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Development of automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence in situ hybridization (FISH).

Nitta H, Hauss-Wegrzyniak B, Lehrkamp M, Murillo AE, Gaire F, Farrell M, Walk E, Penault-Llorca F, Kurosumi M, Dietel M, Wang L, Loftus M, Pettay J, Tubbs RR, Grogan TM - Diagn Pathol (2008)

Bottom Line: Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues.The application also has the potential to be used for other gene targets.The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Office of Medical Affairs, Ventana Medical Systems Inc., Tucson, AZ, USA. hiro.nitta@ventana.roche.com

ABSTRACT

Background: Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas.

Methods: The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark(R) XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatase (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives.

Results: Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 - 1.0000) and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa = 0.8993%, 95% CI = 0.8068 - 0.9919) and without the FISH equivocal cases (100%, Simple Kappa = 1.0000%, 95% CI = 1.0000 - 1.0000).

Conclusion: Automated BDISH applications for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.

No MeSH data available.


Related in: MedlinePlus

Brightfield double in situ hybridization (BDISH) for HER2 and chromosome 17 centromere (CEN 17) on formalin-fixed, paraffin-embedded clinical breast cancer cases. Examples of normal HER2 gene (A), amplified HER2 gene (B), single HER2 gene (C), and chromosome 17 polysomy (D) cases were shown. 100×.
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Figure 3: Brightfield double in situ hybridization (BDISH) for HER2 and chromosome 17 centromere (CEN 17) on formalin-fixed, paraffin-embedded clinical breast cancer cases. Examples of normal HER2 gene (A), amplified HER2 gene (B), single HER2 gene (C), and chromosome 17 polysomy (D) cases were shown. 100×.

Mentions: Representative images of BDISH staining on clinical samples are presented in Figure 3. Cancer cells were easily identified based on the tissue morphology and assessments of HER2/CEN 17 ratios could be readily conducted. Non-amplified HER2 gene cases showed 0–4 copies of HER2 genes and 0–4 copies of CEN 17 depending on cell cycle stage and how each cell was cut within a tissue section (Figure 3A), while amplified HER2 gene cases showed multiple copies or clusters of HER2 genes and a few copies of CEN 17 (Figure 3B). Besides non-amplified and amplified HER2 cases, cases with a single copy of HER2 gene due to centromere 17 monosomy or a monoallelic deletion of HER2 gene (Figure 3C) and multiple copies of CEN 17 due to chromosome 17 polysomy (Figure 3D) were also observed.


Development of automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence in situ hybridization (FISH).

Nitta H, Hauss-Wegrzyniak B, Lehrkamp M, Murillo AE, Gaire F, Farrell M, Walk E, Penault-Llorca F, Kurosumi M, Dietel M, Wang L, Loftus M, Pettay J, Tubbs RR, Grogan TM - Diagn Pathol (2008)

Brightfield double in situ hybridization (BDISH) for HER2 and chromosome 17 centromere (CEN 17) on formalin-fixed, paraffin-embedded clinical breast cancer cases. Examples of normal HER2 gene (A), amplified HER2 gene (B), single HER2 gene (C), and chromosome 17 polysomy (D) cases were shown. 100×.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577627&req=5

Figure 3: Brightfield double in situ hybridization (BDISH) for HER2 and chromosome 17 centromere (CEN 17) on formalin-fixed, paraffin-embedded clinical breast cancer cases. Examples of normal HER2 gene (A), amplified HER2 gene (B), single HER2 gene (C), and chromosome 17 polysomy (D) cases were shown. 100×.
Mentions: Representative images of BDISH staining on clinical samples are presented in Figure 3. Cancer cells were easily identified based on the tissue morphology and assessments of HER2/CEN 17 ratios could be readily conducted. Non-amplified HER2 gene cases showed 0–4 copies of HER2 genes and 0–4 copies of CEN 17 depending on cell cycle stage and how each cell was cut within a tissue section (Figure 3A), while amplified HER2 gene cases showed multiple copies or clusters of HER2 genes and a few copies of CEN 17 (Figure 3B). Besides non-amplified and amplified HER2 cases, cases with a single copy of HER2 gene due to centromere 17 monosomy or a monoallelic deletion of HER2 gene (Figure 3C) and multiple copies of CEN 17 due to chromosome 17 polysomy (Figure 3D) were also observed.

Bottom Line: Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues.The application also has the potential to be used for other gene targets.The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Office of Medical Affairs, Ventana Medical Systems Inc., Tucson, AZ, USA. hiro.nitta@ventana.roche.com

ABSTRACT

Background: Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas.

Methods: The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark(R) XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatase (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives.

Results: Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 - 1.0000) and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa = 0.8993%, 95% CI = 0.8068 - 0.9919) and without the FISH equivocal cases (100%, Simple Kappa = 1.0000%, 95% CI = 1.0000 - 1.0000).

Conclusion: Automated BDISH applications for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation.

No MeSH data available.


Related in: MedlinePlus