Epidermal Growth Factor Receptor FISH Research Articles

Role of anti-EGFR monoclonal antibody in EGFR FISH positive NSCLC patients: A meta-analysis of phase III randomized controlled trials.

e20650Background: Monoclonal antibodies against epidermal growth factor receptor (EGFR) have been developed in combination with chemotherapy for NSCLC management. EGFR gene copy number by fluoresce...

Biomarker Analyses from a Randomized, Placebo-Controlled, Phase IIIb Trial Comparing Bevacizumab with or without Erlotinib as Maintenance Therapy for the Treatment of Advanced Non-Small-Cell Lung Cancer (ATLAS)

ATLAS compared bevacizumab plus erlotinib (B+E) with bevacizumab plus placebo (B+P) as maintenance therapy after first-line bevacizumab plus chemotherapy (B+C) for advanced non-small-cell lung cancer (NSCLC). Prespecified biomarkers were prospectively evaluated. Tumor samples were analyzed for: epidermal growth factor receptor (EGFR) expression (immunohistochemistry [IHC]); EGFR gene copy number (fluorescence in-situ hybridization [FISH]); EGFR mutations (exon 19 deletions/L858R mutations); and KRAS mutations (exons 2/3). Progression-free survival (PFS) and overall survival (OS) were estimated. Of 743 patients randomized to receive maintenance treatment (after four cycles of B+C without progression), 190 (B+E) and 177 (B+P) were evaluable for biomarker status. Median PFS (from randomization) was 4.4 months (B+E) versus 3.7 months (B+P; hazard ratio [HR], 0.75; 95% confidence interval [CI], 0.57-0.99), which was numerically similar to the intent-to-treat PFS. PFS benefit of B+E was observed across most biomarker subgroups. EGFR IHC, EGFR FISH, and EGFR/KRAS mutation status were not predictive of outcome. B+E-treated patients with EGFR mutation-positive NSCLC had longer PFS compared with B+P-treated patients (HR, 0.44; 95% CI, 0.22-0.86; p = 0.0139). Patients with KRAS wild-type disease had significant PFS improvements with B+E, compared with B+P (HR, 0.66; 95% CI, 0.485-0.914; p = 0.0105). No OS benefit of B+E was observed. Patients with KRAS wild-type or EGFR mutation-positive NSCLC derived PFS benefits from B+E. However, EGFR IHC, EGFR FISH, and EGFR or KRAS mutation status were not strongly predictive of survival. A larger sample size would be needed to confirm the initial trends observed in this study.

Design of a phase III clinical trial with prospective biomarker validation: SWOG S0819.

The role of cetuximab in the treatment of advanced non-small cell lung cancer (NSCLC) is currently unclear. The molecular target of cetuximab, epidermal growth factor receptor (EGFR), as measured by FISH, has shown potential as a predictive biomarker for cetuximab efficacy in NSCLC. SWOG S0819 is a phase III trial evaluating both the value of cetuximab in this setting and EGFR FISH as a predictive biomarker. This work describes the decision process for determining the design and interim monitoring plan for S0819. Six possible designs were evaluated in terms of their properties and the hypotheses that can be addressed within the design constraints. A subgroup-focused, multiple-hypothesis design was selected for S0819 that incorporates coprimary endpoints to assess cetuximab in both the overall study population and among EGFR FISH-positive (FISH(+)) patients, with the sample size determined based on evaluation in the EGFR FISH(+) group. The chosen interim monitoring plan specifies interim evaluations of both efficacy and futility in the EGFR FISH(+) group alone. The futility-monitoring plan to determine early stopping in the EGFR FISH-nonpositive group is based on evaluation within the positive group, the entire study population, and the nonpositive group. SWOG S0819 uses a design that addresses both the biomarker-driven and general-efficacy objectives of this study.

Intratumoral heterogeneity of EGFR mutation and clinical significance in Chinese patients with advanced non-small cell lung cancer.

7537 Background: Whether exist intratumoral heterogeneity of epidermal growth factor receptor (EGFR) gene mutation remains controversial. This study aims to evaluate intratumoral heterogeneity of EGFR and its clinical significance in patients with non-small cell lung cancer (NSCLC). Methods: Samples were collected from 85 patients with stage IIIa-IV who had palliative surgery resection. All of the bulk tissues had been routinely detected EGFR mutation, in which contained 30 wild-type and 55 mutant-type EGFR. 28–34 tumor foci for each sample were microdissected. EGFR mutation was detected by ARMS. EGFR copy number was detected by FISH. 23 patients received EGFR-tyrosine kinase inhibitor (TKIs) therapy. Results: 1740 tumor foci from 55 EGFR mutant-type (Group-M) and 959 foci from 30 wild-type cases(Group-W) were fractionated. Mean mutation contents were 73.9%(1285/1740) in Group-M and 0 in Group-W. Accordingly, 28.2%(24/85)consisted of cells with both wild-type and mutated-type EGFR, with the proportion of EGFR mutant cells ranging from 1% to 90%, whereas 71.8%(61/85) samples contained pure mutant-type (n=31) or wild-type EGFR cells(n=30). 31 patients (36.5%) presented EGFR FISH positive. The patients with mutation heterogeneity, regardless of high or low mutation contents, presented lower EGFR copy number than those with pure mutation cases (16.7% vs 71.0%,p<0.05). Among 23 patients who received EGFR-TKIs therapy, the mean mutation content was higher in partial response (71.1%) and stable disease group (43.4%) than in progressive disease group (5.0%) (p=0.005). Conclusions: This study showed that the heterogeneity of EGFR mutation was found in introtumoral region with different degree. The response to EGFR-TKIs was correlated with EGFR mutant content.

Gefitinib, cisplatin, and concurrent radiotherapy for locally advanced head and neck cancer: EGFR FISH, protein expression, and mutational status are not predictive biomarkers

Gefitinib was demonstrated to be synergistic with cisplatin and radiotherapy (RT) in in vitro studies. Biomarkers predictive of response to gefitinib in squamous cell head and neck cancer is still lacking. Thirty-one patients with locally advanced and easily accessible primary tumor sites for biopsies were recruited. Gefitinib was started 3 weeks before the start of cisplatin/concurrent radiotherapy (CTRT) and continued during the CTRT phase and thereafter for 4 months as consolidation phase. Two baselines and a repeat tumor sample were taken after 2 weeks of gefitinib alone to study its impact on tumor gene expression. Epidermal growth factor receptor (EGFR) protein expression, FISH and mutational status, and matrix metallopeptidase 11 (MMP11) protein expression were correlated with response and survival outcome. The overall response rate to gefitinib alone was 9.7%. The survival outcome is as follows: median disease free 1.3 years, median survival time 2.4 years, 3-year disease free 42.9%, and 3-year overall survival 48.4%. EGFR FISH, protein expression, and mutational status did not predict for response nor survival outcome of patients. Although MMP11 overexpression did not predict for response, it predicted significantly for a poorer survival outcome. Gefitinib can be combined safely with cisplatin/RT. More studies are needed to uncover predictive biomarkers of benefit to gefitinib.

Relationship between Epidermal Growth Factor Receptor Status, p16INK4A, and Outcome in Head and Neck Squamous Cell Carcinoma

Human papilloma virus (HPV) infection is a powerful prognostic biomarker in head and neck squamous cell carcinoma (HNSCC). Increased epidermal growth factor receptor (EGFR) gene copy number and protein expression have been reported to be negative predictors of outcome. This study examined the relationship between HPV status, EGFR gene copy number, EGFR protein expression, and clinical outcome in HNSCC patients treated with chemoradiation. HPV status was determined using p16(INK4A) immunohistochemistry (IHC), EGFR gene copy number was evaluated with FISH, and EGFR protein expression by IHC in 212 subjects. EGFR FISH was positive in 41 of 204 (20%) patients and was negatively correlated with failure-free survival (FFS; HR = 1.84, P = 0.027) and overall survival (OS; HR = 1.78, P = 0.082). For p16(INK4A), 85 of 200 (42.5%) patients were found to be p16 positive, including 75 of 131 (57%) with oropharyngeal cancer. Patients with p16-positive oropharyngeal cancer had significantly improved FFS (HR = 0.28, P < 0.001) and OS (HR = 0.31, P = 0.002). Only 2 of 126 (1.6%) oropharyngeal cancer patients were found to be p16+/EGFR FISH+. EGFR IHC was positive in 81 of 93 (87%) of patients and was associated with poorer FFS (HR = 1.98, P = 0.35) and OS (HR = 2.52, P = 0.22). Increased EGFR gene copy number is largely restricted to p16(INK4A)-negative oropharyngeal cancer. Although p16(INK4A) and EGFR FISH are both predictive of outcome in univariate analyses, only p16(INK4A) remains independently predictive. Knowledge of HPV and EGFR status can have implications for treatment options and prognosis in HNSCC.

Comparative Prognostic Value of Epidermal Growth Factor Quantitative Protein Expression Compared with FISH for Head and Neck Squamous Cell Carcinoma

Epidermal growth factor receptor (EGFR) overexpression correlates with recurrence and with treatment resistance in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to evaluate the relationship of EGFR gene copy number utilizing FISH and protein expression with automated quantitative analysis (AQUA) and to correlate those with patient outcome. A tissue microarray composed of 102 HNSCC treated with (chemo)radiation was constructed and analyzed for EGFR copy number by FISH (Vysis; Abbott Laboratories) and EGFR protein expression using AQUA analysis of EGFR staining scored on a scale of 0 to 255. We evaluated associations of EGFR FISH status and AQUA score with clinicopathologic parameters and survival prognosis. Eleven (17.2%) of 64 tumors with FISH results showed EGFR high polysomy and/or gene amplification (FISH positive). Protein levels assessed by AQUA in FISH-positive cases were significantly higher (P = 0.04) than in FISH-negative cases. Using the continuous AQUA scores for EGFR expression, AQUA and FISH showed significant agreement (Pearson's ρ = 0.353, P = 0.04). Patients with high tumor EGFR protein expression had inferior 5-year overall survival (27.7%) compared with those with low tumor EGFR expression (54%; P = 0.029). There was no significant association between EGFR FISH status and overall survival (P = 0.201). In the multivariate model, high tumor EGFR protein expression status remained an independent prognostic factor for overall survival (P = 0.047). EGFR protein content correlates with gene copy number if protein content is quantitated and automatically analyzed, as with AQUA. EGFR protein levels assessed by AQUA strongly predict for patient outcome in HNSCC, whereas EGFR FISH status does not provide prognostic information.

Escalating weekly doses of cetuximab and correlation with skin toxicity: A phase I study

Cetuximab is a chimeric monoclonal antibody targeting the epidermal growth factor receptor (EGFR). The recommended dosage is an initial load of 400 mg/m² intravenously (IV) followed by a weekly maintenance dose of 250 mg/m². It has been reported retrospectively that cetuximab efficacy was correlated with dose-related severity of skin rash. This study was prospectively designed to examine the safety and feasibility of escalating weekly doses of cetuximab, testing the hypothesis of the relationship of dose-dependent skin toxicity and efficacy. Methods Four dose levels were tested: Cetuximab 400 mg/m² IV loading dose and 250, 300, 350, 400 mg/m² weekly IV maintenance. There was no intra-patient dose escalation. Standard dose limiting toxicity criteria were used. Rash was evaluated using two additional validated dermatology methods: global acne grading scale (GAGS) and acne lesion counting (ALC). Tumor specimens and blood samples were obtained for correlative analyses. Twenty seven patients with solid tumors were enrolled: five head and neck, three pancreas, four gall bladder, two each of prostate, breast, colorectal, lung, and esophagus, and five others. Planned dose escalation was completed without reaching dose-limiting toxicity (DLT) or the maximum tolerated dose (MTD). The highest dose level was expanded to a total of 17 patients. Gr 3/4 toxicities included: lymphopenia (2), fatigue (2), and hypomagnesemia (2). One patient experienced a grade 3 rash (350 mg/m²). Sixty five percent of pts had a ≥ Gr 2 rash that was not dose dependent. In 22 evaluable patients, there was one partial response (PR) in a patient with cholangiocarcinoma (400 mg/m²) and seven patients had stable disease (SD). ALC and GAGS demonstrated no correlation with dose or response. Correlative studies evaluating k-ras, EGFR FISH status and immunologic correlatives were conducted on available tumor samples. Cetuximab administered at 400 mg/m² IV as a loading dose with weekly maintenance dose of 400 mg/m² is feasible and well tolerated. There was no direct correlation of the grade of rash with dose in this group of patients with heterogenous solid tumors.

Predictors of gefitinib outcomes in advanced non-small cell lung cancer (NSCLC): Study of a comprehensive panel of molecular markers

A number of different clinical characteristics and molecular markers related to epidermal growth factor receptor (EGFR) activation have been reported to singly correlate with therapeutic activity of EGFR tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC). This study was designed to evaluate the predictive value on gefitinib outcomes of a comprehensive panel of molecular parameters in advanced NSCLC patients. EGFR and K-ras mutations were detected by direct sequencing on tumor DNA from paraffin embedded samples. EGFR and HER2 gene copy number was assessed by FISH. EGFR protein expression was quantified by immunohistochemistry. EGFR gene intron 1 polymorphism was assessed on genomic DNA isolated from venous whole blood samples. Ninety-one patients were prospectively enrolled and the overall gefitinib response rate was 18.7% (2 complete and 15 partial responses). Sex ( p = 0.005), non-smoking status ( p = 0.010), skin toxicity ( p = 0.020), EGFR gene mutations ( p < 0.001) and EGFR FISH positivity ( p = 0.016) were found to be associated with gefitinib response. K-ras mutation was detected in only seven non-responder patients. The median overall survival was of 10 months. Only non-smoking status and EGFR intron 1 polymorphism showed a statistically significant correlation with survival ( p = 0.031 and 0.044, respectively). In conclusion, we have confirmed the role of EGFR gene mutation as predictor of response to EGFR TKIs. Moreover, EGFR gene copy number and, potentially, also EGFR intron 1 polymorphism could aid in better prediction of EGFR TKI responsiveness in advanced NSCLC.

EGFR protein overexpression and gene copy number increases in oral tongue squamous cell carcinoma

New promising therapeutic agents targeting epidermal growth factor receptor (EGFR) have been developed although clinical information concerning EGFR status in oral tongue squamous cell carcinoma (OTSCC) is limited. We investigated EGFR protein expression and gene copy numbers in 78 pretreatment OTSCC paraffin samples. EGFR protein expression was found in all 78 tumours, of which 72% showed an intense staining. Fifty-four percent of the tumours had high (⩾four gene copies) EGFR gene copy numbers. EGFR gene copy number was significantly associated with EGFR protein expression ( P = 0.002). Pretreatment EGFR staining intensity tended to be associated with non-pathological complete remission after preoperative radiotherapy for Stage II OTSCC. No correlation was found between EGFR status and survival. EGFR FISH results were significantly ( P = 0.003) higher in more advanced tumours (Stages II, III and IV) than in the tumours in Stage I. Non-smokers exhibited a significantly higher EGFR gene copy number and protein overexpression in Stages I and II OTSCC than smokers ( P = 0.001, P = 0.009). In conclusion, EGFR was found to be overexpressed in all OTSCCs making this cancer type interesting for exploring new therapeutic agents targeting the EGFR receptor.

An overview of the epidermal growth factor receptor fluorescence in situ hybridisation challenge in tumour pathology

Epidermal growth factor receptor (EGFR) is deregulated in a variety of solid malignant tumours. Due to the availability of specific targeted therapies, the request to evaluate EGFR in neoplastic tissues...

Effect of antityrosyne kinase agents on in vitro tumor cell proliferation induced by wound drainage fluids (WDFs) of head and neck cancer (HNSCC) patients

6077 Background: Recurrence of resected HNSCC is due to outgrowth of unrecognized residual cells, and/or to second field tumors. Epidermal growth factor receptor (EGFR) is overexpressed in HNSCC and in the mucosa surrounding the tumor. Evidence indicates that processes triggered by surgery stimulate recurrence of EGFR family overexpressing tumors. Methods: Tumors collected from 33 resected HNSCC patients were characterized for EGFR expression-activation using EGFR-DAKO Kit, EGFR antibody (528:sc- 120, Santa Cruz) and anti-phosphotyrosine antibody (clone 4G10, Upstate) for immunohistochemistry, immunoprecipitation/Western blott. EGFR FISH was performed on 2 tumors and surrounding dysplastic areas using the LSI EGFR/CEP 7 probe. WDFs at 24–48 hours after surgery of the 33 HNSCC patients and as control, from 23 plastic/reconstructive interventions were collected. WDFs were employed for stimulating the proliferation of two EGFR overexpressing squamous carcinoma cell lines (SCCLs) in which FISH detected gene amplification (A431) and three EGFR gene copies (CAL27). Cell proliferation induced by WDFs was measured spectrophotometrically with sulphorodamine B staining. Results: EGFR overexpression/activation was demonstrated in cancers, dysplasia, metaplasia and normal mucosa surrounding the tumors. In 2 pts both dysplasia and the tumor shared an increased EGFR gene copy number. A significant higher presence of EGFR mRNA in the HNSCC WDFs, as compared to plastic WDFs was observed. Both types of WDFs contain EGF-like growth factors that induce EGFR- mediated proliferation of SCCLs that was similarly inhibited by anti EGF antibody, gefitinib and lapatinib in A431 and CAL27. By contrast, cetuximab resulted to be more efficient to inhibit A431 growth with respect to CAL27. Conclusions: The results provide evidence that in operated HNSCC patients wound processes occur in presence of an EGFR activated enriched environment surrounding the excised tumor area, on which surgery-related release of EGF-like factors might promote cell proliferation leading to tumor recurrence. Clinical implications of our findings would suggest a role for preoperative and early maintenance of anti-EGFR treatment in HNSCC patients. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GlaxoSmithKline, Merck Serono GlaxoSmithKline, Merck Serono

EGFR, DCC, and K-RAS mutations as predictive factors for cetuximab sensitivity in metastatic colorectal cancer (mCRC)

4128 Background: Several studies suggest that increased gene copy number of the Epidermal Growth Factor Receptor (EGFR) or presence of mutations of the K-ras oncogene are associated with cetuximab response or resistance in patients (pts) with mCRC. In this setting data are not yet available regarding deleted in colorectal cancer (DCC), a tumor-suppressor gene located at chromosome 18q21, although DCC could theoretically modulate the EGFR/K-RAS pathway. This study aimed to identify biological predictors for sensitivity and resistance to cetuximab in pts with mCRC. Methods: Paraffin embedded tumor blocks from 31 mCRC pts treated with cetuximab-based regimens (Table 1) were retrospectively analyzed for EGFR (immunohistochemical staining - IHC and fluorescence in situ hybridization - FISH), DCC (Fragment analysis), and K-RAS (exon 12 mutation). Results: EGFR FISH positive patients (n=18), defined as ratio EGFR/nucleus =3, had a significantly higher time to progression -TTP (6.2 versus 3.2 months, p = 0.003) than EGFR FISH negative (n=13). The concordance rate of IHC-positive cases with cases positive for EGFR gene amplification by FISH was 55% (17/31) but EGFR expression assessed by IHC was not associated with any clinical end-point. Allelic losses (LOH) at the DCC locus were observed in 45% of pts. The median TTP was 6.6 months in pts without LOH and 2.7 months in pts with DCC-LOH (log-rank test, p =.04). Compared to pts with wild type K-RAS (n=21), K-RAS mutation carriers (n=10) had a significantly lower objective response rate (PR + SD, 3% versus 42%, p = 0.02), shorter TTP (2.3 versus 6.6 months, p = 0.06) and shorter survival which was not statistically significant (p = 0.2). Multivariate analysis showed that TTP was not correlated with treatment lines, sex, age and objective response rate. Conclusions: This study suggests that there is a significant benefit in TTP for EGFR FISH positive patients treated with cetuximab. DCC allelic losses could predict early resistance to cetuximab therapy and K-RAS mutation analysis identifies a group of pts with the lowest chance of benefiting from therapy. No significant financial relationships to disclose.

A phase II randomized selection trial evaluating concurrent chemotherapy plus cetuximab or chemotherapy followed by cetuximab in patients with advanced non-small cell lung cancer (NSCLC): Final report of SWOG 0342

7545 Background: Randomized clinical trials have failed to show a survival benefit for small molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors plus chemotherapy in unselected patients with metastatic NSCLC. In contrast, pilot trials of EGFR targeted antibodies plus chemotherapy have suggested enhanced anti-tumor activity. This randomized phase II selection trial was designed to select a cetuximab-chemotherapy regimen for future evaluation in a phase III setting. Methods: Untreated patients (pts) with advanced stage NSCLC were randomized to receive paclitaxel (P) 225 mg/m2 and carboplatin (Cb) AUC=6 every 3 weeks plus concurrent cetuximab (C) 400 mg/m2 loading dose followed by 250 mg/m2, weekly for 4 cycles followed by maintenance C or sequential PCb for 4 cycles followed by C. Treatment was continued until disease progression. Eligible patients were required to have stage IIIB (pleural effusion) or IV (without brain metastases) disease, a performance status of 0–1 and adequate organ function. The primary endpoint was overall survival. The regimen with superior median survival would be considered for further evaluation provided it met a 10-month minimum. Given a true hazard ratio of 1.3, the probability of correctly choosing the superior arm would be &gt; 90%. Results: From July 2004 to June 2005, 242 eligible pts were enrolled onto the study, Final results are described below: Conclusions: Both regimens met efficacy criterion for continued evaluation though the concurrent regimen of PCb + C, had numerically higher survival, and was chosen for further study. Some toxicities were significantly increased with concurrent therapy. A phase II trial of PCb + Cetuximab + Bevacizumab is ongoing (SWOG 0536) in anticipation of a phase III trial. Molecular correlative studies of the EGFR signaling pathway including EGFR IHC, FISH and mutation analysis are underway. No significant financial relationships to disclose. [Table: see text]

“Who Should Receive Epidermal Growth Factor Receptor Inhibitors for Non-Small Cell Lung Cancer and When?”

Inhibition of the epidermal growth factor receptor (EGFR) pathway in non-small cell lung cancer (NSCLC) is an exciting and rapidly evolving field. Erlotinib and gefitinib, two tyrosine kinase inhibitors (TKIs) of EGFR, have demonstrated activity in advanced NSCLC in the second- and third-line settings. Subset analyses of phase II and phase III clinical trials lead to the recognition that these two agents had more activity in certain subsets of NSCLC patients including never smokers, people of Asian descent and patients with EGFR FISH-positive or mutation-positive tumors. In particular, never smokers had statistically significant improvements in survival with either erlotinib or gefitinib therapy. Patients with EGFR FISH- or mutation-positive tumors had improved response rates to TKI therapy while those with KRAS mutant tumors did not derive any benefit. In the BR.21 trial treatment with erlotinib resulted in statistically significant improvements in overall survival and quality of life. Thus, while the question of who should receive EGFR TKI therapy is still not completely answered, all patients should be considered for erlotinib therapy in the second- or third-line setting. In daily clinical practice, there is currently no data to support the use of EGFR mutation or FISH status in this decision making process. Prospective trials are ongoing to determine which patient and tumor characteristics are predictive of a clinical benefit from TKI therapy.

Combination of EGFR gene copy number and protein expression predicts outcome for advanced non-small-cell lung cancer patients treated with gefitinib

Biological markers for optimal selection of patient to epidermal growth factor receptor (EGFR)-targeted therapies are not established in advanced non-small-cell lung cancer (NSCLC). EGFR/HER2 gene copy number by FISH, EGFR protein and pAKT expression by immunohistochemistry (IHC) and EGFR and KRAS mutations were tested in 204 gefitinib-treated NSCLC patients. Increased EGFR and HER2 gene copy number (FISH+), EGFR protein overexpression (IHC+), EGFR mutations and pAKT overexpression were all associated with significantly higher response rates (33%, 29%, 22%, 39% and 20% respectively). EGFR FISH+ (32%) and IHC+ (61%) correlated with improved survival, while EGFR mutations (27%), KRAS mutations (26%) and pAKT expression (69%) did not. In multivariate survival analysis EGFR FISH and IHC were independent predictive markers. EGFR FISH+/IHC+ patients (23%) had a median survival of 21 months versus 6 months for double-negative patients (30%). Combination of EGFR FISH and IHC is effective predictor for benefit from gefitinib. Patients with double-negative results are unlikely to benefit in western NSCLC populations.

Clinical and biological effects of gefitinib in EGFR FISH positive/phospho-akt positive or never smoker non-small cell lung cancer (NSCLC): Preliminary results of the ONCOBELL trial

7023 Background: Effectiveness of gefitinib, a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR), in never smokers and/or EGFR FISH positive NSCLC patients has been evaluated only in retrospective studies and few data exist on the biological effects of gefitinib therapy in tumor samples. Methods: The ONCOBELL trial is a multicenter prospective phase II study designed to include 39 patients EGFR FISH positive/phospho-Akt (p-AKT) positive and/or never smokers with advanced or metastatic NSCLC. Gefitinib is given at the daily dose of 250 mg. When feasible, primary tumor biopsy was repeated immediately before therapy, during therapy and at progression. Specimens were evaluated for EGFR and HER2 gene copy number by FISH, EGFR gene mutation by DNA sequencing, and EGFR and p-AKT protein expression by immunohistochemistry. Results: Data from the first 28 enrolled patients are available: median age was 60 years (range, 43–80); male/female: 7/21; stage IIIB/IV: 2/26; ECOG PS 0/1/2: 18/6/4; 25 patients had a diagnosis of adenocarcinoma and/or bronchioloalveolar carcinoma; 13 patients had brain metastases, and in 10 cases gefitinib was offered as first-line therapy. Twenty-four patients were never smokers and 19 were EGFR FISH positive. Overall response rate was 54%, including 1 complete and 14 partial responses. Median time to progression was 6.45 months, and median survival was not reached. Response to therapy was not influenced by previous therapies or presence of brain metastases. Toxicity was mild, and consisted in grade 1–2 skin rash (68%) and diarrhea (57%). One patient developed grade 3 diarrhea and another patient was hospitalized for acute interstitial lung disease. In patients exposed to previous chemotherapy, no difference in EGFR or HER2 FISH results were observed in specimens collected at the time of original diagnosis compared to specimens collected immediately before gefitinib therapy. In 14 cases primary tumor biopsy was repeated during gefitinib therapy. Conclusions: These preliminary data indicate that gefitinib is highly effective in patients with trial characteristics. Clinical and biological data for the entire cohort will be available in June 2006. No significant financial relationships to disclose.