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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

Intratumoral heterogeneity of HER-2 gene status detected by chromogen in situ hybridization in two different areas (areas 1 and 2) from three breast tumors (A, B, and C). Nonamplified HER-2 gene (2–5 copies per nucleus), low-level amplification (6–10 copies or small clusters) and high-level amplification (>10 copies or large clusters) were observed in different areas from the same tumor. FISH results of the same cases are represented on the right side of the figure.
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Figure 2: Intratumoral heterogeneity of HER-2 gene status detected by chromogen in situ hybridization in two different areas (areas 1 and 2) from three breast tumors (A, B, and C). Nonamplified HER-2 gene (2–5 copies per nucleus), low-level amplification (6–10 copies or small clusters) and high-level amplification (>10 copies or large clusters) were observed in different areas from the same tumor. FISH results of the same cases are represented on the right side of the figure.

Mentions: CISH was performed on 43 cytological samples; in three of these cases the spots presented inconclusive results; the brown spots were undistinguished. Twenty-four out of 37 tumors (64.9%) were not amplified by CISH, while 13 samples (35.1%) showed high-level HER-2 amplification. Different areas of the tumor were evaluated and the final analysis showed agreement, except in three cases (Figure 2A–C). Case A presented an equivalent number of nonamplified cells (2 copies and 3–5 copies) in area 1 and more than 50% of the cells with high-level amplification in area 2 (this case presented 0 score by IHC and normal expression level by qRT-PCR). Case B showed nonamplified cells in both areas, but in area 2, low-level and high-level amplification cells were also observed (IHC, 0 score; qRT-PCR, overexpression). Case C showed preferentially nonamplified cells; however, in area 1, cells with 3–5 copies were predominant and in area 2, a prevalence of two HER-2 copies was detected and the presence of sporadic cells with high-level amplification was also found (IHC, 3+ score; qRT-PCR, overexpression). More than 400 cells were evaluated for each of these cases. In the other samples (37 cases), the nuclear features were maintained, the morphological details were apparent and large gene copy clusters were easily detected at low magnification. Normal epithelial cells and lymphocytes showed one or two HER-2 signals per nucleus.


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)

Intratumoral heterogeneity of HER-2 gene status detected by chromogen in situ hybridization in two different areas (areas 1 and 2) from three breast tumors (A, B, and C). Nonamplified HER-2 gene (2–5 copies per nucleus), low-level amplification (6–10 copies or small clusters) and high-level amplification (>10 copies or large clusters) were observed in different areas from the same tumor. FISH results of the same cases are represented on the right side of the figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Intratumoral heterogeneity of HER-2 gene status detected by chromogen in situ hybridization in two different areas (areas 1 and 2) from three breast tumors (A, B, and C). Nonamplified HER-2 gene (2–5 copies per nucleus), low-level amplification (6–10 copies or small clusters) and high-level amplification (>10 copies or large clusters) were observed in different areas from the same tumor. FISH results of the same cases are represented on the right side of the figure.
Mentions: CISH was performed on 43 cytological samples; in three of these cases the spots presented inconclusive results; the brown spots were undistinguished. Twenty-four out of 37 tumors (64.9%) were not amplified by CISH, while 13 samples (35.1%) showed high-level HER-2 amplification. Different areas of the tumor were evaluated and the final analysis showed agreement, except in three cases (Figure 2A–C). Case A presented an equivalent number of nonamplified cells (2 copies and 3–5 copies) in area 1 and more than 50% of the cells with high-level amplification in area 2 (this case presented 0 score by IHC and normal expression level by qRT-PCR). Case B showed nonamplified cells in both areas, but in area 2, low-level and high-level amplification cells were also observed (IHC, 0 score; qRT-PCR, overexpression). Case C showed preferentially nonamplified cells; however, in area 1, cells with 3–5 copies were predominant and in area 2, a prevalence of two HER-2 copies was detected and the presence of sporadic cells with high-level amplification was also found (IHC, 3+ score; qRT-PCR, overexpression). More than 400 cells were evaluated for each of these cases. In the other samples (37 cases), the nuclear features were maintained, the morphological details were apparent and large gene copy clusters were easily detected at low magnification. Normal epithelial cells and lymphocytes showed one or two HER-2 signals per nucleus.

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