Jürgen Reuter

DCR3 locus is a predictive marker for 5-fluorouracil-based adjuvant chemotherapy in colorectal cancer

Adjuvant chemotherapy reduces the incidence of distant metastasis and increases survival of patients with colorectal cancer. However, predictive markers are needed to define subsets of patients with stage II and III disease that may benefit from adjuvant treatment. A secreted member of the TNF receptor superfamily, the decoy receptor 3 (DcR3), was reported to be amplified in colorectal cancer as a negative regulator of Fas‐mediated apoptosis. We analyzed DcR3 gene copy number and protein expression in a large series of tumors from a randomized multicenter trial of 5‐fluorouracil/mitomycin C (FU/MMC) adjuvant chemotherapy of the Swiss Group for Clinical Cancer Research (SAKK 40/81), using real‐time quantitative PCR and immunohistochemistry on tumor microarrays. Results of gene status and protein expression of DcR3 were correlated with disease‐free and overall survival of patients. We observed amplification of the DcR3 gene in 185/294 (63%) and overexpression of the DcR3 protein in 163/223 (73%) of colorectal tumors. Multivariate analysis showed no prognostic effect of DcR3 gene amplification and protein overexpression. However, adjuvant chemotherapy was significantly more beneficial in patients with normal DcR3 gene copy number than in patients with amplification (DFS: HR 2.84, 95% CI 1.16–6.98, p = 0.02; OS: HR 3.15, 95% CI 1.19–8.32, p = 0.02), whereas DcR3 protein overexpression did not influence the effect of adjuvant chemotherapy (DFS: HR 1.02, 95% CI 0.65–1.60, p = 0.95; OS: HR 0.95, 95% CI 0.61–1.49, p = 0.83). We conclude that amplification of the 20q13 locus is a predictive marker for adjuvant chemotherapy in colorectal cancer.

Five putative drug resistance parameters (MDR1/P-glycoprotein, MDR-associated protein, glutathione-S-transferase, bcl-2 and topoisomerase IIα) in 57 newly diagnosed acute myeloid leukaemias

Abstract: Using a modified quantitative reverse transcriptase (RT) PCR assay in 57 patients with acute myeloid leukaemia (AML) from a Swiss Phase III multicentre study (SAKK 30/85), we measured the m‐RNA expression of the genes from the multidrug resistance gene 1 (MDR1), the multidrug resistance associated protein (MRP), glutathione‐S‐transferase (GST) π, bcl‐2 and topoisomerase (topo) IIα. P‐glycoprotein (p‐gp) was measured by Western blot, and GST activity by functional assays. To analyse progression‐free (PFS) and overall survival (OS), parameters were prospectively divided into “low” and “high” groups, according to their median values (exceptions: MDR1 and p‐gp). Median follow‐up was 60 months. Results: MDR1‐ and MRP mRNA levels correlated with each other (r = 0.54, Spearman), FABM4/M5 and extramedullary disease. “Low” bcl‐2‐mRNA predicted longer PFS: 22 months vs. 7 months (median, p = 0.02, log rank), and longer OS: 64 months vs. 14 months (p = 0.06). “Low” topo Hoc predicted poorer outcome: median PFS 9 vs. 19 months (p = 0.03); median survival 12 months vs. “not reached” (p = 0.03). An improved outcome tendency, albeit nonsignificant, was seen in p‐gp‐negative patients. In a Cox model adjusted for age, performance status, presence of Auer rods, FAB type and clinical response, bcl‐2 and topo IIα mRNA levels retained their predictive values.

bcl-2 expression is reciprocal to p53 and c-myc expression in metastatic human colorectal cancer.

Apoptosis (programmed cell death) inhibition may be an important mechanism by which gastrointestinal mucosal cells containing damaged DNA evade normal clearance mechanisms and grow to become invasive tumours. Since bcl-2 is an apoptosis inhibitor, bcl-2 mRNA expression was measured in 21 metastases of colorectal cancer using reverse transcription-polymerase chain reaction analysis. The mean bcl-2 mRNA expression (0.45 U, P < 0.0001) was lower than that of normal mucosal controls (= 1 U). p53 expression was inversely correlated with bcl-2 expression (P = 0.021) in 19 evaluable samples, and in tumours where p53 expression was over twice that of normal colonic mucosal values, bcl-2 mRNA was significantly decreased (mean 0.30, P = 0.0052). c-myc was also inversely correlated with bcl-2 expression (P = 0.025). Decreased bcl-2 expression in metastatic colorectal cancer may be partly due to allelic loss, given the proximity of bcl-2 to the frequently deleted DCC gene on chromosome 18q. However, the inverse correlation to p53/c-myc suggests an active downregulation of bcl-2, possibly following delegation of its apoptosis inhibiting role to other genes.

Multiple drug resistance gene expression in human renal cell cancer is associated with the histologic subtype.

Overexpression of p170 glycoprotein, the product of the multiple drug resistance (mdr) gene, has been associated with resistance to various cytotoxic drugs used in the treatment of human neoplasms. Normal renal epithelial cells express p1 70 as a function of their secretory capacity. Because renal cell carcinomas (RCC) respond poorly to chemotherapeutic regimens, p1 70 expression was studied in primary RCC. Such expression was measured in 40 human RCC and normal kidney tissues using immunohis‐tochemical staining with the monoclonal antibody C‐219. Staining intensities of the whole tumor and of different areas of the cryostat sections were transformed into digital numbers using an algorithm designed for this purpose. In most tumors, an inhomogeneous staining pattern and a correlation between grade of differentiation and C‐219 immunoreactivity was observed. A comparison of the tumors according to their histopathologic subtypes showed clear differences. The means (range) of the staining intensities of the different types of RCC: clear cell carcinoma Grade 1 (n = 3), 2.0 (2.0 to 2.0); clear cell carcinoma Grade 2 (n = 19), 0.8 (0.0 to 2.9); clear cell carcinoma Grade 3 (n = 5), 0.1 (0.0 to 0.2); tubular carcinoma (n = 4), 2.0 (2.0 to 3.0); anaplastic carcinoma (n = 8), 0.05 (0.0 to 0.2); oncocytoma (n = 1), 0.0 (0.0 to 0.0); and normal kidney (n = 40), 0.5 (0.0 to 2.0). The differences between anaplastic, clear cell, and tubular carcinoma were significant (P < 0.001 by Kruskal‐Wallis test). In addition, the difference between the three subgroups of clear cell carcinoma was significant (P < 0.01). It was concluded that the histopathologic subtypes of RCC correlate with the degree of mdr gene expression, as determined by staining with the C‐219 monoclonal antibody.

mRNA Expression, Measured by Quantitative Reverse Transcriptase Polymerase Chain Reaction, of Five Putative Drug Resistance Parameters, in Normal and Leukaemic Peripheral Blood and Bone Marrow

Using a quantitative reverse transcriptase PCR assay, the mRNA expression of five putative drug resistance-related genes were assessed in normal peripheral (n = 14) and bone marrow (n = 4) mononuclear cells from healthy donors and patients with acute myeloid leukaemia (n = 11). The mRNA levels of MDR1, the multidrug resistance-associated protein and glutathione-S-transferase pi were equally expressed in both compartments. Bcl-2 mRNA was slightly higher in the leukaemic marrow samples. However, topoisomerase II alpha mRNA levels were found to be much higher in normal and leukaemic marrow cells compared to peripheral blood (p < 0.01), which may, in part, reflect the different proliferation pattern of the mononuclear cells in the two compartments. Such findings could be important for researchers using bulk assays in a mix of samples from peripheral blood or bone marrow to investigate prognostic factors in patients with leukaemia.

Gene therapy with cytokine-transfected xenogenic cells (Vero-IL-2) in patients with metastatic solid tumors: mechanism(s) of elimination of the transgene-carrying cells

Abstract Eleven patients with advanced cancer were treated in a clinical gene therapy trial by repeated intra- tumoral injections with different doses of xenogenic fibroblasts secreting high amounts of human interleukin-2 (Vero-IL2). Treatments in a total of 14 courses were well tolerated and resulted in clinical responses and measurable biological effects. Together with increases in serum interleukin-2 (IL-2), modifications of the V-β T cell receptor repertoire and induction of intratumoral T-cell infiltration were observed. When the intratumoral expression of endogenous cytokine genes and the persistence of the IL-2 transgene at the application site and in peripheral blood were investigated, rapid disappearance of the transgene at the application site appeared to be the most prominent biological effect. Tests detecting a single Vero-IL2 cell against a background of 105 non-transfected cells were not able to demonstrate significant expression of exogenous IL-2 (i.e. the transgene or transgene-carrying cells) in tumor biopsies or blood at different times. Therefore, further studies were performed to evaluate the mechanism(s) involved in the rapid disappearance of xenogenic carrier cells in more detail. We show here that significant in vitro cytotoxicity against transgene-carrying Vero cells can be observed in peripheral blood of all the patients before treatment as well as in healthy controls. “Cold” target inhibition shows that significant killing of Vero-IL2 cells is mediated by natural killer (NK) cells. This was confirmed by showing that established CD3−/CD16+/CD56+ peripheral blood NK cell clones kill both K562 and Vero-IL2 target cells. The failure of other mechanisms (complement, antibody-dependent cell cytotoxicity or cytotoxic T lymphocytes) to destroy xenogenic, histoincompatible Vero cells in vitro suggests that NK cells also might be responsible for the killing of Vero-IL2 in vivo and for the failure to detect the transgene at the application site. These results might also be of importance for some aspects of the current discussion of xenotransplantation.

MTS1 expression and prognosis in acute myeloid leukemia

MTS1, a tumor supressor gene located on chromosome 9p21, has been shown to be altered in a number of human tumor cell lines, primary solid tumors, and leukemias. In this study we found low expression of MTS1 in lymphocytes from seven of nine healthy donors, but in none of eight granulocyte samples from the same controls, suggesting a physiological role of MTS1 in peripheral blood cells capable of proliferation, but not in end-stage differentiated cells. We detected MTS1 mRNA expression in 38 of 57 patients (66%) with acute myelogenous leukemia (AML) treated in a standardized clinical protocol. No deletion of the MTS1 gene was found in any of the AML samples tested. There was no significant association between expression of MTS1 and response to therapy, progression-free, or overall survival. Except for a negative correlation between MTS1 level and leukocyte count at diagnosis (p=0.03), there was also no association with any of the known prognostic parameters in AML. We conclude that MTS1 shows a significantly higher expression in leukemic than in normal peripheral blood cells, that deletion of MTS1 is not a frequent event in AML, and that its expression is not significantly correlated with outcome of the disease.