined by measuring the proportion of mitotic cells including abnormal mitosis and the nuclear DNA content by a microspectrophotometer. Twelve ratios of mitotic cells calculated in the present study showed a step-wise increase from normal squamous cell epithelium through various grades of dysplasia to carcinoma in situ (CIS). The great difference between moderate and severe dysplasia was observed. All ratios in CIS with bulky outgrowth (CIS(b)) were the highest. The nuclear DNA content in various lesions also indicated the great difference between moderate and severe dysplasias in the DNA histograms. Severe dysplasia had a wider distributed DNA histogram without distinct modes similar to those in CIS and the non-invasive areas of the microinvasive carcinoma. These results may suggest that severe dysplasia but not slight or moderate dysplasia is a direct precursor lesion for uterine cervical epidermoid carcinoma.

[The changes in nuclear DNA amounts in the development and progression of uterine cervical cancer determined by cytofluorometry].

After microsopically-directed sampling of the tissues from various histological sections taken from 16 cases of cervical dysplasia, 6 cases of carcinoma in situ, 11 cases of stage Ib invasive cervical cancer of keratinizing type, 28 cases of large cell non-keratinizing type and 14 cases of small cell type, nuclear DNA levels of the cells dispersed from the tissues were measured by cytofluorometry after Feulgen stain. The DNA levels of cells obtained from normal cervical squamous epithelium and squamous metaplastic epithelium were in 2C(diploid)-4C(tetraploid) regions and those from mild and severe dysplasia were in 2C approximately 4C or high 4C and in low 2C or 2C approximately high 4C or 8C(octaploid) regions respectively with the mode of 2C. In five of 6 cases (83.3%) of carcinoma in situ, the amount of DNA in the neoplastic cells ranged up to the hyperoctaploid region with the mode of 2C or 4C. There were hyperoctaploid cells in 90.9% of cases of the keratinizing type, 96.4% of cases of the large cell non-keratinizing type and 100% of cases of the small cell type. The incidence of hyperoctaploid cells in samples from superficial invasion (stromal invasion less than 3mm) was not different from that of deep invasion (stromal invasion of more than 5mm) in each histological type. When the modal values for nuclear DNA in the superficially invasive lesions were compared with those of the deeply invasive lesions, aneuploidy was more frequently observed in the lesions of deep stromal invasion, irrespective of the histological type. The dominant changes in the mode according to the depth of stromal invasion were 2C to aneuploid in keratinizing type and 4C to aneuploid in cases of large cell non-keratinizing type and small cell type. The results suggest that the hyperoctaploid and aneuploid cells are useful markers for quantitative discrimination among dysplasia, carcinoma in situ and invasive squamous cell carcinoma and that aneuploid stem cells may be generated from 2C and 4C stem cell lines in the progression of stromal invasion.