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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.

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Analysis of case A heterogeneity. (A) Schematic representation of case A with distinction of area 1 (HER2+) and area 2 (HER2−): H&E section, immunohistochemical stainings for HER2, ER, PR, CK5/6, CK8 and SISH evaluation of HER2 (magnification × 250). (B) Analysis of X-chromosome methylation pattern (HUMARA): allelic profiles for the total tumour and microdissected areas 1 and 2 are shown, before HpaII (upper line) and after HpaII (lower line) digestion. Differences between the two areas are represented by arrows.
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fig3: Analysis of case A heterogeneity. (A) Schematic representation of case A with distinction of area 1 (HER2+) and area 2 (HER2−): H&E section, immunohistochemical stainings for HER2, ER, PR, CK5/6, CK8 and SISH evaluation of HER2 (magnification × 250). (B) Analysis of X-chromosome methylation pattern (HUMARA): allelic profiles for the total tumour and microdissected areas 1 and 2 are shown, before HpaII (upper line) and after HpaII (lower line) digestion. Differences between the two areas are represented by arrows.

Mentions: In Case A, H&E examination showed the presence of a 1-cm less differentiated area (area 1) located inside the main tumour component (area 2) (Figure 3A). Area 1 was scored 3+ for HER2, was positive for CK5/6 and negative for ER, PR, CK8 and CK17, while area 2 was negative for HER2, CK5/6 and CK17 and positive for ER, PR and CK8. Silver in situ hybridisation confirmed HER2 amplification only in tumour cells of area 1 (Figure 3A). There was allelic loss at D17S855 in area 1 but not in area 2 (data not shown). Allelic profiles with the other microsatellites were either non-informative or showed no significant difference between the two areas (data not shown). The analysis of the X-chromosome methylation pattern showed important differences between areas 1 and 2 (Figure 3B): before HpaII digestion, allelic profiles showed LOH in each area, but on two distinct alleles. After HpaII digestion, profiles showed inactivation of one X chromosome in both areas 1 and 2, indicating that both areas are populated by monoclonal cells. However, areas 1 and 2 did not inactivate the same X chromosome, demonstrating that they are not deriving from the same clone.


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 case A heterogeneity. (A) Schematic representation of case A with distinction of area 1 (HER2+) and area 2 (HER2−): H&E section, immunohistochemical stainings for HER2, ER, PR, CK5/6, CK8 and SISH evaluation of HER2 (magnification × 250). (B) Analysis of X-chromosome methylation pattern (HUMARA): allelic profiles for the total tumour and microdissected areas 1 and 2 are shown, before HpaII (upper line) and after HpaII (lower line) digestion. Differences between the two areas are represented by arrows.
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Related In: Results  -  Collection

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fig3: Analysis of case A heterogeneity. (A) Schematic representation of case A with distinction of area 1 (HER2+) and area 2 (HER2−): H&E section, immunohistochemical stainings for HER2, ER, PR, CK5/6, CK8 and SISH evaluation of HER2 (magnification × 250). (B) Analysis of X-chromosome methylation pattern (HUMARA): allelic profiles for the total tumour and microdissected areas 1 and 2 are shown, before HpaII (upper line) and after HpaII (lower line) digestion. Differences between the two areas are represented by arrows.
Mentions: In Case A, H&E examination showed the presence of a 1-cm less differentiated area (area 1) located inside the main tumour component (area 2) (Figure 3A). Area 1 was scored 3+ for HER2, was positive for CK5/6 and negative for ER, PR, CK8 and CK17, while area 2 was negative for HER2, CK5/6 and CK17 and positive for ER, PR and CK8. Silver in situ hybridisation confirmed HER2 amplification only in tumour cells of area 1 (Figure 3A). There was allelic loss at D17S855 in area 1 but not in area 2 (data not shown). Allelic profiles with the other microsatellites were either non-informative or showed no significant difference between the two areas (data not shown). The analysis of the X-chromosome methylation pattern showed important differences between areas 1 and 2 (Figure 3B): before HpaII digestion, allelic profiles showed LOH in each area, but on two distinct alleles. After HpaII digestion, profiles showed inactivation of one X chromosome in both areas 1 and 2, indicating that both areas are populated by monoclonal cells. However, areas 1 and 2 did not inactivate the same X chromosome, demonstrating that they are not deriving from the same clone.

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