Real-Time Quantitative PCR of Microdissected Paraffin-Embedded Breast Carcinoma

Abstract

We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods. Study cases (27 carcinomas and 3 ductal breast carcinoma in situ (DCIS) cases) showed varying Her-2 expression as determined by IHC (HercepTest). In carcinomas, there was a good correlation between HER-2 DNA amplification and strong HER-2 protein expression detected by FISH and IHC, respectively. A single DCIS case was amplified in FISH, but not in IHC. Both HER-2 gene amplification and expression could be quantified in microdissected paraffin-embedded tumors using real-time PCR, DNA and RNA being successfully detected in 146 of 150 (97%) and 141 of 150 (94%) samples, respectively. PCR analysis for HER-2 DNA amplification using the LightCycler HER2/neu DNA Quantification kit (Roche Molecular Biochemicals, Mannheim, Germany) correlated fairly well with IHC and FISH. All IHC HER-2 3+ tumors were amplified according to the kit, as was the FISH-amplified DCIS case. DNA-PCR identified five additional tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique for establishing HER-2 status in paraffin-embedded tumors.

Evaluation of the prognostic and predictive value of HER family mRNA expression in high-risk early breast cancer: A Hellenic Cooperative Oncology Group (HeCOG) study

“…Our data confirm previous studies demonstrating a substantial agreement between the results of HER2 status evaluation at the mRNA and protein levels (Ginestier et al, 2004;Gjerdrum et al, 2004;Vinatzer et al, 2005). A recent study that compared four different methods of assessment of HER2 status found a good correlation between RT -PCR and IHC, with an overall concordance that varied from 82 to 93% (Ginestier et al, 2004).…”

“…However, routine methods of histological fixation and tissue processing could potentially damage or destroy RNA. In addition, dilution of tumour genomic material by nucleic acids from nonneoplastic tissue components is also a potential source of imprecision (Gjerdrum et al, 2004). Furthermore, the required equipment for kRT -PCR is not available in all histopathology laboratories and is quite expensive.…”

“…However, a disadvantage of this technique is the specific requirements for handling of the tissue specimens to preserve the integrity of RNA. Recent studies have shown that the small amounts of degraded RNA in archival formalin-fixed paraffin-embedded (FFPE) tissues can be successfully amplified and detected using kRT -PCR techniques (Gjerdrum et al, 2004).…”

An efficient protocol for genomic DNA extraction from formalin-fixed paraffin-embedded tissues

“…Different authors state that a suitable DNA extraction method from FFPET is essential and needs to be chosen in relation to specific endpoints of a precise investigation; for instance, the increased length of amplicon or the increase of effective amplifiable copy number (Wang et al, 1996;Poljak et al, 2000;Gilbert et al,2007b). Therefore diverse nucleic acid extraction methods already described from FFPET, enable the use of DNA extracted for specific approaches: nested PCR-SSCP assay (Wang et al, 1996), RAPD-PCR (Jacobs et al, 2007), real-time quantitative PCR (Gjerdrum et al,2004), Southern blot hybridisation (e.g. Dubeau et al, 1986;Jackson et al, 1990;Rogers et al, 1990), flow cytometry (Leers et al,1999), microarray comparative genomic hybridization (Vékony et al, 2007) and SNP BeadArrays (Oosting et al, 2007).…”

“…In the wide variety of methods for DNA extraction from FFPET, the majority was applied to collections of human samples, fixed with formalin (the fixative generally used in histopathological practice) (Gjerdrum et al, 2004). In veterinary clinics the procedures used are essentially the same.…”