J. H. Van Dierendonck

A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA.

Apoptosis (programmed cell death) can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. We describe a new staining method for formalin-fixed, paraffin-embedded tissue sections that involves an in situ end-labeling (ISEL) procedure. After protease treatment to permeate the tissue sections, biotinylated nucleotides are in situ incorporated into DNA breaks by polymerase and subsequently stained with DAB via peroxidase-conjugated avidin. Staining of cells with the morphological characteristics of apoptosis was demonstrated in tissues known to exhibit programmed cell death, i.e., prostate and uterus after castration, tumors, lymph node follicles, and embryos. Apoptotic cells could be discriminated morphologically from areas of labeled necrotic cells, in which DNA degradation also occurs. Because apoptosis is relatively easily recognized in H&E-stained sections of involuting prostates of castrated rats, we used this model system to validate the ISEL method for the quantification of apoptotic cells. A high correlation was found between the fractions of ISEL-labeled cells and the fractions of apoptotic cells that were morphologically determined in adjacent sections. We conclude that ISEL is a useful technique for quantification of apoptosis in paraffin sections, especially for those tissues in which morphological determination is difficult. Furthermore, this new staining method enables the use of automated image cytometry for evaluating apoptosis.

Expression of Bcl-2 in node-negative breast cancer is associated with various prognostic factors, but does not predict response to one course of perioperative chemotherapy

The aim of this study was to assess relationships between Bcl-2 expression, response to chemotherapy and a number of pathological and biological tumour parameters in premenopausal, lymph node-negative breast cancer patients. Expression of Bcl-2 was determined using immunohistochemistry on paraffin-embedded sections in a series of 441 premenopausal, lymph node-negative breast cancers of patients randomised to receive perioperative chemotherapy (5-fluorouracil, doxorubicin, cyclophosphamide) or no perioperative chemotherapy. Immunohistochemistry of Bcl-2 was evaluated by scoring both staining intensity (0-3) and number of positive cells (0-2). Using these scores tumours were grouped into categories 0-6. It was found that 9.2% of the tumours were completely negative (0), 17.2% weakly (1 + 2), 41.6% moderately (3 + 4) and 31.9% strongly positive (5 + 6) for Bcl-2. A positive correlation was found between high Bcl-2 expression and oestrogen (P < 0.001) and progesterone receptor positivity (P < 0.001) and low tumour grade (P < 0.001), whereas high Bcl-2 expression was negatively correlated with p53 (P < 0.001) and c-erb-B-2 positively (P < 0.001), high Ki-67 index (P < 0.001), mitotic index (P < 0.001) and large tumour size (P = 0.006). Patients with tumours expressing high levels of Bcl-2 (overall score 3-6) had a significantly better disease-free (P = 0.004) and overall (P = 0.009) survival. However, in a multivariate model this association no longer remained significant. There was a trend for an effect of adjuvant chemotherapy on disease-free survival both for patients with Bcl-2-positive (HR-0.61, 95% CI 0.35-1.06, P = 0.07) and negative (HR = 0.55, 95% CI 0.27-1.12, P = 0.09) breast tumours at a median follow-up of 49 months. The level of Bcl-2 expression does not seem to predict response to perioperative chemotherapy in premenopausal, lymph node-negative breast cancer patients. High levels of Bcl-2 are preferentially expressed in well-differentiated tumours and are associated with favourable prognosis. However, Bcl-2 expression is not an independent prognostic factor in this patient series.

Specific tumor-cell killing with adenovirus vectors containing the apoptin gene

Specificity is an essential prerequisite for cancer gene therapy. Recently we described that apoptin, a protein of 121 amino acids which is derived from the chicken anemia virus, induces programmed cell death or apoptosis in transformed and malignant cells, but not in normal, diploid cells (Danen-van Oorschot AAAM et al, Proc Natl Acad Sci USA 1997; 94: 5843–5847). This protein has an intrinsic specificity that allows it to selectively kill tumor cells, irrespective of the p53 or Bcl-2 status of these cells. Hence, it is attractive to explore the use of the apoptin gene for therapeutic applications, viz cancer gene therapy. In this paper, we describe the generation and characterization of an adenovirus vector, AdMLPvp3, for the expression of apoptin. Despite the fact that apoptin ultimately induces apoptosis in the helper cells, which are transformed by the adenovirus type 5 early region 1 (E1), the propagation kinetics and yields of AdMLPvp3 are similar to those of control vec- tors. Infection with AdMLPvp3 of normal rat hepatocytes in cell culture did not increase the frequency of apoptosis. In contrast, in the hepatoma cell lines HepG2 and Hep3b, infection with AdMLPvp3, but not with control vectors, led to a rapid induction of programmed cell death. Experiments in rats demonstrated that AdMLPvp3 could be safely administered by intraperitoneal, subcutaneous or intravenous injection. Repeated intravenous doses of AdMLPvp3 were also well tolerated, indicating that the apoptin-expressing virus can be administered without severe adverse effects. In a preliminary experiment, a single intratumoral injection of AdMLPvp3 into a xenogeneic tumor (HepG2 cells in Balb/Cnu/nu mice) resulted in a significant reduction of tumor growth. Taken together, our data demonstrate that adenovirus vectors for the expression of the apoptin gene may constitute a powerful tool for the treatment of solid tumors.

Loss of Bcl-2 in invasive breast cancer is associated with high rates of cell death, but also with increased proliferative activity.

Bcl-2 has been demonstrated to inhibit apoptosis in breast cancer cells in vitro, and the ratio between Bcl-2 and its proapoptotic homologue Bax seems to be an important determinant of cellular sensitivity to induction of apoptosis. However, little information is available on the relationship between Bcl-2 and the rate of apoptotic and necrotic cell death in breast tumours. From a series of 441 premenopausal, lymphnode-negative breast cancer patients, a subset of 49 tumours was selected in which immunostaining for the 26-kDa isoform of Bcl-2 was either absent (n = 23) or very high (n = 26). High expression of Bcl-2 was found to be strongly associated with low rates of apoptotic (P < 0.001) and necrotic cell death (P < 0.001). The mean value of the apoptotic index was 2.69%+/-1.40% in Bcl-2-negative tumours and 0.68%+/-1.00% in Bcl-2-positive tumours. Expression of the proapoptotic protein Bax correlated neither with Bcl-2 nor with the frequency of apoptotic cells. Immunostaining for the antiapoptotic Bcl-2 homologue BcI-X(L) correlated with Bcl-2 expression (P < 0.001) but not with apoptosis. High proliferation rate and high tumour grade (Bloom-Richardson) were strongly associated with absence of Bcl-2 expression (P< 0.001). p53 accumulation was associated with absence of Bcl-2 expression and increased apoptotic activity. Loss of Bcl-2 expression was strongly correlated with increased apoptotic and necrotic cell death, high proliferation rate and high tumour grade, supporting a model in which Bcl-2 not only mediates cell death, but also cell division in breast cancer tissue, and in which regulation of cell division and cell death are tightly linked.

Outgrowth of BT-474 human breast cancer cells in immune-deficient mice: a new in vivo model for hormone-dependent breast cancer.

The effect of co-inoculation of basement membrane matrix, Matrigel and two human breast cancer cell lines, BT-474 and SK-BR-3, was tested in immune-deficient mice. Both cell lines strongly overexpress c-ErbB-2 protein, whereas only BT-474 is reported to be oestrogen receptor positive. Co-inoculation of Matrigel and BT-474 cells but not of Matrigel and SK-BR-3 cells resulted in tumour formation in bg-nu-xid mice. Oestrogen supplementation greatly enhanced tumorigenicity, but did not seem to be an absolute requirement. In vivo, BT-474 cells grow as a poorly differentiated adenocarcinoma with a doubling time of 9.4 +/- 1.1 days after inoculation into the neck region. A high proliferative activity appears to be compensated by a relatively high rate of cell loss, as BT-474 tumours contain many cells with the typical morphology of apoptotic cell death. Wild-type p53, known to participate in the induction of apoptosis, is absent from the tumours, whereas Bcl-2, known to inhibit apoptosis, is expressed at intermediate levels. BT-474 tumours tend to metastasise to the regional lymph nodes and are capable of forming micrometastatic lesions in the lung. Flow cytometrical analysis of DNA ploidy demonstrated no change in tumours compared with the cell line. Immunohistochemical and flow cytometrical detection of a number of hormone and growth factor receptors, transcription factors, cell adhesion molecules and proteins involved in proliferation and cell death demonstrated no major changes in ploidy and phenotype of tumours compared with the cell line. High expression of the cell-surface molecules c-ErbB-2 and episialin make it a potentially useful model for research in immune therapy.

Increasing the effective concentration of melphalan in experimental rat liver tumours: comparison of isolated liver perfusion and hepatic artery infusion.

Regional chemotherapy allows further exploitation of the steep dose response curve of most chemotherapeutic agents, while systemic toxicity remains tolerable. We investigated the difference in maximally tolerated dose, pharmacokinetics and antitumour effect comparing administration of melphalan as a bolus in isolated liver perfusion (ILP) or via hepatic artery infusion (HAI). For these in vivo studies an experimental model for liver metastases in male WAG/Ola rats is obtained by subcapsular inoculation of CC531 rat colon carcinoma cells. In this system, ILP allowed administration of a two times higher dose than HAI (12 mg kg-1 vs 6 mg kg-1). In both treatment modalities systemic toxicity (leukopenia) was dose limiting. No hepatic toxicity was observed. Bolus administration of the maximally tolerated doses of melphalan in HAI (6 mg kg-1) and ILP (12 mg kg-1) resulted in four times higher concentrations in both liver and tumour tissue of the ILP treated rats. However, the ratio of mean drug concentration in liver vs tumour tissue appeared to be 1.5 times that found for HAI. In the range of the in tumour tissue measured melphalan concentrations the CC531 cells showed a steep dose response relationship in vitro. Whereas HAI resulted in significant tumour growth delay, complete remissions were observed in 90% of the rats treated with ILP. This study shows that with 12 mg kg-1 melphalan in ILP highly effective drug concentrations are achieved in CC531 tumour tissue; although the melphalan concentration in liver tissue shows an even higher increase than in tumour tissue, hepatic toxicity is negligible in this dose range.(ABSTRACT TRUNCATED AT 250 WORDS)

TREATMENT OF COLORECTAL CANCER METASTASES CONFINED TO THE LIVER

Hepatic metastases are a major cause of death in patients with disseminated colorectal cancer. The prognosis of patients with hepatic metastases is very poor and mainly determined by the extent of hepatic disease at presentation. In these patients, the goal of any treatment is to obtain a complete tumour remission in the liver; this is the only way to obtain a significant survival benefit. In this overview, we summarise data from (i) studies comparing survival of patients after primary resection of liver metastases with survival after repeat liver resections, (ii) studies comparing hepatic arterial infusion of fluoropyrimidines with systemic delivery of these anticancer drugs, and (iii) phase I/II studies on isolated liver perfusion (ILP) with alkylating compounds. Furthermore, we discuss alternative strategies to combat liver metastases, including those taking advantage of an ILP setting.

EFFECTIVENESS OF ISOLATED LIVER PERFUSION WITH MITOMYCIN C IN THE TREATMENT OF LIVER TUMOURS OF RAT COLORECTAL CANCER

Dose limiting systemic toxicity prevents sufficient exploitation of the steep dose response relationship of most anticancer agents. In our rat liver tumour model (the CC531 colorectal carcinoma), isolated liver perfusion allows administration of higher doses of mitomycin C than hepatic artery infusion, while systemic toxicity remains minimal. To determine the temporal pattern of mitomycin C induced cytokinetic changes, we analysed flow cytometric DNA histograms of CC531 liver tumours from rats treated with high dose mitomycin C (3.2 mg kg-1) via hepatic artery infusion and sacrificed at different time intervals after treatment. Between 12 and 36 h after treatment, the fraction of cells in late S and G2/M phase had markedly increased. The effects of administration of the respective maximally tolerated doses of mitomycin C in isolated liver perfusion and via hepatic artery infusion on progression of tumour cells through the cell cycle and on gross tumour growth were compared. Isolated liver perfusion with mitomycin C resulted in a significant increase in the proportion of cells in mid and late S, and in some accumulation of cells in early S and G2/M phase at 24 and 48 h after treatment. In contrast, after hepatic artery infusion a significant increase of the fraction of cells in G2/M phase was observed at 24 h after treatment. Monitoring tumour growth after isolated liver perfusion five out of seven rats showed a complete tumour remission, while after hepatic artery infusion only a minimal growth delay was detected. This study demonstrates that isolated liver perfusion in the rat CC531 liver tumour model allows the administration of a well-tolerated dose of mitomycin C being high enough to induce a marked DNA synthesis inhibition and even complete tumour remission.

A prolactin-dependent, metastasising rat mammary carcinoma as a model for endocrine-related tumour dormancy

In order to study the growth kinetics of breast tumours during long-term hormonal withdrawal, we developed a transplantable, invasive mammary carcinoma EMR-86 that originated in a female WAG/Olac rat bearing a subcutaneously implanted oestrogen pellet (EP). Outgrowth of transplanted tumours occurs only in the presence of an EP, and metastases are formed in lungs and regional lymph nodes. Subsequent EP removal induces rapid regression. However, tumours do not disappear completely, as small nodules persist. These dormant tumour remnants can be restimulated even after long periods. Because EP-stimulated tumours regressed after treatment with bromocriptine and dormant tumours in non-oestrogenized rats grew out after treatment with perphenazine, prolactin is the major growth-stimulating hormone in this model. Cell kinetics in the growing, regressing and dormant phase were studied by immunocytochemical detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in tissue sections. BrdUrd labelling indices decreased from 21.6 +/- 3.0% to less than 1% within 7 days after EP removal. After prolonged hormonal withdrawal (up to 90 days) BrdUrd-labelled tumour cells could always be demonstrated (range 0.4-0.8%), without a concomitant increase in tumour volume. Additional treatment either with bromocriptine or with ovariectomy could not significantly reduce this residual proliferative activity, as demonstrated by continuous BrdUrd labelling experiments. The results indicate that in vivo dormancy may represent a steady state of cell division and cell loss, rather than an accumulation of cells in a non-cycling G0 state.

Comparison of the effects of microwave heating and high pressure cooking for antigen retrieval of human and rat Bcl-2 protein in formaldehyde-fixed, paraffin-embedded sections

Immunohistochemical detection of expression of the anti-apoptotic Bcl-2 protein is widely studied as a putative prognostic and predictive factor in various types of cancer. For that purpose, heating for 10 min by microwave (MW) up to 100o C in citrate buffer, pH 6.0, prior to immunostaining is often used to retrieve Bcl-2 antigens in archival formalin-fixed, paraffin-embedded tissue. We recently reported that Bcl-2 is not only a cytoplasmic protein, but that it is present also in interphase nuclei and that it strongly associates with mitotic chromosomes. Furthermore, we showed that binding of the monoclonal antibody (MAb) #124 with nuclear/chromosomal epitopes is diminished by formaldehyde-based fixatives and cannot be restored by MW treatment for 10 min. Here we report that prolonged MW heating or heating up to 130°C in a high pressure cooker (HPC), despite improved cytoplasmic immunostaining, fails to retrieve nuclear/chromosomal Bcl-2 epitopes recognized by the MAb #124 in human tissues. In contrast, these procedures can retrieve nuclear/chromosomal Bcl-2 epitopes detected by polyclonal #15616E antibodies in rat tissues. The specificity of these epitopes was confirmed by Western blot analysis of tissues treated by MW heating or HPC.