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Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma.

Rosa FE, Silveira SM, Silveira CG, Bérgamo NA, Neto FA, Domingues MA, Soares FA, Caldeira JR, Rogatto SR - BMC Cancer (2009)

Bottom Line: The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and HER-2 transcript overexpression (20%).In general, concordance occurred between qRT-PCR and CISH results.Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Institute of Biosciences, UNESP, São Paulo State University, Botucatu, Sao Paulo, Brazil. fabiolarosa7@yahoo.com.br

ABSTRACT

Background: HER-2 gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas.

Methods: To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH.

Results: The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and HER-2 transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and HER-2 downexpression (38.5%) by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350).

Conclusion: Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene.

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Related in: MedlinePlus

Breast cancer cells showing immunohistochemistry and chromogen in situ hybridization results of HER-2. (A-D) IHC: HER-2 protein expression scored as 0 (A), 1+ (B), 2+ (C), and 3+ (D); (E-H) CISH: HER-2 gene detected in nuclei with two signals (E), more than two signals (F), and high-level amplification (G-H).
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Figure 1: Breast cancer cells showing immunohistochemistry and chromogen in situ hybridization results of HER-2. (A-D) IHC: HER-2 protein expression scored as 0 (A), 1+ (B), 2+ (C), and 3+ (D); (E-H) CISH: HER-2 gene detected in nuclei with two signals (E), more than two signals (F), and high-level amplification (G-H).

Mentions: Breast cancer samples were assessed by HER-2 gene amplification and protein expression in histological samples using CISH and IHC, respectively (Figure 1). Transcript expression was evaluated by qRT-PCR in fresh samples after microdissection. HER-2 data obtained in all the procedures are shown in Tables 2 and 3.


Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma.

Rosa FE, Silveira SM, Silveira CG, Bérgamo NA, Neto FA, Domingues MA, Soares FA, Caldeira JR, Rogatto SR - BMC Cancer (2009)

Breast cancer cells showing immunohistochemistry and chromogen in situ hybridization results of HER-2. (A-D) IHC: HER-2 protein expression scored as 0 (A), 1+ (B), 2+ (C), and 3+ (D); (E-H) CISH: HER-2 gene detected in nuclei with two signals (E), more than two signals (F), and high-level amplification (G-H).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667535&req=5

Figure 1: Breast cancer cells showing immunohistochemistry and chromogen in situ hybridization results of HER-2. (A-D) IHC: HER-2 protein expression scored as 0 (A), 1+ (B), 2+ (C), and 3+ (D); (E-H) CISH: HER-2 gene detected in nuclei with two signals (E), more than two signals (F), and high-level amplification (G-H).
Mentions: Breast cancer samples were assessed by HER-2 gene amplification and protein expression in histological samples using CISH and IHC, respectively (Figure 1). Transcript expression was evaluated by qRT-PCR in fresh samples after microdissection. HER-2 data obtained in all the procedures are shown in Tables 2 and 3.

Bottom Line: The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and HER-2 transcript overexpression (20%).In general, concordance occurred between qRT-PCR and CISH results.Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Genetics, Institute of Biosciences, UNESP, São Paulo State University, Botucatu, Sao Paulo, Brazil. fabiolarosa7@yahoo.com.br

ABSTRACT

Background: HER-2 gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas.

Methods: To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH.

Results: The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4%) and HER-2 transcript overexpression (20%). Moreover, 2+ immunostaining cases presented nonamplified status (50%) by CISH and HER-2 downexpression (38.5%) by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350).

Conclusion: Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene.

Show MeSH
Related in: MedlinePlus