Telomerase activity, MIB-1, PCNA, HPV 16 and p53 as diagnostic markers for cervical intraepithelial neoplasia.

The present investigation evaluated the relationship between dysplasia of the uterine cervix and telomerase activity, expression of p53, MIB-1 and PCNA. Telomerase activity was measured on cervical cytobrush material from 126 women suspected of having dysplasia and 61 controls using the telomeric repeat amplification protocol. Immunohistochemistry was used to detect the tumor suppressor protein p53 and cell proliferation, the latter by MIB-1 and PCNA expression. Infection with human papillomavirus 16 was detected by PCR amplification and Southern blot hybridization of DNA extracted from the same brush material. Positive telomerase activity was found in 5 of 43 (11.6%) normal samples, 12 of 57 (21.1%) samples with inflammation or koilocytosis, 7 of 17 (41.2%) CIN 1 (cervical intraepithelial neoplasia, grade 1), 8 of 20 (40.0%) CIN 2, and 25 of 42 (59.5%) CIN 3/ CIS. Telomerase activity was significantly related to the level of dysplasia (p<0.001) and proliferation measured by MIB-1 (p=0.019), but not to the level of PCNA (p=0.445), HPV 16 status (p=0.098) or staining for p53 (p=0.271). Dysplasia was also related to PCNA, MIB1, p53, and presence of HPV 16. A sequential increase in the examined parameters, paralleling the progression of abnormality, was observed. PCNA and telomerase showed an increase in CIN 1, MIB-1 and HPV16 in CIN 2, and finally p53 in CIN 3/CIS.

Combined prognostic importance of CA 125, histopathologic grade and DNA-index in advanced ovarian cancer.

PURPOSE: To analyse the possible prognostic importance of a combination of serum CA 125 levels, tumor cell DNA distribution and histopathologic grade in patients with epithelial ovarian cancer. METHOD: The study included 41 patients with ovarian cancer stages II-IV. CA 125 was measured before the start of chemotherapy and after every course. The DNA distribution was analysed by flow cytometry in biopsy specimens from the primary tumour. The histopathologic grading was performed according to WHO criteria. RESULTS: The CA 125 level was of major prognostic significance. All patients with an abnormal value (> 35 U/ml) after three courses died within 16 months. The DNA distribution did not add significant prognostic information, but the histopathologic grade was of significant prognostic importance as investigated by Cox analysis. CONCLUSION: CA 125 is an important prognostic marker in epithelial ovarian cancer. The value after three courses of chemotherapy is significant. The histopathologic grade adds further prognostic information.

Mean nuclear volume: a supplementary prognostic factor in endometrial cancer.

We evaluated the following nine parameters with respect to their prognostic value in females with endometrial cancer: four stereologic parameters [mean nuclear volume (MNV), nuclear volume fraction, nuclear index and mitotic index], the immunohistochemical expression of cancer antigen (CA125) and the postsurgical stage (P-stage), the malignancy grade, the depth of myometrial tumor invasion and the age of the female. Tissue was obtained from 68 consecutive endometrial cancer patients with clinical FIGO stage I tumors who were registered in the Danish endometrial cancer study (DEMCA). The primary operation included total abdominal hysterectomy and bilateral salpingo-oophorectomy. All patients underwent radical operations. The tumors were classified postsurgically as P-stage I-III. All patients received the same postoperative radiation therapy. The surviving patients were observed for a median of 6.4 years (range 5.0-8.4 years). Cox regression analysis (automatic forward selection) showed the MNV to be the most significant prognostic parameter followed by the P-stage. Patients who had localized tumors or tumors with small nuclei had a better probability of surviving than did women with advanced tumors or tumors with large nuclei. No significant relationship between survival and the remaining seven parameters was demonstrated. This first investigation of the prognostic value of the MNV in females with endometrial cancer demonstrates that it may assist the distinction between those patients who can be cured by hysterectomy alone and those who need adjuvant therapy.

The presence of HPV in cervical biopsies, determined by histology and PCR with 2 different pairs of consensus primers.

The link between human Papillomavirus (HPV) and cervical carcinomas has become increasingly convincing recently, and the detection of infection with certain HPV types may become an important element in screening for cervical pre-cancer. We applied the polymerase chain reaction (PCR) technique to detect and type HPV-DNA in biopsies from the uterine cervix with a histological diagnosis of CIN and condylomatosis. Forty-eight consecutive cervical biopsies were analysed for HPV-DNA by PCR, using two different sets of consensus-primers. Typing was performed with HPV type-specific primers. Histological reevaluation revealed that 42 biopsies (87%) had condylomatous features. HPV was demonstrated in 46/48 (95.8%). HPV type 6/11 was found in seven biopsies, while HPV 16 was present in 23 samples, including two cases of double-infection. HPV type 18 was not found at all, and in 19 cases the HPV type present could not be determined. Apparently, HPV-DNA was absent in two cases. The results indicate that HPV-DNA screening is a valuable supplement to histopathological screening for cervical neoplasia. The value of different primer pairs is discussed.

[Vaginal smears on clinical indication in a county with systematized screening]. / Vaginalcytologisk undersøgelse på klinisk indikation i et amt med systematisk screening.

In Denmark PAP-smear screening is performed by the GPs. The objective of this study is to report the proportion af smears taken on clinical indication over an eight week period in the county of Aarhus, and to obtain a precise reason for making a PAP-smear on clinical indication. Some 27% of all smears taken were on clinical indication. Some 55% of these smears were due to a follow-up on previous PAP-smears, while tests due to subjective symptoms account for the major part of the remaining tests. Testing the target group outside the screening programme is discouraged, but some 43% of tests on indication are not due to follow-up of previous smears. Further studies of the consequences of such testing are recommended.

Value of quantitative pathological variables as prognostic factors in advanced ovarian carcinoma.

AIMS: To evaluate correlations among clinical, pathological, morphometric, stereological, and DNA flow cytometric variables and their prognostic value in advanced ovarian cancer. METHODS: Tissue was collected from 180 patients with advanced ovarian cancer. All 180 had undergone debulking surgery and were being treated with cisplatin. Long term follow up was available for all patients. The mitotic activity index (MAI), volume % of epithelium (VPE), mean nuclear area (MNA), standard deviation of the nuclear area (SDNA), estimates of volume weighted mean nuclear volume (nu v), and variables obtained from minimum spanning tree (MST) analysis were assessed in the least differentiated tumour section in each case. DNA flow cytometry was also performed. RESULTS: Quantitative pathological features differed significantly with respect to histological grade. The MAI, MNA, SDNA, and the number of points connected to three neighbours differed significantly among the different DNA ploidy groups. The VPE and number of points connected to two or three neighbours differed significantly between FIGO stages III and IV. Fifty two (29%) patients survived. FIGO stage, residual disease and SDNA had prognostic significance on both univariate and multivariate survival analysis. In patients with FIGO III stage disease and residual tumour nodes < or = 2 cm in diameter (67 patients, 29 (43%) survivors) a prognostic index was established based on SDNA and of the line length of the MST. The median survival time was not reached in a subgroup of patients with favourable prognosis (overall survival 57%). Median survival was 32 months for patients with an unfavourable index score (overall survival 28%). CONCLUSION: Morphometric variables have important additional value in predicting prognosis in patients with advanced ovarian cancer.

The significance of differences in prognostic value of quantitative pathologic features in FIGO stage III and IV serous adenocarcinoma of the ovary between a group of Danish patients and other groups.

Previous studies have shown DNA cytometric, morphometric and stereologic features to be strong prognosticators in advanced ovarian cancer. However, in one Danish study a new stereologic parameter, the volume-weighted mean nuclear volume ( nuv), was not of prognostic significance. In that study, sampling was performed in the whole tumor section, contrary to that in other studies where measurements were performed in the worst differentiated area (the measurement area). The aim of the present investigation was, therefore, to confirm or reject the hypothesis that random estimates of nuclear {ovbar|nu}v over the whole tumor, rather than in the measurement area, are the cause for the lack of prognostic value in the Danish patients. Additionally, the mitotic activity index (MAI), volume percentage of epithelium (VPE), mean nuclear area (MNA) and standard deviation of the nuclear area (SDNA), which have proved to be of strong prognostic significance in advanced ovarian cancer, were assessed in the measurement area. The MAI, VPE, MNA, SDNA and nuclear {ovbar|nu}v were well reproducible, but prognostically not significant. This study thus excludes differences in microscopic sampling as an explanation for the contrasting findings in the Danish study and other ones. Possible remaining explanations are: (i) differences in sampling at the macroscopic level; (ii) fixation differences; (iii) patient selection bias; and (iv) geographic differences in the degree of malignancy of advanced ovarian cancer in Denmark and the Netherlands. As to the latter, in comparison with two Dutch studies the Danish ovarian cancers in this series had residual disease <2 cm (33% vs 50%) less often, were less often well differentiated (4% vs 19%), and less often had favorable morphometric characteristics (6% vs 15%). Further studies are required to analyze whether other Danish patient groups with advanced ovarian cancers differ from the Dutch.

Prediction of lymph node metastases in patients with early squamous cell carcinoma of the cervix uteri by histopathological grading and flow cytometry.

The present study comprises a retrospective investigation of 126 patients with stage IB squamous cell carcinoma of the cervix, and a similar prospective investigation of 53 patients with stage IB and 6 patients with stage IIA disease. Tumour biopsies from these patients were analysed by means of flow cytometry and a semiquantitative histological grading system. The study showed that a combination of a low tumour cell DNA index and a low score value of the grading system indicated a very low risk of regional lymph node metastases (0% lymph node metastases in patients with low scores vs. 24-46% metastases in patients with high scores, P < 0.001). In order to study the reproducibility of the histological grading 20 randomly selected cases were studied blindly by three of the participating pathologists and after discussion of the grading criteria. A kappa coefficient of 722 demonstrated a substantial agreement between the observers. These results suggest that by combining flow cytometry with semiquantitative histological grading, a subgroup of patients with early squamous cell cancer of the cervix uteri may be selected that could be sufficiently treated with simple hysterectomy instead of radical hysterectomy including lymphadenectomy, which, in many oncology centres, is the standard treatment of this patient category.

Nuclear volume and prognosis in ovarian cancer.

The prognostic value of the volume-weighted mean nuclear volume (MNV) was investigated retrospectively in 100 ovarian cancer patients with FIGO-stage IB-II (n = 51) and stage III-IV (n = 49) serous tumors. No association was demonstrated between the MNV and the survival or between the MNV and two of five additional parameters: age and amount of residual tumor. However, both in stage IB-II and stage III-IV carcinomas, the MNV increased significantly with increasing tumor grade and DNA-index. Furthermore, FIGO-stage IB-II patients had significantly smaller nuclei than patients with stage III-IV disease.

DNA level and stereologic estimates of nuclear volume in squamous cell carcinomas of the uterine cervix. A comparative study with analysis of prognostic impact.

Grading of malignancy in squamous cell carcinomas of the uterine cervix is based on qualitative, morphologic examination and suffers from poor reproducibility. Using modern stereology, unbiased estimates of the three-dimensional, volume-weighted mean nuclear volume (nuclear vv), were obtained in pretreatment biopsies from 51 patients treated for cervical cancer in clinical Stages I through III (mean age of 56 years, follow-up period greater than 5 years). In addition, conventional, two-dimensional morphometric estimates of nuclear and mitotic features were obtained. DNA indices (DI) were estimated by flow cytometry. Finally, the semiquantitative malignancy grade score value (MGS) was determined according to previously published methods. Estimates of nuclear vv were on average increased in euploid lesions (2P = 0.01), but the overall relationship between nuclear vv and DI was poor. Different clinical stages of disease did not differ with regard to nuclear vv (2P = 0.99) and DI (2P = 0.56). No relationship was disclosed between MGS and nuclear vv (2P = 0.85). Single-factor analysis showed prognostic impact of clinical stage of disease (2P = 0.0001) and DI (2P = 0.04), whereas estimates of nuclear vv were only of marginal prognostic significance (2P = 0.07). However, Cox multivariate regression analysis showed independent prognostic value of patient age and nuclear vv along with clinical stage and DI. All other investigated variables were rejected from the model. A prognostic index with highly distinguishing capacity between prognostically poor and favorable cases was constructed (2P = 1.9 x 10(-7)). It is concluded that realistic estimates of nuclear volume are independent of nuclear DNA content and are of prognostic value for objective malignancy grading in patients with squamous cell carcinoma of the uterine cervix.

OC-125 as a diagnostic aid in the cytological evaluation of ascitic cells in patients with ovarian carcinoma.

OC-125 is a monoclonal antibody raised against tumor cells from a patient with serous cystadenocarcinoma and reacting with an antigen CA-125 on the surface of ovarian epithelial cancer cells. We investigated whether immunohistochemical determination of CA-125 in cell samples from the peritoneal cavity could be used to discriminate between non-specific inflammatory changes in the mesothelium and malignant ovarian tumour cells. Three categories of patients were investigated: patients with disseminated serous or endometrioid ovarian carcinomas, patients with non-specific inflammatory changes in the peritoneal cavity and patients subjected to simple hysterectomy with no pathological changes in the mesothelium. In all cases of ovarian cancer CA-125 positive cells could be detected in the peritoneal fluid; none of the other categories showed positive reaction for CA-125. The results suggest that OC-125 may be a valuable adjunct in differentiating neoplastic from other cells in ascitic fluid from patients with some types of ovarian cancer.

Stereological estimates of nuclear volume in squamous cell carcinoma of the uterine cervix and its precursors.

Using modern stereology, this study was carried out to obtain base-line data concerning three-dimensional, mean nuclear size in precancerous and invasive lesions of the uterine cervix. Unbiased estimates of the volume-weighted mean nuclear volume (nuclear vv) were obtained by point-sampling of nuclear intercepts in 51 pre-treatment biopsies from patients with invasive squamous cell carcinomas (SCC). Vertical sections from 27 specimens with cervical intraepithelial neoplasia (CIN) grades I through III were also investigated, along with 10 CIN III associated with microinvasion (CIN III + M). On average, nuclear vv was larger in SCC than in CIN III and CIN III + M together (2 P = 8.9 . 10(-5). A conspicuous overlap of nuclear vv existed between all investigated lesional groups. The reproducibility of estimates of nuclear vv in biopsies with SCC was acceptable (r = 0.85 and r = 0.84 in intra- and inter-observer studies, respectively). The efficiency of the sampling scheme was high, with more than 60% and more than 80% of the total observed variance contributed by differences between individual lesions with CIN and SCC, respectively. Estimates of nuclear vv based on sampling within the whole epithelial thickness and on sampling in the lower one-third in CIN I and the lower two-thirds in CIN II lesions were of the same magnitude. Approximate estimates of the absolute variation of nuclear vv were directly proportional to individual estimates of nuclear vv, whereas the relative variation of nuclear vv tended to decrease with increasing mean nuclear volume. Based on the rather small number of cases investigated, estimates of nuclear vv are unable to distinguish between different grades of CIN. However, the estimation of nuclear vv is well-suited for the purposes of objective grading of malignancy in SCC.

[Treatment of cervix cancer in Denmark]. / Behandlingen af cancer cervicis uteri i Danmark.

The current status of cervical cancer treatment in Denmark is discussed. Diagnostic aspects and problems of classification are presented briefly supplemented with a comment on new prognostic parameters based on a semiquantitative score system and flow cytometry. Surgery is the treatment of election for the early stages whereas radiotherapy is the treatment of choice in advanced stages. Chemotherapy should only be employed in the framework of clinical trials. It is concluded that centralised treatment should be maintained.

Is radical hysterectomy always necessary in early cervical cancer?

A semiquantitative histopathologic grading system was used in combination with flow cytometric measurements of tumor cell DNA content to predict the risk of lymph node metastases in early cervical cancer. A retrospective study of 126 stage IB patients showed that a group with no risk of lymph node involvement could be identified by the use of both the histopathologic score and the DNA index. The results were confirmed in a prospective investigation of 59 new patients. Simple hysterectomy may be advised in low-risk patients.

Ploidy level, histopathological differentiation and response to chemotherapy in serious ovarian cancer.

The ploidy level was measured in tumour specimens from 64 patients with advanced epithelial ovarian cancer (FIGO stages III and IV) by flow cytometric analysis. The same tumours were histopathologically graded based on a score system considering eight histopathological parameters. The results showed that both the histopathological grading index (HGI) and the DNA index were important to the frequency of complete pathological remission as assessed by second look operation. A combination of the two parameters allowed a division of the patients into three groups with different frequencies of complete pathological remission and survival. It is concluded that flow cytometric analysis and extended histopathological grading may be important adjuncts in the evaluation of malignant epithelial ovarian tumours.

A new histologic grading index in ovarian carcinoma.

It is generally believed that the degree of differentiation or tumor grade is of prognostic value in human ovarian carcinoma, but the methods of evaluation are subject to considerable variation. The present work investigates the prognostic value of a new Histologic Grading Index (HGI), containing most of the known systems and taking into account both the usual histologic and cytologic features (tumor structure, nuclear polymorphism, nucleolar features, nuclear-cytoplasmic ratio, frequency of mitosis) and the probably more independent parameters (mode of invasion, capsular penetration, and vascular invasion). This study shows that the index correlates significantly with both presence of tumor at second-look operations and survival.

Prognostic influence of ploidy level and histopathologic differentiation in cervical carcinoma stage Ib.

Flow-cytometric DNA analysis and extended histopathologic grading were performed in specimens from 126 patients with squamous cell carcinoma of the uterine cervix stage Ib. Archival material was used for the measurements and the ploidy level was analysed according to the method described by Hedley with some modifications. The histopathologic grading was based on eight well-defined parameters all scored 1-3. The results showed that the ploidy level held significant prognostic information about the 10 year survival according to a division of DNA indices above and below 1.5. Further prognostic information appeared from a combination of DNA index and histopathologic score value. The combination held its prognostic importance in subgroups of patients with different tumour sizes. It is concluded that flow-cytometric analysis and histopathologic grading can identify subsets of patients who need more aggressive treatment.

Benign nevus cells in the lymph nodes. An immunohistochemical study.

Aggregates of nevus cells in the axillary lymph nodes may give rise to a suspicion of metastatic breast cancer. Usually the nevus cells are confined to the capsule or the trabeculae, but in the present case, clusters of nevus cells were also observed in the peripheral sinus. Immunohistochemical analysis for S-100 protein and epithelial membrane antigen established the true nature of the cells.