Manfred Volm

Association of vascular endothelial growth factor expression with intratumoral microvessel density and tumour cell proliferation in human epidermoid lung carcinoma

Vascular endothelial growth factor (VEGF) expression, vascularisation and tumour cell proliferation were analysed in 91 human epidermoid lung carcinomas using immunohistochemistry. A polyclonal anti-VEGF antibody was used for VEGF expression, a polyclonal antibody directed against human von Willebrand factor (factor VIII) to identify blood vessels and the proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells. Positive staining for VEGF was obtained in 54 out of 91 cases (59%), the number of blood vessels varied from zero to 64 counts (mean 9.4) and the proportion of PCNA-positive cells varied from 1.3% to 72.1% (mean 25.2%). The mean PCNA labelling index and mean microvessel count in VEGF-positive tumours were significantly higher than those in VEGF-negative tumours (Wilcoxon rank sum test, P<0.0001; p<0.05). In addition, PCNA labelling index significantly increased with increasing VEGF expression (Jonckheere test, P<0.0001). In contrast, no association was found between PCNA labelling index and tumour vascularity (r=0.07, P=0.48). The close correlation of VEGF expression with tumour cell proliferation and microvessel density suggests that VEGF acts both as an autocrine growth factor and as stimulator for angiogenesis. However, tumour cell proliferation and microvessel growth and/or density may be regulated by separate mechanisms.

Prognostic value of ERBB-1, VEGF, cyclin A, FOS, JUN and MYC in patients with squamous cell lung carcinomas

Patients with previously untreated squamous cell lung carcinomas were evaluated to see if combining the expression of molecular and cellular factors with the most important clinical prognostic factors could improve the diagnostic ability to predict prognosis. For this reason, immunohistochemistry was used to examine the squamous cell lung carcinomas from 121 patients for their expression of ERBB-1, vascular endothelial growth factor (VEGF), cyclin A, FOS, JUN and MYC. Median survival was shorter for patients with ERBB-1-, VEGF-, cyclin A-, FOS-, or JUN-positive tumours. For those patients with positive lymph node involvement, the survival times were also shorter in the VEGF-positive, cyclin A-positive and FOS-positive groups. Multivariate analysis independently demonstrated a significant prognostic value for lymph node involvement, VEGF and FOS.

Tissue‐factor expression in human non‐small‐cell lung carcinoma measured by immunohistochemistry: Correlation between tissue factor and angiogenesis

Tissue factor (TF) is the physiological initiator of blood coagulation. It has been suggested that TF also regulates tumor growth and angiogenesis. We therefore used immunohistochemistry to analyze the expression of TF and angiogenesis in non‐small‐cell lung carcinomas of 191 patients. A significant association was found between TF expression and microvessel density (MVD): TF‐negative carcinomas more frequently exhibited low MVD. Additionally, a significant relationship between TF expression and the expression of vascular endothelial growth factor (VEGF) was discovered. TF was also compared with the resistance of the carcinomas to doxorubicin, as measured in vitro: TF‐negative tumors were more frequently resistant to doxorubicin than were TF‐positive tumors. Kaplan‐Meier survival analysis revealed that survival times were longer in patients with TF‐negative tumors than in patients with TF‐positive tumors. These data suggest that TF functions as an additional angiogenic factor that could be used as a new prognostic and predictive factor for non‐small‐cell lung carcinomas. Int. J. Cancer (Pred. Oncol.) 79:19–22, 1998.© 1998 Wiley‐Liss, Inc.

P-glycoprotein expression in treated and untreated human breast cancer.

The expression of P-glycoprotein in primary and recurrent human breast cancer was investigated by means of immunohistochemistry, using a monoclonal antibody (C219) and the streptavidin-biotin-peroxidase method. Twelve patients received no chemotherapeutic treatment. The other 11 patients were treated with chemotherapy, and all developed clinical resistance to it. No or only minimal reactivity was found in specimens coming from the untreated patients (12 cases) or from patients treated with substances not involved in the multidrug resistance phenomenon (four cases). In contrast, three out of seven tumours from patients treated with multidrug resistance related substances showed clear reactivity (positive staining in more than 20% of the tumour cells). In one of these cases, where specimens of the tumour could be studied before and after treatment, an association between the latter and expression of P-glycoprotein was suggested. Finally, this marked expression of P-glycoprotein only took place in tumours treated over a longer space of time (five courses or more of multidrug resistance related chemotherapy).

Pretherapeutic detection of tumour resistance and the results of tumour chemotherapy

Abstract A short-term in vitro test is described which can be used to detect proliferation-dependent and induced tumour resistance to cytostatic agents. The test involves incubation of tumour cell suspensions with cytostatic agents for short periods. The resulting effects on nucleic acid metabolism are measured using radioactively labelled precursors of DNA or RNA. The Walker carcinosarcoma and a neurosarcoma respond differently to treatment with adriamycin, in accordance with the relative growth rates of the tumours. This differential sensitivity of the tumours to adriamycin can also be detected in the test. Similarly, an induced resistance to adriamycin which was developed in the Sarcoma 180 was also found using the test, as was cross resistance to other cytostatics. Human lung and ovarian carcinomas which were only slightly inhibited by adriamycin in vitro before starting treatment ( inhibition at a concentration of 10 −2 mg/ml or 1.7 × 10 −5 M ) proved to be resistant to chemotherapy in the clinic. Tumours which showed a clear response to adriamycin in the test ( >30% inhibition) showed in most cases remission on clinical treatment with cytostatics. A clear correlation was observed between the inhibitory effects of adriamycin in vitro and the course of clinical therapy even when a combination therapy was used which did not include adriamycin. A significant correlation was also obtained between the inhibition due to adriamycin in vitro and the survival times of the tumour-bearing patients. The short-term test can therefore be used to distinguish between chemoresistant and chemosensitive tumours.

Expression of FAS (CD95/APO‐1) and FAS ligand in lung cancer, its prognostic and predictive relevance

In order to examine whether or not the expression of the apoptosis‐related receptor Fas (CD‐95/APO‐1) and its ligand (FasL) has relevance for patient survival, immunohistochemistry was used to analyze the proteins of both factors in 164 non‐small cell lung carcinomas. Patients with Fas‐positive tumors exhibited significantly longer survival times than patients with Fas‐negative carcinomas. In contrast, FasL did not significantly influence patient survival time. A multivariate analysis of clinical and biological factors indicated that lymph node status and Fas expression were significant prognostic factors. Carcinoma patients who were negative for both Fas and FasL had a significantly higher incidence of lymph node involvement than did carcinoma patients who were positive for Fas and FasL. Carcinomas that were positive for Fas and FasL demonstrated a greater sensitivity to doxorubicin in vitro. Int. J. Cancer (Pred. Oncol.) 84:239–243, 1999.

Expression of resistance factors (P‐glycoprotein, glutathione S‐transferase‐π, and topoisomerase II) and their interrelationship to proto‐oncogene products in renal cell carcinomas

Background. This study investigates whether or not an interrelationship exists between the expression of resistance‐related proteins (P‐glycoprotein, glutathione S‐transferase, topoisomerase II) and proto‐oncogene products (c‐fos, c‐myc, c‐K‐ras, epidermal growth factor receptor [EGF‐R], and c‐neu proteins).

Prognostic significance of DNA patterns and resistance-predictive tests in non-small cell lung carcinoma.

In a cooperative study, 240 surgical specimens of patients with non‐small cell lung carcinomas (NSCLC) were investigated by means of flow cytometry, xenotransplantation to athymic mice and, an in vitro short‐term test for predicting resistance. Aneuploidy was found in 83% of the tumors, and 20% showed more than one aneuploid DNA stemline. Patients with both aneuploid tumors and tumors with more than one DNA stemline had a significantly shorter survival rate than those with only diploid or only one DNA stemline. Patients whose tumors showed a low G0/G1‐cell proportion or a high proliferation pool (S and G2/M‐cell proportion) died earlier. A relationship could not be discerned between growth of tumors in nude mice or establishment of cell lines and the prognosis for the patients. Patients with in vitro‐resistant tumors died earlier under chemotherapy than those with in vitro‐sensitive tumors. Patients treated by radiation survived longer if the tumors were resistant in vitro. Thus, DNA patterns and in vitro short‐term tests for predicting resistance represent useful tools for prognostic evaluation of patients with NSCLC.

Human tumor xenografts as model for drug testing

This paper reviews the history of xenografts, the endpoints commonly used to evaluate response and chemotherapeutic results obtained with serially maintained human tumor xenografts from different laboratories, and discusses the potential clinical relevance of the heterotransplant model for cancer chemotherapy. Specifically, an attempt is made to correlate the published xenograft data with the clinical data. Drug testing with different types of xenotransplanted tumors has shown that the response of xenografts obtained in immune-deficient animals is comparable to that in clinical practice. In addition, xenografts of a particular tumor type are able to identify agents of known clinical activity against that disease.

Prediction of Broad Spectrum Resistance of Tumors towards Anticancer Drugs

Purpose: Drug resistance is a major obstacle in cancer chemotherapy. Although the statistical probability of therapeutic success is known for larger patient groups from clinical therapy trials, it is difficult to predict the individual response of tumors. The concept of individualized therapy aims to determine in vitro the drug response of tumors beforehand to choose effective treatment options for each individual patient. Experimental Design: We analyzed the cross-resistance profiles of different tumor types (cancers of lung, breast, and colon, and leukemia) towards drugs from different classes (anthracyclines, antibiotics, Vinca alkaloids, epipodophyllotoxins, antimetabolites, and alkylating agents) by nucleotide incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Hierarchical cluster analysis and COMPARE analyses were applied. Results: Tumors exert broad resistance profiles, e.g., tumors resistant to one drug tend to also be resistant to other drugs, whereas sensitive tumors reveal sensitivity towards many drugs. Interestingly, the broad spectrum resistance phenotype could reliably be predicted by doxorubicin alone. Expression of the ATP-binding cassette transporter P-glycoprotein (ABCB1, MDR1) and the proliferative activity of tumors were identified as underlying mechanisms of broad spectrum resistance. To find novel compounds with activity against drug-resistant tumors, a database with 2,420 natural products was screened for compounds acting independent of P-glycoprotein and the proliferative state of tumor cells. Conclusions: Tumors exert cross-resistance profiles much broader than the classical multidrug resistance phenotype. Broad spectrum resistance can be predicted by doxorubicin due to the multifactorial mode of action of this drug. Novel cytotoxic compounds from natural resources might be valuable tools for strategies to bypass broad spectrum resistance.