BRCA1-like profile is not significantly associated with survival benefit of non-myeloablative intensified chemotherapy in the GAIN randomized controlled trial.

PURPOSE: The BRCA1-like profile identifies tumors with a defect in homologous recombination due to inactivation of BRCA1. This profile has been shown to predict which stage III breast cancer patients benefit from myeloablative, DNA double-strand-break-inducing chemotherapy. We tested the predictive potential of the BRCA1-like profile for adjuvant non-myeloablative, intensified dose-dense chemotherapy in the GAIN trial. METHODS: Lymph node positive breast cancer patients were randomized to 3 × 3 dose-dense cycles of intensified epirubicin, paclitaxel, and cyclophosphamide (ETC) or 4 cycles concurrent epirubicin and cyclophosphamide followed by 10 cycles of weekly paclitaxel combined with 4 cycles capecitabine (EC-TX). Only triple negative breast cancer patients (TNBC) for whom tissue was available were included in these planned analyses. BRCA1-like or non-BRCA1-like copy number profiles were derived from low coverage sequencing data. RESULTS: 119 out of 163 TNBC patients (73%) had a BRCA1-like profile. After median follow-up of 83 months, disease free survival (DFS) was not significantly different between BRCA1-like and non-BRCA1-like patients [adjusted hazard ratio (adj.HR) 1.02; 95% confidence interval (CI) 0.55-1.86], neither was overall survival (OS; adj.HR 1.26; 95% CI 0.58-2.71). When split by BRCA1-like status, DFS and OS were not significantly different between treatments. However, EC-TX seemed to result in a trend to an improvement in DFS in patients with a BRCA1-like tumor, while the reverse accounted for ETC treatment in patients with a non-BRCA1-like tumor (p for interaction = 0.094). CONCLUSIONS: The BRCA1-like profile is not associated with survival benefit for a non-myeloablative, intensified regimen in this study population. Considering the limited cohort size, capecitabine might have additional benefit for TNBC patients.

Alternative splicing of p53 and p73: the novel p53 splice variant p53delta is an independent prognostic marker in ovarian cancer.

Similar to p73, the tumor suppressor gene p53 is subject to alternative splicing. Besides p53DeltaE6 and p53beta, we identified p53zeta, p53delta and p53varepsilon, arising from alternative splicing of exon 6 and intron 9, respectively. p53 splice variants were present in 18 of 34 ovarian cancer cell lines (52.9%) and 134 of 245 primary ovarian cancers (54.7%). p53delta expression was associated with impaired response to primary platinum-based chemotherapy (P=0.032). Also, p53delta expression constituted an independent prognostic marker for recurrence-free and overall survival (hazard ratio 1.854, 95% confidence interval 1.121-3.065, P=0.016; and hazard ratio 1.937, 95% confidence interval 1.177-3.186, P=0.009, respectively). p53beta expression was associated with adverse clinicopathologic markers, that is, serous and poorly differentiated cancers (P=0.002 and P=0.008, respectively), and correlated with worse recurrence-free survival in patients exhibiting functionally active p53 (P=0.049). DeltaN'p73 constituted the main N-terminally truncated p73 isoform and was preferentially found in ovarian cancer cell lines showing functionally active p53, supporting our hypothesis that N-terminally truncated p73 isoforms can alleviate the selection pressure for p53 mutations by the inhibition of p53 protein function.

Characterization of a human carcinosarcoma cell line of the ovary established after in vivo change of histologic differentiation.

OBJECTIVES: Cell lines are valuable in vitro models for clinical and basic research. Most ovarian cancer cell lines described are serous cystadenocarcinomas or poorly differentiated adenocarcinomas. The establishment of ovarian cancer cell lines with rare histologic differentiation is especially of interest. We describe the establishment of a carcinosarcoma cell line of the ovary after in vivo selection. METHODS: The cell line OV-MZ-22 was established from a solid tumor mass in the upper abdomen. At the time of establishment, the patient underwent secondary debulking and was pretreated with six cycles of cis-platinum/epirubicin/cyclophosphamide. Features of the cell line studied included morphology, ultrastructure, heterotransplantation, chromosome analysis, and analysis of intermediate filament proteins and actins by immunocytochemistry. RESULTS: The first histologic report of the patient described a papillary cystadenocarcinoma, which changed to a carcinosarcoma with predominantly sarcomatous differentiation at secondary debulking. This cell line is aneuploid and shows no expression of the tumor-associated antigens CA-125 and CEA, but an overexpression of MDR-1, lung resistance protein, p53, and topoisomerase I and II, but not of multidrug-resistance-associated protein. The cell line did not give rise to transplant tumors in nude mice. The histologic and immunocytochemical comparison of the primary and the relapsed tumor proved evidence of an in vivo change of differentiation from predominantly papillary cystadenocarcinoma to carcinosarcoma. Morphological characteristics and intermediate filament pattern underlined the sarcomatous differentiation and origin of this cell line. The differentiation phenotype of OV-MZ-22 cells is that of smooth-muscle cells. CONCLUSION: The change of histologic differentiation was apparently due to a selection process caused by platinum-containing chemotherapy. The origin of the cell line and its rarity make this new line an appropriate tool for further investigation.

Soluble CD44 splice variants and pelvic lymph node metastasis in ovarian cancer patients.

Alternative splicing of CD44 and aberrant levels of soluble CD44 protein in the serum of cancer patients has been correlated to tumor progression and metastasis. To examine the clinical value of CD44 serum levels (sCD44) in ovarian cancer we determined concentrations of the soluble, variable isoforms sCD44std, sCD44v5 and sCD44v6 with a sensitive ELISA. Pre-operative serum samples from 66 patients with histologically diagnosed invasive disease as well as sera taken from 40 healthy blood donors were analyzed. In sera of ovarian cancer patients we detected elevated concentrations of overall CD44 serum levels represented by sCD44std (p=0.001), but decreased levels of the specific isoforms CD44v5 (p=0.0002) and v6 (p=0.0001). This is the first report demonstrating that ovarian cancer patients with pelvic lymph node metastasis at the time of diagnosis showed specifically elevated sCD44v6 (p=0.073) serum concentrations in comparison to patients without lymph node involvement, whereas overall sCD44 serum levels did not differ. Decreased serum levels of sCD44v5 were found in progesterone receptor-positive tumors (p=0. 059) and postmenopausal patients (p=0.032). Increased concentrations of sCD44v6 were detectable in estrogen receptor-positive tumors but not significantly (p=0.138). Serum CD44v5 levels were associated with shortened relapse-free survival time. No association was found between serum CD44 isoforms and the classical clinicopathological parameters stage and grading or overall survival. CD44 splice variants are possibly involved in a complex interaction with the hormonal environment during tumorigenesis and metastasis of ovarian cancer.

Telomere length in human white blood cells remains constant with age and is shorter in breast cancer patients.

BACKGROUND: Telomeres, which are TTAGGG repeats at the end of the eukaryotic chromosome, are essential for complete DNA replication. Telomere length has been reported to decrease in peripheral WBC, unlike the telomerase activity found in these cells. The purpose of this study was to investigate whether telomere length in WBC is indeed age dependent and could serve as a genetic marker in breast or ovarian cancer. METHODS: Five age groups: 20-29; 30-39; 4049; 50-59 and > or = 60 years were examined. The cancer patients were 18 women with ovarian cancer and 18 women with breast cancer. Southern blot analysis of the DNA from peripheral white blood cells (WBC) was performed using 32P-labeled (TTAGGG)3 probe. Blots were scanned in a phosphoimager and analyzed by computer-assisted image analysis. RESULTS: No statistically significant correlation was observed between telomere length and age in either healthy females or cancer patients. However, significantly shorter median telomere length was found in WBC obtained from breast cancer patients as compared to healthy individuals and ovarian cancer patients. CONCLUSIONS: It is concluded that telomere length in WBC is not age dependent, but is significantly shorter in breast cancer patients.

Differential characteristics of two new tumorigenic cell lines of human breast carcinoma origin.

Permanent human tumor cell lines are an important tool for the study of breast cancer. Two new breast cancer cell lines (BrCa-MZ-01 and BrCa-MZ-02) were isolated from a solid tumor and a pleural effusion, respectively. One cell line was established from a medullary carcinoma, the other from a ductal carcinoma. These cells exhibit ultrastructural and immunohistochemical features of epithelial cells of mammary origin. Intermediate filament and cytokeratin typing showed a clear predominance of the simple-epithelial cytokeratins CK 8, CK 18 and CK 19, although the expression was reduced in comparison to the hormone receptor-positive reference cell lines MCF-7 and ZR-75-1. Both cell lines produced slow-growing tumors after subcutaneous (s.c.) transplantation of 1 x 10(7) viable tumor cells into nude mice. The cell line BrCa-MZ-01 expresses the estrogen and progesterone receptor, whereas the cell line BrCa-MZ-02 remains negative. Both cell lines are positive for secretion of platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta), whereas interleukin-6 (IL-6) is only secreted by the cell line BrCa-MZ-02.

Expression of CD44 standard and variant isoforms v5, v6 and v7 in human ovarian cancer cell lines.

The expression of standard CD44 protein (CD44std) and its splice variants v5, v6 and v7 was investigated in 43 human ovarian carcinoma cell lines by flow cytometry and immunocytochemistry using monoclonal antibodies raised against extracellular epitopes. Twenty six (60%) cell lines expressed CD44 std. Variant isoforms of CD44 were expressed in 12 of the 26 CD44 positive cell lines. All 12 cell lines expressed CD44 v5. In addition 6 cell lines expressed CD44 v6 and one of these expressed CD44 v7 simultaneously. No significant differences of CD44 expression were found between cell lines derived from solid tumors or ascites. In ovarian cancer cells splicing of CD44 v5 appears to be a prerequisite for expression of downstream variable exons. New acquisition of variable CD44 exons may be implicated in the tumorigenesis of ovarian cancer. The CD44 gene provides a biological model to study the role of alternative splicing in gynecologic malignancy.

Intron 9 retention in gene transcripts suggests involvement of CD44 in the tumorigenesis of ovarian cancer.

Increased or de novo expression of certain CD44 variant isofoms as well as abnormal processing of pre-mRNA of the CD44 gene have been found in different malignant human tumors. Retention of the non-coding intron 9 sequence in CD44 mRNA especially, appears to be a discriminating aberrant splicing pattern in several cancer tissues and cells. We examined the expression of the CD44 intron 9 sequence in the mRNA of 45 permanent ovarian cancer cell lines and in normal ovarian tissue. Abnormal retention of intron 9 was found in 19 (60%) cell lines expressing CD44 std. Normal ovarian tissue as well as all ovarian cancer cells without CD44 s t d expression were found to be negative for intron 9 retention. Our results indicate that besides newly acquired expression of certain CD44 isoforms abnormal retention of the non-coding intron 9 sequence in CD44 gene transcripts is also a common and maybe crucial event in the tumorigenesis of ovarian cancer. Since intron 9 is not retained in normal ovarian tissue this aberration might serve as a marker for human adenocarcinoma of the ovary.

Chicken ovalbumin upstream promoter--transcription factor (COUP-TF) expression in human endometrial cancer cell lines.

COUP-TF is an orphan member of the steroid receptor superfamily. COUP-TF down-regulates hormonal induction by other steroid receptors involved in cell proliferation and differentiation. Previous study has suggested a role in gynecological adenocarcinoma. In the present study we evaluated COUP-TF expression in endometrial cancer. Fourteen permanent endometrial cancer cell lines were established front the primary site of 14 endometrial cancer patients. Immunocytochemistry for COUP-TF-like activity was performed using an affinity selected polyclonal rabbit-derived antibody in an immunoperoxidase staining technique. The staining intensity and cell surface area were quantified by image analysis. By immunostain 2 cell lines were COUP-TF (+), 6 (+ +) and 6 (+ + +). Quantitative differences in staining intensity and cell surface area were not significant in these groups. All cell lines tested were immunocytochemically negative for estrogen and progesterone receptors. COUP-TF is a new factor involved in endometrial cancer cell differentiation and growth, especially in estrogen receptor negative tumors.

Long-term results after operative correction of vaginal aplasia.

OBJECTIVE: We evaluated the long-term results after operative correction of vaginal aplasia with special interest in sexual and psychologic rehabilitation. STUDY DESIGN: Forty-four patients who had undergone surgical construction of an artificial vagina took part in follow-up. The operative result was assessed by a gynecologic examination. A structured interview and five questionnaires allowed an evaluation of psychosexual rehabilitation. RESULTS: The measured length of the vagina ranged between 3.5 and 15 cm. Unimpaired sexual intercourse could be ensured in a vagina with a minimum length of > or = 6 cm. The early onset of regular sexual intercourse played an important role for the long-term success of the operation. Patients were considered for an operation if they had a steady partner and hence could guarantee early and regular coitus. This concept was substantiated in our follow-up investigation. The increase in self-confidence thanks to sexual rehabilitation was striking. In a preoperative and postoperative survey 27 of the 44 patients displayed an impressive increase in self-confidence. However, infertility still posed a problem after successful surgical intervention. The overwhelming majority of patients were still depressed about infertility many years after the operation. One way to solve this problem may be adoption. CONCLUSIONS: After surgery 82% of patients achieved a functional satisfactory postoperative result combined with overall psychosexual rehabilitation. An intensification of medical and psychologic care after discharge could further improve the long-term results. Special support is needed to solve the problem of infertility.

Subcellular localization of accumulated p53 in ovarian cancer cells.

Inactivation of the tumor suppressor gene p53 is frequently associated with ovarian cancer. Accumulation of stabilized p53 protein is a common feature in this tumor type. Underlying mutations in the p53 core region can lead to loss of the normal conformational state or loss of residues necessary for DNA binding and transcriptional regulation. Five HPV-free ovarian cancer cell lines established in our laboratory with and without immunocytochemically detectable p53 expression were selected for the correlation of subcellular localization of aberrant p53 and the type of gene mutation. The expression level regarding staining intensity and proportion of cells accumulating p53 was characterized employing an immunoreactive score. Two cell lines with point missense mutations in the core region showed strong nuclear or nuclear plus cytoplasmic staining. One cell line with exclusive staining of the cytoplasm contained a deletion of the major nuclear localization signal. Among two cell lines without p53 accumulation, one contained a microdeletion resulting in a frame shift, the other carried the wild-type sequence. The MDM2 oncogene was not amplified and its gene product was not overexpressed. In ovarian cancer, inactivated p53 can accumulate in both major cell compartments depending on the type of the underlying mutation.

[Prognostic differentiation of ovarian carcinomas by immunohistochemical analysis of estrogen receptor expression]. / Die prognostische Differenzierung von Ovarialkarzinomen durch immunhistochemische Analyse der Ostrogenrezeptorexpression.

Analogous to breast cancer, the expression of estrogen receptors (ER) by human ovarian cancers is thought to be of prognostic significance. This prospective study was designed to evaluate the ability of immunohistochemistry (IHC) for ER to define prognostic subgroups in this disease and to possible characterize the group of patients that would benefit from ER analysis. The IHC technique for localising ER was published by Press and Green (1984). In IHC the percentage of cells stained was determined as well as the staining intensity that was graded on a 4 step scale. Tissue from the primary focus of 61 ovarian cancers was sampled during surgery and frozen immediately. FIGO Stage 1 disease was observed in 7 patients, stage 2 in 2, stage 3 in 36 and stage 4 in 16 women. Tumours of histological grade 1 were found in 3 cases, grade 2 in 20 and grade 3 in 38 specimen. Serous carcinoma was diagnosed in 43 lesions, mixed cell types in 10, mucinous and endometrioid each in 4. Minimal follow-up was 8 years. Statistical analysis was performed using the Wilcoxon Rank and Log-Rank test of Kaplan Meier curves. The median survival was 10.62 +/- 1.83 years for stage 1, 5.83 +/- 0.39 years for stage 2, 2.38 +/- 0.39 years for stage 3, and 3.71 +/- 0.76 years for stage 4. Tumour stage (p < 0.05), grade (p < 0.05) and cell type (p < 0.01) were predictors of survival. Age was of borderline significance (p = 0.058). The relative amount of stained cells was no predictor of prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)

[An unusual case: vaginal foreign body (forceps) as an incidental finding during primary operation for breast cancer]. / Der besondere Fall: Vaginaler Fremdkörper (Zange) als Zufallsbefund anlässlich der Primäroperation eines Mammakarzinomes.

A 29 year-old patient was admitted for surgery because of two breast lumps. One of these was suspicious for malignancy by a mammogram and palpation. When her history was taken, the patient mentioned putrid vaginal discharge and denied any previous gynaecologic examination. After initial hesitancy, the patient agreed to have a pelvic examination. This revealed an oxydized iron rod protruding 2 cm out of the vagina. The object was identified by x-ray examination as one handle of iron forceps often used for cutting metal wires. The patient had been carrying this foreign body for fifteen years. She was not willing to relate any information about the cause of the manipulation or any eventual culprit. She was consistent and her personality was dominated by introverted behaviour. The foreign body was removed under the same anaesthesia as that used for the operation on the breast cancer.

p53 mutant His175 identified in a newly established fallopian tube carcinoma cell line secreting interleukin 6.

Fallopian tube carcinoma is a lethal gynecologic malignancy. Etiologic factors are unknown. No experimental data on molecular alterations exist so far. For an in vitro model, we established the permanent human tubal carcinoma cell line FT-MZ-1. The median doubling time was 14 days with 24.2% in S phase. A point missense mutation of the p53 tumor suppressor gene resulting in the His175 mutant was identified. Aberrant p53 protein accumulated in nucleus and cytoplasm. FT-MZ-1 substantially secreted interleukin 6 (Il-6) coinciding with the inactivation of p53 as a transrepressor on the Il-6 gene promoter.

Establishment of new ovarian and colon carcinoma cell lines: differentiation is only possible by cytokeratin analysis.

Two human ovarian (OV-MZ-10, OV-MZ-15) and two colon cancer cell lines (CO-MZ-5, CO-MZ-6) were newly established in permanent cell culture. These cell lines have been maintained in vitro for 5-6 years, the passage number varying from 25 to 228. They were established from ascites or solid tumours at the time of primary surgery. By clinical and histopathological judgement alone all four cell lines would have been interpreted as ovarian cancer cell lines. Morphological criteria or the expression of the tumour-associated antigens CA-125 and CEA allowed no differential diagnosis. Only the analysis of the expression of different cytokeratins and vimentin enabled us to verify the different origin of the cell lines. Ovarian cancer cell lines, in contrast to the colon cancer cell lines, are positive for the expression of cytokeratin (CK) 7 and for vimentin. CK 20 proved to be the marker with the best discrimination. CK 20 was found exclusively in the colon carcinoma cell lines, but not in the ovarian carcinoma cell lines. The evaluation of cytokeratin expression is a helpful diagnostic modality in differentiating between adenocarcinoma cell lines derived from ovarian and colon tumours.

Prognostic significance of estrogen receptor expression in ovarian cancer. Immunoreactive Score (IRS) vs. Composition Adjusted Receptor Level (CARL).

An Immunoreactive Score (IRS) was compared to the Composition Adjusted Receptor Level (CARL) evaluating prognostic significance of estrogen receptor (ER) expression in 61 ovarian cancers after > or = 8 years. CARL of ER allowed for calculating individual risk curves in stage III and IV, Grade 2 and 3, serous ovarian carcinoma after surgical debulking to < or = 2 cm residual and platinum based chemotherapy. In overall analysis and in subsets defined by tumor stage, grade or a combination thereof significant prognostic distinction became possible using a cutoff IRS of 4 vs. 0 or vs. scores < or = 3. This study substantiates ER as a prognostic variable in human ovarian cancer and for the first time establishes the value of an IRS.

Effects of interferon gamma on the proliferation and modulation of cell-surface structures of human ovarian carcinoma cell lines.

Platinum-containing regimens are very effective in the primary treatment of ovarian cancer. However, upon subsequent treatment most tumors develop multidrug resistance. The clinical application of biological response modifiers like interferon gamma (IFN gamma) in advanced ovarian cancer is therefore of increasing interest. Permanent ovarian cancer cell lines are suitable for investigating the mode of action and the potential clinical effectiveness of such response modifiers. IFN gamma is known to modulate many cellular functions. In this study it was compared for its antiproliferative and antigen-modulatory activity on the expression of tumor-associated (CA-125, HMFG, CEA) and major histocompatibility complex (MHC) class I and II antigens as well as of the epidermal growth factor (EGF) receptor on 20 newly established human ovarian carcinoma cell lines. IFN gamma in concentrations of 10, 50 and 100 U/ml was used to study its antigen-modulatory effect, and at additional 1 U/ml and 1000 U/ml to assess its antiproliferative effect on the cells. The cells were incubated with IFN for 4 days. Two cell lines showed strong antiproliferative activity even at minimal doses (up to 50 U/ml). Intermediate growth inhibition between 34% and 84% was observed in 15 cell lines with higher doses. Three lines were resistant to IFN gamma. Independent of the antiproliferative effect, IFN gamma enhanced the expression of MHC class I and MHC class II in nearly all cell lines. Upregulation was also observed for most of the tumor-associated antigens (TAA) and EGF receptor expression. A down-regulation was noticed but rarely. The fact that IFN gamma showed an antiproliferative activity on the majority of the cell lines is of clinical relevance. The in vitro modulation of cell-surface determinants by IFN gamma warrants special attention. The enhanced expression of TAA and MHC antigens can improve immunogenicity of the tumor cells and may explain the therapeutic effects observed under IFN therapy in ovarian cancer. By contrast, enhanced expression of the EGF receptor, often associated with poor patient survival rates, may be an undesirable side-effect of IFN therapy.