Preliminary Evidence on the Diagnostic and Molecular Role of Circulating Soluble EGFR in Non-Small Cell Lung Cancer

Assessment of biological diagnostic factors providing clinically-relevant information to guide physician decision-making are still needed for diseases with poor outcomes, such as non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) is a promising molecule in the clinical management of NSCLC. While the EGFR transmembrane form has been extensively investigated in large clinical trials, the soluble, circulating EGFR isoform (sEGFR), which may have a potential clinical use, has rarely been considered. This study investigates the use of sEGFR as a potential diagnostic biomarker for NSCLC and also characterizes the biological function of sEGFR to clarify the molecular mechanisms involved in the course of action of this protein. Plasma sEGFR levels from a heterogeneous cohort of 37 non-advanced NSCLC patients and 54 healthy subjects were analyzed by using an enzyme-linked immunosorbent assay. The biological function of sEGFR was analyzed in vitro using NSCLC cell lines, investigating effects on cell proliferation and migration. We found that plasma sEGFR was significantly decreased in the NSCLC patient group as compared to the control group (median value: 48.6 vs. 55.6 ng/mL respectively; p = 0.0002). Moreover, we demonstrated that sEGFR inhibits growth and migration of NSCLC cells in vitro through molecular mechanisms that included perturbation of EGF/EGFR cell signaling and holoreceptor internalization. These data show that sEGFR is a potential circulating biomarker with a physiological protective role, providing a first approach to the functional role of the soluble isoform of EGFR. However, the impact of these data on daily clinical practice needs to be further investigated in larger prospective studies.     

Hybrid Pharmacophore- and Structure-Based Virtual Screening Pipeline to Identify Novel EGFR Inhibitors That Suppress Non-Small Cell Lung Cancer Cell Growth

Dysregulated epidermal growth factor receptor (EGFR) expression is frequently observed in non-small cell lung cancer (NSCLC) growth and metastasis. Despite recent successes in the development of tyrosine kinase inhibitors (TKIs), inevitable resistance to TKIs has led to urgent calls for novel EGFR inhibitors. Herein, we report a rational workflow used to identify novel EGFR-TKIs by combining hybrid ligand- and structure-based pharmacophore models. Three types of models were developed in this workflow, including 3D QSAR-, common feature-, and structure-based EGFR-TK domain-containing pharmacophores. A National Cancer Institute (NCI) compound dataset was adopted for multiple-stage pharmacophore-based virtual screening (PBVS) of various pharmacophore models. The six top-scoring compounds were identified through the PBVS pipeline coupled with molecular docking. Among these compounds, NSC609077 exerted a significant inhibitory effect on EGFR activity in gefitinib-resistant H1975 cells, as determined by an enzyme-linked immunosorbent assay (ELISA). Further investigations showed that NSC609077 inhibited the anchorage-dependent growth and migration of lung cancer cells. Furthermore, NSC609077 exerted a suppressive effect on the EGFR/PI3K/AKT pathway in H1975 cells. In conclusion, these findings suggest that hybrid virtual screening may accelerate the development of targeted drugs for lung cancer treatment.     

Diagnostic and Therapeutic Implications of microRNAs in Non-Small Cell Lung Cancer

microRNAs (miRNAs), endogenous suppressors of target mRNAs, are deeply involved in every step of non-small cell lung cancer (NSCLC) development, from tumor initiation to progression and metastasis. They play roles in cell proliferation, apoptosis, angiogenesis, epithelial-to-mesenchymal transition, migration, invasion, and metastatic colonization, as well as immunosuppression. Due to their versatility, numerous attempts have been made to use miRNAs for clinical applications. miRNAs can be used as cancer subtype classifiers, diagnostic markers, drug-response predictors, prognostic markers, and therapeutic targets in NSCLC. Many challenges remain ahead of their actual clinical application; however, when achieved, the use of miRNAs in the clinic is expected to enable great progress in the diagnosis and treatment of patients with NSCLC.     

WISP1 Polymorphisms Contribute to Platinum-Based Chemotherapy Toxicity in Lung Cancer Patients

Platinum-based chemotherapy toxicity is always one of the serious problems from which lung cancer patients suffer. The genetic polymorphism of WISP1 was revealed to be associated with susceptibility and platinum-based chemotherapy response in our previous studies. In this study, we aimed to investigate the relationship of WISP1 genetic polymorphisms with platinum-based chemotherapy toxicity in lung cancer patients. A total of 412 lung cancer patients were enrolled in this study, and 28 polymorphisms of the WISP1 gene were genotyped by SequenomMassARRAY. We found that WISP1 polymorphisms (rs2929965, rs2929969, rs2929970, rs2929973 and rs754958) were related to the overall chemotherapy toxicity of lung cancer in subgroup analyses. Rs16904853, rs2929970, rs2977549 and rs2977551 (p = 0.021, 0.028, 0.024, 0.048, respectively) polymorphisms were significantly associated with hematologic toxicity. Rs2929946, rs2929970, rs2977519, rs2977536, rs3739262 and rs754958 (p = 0.031, 0.046, 0.029, 0.016, 0.042, 0.035, respectively) polymorphisms were significantly associated with the gastrointestinal toxicity of lung cancer. Genotypes of WISP1 may be novel and useful biomarkers for predicting platinum-based chemotherapy toxicity in lung cancer patients.     

CHID1 Is a Novel Prognostic Marker of Non-Small Cell Lung Cancer

There is an urgent need for identification of new prognostic markers and therapeutic targets for non-small cell lung cancer (NSCLC). In this study, we evaluated immune cells markers in 100 NSCLC specimens. Immunohistochemical analysis revealed no prognostic value for the markers studied, except CD163 and CD206. At the same time, macrophage markers iNOS and CHID1 were found to be expressed in tumor cells and associated with prognosis. We showed that high iNOS expression is a marker of favorable prognosis for squamous cell lung carcinoma (SCC), and NSCLC in general. Similarly, high CHID1 expression is a marker of good prognosis in adenocarcinoma and in NSCLC in general. Analysis of prognostic significance of a high CHID1/iNOS expression combination showed favorable prognosis with 20 months overall survival of patients from the low CHID1/iNOS expression group. For the first time, we demonstrated that CHID1 can be expressed by NSCLC cells and its high expression is a marker of good prognosis for adenocarcinoma and NSCLC in general. At the same time, high expression of iNOS in tumor cells is a marker of good prognosis in SCC. When used in combination, CHID1 and iNOS show a very good prognostic capacity for NSCLC. We suggest that in the case of lung cancer, tumor-associated macrophages are likely ineffective as a therapeutic target. At the same time, macrophage markers expressed by tumor cells may be considered as targets for anti-tumor therapy or, as in the case of CHID1, as potential anti-tumor agents.     

Polymorphisms in XPD and ERCC1 Associated with Colorectal Cancer Outcome

Using the comprehensive approach to selecting polymorphisms to date, we sought to examine whether recurrence in colorectal cancer was associated with inherited variation in three genes involved in DNA repair and cell proliferation. Three polymorphisms, which are excision repair cross-complementation 1 (ERCC1), xeroderma pigmentosum group D (XPD) and epidermal growth factor receptor (EGFR), were assessed in 257 postoperative stage II/III CRC patients with 5-fluorouracial chemotherapy in Taiwan. In addition, the correlations between genetic polymorphisms and patients’ clinicopathological features were investigated. Genotypes of XPD codon751 A/A and ERCC1 codon118 T/T were associated with regional recurrence in a statistically significant way (p = 0.018). Patients who carried XPD AA and ERCC1 TT genotypes demonstrated a significantly greater regional recurrence risk (OR = 5.625, 95% CI, 1.557–20.32). Inherited variation in XPD and ERCC1 was associated with outcome in patients with colorectal cancer in Taiwan. As the significant association of single-nucleotide polymorphisms has not been studied previously in colorectal cancer, these findings suggest novel sites of variation, in part explaining the range of treatment responses seen in this disease.     

Targeted Therapy and Immunotherapy in Early-Stage Non-Small Cell Lung Cancer: Current Evidence and Ongoing Trials

The scenario of neoadjuvant and adjuvant settings in non-small cell lung cancer (NSCLC) is rapidly evolving. As already happened for the advanced disease, also early stages have entered the era of precision medicine, with molecular analysis and Programmed death-ligand 1 (PD-L1) evaluation that by now can be considered a routine assessment. New treatment options have been recently approved, with osimertinib now part of clinical practice for Epidermal Growth Factor Receptor mutated (EGFRm) patients, and immune checkpoint inhibitors (ICIs) available after FDA approval both in the adjuvant (atezolizumab) and neoadjuvant (nivolumab) setting. No mature data on overall survival benefits are available yet, though. Several clinical trials with specific-tyrosine kinase inhibitors (TKIs) and ICIs are currently ongoing, both with and without concomitant chemotherapy. As therapeutic strategies are rapidly expanding, quite a few questions remain unsettled, such as the optimal duration of adjuvant targeted therapy or the effective benefit of ICIs in early-stage EGFRm or ALK (Anaplastic Lymphoma Kinase) rearranged patients, or the possibility to individuate high-risk patients after surgical resection assessing minimal residual disease (MRD) by ctDNA evaluation. We hereby report already available literature data and summarize ongoing trials with targeted therapy and immunotherapy in early-stage NSCLC, focusing on practice-changing results and new perspectives for potentially cured patients.     

Novel Emerging Molecular Targets in Non-Small Cell Lung Cancer

In the scenario of systemic treatment for advanced non-small cell lung cancer (NSCLC) patients, one of the most relevant breakthroughs is represented by targeted therapies. Throughout the last years, inhibitors of the epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-Ros oncogene 1 (ROS1), and V-raf murine sarcoma viral oncogene homolog B (BRAF) have been approved and are currently used in clinical practice. However, other promising molecular drivers are rapidly emerging as therapeutic targets. This review aims to cover the molecular alterations with a potential clinical impact in NSCLC, including amplifications or mutations of the mesenchymal–epithelial transition factor (MET), fusions of rearranged during transfection (RET), rearrangements of the neurotrophic tyrosine kinase (NTRK) genes, mutations of the Kirsten rat sarcoma viral oncogene (KRAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), as well as amplifications or mutations of human epidermal growth factor receptor 2 (HER2). Additionally, we summarized the current status of targeted agents under investigation for such alterations. This revision of the current literature on emerging molecular targets is needed as the evolving knowledge on novel actionable oncogenic drivers and targeted agents is expected to increase the proportion of patients who will benefit from tailored therapeutic approaches.     

Protein Kinase C as a Therapeutic Target in Non-Small Cell Lung Cancer

Driver-directed therapeutics have revolutionized cancer treatment, presenting similar or better efficacy compared to traditional chemotherapy and substantially improving quality of life. Despite significant advances, targeted therapy is greatly limited by resistance acquisition, which emerges in nearly all patients receiving treatment. As a result, identifying the molecular modulators of resistance is of great interest. Recent work has implicated protein kinase C (PKC) isozymes as mediators of drug resistance in non-small cell lung cancer (NSCLC). Importantly, previous findings on PKC have implicated this family of enzymes in both tumor-promotive and tumor-suppressive biology in various tissues. Here, we review the biological role of PKC isozymes in NSCLC through extensive analysis of cell-line-based studies to better understand the rationale for PKC inhibition. PKC isoforms α, ε, η, ι, ζ upregulation has been reported in lung cancer, and overexpression correlates with worse prognosis in NSCLC patients. Most importantly, PKC isozymes have been established as mediators of resistance to tyrosine kinase inhibitors in NSCLC. Unfortunately, however, PKC-directed therapeutics have yielded unsatisfactory results, likely due to a lack of specific evaluation for PKC. To achieve satisfactory results in clinical trials, predictive biomarkers of PKC activity must be established and screened for prior to patient enrollment. Furthermore, tandem inhibition of PKC and molecular drivers may be a potential therapeutic strategy to prevent the emergence of resistance in NSCLC.     

KRAS and EGFR Mutations Differentially Alter ABC Drug Transporter Expression in Cisplatin-Resistant Non-Small Cell Lung Cancer

Lung carcinoma is still the most common malignancy worldwide. One of the major subtypes of non-small cell lung cancer (NSCLC) is adenocarcinoma (AC). As driver mutations and hence therapies differ in AC subtypes, we theorized that the expression and function of ABC drug transporters important in multidrug resistance (MDR) would correlate with characteristic driver mutations KRAS or EGFR. Cisplatin resistance (CR) was generated in A549 (KRAS) and PC9 (EGFR) cell lines and gene expression was tested. In three-dimensional (3D) multicellular aggregate cultures, both ABCB1 and ABCG2 transporters, as well as the WNT microenvironment, were investigated. ABCB1 and ABCG2 gene expression levels were different in primary AC samples and correlated with specific driver mutations. The drug transporter expression pattern of parental A549 and PC9, as well as A549-CR and PC9-CR, cell lines differed. Increased mRNA levels of ABCB1 and ABCG2 were detected in A549-CR cells, compared to parental A549, while the trend observed in the case of PC9 cells was different. Dominant alterations were observed in LEF1, RHOU and DACT1 genes of the WNT signalling pathway in a mutation-dependent manner. The study confirmed that, in lung AC-s, KRAS and EGFR driver mutations differentially affect both drug transporter expression and the cisplatin-induced WNT signalling microenvironment.     

MiR-122 Induces Radiosensitization in Non-Small Cell Lung Cancer Cell Line

MiR-122 is a novel tumor suppresser and its expression induces cell cycle arrest, or apoptosis, and inhibits cell proliferation in multiple cancer cells, including non-small cell lung cancer (NSCLC) cells. Radioresistance of cancer cell leads to the major drawback of radiotherapy for NSCLC and the induction of radiosensitization could be a useful strategy to fix this problem. The present work investigates the function of miR-122 in inducing radiosensitization in A549 cell, a type of NSCLC cells. MiR-122 induces the radiosensitization of A549 cells. MiR-122 also boosts the inhibitory activity of ionizing radiation (IR) on cancer cell anchor-independent growth and invasion. Moreover, miR-122 reduced the expression of its targeted genes related to tumor-survival or cellular stress response. These results indicate that miR-122 would be a novel strategy for NSCLC radiation-therapy.     

EGFR-Mutated Non-Small Cell Lung Cancer and Resistance to Immunotherapy: Role of the Tumor Microenvironment

Lung cancer is a leading cause of cancer-related deaths worldwide. About 10–30% of patients with non-small cell lung cancer (NSCLC) harbor mutations of the EGFR gene. The Tumor Microenvironment (TME) of patients with NSCLC harboring EGFR mutations displays peculiar characteristics and may modulate the antitumor immune response. EGFR activation increases PD-L1 expression in tumor cells, inducing T cell apoptosis and immune escape. EGFR-Tyrosine Kinase Inhibitors (TKIs) strengthen MHC class I and II antigen presentation in response to IFN-γ, boost CD8+ T-cells levels and DCs, eliminate FOXP3+ Tregs, inhibit macrophage polarization into the M2 phenotype, and decrease PD-L1 expression in cancer cells. Thus, targeted therapy blocks specific signaling pathways, whereas immunotherapy stimulates the immune system to attack tumor cells evading immune surveillance. A combination of TKIs and immunotherapy may have suboptimal synergistic effects. However, data are controversial because activated EGFR signaling allows NSCLC cells to use multiple strategies to create an immunosuppressive TME, including recruitment of Tumor-Associated Macrophages and Tregs and the production of inhibitory cytokines and metabolites. Therefore, these mechanisms should be characterized and targeted by a combined pharmacological approach that also concerns disease stage, cancer-related inflammation with related systemic symptoms, and the general status of the patients to overcome the single-drug resistance development.     

Safety of Surgery after Neoadjuvant Targeted Therapies in Non-Small Cell Lung Cancer: A Narrative Review

New drugs, including immune checkpoint inhibitors and targeted therapy, have changed the prognosis in a subset of patients with advanced lung cancer, and are now actively investigated in a number of trials with neoadjuvant and adjuvant regimens. However, no phase III randomized studies were published yet. The current narrative review proves that targeted therapies are safe in neoadjuvant approach. Unsurprisingly, administration of therapy is related to an acceptable toxicity profile. Severe adverse events’ rate that rarely compromises outcomes of patients with advanced lung cancer is not that commonly accepted in early lung cancer as it may lead to missing the chance of curative surgery. Among those complications, the most important factors that may limit the use of targeted therapies are severe respiratory adverse events precluding the resection occurring after treatment with some anaplastic lymphoma kinase and rarely after epidermal growth factor receptor tyrosine kinase inhibitors. At this point, in the presented literature assessing the feasibility of neoadjuvant therapies with anaplastic lymphoma kinase and epidermal growth factor receptor tyrosine kinase inhibitors, we did not find any unexpected intraoperative events that would be of special interest to a thoracic surgeon. Moreover, the postoperative course was associated with typical rate of complications.     

Anti-PD1/PD-L1 Immunotherapy for Non-Small Cell Lung Cancer with Actionable Oncogenic Driver Mutations

Anti-PD1/PD-L1 immunotherapy has emerged as a standard of care for stage III-IV non-small cell lung cancer (NSCLC) over the past decade. Patient selection is usually based on PD-L1 expression by tumor cells and/or tumor mutational burden. However, mutations in oncogenic drivers such as EGFR, ALK, BRAF, or MET modify the immune tumor microenvironment and may promote anti-PD1/PD-L1 resistance. In this review, we discuss the molecular mechanisms associated with these mutations, which shape the immune tumor microenvironment and may impede anti-PD1/PD-L1 efficacy. We provide an overview of the current clinical data on anti-PD1/PD-L1 efficacy in NSCLC with oncogenic driver mutation.     

FGFR1–4 RNA-Based Gene Alteration and Expression Analysis in Squamous Non-Small Cell Lung Cancer

While fibroblast growth factor receptors (FGFRs) are involved in several biological pathways and FGFR inhibitors may be useful in the treatment of squamous non-small cell lung cancer (Sq-NSCLC), FGFR aberrations are not well characterized in Sq-NSCLC. We comprehensively evaluated FGFR expression, fusions, and variants in 40 fresh-frozen primary Sq-NSCLC (stage IA3–IV) samples and tumor-adjacent normal tissues using real-time PCR and next-generation sequencing (NGS). Protein expression of FGFR1–3 and amplification of FGFR1 were also analyzed. FGFR1 and FGFR4 median gene expression was significantly (p < 0.001) decreased in tumors compared with normal tissue. Increased FGFR3 expression enhanced the recurrence risk (hazard ratio 4.72, p = 0.029), while high FGFR4 expression was associated with lymph node metastasis (p = 0.036). Enhanced FGFR1 gene expression was correlated with FGFR1 protein overexpression (r = 0.75, p = 0.0003), but not with FGFR1 amplification. NGS revealed known pathogenic FGFR2,3 variants, an FGFR3::TACC3 fusion, and a novel TACC1::FGFR1 fusion together with FGFR1,2 variants of uncertain significance not previously reported in Sq-NSCLC. These findings expand our knowledge of the Sq-NSCLC molecular background and show that combining different methods increases the rate of FGFR aberrations detection, which may improve patient selection for FGFRi treatment.