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There are no validated predictors of benefit from anthracyclines. We compared cyclophosphamide, methotrexate, 5-fluorouracil (CMF), and epirubicin in different sequences with CMF alone in a phase III trial on operable breast cancers. Outcomes were analyzed in relation to tumor biological profiles to identify potential predictors of the efficacy of different treatments/drug combinations. Patients with N- or 1-3N+ tumors, were randomized to receive (a) epirubicin (4 cycles) followed by CMF (4 cycles); (b) CMF (4 cycles) followed by epirubicin (4 cycles), or (c) CMF (6 cycles) alone. Immunohistochemical assessments of estrogen (ER) and progesterone (PgR) receptors, HER2 and Ki67 were available for 705 patients (arm A/B/C: 276/269/160). Prognostic and predictive relevance was analyzed by log-rank tests and Cox models. Ki67 > 20 % and absent/low expression of ER and PgR were associated with worsen disease-free (DFS) and overall survival (OS). In patients with triple negative tumors (ER-, PgR-, HER2-), epirubicin-containing regimens yielded better DFS (HR 0.33, 95 % CI 0.17-0.62, P = 0.0007) and OS (HR 0.24, 95 % CI 0.10-0.57, P = 0.001) compared with CMF alone, whereas no differences were found in patients with HER2-positive (HER2+, ER-, PgR-) subtype. Treatment by subtype interaction (HER2-positive vs. others) was significant for DFS (χ (2) = 6.72, P = 0.009). In triple unfavorable (ER-, PgR-, Ki67 > 20 %) tumors, the use of epirubicin yielded better DFS (HR 0.45,95 % CI 0.26-0.78, P = 0.005) and OS (HR 0.30, 95 % CI 0.15-0.63, P = 0.001). Epirubicin-containing regimens seem to be superior to CMF alone in patients with highly proliferating, triple negative or triple unfavorable tumors.

Adjuvant cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) have proven highly effective in rapidly proliferating breast cancer (RPBC). It has also been seen that sequential administration of doxorubicin and CMF is superior to their alternation, especially in indolent tumors. In a phase III study, we evaluated whether adjuvant epirubicin (E) followed by CMF is superior to the inverse sequence in RPBC. Patients with node-negative or 1-3 node-positive RPBC (Thymidine Labeling Index > 3% or histological grade 3 or S-phase > 10% or Ki67 > 20%) were randomized to receive E (100 mg/m(2) i.v. d1, q21 days for 4 cycles) followed by CMF (600, 40, 600 mg/m(2) i.v. d1 and 8, q28 days for 4 cycles) (E → CMF) or CMF followed by E (CMF → E) or CMF for 6 cycles. From November 1997 to December 2004, 1066 patients were enrolled: E → CMF 440, CMF → E 438, and CMF 188. At a median follow-up of 69 months, 5-year OS was 91% (95% CI 88-94) for E → CMF and 93% (95% CI 90-95) for CMF → E, with adjusted hazard ratio of 0.88 (95% CI 0.58-1.35), and DFS was 80% in both arms, with adjusted hazard ratio of 0.99 (95% CI 0.73-1.33, Cox model). Adverse events were similar, apart from a higher rate of neutropenia in the CMF → E arm. No important differences in clinical outcome were observed between the two different sequences, making both a valid option in early breast cancer. Further molecular characterization of the tumors might help to identify subgroups achieving higher benefit from either sequence.

The randomized multicenter study on rapidly proliferating breast cancer, assessed according to thymidine labelling index (TLI), was activated at the end of the 1980s. The present work investigated whether and to what degree the short-term advantages observed from adjuvant CMF (cyclophosphamide, methotrexate, 5-fluorouracil) were maintained at a longer follow-up. Two hundred and eighty-one patients with node-negative and high TLI tumors were randomized to receive six cycles of CMF or no further treatment. At a median follow-up of 12 years, CMF produced a 25% and 20% relative reduction in relapse and death cumulative incidence, respectively. A breakdown analysis identified a subgroup of patients with intermediate proliferating tumors for whom a 70% and 73% reduction in relapse and death was observed in the intention-to-treat population. An even higher reduction of 80% and 84% in relapse and death was seen for the patients who had received the full CMF dose. We identified a subgroup of patients with intermediate proliferating tumors in whom the high benefit obtained from adjuvant CMF was maintained at a long-term follow up.

Several studies have shown a role of the tyrosine kinase receptor, c-kit, and its ligand, SCF, during organogenesis, normal cell development and growth of some tumor histotypes. In breast cancer, studies using different methodologies have shown conflicting results. In the present study we analyzed c-kit and SCF in 14 normal mammary epithelia samples, in 16 in situ and in 75 invasive breast cancers. The expression of c-kit and SCF protein was analyzed by immunohistochemistry and mRNA expression was evaluated by in situ hybridization and reverse-transcriptase polymerase chain reaction (RT-PCR). The different methodologies gave somewhat different results. Using immunohistochemistry and in situ hybridization, protein and mRNA expression of c-kit and SCF were high in normal mammary gland, significantly lower in in situ and almost completely undetectable in invasive breast cancer. Conversely, using RT-PCR, mRNA expression was observed in normal tissue and in all pathologic lesions of mammary gland, probably due to the high sensitivity of the methodology or to the positivity of elements other than tumor cells expressing the receptor and/or its ligand. These results suggest that the c-kit/SCF pathway plays an important role in the maintenance of normal growth of mammary epithelium and that the process of malignant transformation is accompanied by their progressive loss. Furthermore, we demonstrated that different results are attributable to different methodologies and that morphologic approaches are the most reliable for defining the cellular source of c-kit or SCF expression.

In the last few decades, much effort has been directed towards identifying the phenotypic or functional aspect of tumor cells which can contribute to a biofunctional staging for improving the accuracy of pathologic staging used for identifying patients at different risk. Among known biologic factors, the proliferative capacity of the tumor cell population, a feature common to all tumors, has been widely investigated. Several approaches have been used to measure different aspects of the cell cycle. Among these, the thymidine labeling index (TLI) represents the fraction of cells in S-phase cell fraction and is based on the active incorporation of labelled thymidine into DNA. From basic studies conducted on several thousands of patients, the TLI of primary breast cancers appears closely related to steroid receptor status and generally unrelated to pathologic stage. Retrospective analyses performed on large series of patients treated with local regional therapy alone have consistently shown the relevance of TLI value to clinical aggressiveness in terms of relapse-free survival and overall survival. Moreover, TLI is a prognostic indicator which is independent of tumor size, steroid receptors, and p53 and bc12 protein expression, and which, together with patient age and tumor size, is able to identify patients at different risk of loco-regional or distant metastases. Recently, a direct relationship between TLI and response to polychemotherapy has been shown in patients with operable and advanced breast cancers. This finding, derived from retrospective and recently confirmed in prospective clinical studies, has led to the activation of cell kinetics based therapeutic protocols for patients with node-negative and one to three node-positive operable breast cancers.

The purpose of the present study was to assess prognostic factor for metachronous contralateral recurrence of breast cancer (CBC). Two factors were of particular interest, namely estrogen (ER) and progesterone (PgR) receptors assayed with the biochemical method in primary tumor tissue. Information was obtained from a prospective clinical database for 1763 axillary node-negative women who had received curative surgery, mostly of the conservative type, and followed-up for a median of 82 months. The analysis was performed based on both a standard (linear) Cox model and an artificial neural network (ANN) extension of this model proposed by Faraggi and Simon. Furthermore, to assess the prognostic importance of the factors considered, model predictive ability was computed. In agreement with already published studies, the results of our analysis confirmed the prognostic role of age at surgery, histology, and primary tumor site, in that young patients (< or = 45 years) with tumors of lobular histology or located at inner/central mammary quadrants were at greater risk of developing CBC. ER and PgR were also shown to have a prognostic role. Their effect, however, was not simple in relation to the presence of interactions between ER and age, and between PgR and histology. In fact, ER appeared to play a protective role in young patients, whereas the opposite was true in older women. Higher levels of PgR implied a greater hazard of CBC occurrence in infiltrating duct carcinoma or tumors with an associated extensive intraductal component, and a lower hazard in infiltrating lobular carcinoma or other histotypes. In spite of the above findings, the predictive value of both the standard and ANN Cox models was relatively low, thus suggesting an intrinsic limitation of the prognostic variables considered, rather than their suboptimal modeling. Research for better prognostic variables should therefore continue.

Many biologic prognostic markers are available for patients with breast cancer, and considerable interest has been devoted to confirm preliminary evidence of their role as indicators of treatment response. It remains to be assessed whether such markers are predictors of response only to first-line or also to successive therapies. Proliferative activity, defined by the 3H-thymidine labeling index (TLI), was determined on the primary lesion from 76 patients at time of first diagnosis. At relapse, patients underwent chemotherapy as absolute (48 cases) or relative (28 cases) first-line treatment, and their clinical response was analyzed in relation to the TLI of the primary lesion. The objective clinical response was significantly higher for rapidly (47%; CL, 33-61%) than for slowly proliferating tumors (15%; CL, 1-29%). These findings held true also when adjusted for metastatic site, previous treatment, chemotherapy regimen administered, and hormone receptor status. However, the direct relation between cell proliferation and benefit from chemotherapy held true only when such a treatment was used as an absolute first-line approach. Cell proliferation of primary lesions represents a consistent indicator of response to chemotherapy over time. Previously administered regimens, at least hormone therapy, could alter the proliferation-related chemosensitivity profile of individual tumors.

The ability of lonidamine (LND), an energolytic derivative of indazole-carboxylic acid, to modulate the cytotoxic activity of cisplatin (CDDP) and epidoxorubicin (EPI), singly or in combination, was investigated in two human breast cancer cell lines (MCF7 and T47D). A 72-hr post-incubation with a non-cytotoxic concentration of LND (75 microM) increased the activity of a 1-hr CDDP treatment as well as that of a 1 to 16-hr EPI treatment. A different pattern of interaction among the drugs and modulator was observed as a function of the sequence of drug treatment. Specifically, supra-additive or additive effects of the combination were obtained in the two cell lines according to the different treatment schemes. In particular, the maximum potentiation was observed in MCF7 cells simultaneously exposed to CDDP, EPI and LND for 1 hr and then post-incubated with LND for 72 hr, and in T47 first exposed to EPI and LND, then to CDDP and LND, and finally post-incubated with LND. Flow cytometric analysis of MCF7 cell distribution in the different cycle phases showed that combined treatment with EPI/CDDP/LND was able to stabilize cell cycle perturbations (mainly G2M accumulation) induced by individual agents. The ability of LND to potentiate CDDP and EPI cytotoxicity, and the consideration that LND causes side effects different from those caused by alkylating agents and anthracyclines, make this compound an attractive candidate for multidrug combination therapy in breast cancer.

The expression of cathepsin D was evaluated by immunohistochemistry on histologic sections from formalin-fixed samples in a series of 436 primary breast cancers. The fraction of cathepsin D-positive tumor cells was not related to tumor size or hormone receptor status, and only weakly related to proliferative activity, evaluated as the 3H-thymidine labeling index. Conversely, a higher fraction of positive cells was observed in node-positive than in node-negative tumors (p = 0.05). A matched comparison between immunohistochemical and immunoradiometric results on individual tumors was carried out on 100 cases and showed a significant association but with a correlation coefficient of 0.46. The agreement of results from the two assays was higher in ER- than in ER+ tumors, which sometimes showed an immunostaining limited to macrophages and normal epithelial cells. In situ evaluation has the main advantage of being specifically applicable to detection in tumor cells and allows the simultaneous determination of different biologic aspects for a more complete understanding of breast cancer biology.

A permeable solid support (Transwell Coll.) was used to develop serum-free co-cultures allowing paracrine interactions between hormone-dependent (MCF-7, ZR75.1) and hormone-independent (MDAMB-231, BT20) breast cancer cell lines. Both hormone-independent cell lines were able to stimulate the growth of the hormone-dependent lines, whereas the opposite was observed only in the case of BT20 co-culture with ZR75.1 cells. The cell growth stimulation observed in co-cultures could be abolished by the addition to the culture medium of an excess of transforming growth factor alpha (TGF alpha) or insulin-like growth factor I (IGF-I). Similarly, treatment with a neutralizing anti TGF alpha antibody impaired the growth stimulation exerted by hormone-independent cells on hormone-dependent cells. These results confirm the important role of paracrine interactions in control of the growth of human heterogeneous breast tumors and suggest that the main growth factors involved in such interactions are TGF alpha and probably some growth factors from the insulin-like growth factor family rather than IGF-I itself.

Breast cancers from 476 elderly patients, 70 years and older, operated on since 1972, were analyzed for proliferative activity, hormone receptors, and DNA content. Tumor proliferative activity, expressed as 3H-thymidine labeling index (3H-TdR LI), had a median value of 3.4%, which progressively increased from 1972 to 1990. Estrogen and progesterone receptors were present respectively in 83% and 61% of the cases; the positivity for estrogen receptors slightly increased with time. Aneuploid clones were detected in 74% of the cases, and this incidence was relatively stable during the time of observation. 3H-TdR LI, hormone receptors, and ploidy were generally unrelated to the local-regional extension of the disease in these elderly patients, in agreement with observations on cancer from younger patients. However, the absence of hormone receptors and the presence of aneuploidy were markedly indicative of fast cell proliferation. As in younger patients, these biologic findings in elderly patients could be considered as a complement to clinico-pathologic features in a 'risk-factor profile system' for treatment planning.

The evaluability and reliability of proliferative activity (expressed as 3H-thymidine labeling index, 3H-TdR LI) and ploidy determinations on core needle biopsies were compared with those obtained on surgical material from the same breast cancers. The evaluability of 3H-TdR LI on core needle biopsies was markedly lower than that on surgical material (53% vs 100%), and the association between 3H-TdR LI values in the 16 cases with both evaluable determinations was poor (rs = 0.45). Conversely, determinations of ploidy on core needle biopsy and surgical material provided superimposable results, in terms of evaluability (91% vs 100%) and reliability (rs = 0.99). Further efforts are needed to improve sampling procedures for a reliable assessment of biological markers.

The paper analyzes the relation among tumor size (T), estrogen receptor (ER) status, and labeling index (LI) and their relative merits in predicting the relapse-free (RFS) and overall survival (OS) in 215 node-negative women with primary breast cancer. All patients were subjected to Halsted or modified radical mastectomy; none received postoperative irradiation or systemic adjuvant therapy. The 5-year RFS was 75.3% and OS 89.0%. When singly tested, LI and ER were able to significantly predict RFS and OS. In contrast, T influenced only RFS but not OS. The multivariate analysis showed that, in the presence of the two other variables, only LI retained its prognostic significance both for time to relapse (p = 0.0044) and survival (p = 0.035). From the present findings, LI appears to be a new important prognostic variable in the selection of high risk patients for whom adjuvant systemic therapy should perhaps be part of their primary treatment.

Kinetic and estrogen receptor (ER) determinations were performed on 357 primary breast cancers. The proliferative activity of the cell population was determined by the autoradiographic technique and expressed as [3H]thymidine labeling index (LI): the ER status was assessed by the absorption charcoal technique. From the overall analysis of the relationship between proliferative activity and ER status, by using the median LI value as cutoff of low and high proliferating tumors, a correlation between ER + status and low proliferative activity and between ER- status and high proliferative activity was observed in 64% of the cases. Menopausal status proved to be an important determinant of kinetic and ER features. In fact, the proliferative activity was always higher in ER--tumors than in ER + tumors within the same menopausal group, and a decrease in proliferative activity was observed from premenopause to postmenopause within ER + and ER--tumors. The analysis of the relation between ER levels and proliferative activity in ER + tumors also evidenced three main ER/LI clusters significantly related to premenopause, paramenopause and postmenopause. A particular indication of nodal involvement was evidenced for a cluster of tumors with low ER levels and high LI from postmenopausal patients. The three different, previously proposed kinetic groups of potential relevance for prognostic and therapeutic purposes were confirmed and more precisely defined by considering not only premenopause and postmenopause but also paramenopause.