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

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. 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. Human epidermal growth factor receptor-2 (HER-2) is a proto-oncogene located at 17q21 that encodes a 185-kd transmembrane glycoprotein with tyrosine kinase activity. HER-2 (also known as c-erbB-2 or neu) is a member of the epidermal growth factor receptor family and is now recognized as a key oncogene in several malignancies. Amplification and/or strong expression of HER-2 can be seen in 20% to 30% of invasive breast carcinomas. It is regarded as a marker of adverse clinical outcome and as a predictive marker for reduced response to certain chemotherapy and hormonal treatments.1Revillion F Bonneterre J Peyrat JP ERBB2 oncogene in human breast cancer and its clinical significance.Eur J Cancer. 1998; 34: 791-808Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar Even more importantly, positive HER-2 status predicts for response to therapy with trastuzumab (Herceptin), a humanized monoclonal antibody directed against the external domain of the HER-2 protein which has been shown to be effective in prolonging survival in patients with receptor-positive metastatic breast carcinoma.2Slamon DJ Leyland-Jones B Shak S Fuchs H Paton V Bajamonde A Fleming T Eiermann W Wolter J Pegram M Baselga J Norton L Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.N Engl J Med. 2001; 344: 783-792Crossref PubMed Scopus (9169) Google Scholar Thus, there is a clear need for reliable and robust methods for accurately determining HER-2 status in tumors. Ideally, such methods should also be simple, quantitative, and widely applicable (eg, capable of being performed on formalin-fixed, paraffin-embedded (FFPE) archival biopsies or on small and/or precancerous specimens). However, while the clinical benefit of determining HER-2 status in breast carcinomas is now accepted, there is no consensus on the best diagnostic assay to use for this purpose. HER-2 status can be analyzed at the DNA-, the mRNA-, or the protein level. Various techniques are available for assessing these different target molecules, each with benefits and drawbacks.3Dowsett M Cooke T Ellis I Gullick WJ Gusterson B Mallon E Walker R Assessment of HER2 status in breast cancer: why, when, and how?.Eur J Cancer. 2000; 36: 170-176Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar For practical reasons, immunohistochemistry (IHC) using an anti-HER-2 antibody is by far the most frequently used method for assessing HER-2 status. IHC is available as a standard technique in pathology laboratories, and can be easily used on archival FFPE tissues. Furthermore, it is tumor cell-specific, the observer being able to distinguish signals from neoplastic and non-neoplastic cells. As a result, reports published on the clinical relevance of HER-2 expression have almost all used IHC. However, major disadvantages of HER-2 IHC assessment are that it is at best a semi-quantitative method with considerable inter-observer variations reported in some studies.4Thomson TA Hayes MM Spinelli JJ Hilland E Sawrenko C Phillips D Dupuis B Parker RL HER-2/neu in breast cancer: inter-observer variability and performance of immunohistochemistry with four antibodies compared with fluorescent in situ hybridization.Mod Pathol. 2001; 14: 1079-1086Crossref PubMed Scopus (164) Google Scholar Furthermore, the many available HER-2 antibodies show variable sensitivities and specificities5Press MF Hung G Godolphin W DJ of HER-2/neu antibodies in archival of in of oncogene Google Scholar with in tumor and IHC to considerable assay this is by the use of IHC as the there is that IHC HER-2 at the 2+ need to be with J Mallon E Cooke T clinical of HER-2 which to Pathol. PubMed Scopus Google M J Ellis J M Mallon E Cooke T C between immunohistochemistry and in situ hybridization (FISH) for HER-2 in breast carcinomas from Pathol. PubMed Scopus Google Scholar in situ hybridization (FISH) is a alternative method for assessing HER-2 status. It is a and technique for HER-2 gene amplification at the E S FISH FISH and in and an B T FISH Google Scholar a that to be correlated with strong protein H of in situ hybridization and immunohistochemistry for the of HER-2/neu in breast PubMed Google Scholar and of clinical In to IHC, FISH can a more and quantitative of HER-2 and is widely regarded as the standard for HER-2 status. However, the technique is more and and it more and compared with IHC. techniques and can be used to HER-2 molecules, but are of and are for In these techniques are not tumor Thus, the HER-2 status be by the by the of non-neoplastic and in all in an of gene amplification or can be reduced are for target of by microdissection tumor can be and from or from archival FFPE PubMed Scopus Google A and or PubMed Scopus Google I S microdissection of for of using immunohistochemistry and in situ 2001; Full Text Full Text PDF PubMed Scopus Google Scholar the of in samples, and a more and of tumor DNA and can determine in HER-2 gene and using real-time quantitative PCR, the target can be amplified and detected the assay a and of HER-2 methods of and or or such as to it to T S M of of RNA from and of for such PubMed Scopus Google Scholar However, the of real-time quantitative PCR that even of DNA or RNA be in FFPE T E quantitative mRNA analysis in paraffin-embedded using real-time a on from Full Text Full Text PDF PubMed Scopus Google the of clinical and on the in pathology In this on the feasibility of using real-time quantitative PCR analysis of DNA and mRNA from FFPE archival breast cancer to determine HER-2 and compared these with using IHC and FISH. We show that HER-2 amplification and mRNA expression can be detected by real-time quantitative PCR in archival microdissected FFPE tissues. In that PCR analysis of HER-2 DNA amplification in microdissected using the LightCycler HER2/neu DNA Quantification kit (Roche Molecular Biochemicals, Mannheim, Germany) be used as an alternative to the tumor of HER-2 status. cases of breast tumor were from the of the pathology on HER-2 protein status by the from ductal breast carcinomas and cases of ductal breast carcinoma in situ (DCIS) were All were that been and using standard were for microdissection and FISH were We used the semi-quantitative to HER-2 protein according to the In on were in for using a at by at for and the were with antibody HER2 in a for were and in with and for at and in were in the negative and positive with the kit of of FFPE breast carcinoma of Her-2 performed the for all We scored tumor for Her-2 in with the no or in of detected in more of to in more of strong and in more of was performed by the were using the HER-2/neu DNA kit in with the For each were on was with and and was used as a for tumor for FISH was used for FISH. was in in standard 3 at was in a at in the of HER-2 DNA and in the were with were using a at were in each the by the the on the were to tumor with as or non-neoplastic as In the tumors invasive with DCIS were signals for and were and the for each by the of the IHC HER-2 status of the In with the not with no with signals of or with signals in with to determine the were not We regarded a as being amplified and a as not being amplified. in the between to were were for microdissection using a of the M I H microdissection of J Pathol. Google Scholar of FFPE was using a Molecular as I S microdissection of for of using immunohistochemistry and in situ 2001; Full Text Full Text PDF PubMed Scopus Google Scholar In the were in in with in and in the the of were the of used as an I S microdissection of for of using immunohistochemistry and in situ 2001; Full Text Full Text PDF PubMed Scopus Google Scholar For each five tumor were and using a We tumor to the of on the A of the was as a negative were in an RNA in and Molecular for RNA at at for and at T A M H gene expression analysis in microdissected archival and paraffin-embedded tumor J Pathol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar was for DNA using the were to an with DNA and and at and for at H PCR analysis of DNA and RNA from and paraffin-embedded 2001; PubMed Scopus Google Scholar were at DNA were used RNA was using the according to the by with an of 3 and was in and in RNA from the human B J M H A of human in Google Scholar was with the RNA kit and used for the in a of PCR (also used in for or of each and was performed on a at for by for of HER-2/neu and of gene expression was using the LightCycler (Roche Molecular following the and of real-time quantitative PCR are in of mRNA using real-time 2000; PubMed Scopus Google Scholar We used PCR and T A M H gene expression analysis in microdissected archival and paraffin-embedded tumor J Pathol. 2001; Full Text Full Text PDF PubMed Scopus Google M mRNA expression analysis from formalin-fixed, paraffin-embedded using quantitative 2000; Full Text Full Text PDF PubMed Scopus Google Scholar A standard for gene expression of Her-2 and was using RNA from a human as in T E quantitative mRNA analysis in paraffin-embedded using real-time a on from Full Text Full Text PDF PubMed Scopus Google Scholar In of RNA in were to different each with a known of For a standard was and in each PCR PCR amplification for gene expression was performed in a A DNA Molecular and was used for the PCR according to the and were to this for and of and Quantification was by for at by with the following for at for and at by the amplification We the using the between HER-2/neu and to the between and gene that be the (eg, to variations in mRNA and H PCR analysis of DNA and RNA from and paraffin-embedded 2001; PubMed Scopus Google that no DNA of and for and were used in and in were used in and in were used in and in in a real-time PCR was in with the DNA was used or of the HER-2/neu gene and a gene was performed using the LightCycler HER2/neu DNA Quantification kit (Roche Molecular A of the HER-2/neu gene and a of the gene were amplified the PCR of the HER-2/neu gene and the gene was by using different LightCycler hybridization and in the and DNA were with of in a of of the and of the DNA was performed at for was by at for at for and at for was which the were to kit a to for reliable and to be and to of a HER-2 and are determined using the of of the compared to the in the We additional with known amplification status as positive and with and strong to the of to the a should be regarded as positive for HER-2/neu DNA analysis was performed using the at from real-time quantitative were analyzed with a for is the and is the for analysis of by variations between the patients in the and the to tumor and/or was used to with the DNA was for on the by the DNA were compared using HER-2 protein status was with the cases scored cases cases and 3 cases All DCIS cases scored A All cases were for FISH of tumors were amplified for the HER-2/neu were carcinomas, all of which also showed strong expression by the case of DCIS was also amplified. carcinomas and DCIS cases were not amplified at the DNA In the amplified HER-2 signals were and carcinomas showed In some signals were to and hybridization to be of the which were amplified in IHC and FISH signals for more in at of the tumor cells. In more signals for were in all IHC 2+ in of IHC and in of 3 IHC In the DCIS the single FISH-amplified more signals for in some tumor In tumor in most cases with to Her-2 and of IHC, FISH, and PCR of HER-2 in ductal carcinomas E HER-2 protein expression assay (HercepTest). for HER-2/neu mRNA expression in are as with and HER-2/neu gene amplification in with the LightCycler HER2/neu DNA Quantification kit are as with and mRNA for HER-2 and ductal DCIS ductal carcinoma in IHC, formalin-fixed, FISH, in situ ductal carcinomas E HER-2 protein expression assay for HER-2/neu mRNA expression in are as with and HER-2/neu gene amplification in with the LightCycler HER2/neu DNA Quantification kit are as with and mRNA for HER-2 and in a ductal carcinomas. E DCIS ductal carcinoma in IHC, formalin-fixed, FISH, in situ establishing the compared RNA using RNA kit kit and methods not Furthermore, of the not the was performed with for HER-2 or in a in compared to not We used that amplified (eg, for and for to even from the of RNA available in the FFPE archival used in this of different and and a of at in the not show method to be not the in the in no amplification of HER-2 mRNA could be analyzed by real-time quantitative in all cases in carcinoma there was available for analysis of In all at of the five microdissected from each for HER-2 in the of all five were In 141 of 150 (94%) on the IHC and cases were to five 3+ 2+ no DCIS cases were analyzed in to these We performed analysis for on the between HER-2 and as well as on the from HER-2 and of the HER-2 to the a between A and the was by the between patients in the and by the that from tumor and/or were for HER-2 and to the of in each was performed on the from and showed no between the In this the for and were and respectively. A was to the of cells. the a of for the five case in many of the patients there was a between in the different HER-2 mRNA and DNA Amplification in and are the of by variations between the patients the and the to tumor and/or and are the of by variations between the patients the and the to tumor and/or against against against against against against of not and are the of by variations between the patients the and the to tumor and/or in a of not LightCycler HER2/neu DNA Quantification kit (Roche Molecular was to and in most microdissected from all A of 146 of 150 were between HER-2 and the gene showed a between A and the with and being and respectively. was tumor to for of the for each with five showed in there was considerable in tumors gene amplification and of HER-2 status in the carcinomas using IHC and FISH with that could be on the of published Thus, all carcinomas with amplification determined by FISH, also showed strong expression of HER-2 protein at the 3+ level. DCIS case was to be amplified using FISH, but scored in the cases amplification in FISH and strong protein expression in IHC expression of HER-2 mRNA by real-time quantitative with or of HER-2 protein showed to expression of HER-2 a between these cases and the cases with strong protein expression amplified in FISH and with strong expression of HER-2 protein were all to be amplified using the LightCycler kit In the single DCIS was also to be amplified by DNA-PCR the of this all IHC and tumors were not amplified by of the IHC 2+ cases were amplified in PCR, but were negative with FISH and of by FISH in these five cases was not different to that in the IHC 2+ of IHC and LightCycler PCR of HER-2 in cases and gene gene IHC cases and HER-2 HER-2 in a of FISH and LightCycler PCR of HER-2 in not gene gene HER-2 HER-2 in a in of the the of breast carcinoma have the for of the on the of In an of the clinical of HER-2/neu amplification and/or gene expression is is is the method for HER-2 status in the clinical In the show that by the and of real-time PCR analysis with the of it is to HER-2 gene amplification and gene expression in FFPE tissues. the of that with and real-time PCR to be robust and in the of microdissected Thus, of RNA and DNA were in 141 (94%) and 146 of 150 samples, respectively. assessment of HER-2 amplification in microdissected tumor using the real-time LightCycler HER2/neu DNA kit correlated fairly well with the of the more of FISH and IHC, that this technique be a alternative for assessment of HER-2 status in a clinical IHC of HER-2 protein expression are the most widely used methods for assessing HER-2 status. of these are that are and simple, can be performed in most pathology laboratories, and that compared with are Furthermore, strong HER-2 protein expression is to clinical outcome and However, IHC assay has been variations in and as well as in antibody R S Gullick W J Ellis I HER-2 in breast of clinical and for Full Text Full Text PDF PubMed Scopus Google Scholar and inter-observer variability in can all the of the of IHC assay variability the widely of HER-2 expression in breast carcinomas reported with IHC, have the of FISH for HER-2 gene in cases to be by HER-2 S E L R assessment of the status of protein and oncogene in breast cancer of immunohistochemistry assay with in situ Pathol. 2000; PubMed Scopus Google J A A E B correlation between of fluorescent in situ hybridization and immunohistochemistry for the assessment of the ERBB2 gene status in breast Pathol. 2000; PubMed Scopus Google Scholar FISH has the of being a quantitative method that well with the of IHC H of in situ hybridization and immunohistochemistry for the of HER-2/neu in breast PubMed Google H F H T M M S G of HER-2/neu DNA amplification in breast inter-observer and correlation with HER-2 2001; PubMed Scopus Google Scholar and it is regarded as the standard technique for HER-2 status. are that FISH is to to to and compared to IHC. this technique is available in a of pathology A key by IHC and FISH techniques for HER-2 is that are on In this for in gene and gene expression to be in the tumor this is an of IHC such as the HercepTest, it is in a in most FISH assessment of is in most in situ hybridization assessment is using in situ hybridization M D A D G J in situ a practical alternative for in situ hybridization to HER-2/neu oncogene amplification in archival breast cancer J Pathol. 2000; Full Text Full Text PDF PubMed Scopus Google C in situ a to a practical and method for the of HER2 oncogene in archival human breast PubMed Scopus Google Scholar technique in a in situ hybridization HER-2 status to be in breast carcinomas on a using a However, the clinical and of this technique to be A of with is that are on a of HER-2 by to in to (eg, tumor is a major of and PCR techniques on analysis of published methods to HER-2 amplification have been on PCR with analysis by A V C S H PCR analysis of gene amplification and with protein expression in breast cancer J Cancer. PubMed Scopus Google Scholar However, the of real-time PCR methods it to more quantitative analysis of gene methods are and to can be used to samples, have a of of and can be useful methods for tumors for HER-2 amplification in a clinical However, of tumor with from non-neoplastic is an of in such quantitative in many not in breast carcinoma in which for more of the DJ Godolphin W WJ J A of the HER-2/neu proto-oncogene in human breast and PubMed Scopus Google Scholar In the of HER-2 this to the of an that is and in a In with a has that HER-2 gene amplification was no by real-time PCR the breast carcinoma 30% of the tumor S W H of gene amplification in archival breast cancer by microdissection and quantitative real-time J Pathol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar the of tumor performed microdissection PCR has as a key for this and of tumor from for a of I S microdissection of for of using immunohistochemistry and in situ 2001; Full Text Full Text PDF PubMed Scopus Google Scholar In this to be a reliable technique was and as to as FISH. in this with real-time quantitative and PCR in almost all samples, even the of in was small in and in and is of with the that mRNA be by to in such with RNA being T S M of of RNA from and of for such PubMed Scopus Google T E quantitative mRNA analysis in paraffin-embedded using real-time a on from Full Text Full Text PDF PubMed Scopus Google Scholar As a result, it has been to quantitative on archival several have published in which the small of RNA in FFPE can be successfully amplified and detected using real-time T E quantitative mRNA analysis in paraffin-embedded using real-time a on from Full Text Full Text PDF PubMed Scopus Google T A M H gene expression analysis in microdissected archival and paraffin-embedded tumor J Pathol. 2001; Full Text Full Text PDF PubMed Scopus Google M mRNA expression analysis from formalin-fixed, paraffin-embedded using quantitative 2000; Full Text Full Text PDF PubMed Scopus Google S W H of gene amplification in archival breast cancer by microdissection and quantitative real-time J Pathol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar to these We were able to show a between the different tumor with to the between HER-2 and and HER-2 mRNA In no were between in of mRNA from the gene We considerable in HER-2 mRNA different a of was to in the HER-2 as compared with of mRNA patients the five which to the also for We believe this to be an expression of tumor and of in HER-2 mRNA in there was a clear of gene expression in the different that HER-2 gene expression from FFPE should be used with in the clinical As by Quantification of mRNA using real-time PCR and PubMed Scopus Google Scholar there are several to with to the of real-time quantitative use of is in in as the mRNA of these and in it is not to However, that mRNA expression was in all while there were in HER-2 mRNA different with to HER-2 to HER-2 and HER-2 to that it was to HER-2 to this to real-time quantitative PCR analysis of HER-2 gene of gene amplification using the LightCycler HER2/neu DNA Quantification kit (Roche Molecular showed an even with IHC and FISH DNA it is more DNA also in FFPE the used in the DNA kit for reliable amplification of the DNA available in microdissected to the for HER-2 gene in of gene amplification in the different We believe that this is a of DNA being RNA in FFPE also a of tumor in to this by tumor We that in at should be on tumor from each Furthermore, the was by the between patients in the from FISH analysis show that this should as no of cases showed of and an even (27 of showed of HER-2 the LightCycler HER2/neu DNA Quantification kit, tumors in which the HER-2 was the of of these cases HER-2 protein by the Interestingly, five of the tumors scored in the IHC cases were not FISH amplified. 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