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Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay.

Goud KI, Dayakar S, Vijayalaxmi K, Babu SJ, Reddy PV - Indian J. Med. Res. (2012)

Bottom Line: Thirty of the 90 patients had negative results by IHC and FISH.These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification.There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology & Cytogenetics, Apollo Health City, Hyderabad, India.

ABSTRACT

Background & objectives: Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER 2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis.

Methods: A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy.

Results: Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4.

Interpretation & conclusions: Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.

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Related in: MedlinePlus

Fluorescence in situ hybridization (FISH) images showing negative, equivocal and positive results for HER-2/neu gene amplification. (A) Ratio of HER-2/neu(orange) signal to centromere 17 (green) signal 0.6-1.1 negative by FISH, (B) Ratio 1.6 negative by FISH, (C) Ratio 1.8-2.2, equivocal by FISH, (D) Ratio 3.9 positive by FISH.
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Figure 1: Fluorescence in situ hybridization (FISH) images showing negative, equivocal and positive results for HER-2/neu gene amplification. (A) Ratio of HER-2/neu(orange) signal to centromere 17 (green) signal 0.6-1.1 negative by FISH, (B) Ratio 1.6 negative by FISH, (C) Ratio 1.8-2.2, equivocal by FISH, (D) Ratio 3.9 positive by FISH.

Mentions: Of the 90 patients, 30 with a score of 0/1+ (negative) by IHC were FISH negative for HER-2/neu gene amplification (ratio between 0.6-1.6) (Fig. 1 A, B, Fig. 2 A, B). Of the 28 patients with the score of 2+ by IHC (Fig. 2C), 20 were FISH positive for HER-2/neu gene amplification (>2.2), three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH (Fig. 1C). These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, where all five patients were found eligible for trastuzumab-based therapy. Twenty five patients with the score of 3+ by IHC (Fig. 2D) were FISH positive for HER-2/neu gene amplification with the ratio of >3.9 (>2.2) (Fig. 1D). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2). These cases showed polysomy of chromosome number 17 (spec green signals > 4) which could have been the reason for IHC 3+ (Fig. 3). The association of HER-2/neu status in these 90 patients with histological grade of tumour and estrogen and progesterone receptor status is shown in the Table.


Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay.

Goud KI, Dayakar S, Vijayalaxmi K, Babu SJ, Reddy PV - Indian J. Med. Res. (2012)

Fluorescence in situ hybridization (FISH) images showing negative, equivocal and positive results for HER-2/neu gene amplification. (A) Ratio of HER-2/neu(orange) signal to centromere 17 (green) signal 0.6-1.1 negative by FISH, (B) Ratio 1.6 negative by FISH, (C) Ratio 1.8-2.2, equivocal by FISH, (D) Ratio 3.9 positive by FISH.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3361866&req=5

Figure 1: Fluorescence in situ hybridization (FISH) images showing negative, equivocal and positive results for HER-2/neu gene amplification. (A) Ratio of HER-2/neu(orange) signal to centromere 17 (green) signal 0.6-1.1 negative by FISH, (B) Ratio 1.6 negative by FISH, (C) Ratio 1.8-2.2, equivocal by FISH, (D) Ratio 3.9 positive by FISH.
Mentions: Of the 90 patients, 30 with a score of 0/1+ (negative) by IHC were FISH negative for HER-2/neu gene amplification (ratio between 0.6-1.6) (Fig. 1 A, B, Fig. 2 A, B). Of the 28 patients with the score of 2+ by IHC (Fig. 2C), 20 were FISH positive for HER-2/neu gene amplification (>2.2), three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH (Fig. 1C). These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, where all five patients were found eligible for trastuzumab-based therapy. Twenty five patients with the score of 3+ by IHC (Fig. 2D) were FISH positive for HER-2/neu gene amplification with the ratio of >3.9 (>2.2) (Fig. 1D). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2). These cases showed polysomy of chromosome number 17 (spec green signals > 4) which could have been the reason for IHC 3+ (Fig. 3). The association of HER-2/neu status in these 90 patients with histological grade of tumour and estrogen and progesterone receptor status is shown in the Table.

Bottom Line: Thirty of the 90 patients had negative results by IHC and FISH.These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification.There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology & Cytogenetics, Apollo Health City, Hyderabad, India.

ABSTRACT

Background & objectives: Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER 2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis.

Methods: A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy.

Results: Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4.

Interpretation & conclusions: Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.

Show MeSH
Related in: MedlinePlus