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E. Vollmer
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-015 - Screening EGFR Mutations in NSCLC by Immunohistochemistry (ID 408)
09:30 - 09:30 | Author(s): E. Vollmer
- Abstract
Background:
EGFR mutations are important targets for therapy in NSCLC. EGFR mutations, receptor overexpression and increase gene copy number are main factors inducing EGFR tumorigenic activity. The aim of the present study was to detect EGFR mutations in NSCLC by immunohistochemistry (IHC) and investigate its relation to mutations detected by DNA sequencing, level of wild type EGFR protein overexpression, gene copy number gain and clinicopathological data.
Methods:
The study was performed in a cohort of 216 tumor tissues of primary chemotherapeutic naïve NSCLC. Expression of EGFR mutations was identified by immunohistochemistry (IHC), using the specific antibodies 6B6 and 43B2 (Cell Signaling Technology) followed by DNA sequencing of positive cases for NSCLC-associated EGFR mutations (Applied Biosystems). IHC was scored by two systems: (a) a modified H-score ranging from 0 to 300 (% cancer cells with membranous/cytoplasmic EGFR protein staining multiplied by the staining intensity rank from 0 to 3+) with score 100 as the positive threshold and (b) a qualitative score with cut off for positivity as ≥10% cells with 1+ to 3+ membranous or cytoplasmic staining for the mutation specific antibodies and 2+ and 3+ membranous staining for the wild form of EGFR. Wild EGFR protein expression determined by the 31G7 antibody (Zymed laboratories, CA). Gene copy number was investigated by Fluorescence In Situ Hybridization (FISH) using the SPEC EGFR/CEN7 dual color probe (ZytoLight) and specimens were scored according to the Colorado scoring system with high copy number defined as high polysomy (HP), low amplification (EGFR/CEN7=2.1-3) or high amplification (EGFR/CEN7=3).
Results:
Forty-one cases (19.9%) were positive to mutated EGFR by IHC, and 8 of them showed EGFR specific mutations on exons 18-21 by DNA sequencing. All the mutation confirmed cases had membranous or cytoplasmic staining intensity 2+ and 3+ with the different positive cut-off points of the two scoring systems. 6/10 (60%) of the genotyped NSCLC- associated mutations cases were positive to 43B2 and 4/10 (40%) were positive to 6B6 antibody. 7/10 (70%) of these cases showed 3+ membranous staining in ≥10% of tumor cells, 3/10(30%) showed 2+ cytoplasmic staining in ≥ 10% of tumor cells. All 8 cases (100%) positive for mutations by sequencing had adenocarcinoma histology. Positive correlations were found between EGFR mutations, by IHC and sequencing, and both overexpression of wild EGFR and increase gene copy number (p=0.002 and p<0.001, respectively). Also, positive correlation was detected between EGFR mutations and high tumor grade and clinical stage (p<0.001 for both).
Conclusion:
IHC staining using mutation specific antibodies was demonstrated as a useful sensitive screening test before DNA sequencing. EGFR mutations play synergistic role with EGFR overexpression and increased gene copy number in NSCLC poor prognosis. Follow up of the cases with further evaluation of expression of EGFR wild and mutated forms after chemotherapy and targeted therapy will be performed.
