MIB1 proliferation index in breast infiltrating carcinoma: comparison with other proliferative markers and association with new biological prognostic factors

AIMS: In breast invasive carcinoma our objectives were I) to compare cellular proliferation determined by MIB1 index with S-phase fraction (SPF) assessed by flow cytometry and with mitotic index, and II) to examine the association of MIB1 index with classical and with new biological prognostic factors [bcl-2, p53, c-erbB-2 and cathepsin D (CD)]. METHODS AND RESULTS: From 102 cases of breast invasive carcinoma, 5-microm thick serial sections were cut from formalin-fixed, paraffin-embedded tissue blocks, and processed for detection of CD, c-erbB-2, p53, bcl-2, Ki-67 antigen MIB-1 and estrogen receptors (ER) and progesterone receptors (PR). SPF was measured by flow cytometry in fresh-frozen tissue samples taken from the carcinoma in each patient. MIB1 index was correlated with SPF (rho=0.45, p<0.0001) and with mitotic index (rho=0.42, p<0.0001). The MIB-1 index was positively associated with the histological grade (p=0.001), tumor size (p=0.04) and the presence of metastases in axillary lymph nodes (p=0.01). MIB1 was associated directly with p53 (p=0.045) and inversely with bcl-2 (p=0.0002). The MIB-1 index was not statistically associated with c-erbB-2. There was a weak association between MIBI index and stromal cell CD. The median MIB1 index was higher in tumors with moderate to strong CD staining of stromal cell, but the difference did not reach statistical significance (p=0.09). CONCLUSIONS: MIB1 index correlates with well established methods for assessing tumor proliferation and with parameters of an aggressive phenotype of tumor. MIB1 index is an effective and readily accessible method for assessing tumor proliferation in breast carcinoma.     

Effects of low-dose tamoxifen on breast cancer biomarkers Ki-67, estrogen and progesterone receptors

Breast carcinoma is the most common malignancy among women and it has a major impact on mortality. Studies of primary chemoprevention with tamoxifen have generated high expectations and considerable success rates. The efficacy of lower doses of tamoxifen is similar to that seen with a standard dose of the drug, and there has been a reduction in healthcare costs and side effects.The immune reaction to monoclonal antibody Ki-67 (MIB-1) and the expression of estrogen receptors (1D5) and progesterone receptors (PgR 636) in breast carcinoma were studied in patients treated with 10 mg of tamoxifen for a period of 14 days.A prospective randomized clinical trial was conducted with 38 patients divided into two groups: Group A: N = 20 (control group-without medication) and Group B: N = 18 (tamoxifen/10 mg/day for 14 days). All patients signed an informed consent term previously approved by both institutions. Patients underwent incisional biopsy before treatment and 14 days later a tumor tissue sample was obtained during surgical treatment. Positivity was quantitatively assessed, counting at least 1.000 cells per slide. For statistical data analysis, a Wilcoxon non-parametric test was used, and α was set at 5%.Both groups (A and B) were considered homogeneous regarding control variables. In Group A (control), there was no statistically significant reduction in Ki-67 (MIB-1) (p = 0.627), estrogen receptor (1D5) (p = 0.296) and progesterone receptor positivity (PgR 636) (p = 0.381).In Group B (tamoxifen 10 mg/day), the mean percentage of nuclei stained by Ki-67 (MIB-1) was 24.69% before and 10.43% after tamoxifen treatment. Mean percentage of nuclei stained by estrogen receptor (1D5) was 59.53% before and 25.99% after tamoxifen treatment. Mean percentage of nuclei stained by progesterone receptor (PgR 636), was 59.34 before and 29.59% after tamoxifen treatment. A statistically significant reduction was found with the three markers (p < 0.001).Tamoxifen significantly reduced monoclonal antibody Ki-67 (MIB-1), estrogen receptor (1D5) and progesterone receptor positivity (PgR 636) in the breast epithelium of carcinoma patients treated with a 10 mg dose of tamoxifen for 14 days.     

Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer.

In order to identify the prognostic factors that significantly influence the disease-free survival rate after surgical resection of primary breast cancers, we determined tumour and lymph node grades, and immunohistochemical staining for estrogen and progesterone receptors (ER and PR), c-erbB-2, p53, bcl-2, bax and PCNA in 76 patients. Univariate analysis showed that increased grade of tumour and lymph nodes, negative immunostaining for ER, positive immunostaining for c-erbB-2, and a high PCNA index (> or = 30%) negatively influenced the disease-free survival rate, but PR, p53, bcl-2 and bax had no predictive value. Although p53 was not an independent prognostic factor by itself, the combination of p53, bcl-2, and bax proved to correlate with the disease-free survival, with the best prognosis noted in tumours negative for p53 and positive for both bcl-2 and bax, intermediate prognosis in tumours negative for p53 and positive for either bcl-2 or bax and worst prognosis in tumors negative for p53 as well as bcl-2 and bax. Tumour grade correlated positively with PCNA index, while positive staining for ER correlated negatively with tumour grade as well as with PCNA index, although this was statistically insignificant. Immunostaining of breast cancers for bcl-2 correlated negatively with tumour grade and PCNA index. Immunostaining for c-erbB-2 correlated positively with PCNA but not with tumour grade. Immunostaining for p53 tended to correlate positively with PCNA, but not with tumour grade. Immunostaining for PR and bax did not correlate with tumour grade and PCNA index. These results suggest that in addition to tumour size and lymph node involvement, immunostaining for ER, c-erbB-2, and a high PCNA index are important prognostic factors in human breast cancer. Wild-type p53 with preserved bcl-2 and bax gene products is also a favorable prognostic factor indicating breast cancer at an early stage of cancer progression.     

Limited prognostic value of c-erbB-2 compared to uPA and PAI-1 in primary breast carcinoma

In a retrospective study of 488 women with primary breast cancer, after a median follow-up of 10 years, we sought interactions between disease-free survival (DFS) and overall survival (OS) and tumor antigen levels of two components of the plasminogen system, urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1, and the transmembrane growth factor receptor c-erbB-2. We used ELISAs (American Diagnostica, Greenwich, CT, USA) to quantify uPA and PAI-1 antigen levels in cytosols, and a double monoclonal antibody-based assay (EIA) (Ciba Corning Diagnostics, Alameda, CA, USA) to quantify c-erbB-2 in membrane extracts of the same tissues. Weak positive correlations were found between uPA and c-erbB-2 (r(s) = 0.146; p = 0.001) and between PAI-1 and c-erbB-2 (r(s) = 0.154; p < 0.001). In the overall population, using univariate analyses, c-erbB-2 overexpression and high uPA and PAI-1 antigen levels (> 300 IU/mg, > 1.40 ng/mg and > 5.53 ng/mg, respectively) were significantly associated with shorter DFS (p = 0.003, p < 0.001 and p < 0.001, respectively) and OS (p < 0.001 in all cases). Using multivariate analyses, PAI-1, node status and tumor size were independent predictors of DFS and c-erbB-2 was retained in the model only for OS. In the node-negative subgroup, PAI-1 was the strongest significant survival predictor both for OS (p = 0.003; HR 2.52) and DFS (p < 0.001; HR 2.39). This study shows that in primary breast cancer c-erbB-2 offers no additional prognostic information when uPA and/or PAI-1 are candidates in the multivariate analyses.     

Estrogen and progesterone receptors in colorectal cancer and surrounding mucosa

In this prospective study we have quantified by means of ELISA-methods the cytosolic content of estrogen (ER) and progesterone receptors (PgR) in tumoral tissue and paired normal mucosa from 163 patients with resectable colorectal cancer. Survival analysis was performed in a subgroup of 120 patients and the mean follow-up period was 24.9 months. The cutoff for ER and PgR levels was set at 1 fmol/mg protein. On the basis of this cutoff 20.9% of the cancers were ER positive and 25.8% were PgR positive; normal adjacent tissue presented ER in 18.4% and PgR in 24.5%. Our results did not show any significant correlation between ER and PgR levels in neoplastic tissues. Howewer, a correlation was found in normal mucosa samples (p=0.02). Statistical analysis showed that there was no correlation between tumor ER and PgR content and patient age or sex, tumor location, Dukes' stage, histological differentiation, DNA ploidy status and S-phase fraction. Furthermore, the results did not show any statistical differences in relapse-free and overall survival curves calculated for patients classified according to the hormone receptor content of their tumors. ER and PgR were detected at low levels in normal and neoplastic colorectal tissues without any significant relationship to either clinicopathological tumor characteristics or patient outcome. Their possible role in colorectal cancer remains to be elucidated.     

The Prognostic Value of Mitotic Activity Index (MAI), Phosphohistone H3 (PPH3), Cyclin B1, Cyclin A,and Ki67, Alone and in Combinations,in Node-Negative Premenopausal Breast Cancer

Proliferation, either as the main common denominator in genetic profiles, or in the form of single factors such as Ki67, is recommended for clinical use especially in estrogen receptor-positive (ER) patients. However, due to high costs of genetic profiles and lack of reproducibility for Ki67, studies on other proliferation factors are warranted. The aim of the present study was to evaluate the prognostic value of the proliferation factors mitotic activity index (MAI), phosphohistone H3 (PPH3), cyclin B1, cyclin A and Ki67, alone and in combinations. In 222 consecutive premenopausal node-negative breast cancer patients (87% without adjuvant medical treatment), MAI was assessed on whole tissue sections (predefined cut-off ≥10 mitoses), and PPH3, cyclin B1, cyclin A, and Ki67 on tissue microarray (predefined cut-offs 7th decile). In univariable analysis (high versus low) the strongest prognostic proliferation factor for 10-year distant disease-free survival was MAI (Hazard Ratio (HR)=3.3, 95% Confidence Interval (CI): 1.8-6.1), followed by PPH3, cyclin A, Ki67, and cyclin B1. A combination variable, with patients with MAI and/or cyclin A high defined as high-risk, had even stronger prognostic value (HR=4.2, 95%CI: 2.2-7). When stratifying for ER-status, MAI was a significant prognostic factor in ER-positive patients only (HR=7.0, 95%CI: 3.1-16). Stratified for histological grade, MAI added prognostic value in grade 2 (HR=7.2, 95%CI: 3.1-38) and grade 1 patients. In multivariable analysis including HER2, age, adjuvant medical treatment, ER, and one proliferation factor at a time, only MAI (HR=2.7, 95%CI: 1.1-6.7), and cyclin A (HR=2.7, 95%CI: 1.2-6.0) remained independently prognostic. In conclusion this study confirms the strong prognostic value of all proliferation factors, especially MAI and cyclin A, in all patients, and more specifically in ER-positive patients, and patients with histological grade 2 and 1. Additionally, by combining two proliferation factors, an even stronger prognostic value may be found.     

Prognostic value of CA 15.3 kinetics for metastatic breast cancer

Up to 80% of breast cancer patients developing metastases have high levels of CA 15.3. We studied the prognostic implications of CA 15.3 kinetics in 119 patients before and at first metastasis by univariate and multivariate statistics. At first metastasis, CA 15.3 was elevated in 82.4% of patients, with a lead time (median 162 days) in 42.0% of them. Kaplan-Meier analysis showed overall survival (median 1477 days) to be significantly related to estrogen receptor (ER) and progesterone receptor (PgR) status (p=0.0001) and tumor size (p=0.025). The interval between diagnosis and first abnormal CA 15.3 (p=0.0001), the CA 15.3 concentration (p=0.013), and the presence or absence of a lead time (p=0.001) also had prognostic value. ER and PgR status (p=0.0005 and p=0.0103, respectively), metastasis-free interval (p=0.0003), existence of a CA 15.3 lead time (p=0.0028), and days from diagnosis to first abnormal CA 15.3 (p=0.0055) entered in the Cox model. After first metastasis (median survival 573 days), ER and PgR status (p=0.0001 and p=0.0004, respectively), existence of a lead time for CA 15.3 (p=0.0138), and the concentration of first abnormal CA 15.3 (p=0.0145) had individual prognostic value. In the Cox model ER status (p=0.0001), nodal status (p=0.0191), existence of a lead time for CA 15.3 (p=0.0033), days from diagnosis to first abnormal CA 15.3 (p=0.0132), and concentration of first abnormal CA 15.3 (p=0.0320) were found to be independent prognostic variables. Compared to a matched historical control group that was not monitored by CA 15.3 assaying (n=140), the study group had a significantly longer survival after the first metastasis (p=0.0005). In conclusion, the kinetics of CA 15.3 before the first metastasis is of prognostic value. When associated with 18-fluorodeoxyglucose imaging, serial CA 15.3 assays may help to implement early treatment of metastases.     

Long-term follow-up confirms prognostic impact of PAI-1 and cathepsin D and L in primary breast cancer

After long-term follow-up, the prognostic impact of the following proteolytic factors associated with tumor invasion and metastasis was evaluated in 276 primary breast cancer patients: uPA (urokinase-type plasminogen activator), PAI-1 (uPA inhibitor type 1), and cathepsins B, D and L. The median follow-up of patients still alive at the time of analysis was 109 months. To date 119 patients (43%) have relapsed and 117 (42%) have died. Antigen levels of uPA and PAI-1 were determined by ELISA in detergent extracts; cathepsin B, D, and L content was determined in cytosol fractions of the primary tumor: cathepsin D by ELSA and cathepsin B and L by ELISA. In multivariate analysis (Cox model) for disease-free survival (DFS), lymph node status (p < 0.001; RR = 3.8), cathepsin L (p < 0.001; RR = 2.6) and PAI-1 (p = 0.027; RR = 1.7) were significant factors in all patients. In addition to these factors, grading was significant for overall survival (OS). In another multivariate approach, CART (Classification And Regression Trees) analysis, lymph node status (p < 0.001) turned out to be the strongest discriminator for patients at high risk of relapse. In the node-negative patient subset, PAI-1 was the strongest risk group discriminator (p < 0.001): in this subset, patients with low levels of both PAI-1 and cathepsin D had a very low relapse rate of only 3.2% compared to 39% in the remaining node-negative patients. In node-positive patients cathepsin L gave the best risk group assessment (p = 0.001). In conclusion, tumor-associated PAI-1 and cathepsins D and L provide significant, statistically independent prognostic information for DFS and OS in primary breast cancer, even after a median follow-up period of almost 10 years.     

Age-associated biomarker profiles of human breast cancer

To explore the hypothesis that aging not only increases breast cancer incidence but also alters breast cancer biology, we correlated patient age and diagnosis with tumor histology, stage and biomarkers independently determined from two different tumor archives: an American collection of approximately 800 paraffin-embedded and immunohistochemically analyzed primary breast cancers, and an European collection of approximately 3000 cryobanked primary breast cancers analyzed by ligand-binding and enzyme immunoassay (EIA). The prognostic biomarkers chosen for comparison represented surrogate measures of tumor: (i). proliferation, growth and genetic instability (mitotic and apoptotic indices, Ki-67/MIB-1-positivity, nuclear grade, p53-positivity), (ii). endocrine-dependence (estrogen receptor (ER), progesterone receptors (PR), pS2, Bcl2), (iii). growth factor receptor-dependence (ErbB2, EGFR/ErbB1), and (iv). angiogenic, invasive and proteolytic potential (uPA, PAI-1, Cathepsin D, VEGF). No biomarker reflecting tumor angiogenic, invasive or proteolytic potential showed a significant correlation with patient age at diagnosis. In contrast, significant inverse correlations (|r|>0.1; P< or =0.05) were observed for all measures of tumor growth and genetic instability as well as growth factor receptor overexpression (ErbB2 or EGFR positivity). Only one marker of endocrine-dependence, ER expression, showed a significant positive correlation with patient age at diagnosis. In summary, these findings support the hypothesis that breast cancer biology is significantly affected by patient age. In particular, breast tumors arising in older patients have slower growth rates, are more likely to be ER-positive, and are less likely to be p53-positive, EGFR-positive or ErbB2-positive.     

Insulin-like growth factor II (IGF-II) immunohistochemistry in breast cancer: relationship with the most important morphological and biochemical prognostic parameters

Recent in situ hybridization experiments have shown a high content of IGF-II mRNA in breast cancer stroma. The aim of this study was to examine the relationship between IGF-II protein expression and several prognostic parameters in 75 infiltrating ductal carcinomas (IDC) of the breast. Tissue sections were evaluated for proliferative activity, IGF-II protein, ER, PgR, p53, and p21 expression using immunohistochemical procedures. The degree of stromal proliferation was assessed. Menopausal status, axillary lymph node involvement and nuclear grade were known. Thirty-five patients (44.3%) were premenopausal and 47 (62.6%) had lymph node metastases. Marked stromal proliferation was found in 34 (45.3%) specimens and high nuclear grade in 20 (26.5%). Eighteen tumors (24%) showed no IGF-II immunostaining. In the positive cases, IGF-II was detected both in the tumor stroma and in the cytoplasm of epithelial cancer cells: a high IGF-II content was found in 12 specimens (16.0%), a low content in 14 (18.7%) and a moderate content in 31 (41.3%). Twenty-four tumors (32.0%) showed high proliferative activity. Both ER and PgR were expressed in the nucleus of cancer cells: 49 tumors (65.3%) were ER positive (ER+) and 34 (45.3%) PgR positive (PgR+). p21 protein was detected in 37 tumors (49.6%) and p53 in 12 (16%). IGF-II protein was not correlated with menopausal status, lymph node metastases, nuclear grade, proliferative activity, ER or p53. In contrast, IGF-II correlated strongly with stromal proliferation (p=0.008), PgR (p=0.03) and p21 (p=0.01). This study demonstrates that in IDC of the breast IGF-II protein is expressed in the epithelium and stroma of the majority of tumors and is correlated with stromal amount, PgR and p21 expression. These preliminary results indicate that IGF-II expression in breast cancer is connected with two important regulators of breast cancer growth and differentiation.     

Preliminary study on oncogene product immunohistochemistry (c-erbB-2, c-myc, ras p21, EGFR) in breast pathology.

The expression of oncogene products related to cell growth (c-erbB-2, c-myc, ras p21, EGFR) was investigated in benign (15 cases) and malignant breast lesions (20 cases) by means of immunohistochemistry using the avidin-biotin-peroxidase technique with polyclonal and monoclonal antibodies. The aim of this study was to evaluate the relationship between the staining positivity and various morphological and biological features, such as tumour type, grading, hormone receptor status and cell kinetic parameters. In benign breast lesions, as expected, the kinetic parameters were low, both for Ki-67 and LI. All the specimens showed a diploid condition (the DI being equal to 1) and we found a limited degree of immunoreactivity for all the growth factors and oncogene products. In breast cancer we studied the distribution of immunohistochemical positivity for EGFR, c-erbB-2, c-myc, ras p21 and Ki-67, which was related to age, nodal status, ER and PgR receptor status, LI, DI and histopathological grading. A significant positive correlation was found both between ras p21 expression and nodal status and ER-ICA positivity. We observed a strong correlation between LI and Ki-67 and an inverse relation between Ki-67 and ER expression. These findings suggest the importance of studying the relationship between prognostic factors which may provide preoperative prediction in the biological behaviour of breast cancer, not only on biopsy specimens, but also on fine needle aspirates.     

Prognostic value of pS2 protein and receptors for epidermal growth factor (EGF-R), Insulin-like growth factor-1 (IGF-1-R) and somatostatin (SS-R) in patients with breast and ovarian cancer

The prognostic value of EGF-R, IGF-1-R and SS-R, and of cytosolic estrogen-regulated pS2 protein, was studied in patients (pts) with primary breast and advanced ovarian cancer. Ovarian cancer tissues were negative for pS2 (by immunoradiometric assay) IGF-1-R and EGF-R contents (by ligand binding assay, LBA) were of no or moderate prognostic value for breast cancer pts (n = 214). For advanced ovarian cancer pts, EGF-R content determined by LBA (n = 55) showed no prognostic value, whereas EGF-R status (n = 55) determined by immunohistochemistry (MoAb 2E9) signiificantly correlated with progression of disease (P < 0.05). In breast cancer pts, both SS-R and pS2 showed no association with tumor size, nodal status and grade. For pS2 the best cut-off level with respect to relapse-free (RFS) and overall survival (OS) was found to be 11 ng/mg protein. Both SS-R (1 g% SS-R+, n = 135; P < 0.04) and pS2 (27% pS2+, n = 197; P < 0.001), which were mainly positive in ER+ tumors, were of prognostic value, especially within the subgroups with ER+/PgR+ tumors. Also within N+ and No pts the 5-yr RFS and OS showed a difference between pS2+ and pS2- (33 and 54% for N+, and 31 and 13% difference for No pts). In summary, SS-R and pS2 are valuable pronosticators in breast cancer pts, and prognostic significance of EGF-R in ovarian cancer pts needs further study.     

Cyclin D1 Serves as a Poor Prognostic Biomarker in Stage I Gastric Cancer

TNM stage still serves as the best prognostic marker in gastric cancer (GC). The next step is to find prognostic biomarkers that detect subgroups with different prognoses in the same TNM stage. In this study, the expression levels of epidermal growth factor receptor (EGFR) and cyclin D1 were assessed in 96 tissue samples, including non-tumorous tissue, adenoma, and carcinoma. Then, the prognostic impact of EGFR and cyclin D1 was retrospectively investigated in 316 patients who underwent R0 resection for GC. EGFR positivity increased as gastric tissue became malignant, and cyclin D1 positivity was increased in all the tumorous tissues. However, there was no survival difference caused by the EGFR positivity, while the cyclin D1-postive group had worse overall survival (OS) than the cyclin D1-negative group in stage I GC (10-year survival rate (10-YSR): 62.8% vs. 86.5%, p = 0.010). In subgroup analyses for the propensity score-matched (PSM) cohort, there were also significant differences in the OS according to the cyclin D1 positivity in stage I GC but not in stage II and III GC. Upon multivariate analysis, cyclin D1 positivity was an independent prognostic factor in stage I GC. In conclusion, cyclin D1 may be a useful biomarker for predicting prognosis in stage I GC.     

High levels of cathepsin B predict poor outcome in patients with breast cancer

Cathepsin B (CB) is a thiol-stimulated protease implicated in cancer invasion and metastasis. Other proteases involved in cancer spread such as urokinase-type plasminogen activator (uPA) and cathepsin D have previously been shown to be prognostic markers in breast cancer. CB was assayed by ELISA in 193 patients with primary breast cancer. CB levels were significantly higher in both primary and metastatic breast tumors than in fibroadenomas (p = 0.0001). In the primary carcinomas, CB levels showed no significant correlation with either nodal status, tumor size or estrogen receptor (ER) status. Patients with primary breast cancers containing high levels of CB had a significantly shorter disease-free interval (p = 0.01, chi-square = 6.61) and overall survival (p = 0.014, chi-square = 6.08) than patients with low levels of the protease. However, in multivariate analysis, using nodal status, tumor size, ER status and urokinase plasminogen activator (uPA), CB was not an independent prognostic marker. In contrast, nodal status, ER status and uPA were prognostic in multivariate analysis. In conclusion, CB, like certain other proteases implicated in cancer metastasis, correlates with poor outcome in patients with breast cancer. These results thus support the evidence from model systems linking CB to cancer spread. Inhibition of CB expression or activity might therefore be exploited for anti-metastatic therapies.     

Lymphatic vessel invasion detected by the endothelial lymphatic marker D2-40 (podoplanin) is predictive of regional lymph node status and an independent prognostic factor in patients with resected esophageal cancer

The discovery of markers to lymphatic endothelial cells and the development of novel antibodies to these markers have brought increasing attention to the lymphatics and progress in the understanding of lymphangiogenesis and cancer metastasis. In this study, we investigate the presence of lymphatic vessel invasion (LVI) detected by D2-40 immunohistochemical staining in resected esophageal cancer and correlated with clinicopathologic data and patient survival. Sixty nine patients, who had a primary resection of esophageal cancer, were analyzed by univariate and multivariate logistic regression, and univariate and multivariate survival analysis. The total rate of LVI was 72% (50/69). Positive LVI was significantly correlated with lymph node metastasis (p < 0.001), tumor size (p < 0.001), histological grading (p = 0.017), tumor depth (p = 0.001), and stage (p < 0.001). Multivariate logistic analysis identified LVI (p = 0.036) as a predictor of regional lymph node metastasis. On univariate survival analysis, patients with LVI had a significantly shorter disease-free survival, cancer-specific survival and overall survival. Multivariate analysis proved that LVI diagnosed by D2-40 is an independent prognostic factor of both disease-free survival (p = 0.04) and overall survival (p = 0.032) in resected esophageal cancer. These results show that LVI assessment identifies patients at high risk for regional lymph node metastasis and that LVI is an independent prognostic factor in patients with esophageal cancer.     

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas

p53 protein expression and oestrogen and progesterone receptor status in invasive ductal breast carcinomas The p53 protein expression and oestrogen and progesterone receptors status was investigated in correlation to the grade of malignancy of primary breast carcinomas. Our material constituted imprints from surgical biopsies of 75 invasive ductal breast cancer cases. The p53 protein expression was investigated immunocytologically using the monoclonal antibody p53 DO-7 (DAKO). A biochemical DCC method was applied for the detection of oestrogen and progesterone receptors for all tumours. Fifty-one percent of breast cancer cases were p53 protein positive. A statistically significant association of p53 protein expression and high tumour grade was found (chi2=23.72, d.f.=2, P < 0.001). A statistically significant association was also found between oestrogen and progesterone receptor positive cases and the grade of malignancy (P < 0.001). A negative association between p53 protein expression and oestrogen (ER) and progesterone receptors (PgR) positivity was found. From our results it appears that it is possible to distinguish from grade II tumours two subgroups of cases, one with low malignancy potential and p53 (-), ER (+), PgR (+), and another subgroup with high malignancy potential and phenotype p53 (+), ER (-), PgR (-). The last subset of patients could actually benefit from adjuvant therapy.     

Relationship between steroid receptors (as continuous variables) and response to adjuvant treatments in postmenopausal women with node-positive breast cancer.

In current clinical practice for breast cancer patients, estrogen (ER) and progesterone receptor (PgR) concentrations, quantified by the dextran-coated charcoal assay, are categorized by an arbitrary cutoff into a negative or positive status. However, although the results obtained with this approach are easy to interpret, such a representation could oversimplify the relationship between ER and PgR content and patient outcome and imply an assumption of monotonicity, which is generally expected but rarely proven. We evaluated the relationship between ER and PgR content (considered on a continuous scale) and clinical outcome, using a flexible statistical model, in a group of postmenopausal patients with N-positive operable tumors who were submitted to surgery and different adjuvant treatments (tamoxifen or CMF). Univariate analysis indicated that in the tamoxifen-treated group, ER level, number of metastatic nodes (pN) and age, but not PgR, were significant indicators of clinical outcome (p = 0.032, p = 0.021 and p = 0.029, respectively). Multivariate analysis indicated that in this group of patients there was no interaction between variables, and in the final model for disease-free survival (DFS) only ER and pN were retained with an overall predictive ability of the regression model of 0.723, as evaluated by Harrell's c. However, pN markedly contributed to the predictive ability of the model with respect to ER, since a marked decrease in Harrell's c statistic (c = 0.582) was observed when pN was removed from the model. In the CMF-treated group, only pN affected clinical outcome. When the estimated DFS curves obtained from the final Cox regression models were plotted according to four values of ER (in the tamoxifen-treated group) or three values of pN (in the CMF-treated group) we observed that in the tamoxifen-treated group patients with an ER concentration equal to 0 fmol/mg cytosol protein had the worst prognosis, whereas a marked improvement of the expected DFS was observed for patients with a low but detectable ER level (generally classified as ER-negative because falling below the conventional cutoff value of 10 fmol/mg cytosol protein). Our results seem to suggest that the use of steroid receptor concentrations on a continuous scale, instead of dichotomous "status", is to be preferred in the choice of adequate therapeutic strategies.     

Telomerase activity in breast cancer in Western India (Gujarat)

The purpose of this study was to examine the association of telomerase activity with clinical and histopathological prognostic variables in primary breast cancer (n=64). Telomerase activity in breast cancer was also compared with that in benign (n=10) and non-malignant tissues (n=8; post-lumpectomy tissues histopathologically defined as containing no residual tumor). The parameter was assessed using the Telomerase PCR ELISA kit. Values above OD 0.120 were considered positive. Estrogen and progesterone receptors (ER and PgR) were assayed by the dextran-coated charcoal method and levels >10 fmol/mg cytosol protein were taken as positive. Telomerase activity was detected in 20% and 50% of the patients with benign lesions and primary breast cancer, respectively, and in 50% of post-lumpectomy breast tissues histopathologically defined as containing no residual tumor. Telomerase activity was present in all stages of breast carcinoma and showed a significant inverse correlation with lymph node status (p=0.006), lymphatic invasion (p=0.035) and necrosis (p=0.033). Moreover, when stage II patients were grouped according to nodal involvement, a trend towards significance was observed (p=0.055). No correlation was observed with ER and PgR. The results of our study suggest that telomerase activity might be associated with the presence of cancer cells. Furthermore, telomerase activation may occur early in breast cancer and may be periodically downregulated during subsequent tumor progression.     

Investigation of oncogene amplification or deletion,and oncoprotein expression in papillary thyroid cancer

AIM: Assessment of occurrence and possible prognostic significance of c-myc and Ha-ras amplification, p53 deletion and overexpression of cyclin D1, p53 and p21 in papillary thyroid cancer. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tumor tissue from 24 patients were investigated. Dot-blot DNA hybridization was used to detect oncogene amplification or deletion. The expression of oncoproteins was determined by immunohistochemical method. RESULTS: In our samples neither Ha-ras amplification nor p53 deletion were found. Low c-myc amplification (mean: 2.55) occured in 4 cases (17%). p53 protein was detected in 16 samples (66.6%), with p21 expression (chi(2)=7.02, p<0.01) in 6 cases (25%). The p53 expression did not influence the tumor fenotype. Cyclin D1 overexpression was found in 12 cases (50%), it was often associated with p21 expression (chi2=10.1, p<0.001) and in inverse relation to the tumor lymphocytic infiltration (chi(2)=5.35, p<0.05). Increased expression of estrogen receptor was shown in 4 cyclin D1 positive samples (17%). CONCLUSIONS: The p53 detected in our study is likely not to be mutant protein in all cases because its presence was associated with p21 expression that the mutant protein cannot induce and also it did not mean more aggressive tumor phenotype. The connection of cyclin D1 overexpression with the lymphocytic infiltration of the tumor suggests that the increased expression of cyclin D1 means poor prognosis. The coexpression of cyclin D1 and p21 raises the modulative character of the p21 protein, thought to be a tumor suppressor originally, but we find a CDK-independent, estrogen receptor mediated effect of cyclin D1 more likely, which has been described in breast cancer and is also proved by the coexpression of cyclin D1 and estrogen receptor detected here.