Combinatorial Blood Platelets-Derived circRNA and mRNA Signature for Early-Stage Lung Cancer Detection.

Despite the diversity of liquid biopsy transcriptomic repertoire, numerous studies often exploit only a single RNA type signature for diagnostic biomarker potential. This frequently results in insufficient sensitivity and specificity necessary to reach diagnostic utility. Combinatorial biomarker approaches may offer a more reliable diagnosis. Here, we investigated the synergistic contributions of circRNA and mRNA signatures derived from blood platelets as biomarkers for lung cancer detection. We developed a comprehensive bioinformatics pipeline permitting an analysis of platelet-circRNA and mRNA derived from non-cancer individuals and lung cancer patients. An optimal selected signature is then used to generate the predictive classification model using machine learning algorithm. Using an individual signature of 21 circRNA and 28 mRNA, the predictive models reached an area under the curve (AUC) of 0.88 and 0.81, respectively. Importantly, combinatorial analysis including both types of RNAs resulted in an 8-target signature (6 mRNA and 2 circRNA), enhancing the differentiation of lung cancer from controls (AUC of 0.92). Additionally, we identified five biomarkers potentially specific for early-stage detection of lung cancer. Our proof-of-concept study presents the first multi-analyte-based approach for the analysis of platelets-derived biomarkers, providing a potential combinatorial diagnostic signature for lung cancer detection.

Multiplex Detection of Clinically Relevant Mutations in Liquid Biopsies of Cancer Patients Using a Hybridization-Based Platform.

BACKGROUND: With the advent of precision oncology, liquid biopsies are quickly gaining acceptance in the clinical setting. However, in some cases, the amount of DNA isolated is insufficient for Next-Generation Sequencing (NGS) analysis. The nCounter platform could be an alternative, but it has never been explored for detection of clinically relevant alterations in fluids. METHODS: Circulating-free DNA (cfDNA) was purified from blood, cerebrospinal fluid, and ascites of patients with cancer and analyzed with the nCounter 3 D Single Nucleotide Variant (SNV) Solid Tumor Panel, which allows for detection of 97 driver mutations in 24 genes. RESULTS: Validation experiments revealed that the nCounter SNV panel could detect mutations at allelic fractions of 0.02-2% in samples with ≥5 pg mutant DNA/µL. In a retrospective analysis of 70 cfDNAs from patients with cancer, the panel successfully detected EGFR, KRAS, BRAF, PIK3CA, and NRAS mutations when compared with previous genotyping in the same liquid biopsies and paired tumor tissues [Cohen kappa of 0.96 (CI = 0.92-1.00) and 0.90 (CI = 0.74-1.00), respectively]. In a prospective study including 91 liquid biopsies from patients with different malignancies, 90 yielded valid results with the SNV panel and mutations in EGFR, KRAS, BRAF, PIK3CA, TP53, NFE2L2, CTNNB1, ALK, FBXW7, and PTEN were found. Finally, serial liquid biopsies from a patient with NSCLC revealed that the semiquantitative results of the mutation analysis by the SNV panel correlated with the evolution of the disease. CONCLUSIONS: The nCounter platform requires less DNA than NGS and can be employed for routine mutation testing in liquid biopsies of patients with cancer.

Correction to: The Present and Future of Liquid Biopsies in Non-Small Cell Lung Cancer: Combining Four Biosources for Diagnosis, Prognosis, Prediction, and Disease Monitoring.

The original version of this review article unfortunately contained a mistake in the Funding section.

The Present and Future of Liquid Biopsies in Non-Small Cell Lung Cancer: Combining Four Biosources for Diagnosis, Prognosis, Prediction, and Disease Monitoring.

PURPOSE OF REVIEW: Liquid biopsies have potential as tools for diagnosis, prognosis, and prediction of response to therapy. Herein, we will extensively review four liquid biosources, tumor-educated platelets (TEPs), cell-free DNA (cfDNA), circulating tumor cells (CTCs), and extracellular vesicles (EVs) and we will clarify their optimal application in non-small cell lung cancer (NSCLC) diagnosis and therapy. RECENT FINDINGS: Liquid biopsies are a minimally invasive alternative to tissue biopsies-especially important in NSCLC patients-since tumor tissue is often unavailable or insufficient for complete genetic analysis. The main advantages of liquid biopsies include the possibility for repeated sampling, the lower cost, and the fact that they can reflect the complete molecular status of the patient better than a single-site biopsy. This is specifically important for lung adenocarcinoma patients since the detection of specific genetic alterations can predict response to targeted therapies. Molecular analysis is currently cardinal for therapy decision-making and disease monitoring in lung cancer patients. Liquid biopsies can make easier our daily clinical practice and if prospectively tested and validated may serve as a means for lung cancer early detection.

Clinical applications and challenges in the field of extracellular vesicles.

Body fluids contain cell-derived particles called extracellular vesicles (EVs). EVs are released by cells and are present in all body fluids (i. e. liquid biopsies). EVs contribute to physiology and pathology and offer a plethora of potential clinical applications, ranging from biomarkers to therapeutic applications. In this manuscript we provide an overview of this new and rapidly growing research field, along with its challenges and opportunities.

Platelet removal from human blood plasma improves detection of extracellular vesicle-associated miRNA.

Human blood plasma prepared by centrifugation contains not only extracellular vesicles (EVs) but also platelets and erythrocyte ghosts (ery-ghosts). Here we studied whether analysis of miRNA associated with plasma EVs (EV-miRNA) is affected by the presence of platelets and ery-ghosts. EDTA blood was collected from healthy donors (n = 3), and plasma was prepared by the centrifugation protocol recommended by the International Society on Thrombosis and Haemostasis (ISTH), and by a centrifugation protocol from an EV-miRNA expert lab (non-ISTH protocol). EVs were isolated from plasma by size-exclusion chromatography CL-2B (SEC2B), and concentrations of platelets, activated platelets, ery-ghosts and EVs (150-1000 nm) were measured by calibrated flow cytometry. Two EV-associated miRNAs (let7a-5p and miR-21-5p), and one platelet-associated miRNA (miR-223-3p), were measured by qRT-PCR. Measurements were performed with and without filtration using 0.8 µm track-etched filters to remove platelets and ery-ghosts from plasma and EV-enriched SEC fractions. Plasma prepared by both centrifugation protocols contained platelets and ery-ghosts, which co-migrated with EVs into the EV-enriched SEC2B fractions. Filtration removed platelets and ery-ghosts (>97%; p ≤ 0.05) and did not affect the EV concentrations (p > 0.17). The miRNA concentrations were 2-4-fold overestimated due to the presence of platelets but not ery-ghosts. Thus, filtration of human plasma is expected to improve comparability and reproducibility of quantitative EV-miRNA studies. Therefore, we recommend to measure and report the plasma concentration of platelets for EV-miRNA studies, and to filter plasma before downstream analyses or storage in biobanks.

MicroRNAs in extracellular vesicles released from epicardial adipose tissue promote arrhythmogenic conduction slowing.

Background: Patients with excess epicardial adipose tissue (EAT) are at increased risk of developing cardiac arrhythmias. EAT promotes arrhythmias by depolarizing the resting membrane of cardiomyocytes, which slows down conduction and facilitates re-entrant arrhythmias. We hypothesized that EAT slows conduction by secreting extracellular vesicles (EVs) and their microRNA (miRNA) cargo. Objective: We aimed to determine the role of EAT-derived EVs and their miRNA cargo in conduction slowing. Methods: EAT and subcutaneous adipose tissue (SAT) were collected from patients with atrial fibrillation. Adipose tissue explants were incubated in culture medium and secretome was collected. The numbers of EVs in the EAT and SAT secretome were measured by calibrated flow cytometry. EVs in the EAT secretome were isolated by size exclusion chromatography and miRNAs were sequenced. Pathway analysis was performed to predict candidates involved in cardiac electrophysiology. The candidates were validated in the EAT and SAT by quantitative real-time polymerase chain reaction. Finally, miRNA candidates were overexpressed in neonatal rat ventricular myocytes. Results: The EV concentration was higher in the EAT secretome than in the SAT and control secretomes. miRNA sequencing of EAT-derived EVs detected a total of 824 miRNAs. Pathway analysis led to the identification of 7 miRNAs potentially involved in regulation of cardiac resting membrane potential. Validation of those miRNA candidates showed that they were all expressed in EAT, and that miR-1-3p and miR-133a-3p were upregulated in EAT in comparison with SAT. Overexpression of miR-1-3p and miR-133a-3p in neonatal rat ventricular myocytes led to conduction slowing and reduced Kcnj2 and Kcnj12 expression. Conclusion: miR-1-3p and miR-133a-3p are potential mediators of EAT arrhythmogenicity.

Multiplex Analysis of CircRNAs from Plasma Extracellular Vesicle-Enriched Samples for the Detection of Early-Stage Non-Small Cell Lung Cancer.

BACKGROUND: The analysis of liquid biopsies brings new opportunities in the precision oncology field. Under this context, extracellular vesicle circular RNAs (EV-circRNAs) have gained interest as biomarkers for lung cancer (LC) detection. However, standardized and robust protocols need to be developed to boost their potential in the clinical setting. Although nCounter has been used for the analysis of other liquid biopsy substrates and biomarkers, it has never been employed for EV-circRNA analysis of LC patients. METHODS: EVs were isolated from early-stage LC patients (n = 36) and controls (n = 30). Different volumes of plasma, together with different number of pre-amplification cycles, were tested to reach the best nCounter outcome. Differential expression analysis of circRNAs was performed, along with the testing of different machine learning (ML) methods for the development of a prognostic signature for LC. RESULTS: A combination of 500 µL of plasma input with 10 cycles of pre-amplification was selected for the rest of the study. Eight circRNAs were found upregulated in LC. Further ML analysis selected a 10-circRNA signature able to discriminate LC from controls with AUC ROC of 0.86. CONCLUSIONS: This study validates the use of the nCounter platform for multiplexed EV-circRNA expression studies in LC patient samples, allowing the development of prognostic signatures.

Clinical Activity of Mitogen-Activated Protein Kinase-Targeted Therapies in Patients With Non-V600 BRAF-Mutant Tumors.

PURPOSE: Non-V600 mutations comprise approximately 35% of all BRAF mutations in cancer. Many of these mutations have been identified as oncogenic drivers and can be classified into three classes according to molecular characteristics. Consensus treatment strategies for class 2 and 3 BRAF mutations have not yet been established. METHODS: We performed a systematic review and meta-analysis with published reports of individual patients with cancer harboring class 2 or 3 BRAF mutations from 2010 to 2021, to assess treatment outcomes with US Food and Drug Administration-approved mitogen-activated protein kinase (MAPK) pathway targeted therapy (MAPK TT) according to BRAF class, cancer type, and MAPK TT type. Coprimary outcomes were response rate and progression-free survival. RESULTS: A total of 18,167 studies were screened, identifying 80 studies with 238 patients who met inclusion criteria. This included 167 patients with class 2 and 71 patients with class 3 BRAF mutations. Overall, 77 patients achieved a treatment response. In both univariate and multivariable analyses, response rate and progression-free survival were higher among patients with class 2 compared with class 3 mutations, findings that remain when analyses are restricted to patients with melanoma or lung primary cancers. MEK ± BRAF inhibitors demonstrated greater clinical activity in class 2 compared with class 3 BRAF-mutant tumors than BRAF or EGFR inhibitors. CONCLUSION: This meta-analysis suggests that MAPK TTs have clinical activity in some class 2 and 3 BRAF-mutant cancers. BRAF class may dictate responsiveness to current and emerging treatment strategies, particularly in melanoma and lung cancers. Together, this analysis provides clinical validation of predictions made on the basis of a mutation classification system established in the preclinical literature. Further evaluation with prospective clinical trials is needed for this population.

Analysis of extracellular vesicle mRNA derived from plasma using the nCounter platform.

Extracellular vesicles (EVs) are double-layered phospholipid membrane vesicles that are released by most cells and can mediate intercellular communication through their RNA cargo. In this study, we tested if the NanoString nCounter platform can be used for the analysis of EV-mRNA. We developed and optimized a methodology for EV enrichment, EV-RNA extraction and nCounter analysis. Then, we demonstrated the validity of our workflow by analyzing EV-RNA profiles from the plasma of 19 cancer patients and 10 controls and developing a gene signature to differentiate cancer versus control samples. TRI reagent outperformed automated RNA extraction and, although lower plasma input is feasible, 500 µL provided highest total counts and number of transcripts detected. A 10-cycle pre-amplification followed by DNase treatment yielded reproducible mRNA target detection. However, appropriate probe design to prevent genomic DNA binding is preferred. A gene signature, created using a bioinformatic algorithm, was able to distinguish between control and cancer EV-mRNA profiles with an area under the ROC curve of 0.99. Hence, the nCounter platform can be used to detect mRNA targets and develop gene signatures from plasma-derived EVs.

Non-Small-Cell Lung Cancer Signaling Pathways, Metabolism, and PD-1/PD-L1 Antibodies.

Treatment of advanced (metastatic) non-small-cell lung cancer (NSCLC) is currently mainly based on immunotherapy with antibodies against PD-1 or PD-L1, alone, or in combination with chemotherapy. In locally advanced NSCLC and in early resected stages, immunotherapy is also employed. Tumor PD-L1 expression by immunohistochemistry is considered the standard practice. Response rate is low, with median progression free survival very short in the vast majority of studies reported. Herein, numerous biological facets of NSCLC are described involving driver genetic lesions, mutations ad fusions, PD-L1 glycosylation, ferroptosis and metabolic rewiring in NSCLC and lung adenocarcinoma (LUAD). Novel concepts, such as immune-transmitters and the effect of neurotransmitters in immune evasion and tumor growth, the nascent relevance of necroptosis and pyroptosis, possible new biomarkers, such as gasdermin D and gasdermin E, the conundrum of K-Ras mutations in LUADs, with the growing recognition of liver kinase B1 (LKB1) and metabolic pathways, including others, are also commented. The review serves to charter diverse treatment solutions, depending on the main altered signaling pathways, in order to have effectual immunotherapy. Tumor PDCD1 gene (encoding PD-1) has been recently described, in equilibrium with tumor PD-L1 (encoded by PDCD1LG1). Such description explains tumor hyper-progression, which has been reported in several studies, and poises the fundamental criterion that IHC PD-L1 expression as a biomarker should be revisited.

SRC and PIM1 as potential co-targets to overcome resistance in MET deregulated non-small cell lung cancer.

BACKGROUND: The role of MET alterations in non-small cell lung cancer (NSCLC) is increasing and several targeted agents are under evaluation. MET exon 14 skipping mutations and MET amplifications are associated with potential sensitivity to MET inhibition, though resistance mechanisms are emerging. In MET addicted cells, MET inhibition leads to activation of proviral integration site for Moloney murine leukemia virus-1 (PIM1). PIM1 and proto-oncogene tyrosine-protein kinase Src (SRC) can regulate the expression of receptor tyrosine kinases (RTKs), potentially inducing resistance to MET inhibition through cross-activation. METHODS: We evaluated the activity of class I-II MET inhibitors, the SRC inhibitor dasatinib, and pan-PIM inhibitors in four MET addicted cell lines. We assessed the effect of the dual MET/PIM and MET/SRC inhibition on cell viability and at the protein level. We evaluated RNA expression profiles of the cell lines. Advanced NSCLCs were also screened for MET alterations. RESULTS: All cell lines were sensitive to class I-II MET inhibitors. All cell lines were resistant to single PIM and SRC inhibition. Dual MET/PIM inhibition was synergistic or additive in MET amplified cell lines and dual MET/SRC inhibition was highly synergistic in all MET addicted cell lines. The addition of an SRC inhibitor partially prevents the RTKs cross-activation. MET alterations were found in 9 out of 97 evaluable samples (9.3%); median overall survival in MET altered patients was 5 months (95% CI, 3 m-NA). CONCLUSIONS: We identified a potential role of PIM inhibition in MET amplified tumors and of SRC inhibition in MET addicted tumors. Potential applications of this new treatment strategy warrant further evaluation.

Assessment of the Feasibility and Safety of Durvalumab for Treatment of Solid Tumors in Patients With HIV-1 Infection: The Phase 2 DURVAST Study.

Importance: Therapies targeting the programmed cell death 1 (PD-1) receptor or its ligand (PD-L1), such as the humanized monoclonal antibody durvalumab, have shown durable clinical responses in several tumor types. However, concerns about the safety and feasibility of PD-1/PD-L1 blockade in HIV-1-infected individuals have led to the exclusion of these patients from clinical trials on cancer immunotherapies. Objective: To evaluate the feasibility and safety of durvalumab treatment in patients with advanced cancer and virologically controlled HIV-1 infection. Design, Setting, and Participants: The DURVAST study was a nonrandomized, open-label, phase 2 clinical trial in patients with any solid tumor type in which anti-PD-1 or anti-PD-L1 antibodies have approved indications or for which there are data of antitumoral activity with no other available curative therapy. All patients had basal undetectable plasma viremia while undergoing combination antiretroviral therapy. Interventions: Treatment consisted of intravenous infusion of durvalumab (1500 mg every 4 weeks) until disease progression or unacceptable toxic effects. Main Outcomes and Measures: Adverse events were graded with the use of the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.03. Tumor response was evaluated using the Response Evaluation Criteria in Solid Tumors version 1.1. Results: A total of 20 HIV-1-infected patients with advanced cancer were enrolled; 16 (80%) were male, the median (range) age was 54 (30-73) years, and 12 (60%) had progressed with previous cancer treatment lines. A median (range) of 4 (1-16) cycles of durvalumab were administered. Drug-related adverse events were observed in 50% of patients, and all were grade 1 and 2 (mainly diarrhea, asthenia, and arthromyalgia). Four of 16 response-evaluable patients (25%) had a partial response. Five patients (31%) had stable disease, including 4 with durable stable disease (disease control rate of 50%). CD4+ and CD8+ T-cell counts and plasma HIV-1 viremia remained stable throughout the study. Conclusions and Relevance: Durvalumab treatment was feasible and safe in HIV-1-infected patients with cancer receiving combination antiretroviral therapy. HIV-1-infected patients on suppressive antiretroviral therapy with advanced cancer should have access to cancer immunotherapy treatments. Trial Registration: ClinicalTrials.gov Identifier: NCT03094286.

EGFR first- and second-generation TKIs-there is still place for them in EGFR-mutant NSCLC patients.

Identification of epidermal growth factor receptor (EGFR) as a molecular target has radically changed the treatment of metastatic non-small cell lung cancer (NSCLC) from standard chemotherapy to personalized, targeted therapy. First-, second- and third-generation EGFR tyrosine kinase inhibitors (TKIs) are now available for the treatment of EGFR-mutant NSCLC patients. This review will focus on the clinical development of first- and second-generation EGFR TKIs. We will emphasize on essential points like the head-to-head comparison among EGFR TKIs, their activity on brain metastases, mechanisms of resistance, as well as their combination with anti-angiogenic compounds, other targeted therapies, or immunotherapy. The efficacy of first- and second-generation EGFR TKIs in early-stage EGFR-mutant NSCLC will be also finally reviewed.

Profile of alectinib for the treatment of ALK-positive non-small cell lung cancer (NSCLC): patient selection and perspectives.

Discovered in 2007, anaplastic lymphoma kinase (ALK) gene rearrangements positive (ALK+) lung cancers compose a small subset of non-small cell lung cancer (NSCLC), with rapidly expanded treatments. There are currently several ALK inhibitors, including crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib which have been licensed by the US Food and Drug Administration or the European Medicines Agency for the treatment of ALK+ NSCLC patients. Along with the multiple therapies, the survival of this subtype of NSCLC has been significantly expanded, even for patients whose disease has spread in the brain. Alectinib (Alecensa), a specific ALK and rearranged during transfection tyrosine kinase inhibitor is approved as first-line therapy for metastatic ALK+ NSCLC patients. It is additionally approved for ALK+ NSCLC previously treated with crizotinib. The main aim of this review is to assemble on the efficacy of alectinib for the treatment of ALK+ NSCLC, to elaborate the activity of the drug in the central nervous system, and to debate on which is the position of this compound in the treatment course of ALK+ lung cancer patients.

Integrin-linked kinase (ILK) and src homology 2 domain-containing phosphatase 2 (SHP2): Novel targets in EGFR-mutation positive non-small cell lung cancer (NSCLC).

BACKGROUND: The activation of multiple signaling pathways jeopardizes the clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutation positive non-small cell lung cancer (NSCLC). Integrin-linked kinase (ILK) regulates the interactions between tumor cells and extracellular environment to activate signaling pathways and promote cell proliferation, migration, and epithelial-mesenchymal transition. Src homology 2 domain-containing phosphatase 2 (SHP2) is essential for receptor tyrosine kinase signaling and mitogen-activated protein kinase (MAPK) pathway activation. METHODS: We analyzed tumor ILK, ß-receptor subunit glycoprotein 130 (gp130), SHP2, and stromal hepatocyte growth factor (HGF) and interleukin-6 (IL-6) mRNA expression in baseline tumor specimens of advanced EGFR-mutation positive NSCLC patients treated with EGFR TKIs. RESULTS: ILK, when highly expressed, was an independent poor prognostic factor for the progression-free survival of the patients, both in the univariate (hazard ratio [HR for disease progression, 2.49; 95% CI, 1.37-4.52; P = .0020]) and in the multivariate (HR 3.74; 95% CI, 1.33-10.56; P = .0126) Cox regression model. Patients with high SHP2 expression had an almost 13-month shorter progression-free survival (P = .0094) and an 18-month shorter overall survival (P = .0182) in comparison to those with low SHP2 mRNA expression. INTERPRETATION: The levels of ILK and SHP2 could be predictive for upfront combinatory therapy of EGFR TKIs plus SHP2 or ILK inhibitors. FUND: A grant from La Caixa Foundation, an Instituto de Salud Carlos III grant (RESPONSE, PIE16/00011), an Instituto de Salud Carlos III grant (PI14/01678), a Marie Sklodowska-Curie Innovative Training Networks European Grant (ELBA No 765492) and a Spanish Association Against Cancer (AECC) grant (PROYE18012ROSE).

Cancer Stem Cell Biomarkers in EGFR-Mutation-Positive Non-Small-Cell Lung Cancer.

INTRODUCTION: Epidermal growth factor receptor (EGFR) pathway deregulation promotes the acquisition of stemlike properties in non-small-cell lung cancer. EGFR inhibition through NOTCH enriches lung cancer stem cells (CSCs). Src through Yes-associated protein 1 (YAP1) activates NOTCH. Signal transduction and activator of transcription 3 (STAT3) activation occurs upon EGFR blockade and regulates the generation of CSCs. PATIENTS AND METHODS: Using the Aldefluor assay kit, we investigated the enrichment of aldehyde dehydrogenase (ALDH)-positive cells in EGFR-mutation-positive cells treated with gefitinib, afatinib, and osimertinib. Western blot analysis was performed to evaluate changes in CSC marker expression upon EGFR blockade. We performed gene expression analysis in a cohort of EGFR-mutation-positive non-small-cell lung cancer patients. We evaluated the association of gene expression with treatment outcomes. RESULTS: The cell subpopulation surviving EGFR inhibition had high ALDH activity and elevated CSC marker expression. Concurrent inhibition of EGFR, STAT3, and Src diminished the CSC subpopulation in an EGFR-mutation-positive cellular model. In a cohort of 64 EGFR-mutation-positive patients, 2 ALDH1 isoforms and the NOTCH target hairy and enhancer of split 1 (HES1), when highly expressed, were predictive of worse outcome to EGFR blockade. The gene expression of B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) that maintains the self-renewal of stem cells was also related to treatment outcome. CONCLUSION: Single EGFR inhibitors increase the population of CSCs. Combinatory therapy targeting STAT3 and Src may be of potential benefit. ALDH1, HES1, and Bmi-1 are essential biomarkers in the initial assessment of EGFR-mutation-positive patients.

Characterising acquired resistance to erlotinib in non-small cell lung cancer patients.

Introduction: The therapy of patients with lung adenocarcinoma has significantly changed after the discovery of epidermal growth factor receptor (EGFR) mutations. EGFR mutations occur in 10-15% of Caucasian lung cancer patients and are associated with favorable outcome to orally administered EGFR tyrosine kinase inhibitors (TKIs), like erlotinib. However, as soon as the tumor cells are under the pressure of the specific inhibitor, compensatory signaling pathways are activated and resistance emerges. Areas covered: In this review we will focus on the mechanisms of resistance to the first-generation EGFR TKI, erlotinib, and will mainly summarize the findings throughout the last 10 years in the field of EGFR-mutant lung cancer. Expert opinion: Widespread research has been performed and several mechanisms of resistance to EGFR TKIs, especially first- and second-generation, have been identified. Still, no adequate combinatory therapies have received regulatory approval for the treatment of EGFR-mutant patients at the time of resistance. The third-generation EGFR TKI, osimertinib has been approved for patients whose tumor has become resistant through the secondary T790M resistant EGFR mutation. The identification of the mechanisms of resistance and the application of the adequate therapy to each patient is still an unmet need.

BRAF Mutations Classes I, II, and III in NSCLC Patients Included in the SLLIP Trial: The Need for a New Pre-Clinical Treatment Rationale.

BRAF V600 mutations have been found in 1-2% of non-small-cell lung cancer (NSCLC) patients, with Food and Drug Administration (FDA) approved treatment of dabrafenib plus trametinib and progression free survival (PFS) of 10.9 months. However, 50-80% of BRAF mutations in lung cancer are non-V600, and can be class II, with intermediate to high kinase activity and RAS independence, or class III, with impaired kinase activity, upstream signaling dependence, and consequently, sensitivity to receptor tyrosine kinase (RTK) inhibitors. Plasma cell-free DNA (cfDNA) of 185 newly diagnosed advanced lung adenocarcinoma patients (Spanish Lung Liquid versus Invasive Biopsy Program, SLLIP, NCT03248089) was examined for BRAF and other alterations with a targeted cfDNA next-generation sequencing (NGS) assay (Guardant360®, Guardant Health Inc., CA, USA), and results were correlated with patient outcome. Cell viability with single or combined RAF, MEK, and SHP2 inhibitors was assessed in cell lines with BRAF class I, II, and III mutations. Out of 185 patients, 22 had BRAF alterations (12%) of which seven patients harbored amplifications (32%) and 17 had BRAF mutations (77%). Of the BRAF mutations, four out of 22 (18%) were V600E and 18/22 (82%) were non-V600. In vitro results confirmed sensitivity of class III and resistance of class I and II BRAF mutations, and BRAF wild type cells to SHP2 inhibition. Concomitant MEK or RAF and SHP2 inhibition showed synergistic effects, especially in the class III BRAF-mutant cell line. Our study indicates that the class of the BRAF mutation may have clinical implications and therefore should be defined in the clinical practice and used to guide therapeutic decisions.

Hsp90 inhibitors enhance the antitumoral effect of osimertinib in parental and osimertinib-resistant non-small cell lung cancer cell lines.

BACKGROUND: Osimertinib improve therapy for non-small cell lung cancer (NSCLC). However, invariable acquired resistance appears. METHODS: MTT assay was used to analyze cell viability. Protein expression and activation was detected by Western blotting. In addition, the effects of heat shock protein 90 (Hsp90) inhibitors and osimertinib were studied in colony formation assays. RESULTS: Our laboratory generated osimertinib resistant cell lines from PC9 cell line and overexpression or activation of several proteins was detected. Hsp90 inhibitors, ganetespib and luminespib, inhibited cell viability and colony formation in H1975, PC9 and PC9-derived osimertinib-resistant cell lines and combination of these inhibitors with osimertinib achieved to enhance this cell viability and colony formation inhibition. Luminespib downregulated the expression of the several proteins involved in osimertinib-resistance and the combination of this compound plus osimertinib caused an important decrease of expression of several of these proteins, such as Stat3, Yap, Akt, EGFR and Met. Osimertinib activated the phosphorylation of several membrane receptors and downstream molecules that was partially inhibited by luminespib. In addition, a lung cancer patient with an EGFR eon 20 mutation had a partial radiographic response to ganetespib. CONCLUSIONS: Hsp90 inhibitors and osimertinib exhibits a good efficiency to inhibit cell viability, colony formation and inhibits expression and activation of proteins involved in osimertinib-resistance and may represent an effective strategy for NSCLC with intrinsic resistance to osimertinib inhibition.