High sensitivity and specificity of immunohistochemistry for the detection of hormone receptors in breast carcinoma: comparison with biochemical determination in a prospective study of 793 cases

AIMS: The hormone receptor (HR) status of breast cancer is an important prognostic factor and predictive parameter of the response to hormone therapy. Enzyme immunoassay (EIA) is currently the standard for determination of HR, but immunohistochemistry (IHC) represents a potentially useful alternative. We used IHC to determine HR status in a large prospective study and compared the results to those obtained by EIA. This study was designed to determine which technique should be used in daily practice in our institution which manages a large number of patients. METHODS AND RESULTS: Oestrogen (ER) and progesterone (PgR) receptor status was evaluated in a prospective series of 793 infiltrating breast cancers by IHC in paraffin-embedded tissue sections, using antibodies 6F11 and 1A6, with a rigorous quality control of the methodology. ER were found to be significantly expressed in 81% of cases after IHC analysis and in 78% of cases by EIA. For PgR, the respective rates of positivity were 65% and 69%. The tumour HR level detected by either technique was significantly correlated with the value of tumour size, histological grade and S-phase fraction. A significant link was observed between the percentage of labelled cells after IHC analysis and the amount of protein detected by EIA. Critical analysis of discordance found that, in the group of invasive lobular carcinomas, the rate of HR positivity was higher with IHC (84%) than with EIA (45%) and that, in the overall population, IHC was more specific than EIA, since cases with nonrelevant positivity related to intraductal normal or neoplastic cells expressing HR could be discarded. The cost of IHC analysis was found to be about one-third of that of EIA. CONCLUSIONS: IHC is more sensitive, specific and economical than EIA. It should constitute the new standard technique provided that good quality assurance procedures are respected.     

Evaluation of BrightGen HR RT-qDx assay to detect nuclear receptors mRNA overexpression in FFPE breast cancer tissue samples for selection of tamoxifen therapy

Breast cancer is a significant cause of death in women. Estrogen receptor (ER) and progesterone receptor (PR) are important prognostic factors indicating higher recovery rate in the breast cancer patients. Currently, immunohistochemical (IHC) staining is a conventional method to identify expression of ER and PR. If a breast cancer patient expresses ER or PR, a chemotherapy with estrogen inhibitors such as tamoxifen is supposed to be effective. Although IHC staining is a reliable method, it may not a useful method for continuous monitoring of ER and PR expression changes in multiple breast cancer patients. In the present study, we evaluated an alternative method of IHC for detection of ER and PR expression. A quantitative RT-PCR method called ‘the BrightGen HR RT-qDx assay’ was employed to detect mRNA expression of the nuclear receptors in 199 formalin-fixed paraffin-embedded (FFPE) breast cancer tissue samples. Among the ER/PR positive samples by IHC, 83 were determined positive and 16 were determined negative for the nuclear receptor mRNA by the quantitative RT-PCR method. Among the ER/PR negative samples by IHC, 37 were determined negative and 2 were determined positive by the quantitative RT-PCR method. The overall sensitivity and specificity of the quantitative RT-PCR method were 83.8% and 94.8% (P = 0.0026), respectively. We also optimized the quantitative RT-PCR method by setting up the diagnostic cut-off value using the likelihood ratio. The highest likelihood ratio was when the expression levels of the relative nuclear receptor mRNA passed 103.3 at which sensitivity and specificity was highest. These data suggest that BrightGen HR RT-qDx assay could be an alternative method for detection of the prognostic factors of nuclear receptors expressed in breast cancer patients for providing essential information for therapeutic application of tamoxifen.     

Expression of prognostic factors (EGFR, ER) by immunohistochemical staining method in male breast cancer

Twelve male patients with operable breast cancer were evaluated for the expression of prognostic factors by immunohistochemical staining assay. Seven patients were stage I & II, and five patients were stage III. Axillary lymph node positivity was 42%. Nine patients were nuclear grade I, three were nuclear grade II, and none were nuclear grade III. The expression rate of EGFR (epidermal growth factor receptor), ER (estrogen receptor) were 8.3%, 70.0% respectively. This limited data suggest better tumor behavior in male than in female breast cancer. Adjuvant treatment should be considered in male breast cancer just as in females, based on axillary lymph node and ER states.     

Correlation between immunohistochemical and biochemical estrogen receptors in the prognosis of patients with breast cancer.

To evaluate the reliability of immunohistochemical estrogen receptor (ER) in the prognosis of patients with breast cancer, 83 primary tumors from patients were studied. Immunohistochemical analysis (IHA) was performed using antibody ER 1D5 (Dako) together with microwave treatment for antigen retrieval. ER values obtained using the biochemical steroid binding assay (polyethyleneglycol method, PEG) were available for comparison. Of all tumors, ER positivity was detected in 44.6% by IHA and 36.1% by PEG method. The concordance between the two methods was 69%. No significant correlation was found between the ER status determined by both methods and clinical stage, tumor size, lymph node status or age of patient at diagnosis. However, we found that the immunohistochemical ER is a superior predictor of early recurrence in patients with primary breast cancer to biochemical ER. The findings in the present study emphasize the clinical benefit of the immunohistochemical ER assay as a measure for prognosis.     

Comparison of monoclonal immunocytochemical and immunoenzymatic methods for steroid receptor evaluation in breast cancer

The production of monoclonal antibodies against estrogen receptor (ER) and progesterone receptor (PR) has permitted the development of the enzyme immunoassay (EIA) and immunocytochemical assay (ICA) for steroid receptor determination. The results obtained with these two techniques, using the same monoclonal antibodies, were compared in a large series of breast carcinomas (187 for ER and 100 for PR). The correlation between these methods was significant for ER (rs = 0.54) and PR (rs = 0.55) (P less than 0.001) but was lost when the receptor concentrations determined by EIA were less than or equal to 15 and less than or equal to 30 fmol/mg protein for ER and PR, respectively. When these values are considered as cutoffs, the concordance between the two methods was 84.5% for ER and 73% for PR. An analysis of discordant results revealed that low epithelial cellularity generally was present in ICA-positive, EIA-negative specimens, whereas only focal positivity with ICA, or positivity of only normal peripheral mammary ducts and lobules, frequently was found in ICA-negative, EIA-positive tumors. In conclusion, there is good correlation between the results obtained by EIA and ICA methods for detection of ER and PR. The authors suggest that biochemical and histochemical methods for steroid receptors could be considered complementary and used together for the analysis of breast cancer.     

雌激素受体α阴性乳腺癌雌激素受体α基因启动子区CpG岛甲基化和肼苯哒嗪去甲基化作用

目的 检测雌激素受体(ER)α阴性乳腺癌细胞株MDA-MB-231和MDA-MB-435细胞及ERα阴性乳腺癌组织中ERα基因启动子区CpG岛甲基化状态;探索肼苯哒嗪能否作为去甲基化药物恢复ERα基因表达。方法应用特异性聚合酶链反应(MSP)检测乳腺癌细胞株MDA-MB-231和MDA-MB-435细胞和20例ERα阴性乳腺癌组织ERα基因3个启动子区A、B、CpG岛甲基化情况,肼苯哒嗪处理上述两种乳腺癌细胞,逆转录(RT)-PCR检测不同启动子调控下ERα基因异型体(isoform)ERα-A、ERα-B、ERα-C mRNA和ERα基因公共编码区mRNA表达。结果MDA-MB-231和MDA-MB-435细胞启动子区ERα-A、ERα-B均存在CpG岛甲基化,ERα-C无甲基化,20例ERα阴性乳腺癌组织中,13例(65％)ERα-A、10例(50％)ERα-B CpG岛甲基化阳性。其中9例ERα-A、ERα-BCpG岛甲基化均阳性(45％),仅1例(5％)ERα-C存在CpG岛甲基化。肼苯哒嗪处理上述两种细胞后,检测到ERα-A、ERα-B mRNA和公共编码区mRNA表达。结论乳腺癌组织和细胞ERα基因表达沉默可能与ERα基因启动子区A、B甲基化有关,且肿瘤分期愈晚,甲基化程度愈高。肼苯哒嗪能作为去甲基化药物诱导ERα基因表达。     

A study to determine the incidence of estrogen receptor (ER) and progesterone receptor (PR) expression in different histological grades of breast cancer

Abstract

Carcinoma of the breast is the most common non-skin malignancy in women and second most common cause of death due to cancer in females. Breast cancer is a heterogeneous disease with varied morphological appearances, molecular features, behavior, and response to therapy. This study was conducted with the aim of analysis of steroid receptor status in breast cancer and its association with other prognostic factors of breast cancer such as histological grade, tumor size, and axillary lymph node status. Fifty patients with the diagnosis of breast cancer were included in this study. Detailed clinical and histopathologic data were recorded in all cases. Estrogen receptor (ER) and progesterone receptor (PR) status was evaluated by immunohistochemistry. Histological grading was done with Nottingham modification of the Bloom and Richardson method. Fisher’s exact test and chi-square test was used for statistical analysis. On immunohistochemical staining, 44% cases proved to be ER positive and 48% cases PR positive. The results from the present study in India documented low ER and PR positivity in breast cancer in comparison to results from the western world. An inverse relation was found between hormone receptor (HR) status and histological grading, but no statistically significant relationship was noted between HR expression and tumor size and lymph node status.     

Immunohistochemical double staining with estrogen receptor and HER2 on primary breast cancer

Immunohistochemical double staining with estrogen receptor (ER) and epidermal growth factor receptor 2 (HER2) was conducted in tissue sample of 125 women with invasive breast cancer. The age at the time of surgery ranged from 28 to 82 years. The tumor size was 2 cm or less in 42 patients and larger than 2 cm in 83. Axillary lymph node status was positive in 53 patients and negative in 72. Estrogen receptor (ER) which was measured using enzyme immunoassay (EIA) was positive in 67 patients, negative in 50 and unknown in 8. Of the 125 patients evaluated, 83 (66.4%) were immunohistochemically positive for ER. ER by immunohistochemistry (IHC) (ER-IHC) was significantly (p<0.01) correlated with ER by EIA (ER-EIA). ER-EIA values in ER-IHC scores were 1.4 fmol/mg protein in score 0, 0.0 in score 1, 19.0 in score 3, 21.2 in score 4, 12.2 in score 5, 17.6 in score 6, 30.0 in score 7 and 114.8 in score 8. ER-EIA values in ER-IHC-score 8 were significantly higher than in scores 0, 2, 3, 4, 5, 6 and 7. Of the 125 patients, 35 (28%) were immunohistologically positive for HER2. HER2 expression was inversely correlated with ER expression. When evaluated even in ER-positive patients, HER2 overexpression was associated with lower ER levels. In this study, we conducted immunohistochemical double staining with ER and HER2, and demonstrated that low ER levels might be one factor in the relative resistance of HER2-positive and ER-positive tumors to hormonal therapy.     

Expression of cyclin kinase subunit 2 in human breast cancer and its prognostic significance

Cyclin kinase subunit 2 (CKS2) protein is a small cyclin-dependent kinase-interacting protein, which is essential for the first metaphase/anaphase transition of mammalian meiosis. CKS2 is up-regulated in various malignancies, suggesting that CKS2 maybe an oncogene. However, data on its expression pattern and clinical relevance in breast cancer are unknown. The aim of this study is to investigate CKS2 expression and its prognostic significance in breast cancer. The CKS2 expression was examined at mRNA and protein levels by real-time quantitative polymerase chain reaction (RT-PCR) and Western blotting analysis in paired breast cancer tissues and the adjacent normal tissues. The expression of CKS2 protein in 126 specimens of breast cancer was determined by immunohistochemistry assay. The relations between CKS2 expression and clinicopathological features were analyzed. The result show the expression of CKS2 mRNA and protein was higher in breast cancer than the adjacent normal tissues. Compared with adjacent normal breast tissues, Overexpression of CKS2 was detected in 56.3% (71/126) patients. Overexpression of CKS2 was significantly associated with large tumor size (P = 0.035), poor cellular differentiation (P = 0.016), lack expression of progesterone receptor (P = 0.006), and decreased overall survival (P = 0.001). In multivariate analysis, CKS2 expression was an independent prognostic factor for overall survival (Hazard ratio [HR] = 3.404, 95% confidence interval [CI] 1.482-7.818; P = 0.004). CKS2 is up-regulated in breast cancer and associated with large tumor size, lack expression of progesterone receptor, poor tumor differentiation and survival. CKS2 may serve as a good prognostic indicator for patients with breast cancer.     

Frequency of oestrogen and progesterone receptor positivity by immunohistochemical analysis in 7016 breast carcinomas: correlation with patient age, assay sensitivity, threshold value, and mammographic screening

AIMS: A routine immunohistochemical (IHC) assay is now commonly used for determining the oestrogen receptor (ER) and progesterone receptor (PR) status of women with breast cancer. To date, no large studies have been conducted that report the expected frequency of receptor positivity in relation to patient age and sensitivity of the IHC assay. Data on 7016 breast carcinomas from 71 laboratories were analysed to determine the frequency of receptor positivity and investigate possible causes of the observed variation in detection rates. METHODS: Members of UK NEQAS-ICC (UK National External Quality Assessment Scheme for Immunocytochemistry) provided data on the receptor status of cases routinely assayed in their departments over a period of two to 26 months between June 1996 and September 1998. Data on 7016 breast carcinomas were stratified according to patient age and receptor status. Frequency of receptor positivity was correlated with IHC assay sensitivity, the threshold value used to determine receptor positivity, and the presence or absence of mammographic screening in the hospitals or clinics served by the laboratories. RESULTS: The highest proportion of receptor positive cases occurred in patients in the age ranges > 65 years for ER and 41-50 years for PR. There was a significant positive correlation between frequency of receptor positivity and the sensitivity of the IHC assay, for both ER (rs = 0.346; p = 0.019; two tailed) and PR (rs = 0.561; p = 0.003; two tailed). The mean frequency of receptor positivity for laboratories using the same 10% threshold value was 77% for ER (95% confidence interval (CI), 74% to 80%) in laboratories with high sensitivity and 72% (95% CI, 68% to 76%) for those with low assay sensitivity (p = 0.025). For PR, the mean frequency of receptor positivity for laboratories using the same 10% threshold value and having high assay sensitivity was 63% (95% CI, 57% to 69%), and 51% (95% CI, 38% to 65%) for laboratories with assays of low sensitivity (p = 0.022). The mean frequency of ER positivity for laboratories serving hospitals and clinics where mammographic screening does and does not take place was 73.4% and 75.7%, respectively (p = 0.302; two tailed). CONCLUSIONS: Of the parameters investigated, patient age and IHC assay sensitivity were found to be the main variables influencing the frequency of receptor positivity. We recommend the range of receptor values obtained by laboratories achieving high assay sensitivity as a useful guide against which all laboratories can gauge their own results.     

Prognostic significance of Bcl-2 in invasive mammary carcinomas: a comparative clinicopathologic study between "triple-negative" and non-"triple-negative" tumors

Bcl-2 is a tumorigenic protein that is expressed in 25% to 50% of breast cancers. Although its expression has been widely accepted as a favorable prognostic marker, its protective mechanism of action remains unclear. "Triple-negative" tumors are an aggressive subgroup known to carry a poor prognosis. Studies documenting prognostic significance of Bcl-2 expression in triple-negative in comparison to non-triple-negative breast cancers are limited. Bcl-2 expression was correlated with tumor size, grade, histologic type, lymphovascular invasion, lymph node status, patients' overall survival, estrogen receptor, progesterone receptor, Her-2, p53, and epidermal growth factor receptor in 124 triple-negative and 458 non-triple-negative tumors. There were significant differences between triple-negative and non-triple-negative tumors in their relationship to Bcl-2 expression (81% versus 29%, respectively) and tumor aggression. As previously reported, in non-triple-negative tumors, Bcl-2 positivity correlated with less aggressive tumors (94% of grade I tumors were Bcl-2+ versus 62% of grade III tumors, P < .011) and overall survival (P = .008). However, the opposite was true in patients with triple-negative tumors, where Bcl-2 positivity was associated with poorer survival (P = .64). In triple-negative tumors, Bcl-2 positivity was not associated with any of the aforementioned parameters except for a lower incidence of lymph node metastasis. Moreover, by Cox regression analysis of all variables, in patients with triple-negative tumors, lymphovascular invasion (P = .009) and Bcl-2 expression (P = .028) were predictors of poor survival. In conclusion, there are major clinicopathologic differences between breast cancer phenotypes. Our results establish the value of using Bcl-2 in prognostic stratification of patients and its potential therapeutic implications in selecting patients for treatment.     

The significance of c-erbB-2 overexpression and p53 expression in patients with axillary lymph node--negative breast cancer: a tissue microarray study

We conducted this study to examine whether the expression of c-erbB-2 and p53 is the prognostic indicator for patients with early-stage breast cancer in which axillary lymph node metastasis is absent. We examined 326 patients with early-stage breast cancer in which axillary lymph node metastasis is absent. Tissue microarrays were constructed. Following this, immunohistochemical staining was done for estrogen receptor (ER), progesterone receptor (PR), c-erbB2, and p53. The results were as follows: (1) expression of c-erbB-2 was correlated with other clinicopathologic factors (eg, patient's age, presence of menopause, tumor size, histologic and nuclear grade, and presence of hormone receptors such as ER and PR); and (2) expression of p53 was correlated with survival rate, patient's age, presence of menopause, and tumor size. However, these results were not statistically significant. In conclusion, our results indicate that expression of c-erbB-2 and p53 did not have any prognostic value in patients with early-stage breast cancer in which axillary lymph node metastasis is absent.     

Studies on p53 immunolocalisation in breast cancer and its prognostic significance

Immunocytochemical localisation of mutant p53 in breast tumours serves as a potential prognostic molecular marker. In order to study the expression of p53 protein in breast cancer which constitutes the second most common malignancy in the South Indian female population, MAb CIBCVMC12 has been generated against human p53 protein isolated and purified from bacterial cell lysate of E.coli carrying the plasmid T 7-7 Hup53 grown in Luria broth to induce the expression of p53. The positive clones selected by ELISA were found to exhibit strong staining of nuclear p53 in both fresh and archival paraffin embedded breast tumour tissue sections. Commercial MAb D 07 against p53 was used as control. In immunoprecipitation, this MAb of IgG2b isotype was found to bind specifically to a protein of 53 kD. Immuno cyto chemical assay of normal, benign and malignant breast tissues of different histological types revealed that the majority of tumour cells were strong positive in the case of infiltrating ductal and lobular carcinomas, the staining being less intense for in situ carcinoma. The test for normal and benign tissues was negative. The staining patterns were comparable with those of control antibody. These results suggest that the MAb generated is specific to p53. The p53 protein expression was compared with the estrogen receptor (ER) status for 50 breast tumours which revealed that 38% of these were p53 positive and of these two were ER+. Among the p53 negative tumours, 48% were found to be ER+. A comparison of the p53 expression for 100 breast cancer patients indicated that 57% of the tumours were p53 negative and these patients had a longer overall 5 year survival rate and recurrent free interval which is statistically very significant. These results might suggest that p53 positive tumours are more aggressive biologically with poor prognosis.     

Estrogen receptor analysis on biopsies and fine-needle aspirates from human breast carcinoma. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies.

Monoclonal antibodies against estrogen receptor (ER) were used for determination of ER status immunocytochemically in histologic specimens from 192 primary breast carcinomas. All tumors were also assayed biochemically for ER with the dextran-coated charcoal method (DCC). The comparison of biochemically and immunocytochemically determined ER status showed concordant results in 80% (P less than 0.0001). In only 2 cases (1%) with low ER levels (less than 20 fmol/mg protein) immunocytochemistry failed to detect ER. ER positivity determined with a semiquantified approach based on intensity and heterogeneity of immunocytochemical staining correlated significantly with biochemically determined ER levels (P = 0.0001). In a series of fine-needle aspirates of 34 breast carcinomas sufficient cell material was available for ER immunocytochemistry (ER-ICA). Overall, the results of ER-ICA in fine-needle aspirates were concordant with ER-ICA in histologic specimens in 88% of the samples. In a few cases with weak positivity of ER-ICA in histologic specimens, ER-ICA was negative in fine-needle aspirates. In no case was there a false-positive immunocytochemical ER determination in a tumor aspirate. Thus, ER-ICA seems to be a reliable assay which can be performed in histologic and cytologic specimens.     

Immunohistochemical profile of invasive lobular carcinoma of the breast: Predominantly vimentin and p53 protein negative,cathepsin D and oestrogen receptor positive

Vimentin, p53 protein and cathepsin D positivity were assessed by immunohistochemistry, and oestrogen receptor (ER) by an enzyme immunoassay, in invasive lobular carcinomas (LC) of the breast. While vimentin was positive in only 5% (3/57) and p53 protein was positive only in 3% (2/63), cathepsin D was expressed in 86% (48/56) and ER in 78% (25/32). Classical LC were negative for p53 protein and all except one were cathepsin D positive. These results are in contrast to invasive ductal breast carcinomas (DC), where the reported average incidence of vimentin and p53 protein is much higher (19% and 33% respectively) and that of cathepsin D and ER lower (63% and 67% respectively). Thus lack of expression of vimentin and lack of p53 positivity together with high incidence of expression of cathepsin D and ER are more often associated with lobular than with ductal differentiation of invasive breast cancer. The results show that LC, distinguished morphologically, can further be defined by its immunohistochemical profile. This in turn may point to underlying biological differences between LC and DC.     

Estrogen receptor β– an independent prognostic marker in estrogen receptor α and progesterone receptor‐positive breast cancer?

Both subtypes of estrogen receptor (ER), ERα and ERβ, are normally present in the mammary gland. The role of ERα as a prognostic marker in breast cancer is well established due to the beneficial effect of providing tamoxifen as adjuvant therapy. The role of ERβ, however, is less clear. To gain insight into the importance of ERβ in breast cancer, 145 primary breast cancers were examined by immunohistochemistry for ERβ, and the expression level was compared with ERα and progesterone receptor (PR) status. Especially, we wanted to examine the significance of ERβ in the contrasting ERα+/PR+ and ERα?/PR? subgroups. In the ERα+/PR+ subgroup (dual positive), the survival difference between patients with low, medium and high ER β level was statistically significant (p = 0.004), with more than 70% of patients with medium and high ERβ levels surviving 100 months, compared with less than 30% in the group with low ERβ level. Further, for ERα+/PR+ patients there was a reduced risk of fatal outcome by multivariate analysis with increasing ERβ levels (p(trend) < 0.01 [univariate analysis]; p(trend) = 0.05 [multivariate analysis]). The risk was 31% and 27% for medium and high ERβ levels, respectively, compared with low ERβ level, adjusting for standard prognostic factors such as tumor diameter, nuclear tumor grade (quantified by mean nuclear area), lymph node status, and patient age at operation. For patients with ERα?/PR? tumors (dual negative), however, there was no association between ERβ levels and patient outcome. Our findings indicate that ERβ expression provides independent prognostic information for breast cancers with ERα/PR‐positive status, a feature typical among screen‐detected breast cancers. The role of ERβ needs to be further evaluated especially in this group of breast cancers.     

Expression of steroid receptors and DNA synthesis in male breast cancer

Using a double-labeling immunocytochemical-autoradiographic assay we studied 18 male breast carcinomas to evaluate the cell kinetic and receptor status in neoplastic cells during S-Phase and to detect possible differences with respect to 21 female breast cancers, from a previously, published study. In male breast cancer, the tumor receptor content and ER/PgR expression in neoplastic cells during S-Phase was higher (p = 0.01) than that in corresponding female tumor while tumoral cell proliferation was lower, but not significantly. In the previous reported study on female breast cancer we demonstrated that proliferative activity was higher in receptor negative cell population both for ER and PgR. Conversely, in male tumor, that difference was only present in relation to the expression of PgR: in fact, the proliferative activity was higher in PgR negative than in PgR positive cells (Anova Test: p = 0.04) while no difference was evidenced between ER negative versus ER positive cells. Moreover, the arrest of DNA synthesis, expressed as percentage of cells without 3H-Tdr labeling, was not related to either the ER or PgR expression, while in female breast cancer it was higher in PgR positive than ER positive cell population. Our data confirmed differences between males and females regarding the receptor status and cell cycle S-Phase in breast cancer. The poorer prognosis of the male breast carcinoma might be related to ineffective therapies which do not consider these differences in the biological profile of the male tumor. The Authors indicate that prognostic and predictive tissutal markers, detected by immunocytochemical methods and useful for therapeutic programming in the female breast cancer have a different significance in the male breast cancer and stress the need for different therapeutic strategies specific for male breast cancer.     

Prognostic value of combined analysis of cyclin D1 and estrogen receptor status in breast cancer patients

The amplification of cyclin D1, located on chromosome 11q13, in breast cancer patients has been found to be associated with reduced relapse-free and overall survival; however, there still exists strong controversy about these findings. In order to evaluate the prognostic value of cyclin D1 and other prognostic variables in human breast cancers, we have assessed estrogen receptor (ER) status, cyclin D1, c-erbB2 and p53 overexpression in 175 primary breast carcinomas, and investigated the relationships of prognostic variables to the patient clinical outcome and the association between cyclin D1 overexpression and other prognostic variables. There was some degree of variability in staining intensities and proportions within the same tumor. The overexpression of both cyclin D1 and ER revealed a significantly prolonged survival in univariate analysis (P = 0.020). Among the various prognostic variables, distant metastasis showed a statistically significant association with overall survival. A significant correlation was observed between cyclin D1 overexpression and small size of the primary tumor (P = 0.031), low Bloom and Richardson's histological grade (P = 0.001), and positive ER status (P = 0.000). In contrast to what was previously expected, the present study suggests that the overexpression of cyclin D1 has a tendency to have a positive clinical outcome and a potential role in identifying a subset of patients predicting a good prognosis, particularly when ER is coexpressed.     

p21WAF1 expression in invasive breast cancer and its association with p53, AP-2, cell proliferation,and prognosis

AIMS: To evaluate the expression and prognostic relevance of p21(WAF1) in breast cancer and to investigate its association with p53, activator protein 2 (AP-2), and cell proliferation (as assessed by Ki-67 expression). METHODS: p21(WAF1) expression was analysed immunohistochemically in a large prospective, consecutive series of 420 patients with breast cancer diagnosed and treated between 1990 and 1995 at Kuopio University Hospital, Kuopio, Finland. Inter-relations between p21(WAF1) expression and p53, AP-2, and Ki-67 were evaluated. The expression of p21(WAF1) was also compared with clinicopathological parameters and the patients' survival. RESULTS: In general, nuclear p21(WAF1) expression was low in carcinomas (median, 2.5%; range, 0-70%). Expression was lowest in lobular carcinomas (chi(2) = 7.4; p = 0.025). p21(WAF1) positive tumours were more often p53 positive (chi(2) = 4.2; p = 0.041) but expression of p21(WAF1) did not correlate with AP-2 expression or Ki-67 in the whole patient group. In addition, the combined expression of p21 and p53 was not associated with AP-2 expression. High nuclear p21(WAF1) positivity (n = 160; 38%) was associated with poor differentiation (chi(2) = 8.1; p = 0.017). In the univariate analyses, p21(WAF1) expression had no prognostic value for predicting breast cancer related survival (BCRS) or recurrence free survival (RFS) in the whole patient group or in the subgroups investigated. However, in postmenopausal patients with lymph node metastases, and oestrogen receptor (ER) and/or progesterone receptor (PR) positive tumours, high p21(WAF1) expression predicted response to adjuvant hormonal treatment with antioestrogens. In the univariate analysis, the significant factors for predicting BCRS were Ki-67 expression, stage, lymph node status, histological grade, ER and PR status, and those for RFS were Ki-67 expression, stage, and lymph node status. In the multivariate analysis, the independent predictors of shorter BCRS were high cell proliferation activity measured by Ki-67 expression (p < 0.001), advanced stage (p < 0.001), and poor differentiation (p = 0.048). Shorter RFS was independently predicted by high cell proliferative activity (p < 0.001) and advanced stage (p < 0.001). CONCLUSIONS: The regulation of p21(WAF1) seems to occur independently of p53 or AP-2 and analysing p21(WAF1) expression provided no prognostic information for patients with breast cancer.