This retrospective study included patients with histologically confirmed stage IIIb, stage IV, or recurrent NSCLC who underwent EGFR mutation testing between February 2009 and December 2013 at a Korean Cancer Center Hospital. Eighty-four patients with EGFR wild-type lung adenocarcinoma received an EGFR TKI (either gefitinib or erlotinib). Patients who had received EGFR TKI for less than 2 weeks (n=7), those who did not undergo follow-up chest tomography after EGFR TKI administration (n=8), or those who had other primary cancers (n=3) were excluded from the study. Ultimately, 66 patients were enrolled. Gefitinib or erlotinib was administered orally (250 mg and 150 mg, respectively) once daily until disease progression or unacceptable toxicity.

Demographic and clinical characteristics including age, sex, smoking status, stage at EGFR TKI use, Eastern Cooperative Oncology Group (ECOG) performance status (PS) at EGFR TKI administration, the EGFR TKI agent administered (gefitinib or erlotinib), the number of previous chemotherapy regimens received, and extrathoracic metastatic sites (bones, brain, adrenal gland, liver, lymph nodes, and others locations as evaluated on the TKI administration start date) were retrieved from the medical records.

The study was approved by the institutional review board of Korea Cancer Center Hospital (IRB No. K-1504-001-003) and was conducted in accordance with the Declaration of Helsinki. The patients provided written informed consent to undergo the EGFR mutation and SISH tests.

Tumor response was assessed according to the Response Evaluation Criteria in Solid Tumors. The patients underwent chest computed tomography scans 4–8 weeks after initiating of EGFR TKI treatment, and then every 8–12 weeks thereafter. Brain magnetic resonance imaging or radionuclide bone scanning was also performed if brain or bone metastases were suspected, respectively. Patients who had complete response, partial response, or stable disease were categorized as responders; those who had progressed were categorized as non-responders. Progression-free survival (PFS) and overall survival (OS) were calculated from the first date of EGFR TKI treatment to the date of disease progression and the date of death or last follow-up, respectively.

Genomic DNA was extracted from paraffin-embedded tissues, as described previously18. In patients whose only available tissue was the cytological sample obtained at initial diagnosis, methanol-fixed cytological specimens were used to extract DNA19. EGFR mutation analysis was performed by pyrosequencing using previously described methods20. The presence of EGFR mutations was determined by evaluating exons 18, 19, 20, and 21.

Formalin-fixed paraffin-embedded (FFPE) tumor tissue samples and cell blocks of aspiration specimens were selected from a tissue and cytology archive of samples at the Department of Pathology at Korea Cancer Center Hospital. Hematoxylin and eosin staining was performed to assess sample adequacy and to confirm the histological diagnosis. Thirty-one tissue samples from the 66 patients were available for EGFR SISH analysis.

Dual-color dual-hapten SISH was performed according to the EGFR dinitrophenol (DNP) probe with the chromosome 7 digoxigenin (DIG) probe and ultraVIEW SISH DNP Detection Kit/ultraVIEW RED ISH DIG Detection Kit (Ventana Medical System Inc., Tucson, AZ, USA) for EGFR and chromosome 7 quantitation. The procedure was performed on an automated Ventana Benchmark XT (Ventana/Roche; Ventana Medical System Inc.) according to the manufacturer's standard protocols. Four-micrometer-thick FFPE samples from each case were deparaffinized, denatured, and pretreated with a Tris-based reaction buffer (pH 7.6) in 3 cycles of 12 minutes each at 90℃, and were then incubated with ISH Protease (Ventana/Roche) for 24 minutes. The DNP-labeled EGFR DNA probe and DIG-labeled chromosome 7 probe were co-denatured for 12 minutes at 80℃; slides were hybridized for 7 hours at 44℃ and were then washed stringently 3 times for 8 minutes at 72℃. EGFR signals were detected using the ultraVIEW SISH DNP Detection Kit. The signal was detected as silver deposits with silver acetate, hydroquinone, and hydrogen peroxide. The chromosome 7 signals were detected using the ultraVIEW RED ISH DIG Detection Kit, and were developed as stained red dots with fast red and naphthol phosphate. The slides were finally counterstained with hematoxylin II and bluing reagent before mounting. EGFR SISH scores were defined according to the Colorado scoring system as score 1 (1 or 2 signals in ≥90% of the counted nuclei [disomy]); score 2 (3 signals in 10–40%, ≥4 signals in <10% [low trisomy]); score 3 (3 signals in ≥40%, ≥4 signals in <10% [high trisomy]); score 4 (≥4 signals in 10%–40% [low polysomy]); score 5 (≥4 signals in ≥40% [high polysomy]); and score 6 (amplification [EGFR gene cluster ≥10%, EGFR/chromosome 7 ratio of ≥2, and ≥15 signals in ≥10%])14.

Categorical variables were analyzed using Fisher exact test, while continuous variables were analyzed using the Mann-Whitney test. The associated factors with treatment response of EGFR TKI were identified by univariate and multivariate logistic regression. PFS and OS were estimated by using the Kaplan-Meier method. Comparisons between groups were performed using the log-rank test for time-to-event variables. Cox proportional hazards models were used to evaluate independent predictive factors for survival. All statistical analyses were performed using SPSS software version 18.0 (SPSS Inc., Chicago, IL, USA), and p-values of <0.05 were considered statistically significant.

A total of 66 patients who received EGFR TKIs for lung adenocarcinoma with EGFR wild-type were identified for the analyses. The median follow-up time was 17.0 months (range, 1.9–81.3 months). The patients' median age was 57 years, and 66.7% were men (Table 1). The proportion of ever smokers was 69.1%. Approximately half of all patients were treated with EGFR TKIs beyond second-line therapy. Most patients (97.0%) had stage IV disease; 78.8% had extrathoracic metastasis.

The tumor responses to EGFR TKIs are shown in Table 1; the objective response rate was 12.1% while the disease control rate was 40.9%.

We performed EGFR SISH in 31 NSCLC specimens; analysis was possible in 23 of these cases. Disomy was present in one patient (4.3%), low trisomy in three (13.0%), high trisomy in three (13.0%), low polysomy in five (21.7%), high polysomy in 10 (43.5%), and gene amplification in one (4.3%). SISH positivity was defined as high polysomy or gene amplification. Twelve patients (52.2%) were categorized as EGFR SISH positive (high EGFR copy number), and 11 (47.8%) were characterized as EGFR SISH negative (low EGFR copy number). There was no significant difference in clinical characteristics between EGFR SISH-positive and -negative patients (Table 2). There was a trend toward higher response and disease control rates in SISH-positive patients compared to SISH-negative patients (16.7% vs. 0%, p=0.478 and 33.4% vs. 18.2%, p=0.640, respectively). The median PFS and OS were longer in SISH-positive patients than in SISH-negative patients (PFS: 2.0 months, vs. 1.0 month respectively; hazard ratio [HR], 0.57; 95% confidence interval [CI], 0.24–1.35; p=0.201 and OS: 7.5 months vs. 3.0 months, respectively; HR, 0.55; 95% CI, 0.23–1.30; p=0.173), but the differences were not statistically significant.

To investigate the baseline clinical variables that could predict treatment response to EGFR TKIs, logistic regression models were used to evaluate the correlation between these factors and treatment response. On univariate analyses (Table 3), female sex and never smoker were significantly associated with better treatment response (odds ratio [OR], 3.10; 95% CI, 1.07–8.94; p=0.037 and OR, 3.10; 95% CI, 1.05–9.19; p=0.041). But, there was no clinical factor to predict treatment response on multivariate analysis.

The overall median PFS and OS rates of all 66 patients were 2.1 months (95% CI, 1.77–2.43) and 9.7 months (95% CI, 6.35–13.05), respectively. Univariate analysis of PFS showed that female sex and ECOG PS scores of 0–1 had significantly positive effects on PFS; both factors were also significant independent predictors of PFS on multivariate analysis (Table 4). Furthermore, female sex, never smoker status, ECOG PS 0–1, and a low number of extrathoracic metastasis sites were significant predictors of longer OS on univariate analysis (Table 5). Treatment with EGFR TKIs beyond second-line therapy was associated with poor OS. On multivariate analysis, an ECOG PS 0–1 and a low tumor burden of extrathoracic metastases were independent predictors of longer OS.