Quantitation of c-&lt;i&gt;erb&lt;/i&gt;B-2 Gene Amplification in Breast Cancer Tissue by Competitive PCR

Okuyama, Nobuo; Hatano, Yoshinori; Park, Youngjin; Shimatani, Shinji; Sasamoto, Shigemi; Katou, Nobuhide; Takagi, Keigo; Yamazaki, Shojiro; Inoue, Akira; Hemmi, Hiromichi; Shimatake, Hiroyuki; Yanagida, Maki; Miura, Myota

doi:10.1159/000030058

Cited by 7 publications

(5 citation statements)

References 18 publications

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“…In previously reported competitive methods, exact amounts of input competitors and sample DNAs were essential [25], [26], and so clinical application was difficult. However, in our present mrcPCR assay, the use of various amounts of genomic DNA (2.5–40 ng) or competitor mixture (6.25–25.0 fg) yielded consistent results (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, its requirement for meticulously accurate measurements of input genomic DNA amounts greatly hampers its clinical application [25], [26]. Another modified method of the competitive PCR employing multiple modified exogenous reference sequences was applied to measure mRNA quantification, as previously published as real competitive PCR [27].…”

Section: Introductionmentioning

confidence: 99%

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Simple and Versatile Molecular Method of Copy-Number Measurement Using Cloned Competitors

et al. 2013

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Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRT's limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Simple and Versatile Molecular Method of Copy-Number Measurement Using Cloned Competitors

et al. 2013

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“…HER2 testing methodologies measure HER2 overexpression at the DNA level by fluorescence in situ hybridization (FISH), Southern blot [Tandon et al 1989;Thor et al 1989] and polymerase chain reaction (PCR), which can be competitive [Deng et al 1999;Okuyama et al 1999;Vona et al 1999], differential [Deng et al 1999;Frye et al 1989;Gramlich et al 1994] or real-time PCR [Bieche et al 1999;Higuchi et al 1993]; at the RNA level by Northern blot [Slamon et al 1989]; or at the protein level by IHC, enzyme-linked immunosorbent assay (ELISA) and Western blot [Slamon et al 1989]. Some of these methods require resources and expertise exceeding those available in the average pathology laboratory and preclude the use of archival material.…”

Section: Her2 Testing Methodsmentioning

confidence: 99%

“…However, its drawbacks are that it is time-consuming, tedious and expensive. Competitive PCR is another suitable HER2 testing option that has received increasing attention recently [Deng et al 1999;Okuyama et al 1999;Vona et al 1999]. …”

Section: Pcrmentioning

confidence: 99%

Testing for HER2 Status

Hanna¹

2001

Oncology

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Incorporation of a new biological marker such as HER2 into routine clinical practice requires proof that it provides reproducible information independent of, and better than, conventional pathologic criteria, and that it influences treatment decisions. In breast cancer, HER2 amplification/overexpression predicts for a poor clinical outcome and an enhanced survival benefit from the HER2-targeted therapy, Herceptin^®, and may predict for resistance to some conventional therapies. Thus, HER2 is considered to be a clinically important molecule and testing for HER2 abnormalities is already part of routine patient assessment in many parts of the world. There is currently no gold standard for HER2 testing. The main challenge is to standardize and technically validate HER2 testing methodologies. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are the most common HER2 tests used, and show a high level of concordance. HER2 testing approaches based on the polymerase chain reaction (PCR) are under extensive investigation and appear promising. A Canadian HER2 testing algorithm designed to increase the validity and reproducibility of HER2 testing has been compiled. HER2-positive cases are defined as those with >10% of tumor cells with moderate/strong, complete membrane staining in the invasive component, by IHC. Confirmatory HER2 testing using either FISH or quantitative PCR is recommended for indeterminate cases. Additional studies are required to calibrate HER2 testing results to clinical outcome.

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“…These methods can be semi-quantitative or quantitative depending on the method of product quantification applied. Several investigators have noted a high level of concordance between the different PCR assays and IHC for HER2 testing [34, 35, 36, 37, 38, 39, 40]. …”

Section: Her2 Testingmentioning

confidence: 99%

Emerging Technologies for HER2 Testing

Vijver

2002

Oncology

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HER2-positive status is the sole criterion for identifying patients with breast cancer for Herceptin^® therapy, which has known efficacy in women with metastatic breast cancer (MBC). Immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH), which measure the HER2 protein and gene, respectively, are currently the most widely used HER2 tests in the clinical setting. However, results from these assays are influenced by many variables including choice of antibody or probe, methodology, level of user experience and interlaboratory variability. Although there is no widespread standard testing algorithm, the importance of HER2 in clinical practice demands accurate and reproducible tests. Polymerase chain reaction (PCR) and chromogenic in-situ hybridization (CISH) represent upcoming methods for assessing HER2 gene amplification. Enzyme-linked immunosorption assay (ELISA), a semi-automated technique that can be used to measure the level of the HER2 extracellular domain (ECD) in serum, may also prove useful. While such technologies show great promise, they will at least have to be validated against IHC or FISH before being accepted into routine clinical practice.

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Quantitation of c-<i>erb</i>B-2 Gene Amplification in Breast Cancer Tissue by Competitive PCR

Cited by 7 publications

References 18 publications

Simple and Versatile Molecular Method of Copy-Number Measurement Using Cloned Competitors

Simple and Versatile Molecular Method of Copy-Number Measurement Using Cloned Competitors

Testing for HER2 Status

Emerging Technologies for HER2 Testing

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