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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.

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.

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.

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 (~106 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.

Array comparative genomic hybridisation (CGH) is a powerful method for the genetic analysis of lesional and normal tissues to identify genomic imbalances associated with malignancies. However, the use of this technique with DNA extracted from archival formalin fixed, paraffin embedded (FFPE) tissue specimens, the most widely available resource for retrospective studies, is subject to quantitative and qualitative limitations. In this report, the suitability and integrity of the DNA extracted from FFPE MCF7 breast cancer cells fixed for different periods of time for array CGH applications were examined.Using our established cDNA microarray protocol in conjunction with whole genome amplification methods, the genetic profiles of freshly harvested MCF7 cells and their matched FFPE counterparts were analysed. Congruent profiles between FFPE MCF7 cells and their fresh counterpart and between amplified and non-amplified FFPE MCF7 cells were observed. Our results demonstrate that formalin fixation of20 hours has no significant adverse effect on the integrity of DNA for array CGH studies.Our findings attest to the fidelity of our array CGH methods to effectively examine material recovered from FFPE tissue specimens for microarray applications. This in turn has great potential to identify novel diagnostic and prognostic markers for 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.

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

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.