Usefulness of tissue microarrays for assessment of protein expression, gene copy number and mutational status of EGFR in lung adenocarcinoma

Abstract

Specific inhibitors targeting the epidermal growth factor receptor (EGFR) can increase survival rates in certain lung adenocarcinoma patients with mutations in the EGFR gene. Although such EGFR-targeted therapies have been approved for use, there is no general consensus among surgical pathologists on how the EGFR status should be tested in lung adenocarcinoma tissues and whether the results of immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and mutational analysis by molecular methods correlate. We evaluated the EGFR status in 61 lung adenocarcinomas by IHC (using total and mutant-specific antibodies against EGFR), by FISH analysis on tissue microarrays (TMAs), and by direct sequencing. The results of each method were compared using χ² and κappa statistics. The sensitivity and negative predictive value estimating the presence of abnormal EGFR for each test was calculated. The results show that, with respect to expression patterns and clinicopathological parameters, the total and mutant-specific EGFR detected by immunohistochemistry and FISH analysis on TMAs are valid and are equivalent to conventional methods performed on whole-tissue sections. Abnormal EGFR was detected in 52.4% of patients by IHC, FISH, and sequencing. The best sensitivity (100%) and negative predictive value (100%) was determined by evaluating the EGFR status with all methods. Testing for molecular changes in EGFR using a single test is likely to underestimate the presence of EGFR abnormalities. Taken together, these results demonstrate the high potential of TMAs to test for the major mechanisms of EGFR activation in patients with lung adenocarcinoma.

High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer

“…[14][15][16][17][18][19][20][21][22] All these studies used the same mutation-specific antibodies from Cell Signaling Technology and the methods were comparable. Simonetti et al found specificity of 100% for both antibodies in 78 unselected Caucasian patients.…”

“…21 Previous studies using the mutation-specific antibodies from Cell Signaling Technology have found sensitivity ranging from 23 to 80% for exon19 deletions and 36 to 100% for exon21 mutations. [14][15][16][17][18][19][20][21][22] These studies used different scores for intensity and cutoff point for positivity making comparisons between the individual studies difficult, and standardization is warranted. 23 No gold standard method is approved for detecting EGFR mutations in NSCLC.…”

“…Antibody dilutions tested were 1:50, 1:100, and 1:150 according to the literature. [14][15][16][17][18][19][20][21][22] The included sections were incubated with primary monoclonal rabbit antibodies against exon19 deletions (clone 6B6, Cell Signaling Technology, Boston, MA, USA) diluted to 1:150 (optimal dilution), against point mutation of exon21, L858R (clone 43B2, Cell Signaling Technology) diluted to 1:100 amplified by linker (optimal dilution), and with primary monoclonal mouse antibody against EGFR variantIII (clone 218C9, EGFR pharmDx kit, Dako, DK) in 20 min.…”

“…For these reasons, mutation-specific antibodies might be a relevant alternative for determining EGFR status in NSCLC. [14][15][16][17][18][19][20][21][22] So far, no standardized protocol exists for EGFR immunohistochemistry in these tumors. 9,23,24 The aim of this study was to determine sensitivity and specificity of mutation-specific antibodies in detection of EGFR mutations in an unselected cohort of Caucasian patients with NSCLC.…”

Use of mutation specific antibodies to detect EGFR status in small biopsy and cytology specimens of lung adenocarcinoma

“…Recently two monoclonal antibodies, specific to the two most common forms of mutated EGFR protein have become commercially available [19]. Early studies have shown promising results regarding the use of these antibodies in screening patients for TKI therapy [20][21][22][23][24][25][26][27][28].…”