INSIGHT 2: a phase II study of tepotinib plus osimertinib in MET-amplified NSCLC and first-line osimertinib resistance.

MET amplification (METamp), a mechanism of acquired resistance to EGFR tyrosine kinase inhibitors, occurs in up to 30% of patients with non-small-cell lung cancer (NSCLC) progressing on first-line osimertinib. Combining osimertinib with a MET inhibitor, such as tepotinib, an oral, highly selective, potent MET tyrosine kinase inhibitor, may overcome METamp-driven resistance. INSIGHT 2 (NCT03940703), an international, open-label, multicenter phase II trial, assesses tepotinib plus osimertinib in patients with advanced/metastatic EGFR-mutant NSCLC and acquired resistance to first-line osimertinib and METamp, determined centrally by fluorescence in situ hybridization (gene copy number ≥5 and/or MET/CEP7 ≥2) at time of progression. Patients will receive tepotinib 500 mg (450 mg active moiety) plus osimertinib 80 mg once-a-day. The primary end point is objective response, and secondary end points include duration of response, progression-free survival, overall survival and safety. Trial registration number: NCT03940703 (clinicaltrials.gov).

Osimertinib is used to treat a type of lung cancer that has specific changes (mutations) in a gene called EGFR. Although tumors will usually shrink (respond) during treatment with osimertinib, they can stop responding, or become resistant, to osimertinib. A common cause of resistance is 'MET amplification', which describes when extra copies of a gene called MET are present. Lung cancer that is resistant to osimertinib due to MET amplification could be treated by combining osimertinib with a treatment that blocks MET, such as tepotinib. INSIGHT 2 is an ongoing study that is designed to learn about the effects and safety of tepotinib combined with osimertinib, in patients with lung cancer that has stopped responding to osimertinib because of MET amplification. A plain language version of this article is available and is published alongside the paper online: www.futuremedicine.com/doi/suppl/10.2217/fon-2021-1406.

Novel molecular targets for the treatment of lung cancer.

PURPOSE OF REVIEW: The mutational landscape in lung adenocarcinoma (LADC) is broadly recognized, particularly regarding the presence of the epidermal growth factor receptor (EGFR) mutation in non-smokers. However, even in the EGFR canonical-mutant LADC, other accompanying alterations surface which may have a major impact in prognosis and open possibilities to explore new therapeutic approaches. RECENT FINDINGS: Complex genomic rearrangements, including chromothripsis and chromoplexy, are the origin of most-known fusion oncogenes, including echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase, Cluster of Differentiation 74-c-ros oncogene 1, and kinesin-1 heavy chain- rearranged during transfection. Quite often LADCs driven by fusion oncogenes are accompanied by SET domain containing 2 (SETD2) mutations. SETD2 mutations have been described in renal cancer and have been related to cisplatin resistance in LADCs. Suppression of the SETD2 function inhibits the signal transducer and transcription activator function and the interferon-signaling pathway, which could partially explain the lack of effectiveness of immunotherapy in LADCs driven by fusion oncogenes. SUMMARY: Targeted next-generation sequencing of DNA in the tumor tissue or in the circulating plasma of LADC is becoming indispensable for the accurate classification of LADCs that can receive appropriate targeted therapy. It is unquestionable that additional techniques, like RNA sequencing or the nCounter technology, can accomplish accurate assessment of an ample array of fusion oncogenes involved in LADCs.

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.

Targeting PKCι-PAK1 signaling pathways in EGFR and KRAS mutant adenocarcinoma and lung squamous cell carcinoma.

INTRODUCTION: p21-activated kinase 1 (PAK1) stimulates growth and metastasis in non-small cell lung cancer (NSCLC). Protein kinase C iota (PKCι) is an enzyme highly expressed in NSCLC, regulating PAK1 signaling. In the present study we explored whether the PKCι-PAK1 signaling pathway approach can be an efficient target in different types of NSCLC cell and mouse models. METHODS: The effect of IPA-3 (PAK1 inhibitor) plus auranofin (PKCι inhibitor) combination was evaluated by cell viability assay, colony formation and western blotting assay, using three types of NSCLC cell lines: EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma with PAK1 amplification. In addition, for clinical availability, screening for new PAK1 inhibitors was carried out and the compound OTSSP167 was evaluated in combination with auranofin in cell and mice models. RESULTS: The combination of IPA-3 or OTSSP167 plus auranofin showed high synergism for inhibiting cell viability and colony formation in three cell lines. Mechanistic characterization revealed that this drug combination abrogated expression and activation of membrane receptors and downstream signaling proteins crucial in lung cancer: EGFR, MET, PAK1, PKCι, ERK1/2, AKT, YAP1 and mTOR. A nude mouse xenograft assay demonstrated that this drug combination strongly suppressed tumor volume compared with single drug treatment. CONCLUSIONS: Combination of IPA-3 or OTSSP167 and auranofin was highly synergistic in EGFR or KRAS mutant adenocarcinoma and squamous cell carcinoma cell lines and decreased tumor volume in mice models. It is of interest to further test the targeting of PKCι-PAK1 signaling pathways in EGFR mutant, KRAS mutant and squamous NSCLC patients.

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.

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.

Identification of ALK, ROS1, and RET Fusions by a Multiplexed mRNA-Based Assay in Formalin-Fixed, Paraffin-Embedded Samples from Advanced Non-Small-Cell Lung Cancer Patients.

BACKGROUND: Anaplastic lymphoma receptor tyrosine kinase (ALK), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1), and ret proto-oncogene (RET) fusions are present in 5%-7% of patients with advanced non-small-cell lung cancer (NSCLC); their accurate identification is critical to guide targeted therapies. FISH and immunohistochemistry (IHC) are considered the gold standards to determine gene fusions, but they have limitations. The nCounter platform is a potentially useful genomic tool for multiplexed detection of gene fusions, but has not been validated in the clinical setting. METHODS: Formalin-fixed, paraffin embedded (FFPE) samples from 108 patients with advanced NSCLC were analyzed with an nCounter-based assay and the results compared with FISH, IHC, and reverse transcription PCR (RT-PCR). Data on response to fusion kinase inhibitors was retrospectively collected in a subset of 29 patients. RESULTS: Of 108 FFPE samples, 98 were successfully analyzed by nCounter (91%), which identified 55 fusion-positive cases (32 ALK, 21 ROS1, and 2 RET). nCounter results were highly concordant with IHC for ALK (98.5%, CI = 91.8-99.7), while 11 discrepancies were found compared with FISH (87.5% concordance, CI = 79.0-92.9). For ROS1, nCounter showed similar agreement with IHC and FISH (87.2% and 85.9%), but a substantial number of samples were positive only by 1 or 2 techniques. Of the 25 patients deriving clinical benefit from fusion kinase inhibitors, 24 were positive by nCounter and 22 by FISH. CONCLUSIONS: nCounter compares favorably with IHC and FISH and can be used for identifying patients with advanced NSCLC positive for ALK/ROS1/RET fusion genes.

Relationship between gene mutation and lung cancer metastasis.

Cancer is a genetic disease occurring through a multi-step process. Many important genes responsible for the genesis of various cancers have been discovered, their mutations precisely identified and the pathways through which they act characterized. One question that remains unanswered is whether the development of new, more specific therapeutic agents is the best way to minimize cancer morbidity and mortality in the long-term. Metastasis is the relentless pursuit of cancer to escape its primary site and colonize distant organs. Phenotypic changes during cancer progression reflect the sequential accumulation of genetic alterations, which endow cancer cells with the ability to undergo their own divergent evolution and create distinct metastatic species. In order to understand this process, it is crucial to identify genes whose alterations accumulate during cancer progression and correlate with metastatic phenotypes of cancer cells.

Genetics and biomarkers in personalisation of lung cancer treatment.

Non-small-cell lung cancer is often diagnosed at the metastatic stage, with median survival of just 1 year. The identification of driver mutations in the epidermal growth factor receptor (EGFR) as the primary oncogenic event in a subset of lung adenocarcinomas led to a model of targeted treatment and genetic profiling of the disease. EGFR tyrosine kinase inhibitors confer remission in 60% of patients, but responses are short-lived. The pre-existing EGFR Thr790Met mutation could be a subclonal driver responsible for these transient responses. Overexpression of AXL and reduced MED12 function are hallmarks of resistance to tyrosine kinase inhibitors in EGFR-mutant non-small-cell lung cancer. Crosstalk between signalling pathways is another mechanism of resistance; therefore, identification of the molecular components involved could lead to the development of combination therapies cotargeting these molecules instead of EGFR tyrosine kinase inhibitor monotherapy. Additionally, novel biomarkers could be identified through deep sequencing analysis of serial rebiopsies before and during treatment.

The Preclinical Pharmacology of Tepotinib-A Highly Selective MET Inhibitor with Activity in Tumors Harboring MET Alterations.

The mesenchymal-epithelial transition factor (MET) proto-oncogene encodes the MET receptor tyrosine kinase. MET aberrations drive tumorigenesis in several cancer types through a variety of molecular mechanisms, including MET mutations, gene amplification, rearrangement, and overexpression. Therefore, MET is a therapeutic target and the selective type Ib MET inhibitor, tepotinib, was designed to potently inhibit MET kinase activity. In vitro, tepotinib inhibits MET in a concentration-dependent manner irrespective of the mode of MET activation, and in vivo, tepotinib exhibits marked, dose-dependent antitumor activity in MET-dependent tumor models of various cancer indications. Tepotinib penetrates the blood-brain barrier and demonstrates strong antitumor activity in subcutaneous and orthotopic brain metastasis models, in-line with clinical activity observed in patients. MET amplification is an established mechanism of resistance to EGFR tyrosine kinase inhibitors (TKI), and preclinical studies show that tepotinib in combination with EGFR TKIs can overcome this resistance. Tepotinib is currently approved for the treatment of adult patients with advanced or metastatic non-small cell lung cancer harboring MET exon 14 skipping alterations. This review focuses on the pharmacology of tepotinib in preclinical cancer models harboring MET alterations and demonstrates that strong adherence to the principles of the Pharmacological Audit Trail may result in a successful discovery and development of a precision medicine.

Overcoming MET-mediated resistance in oncogene-driven NSCLC.

This study evaluates the efficacy of combining targeted therapies with MET or SHP2 inhibitors to overcome MET-mediated resistance in different NSCLC subtypes. A prevalence study was conducted for MET amplification and overexpression in samples from patients with NSCLC who relapsed on ALK, ROS1, or RET tyrosine kinase inhibitors. MET-mediated resistance was detected in 37.5% of tissue biopsies, which allow the detection of MET overexpression, compared to 7.4% of liquid biopsies. The development of drug resistance by MET overexpression was confirmed in EGFRex19del-, KRASG12C-, HER2ex20ins-, and TPM3-NTRK1-mutant cell lines. The combination of targeted therapy with MET or SHP2 inhibitors was found to overcome MET-mediated resistance in both in vitro and in vivo assays. This study highlights the importance of considering MET overexpression as a resistance driver to NSCLC targeted therapies to better identify patients who could potentially benefit from combination approaches with MET or SHP2 inhibitors.

Brief Report: Clinical Response, Toxicity, and Resistance Mechanisms to Osimertinib Plus MET Inhibitors in Patients With EGFR-Mutant MET-Amplified NSCLC.

Introduction: MET amplification is a known resistance mechanism to EGFR tyrosine kinase inhibitor (TKI) treatment in EGFR-mutant NSCLC. Dual EGFR-MET inhibition has been reported with success in overcoming such resistance and inducing clinical benefit. Resistance mechanisms to dual EGFR-MET inhibition require further investigation and characterization. Methods: Patients with NSCLC with both MET amplification and EGFR mutation who have received crizotinib, capmatinib, savolitinib, or tepotinib plus osimertinib (OSI) after progression on OSI at MD Anderson Cancer Center were included in this study. Molecular profiling was completed by means of fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS). Radiological response was assessed on the basis of Response Evaluation Criteria in Solid Tumors version 1.1. Results: From March 2016 to March 2022, 23 treatments with dual MET inhibitor and osi were identified with a total of 20 patients included. Three patients received capmatinib plus OSI after progression on crizotinib plus OSI. Median age was 64 (38-89) years old and 75% were female. MET amplification was detected by FISH in 14 patients in the tissue, NGS in 10 patients, and circulating tumor DNA in three patients. Median MET gene copy number was 13.6 (6.4-20). Overall response rate was 34.8% (eight of 23). In assessable patients, tumor shrinkage was observed in 82.4% (14 of 17). Median time on treatment was 27 months. Two of three patients responded to capmatinib plus OSI after progression on crizotinib plus OSI. Dual EGFR-MET inhibition was overall well tolerated. Two patients on crizotinib plus OSI and one pt on capmatinib plus OSI discontinued therapy due to pneumonitis. One pt discontinued crizotinib plus OSI due to gastrointestinal toxicity. Six patients were still on double TKI treatment. At disease progression to dual EGFR-MET inhibition, FISH and NGS on tumor and plasma were completed in six patients. Notable resistance mechanisms observed include acquired MET D1246H (n = 1), acquired EGFR C797S (n = 2), FGFR2 fusion (n = 1, concurrent with C797S), and EGFR G796S (n = 1, concurrent with C797S). Four patients lost MET amplification. Conclusions: Dual EGFR and MET inhibition yielded high clinical response rate after progression on OSI. Resistance mechanisms to EGFR-MET double TKI inhibition include MET secondary mutation, EGFR secondary mutation, or loss of MET amplification.

Effect of Osimertinib on CTCs and ctDNA in EGFR Mutant Non-Small Cell Lung Cancer Patients: The Prognostic Relevance of Liquid Biopsy.

INTRODUCTION: Liquid biopsy is a useful tool for monitoring treatment outcome in solid tumors, including lung cancer. The relevance of monitoring CTCs and plasma ctDNA as predictors of clinical outcome was assessed in EGFR-mutant NSCLC patients treated with osimertinib. METHODS: Forty-seven EGFR-mutant NSCLC patients who had progressed on prior first- or second-generation EGFR inhibitors were enrolled in the study and treated with osimertinib, irrespective of the presence of the T790M mutation in the primary tumor or the plasma. Peripheral blood was collected at baseline (n = 47), post-Cycle 1 (n = 47), and at the end of treatment (EOT; n = 39). CTCs were evaluated in 32 patients at the same time points (n = 32, n = 27, and n = 21, respectively) and phenotypic characterization was performed using triple immunofluorescence staining (CK/VIM/CD45). RESULTS: Osimertinib resulted in an ORR of 34% (2 CR) and a DCR of 76.6%. The median PFS and OS values were 7.5 (range, 0.8-52.8) and 15.1 (range, 2.1-52.8) months, respectively. ctDNA was detected in 61.7%, 27.7%, and 61.5% of patients at baseline, post-Cycle 1, and EOT, respectively. CTCs (CK+/CD45-) were detected in 68.8%, 48.1%, and 61.9% of patients at the three time points, respectively. CTCs expressing both epithelial and mesenchymal markers (CK+/VIM+/CD45-) were detected in 56.3% and 29.6% of patients at baseline and post-Cycle 1, respectively. The detection of ctDNA at baseline and post-Cycle 1 was associated with shorter PFS and OS, whereas the ctDNA clearance post-Cycle 1 resulted in a significantly longer PFS and OS. Multivariate analysis revealed that male sex and the detection of ctDNA at baseline were independent predictors of shorter PFS (HR: 2.6, 95% C.I.: 1.2-5.5, p = 0.015 and HR: 3.0, 95% C.I.: 1.3-6.9; p = 0.009, respectively). CONCLUSIONS: The decrease in both CTCs and ctDNA occurring early during osimertinib treatment is predictive of better outcome, implying that liquid biopsy monitoring may be a valuable tool for the assessment of treatment efficacy.

Deficiency of the splicing factor RBM10 limits EGFR inhibitor response in EGFR-mutant lung cancer.

Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.