A. Mangia

HER-2 Expression and Cell Proliferation: Prognostic Markers in Patients With Node-Negative Breast Cancer

PURPOSE We analyzed the clinical relevance of HER-2 expression, widely investigated in breast cancer but with contradictory results, in the largest case series of node-negative breast cancer patients investigated to date. PATIENTS AND METHODS The pure prognostic value of HER-2 expression was investigated in 529 patients treated with locoregional therapy alone until early relapse. Proliferative activity was evaluated as [3H]thymidine labeling index and HER-2 expression by immunohistochemistry. All biologic determinations were conducted within the context of an intra- and interlaboratory National Quality Control Program. RESULTS HER-2 expression was not related to relapse-free survival in the overall series but was a significant discriminant of prognosis in the subgroup of patients with rapidly proliferating tumors. Six-year rate of relapse was 40% for patients with highly (> or =30%) positive tumors and 26% for those with weakly HER-2-expressing tumors (P =.039). CONCLUSION HER-2 expression in association with proliferative activity identifies a subgroup of node-negative breast cancer patients with the worst prognosis, who are candidates for specific intensive adjuvant therapy.

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.

Heterogeneity of Intratumour Proliferative Activity in Primary Breast Cancer: Biological and Clinical Aspects

The present retrospective study was undertaken to verify whether the extent of intratumour proliferative activity variation or the method of quantifying tumour proliferative activity is related to biological characteristics and clinical outcome in a series of operable node-negative breast cancer patients. For tumour proliferative activity evaluation, the 3H-thymidine autoradiographic assay was used. After incubation of 3-8 samples from different areas of the equatorial section of each tumour for 1 h at 37 degrees C with 3H-thymidine, the following methods were used for evaluation of tumour cell labelling: mean tumour labelling index (LI), the highest labelling value from a specific area (LI-max), and the extent of intratumour labelling variation from several samples (LI-CV). LI-max was related to ER and PgR status, and linearly correlated with LI (c.c. = 0.92, P < 10(-6)) whereas LI-CV was independent of tumour size, grade ER and PgR status, but dependent on the number of tumour samples analysed for each tumour. After 5 years of median follow-up, disease-free survival was only related to tumour size (T1 versus T2: 84 versus 64%, P < 0.04 by log rank analysis) and different LI values (low versus high 3H-Tdr-LI:86 versus 61%, P < 0.03 by log rank analysis). LI-max and LI-CV values were not significantly related to clinical outcome. Cox multivariate analysis confirmed the independent prognostic value of LI and tumour size on disease-free survival.

Overexpression of nuclear NHERF1 in advanced colorectal cancer: association with hypoxic microenvironment and tumor invasive phenotype.

Over 57% of colorectal cancer (CRC) patients have regional or distant spread of their disease at the time of diagnosis. Despite recent advances, there is a compelling need to better characterize prognostic markers for advanced CRC. The present study investigates protein expression of NHERF1, HIF-1α and TWIST1 and their relationship in distant normal mucosa (DNM), tumor (T) and adjacent normal mucosa (ANM), lymph node metastasis (LNM) and liver metastasis (LM), determining their role as potential markers in advanced stages of human CRC. Overexpression of nuclear NHERF1 was shown in 47% of tumors, which exhibited a significant association with poor histological grade (P=0.0346). Nuclear NHERF1 showed a higher expression in T, LNM and LM than both DNM (P<0.0001) and ANM (P<0.05). Nuclear HIF-1α was significantly higher in T, LNM and LM than DNM and ANM (P<0.05, P<0.001, P<0.0001, respectively). A positive correlation between nuclear NHERF1 and nuclear HIF-1α was found in LNM (r=0.331, P=0.020), where an extended co-localization of the two proteins was demonstrated. TWIST1 was more expressed in T than DNM and ANM (P<0.0001) and was higher in T than LNM and LM (P<0.0001). Moreover, nuclear NHERF1 was directly correlated to TWIST1 (r=0.339, P=0.015) in T samples, where a high co-expression of the two proteins was demonstrated both in no longer polarized epithelial cells and in invasive mesenchymal elements adjacent to hypoxic and perinecrotic colonic areas. Overall, nuclear NHERF1 expression was associated with poorer differentiation grade and with higher expression both of HIF-1α in lymphatic metastasis and TWIST1 in invasive front of tumor. Our results support the oncogenic role of NHERF1 and promote nuclear NHERF1 as a potential new biomarker of advanced CRC.

Peritumoral vascular invasion and NHERF1 expression define an immunophenotype of grade 2 invasive breast cancer associated with poor prognosis

BackgroundTraditional determinants proven to be of prognostic importance in breast cancer include the TNM staging, histological grade, proliferative activity, hormone receptor status and HER2 overexpression. One of the limitations of the histological grading scheme is that a high percentage of breast cancers are still classified as grade 2, a category with ambiguous clinical significance. The aim of this study was to best characterize tumors scored as grade 2.MethodsWe investigated traditional prognostic factors and a panel of tumor markers not used in routine diagnosis, such as NHERF1, VEGFR1, HIF-1α and TWIST1, in 187 primary invasive breast cancers by immunohistochemistry, stratifying patients into good and poor prognostic groups by the Nottingham Prognostic Index.ResultsGrade 2 subgroup analysis showed that the PVI (p = 0.023) and the loss of membranous NHERF1 (p = 0.028) were adverse prognostic factors. Relevantly, 72% of grade 2 tumors were associated to PVI+/membranous NHERF1- expression phenotype, characterizing an adverse prognosis (p = 0.000). Multivariate logistic regression analysis in the whole series revealed poor prognosis correlated with PVI and MIB1 (p = 0.000 and p = 0.001, respectively). Furthermore, in the whole series of breast cancers we found cytoplasmic NHERF1 expression positively correlated to VEGFR1 (r = 0.382, p = 0.000), and in VEGFR1-overexpressing tumors the oncogenic receptor co-localized with NHERF1 at cytoplasmic level.ConclusionsThe PVI+/membranous NHERF1- expression phenotype identifies a category of grade 2 tumors with the worst prognosis, including patient subgroup with a family history of breast cancer. These observations support the idea of the PVI+/membranous NHERF1- expression immunophenotype as a useful marker, which could improve the accuracy of predicting clinical outcome in grade 2 tumors.

Failure of primary breast cancer neoangiogenesis to predict pattern of distant metastasis.

Abstract In the present study, the primary tumor angiogenesis characteristics of 81 stage IV previously untreated breast cancers with synchronous metastasis to different distant sites (10 patients with soft tissue metastases, 31 with bone metastases, and 40 with visceral metastases) were analyzed. The primary intratumor microvessel density was assessed by immunohistochemical assay on paraffin-embedded primary tumor samples, using a monoclonal anti-CD34 antibody. The mean primary intratumor microvessel density (at 400× fields) was 78±39 (SD) microvessels per field. The microvessel density was not significantly related to the main clinical/pathological features of the tumor (age, cytohistological grade, DNA ploidy, diameter, and receptor status). The percentage of tumor cases with high primary intratumor microvessel density (cut-off median value of the series 73±39 microvessels/field) did not significantly differ in patients with bone, soft tissue, or visceral metastatic disease. Aanalysis of clinical outcome showed a significantly shorter time to progression and overall survival for patients with visceral metastases (P<0.001 and P<0.0002 by log-rank, respectively). Presence of visceral metastases was confirmed to be the only independent prognostic factor related to a worse TTP (hazard risk 2.15, 95% confidence interval 1.14–4.03, P<0.02) and overall survival (hazard risk 1.81, 95% confidence interval 0.98–3.35, P<0.06) by multivariate analysis. In conclusion, the assessment of neoangiogenesis of primary breast cancer by CD34 expression does not provide information predictive of different distant sites of metastasis.

Cytosolic levels of estrogen-regulated pS2 protein in breast cancer: correlation with tumor proliferative activity.

Using a new immunoradiometric assay, a total of 100 cytosols obtained from primary breast tumors were examined for content of pS2, an estrogen-regulated protein. In our series, the median pS2 value was 5 ng/mg protein cytosol which was used as the cut-off. pS2 content was not correlated with menopausal status, tumor size, nodal involvement or tumor proliferative activity expressed as labeling index (tritiated thymidine LI). A positive correlation was found between pS2 and estrogen (ER) and progesterone (PgR) hormone receptors. pS2 in association with ER and PgR seems to identify tumor subgroups with diverse hormone responsiveness. Moreover, in favorable and unfavorable prognostic subgroups, LI and pS2 further emphasize this prognostic diversity.

Randomised Clinical Trial of Adjuvant Chemotherapy in Patients with Node-Negative, Fast-Proliferating Breast Cancer

SummaryA prospective randomised clinical trial in patients with node-negative, fast-proliferating breast cancer was initiated in January 1990 to verify the feasibility and reliability of a 3H-thymidine (3H-Tdr) autoradiographic assay in a prospective and consecutive series of node-negative patients and the therapeutic effect of adjuvant chemotherapy in patients with node-negative breast cancer and high tumour proliferative activity. Node-negative patients with a high 3H-Tdr Labelling Index (3H-Tdr-LI) were randomised to receive either no further treatment or combination chemotherapy consisting of fluorouracil, epirubicin plus cyclophosphamide (FEC) for 6 cycles. The autoradiographic assay was performed in 307 of 317 patients (97%) and was evaluable in 291 of 317 patients (92%). A total of 176 patients with a high 3H-Tdr-LI entered the clinical randomised study: 91 in the FEC arm and 85 in the control arm. Patient groups were fairly well balanced regarding the most important clinical and pathological characteristics. In total, 530 FEC cycles have been administered with an actual dose intensity of 90%. Patients receiving FEC demonstrated leucopenia in 35% of cases, alopecia in 70%, and loss of menses in premenopausal patients in an age-dependent manner. Patients are still being entered into the study.

Predictability of Monthly and Yearly Rhythms of Breast Cancer Features

In order to evaluate if breast cancer biological characteristics undergo significant menstrual and seasonal variations, we analysed in a consecutive series of 905 breast cancer patients, steroid receptor level (ER and PgR by DCC assay), proliferative activity (3H-Thymidine Labeling Index, 3H-TLI) and size of primary tumour in relation to calendar date and day of menstrual cycle at the time of the surgical procedure. For data analysis, the method of time series construction and classical spectral analyses with Bartlett Kolmogorov–Smirnov test for white noise (BKS test) was utilised. For what concerns menstrual variations, 3H-TLI showed a significant periodicity (t=0.3146, p<0.01 by BKS test) with peaks at day 12nd and day 18th; ER showed a significant periodicity (t=0.3605, p<0.01 by BKS test) with more evident peak at day 27th; PgR, a significant periodicity (t=0.160, p=0.05 by BKS test) with peaks at day 15th and day 24th, similar to that observed for tumour size (t=0.19, p<0.05 by BKS test). With respect to yearly fluctuations, 3H-TLI showed only a trend for a significant rhythm (t=0.16, p=0.06 by BKS test) with peaks in May and November; ER a significant periodicity (t=0.2099, p<0.05 by BKS test) with two evident peaks in January and April; also for PgR a significant periodicity (t=0.3161, p<0.05 by BKS test) was demonstrated with a peak in July; finally, tumour size showed a significant rhythm (t=0.335, p<0.01) paralleling 3H-TLI behaviour. Finally, the analysis of variance with interaction of menstrual and seasonal timings showed that only the seasonal timing was able to independently influence the 3H-TLI variations (3H-TLI higher in spring). We confirmed that breast biology has significant menstrual and seasonal variations and that the seasonality is probably the timing factor more relevant in periodicity determination.

Expression of proteins involved in DNA damage response in familial and sporadic breast cancer patients.

Understanding the expression of proteins involved in DNA damage response could improve knowledge of the pathways that contribute to familial and sporadic breast cancer (BC). We aimed to assess the different roles of BRCA1, poly(ADP‐ribose) polymerase‐1 (PARP1), BRCT‐repeat inhibitor of hTERT expression (BRIT1) and novel SWItch 5 (SWI5) expression in 130 sporadic and 73 familial BC samples, by immunohistochemistry. In the sporadic group, negative nuclear BRCA1 (nBRCA1) expression was associated with positive PgR (p = 0.037). Negative association was found between nBRCA1 expression and HER2 (p = 0.001). In the familial group, nBRCA1 expression was associated with ER (p = 0.002). Reduced nBRCA1 expression was associated with higher histological grade and positive Ki67 both in sporadic (p = 0.0010, p = 0.047) and familial groups (p < 0.001, p = 0.001). Nuclear PARP1 (nPARP1) expression was associated with histological grade (p = 0.035) and positive PgR (p = 0.047) in sporadic cases. High cytoplasmic and low nuclear BRIT1 (cBRIT1 and nBRIT1) expression were associated with high histological grade in the familial group (p = 0.013, p = 0.025). Various statistical associations between the protein expressions were observed in the sporadic group, while in familial group only few associations were found. Univariate analyses showed that nPARP1 expression is able to discriminate between sporadic and familial tumors (OR 2.80, p = 0.002). Multivariate analyses proved that its overexpression is an independent factor associated with a high risk of sporadic tumor (OR 2.96, p = 0.017). Our findings indicate that nPARP1 expression is an independent factor for sporadic BCs and PARP1 inhibitors could be a promising therapy for different phenotypes.