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Aberrant expression of microRNA-146a (miR-146a) has been reported to be involved in the development and progression of various types of cancers. However, its role in non-small cell lung cancer (NSCLC) has not been elucidated. The aim of this study was to investigate the contribution of miR-146a to various aspects of the malignant phenotype of human NSCLCs. In functional experiments, miR-146a suppressed cell growth, induced cellular apoptosis and inhibited EGFR downstream signaling in five NSCLC cell lines (H358, H1650, H1975, HCC827 and H292). miR-146a also inhibited the migratory capacity of these NSCLC cells. On the other hand, miR-146a enhanced the inhibition of cell proliferation by drugs targeting EGFR, including both TKIs (gefitinib, erlotinib, and afatinib) and a monoclonal antibody (cetuximab). These effects were independent of the EGFR mutation status (wild type, sensitizing mutation or resistance mutation), but were less potent compared to the effects of siRNA targeting of EGFR. Our results suggest that these effects of miR-146a are due to its targeting of EGFR and NF-κB signaling. We also found, in clinical formalin fixed paraffin embedded (FFPE) lung cancer samples, that low expression of miR-146a was correlated with advanced clinical TNM stages and distant metastasis in NSCLC (P<0.05). The patients with high miR-146a expression in their tumors showed longer progression-free survival (25.6 weeks in miR-146a high patients vs. 4.8 weeks in miR-146a low patients, P<0.05). miR-146a is therefore a strong candidate prognostic biomarker in NSCLC. Thus inducing miR-146a might be a therapeutic strategy for NSCLC.

Background In the majority of familial breast cancer (BC) families, the etiology of the disease remains unresolved. To identify missing BC heritability resulting from relatively rare variants (minor allele frequency ≤ 1%), we have performed whole exome sequencing followed by variant analysis in a virtual panel of 492 cancer-associated genes on BC patients from BRCA1 and BRCA2 negative families with elevated BC risk. Methods BC patients from 54 BRCA1 and BRCA2 -negative families with elevated BC risk and 120 matched controls were considered for germline DNA whole exome sequencing. Rare variants identified in the exome and in a virtual panel of cancer-associated genes [492 genes associated with different types of (hereditary) cancer] were compared between BC patients and controls. Nonsense, frame-shift indels and splice-site variants (strong protein-damaging variants, called PDAVs later on) observed in BC patients within the genes of the panel, which we estimated to possess the highest probability to predispose to BC, were further validated using an alternative sequencing procedure. Results Exome- and cancer-associated gene panel-wide variant analysis show that there is no significant difference in the average number of rare variants found in BC patients compared to controls. However, the genes in the cancer-associated gene panel with nonsense variants were more than two-fold over-represented in women with BC and commonly involved in the DNA double-strand break repair process. Approximately 44% (24 of 54) of BC patients harbored 31 PDAVs, of which 11 were novel. These variants were found in genes associated with known or suspected BC predisposition ( PALB2, BARD1, CHEK2, RAD51C and FANCA ) or in predisposing genes linked to other cancer types but not well-studied in the context of familial BC ( EXO1, RECQL4, CCNH, MUS81, TDP1, DCLRE1A, DCLRE1C, PDE11A and RINT1 ) and genes associated with different hereditary syndromes but not yet clearly associated with familial cancer syndromes ( ABCC11, BBS10, CD96, CYP1A1, DHCR7, DNAH11, ESCO2, FLT4, HPS6, MYH8, NME8 and TTC8 ). Exome-wide, only a few genes appeared to be enriched for PDAVs in the familial BC patients compared to controls. Conclusions We have identified a series of novel candidate BC predisposition variants/genes. These variants/genes should be further investigated in larger cohorts/case-control studies. Other studies including co-segregation analyses in affected families, locus-specific loss of heterozygosity and functional studies should shed further light on their relevance for BC risk.

Background Among the 10% of pancreatic cancers that occur in a familial context, around a third carry a pathogenic variant in a cancer predisposition gene. Genetic studies of pancreatic cancer predisposition are limited by high mortality rates amongst index patients and other affected family members. The genetic risk for pancreatic cancer is often shared with breast cancer susceptibility genes, most notably BRCA2, PALB2, ATM and BRCA1 . Therefore, we hypothesized that additional shared genetic etiologies might be uncovered by studying families presenting with both breast and pancreatic cancer. Methods Focusing on a multigene panel of 276 DNA Damage Repair (DDR) genes, we performed next-generation sequencing in a cohort of 41 families with at least three breast cancer cases and one pancreatic cancer. When the index patient with pancreatic cancer was deceased, close relatives (first or second-degree) affected with breast cancer were tested (39 families). Results We identified 27 variants of uncertain significance in DDR genes. A splice site variant (c.1605 + 2T > A) in the RAD17 gene stood out, as a likely loss of function variant. RAD17 is a checkpoint protein that recruits the MRN (MRE11-RAD50-NBS1) complex to initiate DNA signaling, leading to DNA double-strand break repair. Conclusion Within families with breast and pancreatic cancer, we identified RAD17 as a novel candidate predisposition gene. Further genetic studies are warranted to better understand the potential pathogenic effect of RAD17 variants and in other DDR genes.

Non-small cell lung cancer (NSCLC) patients harboring activating mutations in epidermal growth factor receptor (EGFR) are sensitive to therapy with EGFR tyrosine kinase inhibitors (TKI). Despite remarkable clinical responses using EGFR TKI, surviving drug tolerant cells serve as a reservoir from which drug resistant tumors may emerge. This study addresses the need for improved efficacy of EGFR TKI by identifying targets involved in functional drug tolerance against them. To this aim, a high-throughput siRNA kinome screen was performed using two EGFR TKI-sensitive EGFR-mutant NSCLC cell lines in the presence/absence of the second-generation EGFR TKI afatinib. From the screen, Serine/Threonine/Tyrosine Kinase 1 (STYK1) was identified as a target that when downregulated potentiates the effects of EGFR inhibition in vitro. We found that chemical inhibition of EGFR combined with the siRNA-mediated knockdown of STYK1 led to a significant decrease in cancer cell viability and anchorage-independent cell growth. Further, we show that STYK1 selectively interacts with mutant EGFR and that the interaction is disrupted upon EGFR inhibition. Finally, we identified fibroblast growth factor 1 (FGF1) as a downstream effector of STYK1 in NSCLC cells. Accordingly, downregulation of STYK1 counteracted the afatinib-induced upregulation of FGF1. Altogether, we unveil STYK1 as a valuable target to repress the pool of surviving drug tolerant cells arising upon EGFR inhibition. Co-targeting of EGFR and STYK1 could lead to a better overall outcome for NSCLC patients.

Background The epidermal growth factor receptor (EGFR) is a validated therapeutic target in non-small cell lung cancer (NSCLC). However, current single agent receptor targeting does not achieve a maximal therapeutic effect, and some mutations confer resistance to current available agents. In the current study we have examined, in different NSCLC cell lines, the combined effect of RNA interference targeting the EGFR mRNA, and inactivation of EGFR signaling using different receptor tyrosine kinase inhibitors (TKIs) or a monoclonal antibody cetuximab. Methods NSCLC cells (cell lines HCC827, H292, H358, H1650, and H1975) were transfected with EGFR siRNA and/or treated with the TKIs gefitinib, erlotinib, and afatinib, and/or with the monoclonal antibody cetuximab. The reduction of EGFR mRNA expression was measured by real-time quantitative RT-PCR. The down-regulation of EGFR protein expression was measured by western blot, and the proliferation, viability, caspase3/7 activity, and apoptotic morphology were monitored by spectrophotometry, fluorimetry, and fluorescence microscopy. The combined effect of EGFR siRNA and different drugs was evaluated using a combination index. Results EGFR-specific siRNA strongly inhibited EGFR protein expression almost equally in all cell lines and inhibited cell growth and induced cell apoptosis in all NSCLC cell lines studied, albeit with a different magnitude. The effects on growth obtained with siRNA was strikingly different from the effects obtained with TKIs. The effects of siRNA probably correlate with the overall oncogenic significance of the receptor, which is only partly inhibited by the TKIs. The cells which showed weak response to TKIs, such as the H1975 cell line containing the T790M resistance mutation, were found to be responsive to siRNA knockdown of EGFR, as were cell lines with downstream TKI resistance mutations. The cell line HCC827, harboring an exon 19 deletion mutation, was more than 10-fold more sensitive to TKI proliferation inhibition and apoptosis induction than any of the other cell lines. Cetuximab alone had no relevant in vitro activity at concentrations obtainable in the clinic. The addition of EGFR siRNA to either TKIs or cetuximab additively enhanced growth inhibition and induction of apoptosis in all five cell lines, independent of the EGFR mutation status (wild-type or sensitizing mutation or resistant mutation). The strongest biological effect was observed when afatinib was combined with an EGFR-specific siRNA. Conclusions EGFR knockdown by siRNA further decreases the cell growth of lung cancer cells that are treated with TKIs or cetuximab alone, confirming that single agent drug targeting does not achieve a maximal biological effect. The siRNA inhibits EGFR oncogenic activity that bypasses downstream \"resistance\" mutations such as KRAS and PTEN. The combined treatment of siRNA and EGFR inhibitory agents is additive. The combination of a potent, irreversible kinase inhibitor such as afatinib, with EGFR-specific siRNAs should be further investigated as a new strategy in the treatment of lung cancer and other EGFR dependent cancers, including those with downstream resistance mutations.

The Belgian Approach for Local Laboratory Extensive Tumor Testing (BALLETT) study assessed the feasibility of using comprehensive genomic profiling (CGP) in clinical decision-making for patients with advanced cancers. This multi-center study enrolled 872 patients from 12 Belgian hospitals. CGP was performed on tumor tissues using a standardized CGP panel (523 genes) across nine laboratories with success in 93% of patients and a median turnaround time of 29 days. Actionable genomic markers were identified in 81% of patients, substantially higher than the 21% using nationally reimbursed, small panels. A national molecular tumor board (nMTB) recommended treatments for 69% of patients, with 23% receiving matched therapies. Reasons for non-compliance were highly variable across clinical sites. Overall, BALLETT demonstrates the feasibility of implementing decentralized CGP and its potential to identify actionable targets in most patients with advanced cancers. BALLETT reinforces CGP’s utility and emphasizes the importance of collaboration, standardization, and addressing implementation challenges.

The combination of radiotherapy (RT) with immunotherapy represents a promising treatment modality for non-small cell lung cancer (NSCLC) patients. As only a minority of patients shows a persistent response today, a spacious optimization window remains to be explored. Previously we showed that fractionated RT can induce a local immunosuppressive profile. Based on the evolving concept of an immunomodulatory role for vagal nerve stimulation (VNS), we tested its therapeutic and immunological effects alone and in combination with fractionated RT in a preclinical-translational study. Lewis lung carcinoma-bearing C57Bl/6 mice were treated with VNS, fractionated RT or the combination while a patient cohort with locally advanced NSCLC receiving concurrent radiochemotherapy (ccRTCT) was enrolled in a clinical trial to receive either sham or effective VNS daily during their 6 weeks of ccRTCT treatment. Preclinically, VNS alone or with RT showed no therapeutic effect yet VNS alone significantly enhanced the activation profile of intratumoral CD8 + T cells by upregulating their IFN-γ and CD137 expression. In the periphery, VNS reduced the RT-mediated rise of splenic, but not blood-derived, regulatory T cells (Treg) and monocytes. In accordance, the serological levels of protumoral CXCL5 next to two Treg-attracting chemokines CCL1 and CCL22 were reduced upon VNS monotherapy. In line with our preclinical findings on the lack of immunological changes in blood circulating immune cells upon VNS, immune monitoring of the peripheral blood of VNS treated NSCLC patients (n=7) did not show any significant changes compared to ccRTCT alone. As our preclinical data do suggest that VNS intensifies the stimulatory profile of the tumor infiltrated CD8 + T cells, this favors further research into non-invasive VNS to optimize current response rates to RT-immunotherapy in lung cancer patients.

Epidermal growth factor receptor (EGFR) and c-MET receptors are expressed on many non-small cell lung cancer (NSCLC) cells. Current single agent therapeutic targeting of a mutant EGFR has a high efficacy in the clinic, but is not curative. Here, we investigated the combination of targeting EGFR and c-MET pathways in NSCLC cells resistant to receptor tyrosine kinase inhibitors (TKIs), using RNA interference and inhibition by TKIs. Different NSCLC cell lines with various genomic characteristics (H358, H1650 and H1975) were transfected with EGFR-specific-siRNA, T790M-specific-siRNA, c-MET siRNA or the combination. Subsequently EGFR TKIs (gefitinib, erlotinib or afatinib) or monoclonal antibody cetuximab were combined respectively with the c-MET-specific TKI su11274 in NSCLC cell lines. The cell proliferation, viability, caspase-3/7 activity and apoptotic morphology were monitored by spectrophotometry, fluorimetry and fluorescence microscopy. The combined effect of EGFR TKIs, or cetuximab and su11274, was evaluated using a combination index. The results showed that the cell lines that were relatively resistant to EGFR TKIs, especially the H1975 cell line containing the resistance T790M mutation, were found to be more sensitive to EGFR-specific-siRNA. The combination of EGFR siRNA plus c-MET siRNA enhanced cell growth inhibition, apoptosis induction and inhibition of downstream signaling in EGFR TKI resistant H358, H1650 and H1975 cells, despite the absence of activity of the c-MET siRNA alone. EGFR TKIs or cetuximab plus su11274 were also consistently superior to either agent alone. The strongest biological effect was observed when afatinib, an irreversible pan-HER blocker was combined with su11274, which achieved a synergistic effect in the T790M mutant H1975 cells. In a conclusion, our findings offer preclinical proof of principle for combined inhibition as a promising treatment strategy for NSCLC, especially for patients in whom current EGFR-targeted treatments fail due to the presence of the T790M-EGFR-mutation or high c-MET expression.

Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibition is the preferred first-line treatment of advanced adenocarcinoma of the lung that harbors EGFR activating tyrosine kinase domain mutations. Most data available pertain to Asian populations in which such mutations are more prevalent. We report on the long-term results of first-line treatment with erlotinib in Caucasian patients with advanced adenocarcinoma of the lung that have a somatic EGFR mutation in their tumor. Multicenter academic prospective phase II study with erlotinib in patients with an activating EGFR tyrosine kinase (TK) domain somatic mutation (any exon encoding the kinase domain) in the tumor and no prior treatment for their advanced disease. Phenotypic preselecting of 229 patients led to a high EGFR mutation detection rate of 24% of which 46 patients were included in the phase II study. With a progression free survival (PFS) of 81% at three months the study met its primary endpoint for presumed superiority over chemotherapy. With an overall median PFS of 11 months and a median overall survival (OS) of 23 months, the results compare favorably with results obtained in randomized studies using TKI in first line in EGFR mutation positive adenocarcinoma of the lung. The present study reinforces the use of EGFR tyrosine kinase inhibition (TKI) as a first line treatment of choice for advanced adenocarcinoma of the lung carrying an activating EGFR mutation. The mutation rate in preselected Caucasian patients is higher than previously reported. Issues relevant for clinical practice are discussed. ClinicalTrials.gov NCT00339586.

SummaryBackground In the first part of this extensive phase I study (NCT00516724), continuous olaparib twice daily (bid) with carboplatin and/or paclitaxel resulted in myelosuppression and dose modifications. Here, we report the safety, tolerability, and efficacy of intermittent olaparib dosing combined with carboplatin and paclitaxel. Methods Patients with advanced solid tumors (part D) and enriched for ovarian and breast cancer (part E) received olaparib (capsule and tablet formulations) using intermittent schedules (2 to 10 days of a 21-day cycle) combined with carboplatin/paclitaxel. Safety assessments included evaluation of dose-limiting toxicities (DLTs; cycle 1 only), adverse events (AEs), and physical examinations. Pharmacokinetic assessments of olaparib capsule and tablet combined with carboplatin/paclitaxel were performed. Tumor responses (RECIST) were assessed every 2 cycles. Results In total, 132 heavily pre-treated patients were included. One DLT of grade 3 elevated alanine aminotransferase lasting for 8 days was reported (olaparib tablet 100 mg bid days 3–12, carboplatin area under the curve 4 and paclitaxel 175 mg/m2). The most common hematological AEs were neutropenia (47%) and thrombocytopenia (39%), which frequently led to dose modifications. Non-hematological AEs were predominantly grade 1–2, including alopecia (89%) and fatigue (84%). Overall objective response rate was 46%. Conclusions Discontinuous dosing of olaparib resulted in significant myelosuppression leading to dose interruptions and/or delays. Anti-tumor activity was encouraging in patients enriched with BRCA-mutated breast and ovarian cancer. The most appropriate olaparib tablet dose for use in further studies evaluating olaparib in combination with carboplatin and paclitaxel is 50 mg bid (days 1–5).