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Immunohistochemistry and fluorescence in situ hybridization assessment of HER2 in clinical trials of adjuvant therapy for breast cancer (NCCTG N9831, BCIRG 006, and BCIRG 005).

Perez EA, Press MF, Dueck AC, Jenkins RB, Kim C, Chen B, Villalobos I, Paik S, Buyse M, Wiktor AE, Meyer R, Finnigan M, Zujewski J, Shing M, Stern HM, Lingle WL, Reinholz MM, Slamon DJ - Breast Cancer Res. Treat. (2013)

Bottom Line: Upon adjudication, consensus was reached on 16/30 and 18/30 of discordant IHC and FISH cases, respectively, resulting in overall concordance rates of 96 and 97 %.Among 53 N9831 patients with HER2-normal disease adjudicated as IHC-/FISH-(although locally positive), there was a non-statistically significant improvement in disease-free survival with concurrent trastuzumab compared to chemotherapy alone (adjusted hazard ratio 0.34; 95 % CI, 0.11-1.05; p = 0.06).Agreement was improved at adjudication (96 %).

View Article: PubMed Central - PubMed

Affiliation: Mayo Clinic, 4500 San Pablo Road S., Jacksonville, FL 32224, USA. perez.edith@mayo.edu

ABSTRACT
A comprehensive, blinded, pathology evaluation of HER2 testing in HER2-positive/negative breast cancers was performed among three central laboratories. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analyses were performed on 389 tumor blocks from three large adjuvant trials: N9831, BCIRG-006, and BCIRG-005. In 123 cases, multiple blocks were examined. HER2 status was defined according to FDA-approved guidelines and was independently re-assessed at each site. Discordant cases were adjudicated at an on-site, face-to-face meeting. Results across three independent pathologists were concordant by IHC in 351/381 (92 %) and FISH in 343/373 (92 %) blocks. Upon adjudication, consensus was reached on 16/30 and 18/30 of discordant IHC and FISH cases, respectively, resulting in overall concordance rates of 96 and 97 %. Among 155 HER2-negative blocks, HER2 status was confirmed in 153 (99 %). In the subset of 102 HER2-positive patients from N9831/BCIRG-006, primary blocks from discordant cases were selected, especially those with discordant test between local and central laboratories. HER2 status was confirmed in 73 (72 %) of these cases. Among 118 and 113 cases with IHC and FISH results and >1 block evaluable, block-to-block variability/heterogeneity in HER2 results was seen in 10 and 5 %, respectively. IHC-/FISH- was confirmed for 57/59 (97 %) primary blocks from N9831 (locally positive, but centrally negative); however, 5/22 (23 %) secondary blocks showed HER2 positivity. Among 53 N9831 patients with HER2-normal disease adjudicated as IHC-/FISH-(although locally positive), there was a non-statistically significant improvement in disease-free survival with concurrent trastuzumab compared to chemotherapy alone (adjusted hazard ratio 0.34; 95 % CI, 0.11-1.05; p = 0.06). There were similar agreements for IHC and FISH among pathologists (92 % each). Agreement was improved at adjudication (96 %). HER2 tumor heterogeneity appears to partially explain discordant results in cases initially tested as positive and subsequently called negative.

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Intratumoral HER2 heterogeneity. HER2 protein and gene/chromosome heterogeneity in the same tumor. a HER2 gene amplification. Representative FISH staining demonstrating a focal HER2 amplified clone that corresponds to the area of HER2 protein over-expression in b. b Variable HER2 IHC Protein Immunostaining. The area identified shows IHC 3+ immunostaining, while the remainder of the microscopic field shows IHC 2+ immunostaining heterogeneity. c Representative FISH staining demonstrating polysomy 17 in the same tumor as in a and b
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Fig2: Intratumoral HER2 heterogeneity. HER2 protein and gene/chromosome heterogeneity in the same tumor. a HER2 gene amplification. Representative FISH staining demonstrating a focal HER2 amplified clone that corresponds to the area of HER2 protein over-expression in b. b Variable HER2 IHC Protein Immunostaining. The area identified shows IHC 3+ immunostaining, while the remainder of the microscopic field shows IHC 2+ immunostaining heterogeneity. c Representative FISH staining demonstrating polysomy 17 in the same tumor as in a and b

Mentions: Among 121 patients with two tissue blocks and two patients with three tissue blocks for analysis (Tables 1, 3), IHC results were obtained in 118 patients with the adjudicated IHC result agreeing across blocks in 106 (90 %) (Table 3). Among 113 patients with FISH results for >1 block, the adjudicated FISH result agreed across blocks in 107 (95 %) (Table 3). Of the 22 N9831 patients with HER2-normal (IHC-negative/FISH-negative) disease with duplicate blocks, 5/22 (23 %) tested positive (by IHC and/or FISH) in at least one of the duplicate blocks (Table 3), clearly demonstrating heterogeneous HER2 gene copy number and/or protein immunostaining in the same tumor (Fig. 2). Moreover, the focal HER2-amplified region corresponded to the areas of HER2 protein over-expression, confirming the presence of a population of HER2-positive cells in these tumors that had been called HER2-normal (Fig. 2a, b).Table 3


Immunohistochemistry and fluorescence in situ hybridization assessment of HER2 in clinical trials of adjuvant therapy for breast cancer (NCCTG N9831, BCIRG 006, and BCIRG 005).

Perez EA, Press MF, Dueck AC, Jenkins RB, Kim C, Chen B, Villalobos I, Paik S, Buyse M, Wiktor AE, Meyer R, Finnigan M, Zujewski J, Shing M, Stern HM, Lingle WL, Reinholz MM, Slamon DJ - Breast Cancer Res. Treat. (2013)

Intratumoral HER2 heterogeneity. HER2 protein and gene/chromosome heterogeneity in the same tumor. a HER2 gene amplification. Representative FISH staining demonstrating a focal HER2 amplified clone that corresponds to the area of HER2 protein over-expression in b. b Variable HER2 IHC Protein Immunostaining. The area identified shows IHC 3+ immunostaining, while the remainder of the microscopic field shows IHC 2+ immunostaining heterogeneity. c Representative FISH staining demonstrating polysomy 17 in the same tumor as in a and b
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585916&req=5

Fig2: Intratumoral HER2 heterogeneity. HER2 protein and gene/chromosome heterogeneity in the same tumor. a HER2 gene amplification. Representative FISH staining demonstrating a focal HER2 amplified clone that corresponds to the area of HER2 protein over-expression in b. b Variable HER2 IHC Protein Immunostaining. The area identified shows IHC 3+ immunostaining, while the remainder of the microscopic field shows IHC 2+ immunostaining heterogeneity. c Representative FISH staining demonstrating polysomy 17 in the same tumor as in a and b
Mentions: Among 121 patients with two tissue blocks and two patients with three tissue blocks for analysis (Tables 1, 3), IHC results were obtained in 118 patients with the adjudicated IHC result agreeing across blocks in 106 (90 %) (Table 3). Among 113 patients with FISH results for >1 block, the adjudicated FISH result agreed across blocks in 107 (95 %) (Table 3). Of the 22 N9831 patients with HER2-normal (IHC-negative/FISH-negative) disease with duplicate blocks, 5/22 (23 %) tested positive (by IHC and/or FISH) in at least one of the duplicate blocks (Table 3), clearly demonstrating heterogeneous HER2 gene copy number and/or protein immunostaining in the same tumor (Fig. 2). Moreover, the focal HER2-amplified region corresponded to the areas of HER2 protein over-expression, confirming the presence of a population of HER2-positive cells in these tumors that had been called HER2-normal (Fig. 2a, b).Table 3

Bottom Line: Upon adjudication, consensus was reached on 16/30 and 18/30 of discordant IHC and FISH cases, respectively, resulting in overall concordance rates of 96 and 97 %.Among 53 N9831 patients with HER2-normal disease adjudicated as IHC-/FISH-(although locally positive), there was a non-statistically significant improvement in disease-free survival with concurrent trastuzumab compared to chemotherapy alone (adjusted hazard ratio 0.34; 95 % CI, 0.11-1.05; p = 0.06).Agreement was improved at adjudication (96 %).

View Article: PubMed Central - PubMed

Affiliation: Mayo Clinic, 4500 San Pablo Road S., Jacksonville, FL 32224, USA. perez.edith@mayo.edu

ABSTRACT
A comprehensive, blinded, pathology evaluation of HER2 testing in HER2-positive/negative breast cancers was performed among three central laboratories. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analyses were performed on 389 tumor blocks from three large adjuvant trials: N9831, BCIRG-006, and BCIRG-005. In 123 cases, multiple blocks were examined. HER2 status was defined according to FDA-approved guidelines and was independently re-assessed at each site. Discordant cases were adjudicated at an on-site, face-to-face meeting. Results across three independent pathologists were concordant by IHC in 351/381 (92 %) and FISH in 343/373 (92 %) blocks. Upon adjudication, consensus was reached on 16/30 and 18/30 of discordant IHC and FISH cases, respectively, resulting in overall concordance rates of 96 and 97 %. Among 155 HER2-negative blocks, HER2 status was confirmed in 153 (99 %). In the subset of 102 HER2-positive patients from N9831/BCIRG-006, primary blocks from discordant cases were selected, especially those with discordant test between local and central laboratories. HER2 status was confirmed in 73 (72 %) of these cases. Among 118 and 113 cases with IHC and FISH results and >1 block evaluable, block-to-block variability/heterogeneity in HER2 results was seen in 10 and 5 %, respectively. IHC-/FISH- was confirmed for 57/59 (97 %) primary blocks from N9831 (locally positive, but centrally negative); however, 5/22 (23 %) secondary blocks showed HER2 positivity. Among 53 N9831 patients with HER2-normal disease adjudicated as IHC-/FISH-(although locally positive), there was a non-statistically significant improvement in disease-free survival with concurrent trastuzumab compared to chemotherapy alone (adjusted hazard ratio 0.34; 95 % CI, 0.11-1.05; p = 0.06). There were similar agreements for IHC and FISH among pathologists (92 % each). Agreement was improved at adjudication (96 %). HER2 tumor heterogeneity appears to partially explain discordant results in cases initially tested as positive and subsequently called negative.

Show MeSH
Related in: MedlinePlus