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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 in situ hybridization and dual color fluorescence in situ hybridization (FISH) for HER2 and CEN 17. HER2 and CEN 17 detection with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene and chromosome 17 polysomy) (A, C, E, G) and BT-474 (amplified HER2 gene and chromosome 17 polysomy) (B, D, F, H). Normal HER2 gene signal is seen as black dots in the nuclei of MCF7 xenograft tumor (A) while amplified HER2 gene signal is seen as clusters of black dots in the nuclei of BT-474 tumor (B). CEN 17 signal is detected as red dots that are slightly larger than silver black dots (C, D). Double staining of HER2 gene and CEN 17 is obtained with silver grains and red dots (E, F). Individual HER2 gene and CEN 17 signals can be still recognized when both targets are co-localized (arrow heads, E). HER2 FISH signal is red-orange and CEN 17 FISH signal is green in the blue nuclei counterstained with DAPI (G, H). 100×.
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Figure 2: Brightfield in situ hybridization and dual color fluorescence in situ hybridization (FISH) for HER2 and CEN 17. HER2 and CEN 17 detection with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene and chromosome 17 polysomy) (A, C, E, G) and BT-474 (amplified HER2 gene and chromosome 17 polysomy) (B, D, F, H). Normal HER2 gene signal is seen as black dots in the nuclei of MCF7 xenograft tumor (A) while amplified HER2 gene signal is seen as clusters of black dots in the nuclei of BT-474 tumor (B). CEN 17 signal is detected as red dots that are slightly larger than silver black dots (C, D). Double staining of HER2 gene and CEN 17 is obtained with silver grains and red dots (E, F). Individual HER2 gene and CEN 17 signals can be still recognized when both targets are co-localized (arrow heads, E). HER2 FISH signal is red-orange and CEN 17 FISH signal is green in the blue nuclei counterstained with DAPI (G, H). 100×.

Mentions: Images of HER2 single ISH, CEN 17 single ISH, and HER2 and CEN 17 BDISH results with formalin-fixed, paraffin-embedded xenograft tumor sections are presented in Figure 2. Single copies of HER2 signal were recognized as black discrete dots in the nuclei with MCF7 xenograft tumor (Figure 2A) while amplified HER2 gene signals were visualized as either an increased number of HER2 signals, clusters of black dots with BT-474 tumor, and/or both (Figure 2B). Single CEN 17 copies were observed as red dots that were slightly larger than the black dots for HER2 genes in the nuclei with MCF7 tumor (Figure 2C) and BT-474 tumor (Figure 2D). After single staining for HER2 gene or CEN 17 was optimized, the BDISH application with sequential detection for HER2 targets followed by CEN 17 targets was tested on xenograft tumors. Single copies of HER2 genes and CEN 17 were stained in the nuclei of MCF7 tumor cells (Figure 2E) and amplified HER2 genes and single copies of CEN 17 were visualized in the nuclei of BT-474 tumor cells (Figure 2F). Because of the size difference and color contrast of black dots for HER2 gene and red dots for CEN 17, they could be visually separated even when red and black signals were co-localized in the nuclei of MCF7 tumor cells (arrowheads, Figure 2E). When CEN 17 probe was omitted from the complete BDISH assay, there was no fast red staining on xenograft tumor sections (data not shown). Thus, the anti-DNP antibody used for CEN 17 signal detection (the second ISH detection) didn't recognize the DNP-hapten of HER2 probe signal (the first ISH detection) even though the same hapten was used for the sequential hybridization method. For image comparison of BDISH and FISH for HER2 gene and CEN 17, FISH images with MCF7 tumor and BT-474 tumor are presented in Figure 2G and Figure 2H, respectively. HER2 genes are seen as red-orange dots and CEN 17 targets are seen as green dots.


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 in situ hybridization and dual color fluorescence in situ hybridization (FISH) for HER2 and CEN 17. HER2 and CEN 17 detection with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene and chromosome 17 polysomy) (A, C, E, G) and BT-474 (amplified HER2 gene and chromosome 17 polysomy) (B, D, F, H). Normal HER2 gene signal is seen as black dots in the nuclei of MCF7 xenograft tumor (A) while amplified HER2 gene signal is seen as clusters of black dots in the nuclei of BT-474 tumor (B). CEN 17 signal is detected as red dots that are slightly larger than silver black dots (C, D). Double staining of HER2 gene and CEN 17 is obtained with silver grains and red dots (E, F). Individual HER2 gene and CEN 17 signals can be still recognized when both targets are co-localized (arrow heads, E). HER2 FISH signal is red-orange and CEN 17 FISH signal is green in the blue nuclei counterstained with DAPI (G, H). 100×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Brightfield in situ hybridization and dual color fluorescence in situ hybridization (FISH) for HER2 and CEN 17. HER2 and CEN 17 detection with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene and chromosome 17 polysomy) (A, C, E, G) and BT-474 (amplified HER2 gene and chromosome 17 polysomy) (B, D, F, H). Normal HER2 gene signal is seen as black dots in the nuclei of MCF7 xenograft tumor (A) while amplified HER2 gene signal is seen as clusters of black dots in the nuclei of BT-474 tumor (B). CEN 17 signal is detected as red dots that are slightly larger than silver black dots (C, D). Double staining of HER2 gene and CEN 17 is obtained with silver grains and red dots (E, F). Individual HER2 gene and CEN 17 signals can be still recognized when both targets are co-localized (arrow heads, E). HER2 FISH signal is red-orange and CEN 17 FISH signal is green in the blue nuclei counterstained with DAPI (G, H). 100×.
Mentions: Images of HER2 single ISH, CEN 17 single ISH, and HER2 and CEN 17 BDISH results with formalin-fixed, paraffin-embedded xenograft tumor sections are presented in Figure 2. Single copies of HER2 signal were recognized as black discrete dots in the nuclei with MCF7 xenograft tumor (Figure 2A) while amplified HER2 gene signals were visualized as either an increased number of HER2 signals, clusters of black dots with BT-474 tumor, and/or both (Figure 2B). Single CEN 17 copies were observed as red dots that were slightly larger than the black dots for HER2 genes in the nuclei with MCF7 tumor (Figure 2C) and BT-474 tumor (Figure 2D). After single staining for HER2 gene or CEN 17 was optimized, the BDISH application with sequential detection for HER2 targets followed by CEN 17 targets was tested on xenograft tumors. Single copies of HER2 genes and CEN 17 were stained in the nuclei of MCF7 tumor cells (Figure 2E) and amplified HER2 genes and single copies of CEN 17 were visualized in the nuclei of BT-474 tumor cells (Figure 2F). Because of the size difference and color contrast of black dots for HER2 gene and red dots for CEN 17, they could be visually separated even when red and black signals were co-localized in the nuclei of MCF7 tumor cells (arrowheads, Figure 2E). When CEN 17 probe was omitted from the complete BDISH assay, there was no fast red staining on xenograft tumor sections (data not shown). Thus, the anti-DNP antibody used for CEN 17 signal detection (the second ISH detection) didn't recognize the DNP-hapten of HER2 probe signal (the first ISH detection) even though the same hapten was used for the sequential hybridization method. For image comparison of BDISH and FISH for HER2 gene and CEN 17, FISH images with MCF7 tumor and BT-474 tumor are presented in Figure 2G and Figure 2H, respectively. HER2 genes are seen as red-orange dots and CEN 17 targets are seen as green dots.

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