W. Meier

20q13 and cyclin D1 in ovarian carcinomas. Analysis by fluorescence in situ hybridization

In ovarian carcinomas, alterations of the chromosomal region 20q13 and the cyclin D1 gene have been described. This study has sought to determine their prognostic significance. Fluorescence in situ hybridization (FISH) on dissociated nuclei and paraffin sections with DNA probes for 20q13.2 and cyclin D1, as well as immunohistochemistry (cyclin D1), were applied to formalin‐fixed tissue of 69 invasive ovarian carcinomas, mainly of serous type. On dissociated nuclei 33/47 cases (70%) and on tissue sections 13/66 cases (20%) demonstrated an increase of 20q13.2 copies. The presence of ≥4 copies per nucleus (isolated nuclei) and ≥3 copies per nucleus (sections) was associated with an adverse prognosis (Kaplan–Meier for FIGO stage III after stratification for residual tumour: p=0.0049 and p=0.03, respectively). Thirty‐four out of 47 cases (72%) showed an increase of cyclin D1 copies. Kaplan–Meier analysis for FIGO stage III after stratification for residual tumour>2u2009cm or ≤2u2009cm revealed an unfavourable outcome for cases with more than two cyclin D1 copies (p=0.04). No correlation was seen between FISH and immunohistochemistry. Multivariate analysis identified residual tumour (p=0.0002), 20q13.2 gain (p=0.0004) and cyclin D1 gain (p=0.0343) as independent prognostic factors. It is concluded that gains of chromosomal region 20q13.2 and the cyclin D1 gene are frequent and biologically important events, with prognostic relevance, in advanced ovarian carcinomas. Copyright

Immunohistochemical Analysis of Drug Resistanceassociated Proteins in Ovarian Carcinomas

Loss of function of the tumor suppressor gene p53, increased expression of glutathione-S-transferase pi (GST7pi) and the major vault protein are involved in drug resistance of ovarian carcinomas. However, a study comparing these factors has not yet been performed. Therefore, paraffin-embedded material of 213 ovarian tumors with well-documented follow-up was used for immunohistochemical analysis of p53 protein, GSTpi, and major vault protein (antibodies LRP-56, LMR-5). Forty-six percent of the cases showed nuclear p53 accumulation. Strong immunoreactivity for GSTpi, LRP-56, and LMR-5 was seen in 50%, 36%, and 47%, respectively. p53 positivity was most often found in serous carcinomas (p < 0.05). Strong GSTpi expression was the only factor that correlated with clinical resistance to chemotherapy (p = 0.04). In the whole group, as well as in FIGO III cases stratified for residual disease < or = and >2 cm, p53 and GSTpi correlated with an adverse outcome (p = 0.01 for p53 and p = 0.04 for GSTpi). Strong LRP-56 or LMR-5 staining was associated with a tendency towards poorer prognosis, without reaching statistical significance. In multivariate analysis for FIGO III, only residual disease and p53 proved to be independent prognostic factors. Our observations confirm the prognostic significance of p53 accumulation in ovarian carcinomas. Only GSTpi immunoreactivity was significantly correlated with drug resistance.

Experiences with SCC antigen, a new tumor marker for cervical carcinoma

Squamous cell carcinoma (SCC) antigen was first described by Kato et al. in patients with carcinoma of the cervix uteri. SCC serum levels can be measured with a radioimmunoassay. In our investigation, 2.0 ng/ml was taken as the upper limit of the standard range. In 35 healthy women there were no elevated SCC serum levels. Eight of 40 patients with breast, endometrial and ovarian cancer had raised SCC levels. In only two of 12 patients with benign gynecological diseases, SCC was also elevated. Sixty per cent of the patients with primary and 73% of the patients with recurrent cervical cancer showed pathological values; CEA was elevated in 31% and 51% respectively. The absolute values increased with the stage of the disease. Sixty-nine per cent of patients with squamous cell carcinoma had elevated levels. In five of nine adenosquamous carcinomas SCC was pathological. SCC shows a high sensitivity for squamous cell carcinomas of the cervix uteri. The tumor marker might be helpful in the control of primary therapy and follow-up of cervical cancer patients.

CA-125 in gynecological malignancies

CA-125 is an antigenic determinant that can be demonstrated in the majority of epithelial ovarian carcinomas. It can be measured in the serum with a radioimmunoassay by means of a monoclonal antibody. The tumor marker has a low specificity but high sensitivity for ovarian cancer, especially for serous cystadenocarcinoma. In our investigation we were interested in particular in the correlation between CA-125 and the histological findings at second-look operation. In 22 patients, second-look was performed after 6 cycles of chemotherapy, in 16 patients active tumor was demonstrated. In 6 patients with negative CA-125 values, residual tumor less than 1 cm was demonstrated. In order to verify a complete remission, a second-look operation has to be performed. No false-positive CA-125 levels were found. In all patients with elevated CA-125 serum values, residual tumor was histologically confirmed at second look.

Clinical significance of the tumour markers CA 125 II and CA 72-4 in ovarian carcinoma.

In a retrospective study we compared the usefulness of the tumour marker CA 72‐4 with the established marker CA 125 II (both EIA on Cobas‐Core, Hoffmann LaRoche, Basel Switzerland) at the time of primary diagnosis of ovarian carcinoma (n = 123) in order to discriminate between ovarian carcinomas of different histological type. We compared their diagnostic value, behaviour in follow‐up care and evaluated possible combinations. Fixing specificity at 95% vs. benign gynaecological diseases (n = 37) as the clinically relevant reference group, we found cut‐off values of 160 U/mL for CA 125 II and 3.0 U/mL for CA 72‐4. On the basis of this specificity, we found comparable sensitivity for CA 125 II and CA 72‐4 for all kinds of ovarian carcinoma at the time of primary diagnosis. With regard to histology, we found best sensitivity for CA 125 II in serous ovarian cancer and for CA 72‐4 in mucinous ovarian cancer. Additional sensitivities were found in ovarian carcinoma in general but little in serous ones. No additive sensitivity was found in mucinous ovarian carcinomas with CA 72‐4 as leading marker. In follow‐up care, CA 72‐4 was the leading marker in II cases and CA 125 II in 16, while in one case both markers were negative. In 6 cases the change of values reflecting clinical follow‐up‐care was within the so‐called reference range. According to our results, at the time of primary diagnosis because of lack of histological findings the combined determination of CA 125 II and CA 72‐4 can be recommended. In follow‐up care and control of efficacy of therapy the preoperative positive or leading marker is generally sufficient. The determination of both markers in follow‐up care is indicated only if they both are negative at primary diagnosis and until one of them becomes clearly positive.

Squamous cell carcinoma antigen and carcinoembryonic antigen levels as prognostic factors for the response of cervical carcinoma to chemotherapy

Between January 1986 and December 1988, 36 patients with primary advanced or recurrent cervical carcinoma were treated with cytostatic drugs in our department. Treatment at first was a combination of cisplatin and etoposide. After August 1987, a combination of carboplatin and ifosfamide was used. In all patients showing primary response to therapy, the squamous cell carcinoma antigen (SCC) and carcinoembryonic antigen (CEA) levels fell rapidly to normal after one or two cycles. In contrast, clinical remission was not obtained in those patients with levels which remained high or rose again following an initial decrease. Chemotherapy is often the only available therapy for advanced cervical carcinoma or recurrent disease, although the results of treatment, especially in squamous cell carcinoma, remain poor. The course of the SCC or CEA levels can help to decide whether the patient would profit from a continuation of the therapy. With the tumor markers, treatment can be individualized so that, above all, cases of therapy failure or further tumor progression can be detected early and the patient can be spared the severe side effects of the treatment.

DNA ploidy and MYC DNA amplification in ovarian carcinomas

There is increasing evidence that DNA ploidy is a prognostic factor in ovarian carcinomas, but it is uncertain whether MYC DNA amplification is an epiphenomenon of DNA nondiploidy or a distinct biological change with an impact on the clinical course of the disease. To clarify these issues we analysed DNA ploidy by flow and image cytometry and MYC copy number by polymerase chain reaction in archival material from ovarian carcinomas with known follow up. The results were compared with proliferative activity (Ki67 index) and p53 and bcl-2 expression. DNA cytometry revealed nondiploidy in 84 of 144 cases (58.3%). Nondiploidy was statistically significantly correlated with histological tumour type, histological grade, Ki67 index >10%, FIGO stage, presence of residual tumour after debulking surgery and adverse postoperative outcome. Furthermore, DNA nondiploidy was associated with p53 accumulation. We found that 84.9% of the p53-positive cases were nondiploid. This points to the paramount importance of wild type p53 for the maintenance of genome integrity in this tumour type. MYC DNA amplification was seen in 33.8% (26/77 cases) of ovarian carcinoma. There was no correlation between MYC DNA amplification and histological tumour type, histological grade, FIGO stage, DNA ploidy, proliferative activity or prognosis. However, when p53 and bcl-2 expression was taken into account, a statistically significant correlation between gene alteration or expression patterns and histological tumour type was revealed. The group of mucinous carcinomas demonstrated both MYC DNA amplification and strong bcl-2 expression in 50% and contained the largest fraction of cases without aberration (37.5%). Endometrioid carcinomas were characterized by strong bcl-2 expression in 85%, whereas serous and undifferentiated carcinomas predominantly exhibited p53 alterations, frequently accompanied by bcl-2 overexpression or MYC DNA amplification. Thus, in interaction with other genes MYC DNA amplification may play a role in the determination of the varying differentiation patterns of ovarian carcinomas.

Serum levels of CA 125 and histological findings at second-look laparotomy in ovarian carcinoma.

In a prospective study, the serum levels of CA 125 were estimated at regular intervals in 139 patients with ovarian carcinoma. Seventy-two of 78 patients with a second-look laparotomy had elevated CA 125 levels initially. The main aim of our investigation was the correlation of CA 125 levels with the histological findings at second-look laparotomy. A total of 26 patients were free from tumor. In each case CA 125 lay within the normal range. From the 46 patients where residual tumor was found, CA 125 levels were elevated in 23 cases, so that in 23 women with residual tumor, false negative levels were found. There were no false positive CA 125 levels. In all women with raised tumor marker levels at the time of the second-look laparotomy, tumor was found despite the often negative clinical or technical preoperative screening. A negative tumor marker at the time of the second look does not exclude residual tumor. For histological proof of complete remission, a second-look operation is imperative. If the CA 125 level is raised, the relevance of the planned second-look laparotomy is open to discussion.

CA 12–5 serum levels and histological findings at second look in patients with ovarian cancer

Ce l l u l a r p r o l i f e r a t i o n i s potent ly st imulated by a fami ly of hormonally act ive polypept ides, the growth fac tors . Growth f_actors have the po ten t ia l to induce c e l l u l a r t ranstormat ion i f they act at the wrong time or in the wrong place. Factors inducing c e l l t ransformat ion have been shown to be a r e l a t i v e to epidermal growth fac to r (EGF) and in te rac t with c e l l u ] a r receptors .f_or EGF (EGF Like f ac to r s ) , lhe aim o• t h i s study was to inves t iga te ovarian carcinomas f o r the presence o• EGF l i k e fac to rs (EGF-F) in co r re la t i on to c l i n i c a l parameters. Specimens of ovarian carcinomas and nonmalignant t i ssues were ext rac ted with 1 M acet ic acid, centri• end the supernmten ts analyzed • the presence o• EGF F by a EGF radio receptorassay, lhe ~actor content i s expressed as EGF competing a c t i v i t y in n~ EGF units/mg pro te in . In ex t rac ts of nonmalignant t i ssues, i . e . normal ovar ies and myometrJum, and ovarian carcinomas EGF-F could be detected. However, the fac to r contents o f the d i f f e r e n t ex t rac ts var ied w ide l y . . I n nonmalignant t issues fac to r leve ls did not exceed 6 ng EGF units/mg. 18/42 ovarian carcinomas contained high fac to r concentrat ion between 6-17 EGF units/mg. Pat ients were separated in two groups with low (<6 ng) and high (>6 n~) EGF-F t i ssue leve ls . Both groups were cor re la ted with c l i n i c a l date. No d i f fe rences were noticed to h i s t o l o g i c a l subtype and steroidhormonereceptorstatus. Low res idua l tumorrest (< 2 cm) a f t e r primary surgery was found in 11/20 cases with low • content compared to 4/15 cases with high • content. The responserate to a c is-p lat inum combinat ion chemotherapy in the group with low fac to r concent r a t i o n was: 4/21 progressive disease (PD) 3/21 no change (n.c. ) and 14/21 remission (CR+PR). The resu l t s in the group with high fac to r concentrat ion were: 9/24 PD, 5/14 n.c. and 0/14 CR+PR. Dit• in the su rv i va l t ime of both groups were also not iced. However, the case number is too low f o r de ta i led s t a t i s t i c a l analysis, lhese results let assume that the bioIooicaI be havieur of ovarian carcinomas couId be influenced by its content of EGF like grovrch factors.

Lymphocytensubpopulationen gynäkologischer Malignompatientinnen in Korrelation zu bekannten Prognosefaktoren und als Verlaufsparameter unter Cytokintherapie

Es ist bekannt, das bei Malignompatienten eine enge Wechselwirkung zwischen Tumor und Immunsystem besteht, die sich unter anderem auch in der veranderten Zusammensetzung von Leukozyten- und Lymphozytenuntergruppen (1) zeigt. Unsere Arbeit hatte zwei Zielsetzungen: Zum einen sollte gezeigt werden, inwieweit diese Untergruppen sich durch Tumorwachstum verandern. Zum anderen wurde untersucht, welche Auswirkungen eine Therapie mit Endoxan und Interleukin-2 bei austherapierten Ovarialkarzinompatientinnen auf die Verteilung der Lymphozytensubpopulationen hat.