F. Barzanti

In vitro preclinical models for a rational design of chemotherapy combinations in human tumors

Today, drug combinations are frequently used in the treatment of cancer to increase therapeutic efficacy. Currently used clinical protocols for cancer combination therapies are mainly obtained empirically or on the basis of results from previous clinical trials. Information obtained from clinical protocols is invaluable, but it is time-consuming, expensive and does not provide data on the biochemical and molecular mechanisms of interaction of the drugs used in combination treatments at cellular level. Therefore, in vitro drug combination studies on established cell lines or primary cell cultures play an important role in designing and optimising combination protocols. A variety of in vitro assays and different mathematics models have been developed to investigate cytotoxic effects and to analyse the type of drug interactions. Increased knowledge of the cellular targets of traditional and new drugs and the development of new technologies have resulted in a new role for the in vitro tests which are no longer used only to evaluate the cytotoxic effects of drugs, but also to investigate the interference on cell cycle, induction of apoptosis and molecular or biochemical interactions. A review on in vitro preclinical tests used to evaluate the effects of drug combinations and to design the rationale of combined chemotherapy protocols is presented.

Schedule-dependent interaction of doxorubicin, paclitaxel and gemcitabine in human breast cancer cell lines

We showed previously that a sequential treatment with doxorubicin (4 hr) followed by paclitaxel (24 hr) (Dox→Pacl) induces a synergistic cytotoxic effect in the BRC‐230 breast cancer cell line and in human primary breast cancer cultures. The validity of this experimental finding was confirmed in a clinical phase I/II study on advanced breast cancer patients. To improve the cytotoxic effect obtained by the Dox→Pacl sequence, we analyzed the effect of adding gemcitabine (Gem) to the Dox→Pacl sequence in a preclinical study. Our study was performed on BRC‐230 and MCF‐7 cell lines, and cytotoxic activity was evaluated by the sulforhodamine B assay and the type of drug interaction by Drewinkos test. When Gem (0.01 μg/ml for 24 hr) was given immediately or 24 hr after Dox→Pacl, an antagonistic cytotoxic effect was observed. Conversely, a synergistic effect was found when Gem was given 48 hr after Dox→Pacl. From results of flow cytometric analysis, the synergistic effect was attributed to cell cycle perturbation. Cells were arrested in G2‐M (95% in treated vs. 21% in control samples) 24 hr after Dox→Pacl treatment. The block progressively recovered thereafter, and after a further 24 hr, at the time of Gem treatment, the cells progressed into the G1‐S phase boundary (the cell cycle phase susceptible to the cytocidal effect of the drug). Our findings suggest that the interactions of Dox, Pacl and Gem are highly schedule‐ and time‐dependent and should be taken into consideration in the planning of clinical protocols. Int. J. Cancer 80:413–416, 1999.

Docetaxel and gemcitabine activity in NSCLC cell lines and in primary cultures from human lung cancer.

The activity of the following drugs was investigated in two established NSCLC cell lines: docetaxel, gemcitabine, vinorelbine, paclitaxel, doxorubicin (0.01, 0.1, 1 μg ml–1), cisplatin, ifosfamide (1, 2, 3 μg ml–1) and carboplatin (2, 4, 6 μg ml–1). The cytotoxic activity was evaluated by the sulphorhodamine B assay. The two most active drugs, docetaxel and gemcitabine, used singly and in association, were investigated as a function of treatment schedule. The sequence docetaxel→gemcitabine produced only a weak synergistic interaction in RAL but a strong synergism in CAEP cells. The synergistic interaction increased in both cell lines after a 48-h washout between the drug administrations. Flow cytometric analysis showed that in docetaxel→gemcitabine sequence, docetaxel produced a block in G2/M phase and, after 48 h, provided gemcitabine with a large fraction of recovered synchronized cells in the G1/S boundary, which is the specific target phase for gemcitabine. Conversely, simultaneous treatment induced an antagonistic effect in both cell lines, and the sequential scheme gemcitabine→docetaxel produced a weak synergistic effect only in RAL cells. Moreover, the synergistic interaction disappeared when washout periods of 24 or 48 h between two drug administrations were adopted. The synergistic activity of docetaxel→ 48-h washout→gemcitabine was confirmed in 11 of 14 primary cultures, which represents an important means of validating experimental results before translating them into clinical practice.

Comparison between differentcell kinetic variables in human breast cancer

Cell kinetics holds a prominent role among biological factors in predicting clinical outcome and response to treatment in neoplastic patients. Different cell kinetic variables are often considered as valid alternatives to each other, but the limited size of case series analysed in several studies and the lack of simultaneous determinations of all the variables on the same tumours do not justify this conclusion. In the present study, the correlation between [3H]thymidine labelling index ([3H]dT LI), flow cytometric S phase cell fraction (FCM‐S) and Ki‐67 immunoreactivity (Ki‐67/MIB‐1) was verified and the type of correlation with the most important clinical, pathological and biological patient and tumour characteristics was investigated in a very large series of breast cancer patients. Ki‐67/MIB‐1, FCM‐S and [3H]dT LI were determined in 609, 526 and 485 patients, respectively, and all three cell proliferation indices were evaluated in parallel on the same tumour in a series of 330 breast cancer patients. All the cell kinetic determinations were performed within the context of National Quality Control Programmes. Very poor correlation coefficients (ranging from 0.37 to 0.18) were observed between the different cell kinetic variables determined in parallel on the same series of breast cancers. Moreover, Ki‐67/MIB‐1 and FCM‐S showed a significant relationship with histological type, grade and tumour size, whereas statistically significant correlations were not observed for [3H]dT LI. In conclusion, the results show that the different cell kinetic variables provide different biological information and cannot be considered as alternatives to each other.

Flow-cytometric determination of tumor cells in lymph nodes.

In solid tumors, metastasis occurs through the dissemination of tumor cells in the bloodstream and the lymphatic system. In particular, lymph node infiltration gives useful prognostic information and represents one of the most important factors for selecting the type of clinical treatment in disease management. Furthermore, the analysis of lymph node infiltration has become important for identifying patients with breast cancer or malignant melanoma who may be candidates for regional lymph node dissection. Tumor cells in lymph nodes are currently identified in tissue sections using morphological and immunohistochemical analyses, but these approaches are time-consuming, and micrometastases may escape detection. The aim of the present study was to define the potential of a flow cytometric (FCM) determination based on cell size and autofluorescence to shorten the time required for lymph node analysis. The sensitivity of the FCM approach, defined on mixtures of tumor cells from established cell lines and peripheral blood lymphocytes (PBLs) at different concentrations, was 1 tumor cell/1,000 PBLs. FCM analysis was performed on 89 lymph nodes, 29 from breast, 41 from lung and 19 from colon cancer patients. Agreement between FCM and morphological results, used as gold standard, was observed in 83% of the cases, and there was a 90% sensitivity to the FCM approach for each tumor type. Disagreement was observed for 15 lymph nodes and was due, in the majority of cases (80%), to FCM-positive and morphologically negative results. A large number of patients and a more accurate pathological examination of consecutive histological sections of lymph nodes are needed to further evaluate the validity of the FCM approach.

Establishment and characterization of two new cell lines derived from human metastatic breast carcinomas

Two human cancer cell lines (MA 2 and MA 3) were established from pleural effusions of infiltrating ductal carcinomas of the breast.The lines were maintained in continuous monolayer culture withdoubling times of 70 (MA 2) and 78 (MA 3) hr for more than two years and possessed extensively rearranged abnormal karyotypeswith modal chromosome number of 83 (MA 2) and 81 (MA 3) and DNA index values of 1.65 and 1.77, respectively. No amplifications or rearrangements were evident in the c-myc, int-2, c-erb B2, c-Ha-ras, or hst 1 genes in MA 2 and MA 3 cell lines.The clinical histories of the patients from whom the cell lines were derived are reported and compared with the results observed inthe cell lines in vitro. The presence of CEA, CA 15-3, and MCA tumormarkers observed in the primary tumor tissues was retained by the established cell lines. While the primary tumor tissueswere ER+/PgR borderline + (MA 2) and ER−/PgR+(MA 3), the MA 2 line was ER+/PgR− and the MA 3 line remained ER−/PgR+.The MDR P-glycoprotein was not expressed either in primary tumor tissues or in the respective cell lines. High expression of cytokeratins7, 18, and 19 was evident by immunohistochemical analysis in each cell line, whereas cytokeratins 8 and 17 were poorly or not at allexpressed. The treatment history of the patients fromwhom the cell lines were derived involved CMF followed six monthslater by novantrone and cisplatin plus VP 16 (MA 2) and FEC followedfour years later by CMF (MA 3). The chemosensitivitypattern assay of the cell lines indicated that the MA 2 linewas sensitive to doxorubicin, cisplatin, and vinblastine, whereas theMA 3 line was sensitive to doxorubicin and cisplatin.The characteristics of these cell lines indicate them to be a goodexperimental model to investigate breast cancer biology and anticancerdrug response.

Combined 4-hydroxy-ifosfamide and vinorelbine treatment in established and primary human breast cell cultures

BACKGROUND Vinorelbine and ifosfamide are active drugs against breast cancer, but the best treatment schedule has yet to be defined by preclinical or clinical studies. The antitumor activity of 4-hydroxy-ifosfamide (4-OH-IF), the active form of ifosfamide, and vinorelbine (VNB) and their interaction were investigated in two established breast cancer cell lines (MCF-7 and BRC-230) and in 10 primary breast cancer cultures. MATERIALS AND METHODS Cytotoxic activity was evaluated by a highly efficient clonogenic assay (HECA). The median-effect principle was applied to evaluate synergistic and antagonistic interactions and the corresponding combination index values were calculated. Cell cycle perturbations were analysed by flow cytometry. RESULTS In MCF-7 and BRC-230 cell lines the sequence VNB for 4 hours followed by 4-OH-IF for 24 hours produced an antagonistic effect. Conversely, the inverse sequential scheme, 4-OH-IF-->VNB provided synergistic effects on both cell lines. The synergism was associated with a strong block in the G2-M phase. Synergistic activity of 4-OH-IF-->VNB sequence was confirmed in 7 of 10 primary breast cancer cultures. CONCLUSIONS In conclusion, the sequence 4-OH-IF-->VNB appeared to be the most effective scheme both in established cell lines and in primary breast cancer cultures.