Annamaria Molino

Ki-67 immunostaining in 322 primary breast cancers: Associations with clinical and pathological variables and prognosis

Cell‐proliferation markers are very important in the clinical management of cancer patients, and the identification of Ki‐67 (a monoclonal antibody that recognizes proliferating cells) can make it easier to define the level of proliferative activity. This study investigated the associations between the Ki‐67 levels measured by means of immunohistochemistry, and other clinical and pathological variables and prognosis in 322 breast‐cancer patients. A significant association was found (p < 0.001) between Ki‐67 values and tumor size, nodal status, estrogen and progesterone receptor status; multivariate analysis showed that Ki‐67 levels were associated with disease‐free and overall survival, thus confirming that it is an independent prognostic variable. Various statistical approaches were used in an attempt to establish the best cut‐off point for dividing patients into groups at high or low risk of relapse but, in this series, we could find no evidence leading to a single “best” cut‐off point. We conclude that the quantitative level of Ki‐67 could be used as a prognostic factor in breast‐cancer patients. Int. J. Cancer 74:433–437, 1997.

A comparative analysis of three different techniques for the detection of breast cancer cells in bone marrow

Three different methods, morphologic, immunocytochemic, and fluorescence activated cell sorter (FC) analysis, were compared with respect to their efficiency in detecting breast cancer cells in bone marrow. In the first series of experiments, the three techniques were compared using bone marrow cells artificially mixed with a known amount of breast cancer cells, whereas in a second series bone marrow from breast cancer patients with bone metastases were used. The following results were obtained: When mixtures of the first series were analyzed, FC analysis detected from 1% to 10% of breast cancer cells in bone marrow (0.2% was a border line value), the morphologic method detected from 0.05% to 10%, and the immunocytochemic method, which was clearly superior, detected breast cancer cells in all mixtures (from 0.00025% to 10%), It was noted that, with both the morphologic and immunocytochemic methods, the percentage of breast cancer cells detected was 2 to 360 times higher than the percentage of added cells, and enrichment was inversely proportional to the percentage of added cells. This result could be a result of different separation of cells during centrifugation due to the different density of breast cancer cells. The superiority of the immunocytochemic method was confirmed in the second series of experiments.

Efficacy of a leptin receptor antagonist peptide in a mouse model of triple-negative breast cancer

Triple-negative breast cancers, which represent 10-20% of all mammary tumours, are characterised by the aggressive phenotype, are often found in younger women and have been associated with poor prognosis. Obesity increases the risk for triple-negative breast cancer development. Because triple-negative breast cancer patients are unresponsive to current targeted therapies and other treatment options are only partially effective, new pharmacological modalities are urgently needed. Here we examined if the leptin (obesity hormone) receptor is a viable target for the treatment of this cancer subtype. In human triple-negative breast cancer tissues, the leptin receptor was expressed in 92% (64/69) and leptin in 86% (59/69) of cases. In a model triple-negative breast cancer cell line MDA-MB-231, the leptin receptor antagonist peptide Allo-aca inhibited leptin-induced proliferation at 50 pM concentration. In an MDA-MB-231 orthotopic mouse xenograft model, Allo-aca administered subcutaneously significantly extended the average survival time from 15.4 days (untreated controls) to 24 and 28.1 days at 0.1 and 1mg/kg/day doses, respectively. In parallel, conventional treatment with 1mg/kg/day intraperitoneal cisplatin prolonged the average survival time to 18.6 days, while administration of 20mg/kg/day oral Tamoxifen (negative control) had no significant survival effects relative to controls. In normal CD-1 mice, Allo-aca produced no systemic toxicity up to the highest studied subcutaneous bolus dose of 50mg/kg, while, as expected, it induced a modest 6-10% body weight increase. Our results indicate that leptin receptor antagonists could become attractive options for triple-negative breast cancer treatment, especially in the obese patient population.

Leptin/HER2 crosstalk in breast cancer: in vitro study and preliminary in vivo analysis

BackgroundObesity in postmenopausal women is associated with increased breast cancer risk, development of more aggressive tumors and resistance to certain anti-breast cancer treatments. Some of these effects might be mediated by obesity hormone leptin, acting independently or modulating other signaling pathways. Here we focused on the link between leptin and HER2. We tested if HER2 and the leptin receptor (ObR) can be coexpressed in breast cancer cell models, whether these two receptors can physically interact, and whether leptin can transactivate HER2. Next, we studied if leptin/ObR can coexist with HER2 in breast cancer tissues, and if presence of these two systems correlates with specific clinicopathological features.MethodsExpression of ObR, HER2, phospo-HER2 was assessed by immonoblotting. Physical interactions between ObR and HER2 were probed by immunoprecipitation and fluorescent immunostaining. Expression of leptin and ObR in breast cancer tissues was detected by immunohistochemistry (IHC). Associations among markers studied by IHC were evaluated using Fishers exact test for count data.ResultsHER2 and ObR were coexpressed in all studied breast cancer cell lines. In MCF-7 cells, HER2 physically interacted with ObR and leptin treatment increased HER2 phosphorylation on Tyr 1248. In 59 breast cancers, the presence of leptin was correlated with ObR (the overall association was about 93%). This result was confirmed both in HER2-positive and in HER2-negative subgroups. The expression of leptin or ObR was numerically more frequent in larger (> 10 mm) tumors.ConclusionCoexpression of HER2 and the leptin/ObR system might contribute to enhanced HER2 activity and reduced sensitivity to anti-HER2 treatments.

Bone marrow micrometastases in 109 breast cancer patients: correlations with clinical and pathological features and prognosis

Background: The presence in bone marrow of cells which react with monoclonal antibodies against tumor-associated antigens has been proposed over the last few years as a new prognostic factor in breast cancer patients. Patients and methods: Bone marrow aspirates were obtained from 109 stage I and II breast cancer patients during or 2–4 weeks after primary surgery. The samples were processed for leukocyte separation on a Ficoll-Hypaque gradient and then used to prepare cytospin slides for immunocytochemical analysis. The slides were stained with a pool of monoclonal antibodies (MoAbs) which recognize tumor associated antigens, using the alkaline phosphatase anti-alkaline phosphatase method. The median follow-up was 36 months (range 15–62); 22 patients relapsed and 7 died. Results: Thirty-four of the 109 patients (31.1%) had MoAb positive bone marrow cells. The bone marrow was positive in 28/74 (37.9%) patients who had the aspirate taken during surgery and in 6/35 (17.1%) who had it taken after surgery (p = 0.055). No association was found between bone marrow positivity and tumour size, nodal status, menopausal status, estrogen receptor positivity or the proliferative index. No association was found between bone marrow and prognosis: the log-rank test was 0.291 (p > 0.5) for OS and 0.023 for DFS; the hazard ratio (positive vs negative) was 1.51 for OS (95% CI: 0.33–6.86) and 0.93 for DFS (95% CI: 0.35–2.45). Conclusions: In our series, bone marrow positivity did not correlate with prognostic parameters or prognosis. Of interest is the relative excess of positivity when the bone marrow was obtained during surgery.

Preoperative staging of primary breast cancer. A multicentric study.

This article reports on a consecutive series of 3627 breast cancer (BC) patients undergoing preoperative staging by chest x‐ray (CXR), bone x‐ray (BXR) or bone scintigraphy (BS), and liver ecography (LE) or liver scintigraphy (LS). The detection rate (DR) of preclinical asymptomatic distant metastases depended on the T and N category (TNM classification system), and was very low (CXR: 0.30%, BXR: 0.64%, BS: 0.90%, LE: 0.24%, LS: 0.23%). The sensitivity, determined after a 6‐month follow‐up, was below 0.50% for all tests. The highest value (0.48%) was recorded for BS, which also had the lowest specificity (0.95%). The entire preoperative staging policy using the studied tests seems questionable due to poor sensitivity and an extremely low DR of distant metastases.

Male Breast Carcinoma: Review of a Multicenter Series of 150 Cases

The authors report on a consecutive retrospective series of 150 male breast cancers. Clinical, diagnostic and therapeutic features are compared over time and with respect to a large consecutive series of female breast cancers. Both age at diagnosis and tumor stage were more advanced in males than in females. Poor alertness of both men and doctors for this unfrequent disease may account for such a delay in diagnosis. The use of mammography increased over time and sonography or cytology were frequently and successfully employed in the last decade. Unfortunately no improvement of tumor stage at diagnosis was observed over time in the present series. A time trend was also evident for the type of surgical and postoperative treatment. Modified radical mastectomy and adjuvant chemo- or hormone therapy were increasingly adopted, although Halsted operation and postoperative radiotherapy were still common in the last decade due to the relatively high proportion of locally advanced T3-4 cancers. Both disease-free and overall survival were worse in men than in women, even after adjustment by stage at diagnosis. This study suggests that male breast cancer has a worse prognosis with respect to female breast cancer and provides no complete explanation of this finding, except for an intrinsic higher aggressivity. No evidence was found which may justify a different diagnostic or therapeutic approach with respect to female breast cancer.

Prognostic significance of estrogen receptors in 405 primary breast cancers: a comparison of immunohistochemical and biochemical methods

Over the last few years, estrogen receptor determinationby means of immunohistochemistry has been extensively used.The aim of this study was to comparethis technique with estrogen receptor determination by meansof dextran-coated charcoal, and to evaluate whether oneof the two methods is more predictive ofprognosis. Estrogen receptors were determined by means ofboth the dextran-coated charcoal method and immunohistochemistry in405 patients with primary breast cancer; age, pathologicaltumor size, nodal status, and progesteron receptors bydextran-coated charcoal method were also recorded. The disease-freeand overall survival probabilities were estimated using theproduct-limit method; Coxs proportional hazard model was usedto evaluate the prognostic role of estrogen receptorsas determined by the two methods.There appears to be a close association betweenestrogen receptor determination by the two methods (81.5%of concordant results) and their prognostic role wassimilar, even when the patients were divided intodifferent groups (on the basis of their estrogenreceptor status) and adjustments for the effect ofother prognostic variables were taken into account. Ourstudy shows that the two methods can beused indifferently to evaluate estrogen receptor status asa prognostic factor in breast cancer patients.

Multicentric, randomized phase III trial of two different adjuvant chemotherapy regimens plus three versus twelve months of trastuzumab in patients with HER2- positive breast cancer (Short-HER Trial; NCT00629278).

Trastuzumab, a monoclonal antibody against the HER2 receptor, is currently approved as a part of adjuvant therapy for patients with HER2-overexpressing breast tumors. The Short-HER study is a phase III randomized, multicentric Italian trial aimed at testing the optimal duration of adjuvant trastuzumab. In this trial, 2500 patients with HER2-positive breast cancer will be randomized to receive the following: (arm A, long) 4 courses of anthracycline- based chemotherapy (doxorubicin/cyclophosphamide or epidoxorubicin/cyclophosphamide) followed by 4 courses of docetaxel or paclitaxel in combination with trastuzumab, followed by 14 additional courses of trastuzumab administered every 3 weeks (for a total of 18 3-weekly doses of trastuzumab); or (arm B, short) 3 courses of 3-weekly docetaxel in combination with weekly trastuzumab (for a total of 9 weekly doses of trastuzumab) followed by 3 courses of 5-fluorouracil/epirubicin/cyclophosphamide. The primary objective is disease-free survival.

Tumor proliferative activity and response to first-line chemotherapy in advanced breast carcinoma

SummaryThe relationship between tumor proliferative activity and response to first-line chemotherapy and survival was investigated in 76 advanced breast cancer patients. Proliferative activity was determined by means of Ki-67 immunohistologic staining on primary tumors (55 patients) or at the relapse site (21 patients), and was classified as low (≤ 25% of stained cells) or high (> 25% of stained cells). The usual WHO response criteria were used. The median duration of follow-up was 18 months (range 3–58).Forty-seven patients (62%) had tumors with low, and 29 (38%) had tumors with a high rate of proliferative activity. The two groups were well balanced in terms of important variables such as disease-free survival, performance status, age, menopausal status, and the type of first-line chemotherapy (anthracycline-based regimens versus cyclophosphamide-methotrexate-5-fluorouracil). The estrogen receptor (ER) content, measured by means of immunohistochemical assay, was markedly different in the two groups, with 27/47 tumors with low proliferative activity (57%) and 6/29 with high-proliferative activity (21%) being ER positive (≥ 45% of stained cells) (p = 0.003). Moreover, a significant difference in the metastatic pattern was also evident, with a higher incidence of bone and a lower incidence of soft tissue metastases in the group of patients with tumors with low proliferative activity (p = 0.004). Overall, 10/47 responses (21%: PR = 7, and CR = 3) were observed in the group with a low rate of proliferative activity, versus 14/29 (48%: PR = 9, and CR = 5) in the group with highly proliferative tumors, the difference being statistically significant (p = 0.03). When a multivariate analy-sis was performed, the only factor that retained independent prognostic significance was the predominant site of disease, particularly soft tissues (p = 0.003). Despite the difference in response rate, when survival analysis was performed according to the Kaplan-Meier method, no significant difference was observed in the two groups, but when the analysis was limited to responsive patients, the median survival observed in those with a low and those with a high rate of proliferation was 35 and 19 months respectively (p = 0.02). The same results were obtained when multivariate survival analysis was carried out using Coxs regression model. These data suggest that there is a link between tumor proliferative activity and response to chemotherapy in advanced breast cancer, and may indicate the need to use more intensive treatments in selected patients with highly proliferative tumors.