Genetic alterations of the epidermal growth factor receptor pathway in a series of non-small cell lung cancer characterized by morphology and immunophenotype

Verdú M,1 Colomer A,1 Erill N,1 Roman R,2 Górriz M, 1,2 Ibáñez R,1 Cordon-Cardo C,1 Puig X.1,2

1HISTOPAT Laboratoris, Barcelona, Catalonia, Spain & 2BIOPAT. Biopatologia Molecular, Grup Assistència.

Background: Non-small cell lung cancer (NSCLC) is classified in two main categories: adenocarcinoma (AC), which includes bronchioloalveolar carcinoma (BAC), and squamous cell carcinoma (SCC). Activating mutations and increased copy number of the EGFR gene have been reported in tumors from patients with NSCLC with increased response rate to tyrosine kinase inhibitors (TKIs). There is a need to tailor certain targeted treatments based on their molecular profile.

Design: The study was performed on formalin-fixed, paraffin-embedded specimens from area of Barcelona, Catalonia, Spain. The cohort included 90 NSCLC patients (78 men and 12 female); 40 cases were diagnosed as AC (including 6 BAC), 38 as SCC, 2 as adenosquamous carcinomas and 10 as large cell undifferentiated carcinomas (LCUC). A panel of six monoclonal antibodies (CK7, CK20, CK5/6, CK903, TTF-1 and P63) was used to discern glandular versus squamous immunophenotypic profile. EGFR expression was assessed by IHC using the antibody clone 31G7, and tumors were considered positive when displaying >10% immunoreactive cells. EGFR gene copy number was assessed by dual-color FISH using both a centromeric probe (CEP7) and a locus specific probe (EGFR LSI), and tumors were classified according to Cappuzzo’s criteria. Mutational study of exons 18, 19 and 21 EGFR, and exon 1 KRAS was conducted by PCR followed by bidirectional sequencing.

Results: Using the combined panel of six antibodies, tumor patterns were assigned as glandular (n=36), squamous (n=38), mixed (n=1), and undetermined (n=15). Tumors previously classified as LCUC were re-classified as AC (n=5) or SCC (n=3). A positive EGFR phenotype by IHC was identified in 68 out of 82 evaluable cases (83%). Regarding mutational status, large exon 19 deletions previously described in the EGFR gene were detected in 3 out of 76 evaluable cases (4%), 2 cases harboring a delE746-A750 and one case with a delL747-P753insS. Mutations in the KRAS gene were found in 8 out of 81 evaluable cases (10%), corresponding to 6 G12C, 2 G12V, and one G13D. All mutated cases were AC. Increased EGFR copy number by FISH (ratio LSI/CEP7 ≥2 or gene copies ≥4 per tumoral nuclei) was detected in 43 out of 76 evaluable cases (57%), which histologically corresponded to 23 AC and 20 SCC. The 3 EGFR mutated patients were females carrying increased EGFR copy number, and without KRAS mutations. Neither histologic type (AC including BAC versus SCC) nor gender were found to be significantly associated with EGFR status.

Conclusions: Glandular and squamous immunophenotypic profiles by IHC can assist in discerning AC and/or SCC in the majority of LCUC cases. In our series, FISH seems to be a more appropriate technique to determine genetic alterations of the EGFR pathway since 91% of mutations (including EGFR and KRAS point mutations) occurred in tumors harboring high EGFR copy number.

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Abstract exhibited at the:

International Academy of Pathology Centennial Congress
Montreal, Québec, Canada
September 16-21, 2006

Abstract in:

Modern Pathology 2006; 19 (suppl. 3): 142.