Epidermal Growth Factor Receptor Fluorescence In Situ Hybridization Research Articles

Immunohistochemical Expression (IHC) and Fluorescence In Situ Hybridization (FISH) of EGFR in Colorectal Carcinoma

Adenocarcinoma of the colon is the most common malignancy of the gastrointestinal tract and is a major cause of morbidity and mortality worldwide. Epidermal growth factor receptor (EGFR) is also known as HER-1 or erb-B1. The beginning of EGF to EGFR produces a biological signal to the cell that initiates several functions that promote tumor growth, including cell invasion and metastasis, repair and new blood vessel formation. Thus, EGFR is recognized as an important player in colorectal cancer (CRC) initiation and progression. In this study, total 61 cases of colorectal carcinoma were included and histological grading, immuno-histochemical (IHC) expression and fluorescence in situ hybridization of EGFR were conducted. For the grading, 11/61(18%) cases were well differentiated, 38/61(62%) cases were moderately differentiated and 12/61(20%) were poorly differentiated colorectal carcinoma. EGFR IHC immune expression was positive in 50/61(82%) cases and negative in 11/61(18%) cases. All 11 cases of well differentiated cases gave EGFR IHC positive immunoreaction. Among the 38 cases of moderately differentiated adenocarcinoma, 30 cases showed EGFR IHC positivity and 8 cases gave no reaction. Nine out of 12 cases of poorly differentiated adenocarcinoma showed EGFR IHC positive and 3 cases gave no reaction. Half (10/20, 50%) of the EGFR IHC highly positive cases showed FISH positive and other half cases give FISH negative reaction. Detection of EGFR is mainly for anti-EGFR targeted therapy. Therefore, this study aids in selection of patients for anti-EGFR targeted therapy and helpful in treatment options and disease management. Moreover, EGFR FISH can be tested together with KRAS mutation and can predict the treatment response and the disease outcome.

Financially effective test algorithm to identify an aggressive, EGFR-amplified variant of IDH-wildtype, lower-grade diffuse glioma.

Update 3 of the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) recognizes amplification of epidermal growth factor receptor (EGFR) as one important aberration in diffuse gliomas (World Health Organization [WHO] grade II/III). While these recommendations endorse testing, a cost-effective, clinically relevant testing paradigm is currently lacking. Here, we use real-world clinical data to propose a financially effective diagnostic test algorithm in the context of new guidelines. To determine the prevalence, distribution, neuroradiographic features (Visually Accessible REMBRANDT Images [VASARI]), and prognostic relevance of EGFR amplification in lower-grade gliomas, we assembled a consecutive series of diffuse gliomas. For validation we included publicly available data from The Cancer Genome Atlas. For a cost-utility analysis we compared combined EGFR and isocitrate dehydrogenase (IDH) testing, EGFR testing based on IDH results, and no EGFR testing. In n = 71 WHO grade II/III gliomas, we identified EGFR amplification in 28.2%. With one exception, all EGFR amplifications occurred in IDH-wildtype gliomas. Comparison of overall survival showed that EGFR amplification denotes a significantly more aggressive subset of tumors (P < 0.0001, log-rank). The radiologic phenotype in the EGFR-amplified tumors includes diffusion restriction (15%, P = 0.02), >5% tumor contrast enhancement (75%, P = 0.016), and mild (not avid) enhancement (P = 0.016). The proposed testing algorithm reserves EGFR fluorescence in situ hybridization (FISH) testing for IDH-wildtype cases. Implementation would result in ~37.9% cost reduction at our institution, or about $1.3-4 million nationally. EGFR-amplified diffuse gliomas are "glioblastoma-like" in their behavior and may represent undersampled glioblastomas, or subsets of IDH-wildtype diffuse gliomas with inherently aggressive biology. EGFR FISH after IDH testing is a financially effective and clinically relevant test algorithm for routine clinical practice.

PATH-06. A NEW DIAGNOSTIC ASSAY FOR THE DETECTION OF EGFR AMPLIFICATION IN GLIOBLASTOMA TISSUE SPECIMENS

Glioblastoma (GBM) is the most common malignant brain tumor among adults with poor prognosis. Epidermal Growth Factor Receptor (EGFR) gene is found to be amplified in approximately 50% of GBM tumors. Drugs that target amplified EGFR are being evaluated in the clinical trials for the treatment of GBM patients. A new Vysis EGFR FISH (Fluorescence in situ Hybridization) assay was developed to detect amplification of the EGFR gene in glioblastoma tissue samples. The assay is comprised of two DNA probes labeled with spectrally distinct fluorophores: orange EGFR probe that hybridizes to 7p11.2-7p12 region, and green CEP 7 probe that hybridizes to a centromere of chromosome 7. Analytical sensitivity and specificity of the probes was tested on 100 metaphase spreads from 5 donors and was shown to be 100%. The assay conditions were optimized in guardbanding studies using universal reagents kits with both manual and automated VP 2000 processing methods. In feasibility study the assay demonstrated 100% (142/142) success rate for manual and 99.3% (141/142) for automated VP 2000 slide processing methods. Analytical reproducibility showed no significant differences between reviewers, slides and laboratories. The positive percent agreement for EGFR amplified specimens was 100% (480/480); negative percent agreement for non-amplified specimens was 99.8% (479/480), and overall percent agreement was 99.9% (959/960). Vysis EGFR CDx FISH Kit demonstrated high photostability, in-use stability, and real time stability for at least 19 months. Stability of FFPE tissue slides was demonstrated that allows using tissue blocks and tissue sections archived for up to 32 months without loss of performance. Fully characterized Vysis EGFR FISH assay proved to be an effective tool in detecting EGFR gene amplification in GBM FFPE tumor tissue with manual and automated assay formats. Vysis EGFR CDx FISH Kit* is being validated in the ongoing clinical trials. *In Development and not Commercially Available

OA23.01 Anti-EGFR Monoclonal Antibodies plus Chemotherapy in the First-Line Treatment of Advanced NSCLC: A Meta-Analysis

Monoclonal Antibodies (mAbs) against the Epidermal Growth Factor Receptor (EGFR) in association with platinum-based doublet chemotherapy have emerged as a potential first-line treatment option for advanced non-small cell lung cancer (NSCLC). This study was conducted to systematically review available data and evaluate the efficacy and toxicity of anti-EGFR mAbs plus chemotherapy vs chemotherapy alone for advanced NSCLC. We carried out a search on network databases and oncology conference abstracts for studies between 1990 and January 2016. Only prospective randomized clinical trials were included. Primary endpoints were overall survival (OS) and toxicity frequency. Secondary endpoints were progression-free survival (PFS) and overall response rate (ORR). Subgroup analysis was performed assessing histological subtypes, EGFR protein expression by immunohistochemistry (IHC), EGFR gene copy number by fluorescence in-situ hybridization (FISH), EGFR mutation status, and smoking status. Seven studies (2 with necitumumab and 5 with cetuximab) were included with 5,057 patients. Compared to chemotherapy alone, significant benefits were demonstrated by the addition of anti-EGFR mAb to chemotherapy in OS (HR 0.90; 95%CI 0.84-0.95), PFS (HR 0.93; 95%CI 0.87-0.98), and ORR (OR 1.27; 95%CI 1.06-1.51). In subgroup analyses, the association of anti-EGFR mAb was associated with improved OS among patients with squamous histology (HR 0.84; 95%CI 0.76-0.92), tumours with high EGFR expression by IHC (HR 0.83; 95%CI 0.70-0.98), and smokers (HR 0.87; 95%CI 0.79-0.96). Patients with squamous histology and high EGFR expression by IHC achieved the highest benefit with the association (HR 0.71; 95%CI 0.59-0.86). The OS with the association also seemed to be higher in EGFR FISH negative and in EGFR wild-type tumours, but without statistical significance. Chemotherapy plus anti-EGFR mAb caused more grade 3 or worse adverse events (OR 1.73; 95%CI 1.50-2.00), remarkedly these known to be associated with anti-EGFR therapy, such as acne-like rash (OR 34.13; 95%CI 16.40-71.00) and hypomagnesemia (OR 6.23; 95%CI 3.04-12.77). Anti-EGFR therapy plus platinum-based doublet chemotherapy as first-line treatment demonstrated significant efficacy benefits with acceptable toxicity for advanced NSCLC. This benefit is more expressive among squamous histology with high EGFR expression. EGFR protein expression by IHC seems to be a predictive marker for survival in the association group. Further research is needed to corroborate these findings.

Détection de mutations de BRAF, dans le plasma de patients atteints de mélanome métastatique

Despite growing evidence that epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation analysis is the most reliable predictor of the lung carcinoma response to EGFR-targeted therapies, there is still discussion about the role of EGFR fluorescence in situ hybridization (FISH). Studies focusing on EGFR FISH as a predictor of response to EGFR-targeted therapies mostly focused on the relationship between EGFR FISH and EGFR mutations. The incidence of KRAS and V-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations in EGFR-amplified or EGFR FISH-positive lung adenocarcinomas remains unknown. The aim of this study was to prospectively characterize the incidence of KRAS and BRAF mutations in EGFR FISH-positive surgically treated lung adenocarcinomas. Of 386 primary lung adenocarcinomas, 77 (20%) were EGFR FISH positive by University of Colorado criteria. The incidence of KRAS mutations in EGFR FISH-positive lung adenocarcinomas was 23% and was not significantly different from the incidence of KRAS mutations in EGFR FISH-negative subsets of adenocarcinoma (32%). A higher mean ratio between EGFR and chromosome 7 enumeration probe (EGFR/CEP7) was observed in EGFR-mutated tumors when compared to cases with KRAS mutation (13 versus 4.5, respectively). Our results showed significant number of EGFR FISH positive/amplified lung adenocarcinomas harboring KRAS mutation. It appears that an increase in EGFR/CEP7 ratio to cutoff point of 4.5 may distinguish between coexisting EGFR (FISH ratio of >5) or KRAS (FISH ratio of 2 to 5) mutations. Observations presented here indicate that the patient selection for EGFR-targeted therapies should include EGFR and KRAS mutational analysis, probably complemented by EGFR FISH studies.

High EGFR copy number predicts benefits from tyrosine kinase inhibitor treatment for non-small cell lung cancer patients with wild-type EGFR.

BackgroundThis study was designed to determine whether advanced non-small-cell lung cancer (NSCLC) patients with high copy number of epidermal growth factor receptor (EGFR) can benefit from treatment with EGFR-tyrosine kinase inhibitors (TKIs).MethodsEGFR gene copy number was assessed by fluorescence in situ hybridization (FISH) and EGFR mutations was tested using Luminex xTAG technology in 502 TKI-treated NSCLC patients. The association between both biomarkers and clinical benefit from EGFR-TKI were analyzed.ResultsEGFR FISH + and EGFR mutations were significantly associated with higher response rates (37.2% and 43.7%, respectively), superior progression-free survival (PFS) (FISH+, 11.2 months; hazard ratio [HR], 0.51; 95% CI, 0.42 to 0.62; p < 0.001; mutation+, 11.7 months; HR, 0.37; 95% CI, 0.31 to 0.45; p < 0.001) and overall survival (OS) (FISH+, 30.2 months; HR, 0.51; 95% CI, 0.40 to 0.65; p < 0.001; mutation+, 30.2 months; HR, 0.45; 95% CI, 0.36 to 0.58; p < 0.001). In patients with wild-type EGFR, EGFR FISH + correlated with longer PFS than EGFR FISH- status (4.4 months vs. 2.0 months; HR, 0.56; 95% CI, 0.41 to 0.75; p < 0.001), so did amplification (5.0 months vs. 2.0 months; HR, 0.43; 95% CI, 0.24 to 0.76; p = 0.003). However, FISH + had no association with improved PFS in EGFR-mutated patients (HR, 0.77; 95% CI, 0.57 to 1.03; p = 0.076).ConclusionsA combined analysis of EGFR FISH and mutation is an effective predictor of EGFR-TKI therapy. Specifically, a high EGFR copy number may predict benefit from TKIs treatment for NSCLC patients with wild-type EGFR.

Concomitant high gene copy number and protein overexpression of IGF1R and EGFR negatively affect disease-free survival of surgically resected non-small-cell-lung cancer patients.

Insulin-like growth factor 1 receptor (IGF1R) represents a novel molecular target in non-small-cell-lung cancer (NSCLC). IGF1R and epidermal growth factor receptor (EGFR) activation are essential to mediate tumor cell survival, proliferation, and invasion. This study investigates the prognostic role of IGF1R and EGFR in surgically resected NSCLC. IGF1R and EGFR copy number gain (CNG) were tested by fluorescence in situ hybridization (FISH) and protein expression by immunohistochemistry (IHC) in 125 stage I-II-IIIA NSCLC patients. Fourty-six tumors (40.3%) were IGF1R FISH-positive (FISH+), and 76 (67.2%) were EGFR FISH+. Tumors with concomitant IGF1R/EGFR FISH+ were observed in 34 cases (30.1%). IGF1R and EGFR FISH+ were associated with SCC histology (p = 0.01 and p = 0.04, respectively). IGF1R and EGFR protein over-expression (IHC+) were detected in 45 (36.0%) and 69 (55.2%) cases, respectively. Tumors with concomitant IGF1R/EGFR IHC+ were detected in 31 (24.8%) patients. IGF1R/EGFR FISH+ and IGF1R/EGFR IHC+ were significantly associated (χ(2) = 4.02, p = 0.04). Patients with IGF1R/EGFR FISH+ and IGF1R/EGFR IHC+ were associated with shorter disease-free survival (DFS) (p = 0.05 and p = 0.05, respectively). Patients with concomitant IGF1R/EGFR FISH+/IHC+ had a worse DFS and overall survival (p = 0.005 and p = 0.01, respectively). The multivariate model confirmed that IGF1R/EGFR FISH+/IHC+ (hazard ratio (HR), 4.08; p = 0.01) and tumor stage (II-III vs I) (HR, 4.77; p = 0.003) were significantly associated with worse DFS. IGF1R/EGFR FISH+ correlates with IGF1R/EGFR IHC+. IGF1R/EGFR FISH+/IHC+ is an independent negative prognostic factor for DFS in early NSCLC. These features may have important implications for future anti-IGF1R therapeutic approaches.

Relationship between epidermal growth factor receptor gene mutation and copy number in Chinese patients with non-small cell lung cancer

Epidermal growth factor receptor (EGFR) gene mutation and copy number are useful predictive markers that guide the selection of non-small cell lung cancer (NSCLC) patients for EGFR-targeting therapy. This study aimed to investigate the correlation between EGFR gene mutation and copy number and clinicopathologic characteristics of Chinese patients with NSCLC. NSCLC specimens collected from 205 patients between November 2009 and January 2011 were selected to detect EGFR gene mutations with real-time polymerase chain reaction (RT-PCR) and to detect EGFR gene copy number with fluorescence in situ hybridization (FISH). EGFR mutations primarily occurred in females, non-smokers, and patients with adenocarinomas (all P < 0.001). Tissues from 128 (62%) patients were FISH-positive for EGFR, including 37 (18%) with gene amplification and 91 (44%) with high polysomy. EGFR gene mutation was correlated with FISH-positive status (R = 0.340, P < 0.001). Multivariate analysis showed that not smoking (OR = 5.910, 95% CI = 2.363–14.779, P < 0.001) and having adenocarcinoma (OR = 0.122, 95% CI = 0.026–0.581, P = 0.008) were favorable factors for EGFR gene mutation. These results show a high frequency of EGFR FISH positivity in NSCLC tissues from Chinese patients and a significant relevance between EGFR gene mutations and FISH-positive status. Among the FISH-positive samples, EGFR gene mutation occurred more frequently in samples with gene amplification compared to those with high polysomy, suggesting that EGFR mutation and gene amplification should be used as clinical decision parameters to predict response to EGFR-targeting therapy.

EGFR FISH analysis in colorectal cancer as a tool in selecting patients for antiEGFR monoclonal antibodies therapy

The recent introduction of targeted therapies in the treatment of patients with metastatic colorectal cancer (mCRC) not only improved efficacy but also toxicity and costs of the therapy, therefore requiring the identification of decision-making tools to select patients who are likely to benefit from them. By now, several studies have demonstrated an association between epidermal growth factor receptor (EGFR) non-increased gene copy number, evaluated by fluorescence in situ hybridization (FISH), and resistance to the treatment with antiEGFR monoclonal antibodies (moAbs) in patients with mCRC. However, the reproducibility of data by standardization of methods still remains an obstacle to be faced for clinical application of the test. We present a review of studies pertaining EGFR FISH analysis as a predictive test of clinical outcome to the treatment with antiEGFR moAbs in mCRC to point out the existing knowledge and the open questions about this issue.

Insulin-like growth factor receptor-1 (IGF1R) and epidermal growth factor receptor (EGFR) amplification and expression in surgically resected NSCLC.

10519 Background: IGF1R represents a novel molecular target in non-small cell lung cancer (NSCLC). IGF1R and EGFR activation is essential to mediate tumor cell survival, proliferation and invasion. This study investigates the prognostic role of IGF1R and EGFR copy number gain (CNG) by fluorescence in situ hybridization (FISH) and protein overexpression by immunohistochemistry (IHC) in surgically resected NSCLC. Methods: 114 NSCLC patients were evaluated; median age was 66y (range 40–84), Male/Female:96/18; squamous (SCC)/adeno/BAC/other:59/34/9/12; smoker/never smoker:105/9, and stage I/II/III:71/18/25. IGF1R and EGFR FISH were tested by customized and commercial probes, respectively; positive specimens showed gene amplification or polysomy (≥4 copies in ≥10% of tumor cells). IGF1R and EGFR protein expression were evaluated using mouse antibodies (clones 24-31 and 3147, respectively); overexpression was defined by ≥10% positive cells. Kaplan-Meier estimates of survival and time to recurrence were calculated for clinical and biologic variables using Cox model for multivariate analysis. Results: 46 tumors (40%) were IGF1R FISH+ and 76 (77%) were EGFR FISH+. IGF1R FISH+ was associated with EGFR FISH+ (p=0.03) and co-amplification was observed in 34 cases (30%). IGF1R and EGFR FISH+ were associated with SCC (p=0.01 and p=0.05, respectively). IGF1R and EGFR overexpression was detected in 36% and 55% of NSCLC patients and co-expression was detected in 25%. Co-amplification and co-expression of both receptors were significantly associated (p=0.045). IGF1R and EGFR co-amplification and co-expression associated with shorter disease free survival (DFS; p=0.05, p=0.05 respectively), also at multivariate analysis adjusting for stage (p=0.0002). Conclusions: IGF1R and EGFR are frequently co-amplified in NSCLC and CNG correlates with protein overexpression. Both co-amplification and co-expression of IGF1R and EGFR predicts shorter DFS. These results provide a strong rational for targeting simultaneously EGFR and IGF1R in clinical trials for NSCLC. We thank the Italian Association for Cancer Research (AIRC) for supporting the study (AF Fellowship).

Standardization of EGFR FISH in colorectal cancer: Results of an international, interlaboratory reproducibility ring study.

3597 Background: Epidermal growth factor receptor (EGFR) gene copy number (GCN) evaluated by fluorescence in situ hybridization (FISH) has been shown to discriminate among KRAS wild type patients those with better outcome to EGFR-targeted therapy in metastatic colorectal cancer (CRC), further enhancing selection of patients. Nevertheless, enumeration of gene copies in CRC is challenging and the lack of analytical standardisation has limited incorporation of the test into the clinical practice. Methods: A ring study was designed in order to assess EGFR FISH inter-laboratory consensus among 5 pathology reference centres located in Belgium, Italy, Switzerland, and United States. A set of 12 CRC samples circulated among laboratories and samples were scored according to commonly agreed guidelines. Reproducibility was quantified using the standard error of measurement (SEM). Results: A SEM of 0.865 and a within-subject coefficient of variation (WSCV) of 26.8% for mean EGFR gene per nuclei and a SEM of 0.235 and a WSCV of 19.4% for the mean EGFR gene/CEP7 ratio were observed, indicating low level of agreement. Measurement of the fraction of cells displaying chromosome 7 polysomy within tumor specimens was also performed, showing WSCV of 46.6%, 34.0% and 51.0% for % of cells displaying ≤2, ≥3 and ≥4 EGFR signals, respectively. Among different slides of the same specimen, the WSCV was 6.1% for mean EGFR gene per nuclei and 3.9% for mean of EGFR gene/CEP7 ratios and 10.5%, 12.4% and 24.0% for the % of cells displaying ≤2, ≥3 and ≥4 EGFR signals, respectively. Conclusions: Molecular diagnosis of EGFR GCN by FISH varied largely among experienced international pathology centres, with fluctuations covering the whole range of proposed cut-offs of predictive usefulness from literature. Definition of a detailed scoring system and implementation of a comprehensive training program for molecular diagnostic laboratories are therefore necessary before including the test into clinical practice as a standard assay for predicting outcome to EGFR-targeted therapies in metastatic CRC.

EGFR Fluorescence In situ Hybridization Pattern of Chromosome 7 Disomy Predicts Resistance to Cetuximab in KRAS Wild-type Metastatic Colorectal Cancer Patients

Metastatic colorectal cancer patients with low epidermal growth factor receptor (EGFR) gene copy number are unlikely to respond to anti-EGFR monoclonal antibody (mAb) treatment. The objective of this study was to investigate EGFR fluorescence in situ hybridization (FISH) patterns of chromosome 7 disomy with efficacy of cetuximab therapy in metastatic colorectal cancer patients. We detected the EGFR FISH patterns and KRAS status in 74 tumors from cetuximab-treated metastatic colorectal cancer patients and analyzed with response rate (RR) and progression-free survival (PFS). One of the 16 (6.25%) patients with chromosome 7 homogeneous disomy (defined as FISH negative) had objective response to cetuximab. A total of 53(76.8%) patients with chromosome 7 pattern of variable ratios of disomy versus polysomy (defined as FISH positive) had a significantly higher RR (37.7% versus 6.25%; P = 0.01), a trend towards longer PFS (4.5 versus 2.9 months; P = 0.07). Among 54 KRAS wild-type patients, EGFR FISH-positive patients had significantly higher RR (51.3% versus 9%; P = 0.01) and longer PFS (5.0 versus 2.3 months; P = 0.02) than EGFR FISH-negative patients. However, among 20 KRAS mutant-type patients, there was no difference in RR (0% versus 0%) and PFS (2.5 versus 3.8 months; P = 0.51) between EGFR FISH-positive and -negative patients. Our results show firstly that patients with EGFR FISH pattern of chromosome 7 disomy have a very low chance to benefit from cetuximab-based therapy. EGFR FISH pattern of chromosome 7 disomy may be as a negative predicative factor for cetuximab response in KRAS wild-type metastatic colorectal cancer patients.

Increased ALK Gene Copy Number and Amplification are Frequent in Non-small Cell Lung Cancer

Translocation of the anaplastic lymphoma kinase (ALK) gene is involved in the tumorigenesis of a subset of non-small cell lung carcinomas (NSCLCs) and identifies patients sensitive to ALK inhibitors. ALK copy number changes and amplification, which plays an oncogenic role in tumors such as neuroblastoma, are poorly characterized in NSCLC. We aimed to study the prevalence of ALK copy number changes and their correlation to ALK protein expression, epidermal growth factor receptor (EGFR) status, and clinicopathological data in patients with NSCLC. ALK status was evaluated by fluorescence in situ hybridization (FISH). Specimens with ALK translocation were studied for echinoderm microtubule-associated protein-like 4 (EML4), KIF5B, and TFG status. ALK expression was assessed by immunohistochemistry. EGFR gene and protein status were evaluated in adenocarcinomas. Survival analysis was performed. One hundred seven NSCLC cases were evaluated. There were two cases of EML4-ALK translocation and one with an atypical translocation of ALK. Both cases of EML4-ALK translocation had ALK protein expression, whereas in the rest, ALK was undetected. Eleven cases (10%) exhibited ALK amplification and 68 (63%) copy number gains. There was an association between ALK amplification and EGFR FISH positivity (p < 0.0001) but not with prognosis. In conclusion, EML4-ALK translocation is a rare event in NSCLC. The study reveals a significant frequency of ALK amplification and its association with EGFR FISH positivity in lung adenocarcinomas. Based on these findings, a potential role of ALK amplification in the response to ALK inhibitors alone or combined with EGFR inhibitors in NSCLC merits further studies.

Molecular predictors of outcome in a phase 3 study of gemcitabine and erlotinib therapy in patients with advanced pancreatic cancer

National Cancer Institute of Canada Clinical Trials Group PA.3 (NCIC CTG PA.3) was a phase 3 study (n = 569) that demonstrated benefits for overall survival and progression-free survival with the addition of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib to gemcitabine in patients with advanced pancreatic carcinoma (APC). Mutation status of the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and EGFR gene copy number (GCN) were evaluated as predictive markers in 26% of patients who had tumor samples available for analysis. KRAS mutation status was evaluated by direct sequencing of exon 2, and EGFR GCN was determined by fluorescence in situ hybridization (FISH) analysis. The results were correlated with survival, which was the primary endpoint of the trial. KRAS analysis was successful in 117 patients, and EGFR FISH analysis was successful in 107 patients. KRAS mutations were identified in 92 patients (78.6%), and EGFR amplification or high polysomy (FISH-positive results) was identified in 50 patients (46.7%). The hazard ratio of death between gemcitabine/erlotinib and gemcitabine/placebo was 0.66 (95% confidence interval [CI], 0.28-1.57) for patients with wild-type KRAS and 1.07 (95% CI, 0.68-1.66) for patients with mutant KRAS (P value for interaction = .38), and the hazard ratio was 0.6 (95% CI, 0.34-1.07) for FISH-negative patients and 0.90 (95% CI, 0.49-1.65) for FISH-positive patients (P value for interaction = .32). In a molecular subset analysis of patients from NCIC CTG PA.3, EGFR GCN and KRAS mutation status were not identified as markers predictive of a survival benefit from the combination of erlotinib with gemcitabine for the first-line treatment of APC.

Molecular Testing in Lung Carcinoma

The development of molecular targeted therapies with small-molecule inhibitors of the epidermal growth factor receptor (EGFR) has resulted in new therapeutic options for patients with advanced stage lung cancer. Certain clinical characteristics have been associated with an increased likelihood of response to EGFR tyrosine kinase inhibitors (TKIs; women, never smokers, East Asian ethnicity, and adenocarcinoma histology). Furthermore, several molecular events can also affect the efficacy of these therapies. Activating mutations in EGFR exons 18 through 21 (insert deletions in exon 19 and the single L858R point mutation in exon 21 being the most common) seem to be more reliable predictors of response than clinical features and are associated with increased response and survival after TKI therapy.1–3 In contrast, K- ras mutations, insertion mutations or T790M point mutations in exon 20 of EGFR , and MET amplification are negative predictors of response.4–6 These observations have resulted in numerous retrospective studies that correlate patient response to the molecular profile of lung adenocarcinomas. Unfortunately, different methodological approaches have been used for EGFR assessment, including DNA mutational analysis, fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) for gene copy number changes, and immunohistochemical analysis for protein expression. Not surprisingly, reported results are inconsistent, reflecting the lack of standardization of the method and interpretation criteria. In general, EGFR FISH and DNA mutation analyses are the 2 most extensively studied methods for selection of candidates for …

Relationship between epidermal growth factor receptor (EGFR) gene copy number, p16 status, and outcome in locally advanced squamous cell carcinoma of the head and neck (LASCCHN).

5528 Background: Human papilloma virus infection (HPV) is a powerful prognostic biomarker in LASCCHN and can be indirectly identified by p16 immunohistochemistry (IHC). Increased EGFR gene copy number has been reported to be a negative predictor of outcome in LASCCHN. In this study the relationship of p16 to EGFR gene copy number in patients treated with chemoradiation was assessed. Methods: Formalin-fixed paraffin embedded tissue was obtained from 110 patients with LASCCHN treated on three clinical trials with conventional chemoradiation or with a tirapazamine containing regimen. EGFR gene copy number was assessed by Fluorescence in situ hybridization (FISH) using LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probes (Vysis/Abbott). Tumors with high polysomy or amplification of EGFR were scored as positive. p16 was assessed by IHC using a validated antibody (Neomarkers). Markers were correlated with outcome i.e., failure-free survival (FFS) and overall survival (OS) using Cox regression, adjusting for trial. Results: Results for both EGFR FISH and p16 IHC were obtained in 97 patients. EGFR FISH was positive in 21/97 (22%) tumors including 12/66 (18%) oropharyngeal tumors. p16 was positive in 40/97 (41%) tumors including 36/66 (55%) oropharyngeal tumours. Only 1/40 p16 positive tumors were EGFR FISH positive compared with 20/57 p16 negative tumors (p<0.001). EGFR FISH positivity was associated with inferior FFS (HR 2.48 (95%CI 1.14 to 5.38), p = 0.022) and OS (HR 2.33 (95%CI 0.98 to 5.57), p = 0.057). p16 positivity was associated with improved FFS (HR 0.19 (95%CI 0.07 to 0.56), p = 0.002) and OS (HR 0.18 (95%CI 0.05 to 0.62), p = 0.006). On multivariable Cox regression analysis only p16 was independently predictive of outcome (p = 0.007 and 0.0014 for FFS and OS respectively). Conclusions: EGFR FISH positivity and p16 expression were found to be almost mutually exclusive in LASCCHN with only 1 of 97 tumors positive for both markers suggesting that increased EGFR copy number is largely restricted to HPV-negative LASCCHN. EGFR FISH and p16 were predictive of outcome in univariate analyses, but only p16 remained predictive on multivariable analysis. No significant financial relationships to disclose.

EGFR fluorescence in situ hybridization-positive lung adenocarcinoma: incidence of coexisting KRAS and BRAF mutations

Despite growing evidence that epidermal growth factor receptor (EGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation analysis is the most reliable predictor of the lung carcinoma response to EGFR-targeted therapies, there is still discussion about the role of EGFR fluorescence in situ hybridization (FISH). Studies focusing on EGFR FISH as a predictor of response to EGFR-targeted therapies mostly focused on the relationship between EGFR FISH and EGFR mutations. The incidence of KRAS and V-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations in EGFR-amplified or EGFR FISH-positive lung adenocarcinomas remains unknown. The aim of this study was to prospectively characterize the incidence of KRAS and BRAF mutations in EGFR FISH-positive surgically treated lung adenocarcinomas. Of 386 primary lung adenocarcinomas, 77 (20%) were EGFR FISH positive by University of Colorado criteria. The incidence of KRAS mutations in EGFR FISH-positive lung adenocarcinomas was 23% and was not significantly different from the incidence of KRAS mutations in EGFR FISH-negative subsets of adenocarcinoma (32%). A higher mean ratio between EGFR and chromosome 7 enumeration probe (EGFR/CEP7) was observed in EGFR-mutated tumors when compared to cases with KRAS mutation (13 versus 4.5, respectively). Our results showed significant number of EGFR FISH positive/amplified lung adenocarcinomas harboring KRAS mutation. It appears that an increase in EGFR/CEP7 ratio to cutoff point of 4.5 may distinguish between coexisting EGFR (FISH ratio of >5) or KRAS (FISH ratio of 2 to 5) mutations. Observations presented here indicate that the patient selection for EGFR-targeted therapies should include EGFR and KRAS mutational analysis, probably complemented by EGFR FISH studies.

9LBA A randomized phase III study comparing gefitinib with carboplatin (CBDCA) plus paclitaxel (TXL) for the first-line treatment of non-small cell lung cancer (NSCLC) with sensitive EGFR mutations: NEJ002 study

prognostic/predictive value of several biomarkers (BM): epidermal growth factor receptor (EGFR) immunohistochemistry (IHC), EGFR fluorescence in situ hybridization (FISH), and EGFR and Kras mutation status. Methods: 743 pts with advanced NSCLC whose disease had not progressed following 4 cycles of 1-line chemotherapy with B were randomized by the interim analysis data cut-off to receive B+E or B+P. B dosing was 15mg/kg every 3 weeks; E dosing was 150mg daily. Maintenance therapy continued until unacceptable toxicity or disease progression. Archival tumor tissue was collected for BM analysis. EGFR FISH and IHC analyses were performed using the PathVysion and PharmDx kits from Abbott-Vysis and DAKO. Analyses of EGFR tyrosine kinase mutations in exons 18−21 and Kras mutations in exons 2 and 3 were performed using DHPLC (denaturing high-performance liquid chromatography) and mutations were validated by sequencing. Results: Biomarker assay results were available for 49.4% of randomized patients. Their demographics were similar to the overall trial population. The PFS HRs for the BM defined subgroups are presented in the table.

EGFR FISH analysis in colorectal cancer as a tool in selecting patients for antiEGFR monoclonal antibodies therapy

The recent introduction of targeted therapies in the treatment of patients with metastatic colorectal cancer (mCRC) not only improved efficacy but also toxicity and costs of the therapy, therefore requiring the identification of decision-making tools to select patients who are likely to benefit from them. By now, several studies have demonstrated an association between epidermal growth factor receptor (EGFR) non-increased gene copy number, evaluated by fluorescence in situ hybridization (FISH), and resistance to the treatment with antiEGFR monoclonal antibodies (moAbs) in patients with mCRC. However, the reproducibility of data by standardization of methods still remains an obstacle to be faced for clinical application of the test. We present a review of studies pertaining EGFR FISH analysis as a predictive test of clinical outcome to the treatment with antiEGFR moAbs in mCRC to point out the existing knowledge and the open questions about this issue.

Assessment of mean EGFR gene copy number is a highly reproducible method for evaluating FISH in histological and cytological cancer specimens

The aims of this study were first, to systematically assess the inter-observer reproducibility of mean Epidermal Growth Factor Receptor (EGFR) gene copy number (MCN) in histological and cytological specimens from lung and non-lung cancers, second to compare the performance of this quantitative approach to the current Colorado criteria for the assessment of fluorescence in situ hybridization (FISH) positivity and third to develop a model to convert cytology into histology MCN. EGFR FISH analysis was performed on 170 histological and 153 cytological specimens. The MCN and Colorado criteria were assessed by two independent observers and agreement evaluated using Bland–Altman plots and kappa values. Conversion of cytology into histology MCN was tested on two randomized subgroups of specimens. Applying the Colorado criteria, agreement between observers was moderate with histology ( k = 0.5) and excellent with cytology ( k = 0.94). Positivity in histology versus cytology led to fair agreement ( k = 0.33). MCN was significantly greater in cytology compared to histology ( p < 0.001) with excellent inter-observer agreement in both sample types ( r = 0.99 and 0.89, respectively). The average difference in MCN between observers was −0.003 (95%CI: −0.05 to 0.05) and 0.008 (95%CI: −0.09 to 0.11) for cytology and histology, respectively. A reliable conversion model with an R 2-value of 0.91 in the validation subgroup ( p < 0.001) was obtained. The MCN is a reproducible scoring method for evaluating EGFR FISH in samples from lung and non-lung cancers. This quantitative scoring system may constitute an alternative to current methods of gene copy number assessment and will further help to optimize patient selection for targeted therapies.