Amplification of the prolactin receptor gene in mammary lobular neoplasia.

The identification of lobular carcinoma in situ (LCIS) in a patient's specimen confers an appreciable increased risk of development of future invasive mammary carcinoma. However, the study of LCIS presents a challenge as it is usually only recognized in fixed specimens. Recent advances in high throughput genomics have made possible comprehensive copy number analysis of lesions such as this. Using array comparative genomic hybridization (aCGH), we characterized eight cases of lobular carcinoma (four invasive and four non-invasive) from microdissected samples of archival specimens and validated our results by quantitative real-time PCR (qRT-PCR). Immunohistochemistry (IHC) was performed on an independent set of 80 in situ ductal (DCIS) and lobular breast lesions to confirm our results. Amplification of the prolactin receptor gene (PRLr) was identified in 4/4 cases of LCIS by aCGH. We confirmed this amplification by qRT-PCR and demonstrated PRLr expression in 29/40 (73%) cases of lobular neoplasia by IHC. Amplification of PRLr was neither detected in 10 cases of DCIS nor in 5 areas of normal breast tissue by qRT-PCR and only 14/40 (35%) cases of DCIS showed PRLr expression by IHC (P = 0.0008). Our study suggests the prolactin receptor gene is a molecular target that may be important in the pathogenesis and progression of lobular neoplasia. Investigation of the status of this gene in cases of DCIS has indicated that it may not be as important in the progression of this type of breast cancer, supporting the view that lobular and ductal carcinomas may evolve along separate pathways.

Genomic alterations in primary breast cancers compared with their sentinel and more distal lymph node metastases: an aCGH study.

Metastatic potential of breast cancer may be associated with specific genomic alterations and the earliest metastases are likely to be found in the sentinel lymph nodes (SLN). Using array comparative genomic hybridization (aCGH), we compared the genomes of primary breast invasive duct carcinomas (IDCs), their sentinel and more distal lymph node metastases, and IDCs without nodal metastasis. Thirty-three samples from 22 patients with IDC were subjected to aCGH: 8 IDC samples from patients without lymph node metastasis, 11 IDCs associated with SLN metastases out of which 7 had paired samples of metastases, and 14 samples of lymph node metastases out of which 8 were sentinel-distal pairs from 4 patients. aCGH data were analyzed by correlation of genomic profiles, cluster analysis, segmentation, and peak identification. Quantitative real-time PCR was used for data validation. We observed high genomic similarity between primary tumors and their nodal metastases as well as between metastases to the sentinel and distal lymph nodes. Several recurrent alterations were detected preferentially in IDC associated with SLN metastases compared to IDCs without metastasis. Amplification within the 17q24.1-24.2(59.96-62.76 Mb) region was associated with presence of sentinel or distal lymph node metastases; larger tumor size and higher histological grade. In our samples, there were genomic events associated with metastatic progression, which could be detected in both primary tumors and LN metastases. Gain on 17q24.1-24.2 is a candidate region for further testing as a predictor of nodal metastasis.

Genomic differences between pure ductal carcinoma in situ of the breast and that associated with invasive disease: a calibrated aCGH study.

PURPOSE: In the quest for new targets, genomes of ductal carcinoma in situ (DCIS) and infiltrating duct carcinoma (IDC) have been compared previously; however, genomic alterations associated with cancer progression were difficult to identify. We hypothesized that significant events can be detected by comparing lesions with a broader range of behavior: from pure DCIS to IDC associated with lymph node metastasis. EXPERIMENTAL DESIGN: Array comparative genomic hybridization, calibrated by self-self hybridization tests, was used to study 6 cases of pure DCIS and 17 cases of DCIS paired with IDC where 8 tumors had spread to the local lymph nodes. RESULTS: Pure DCIS exhibited a marginally higher degree of genomic complexity than DCIS and IDC components of invasive tumors. The latter two showed similarity between tumors and between components of the same tumor with several regions detected preferentially compared with pure DCIS. IDC associated with lymph node metastases showed similarity of genomic profiles as a group. Gain on 17q22-24.2 was associated with higher histologic grade, large IDC size, lymphatic/vascular invasion, and lymph node metastasis (P < 0.05). CONCLUSIONS: Our findings suggest that DCIS and IDC are associated with specific genomic events. DCIS associated with IDC is genomically similar to the invasive component and therefore may represent either a clone with high invasive potential or invasive cancer spreading through the ducts. Specifically, gain on 17q22-24.2 is a candidate region for further testing as a predictor of invasion when detected in DCIS and predictor of nodal metastasis when detected in DCIS or IDC.

Quantitative detection of circulating epithelial cells by Q-RT-PCR.

INTRODUCTION: It has been shown that the quantity of circulating tumor cells (CTCs) in breast cancer patients is an independent predictor of survival and treatment response. Real time quantitative reverse transcriptase PCR (Q-RT-PCR) is a sensitive technique for detection of CTCs. Our aim was to investigate whether the technique can be used also to quantitate these CTCs. METHODS: We tested cytokeratin 19 (CK19), maspin, mammaglobin, GAPDH and RPL19 genes for their level of expression and linearity of amplification in serial dilutions of RNA extracted from the MDA-MB-231, UACC-812, T47D and HS578T breast cancer cell lines. To simulate CTCs, serial dilutions of cultured T47D and HS578T cells were added to peripheral blood from healthy volunteers. The samples were subjected to enrichment, RNA extraction and Q-RT-PCR. RESULTS: CK19 was reliably expressed in all four cell lines with a linear relationship between the quantity of added cells and the amount of CK19 RNA. The lower limit of reliable detection was 5 cells per sample, which corresponds to a concentration of 0.7 cell/ml in 7.5 ml of blood or would translate to a lower CTC concentration in a larger volume of blood. CONCLUSION: This technique may prove useful for high throughput comparative quantification of CTCs in individual patients during treatment and subsequent follow up for research and clinical management purposes.