HER2/neu overexpression/amplification is seen more frequently in ductal carcinoma in situ, particularly high-grade ductal carcinoma in situ (50 -60%), than in invasive ductal carcinoma of the breast (25-30%). To date, however, the role of HER2/neu in the progression of in situ to invasive disease has not been clarified. Two hundred fiftyone breast tumors were retrieved from the pathology files at Mount Sinai Hospital. These included 91 cases of ductal carcinoma in situ, 136 cases of invasive ductal carcinomas with associated ductal carcinoma in situ, and 24 cases of pure invasive carcinomas. All cases were reviewed and stained with two monoclonal antibodies to HER2/neu (CB11 and TAB250). Immunohistochemical staining was recorded using a semiquantitative scoring system (1). Representative cases were also investigated using fluorescence in situ hybridization. HER2/neu protein overexpression (defined as immunohistochemical staining with score of >5) was seen in 34% of cases of pure ductal carcinoma in situ, 17% of invasive carcinomas with associated ductal carcinoma in situ, and 12.5% of pure invasive carcinomas (P ‫؍‬ .01). Sixty percent of cases of high-grade ductal carcinoma in situ showed HER2/neu protein overexpression, versus 29% of high-grade invasive carcinomas with associated ductal carcinoma in situ and 22% of high-grade pure invasive ductal carcinomas (P ‫؍‬ . The importance of HER2/neu in cancer has been a topic of considerable interest of late, both in its role as a prognostic indicator and as a predictor of response to therapy (2-8). With the advent of the drug Herceptin, assessment of HER2/neu status in patients with metastatic breast carcinoma has become an even more important clinical consideration. Previous studies have shown that approximately 25-30% of invasive ductal carcinomas of breast show HER2/neu overexpression/amplification (9). In contrast, the incidence of HER2/neu overexpression/amplification in ductal carcinoma in situ is Ն60% (10 -12), whereas benign and atypical breast lesions generally do not show any evidence of HER2/neu overexpression (13). To date, there have

Tumor development from an early lesion through to invasive disease is not a clearly defined progression in the breast. Studies of invasive lobular carcinoma have reported mutations, loss of heterozygosity (LOH) and loss of protein expression in epithelial (E)-cadherin, a protein involved in cell adhesion. Our study examines in situ lobular neoplastic lesions without concurrent invasive carcinoma for E-cadherin gene alterations and protein expression, beta-catenin, alpha-catenin and p120-catenin protein expression, and LOH at the chromosome 16q locus, with the goal of determining the events occurring at the stage of lobular neoplasia. In all, 13 atypical lobular hyperplasia lesions and 13 lobular carcinoma in situ lesions from archived cases were examined. E-cadherin sequence alterations were evaluated using single strand conformation polymorphism and DNA sequencing, and PCR-based LOH analysis was carried out for the 16q locus. Using immunohistochemistry, we assessed protein expression. A total of 23 of 24 lesions evaluated by immunohistochemistry were negative for both E-cadherin and beta-catenin protein expression, and 21 of 23 lesions were negative for alpha-catenin. Cytoplasmic (rather than membrane) localization of p120-catenin was observed in 20 of 21 cases. Lobular carcinoma in situ cases were characterized by mutations; however, atypical lobular hyperplasia cases were not. LOH at 16q was an infrequent event. From our study, we conclude that an altered E-cadherin adhesion complex is an early event affecting atypical lobular hyperplasia as well as lobular carcinoma in situ and occurs prior to progression to invasive disease. However, the loss of protein expression is accompanied by E-cadherin DNA alterations in lobular carcinoma in situ but not in atypical lobular hyperplasia. These cases lacking both protein expression and gene alterations suggest that another mechanism is involved, possibly as early as at the hyperplastic stage, causing silencing of the E-cadherin complex.

We prospectively evaluated a series of 254 breast cancers by quantitative polymerase chain reaction (PCR) and immunohistochemistry using 3 antibodies: HercepTest, CB11, and TAB250. DNA was extracted from a 10-micron tumor section for PCR, and 4-micron serial sections were taken from the same block for immunohistochemistry. The immunohistochemical results were scored using a semiquantitative immunohistochemical system. A positive tumor by immunohistochemistry had a score of 5 or more. The manufacturer's recommended scoring system was used for the HercepTest. Tumors were positive for gene amplification if the ratio of the HER2/neu gene to control gene after normalization was 2 or more. Of 254 cases, 61 showed gene amplification. For immunohistochemistry, 23% of tumors were positive with CB11, 27% with TAB250, and 37% with the HercepTest. Results for each antibody were compared with PCR results. The overall concordance for the HercepTest was 82%, which was significantly lower than that for CB11 (88%) or TAB250 (87%). The specificity for the HercepTest was 80% compared with 90% for TAB250 and 93% for CB11, while the positive predictive value for the HercepTest was 57% compared with 71% and 76% for TAB250 and CB11, respectively.