Jordi Bertran-Alamillo

Screening for Epidermal Growth Factor Receptor Mutations in Lung Cancer

BACKGROUND Activating mutations in the epidermal growth factor receptor gene (EGFR) confer hypersensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in patients with advanced non-small-cell lung cancer. We evaluated the feasibility of large-scale screening for EGFR mutations in such patients and analyzed the association between the mutations and the outcome of erlotinib treatment. METHODS From April 2005 through November 2008, lung cancers from 2105 patients in 129 institutions in Spain were screened for EGFR mutations. The analysis was performed in a central laboratory. Patients with tumors carrying EGFR mutations were eligible for erlotinib treatment. RESULTS EGFR mutations were found in 350 of 2105 patients (16.6%). Mutations were more frequent in women (69.7%), in patients who had never smoked (66.6%), and in those with adenocarcinomas (80.9%) (P<0.001 for all comparisons). The mutations were deletions in exon 19 (62.2%) and L858R (37.8%). Median progression-free survival and overall survival for 217 patients who received erlotinib were 14 months and 27 months, respectively. The adjusted hazard ratios for the duration of progression-free survival were 2.94 for men (P<0.001); 1.92 for the presence of the L858R mutation, as compared with a deletion in exon 19 (P=0.02); and 1.68 for the presence of the L858R mutation in paired serum DNA, as compared with the absence of the mutation (P=0.02). The most common adverse events were mild rashes and diarrhea; grade 3 cutaneous toxic effects were recorded in 16 patients (7.4%) and grade 3 diarrhea in 8 patients (3.7%). CONCLUSIONS Large-scale screening of patients with lung cancer for EGFR mutations is feasible and can have a role in decisions about treatment.

Pretreatment EGFR T790M Mutation and BRCA1 mRNA Expression in Erlotinib-Treated Advanced Non–Small-Cell Lung Cancer Patients with EGFR Mutations

Purpose: Advanced non–small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) mutations (deletion in exon 19 or L858R) show an impressive progression-free survival of 14 months when treated with erlotinib. However, the presence of EGFR mutations can only imperfectly predict outcome. We hypothesized that progression-free survival could be influenced both by the pretreatment EGFR T790M mutation and by components of DNA repair pathways. Experimental Design: We assessed the T790M mutation in pretreatment diagnostic specimens from 129 erlotinib-treated advanced NSCLC patients with EGFR mutations. The expression of eight genes and two proteins involved in DNA repair and four receptor tyrosine kinases was also examined. Results: The EGFR T790M mutation was observed in 45 of 129 patients (35%). Progression-free survival was 12 months in patients with and 18 months in patients without the T790M mutation (P = 0.05). Progression-free survival was 27 months in patients with low BRCA1 mRNA levels, 18 months in those with intermediate levels, and 10 months in those with high levels (P = 0.02). In the multivariate analysis, the presence of the T790M mutation (HR, 4.35; P = 0.001), intermediate BRCA1 levels (HR, 8.19; P < 0.0001), and high BRCA1 levels (HR, 8.46; P < 0.0001) emerged as markers of shorter progression-free survival. Conclusions: Low BRCA1 levels neutralized the negative effect of the T790M mutation and were associated with longer progression-free survival to erlotinib. We advocate baseline assessment of the T790M mutation and BRCA1 expression to predict outcome and provide alternative individualized treatment to patients based on T790M mutations and BRCA1 expression. Clin Cancer Res; 17(5); 1–9. ©2011 AACR.

The Impact of EGFR T790M Mutations and BIM mRNA Expression on Outcome in Patients with EGFR-Mutant NSCLC Treated with Erlotinib or Chemotherapy in the Randomized Phase III EURTAC Trial

Purpose: Concomitant genetic alterations could account for transient clinical responses to tyrosine kinase inhibitors of the EGF receptor (EGFR) in patients harboring activating EGFR mutations. Experimental Design: We have evaluated the impact of pretreatment somatic EGFR T790M mutations, TP53 mutations, and Bcl-2 interacting mediator of cell death (BCL2L11, also known as BIM) mRNA expression in 95 patients with EGFR-mutant non–small-cell lung cancer (NSCLC) included in the EURTAC trial (trial registration: NCT00446225). Results: T790M mutations were detected in 65.26% of patients using our highly sensitive method based on laser microdissection and peptide-nucleic acid-clamping PCR, which can detect the mutation at an allelic dilution of 1 in 5,000. Progression-free survival (PFS) to erlotinib was 9.7 months for those with T790M mutations and 15.8 months for those without, whereas among patients receiving chemotherapy, it was 6 and 5.1 months, respectively (P < 0.0001). PFS to erlotinib was 12.9 months for those with high and 7.2 months for those with low/intermediate BCL2L11 expression levels, whereas among chemotherapy-treated patients, it was 5.8 and 5.5 months, respectively (P = 0.0003). Overall survival was 28.6 months for patients with high BCL2L11 expression and 22.1 months for those with low/intermediate BCL2L11 expression (P = 0.0364). Multivariate analyses showed that erlotinib was a marker of longer PFS (HR = 0.35; P = 0.0003), whereas high BCL2L11 expression was a marker of longer PFS (HR = 0.49; P = 0.0122) and overall survival (HR = 0.53; P = 0.0323). Conclusions: Low-level pretreatment T790M mutations can frequently be detected and can be used for customizing treatment with T790M-specific inhibitors. BCL2L11 mRNA expression is a biomarker of survival in EGFR-mutant NSCLC and can potentially be used for synthetic lethality therapies. Clin Cancer Res; 20(7); 2001–10. ©2014 AACR.

A Sensitive Method for Detecting EGFR Mutations in Non-small Cell Lung Cancer Samples with Few Tumor Cells

Background: Detection of epidermal growth factor receptor (EGFR) mutations in advanced non-small cell lung cancer (NSCLC) patients has relied on DNA purification from biopsies, amplification, and sequencing. However, the number of tumor cells in a sample is often insufficient for EGFR assessment. Methods: We prospectively screened 1380 NSCLC patients for EGFR mutations but found that 268 were not evaluable because of insufficient tumor tissue. We therefore developed and validated a method of detecting EGFR mutations in these samples. Tumor cells were microdissected into polymerase chain reaction buffer and amplified. EGFR mutations were detected by length analysis of fluorescently labeled polymerase chain reaction products and TaqMan assay. Results: We determined EGFR status in 217 (81%) of the 268 primary NSCLC samples not evaluable in our original study—fresh and paraffin-embedded with less than 150 cells. Exon 19 deletions were detected in 11.5% of patients and exon 21 L858R mutations in 5.5%. In addition, the exon 20 T790M mutation was detected in 6 of 15 (40%) patients at the time of progression to erlotinib. The primary, sensitive mutation was present in all tumor cells, whereas the T790M mutation was absent in some groups. Conclusions: The method presented here eliminates the need for DNA purification and allows for detection of EGFR mutations in samples containing as few as eight cancer cells.

Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer.

BackgroundImmunohistochemistry (IHC) with mutation-specific antibodies may be an ancillary method of detecting EGFR mutations in lung cancer patients.MethodsEGFR mutation status was analyzed by DNA assays, and compared with IHC results in five non-small-cell lung cancer (NSCLC) cell lines and tumor samples from 78 stage IV NSCLC patients.ResultsIHC correctly identified del 19 in the H1650 and PC9 cell lines, L858R in H1975, and wild-type EGFR in H460 and A549, as well as wild-type EGFR in tumor samples from 22 patients. IHC with the mAb against EGFR with del 19 was highly positive for the protein in all 17 patients with a 15-bp (ELREA) deletion in exon 19, whereas in patients with other deletions, IHC was weakly positive in 3 cases and negative in 9 cases. IHC with the mAb against the L858R mutation showed high positivity for the protein in 25/27 (93%) patients with exon 21 EGFR mutations (all with L858R) but did not identify the L861Q mutation in the remaining two patients.ConclusionsIHC with mutation-specific mAbs against EGFR is a promising method for detecting EGFR mutations in NSCLC patients. However these mAbs should be validated with additional studies to clarify their possible role in routine clinical practice for screening EGFR mutations in NSCLC patients.

Pharmacogenetics of EGFR in lung cancer: perspectives and clinical applications

Lung cancer is a lethal disease, and most cases have already disseminated at the time of diagnosis. Driver mutations in the EGFR tyrosine kinase domain (mainly deletions in exon 19 and L858R mutation in exon 21) have been identified in lung adenocarcinomas, mostly in never smokers, at frequencies of 20-60%. The EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib attain a response rate of 70% and progression-free survival of 9-13 months, although there are subgroups of patients with long-lasting remissions. No significant correlation between EGFR overexpression and response to treatment has been found, while controversial results have been reported regarding EGFR gene amplification. The pretreatment presence of the T790M mutation, initially identified as an acquired resistance mutation to treatment with EGFR TKIs, has also been reported and may indicate a genetically distinct disease. Finally, other genetic factors, such as mRNA expression of BRCA1 and components of the NF-κB pathway, can modulate response to EGFR TKIs in EGFR-mutated patients.

Nondisruptive p53 Mutations Are Associated with Shorter Survival in Patients with Advanced Non–Small Cell Lung Cancer

Purpose: TP53 mutations in early-stage non–small cell lung cancer (NSCLC) may be associated with worse survival but their prognostic role in advanced NSCLC is controversial. In addition, it remains unclear whether mutated patients represent a clinically homogeneous group. Experimental Design: We retrospectively examined TP53 mutations and outcome in a training cohort of 318 patients with stage IIIB–IV NSCLC: 125 epidermal growth factor receptor (EGFR) wild-type (wt) and 193 EGFR mutated (mut). An independent validation cohort of 64 EGFR-mut patients was subsequently analyzed. Mutations were classified as “disruptive” and “nondisruptive” according to their predicted degree of disturbance of the p53 protein structure and function. Results: In the training cohort, TP53 mutations were found in 43 of the 125 EGFR-wt patients (34.4%). Of these, 28 had nondisruptive TP53 mutations and a median overall survival (OS) of 8.5 months, compared with 15.6 months for the remaining 97 patients (P = 0.003). In the EGFR-mut group, TP53 mutations were found in 50 of the 193 patients (25.9%). The OS for the 26 patients with TP53 nondisruptive mutations was 17.8 months versus 28.4 months for the remaining 167 patients (P = 0.04). In the validation cohort, the 11 patients with nondisruptive TP53 mutations had a median OS of 18.1 months compared with 37.8 months for the 53 remaining patients (P = 0.006). In multivariate analyses, nondisruptive TP53 mutations had an independent, significant association with a shorter OS. Conclusions: Nondisruptive mutations in the TP53 gene are an independent prognostic factor of shorter survival in advanced NSCLC. Clin Cancer Res; 20(17); 4647–59. ©2014 AACR.

mRNA expression levels and genetic status of genes involved in the EGFR and NF-κB pathways in metastatic non-small-cell lung cancer patients

BackgroundMetastatic non-small-cell lung cancer (NSCLC) has a dismal prognosis. EGFR is overexpressed or mutated in a large proportion of cases. Downstream components of the EGFR pathway and crosstalk with the NF-κB pathway have not been examined at the clinical level. We explored the prognostic significance of the mRNA expression of nine genes in the EGFR and NF-κB pathways and of BRCA1 and RAP80 in patients in whom EGFR and K-ras gene status had previously been determined. In addition, NFKBIA and DUSP22 gene status was also determined.MethodsmRNA expression of the eleven genes was determined by QPCR in 60 metastatic NSCLC patients and in nine lung cancer cell lines. Exon 3 of NFKBIA and exon 6 of DUSP22 were analyzed by direct sequencing. Results were correlated with outcome to platinum-based chemotherapy in patients with wild-type EGFR and to erlotinib in those with EGFR mutations.ResultsBRCA1 mRNA expression was correlated with EZH2, AEG-1, Musashi-2, CYLD and TRAF6 expression. In patients with low levels of both BRCA1 and AEG-1, PFS was 13.02 months, compared to 5.4 months in those with high levels of both genes and 7.7 months for those with other combinations (P = 0.025). The multivariate analysis for PFS confirmed the prognostic role of high BRCA1/AEG-1 expression (HR, 3.1; P = 0.01). Neither NFKBIA nor DUSP22 mutations were found in any of the tumour samples or cell lines.ConclusionsThe present study provides a better understanding of the behaviour of metastatic NSCLC and identifies the combination of BRCA1 and AEG-1 expression as a potential prognostic model.

BRAF mutation analysis in circulating free tumor DNA of melanoma patients treated with BRAF inhibitors

BRAFV600E is a unique molecular marker for metastatic melanoma, being the most frequent somatic point mutation in this malignancy. Detection of BRAFV600E in blood could have prognostic and predictive value and could be useful for monitoring response to BRAF-targeted therapy. We developed a rapid, sensitive method for the detection and quantification of BRAFV600E in circulating free DNA (cfDNA) isolated from plasma and serum on the basis of a quantitative 5′-nuclease PCR (Taqman) in the presence of a peptide−nucleic acid. We validated the assay in 92 lung, colon, and melanoma archival serum and plasma samples with paired tumor tissue (40 wild-type and 52 BRAFV600E). The correlation of cfDNA BRAFV600E with clinical parameters was further explored in 22 metastatic melanoma patients treated with BRAF inhibitors. Our assay could detect and quantify BRAFV600E in mixed samples with as little as 0.005% mutant DNA (copy number ratio 1 : 20 000), with a specificity of 100% and a sensitivity of 57.7% in archival serum and plasma samples. In 22 melanoma patients treated with BRAF inhibitors, the median progression-free survival was 3.6 months for those showing BRAFV600E in pretreatment cfDNA compared with 13.4 months for those in whom the mutation was not detected (P=0.021). Moreover, the median overall survival for positive versus negative BRAFV600E tests in pretreatment cfDNA differed significantly (7 vs. 21.8 months, P=0.017). This finding indicates that the sensitive detection and accurate quantification of low-abundance BRAFV600E alleles in cfDNA using our assay can be useful for predicting treatment outcome.

First-line therapy and methylation status of CHFR in serum influence outcome to chemotherapy versus EGFR tyrosine kinase inhibitors as second-line therapy in stage IV non-small-cell lung cancer patients

The potential differential effect of first-line treatment and molecular mechanisms on survival to second-line chemotherapy or EGFR tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC) has not been fully investigated. In particular, CHFR is frequently methylated in NSCLC and may influence outcome. We analyzed the outcome of second-line chemotherapy or EGFR TKIs in 179 of 366 patients who had been treated in an ERCC1 mRNA-based customized cisplatin trial and correlated the results with CHFR methylation status. CHFR methylation in circulating DNA was examined by methylation-specific assay. A panel of seven human EGFR wild-type NSCLC cell lines was characterized for their sensitivity to sequential treatment with cisplatin and erlotinib, and the results were correlated with CHFR. Patients who had received first-line docetaxel/cisplatin attained an overall survival of 19.2 months when treated with second-line EGFR TKIs, in comparison with 10.7 months when treated with second-line chemotherapy (P = 0.0002). However, for patients who had received first-line docetaxel/gemcitabine, overall survival was 14.8 months with EGFR TKIs and 10.8 months with chemotherapy (P = 0.29). For patients with unmethylated CHFR overall survival to EGFR TKIs was 21.4 months, and 11.2 months for those with treated with chemotherapy (P = 0.0001). In the only lung tumor cell line not expressing CHFR, pretreatment with cisplatin was antagonistic to erlotinib, while it was synergistic in the other six lines. Second-line EGFR TKIs improved survival in patients receiving first-line cisplatin-based treatment. Unmethylated CHFR predicts increased survival to EGFR TKIs.