Berno Tanner

Surgery in Recurrent Ovarian Cancer: The Arbeitsgemeinschaft Gynaekologische Onkologie (AGO) DESKTOP OVAR Trial

BackgroundThe role of cytoreductive surgery in relapsed ovarian cancer is not clearly defined. Therefore, patient selection remains arbitrary and depends on the center’s preference rather than on established selection criteria. The Descriptive Evaluation of preoperative Selection KriTeria for OPerability in recurrent OVARian cancer (DESKTOP OVAR) trial was undertaken to form a hypothesis for a panel of criteria for selecting patients who might benefit from surgery in relapsed ovarian cancer.MethodsThe DESKTOP trial was an exploratory study based on data from a retrospective analysis of hospital records. Twenty-five member institutions of the Arbeitsgemeinschaft Gynaekologische Onkologie Ovarian Committee (AGO OC) and AGO-OVAR boards collected data on their patients with cytoreductive surgery for relapsed invasive epithelial ovarian cancer performed in 2000–2003.ResultsTwo hundred and sixty-seven patients were included. Complete resection was associated with significantly longer survival compared with surgery leaving any postoperative residuals [median 45.2 vs. 19.7 months; hazard ratio (HR) 3.71; 95% confidence interval (CI) 2.27–6.05; P < .0001]. Variables associated with complete resection were performance status (PS) [Eastern Cooperative Oncology Group (ECOG) 0 vs. > 0; P < .001], International Federation of Gynecology and Obstetrics (FIGO) stage at initial diagnosis (FIGO I/II vs. III/IV, P = .036), residual tumor after primary surgery (none vs. present, P <.001), and absence of ascites > 500 ml (P < .001). A combination of PS, early FIGO stage initially or no residual tumor after first surgery, and absence of ascites could predict complete resection in 79% of patients.ConclusionsOnly complete resection was associated with prolonged survival in recurrent ovarian cancer. The identified criteria panel will be verified in a prospective trial (AGO-DESKTOP II) evaluating whether it will render a useful tool for selecting the right patients for cytoreductive surgery in recurrent ovarian cancer.

Choline-releasing glycerophosphodiesterase EDI3 drives tumor cell migration and metastasis

Metastasis from primary tumors remains a major problem for tumor therapy. In the search for markers of metastasis and more effective therapies, the tumor metabolome is relevant because of its importance to the malignant phenotype and metastatic capacity of tumor cells. Altered choline metabolism is a hallmark of cancer. More specifically, a decreased glycerophosphocholine (GPC) to phosphocholine (PC) ratio was reported in breast, ovarian, and prostate cancers. Improved strategies to exploit this altered choline metabolism are therefore required. However, the critical enzyme cleaving GPC to produce choline, the initial step in the pathway controlling the GPC/PC ratio, remained unknown. In the present work, we have identified the enzyme, here named EDI3 (endometrial differential 3). Purified recombinant EDI3 protein cleaves GPC to form glycerol-3-phosphate and choline. Silencing EDI3 in MCF-7 cells decreased this enzymatic activity, increased the intracellular GPC/PC ratio, and decreased downstream lipid metabolites. Downregulating EDI3 activity inhibited cell migration via disruption of the PKCα signaling pathway, with stable overexpression of EDI3 showing the opposite effect. EDI3 was originally identified in our screening study comparing mRNA levels in metastasizing and nonmetastasizing endometrial carcinomas. Both Kaplan–Meier and multivariate analyses revealed a negative association between high EDI3 expression and relapse-free survival time in both endometrial (P < 0.001) and ovarian (P = 0.029) cancers. Overall, we have identified EDI3, a key enzyme controlling GPC and choline metabolism. Because inhibition of EDI3 activity corrects the GPC/PC ratio and decreases the migration capacity of tumor cells, it represents a possible target for therapeutic intervention.

Activity of O6-methylguanine-DNA methyltransferase in relation to p53 status and therapeutic response in ovarian cancer

The DNA‐repair protein O6‐methylguanine‐DNA methyltransferase (alkyltransferase; MGMT) is a major determinant of resistance of cells to various alkylating cytostatic drugs. Its expression in tissues is highly variable, indicating complex regulatory mechanisms involved. Transfection‐mediated expression of wild‐type p53 has been shown to negatively regulate basal promoter activity of MGMT in vitro. To elucidate whether p53 is involved in regulation of MGMT in tumor tissue, we examined MGMT expression and the p53 status of 140 primary ovarian carcinomas and analyzed the data as to the correlation between MGMT and p53, as well as the survival response of the patients after chemotherapy. We show that MGMT expression is highly variable in ovarian carcinomas, ranging from zero level up to 2500 fmol/mg protein. MGMT activity was significantly lower in tumors with wild‐type p53 (p53wt) than in tumors with mutant p53 (p53mt) (p = 0.045). As expected, the percentage of tumors with p53mt increased with increasing histologic grade of the tumors. Thus, p53mt was observed in 4, 45 and 64% of grades 1, 2 and 3 tumors, respectively (p = 0.001). Increase in p53mt was accompanied by an increase in MGMT activity, which was, on average, 460 ± 66, 624 ± 63 and 662 ± 60 fmol/mg protein in grades 1, 2 and 3 tumors, respectively (p = 0.047). In addition, MGMT activity as well as p53mt were associated with the FIGO stage of the tumors. Mean MGMT activity was 472 ± 48 fmol/mg for patients with FIGO stages I and II, as compared with 675 ± 50 fmol/mg for patients with FIGO stages III and IV, (p = 0.0179). The percentage of p53mt was 27% and 54% in ovarian tumors with FIGO stages I/II and FIGO stages III/IV, respectively (p = 0.004). Thus, progression of ovarian tumors was clearly associated with increase of both MGMT activity and the percentage of p53mt. In tumors expressing low MGMT (<100 fmol/mg), p53mt was very rarely found. No significant association was observed between MGMT level in ovarian carcinomas and the survival of patients treated with cyclophosphamide and carboplatin. On the other hand, a clear correlation was found between histological type, grading, residual tumor mass and p53wt expression and duration of the patients survival. The finding that p53wt expression was associated with low MGMT level in primary ovarian cancer supports the view that down‐regulation of basal MGMT promoter activity by p53wt is also relevant in tumor cells in vivo. Int. J. Cancer (Pred. Oncol.) 84:388–395, 1999.

Induction of DNA crosslinks and DNA strand lesions by cyclophosphamide after activation by cytochrome P450 2B1

Cyclophosphamide requires metabolic activation by cytochrome P450 to exert its genotoxic effects. Therefore in vitro studies on its mechanism of action have been limited to the use of self-activating derivatives of cyclophosphamide or to hepatocytes as an activating system. In this study we used a cell line of Chinese hamster lung fibroblasts (V79 cells), genetically engineered to express active cytochrome P450 2B1 as the sole observable cytochrome P450 (SD1 cells). An increase in DNA strand lesions (SL: DNA single-strand breaks and alkali labile sites) was observed between 0.5 and 1.5 mM cyclophosphamide (24 h incubation) which could be classified as alkali labile sites using a modified alkaline elution assay. Compared to cyclophosphamide, its active metabolite 4-hydroperoxycyclophosphamide (4-OOH-CY) was about 250-fold more effective in induction of SL. Equimolar concentrations of phosphoramide mustard (50 microM), the ultimate DNA binding metabolite of cyclophosphamide, caused only about 50% of SL compared to 4-OOH-CY. A minimum of 12 h of incubation of SD1 cells was needed for cyclophosphamide (1 mM) until SL were detectable, compared to only 2 h for 4-OOH-CY and 1.5 h for phosphoramide mustard (50 microM). DNA crosslinks were observable after shorter incubation periods than single-strand breaks (6 h for cyclophosphamide and 1 h for 4-OOH-CY and phosphoramide mustard) and were no longer detectable at incubation periods of more than 20 h. Treatment of SD1 cells with ionizing radiation only, cyclophosphamide only, and radiation plus cyclophosphamide showed that SL induced by cyclophosphamide were not repaired during incubation with fresh culture medium (24 h). However, an efficient repair of SL caused by ionizing radiation was observed and was not inhibited by cyclophosphamide. These observations give strong evidence that different types of SL were induced by cyclophosphamide and radiation. SD1 cells were able to repair the special kind of SL induced by radiation but not the SL caused by cyclophosphamide.

Metallothionein expression in ovarian cancer in relation to histopathological parameters and molecular markers of prognosis

Metallothioneins (MTs) and glutathione constitute the major fractions of intracellular thiol factors. Abundant nucleophilic sulfhydryl groups can interact with many electrophilic substances, including several anti‐neoplastic agents, participate in controlling intracellular redox potential, and act as scavengers of reactive oxygen species. In the present study, we examined the relation of MTs (alone and in combination with glutathione) to histopathological parameters and survival time of ovarian cancer patients. Expression of the major MT isoforms (MT‐1 and MT‐2) was determined by immunohistochemistry on paraffin‐embedded tumor specimens from 189 patients, 151 suffering from primary epithelial ovarian cancer and 38 from recurrences. MT was negatively associated with survival time when all patients with primary carcinomas (n = 151) were analyzed (p = 0.049, log‐rank test). However, no significant association between MT expression and survival was obtained when subgroups of patients with histological grade 1, 2 or 3 carcinomas were analyzed. Similarly, no significant association of MT expression and survival was obtained with the proportional hazards model adjusted for histological grade. This scenario can be explained by a correlation between MT expression and histological grade: MT was detectable in 26%, 48% and 62% of grade 1, 2 and 3 carcinomas, respectively (p = 0.008, χ2 test). An interesting hypothesis is generated by combined analysis of MT and total glutathione content (GSH). The product of MT and GSH levels (MT × GSH) was negatively associated with survival of grade 1 carcinomas (p = 0.021, log‐rank test) but not with grade 2 and 3 carcinomas (p = 0.176 and 0.403, respectively). When MT × GSH was greater than the median, 25% of patients with grade 1 carcinomas died within 235 days. In contrast, all patients with grade 1 carcinomas survived when MT × GSH in tumor tissue was smaller than the median. This suggests that high expression of sulfhydryl factors might facilitate survival and progression of low‐grade ovarian cancer cells. A significant correlation was obtained between MT expression and mutant p53 (p = 0.037, χ2 test). However, this might be an indirect effect since both MT (p = 0.008) and mutant p53 (p = 0.000) were associated with histological grade. In conclusion, MT expression as well as the product of MT and GSH were associated with histological grade of primary ovarian carcinomas. High expression of both sulfhydryl factors may identify a subgroup of low‐grade carcinomas with an increased risk of progression.

mdm2 mRNA expression is associated with survival in ovarian cancer

Expression of mdm‐2 mRNA was measured in 90 ovarian‐cancer tissue specimens using the S1 nuclease assay, to investigate a possible association between MDM2 expression and prognosis. mdm‐2 mRNA expression was an independent prognostic factor for patients with primary ovarian cancer, FIGO (International Federation of Gynecology and Obstetrics) stages III and IV (n = 57), who all received chemotherapy with carboplatin or cisplatin and cyclophosphamide. Median survival time for patients (FIGO stages III and IV) with no detectable expression of mdm‐2 mRNA (n = 14) was 171 days, as compared with 839 days for patients (n = 43) with detectable mdm‐2 mRNA (p = 0.0194; log‐rank test). However, no association between mdm‐2 mRNA expression and survival was observed for patients with FIGO stages I and II who did not receive chemotherapy. mdm‐2 expression was not associated with FIGO stage, residual disease, histologic grade and type. Our results suggest that mdm‐2, which is known to disrupt p53 function, sensitizes ovarian‐cancer cells to cisplatin/cyclophosphamide, possibly by inhibition of p53‐mediated G1 cell‐cycle arrest and p53‐stimulated nucleotide‐excision repair. Int. J. Cancer 74:438–442, 1997.

Glutathione S-transferase T1 and M1 gene defects in ovarian carcinoma

Glutathione S-transferases (GSTs) M1 and T1 are known to be polymorphic in humans. Both polymorphisms are due to gene deletions, which are responsible for the existence of null genotypes. The gene defect of GSTT1 has been reported to be associated with an increased risk of myelodysplastic syndromes, astrocytoma and meningioma. A lack of GSTM1 was associated with tobacco smoke-induced lung and bladder cancer. In this study we examined whether the GSTT1 and/or GSTM1 homozygous null genotypes were associated with an increased risk of ovarian cancer using a multiplex polymerase chain reaction protocol. The GSTT1 null genotype was observed in 14% of the control subjects that had never suffered from neoplastic disease (n = 115) and in 16% of the patients affected with ovarian cancer (n = 103, OR 0.87, 95% CI 0.39-1.92, P = 0.73). A lack of GSTM1 was observed in 38% of the control subjects and in 46% of the patients (OR 0.77, 95% CI 0.44-1.32). This difference was not significant (P = 0.34). Similarly, no significant differences were obtained if GSTT1 and/or GSTM1 null genotypes were analyzed in subgroups of control subjects and ovarian cancer patients between the ages of 20-40, 41-70 and 71-90 years and in individuals with a positive family history of neoplastic disease. GSTT1 and/or GSTM1 null genotypes were not significantly associated with the histologic type and grade or FIGO (International Federation of Gynecology and Obstetrics) stages of the ovarian carcinomas. In conclusion, GSTT1 and/or GSTM1 null genotypes are not markers for an increased risk of ovarian cancer.

Long-term prognostic significance of HER-2/neu in untreated node-negative breast cancer depends on the method of testing.

IntroductionThe prognostic significance of HER-2/neu in breast cancer is a matter of controversy. We have performed a study in 101 node-negative breast cancer patients with long-term follow-up not treated in the adjuvant setting, and analysed the prognostic significance of immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH), both separately and in combination, in comparison with traditional prognostic factors.MethodsOverexpression was classified semiquantitatively according to a score (0 to 3+) (HER-2_SCO). FISH was used to analyse HER2/neu amplification (HER-2_AMP). Patients classified 2+ by IHC were examined with FISH for amplification (HER-2_ALG). Patients with 3+ overexpression as well as amplification of HER-2/neu were positive for the combined variable HER2_COM. These variables were compared with tumour size, histological grade and hormone receptor status.ResultsHER-2_SCO was 3+ in 20% of all tumours. HER-2_ALG was positive in 22% and amplification (HER-2_AMP) was found in 17% of all tumours. Eleven percent of the tumours showed simultaneous 3+ overexpression and amplification. Only histological grade (relative risk [RR] 3.22, 95% confidence interval [CI] 1.73–5.99, P = 0.0002) and HER-2_AMP (RR 2.47, 95% CI 1.12–5.48, P = 0.026) were significant for disease-free survival in multivariate analysis. For overall survival, both histological grade (RR 3.89, 95% CI 1.77–8.55, P = 0.0007) and HER-2_AMP (RR 3.08, 95% CI 1.24–7.66, P = 0.016) retained their independent significance.ConclusionThe prognostic significance of HER-2/neu in node-negative breast cancer depends on the method of testing: only the amplification of HER-2/neu is an independent prognostic factor for the long-term prognosis of untreated node-negative breast cancer.

Resistance factors in colon cancer tissue and the adjacent normal colon tissue: glutathione S-transferases α and π, glutathione and aldehyde dehydrogenase

Glutathione S-transferases (GST) alpha and pi, glutathione (GSH) and aldehyde dehydrogenase (ADH) were determined in colorectal cancer tissue specimens and in the adjacent normal colon tissue. The median contents in normal and cancer tissue were 8.1 (2.3-30.3) (5-95% quantiles) and 15.1 (5.3-50.3) microg/mg protein for GST pi (P = 0.035), 0.0 (0.0-1.4) and 0.4 (0.0-3.5) microg/mg protein for GST alpha (P = 0.019), 7.3 (1.3-22.7) and 5.6 (2.3-26.0) microg/mg protein for GSH (P = 0.171) and 30.8 (13.0-42.0) and 23.2 (9.0-32.9) microg/mg protein for ADH (P = 0.0017), respectively. Thus, the mean GST alpha and pi both significantly increased in colon cancer compared to the adjacent normal tissue, which underlines their importance as possible resistance factors. A highly significant correlation was obtained between the GSH content in colon cancer and normal tissue (P = 0.0017). Thus, the constitutive GSH expression seems to be maintained during tumor development. A similar correlation was obtained for ADH (P = 0.0075), but the median ADH was lower in cancer tissue compared to the adjacent normal tissue (P = 0.0017). Contrary to GSH and ADH, GST pi did not correlate between normal and colon cancer tissue. Whereas GSH and ADH correlated in normal colon tissue (P = 0.014), no significant correlation for GSH and ADH was observed in colon cancer tissue (P = 0.109). In conclusion, significant correlations between colon cancer and normal tissue were obtained, suggesting that the expression levels of these resistance factors are maintained during carcinogenesis in most patients.

SPOC1, a novel PHD-finger protein: Association with residual disease and survival in ovarian cancer

We report the identification of a novel human gene (SPOC1) which encodes a protein with a PHD‐finger domain. The gene is located in chromosomal region 1p36.23, a region implicated in tumor development and progression. RNA in situ hybridization experiments showed strong SPOC1 expression in some rapidly proliferating cell types, such as spermatogonia, but not in nonpro‐liferating mature spermatocytes. In addition, high SPOC1 mRNA expression was observed in several ovarian cancer cell lines. This prompted us to systematically examine SPOC1 expression in ovarian cancer in relation to prognosis. SPOC1 mRNA expression was quantified in tumor tissue of 103 patients with epithelial ovarian cancer. Interestingly, SPOC1 was associated with residual disease, whereby patients with unresectable tumors showed higher levels compared to patients without residual tumor tissue after surgery (p = 0.029). The univariable proportional hazards model showed an association between SPOC1 expression and survival (p = 0.043, relative risk = 1.535). Median survival time was 1,596 days for patients with low SPOC1 expression vs. only 347 days for patients with high expression, using Kaplan‐Meier analysis. However, SPOC1 was not associated with survival when multivariable analysis was adjusted for residual disease. This can be explained by the correlation between residual disease and SPOC1 expression. In conclusion, SPOC1 is a novel PHD‐finger protein showing strong expression in spermatogonia and ovarian cancer cells. SPOC1 overexpression was associated with unresectable carcinomas and shorter survival in ovarian cancer.