Donata Casadei Giunchi

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.

Disease-Free Survival Advantage of Adjuvant Cyclophosphamide, Methotrexate, and Fluorouracil in Patients With Node-Negative, Rapidly Proliferating Breast Cancer: A Randomized Multicenter Study

PURPOSE According to one of the most recent key scientific questions concerning the use of biomarkers in clinical trials, we investigated whether node-negative breast cancer patients, defined as high-risk cases on the basis of tumor cell proliferation, could benefit from cyclophosphamide, methotrexate, and fluorouracil (CMF) adjuvant therapy. PATIENTS AND METHODS Two hundred eighty-one patients with negative nodes and rapidly proliferating tumors, defined according to thymidine labeling index (TLI), were randomized to receive six cycles of CMF or no further treatment after surgery +/- radiotherapy. RESULTS The 5-year disease-free survival (DFS) was 83% for patients treated with CMF compared with 72% in the control group (P: =.028). Adjuvant treatment reduced both locoregional and distant metastases. When clinical outcome was analyzed in cell kinetic subgroups characterized according to tertile criteria, compared with patients in the control arm, 5-year DFS was significantly higher after adjuvant CMF in patients with TLI values in the second (78% v 88%, respectively; P: =.037) and third tertiles (58% v 78%, respectively; P: =.024). CONCLUSION The results from this randomized clinical study indicate that patients with node-negative, rapidly proliferating tumors significantly benefit from adjuvant CMF.

Addition of 5-fluorouracil to doxorubicin-paclitaxel sequence increases caspase-dependent apoptosis in breast cancer cell lines

IntroductionThe aim of the study was to evaluate the activity of a combination of doxorubicin (Dox), paclitaxel (Pacl) and 5-fluorouracil (5-FU), to define the most effective schedule, and to investigate the mechanisms of action in human breast cancer cells.MethodsThe study was performed on MCF-7 and BRC-230 cell lines. The cytotoxic activity was evaluated by sulphorhodamine B assay and the type of drug interaction was assessed by the median effect principle. Cell cycle perturbation and apoptosis were evaluated by flow cytometry, and apoptosis-related marker (p53, bcl-2, bax, p21), caspase and thymidylate synthase (TS) expression were assessed by western blot.Results5-FU, used as a single agent, exerted a low cytotoxic activity in both cell lines. The Dox→Pacl sequence produced a synergistic cytocidal effect and enhanced the efficacy of subsequent exposure to 5-FU in both cell lines. Specifically, the Dox→Pacl sequence blocked cells in the G2-M phase, and the addition of 5-FU forced the cells to progress through the cell cycle or killed them. Furthermore, Dox→Pacl pretreatment produced a significant reduction in basal TS expression in both cell lines, probably favoring the increase in 5-FU activity. The sequence Dox→Pacl→48-h washout→5-FU produced a synergistic and highly schedule-dependent interaction (combination index < 1), resulting in an induction of apoptosis in both experimental models regardless of hormonal, p53, bcl-2 or bax status. Apoptosis in MCF-7 cells was induced through caspase-9 activation and anti-apoptosis-inducing factor hyperexpression. In the BRC-230 cell line, the apoptotic process was triggered only by a caspase-dependent mechanism. In particular, at the end of the three-drug treatment, caspase-8 activation triggered downstream executioner caspase-3 and, to a lesser degree, caspase-7.ConclusionIn our experimental models, characterized by different biomolecular profiles representing the different biology of human breast cancers, the schedule Dox→Pacl→48-h washout→5-FU was highly active and schedule-dependent and has recently been used to plan a phase I/II clinical protocol.

Cell proliferation as a predictor of response to chemotherapy in metastatic breast cancer: A prospective study

Many biologic prognostic markers are available for patientswith breast cancer, and considerable interest has beendevoted to confirm preliminary evidence of their roleas indicators of treatment response. It remains tobe assessed whether such markers are predictors ofresponse only to first-line or also to successivetherapies. Proliferative activity, defined by the3H-thymidine labelingindex (TLI), was determined on the primary lesionfrom 76 patients at time of first diagnosis.At relapse, patients underwent chemotherapy as absolute (48cases) or relative (28 cases) first-line treatment, andtheir clinical response was analyzed in relation tothe TLI of the primary lesion. The objectiveclinical response was significantly higher for rapidly (47%;CL, 33–61%) than for slowly proliferating tumors (15%;CL, 1–29%). These findings held true also whenadjusted for metastatic site, previous treatment, chemotherapy regimenadministered, and hormone receptor status. However, the directrelation between cell proliferation and benefit from chemotherapyheld true only when such a treatment wasused as an absolute first-line approach. Cell proliferationof primary lesions represents a consistent indicator ofresponse to chemotherapy over time. Previously administered regimens,at least hormone therapy, could alter the proliferation-relatedchemosensitivity profile of individual tumors.

Prognostic Relevance of Mitotic Activity in Patients with Node-Negative Breast Cancer

The prognostic relevance of mitotic activity was analyzed in a series of 306 patients with node-negative breast cancer treated with locoregional therapy alone, until early relapse. Mitotic activity was evaluated as the number of mitotic figures per 10 high-power fields (mitotic activity index) or per 1000 tumor cells (mitotic index). Counting was carried out blindly by two observers. A high correlation was observed between the two determinations (rs = .96, P < .001). For clinical analysis, three mitotic activity index subgroups (mitotic figures/field ≤ 9, 10–19 and more than 19, according to grading criteria) and three mitotic index subgroups (percentage of mitotic figures less than 0.10, 0.11–0.50 and more than 0.50, according to tertile criteria) were considered. No relation was observed between mitotic variables and 6-year disease-free survival, whereas distant disease-free survival was strongly related to mitotic figures per 10 fields (85%, 89% and 70%, P = .012) and to the percentage of mitotic figures out of a total 1000 tumor cells (87%, 86% and 75%, P = .017). Similarly, both mitotic indices were significantly related to 6-year overall survival (99%, 95% and 77%, P < .001, for mitotic figures per 10 fields and 99%, 93% and 82%, P < .001, for the percentage of mitotic figures). These findings were particularly evident in patients with tumors of 1–2 cm. In conclusion, a high number of mitotic figures is associated with a higher probability of developing distant metastases and a shorter survival. The critical point remains the standardization of the preanalytical and analytical steps within quality control programs.

Breast carcinoma stage in relation to time interval since last mammography: A registry-based study

In the Romagna Region of Italy, mammography screening for breast carcinoma (BC) was implemented as a routine practice in the regular healthcare system. The Romagna Cancer Registry evaluated the effects of self‐selection for mammography on the stage of BC at the time of presentation.

Combination chemotherapy in advanced ovarian carcinoma.

Ovarian carcinoma is the fifth most common cause of death among women in western countries. It is often diagnosed in an advanced stage (FIGO Stage III and IV) and requires effective chemotherapy as first-line treatment. The advent of cisplatin combined with adriamycin and cyclophosphamide has remarkably increased the response rate in advanced disease. The authors report 31 cases of epithelial ovarian neoplasia, without prior chemotherapy, treated with cisplatin, adriamycin and cyclophosphamide (PAC I). Of the 30 evaluable patients, 15 had clinical complete remissions (cCR = 50%), 10 clinical partial remissions (cPR = 33%) and 5 no response (NR = 17%). The total response (cCR + cPR) was equal to 83%. Twelve of the 15 patients in cCR underwent second-look laparotomy; in 8 of these cases, histologic and cytologic confirmation of CR was obtained. PAC I was found to be a highly effective therapeutic regimen with moderate toxicity. The individual toxicity reported was gastroenteric (nausea and vomiting), but transitory. No chronic toxic side-effects from cisplatin or adriamycin were noted. However, more definitive results must be obtained to verify its impact on the prolongation of survival.