Daniela Morales-Espinosa

Association of EGFR L858R Mutation in Circulating Free DNA With Survival in the EURTAC Trial

IMPORTANCE The EURTAC trial demonstrated the greater efficacy of erlotinib compared with chemotherapy for the first-line treatment of European patients with advanced non-small-cell lung cancer (NSCLC) harboring oncogenic epidermal growth factor receptor (EGFR) mutations (exon 19 deletion or L858R mutation in exon 21) in tumor tissue. OBJECTIVE To assess the feasibility of using circulating free DNA (cfDNA) from blood samples as a surrogate for tumor biopsy for determining EGFR mutation status and to correlate EGFR mutations in cfDNA with outcome. DESIGN, SETTING, AND PARTICIPANTS This prespecified analysis was a secondary objective of the EURTAC trial using patients included in the EURTAC trial from 2007 to 2011 with available baseline serum or plasma samples. Patients had advanced NSCLC, oncogenic EGFR mutations in the tumor, and no prior chemotherapy for metastatic disease and were treated with erlotinib or chemotherapy. EGFR mutations were examined in cfDNA isolated from 97 baseline blood samples by our novel peptide nucleic acid-mediated 5´ nuclease real-time polymerase chain reaction (TaqMan) assay. MAIN OUTCOMES AND MEASURES Overall survival (OS), progression-free survival (PFS), and response to therapy were correlated with type of EGFR mutations in cfDNA. RESULTS In samples from 76 of 97 (78%) patients with usable blood samples, EGFR mutations in cfDNA were detected. Median OS was shorter in patients with the L858R mutation in cfDNA than in those with the exon 19 deletion (13.7 [95% CI, 7.1-17.7] vs 30.0 [95% CI, 19.3-37.7] months; P < .001). Univariate analyses of patients with EGFR mutations in cfDNA identified the L858R mutation in tumor tissue or in cfDNA as a marker of shorter OS (hazard ratio [HR], 2.70 [95% CI, 1.60-4.56]; P < .001) and PFS (HR, 2.04 [95% CI, 1.20-3.48]; P = .008). For patients with the L858R mutation in tissue, median OS was 13.7 (95% CI, 7.1-17.7) months for patients with the L858R mutation in cfDNA and 27.7 (95% CI, 16.1-46.2) months for those in whom the mutation was not detected in cfDNA (HR, 2.22 [95% CI, 1.09-4.52]; P = .03). In the multivariate analysis of the 76 patients with EGFR mutations in cfDNA, only erlotinib treatment remained an independent predictor of longer PFS (HR, 0.41 [95% CI, 0.23-0.74]; P = .003). CONCLUSIONS AND RELEVANCE The peptide nucleic acid-mediated 5´ nuclease real-time polymerase chain reaction (TaqMan) assay used in this study can be used to efficiently assess EGFR mutations in cfDNA. The L858R mutation in cfDNA may be a novel surrogate prognostic marker. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00446225.

Treatment for small cell lung cancer, where are we now?-a review.

Small cell lung cancer (SCLC) represents between 13% and 15% of all diagnosed lung cancers worldwide. It is an aggressive neoplasia, with a 5-year mortality of 90% or more. It has historically been classified as limited disease (LD) and extensive disease (ED) in most study protocols. The cornerstone of treatment for any stage of SCLC is etoposide-platinum based chemotherapy; in limited stage (LS), concomitant radiotherapy to thorax and mediastinum. Prophylactic radiotherapy to the central nervous system (CNS) [prophylactic cerebral irradiation (PCI)] has diminished the incidence of brain metastasis as the site for relapse in LD and ED patients, therefore it should be offered to patients with complete response to induction first-line treatment. Regarding second-line treatment, results are more modest and topotecan is accepted as treatment for this scenario offering a modest benefit.

Development of a gene panel for next-generation sequencing of clinically relevant mutations in cell-free DNA from cancer patients

Background:When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA.Methods:Our panel (‘SiRe’) covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice.Results:SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression.Conclusions:SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.

Large scale, prospective screening of EGFR mutations in the blood of advanced NSCLC patients to guide treatment decisions

Background In a significant percentage of advanced non-small-cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses. We prospectively analyzed if circulating-free DNA (cfDNA) purified from blood can be used as a surrogate in this setting to select patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). Patients and methods Blood samples were collected in 119 hospitals from 1138 advanced NSCLC patients at presentation (n = 1033) or at progression to EGFR-TKIs (n = 105) with no biopsy or insufficient tumor tissue. Serum and plasma were sent to a central laboratory, cfDNA purified and EGFR mutations analyzed and quantified using a real-time PCR assay. Response data from a subset of patients (n = 18) were retrospectively collected. Results Of 1033 NSCLC patients at presentation, 1026 were assessable; with a prevalence of males and former or current smokers. Sensitizing mutations were found in the cfDNA of 113 patients (11%); with a majority of females, never smokers and exon 19 deletions. Thirty-one patients were positive only in plasma and 11 in serum alone and mutation load was higher in plasma and in cases with exon 19 deletions. More than 50% of samples had <10 pg mutated genomes/µl with allelic fractions below 0.25%. Patients treated first line with TKIs based exclusively on EGFR positivity in blood had an ORR of 72% and a median PFS of 11 months. Of 105 patients screened after progression to EGFR-TKIs, sensitizing mutations were found in 56.2% and the p.T790M resistance mutation in 35.2%. Conclusions Large-scale EGFR testing in the blood of unselected advanced NSCLC patients is feasible and can be used to select patients for targeted therapy when testing cannot be done in tissue. The characteristics and clinical outcomes to TKI treatment of the EGFR-mutated patients identified are undistinguishable from those positive in tumor.

Assays for predicting and monitoring responses to lung cancer immunotherapy.

Immunotherapy has become a key strategy for cancer treatment, and two immune checkpoints, namely, programmed cell death 1 (PD-1) and its ligand (PD-L1), have recently emerged as important targets. The interaction blockade of PD-1 and PD-L1 demonstrated promising activity and antitumor efficacy in early phase clinical trials for advanced solid tumors such as non-small cell lung cancer (NSCLC). Many cell types in multiple tissues express PD-L1 as well as several tumor types, thereby suggesting that the ligand may play important roles in inhibiting immune responses throughout the body. Therefore, PD-L1 is a critical immunomodulating component within the lung microenvironment, but the correlation between PD-L1 expression and prognosis is controversial. More evidence is required to support the use of PD-L1 as a potential predictive biomarker. Clinical trials have measured PD-L1 in tumor tissues by immunohistochemistry (IHC) with different antibodies, but the assessment of PD-L1 is not yet standardized. Some commercial antibodies lack specificity and their reproducibility has not been fully evaluated. Further studies are required to clarify the optimal IHC assay as well as to predict and monitor the immune responses of the PD-1/PD-L1 pathway.

Adaptive resistance to targeted therapies in cancer

It is widely acknowledged that there is a need for molecular profiling in non-small-cell lung cancer. For example, treatment based on EGFR mutation status has attained successful results. However, in spite of excellent initial response to oral EGFR tyrosine kinase inhibitors (TKIs), progression-free survival is still limited. Current research has focused mostly on acquired resistance mechanisms, such as overexpression of AXL and loss of the Mediator MED12. In this review, in contrast, we discuss adaptive, rather than acquired, resistance. Adaptive resistance can occur almost immediately after starting targeted therapy through a rapid rewiring of cancer cell signaling. By losing ERK negative feedback on receptor tyrosine kinase (RTK) expression, cancer cells are exposed to the stimuli of several ligands, and the ensuing activation of several RTKs reprograms all the canonical signaling pathways. The overexpression of several RTKs was observed in breast cancer cell lines treated with a MEK inhibitor and in BRAF(V600E) melanoma cell lines treated with BRAF inhibitors. This rebound effect of overexpression of several RTKs, including ERBB3, also occurs in lung cancers driven by Kras or EGFR mutations when treated with MEK, PI3K or dual PI3K/mTOR inhibitors. Synthetic lethality can be effectively induced by co-targeting these overexpressed RTKs. We speculate that in patients with EGFR mutations, adaptive resistance occurs in a significant proportion of patients. Rebiopsies performed hours after starting treatment with EGFR TKIs can identify which RTKs are overexpressed after treatment. Efficient co-targeting of these RTKs can induce synthetic lethality and help overcome the limited effect of EGFR TKI monotherapy.

BIM and mTOR expression levels predict outcome to erlotinib in EGFR-mutant non-small-cell lung cancer

BIM is a proapoptotic protein that initiates apoptosis triggered by EGFR tyrosine kinase inhibitors (TKI). mTOR negatively regulates apoptosis and may influence response to EGFR TKI. We examined mRNA expression of BIM and MTOR in 57 patients with EGFR-mutant NSCLC from the EURTAC trial. Risk of mortality and disease progression was lower in patients with high BIM compared with low/intermediate BIM mRNA levels. Analysis of MTOR further divided patients with high BIM expression into two groups, with those having both high BIM and MTOR experiencing shorter overall and progression-free survival to erlotinib. Validation of our results was performed in an independent cohort of 19 patients with EGFR-mutant NSCLC treated with EGFR TKIs. In EGFR-mutant lung adenocarcinoma cell lines with high BIM expression, concomitant high mTOR expression increased IC50 of gefitinib for cell proliferation. We next sought to analyse the signalling pattern in cell lines with strong activation of mTOR and its substrate P-S6. We showed that mTOR and phosphodiesterase 4D (PDE4D) strongly correlate in resistant EGFR-mutant cancer cell lines. These data suggest that the combination of EGFR TKI with mTOR or PDE4 inhibitors could be adequate therapy for EGFR-mutant NSCLC patients with high pretreatment levels of BIM and mTOR.

Understanding the function and dysfunction of the immune system in lung cancer: the role of immune checkpoints

Survival rates for metastatic lung cancer, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), are poor with 5-year survivals of less than 5%. The immune system has an intricate and complex relationship with tumorigenesis; a groundswell of research on the immune system is leading to greater understanding of how cancer progresses and presenting new ways to halt disease progress. Due to the extraordinary power of the immune system—with its capacity for memory, exquisite specificity and central and universal role in human biology—immunotherapy has the potential to achieve complete, long-lasting remissions and cures, with few side effects for any cancer patient, regardless of cancer type. As a result, a range of cancer therapies are under development that work by turning our own immune cells against tumors. However deeper understanding of the complexity of immunomodulation by tumors is key to the development of effective immunotherapies, especially in lung cancer.

Liquid Biopsy in Non-Small Cell Lung Cancer

Liquid biopsy analyses are already incorporated in the routine clinical practice in many hospitals and oncology departments worldwide, improving the selection of treatments and monitoring of lung cancer patients. Although they have not yet reached its full potential, liquid biopsy-based tests will soon be as widespread as “standard” biopsies and imaging techniques, offering invaluable diagnostic, prognostic, and predictive information. This review summarizes the techniques available for the isolation and analysis of circulating free DNA and RNA, exosomes, tumor-educated platelets, and circulating tumor cells from the blood of cancer patients, presents the methodological challenges associated with each of these materials, and discusses the clinical applications of liquid biopsy testing in lung cancer.

Melanoma: oncogenic drivers and the immune system.

Advances and in-depth understanding of the biology of melanoma over the past 30 years have contributed to a change in the consideration of melanoma as one of the most therapy-resistant malignancies. The finding that oncogenic BRAF mutations drive tumor growth in up to 50% of melanomas led to a molecular therapy revolution for unresectable and metastatic disease. Moving beyond BRAF, inactivation of immune regulatory checkpoints that limit T cell responses to melanoma has provided targets for cancer immunotherapy. In this review, we discuss the molecular biology of melanoma and we focus on the recent advances of molecularly targeted and immunotherapeutic approaches.