Limits...
Immunohistochemical and molecular analyses of HER2 status in breast cancers are highly concordant and complementary approaches.

Lehmann-Che J, Amira-Bouhidel F, Turpin E, Antoine M, Soliman H, Legres L, Bocquet C, Bernoud R, Flandre E, Varna M, de Roquancourt A, Plassa LF, Giacchetti S, Espié M, de Bazelaire C, Cahen-Doidy L, Bourstyn E, Janin A, de Thé H, Bertheau P - Br. J. Cancer (2011)

Bottom Line: Twelve out of 466 cases (3%) revealed discordances between the two methods.The power of Q-RT-PCR to predict HER2 status (defined by seven methods) was similar to that of IHC.Q-RT-PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT-PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

View Article: PubMed Central - PubMed

Affiliation: AP-HP, Hosp Saint-Louis, Department of Biochemistry, Paris 75010, France. jacqueline.lehmann-che@sls.aphp.fr

ABSTRACT

Background: Immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) are currently the most commonly used methods to assess HER2 status. PCR-based assays allow quantitative determination of HER2 amplification (Q-PCR) or overexpression (Q-RT-PCR), but are not routinely used. We evaluated the relevance of Q-RT-PCR for HER2 status determination.

Methods: We compared IHC and Q-RT-PCR in 466 breast tumours. In discordant or equivocal cases, five additional methods (IHC with two other antibodies, FISH, silver in situ hybridisation (SISH) and Q-PCR) were combined to determine HER2 status. Two cases with HER2 intra-tumour heterogeneity were further explored by allelic profiles analysis and HUMARA clonality determination after microdissection.

Results: We observed 97.3% concordance between Q-RT-PCR and non-equivocal IHC. Twelve out of 466 cases (3%) revealed discordances between the two methods. The power of Q-RT-PCR to predict HER2 status (defined by seven methods) was similar to that of IHC. Although rare, some discordances between techniques might be due to HER2 intra-tumour heterogeneity and we report two examples, one tumour containing two distinct clones, another tumour consisting of HER2 amplified and non-amplified subclones.

Conclusion: Q-RT-PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT-PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

Show MeSH

Related in: MedlinePlus

Analysis of 14 equivocal and 12 discordant cases (n=26). (A) Comparison of seven different methods (columns) in the 26 discordant or equivocal cases (lines): determination at the protein level by IHC – CB11, polyclonal A0485 and 4B5 antibodies, at the mRNA level by Q-RT–PCR, at the DNA level by SISH, FISH and Q-PCR. A negative result is symbolised in blue, positive in red and equivocal in green. Final HER2 status is based on the result of all seven methods: positive if there were majority of positive results, negative if there were majority of negative results, and unclassified in other situations. (B) Illustration of discordant case #17: Upper panel: immunohistochemical staining for HER2 with CB11, A0485 and 4B5 antibodies and indirect immunoperoxydase visualisation (magnification × 250). Lower panel: SISH staining with HER2 probe, CEN17 probe and FISH staining with HER2 probe in green and CEN17 probe in orange. (magnification × 400).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3111154&req=5

fig2: Analysis of 14 equivocal and 12 discordant cases (n=26). (A) Comparison of seven different methods (columns) in the 26 discordant or equivocal cases (lines): determination at the protein level by IHC – CB11, polyclonal A0485 and 4B5 antibodies, at the mRNA level by Q-RT–PCR, at the DNA level by SISH, FISH and Q-PCR. A negative result is symbolised in blue, positive in red and equivocal in green. Final HER2 status is based on the result of all seven methods: positive if there were majority of positive results, negative if there were majority of negative results, and unclassified in other situations. (B) Illustration of discordant case #17: Upper panel: immunohistochemical staining for HER2 with CB11, A0485 and 4B5 antibodies and indirect immunoperoxydase visualisation (magnification × 250). Lower panel: SISH staining with HER2 probe, CEN17 probe and FISH staining with HER2 probe in green and CEN17 probe in orange. (magnification × 400).

Mentions: According to guidelines, all IHC score 2+ (n=14) were analysed not only by hybridisation methods (FISH and SISH), but also with two other IHC–HER2 antibodies and Q-PCR (Figure 2). These additional methods were also performed in all discordant cases (n=12). An overall HER2 status was defined for each of these 26 discordant or equivocal cases, based on the results of all seven techniques used, a case being HER2 positive if there were more positive than negative results (12 out of 26 cases), being negative if there were more negative than positive results (12 out of 26 cases) and being HER2 unclassified in other situations (2 out of 26 cases). Among the 14 equivocal cases, 4 were finally scored positive by the overall HER2 status, 9 negative and 1 remained undefined. In these equivocal cases, Q-RT–PCR analysis predicted the final HER2 status in 10 cases and failed in only 3 cases. However, in the 12 discordant cases, Q-RT–PCR predicted final HER2 status in only two cases and failed in nine cases.


Immunohistochemical and molecular analyses of HER2 status in breast cancers are highly concordant and complementary approaches.

Lehmann-Che J, Amira-Bouhidel F, Turpin E, Antoine M, Soliman H, Legres L, Bocquet C, Bernoud R, Flandre E, Varna M, de Roquancourt A, Plassa LF, Giacchetti S, Espié M, de Bazelaire C, Cahen-Doidy L, Bourstyn E, Janin A, de Thé H, Bertheau P - Br. J. Cancer (2011)

Analysis of 14 equivocal and 12 discordant cases (n=26). (A) Comparison of seven different methods (columns) in the 26 discordant or equivocal cases (lines): determination at the protein level by IHC – CB11, polyclonal A0485 and 4B5 antibodies, at the mRNA level by Q-RT–PCR, at the DNA level by SISH, FISH and Q-PCR. A negative result is symbolised in blue, positive in red and equivocal in green. Final HER2 status is based on the result of all seven methods: positive if there were majority of positive results, negative if there were majority of negative results, and unclassified in other situations. (B) Illustration of discordant case #17: Upper panel: immunohistochemical staining for HER2 with CB11, A0485 and 4B5 antibodies and indirect immunoperoxydase visualisation (magnification × 250). Lower panel: SISH staining with HER2 probe, CEN17 probe and FISH staining with HER2 probe in green and CEN17 probe in orange. (magnification × 400).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111154&req=5

fig2: Analysis of 14 equivocal and 12 discordant cases (n=26). (A) Comparison of seven different methods (columns) in the 26 discordant or equivocal cases (lines): determination at the protein level by IHC – CB11, polyclonal A0485 and 4B5 antibodies, at the mRNA level by Q-RT–PCR, at the DNA level by SISH, FISH and Q-PCR. A negative result is symbolised in blue, positive in red and equivocal in green. Final HER2 status is based on the result of all seven methods: positive if there were majority of positive results, negative if there were majority of negative results, and unclassified in other situations. (B) Illustration of discordant case #17: Upper panel: immunohistochemical staining for HER2 with CB11, A0485 and 4B5 antibodies and indirect immunoperoxydase visualisation (magnification × 250). Lower panel: SISH staining with HER2 probe, CEN17 probe and FISH staining with HER2 probe in green and CEN17 probe in orange. (magnification × 400).
Mentions: According to guidelines, all IHC score 2+ (n=14) were analysed not only by hybridisation methods (FISH and SISH), but also with two other IHC–HER2 antibodies and Q-PCR (Figure 2). These additional methods were also performed in all discordant cases (n=12). An overall HER2 status was defined for each of these 26 discordant or equivocal cases, based on the results of all seven techniques used, a case being HER2 positive if there were more positive than negative results (12 out of 26 cases), being negative if there were more negative than positive results (12 out of 26 cases) and being HER2 unclassified in other situations (2 out of 26 cases). Among the 14 equivocal cases, 4 were finally scored positive by the overall HER2 status, 9 negative and 1 remained undefined. In these equivocal cases, Q-RT–PCR analysis predicted the final HER2 status in 10 cases and failed in only 3 cases. However, in the 12 discordant cases, Q-RT–PCR predicted final HER2 status in only two cases and failed in nine cases.

Bottom Line: Twelve out of 466 cases (3%) revealed discordances between the two methods.The power of Q-RT-PCR to predict HER2 status (defined by seven methods) was similar to that of IHC.Q-RT-PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT-PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

View Article: PubMed Central - PubMed

Affiliation: AP-HP, Hosp Saint-Louis, Department of Biochemistry, Paris 75010, France. jacqueline.lehmann-che@sls.aphp.fr

ABSTRACT

Background: Immunohistochemistry (IHC) and fluorescent in situ hybridisation (FISH) are currently the most commonly used methods to assess HER2 status. PCR-based assays allow quantitative determination of HER2 amplification (Q-PCR) or overexpression (Q-RT-PCR), but are not routinely used. We evaluated the relevance of Q-RT-PCR for HER2 status determination.

Methods: We compared IHC and Q-RT-PCR in 466 breast tumours. In discordant or equivocal cases, five additional methods (IHC with two other antibodies, FISH, silver in situ hybridisation (SISH) and Q-PCR) were combined to determine HER2 status. Two cases with HER2 intra-tumour heterogeneity were further explored by allelic profiles analysis and HUMARA clonality determination after microdissection.

Results: We observed 97.3% concordance between Q-RT-PCR and non-equivocal IHC. Twelve out of 466 cases (3%) revealed discordances between the two methods. The power of Q-RT-PCR to predict HER2 status (defined by seven methods) was similar to that of IHC. Although rare, some discordances between techniques might be due to HER2 intra-tumour heterogeneity and we report two examples, one tumour containing two distinct clones, another tumour consisting of HER2 amplified and non-amplified subclones.

Conclusion: Q-RT-PCR and IHC are highly concordant methods for HER2 status assessment, and Q-RT-PCR allows a highly reliable quantitative assessment and could be a useful adjunct to IHC.

Show MeSH
Related in: MedlinePlus