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.

Mapping genomic and transcriptomic alterations spatially in epithelial cells adjacent to human breast carcinoma.

Almost all genomic studies of breast cancer have focused on well-established tumours because it is technically challenging to study the earliest mutational events occurring in human breast epithelial cells. To address this we created a unique dataset of epithelial samples ductoscopically obtained from ducts leading to breast carcinomas and matched samples from ducts on the opposite side of the nipple. Here, we demonstrate that perturbations in mRNA abundance, with increasing proximity to tumour, cannot be explained by copy number aberrations. Rather, we find a possibility of field cancerization surrounding the primary tumour by constructing a classifier that evaluates where epithelial samples were obtained relative to a tumour (cross-validated micro-averaged AUC = 0.74). We implement a spectral co-clustering algorithm to define biclusters. Relating to over-represented bicluster pathways, we further validate two genes with tissue microarrays and in vitro experiments. We highlight evidence suggesting that bicluster perturbation occurs early in tumour development.

The use of whole genome amplification in the study of human disease.

The availability of large amounts of genomic DNA is of critical importance for many of the molecular biology assays used in the analysis of human disease. However, since the amount of patient tissue available is often limited and as particular foci of interest may consist of only a few hundred cells, the yield of DNA is often insufficient for extensive analysis. To address this problem, several whole genome amplification (WGA) methodologies have been developed. Initial WGA approaches were based on the polymerase chain reaction (PCR). However, recent reports have described the use of non-PCR-based linear amplification protocols for WGA. Using these methods, it is possible to generate microgram quantities of DNA starting with as little as 1mg of genomic DNA. This review will provide an overview of WGA approaches and summarize some of the uses for amplified DNA in various high-throughput genetic applications.

Comparison of whole genome amplification methods for analysis of DNA extracted from microdissected early breast lesions in formalin-fixed paraffin-embedded tissue.

To understand cancer progression, it is desirable to study the earliest stages of its development, which are often microscopic lesions. Array comparative genomic hybridization (aCGH) is a valuable high-throughput molecular approach for discovering DNA copy number changes; however, it requires a relatively large amount of DNA, which is difficult to obtain from microdissected lesions. Whole genome amplification (WGA) methods were developed to increase DNA quantity; however their reproducibility, fidelity, and suitability for formalin-fixed paraffin-embedded (FFPE) samples are questioned. Using aCGH analysis, we compared two widely used approaches for WGA: single cell comparative genomic hybridization protocol (SCOMP) and degenerate oligonucleotide primed PCR (DOP-PCR). Cancer cell line and microdissected FFPE breast cancer DNA samples were amplified by the two WGA methods and subjected to aCGH. The genomic profiles of amplified DNA were compared with those of non-amplified controls by four analytic methods and validated by quantitative PCR (Q-PCR). We found that SCOMP-amplified samples had close similarity to non-amplified controls with concordance rates close to those of reference tests, while DOP-amplified samples had a statistically significant amount of changes. SCOMP is able to amplify small amounts of DNA extracted from FFPE samples and provides quality of aCGH data similar to non-amplified samples.

Whole-Genome Amplification by Degenerate Oligonucleotide Primed PCR (DOP-PCR).

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. Unlike other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). The degenerate-oligonucleotide-primed PCR (DOP-PCR) method described here allows complete genome coverage in a single reaction. In contrast to the pairs of target-specific primer sequences used in traditional PCR, only a single primer, which has defined sequences at its 5'-end (containing an XhoI restriction site) and 3'-end and a random hexamer sequence between them, is used here. DOP-PCR comprises two different cycling stages. In stage 1 (low stringency), low-temperature annealing and extension in the first five to eight cycles occurs at many binding sites in the genome. The 3'-end of the primer binds at sites in the genome complementary to the 6-bp well-defined sequence at the 3'-end of the primer (~10(6) sites in the human genome). The adjacent random hexamer sequence (displaying all possible combinations of the nucleotides A, G, C, and T) can then anneal and tags these sequences with the DOP primer. In stage 2 (high stringency; >25 cycles), the PCR annealing temperature is raised, which increases priming specificity during amplification of the tagged sequence. DOP-PCR generates a smear of DNA fragments (200-1000 bp) that are visible on an agarose gel.

GenomePlex Whole-Genome Amplification.

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). GenomePlex WGA, described in this protocol, is a proprietary amplification technology based on nonenzymatic random fragmentation of genomic DNA. The protocol involves conversion of the genome into an in vitro molecular library of DNA fragments, followed by incubation at various temperatures to add adaptor sequences with specific PCR priming sites to both ends of every fragment. The fragment library can then be amplified several 1000-fold to generate milligram quantities of DNA starting with as little as 10-100 ng.

Whole-Genome Amplification by Adaptor-Ligation PCR of Randomly Sheared Genomic DNA (PRSG).

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). Ligation-mediated PCR techniques involve ligating an adaptor sequence onto a "representation" of DNA molecules, generated following enzymatic digestion, random shearing, or chemical cleavage. Adaptor-ligation PCR of randomly sheared genomic DNA (PRSG), described here, is based on ligation-mediated PCR and was designed to improve genome coverage. Rather than using enzymatically generated fragments, this method uses randomly fragmented DNA as the template. The process involves three steps: (1) the hydrodynamic shearing of genomic DNA to a 0.5-2-kb size range, (2) end filling and adaptor ligation, and (3) high-stringency PCR for faithful replication of the resulting fragments.

Whole-Genome Amplification by Improved Primer Extension Preamplification PCR (I-PEP-PCR).

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). Primer extension preamplification PCR (PEP-PCR), in contrast to degenerate oligonucleotide primed PCR (DOP-PCR), uses totally degenerate 15-mer PCR primers. An additional difference is that in PEP-PCR, the number of potential priming sites is orders of magnitude larger. The effectiveness of PEP-PCR has been increased by several alterations. The improved PEP (I-PEP) PCR approach, described in this protocol, uses a DNA polymerase cocktail that includes Taq DNA polymerase (to carry out the primer extension as in a traditional PCR) and a proofreading DNA polymerase (to provide 3'-to-5'-exonuclease activity, excising misincorporated nucleotides that slow the progression of Taq DNA polymerase). The result is far more efficient WGA, with increased fidelity due to the removal of the misincorporated nucleotides. Similar to DOP-PCR, PEP-PCR generates a smear of DNA fragments that are visible on an agarose gel.

Whole-Genome Amplification by Single-Cell Comparative Genomic Hybridization PCR (SCOMP).

INTRODUCTIONPCR-based whole-genome amplification (WGA) has the goal of generating microgram quantities of genome-representative DNA from picogram or nanogram amounts of starting material. This amplification should introduce little, or ideally no, representational bias. In contrast to other techniques for WGA, PCR-based methods are generally less affected by DNA quality and are more applicable to DNA extracted from various sources (fixed and fresh tissues). Ligation-mediated PCR techniques involve ligating an adaptor sequence onto a "representation" of DNA molecules, generated following enzymatic digestion, random shearing, or chemical cleavage. Single-cell comparative genomic hybridization (SCOMP), described in this protocol, is a form of ligation-mediated PCR that was specifically designed for WGA of extremely limited sources of genomic DNA. The reaction volume is purposely kept to a minimum, and all buffers are optimized to eliminate the need to purify the reaction between steps. In addition, the entire reaction is performed in a single tube. This avoids initial template loss and reduces the risk of PCR contamination. SCOMP begins by converting the genome to a high-complexity representation with a fragment size of <2 kb by digesting with the restriction enzyme MseI. This results in a smear in the range of 100-1500 bp. Following enzyme digestion, adaptors containing specific primer sequences (specific to the restriction enzyme used) are ligated onto the ends of the genomic DNA and amplified in a high-stringency PCR. This results in a smear of PCR products in the range of 100-1500 bp, which can be visualized by agarose gel electrophoresis.

Genomic alterations in sporadic synchronous primary breast cancer using array and metaphase comparative genomic hybridization.

Synchronous primary breast cancer describes the occurrence of multiple tumors affecting one or both breasts at initial diagnosis. This provides a unique opportunity to identify tissue-specific genomic markers that characterize each tumor while controlling for the individual genetic background of a patient. The aim of this study was to examine the genomic alterations and degree of similarity between synchronous cancers. Using metaphase comparative genomic hybridization and array comparative genomic hybridization (aCGH), the genomic alterations of 23 synchronous breast cancers from 10 patients were examined at both chromosomal and gene levels. Synchronous breast cancers, when compared to their matched counterparts, were found to have a common core set of genetic alterations, with additional unique changes present in each. They also frequently exhibited features distinct from the more usual solitary primary breast cancers. The most frequent genomic alterations included chromosomal gains of 1q, 3p, 4q, and 8q, and losses of 11q, 12q, 16q, and 17p. aCGH identified copy number amplification in regions that are present in all 23 tumor samples, including 1p31.3-1p32.3 harboring JAK1. Our findings suggest that synchronous primary breast cancers represent a unique type of breast cancer and, at least in some instances, one tumor may give rise to the other.