Immunocytochemical demonstration of down regulation of HLA class-I molecule expression in human metastatic breast carcinoma.

Deficient expression of HLA class-I molecules observed in many cancers is suggested to influence both disease progression and potential efficacy of T cell-mediated immune therapy. Previous studies have attempted to correlate either primary breast cancer tumor grade with HLA class-I levels or the presence of HLA class-I-deficient cells in metastatic lesions with survival. In this study we evaluated the HLA class-I status of matched primary and secondary breast cancer lesions in order to ask the question: is metastasis of breast cancer associated with down-regulation of HLA class-I expression? Immunocytochemistry analysis shows a definitive correlation between diminished HLA class-I expression and dissemination of breast cancer to tumor-draining lymph nodes: both the total number of HLA class-I+ cells per sample and the levels of expression are dramatically decreased in secondary versus primary tumor lesions. These findings are consistent with the contention that the ability of breast cancer cells to escape the confines of the original tumor lesion requires down-regulation of HLA class-I expression and implies that enhancing HLA class-I expression in secondary breast cancer may have a beneficial effect on T-cell-mediated immunotherapy.

Multiplex loss of heterozygosity analysis by using single or very few cells.

Loss of heterozygosity (LOH) is an indication of tumor suppressor gene inactivation. However, loss of heterozygosity analysis has been limited to either a small scale or to very few genetic markers. To significantly increase the scale of study and to include a large number of markers in the analysis, experimental conditions were established for using single cells or single cell equivalent with 10 markers typed simultaneously. Under these conditions, the allele amplification failure rate was 3.7% when single tissue cultured human cells were used. When 30 cells from a 5- micro m paraffin-archived breast tumor tissue section were used, the failure rates were 0% for four of the five heterozygous loci and 10% for the fifth. Small amplification failure rates (6.1% and 6.7% on average) were observed when 5 or 10 cells from paraffin-archived breast tissue were used. These results indicate that with polymerase chain reaction (PCR) primers of high quality, it is possible to obtain reliable results by using single cells from fresh tissue or very few cells from paraffin-archived specimens. The results also show the importance of including replicates, using primers of high quality, and optimizing PCR conditions when a limited amount of material is used for the assay. The feasibility of LOH analysis with very few paraffin-embedded breast cancer tissues was demonstrated.

Tubular Carcinoma of the Breast: Immunohistochemical and DNA Flow Cytometric Profile.

Molecular markers of ordinary invasive ductal carcinoma of the breast have been extensively studied and their prognostic significance has been assessed. A common variant of breast cancer, tubular carcinoma, has an excellent prognosis as judged from several clinicopathologic studies. One would assume that tubular carcinomas have "favorable" molecular markers, however, published series of tubular carcinomas do not include molecular markers. We describe the molecular markers of 39 consecutive tubular carcinomas collected between January 1995 and July 1997. DNA ploidy, S-phase, estrogen and progesterone receptor (ER and PR, respectively) expression, and immunoreactivity for MIB-1, p53, and erbB2 were evaluated. Seventy-two percent of tubular carcinomas were DNA diploid, 49% had an S-phase less than 5%, 95% were ER positive, 69% were PR positive, 88% had less than 10% MIB-1-positive cells, 97% were p53 negative, and 97% did not overexpress erbB2 protein. Thus tubular carcinomas exhibit favorable molecular characteristics, which may play a role in their good prognosis.

Amplification of Her-2/neu gene in Her-2/neu-overexpressing and -nonexpressing breast carcinomas and their synchronous benign, premalignant, and metastatic lesions detected by FISH in archival material.

Amplification of Her-2/neu in breast carcinoma is associated with poor prognosis, short disease-free interval, and short survival time in both node-negative and -positive patients. Little is known about the starting point of amplification of Her-2/neu and how it progresses from benign to malignant breast lesions. We attempted to address these questions by evaluating amplification of Her-2/neu in benign, premalignant, and malignant lesions using fluorescence in situ hybridization (FISH). Twenty-six patients with Her-2/neu-overexpressing invasive ductal carcinomas (as judged by strong immunoreactivity with Her-2/neu antibody) and coexisting lesions of ductal hyperplasia (DH), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) in the vicinity of the invasive tumor (as judged by review of the hematoxylin and eosin-stained sections), as well as metastatic carcinoma in axillary lymph nodes (mets) were selected for this study. In the primary carcinomas, a close relationship was present between overexpression as detected by immunohistochemistry (IHC) and amplification as demonstrated by FISH (85% concordance). Among these patients, amplification of Her-2/neu in ADH was demonstrated in 7 of 13 cases with ADH, and in DCIS, in 21 of 22 cases with DCIS. There was no amplification in DH or normal ductal epithelium. Significantly, in all 12 patients with synchronous positive axillary lymph nodes, there was concordant amplification of Her-2/neu in the primary and metastatic carcinoma. Amplification was consistent in multifocal metastases, despite morphological heterogeneity in some patients. Amplification ratios increased from ADH to DCIS to invasive carcinoma (P <.01, ADH versus DCIS; P <.05, DCIS versus invasive cancer), but there was no difference in amplification ratios between primary cancers and synchronous axillary metastases (P >.05). We also evaluated Her-2/neu amplification in 21 patients without Her-2/neu overexpression in their primary carcinomas (as judged by absent immunoreactivity with Her-2/neu antibody). Three showed amplification in both primary and metastatic lesions, with a low amplification ratio (approximately 2). One patient had amplification in the primary tumor but not in an axillary metastasis. Two patients exhibited slight amplification in the metastatic carcinoma (ratios 1.6 and 2), but not in their primary cancers. This FISH study indicates that amplification of Her-2/neu can emerge de novo in any stage of the disease process, from ADH to metastatic lesions, but most often appears first in ADH or DCIS. The degree of Her-2/neu amplification increases with progression to invasive carcinoma, there being no further increase in synchronous metastasis. Our data suggest that amplification of Her-2/neu appears to be mainly involved in initiation of breast oncogenesis and that its role in progression of breast cancers is uncertain.

Differential amplification and overexpression of HER-2/neu, p53, MIB1, and estrogen receptor/progesterone receptor among medullary carcinoma, atypical medullary carcinoma, and high-grade invasive ductal carcinoma of breast.

CONTEXT: Medullary carcinoma (MC) is a special type of breast cancer that has a better prognosis than atypical medullary carcinoma (AMC) and high-grade invasive ductal carcinoma (HGIDC) with prominent lymphocytic infiltrates. What accounts for the different clinical courses of these carcinomas, despite their similar histology, is unknown. To address this issue, we performed a comparative study of amplification and overexpression of HER-2/neu and expression of several other important biochemical markers (p53, MIB1, and estrogen receptor [ER]/progesterone receptor [PR]) in these 3 cancer groups. OBJECTIVE: To evaluate HER-2/neu, p53, MIB1, and ER/PR as markers in the differential diagnosis of MC, AMC, and HGIDC.Design.-Nine cases of MC, 13 cases of AMC, and 16 cases of HGIDC with prominent lymphocytic infiltrates were identified according to strict histologic criteria. All tests were performed on formalin-fixed, paraffin-embedded archival tissues. HER-2/neu gene amplification was examined by fluorescence in situ hybridization using PathVysion HER-2 DNA probes. Expression of HER-2/neu, p53, MIB1, and ER/PR was detected by immunohistochemistry. chi2 and Student t tests were applied for statistical analyses. RESULTS: None of 9 cases of MC examined had either amplification or overexpression of HER-2/neu (0%). In contrast, HER-2/neu amplification was observed in AMC (46%, P <.025) and HGIDC (56%, P <.005). All 3 categories of tumors had similar percentages of expression of p53 (78% of MC, 77% of AMC, and 69% of HGIDC) and MIB1 (89% of MC, 92% of AMC, and 94% of HGIDC). Immunostaining for ER/PR was rarely positive in either MC or AMC, and there were no significant differences of expression of ER/PR between these 2 lesions (P >.05). However, the expression rate of ER/PR (31%/44%) in HGIDC is higher than in both MC (P =.05) and AMC (P =.01). CONCLUSIONS: Medullary carcinoma of breast is distinct from AMC and HGIDC with prominent lymphocytic infiltrates in amplification and overexpression of HER-2/neu. This difference may account for its different clinical and biological behavior, and may potentially aid in diagnosis and management of these groups of patients.