Automated and quantitative immunocytochemical assays of Bcl-2 protein in breast carcinomas

Abstract

Summary Expression of the bcl-2 gene was investigated in 218 human breast carcinomas by immunohistochemical analysis. Immunodetections were assessed using (1) frozen sections, (2) documented commercially available monoclonal antibody (bcl-2/124, Dako), (3) automation of immunoperoxidase technique (Ventana) and (4) quantitative evaluation of results by image analysis (SAMBA) and statistical analysis of quantitative data (BMDP software). Bcl-2 protein expression was correlated with current prognostic indicators and with molecular markers detected by the same procedure as for Bcl-2. It was shown that Bcl-2 expression is not related to patients' age, tumour size and type or lymph node status, but an inverse relationship was observed between Bcl-2 and tumour grade (P < 0.0001). An inverse relationship was also observed between Bcl-2 expression and p53 (P<0.0001), Ki67/MIB1 antigen-(P=0.0012), and P-gp-(P=0.002) positive immunoreactions. In contrast, anti-Bcl-2 positive reaction was significantly associated with ER-positive (P < 0.001) and with ER/PR-positive or ER/PR/pS2-positive immunoreactions (P < 0.005). Bcl-2 expression was independent of CD31 and cathepsin D expression. Thus, Bcl-2 protein, thought to be antiapoptotic, exhibits parodoxical expression in human breast carcinomas. It is strongly detected in low-grade tumours (well-differentiated) with low (MIB13) growth fraction, but is independent of the tumour progression (size, node status, CD31, and cathepsin D). Bcl-2 acting on apoptosis is related to p53 gene abnormalities in breast carcinomas. Bcl-2 protein expression may also be involved in response to endocrine therapy (associated to ER/PR/pS2 positive immunoreactions) and probably with chemoresistance mechanisms (inverse relationship with P-gp).

Bcl‐2 expression on fine‐needle aspirates from primary breast carcinoma

“…Our data regarding FNAs confirm the significant inverse association between Bcl-2 expression and p53 positivity, in keeping with the results reported by other authors on histologic sections. 7,9,12,15,[17][18][19]21 We found a significant inverse relation between Bcl-2 expression and Ki-67 growth fraction, in agreement with other studies regarding histology that correlate Bcl-2 expression with proliferative activity assessed either by Ki-67, 17,19 S-phase fraction, 9,21 or 3 Hthymidine labeling index. 12 Several studies support the reliability of nuclear grade evaluated on cytologic smears from FNAs of breast carcinoma.…”

“…In agreement with data obtained regarding histologic samples, a strong positive relation between Bcl-2 immunoreactivity and ER and PgR status was found. 7,9,[11][12][13][14][15][16][17][18][19][20] This observation supports the hypothesis that Bcl-2 in breast carcinoma may be an ERregulated protein, like PgR. 34 Accordingly, in a series of patients treated with first-line endocrine therapy for locally advanced or metastatic disease, Bcl-2 immunostaining was reported as a more accurate predictor of response than ER status.…”

Bcl-2 expression on fine-needle aspirates from primary breast carcinoma

“…In agreement with data obtained regarding histologic samples, a strong positive relation between Bcl‐2 immunoreactivity and ER and PgR status was found 7, 9, 11–20. This observation supports the hypothesis that Bcl‐2 in breast carcinoma may be an ER‐regulated protein, like PgR 34.…”

“…Bcl‐2 protein has been shown to be expressed frequently in breast carcinoma7–12 and its expression is associated with several favorable prognostic factors such as estrogen receptor (ER) and progesterone receptor (PgR) positivity,7–9, 11–20 low cell proliferation rate,9, 12, 17, 19, 21 lack of mutated p53 expression,7, 9, 12, 15, 17–19, 21 c‐ erb B‐2 negativity,7, 17 and high histologic grade of differentiation 17–19, 21. High expression of Bcl‐2 also has been found to be associated strongly with low rates of apoptotic cell death 22, 23…”

Testing for tumor drug resistance in the age of molecular medicine. A contribution to the Debate Round-Table on phenotypic and genotypic analyses of multidrug resistance (MDR) in clinical hospital practice

“…Automated robotic workstations are now commercially available devices for immunohistochemistry. 14 16 the quantitation of RT-PCR products has been a matter of discussion as well. Real-time PCR techniques have recently been developed which may improve precise quantitation of mRNA expression and the determination of thresholds of gene expression.…”