KRAS-mutant non-small cell lung cancer (NSCLC) therapy based on tepotinib and omeprazole combination.

BACKGROUND: KRAS-mutant non-small cell lung cancer (NSCLC) shows a relatively low response rate to chemotherapy, immunotherapy and KRAS-G12C selective inhibitors, leading to short median progression-free survival, and overall survival. The MET receptor tyrosine kinase (c-MET), the cognate receptor of hepatocyte growth factor (HGF), was reported to be overexpressed in KRAS-mutant lung cancer cells leading to tumor-growth in anchorage-independent conditions. METHODS: Cell viability assay and synergy analysis were carried out in native, sotorasib and trametinib-resistant KRAS-mutant NSCLC cell lines. Colony formation assays and Western blot analysis were also performed. RNA isolation from tumors of KRAS-mutant NSCLC patients was performed and KRAS and MET mRNA expression was determined by real-time RT-qPCR. In vivo studies were conducted in NSCLC (NCI-H358) cell-derived tumor xenograft model. RESULTS: Our research has shown promising activity of omeprazole, a V-ATPase-driven proton pump inhibitor with potential anti-cancer properties, in combination with the MET inhibitor tepotinib in KRAS-mutant G12C and non-G12C NSCLC cell lines, as well as in G12C inhibitor (AMG510, sotorasib) and MEK inhibitor (trametinib)-resistant cell lines. Moreover, in a xenograft mouse model, combination of omeprazole plus tepotinib caused tumor growth regression. We observed that the combination of these two drugs downregulates phosphorylation of the glycolytic enzyme enolase 1 (ENO1) and the low-density lipoprotein receptor-related protein (LRP) 5/6 in the H358 KRAS G12C cell line, but not in the H358 sotorasib resistant, indicating that the effect of the combination could be independent of ENO1. In addition, we examined the probability of recurrence-free survival and overall survival in 40 early lung adenocarcinoma patients with KRAS G12C mutation stratified by KRAS and MET mRNA levels. Significant differences were observed in recurrence-free survival according to high levels of KRAS mRNA expression. Hazard ratio (HR) of recurrence-free survival was 7.291 (p = 0.014) for high levels of KRAS mRNA expression and 3.742 (p = 0.052) for high MET mRNA expression. CONCLUSIONS: We posit that the combination of the V-ATPase inhibitor omeprazole plus tepotinib warrants further assessment in KRAS-mutant G12C and non G12C cell lines, including those resistant to the covalent KRAS G12C inhibitors.

Distribution and prognostic impact of EGFR and KRAS mutations according to histological subtype and tumor invasion status in pTis-3N0M0 lung adenocarcinoma.

BACKGROUND: The prognostic impact of EGFR mutation as major targetable somatic gene variant on lung adenocarcinoma is controversial. KRAS is another major somatic variant in lung adenocarcinoma, and a therapeutic agent for KRAS G12C became available in clinical settings. These mutations represent clinicopathological features of lung adenocarcinoma and can guide the treatment choice after recurrence. We evaluated the prognostic impact of EGFR and KRAS mutations by considering other clinicopathological recurrence risks in resected pTis-3N0M0 lung adenocarcinoma. METHODS: Clinicopathological features related to recurrence and genetic status were estimated in consecutive 877 resected cases. Recurrence-free survival (RFS), cumulative recurrence rate (CRR), and overall survival (OS) were compared. Uni- and multivariate analyses for RFS were performed after excluding cases with little or no recurrence risks. RESULTS: EGFR mutation was more likely to be harbored in female, never-smoker, or patients accompanied by > 5% lepidic component. KRAS mutation was more likely to be harbored in patients with current/ex-smoking history, International Association for the Study of Lung Cancer (IASLC) grade 3, or accompanied lymphatic or vascular invasion. In IASLC grade 2 and 3 patients, EGFR or KRAS mutation cases had significantly worse 5-year RFS than wild type patients (76.9% vs. 85.0%, hazard ratio [HR] = 1.55, 95% confidence interval [CI] = 1.62-6.41, P < 0.001). EGFR or KRAS mutation cases had significantly higher 5-year CRR than wild type patients (17.7% vs. 9.8%, HR = 1.69, 95% CI = 1.44-6.59, P = 0.0038). KRAS mutation cases had higher 5-year CRR than EGFR mutation cases (16.7% vs. 21.4%, HR = 1.62, 95% CI = 0.96-7.19, P = 0.061). There was no significant difference in OS between cohorts. Multivariate analysis revealed that a positive EGFR/KRAS mutation status was risk factor for worse RFS (HR = 2.007, 95% CI = 1.265-3.183, P = 0.003). CONCLUSION: Positive EGFR and KRAS mutation statuses were risk factors for recurrence in resected IASLC grade 2 and 3 patients. KRAS mutations were more likely to be confirmed in cases with an increased risk of recurrence. EGFR and KRAS mutation statuses should be evaluated simultaneously when assessing the risk of recurrence.

Coregulation of pathways in lung cancer patients with EGFR mutation: therapeutic opportunities.

Epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma are a frequent class of driver mutations. Single EGFR tyrosine kinase inhibitor (TKI) provides substantial clinical benefit, but almost nil radiographic complete responses. Patients invariably progress, although survival can reach several years with post-treatment therapies, including EGFR TKIs, chemotherapy or other procedures. Endeavours have been clinically oriented to manage the acquisition of EGFR TKI-resistant mutations; however, basic principles on cancer evolution have not been considered in clinical trials. For years, evidence has displayed rapidly adaptive mechanisms of resistance to selective monotherapy, posing several dilemmas for the practitioner. Strict adherence to non-small cell lung cancer (NSCLC) guidelines is not always practical for addressing the clinical progression that EGFR-mutant lung adenocarcinoma patients suffer. The purpose of this review is to highlight regulatory mechanisms and signalling pathways that cause therapy-induced resistance to EGFR TKIs. It suggests combinatorial therapies that target EGFR, as well as potential mechanisms underlying EGFR-mutant NSCLC, alerting the reader to clinical opportunities that may lead to a deeper and more durable response. Molecular reprogramming contributes to EGFR TKI resistance, and the compiled information is relevant in understanding the development of new combined targeted strategies in EGFR-mutant NSCLC.

Intense Expression of EGFR L858R Characterizes the Micropapillary Component and L858R Is Associated with the Risk of Recurrence in pN0M0 Lung Adenocarcinoma with the Micropapillary Component.

BACKGROUND: Lung adenocarcinoma with the micropapillary (MP) component poses a higher risk of recurrence even when the MP component is not predominant. This study explored genetic features associated with highly malignant behavior of lung adenocarcinoma with the MP component. METHODS: The MP and papillary (PaP) components were captured separately in three patients. Comprehensive mRNA expressions of somatic variants were compared between the MP and PaP components of each patient using next-generation sequencing (NGS). The protein expression of the NGS-detected variant was validated by immunohistochemistry. The prognostic impact of the detected variant was evaluated in 288 adenocarcinoma patients with resection of pN0M0. RESULTS: In two cases, NGS suggested higher RNA expression of EGFR L858R in the MP component than in the PaP component (allele frequency, 0.485 vs. 0.155 and 1.000 vs. 0.526, respectively; P < 0.001 for both). Immunohistochemistry validated intense expression of L858R in the MP component of 27 MP-positive (MP+) patients. Among 288 pN0M0 patients, L858R was more frequently harbored in the MP+ patients than in the MP-negative (MP-) patients. The MP+ patients harboring L858R showed significantly worse recurrence-free survival (RFS) than the MP+ patients without L858R (median RFS 38.7 and 55.0 months, respectively; hazard ratio [HR] 3.004; 95% confidence interval [CI] 1.306-9.132; P = 0.012). Multivariate analysis of the MP+ patients showed that positive L858R status was associated with poorer RFS (HR 2.976; 95% CI 1.190-7.442; P = 0.020). CONCLUSIONS: EGFR L858R was more frequently harbored in the MP+ adenocarcinoma patients than in the MP- adenocarcinoma patients. Intense expression of L858R in the MP component was suggested, and the MP+ patients harboring L858R were at comparatively higher risk of recurrence in the group with pN0M0 lung adenocarcinoma.

Prospective, randomized, cross-over pilot study of the effects of Rikkunshito, a Japanese traditional herbal medicine, on anorexia and plasma-acylated ghrelin levels in lung cancer patients undergoing cisplatin-based chemotherapy.

Purpose Anorexia induced by cytotoxic chemotherapy on delayed phase is a highly frequent adverse event. We aimed to determine the effects of rikkunshito (RKT) on chemotherapy-induced anorexia (CIA) in patients with lung cancer. Methods This prospective, randomized, cross-over pilot trial included 40 lung cancer patients scheduled to undergo cisplatin-based chemotherapy and randomized to either a group given RKT 7.5 g/day for 14 days (Group A, N = 20) or not (Group B, N = 20), then the treatments were switched. All patients received dexamethasone, palonosetron hydrochloride and aprepitant regardless of group assignment. Rescue drugs were allowed as required. The primary and key secondary endpoints were changes in caloric intake and in plasma acylated ghrelin (AG) levels, respectively. Average daily caloric intake during days 3 to 5 was compared with that on day 1 of each course. Results The primary and key secondary endpoints were analyzed in 31 patients (per protocol population) completing the study. Reduction rate of caloric intake was lower in RKT, than in control courses (18% vs. 25%, P = 0.025). Plasma AG levels significantly declined between days 1 and 3 in RKT (12.3 vs. 7.5 fmol/mL, P < 0.001) and control (10.8 vs. 8.6 fmol/mL, P < 0.001) courses. However, those obviously increased to 8.5 fmol/mL (P = 0.025) by day 5 in RKT course but not in control course (7.7 fmol/mL, P = 0.28). Conclusions Rikkunshito could mitigate CIA and ameliorate plasma AG levels during the delayed phase of CDDP-based chemotherapy in lung cancer patients. Clinical trial registration numbers: UMIN000010748.

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.

α-Parvin, a pseudopodial constituent, promotes cell motility and is associated with lymph node metastasis of lobular breast carcinoma.

Invasive lobular carcinoma (ILC) is more frequently lymph node positive than is invasive ductal carcinoma (IDC), and ILC cell infiltration shows distinctive histological characteristics, suggesting the action of ILC-specific invasion molecules. To identify such a molecule, we used a proteomic approach in the pseudopodia of MDA-MB-231 breast cancer cells. A pseudopodial constituent was identified using excimer laser ablation, two-dimensional difference gel electrophoresis, mass spectroscopy, and immunocytofluorescence. MDA-MB-231 cells were modified to express various levels of this constituent by transient transfection and were examined for pseudopodia formation and migratory abilities using wound healing and two-chamber assays. Immunohistochemical positivity of human breast cancer cells (56 ILCs and 21 IDCs) was compared with clinicopathological variables. An actin-binding adaptor protein, α-parvin, was found to localize to pseudopodia and to form focal adhesions in cells not induced to extend pseudopodia. Pseudopodial length and density and migratory abilities correlated with α-parvin expression. Twenty-one (37.5 %) ILCs stained positive for α-parvin, whereas the results were negative for all 21 IDCs (P < 0.001). α-Parvin positivity in ILC was significantly associated with lymphatic invasion (P = 0.038) and lymph node metastasis (P = 0.003) in univariate analyses and to lymph node metastasis (P = 0.020) in multivariate analyses. α-Parvin, a pseudopodial constituent, was found to promote migration of breast cancer cells and to be expressed exclusively by ILC, suggesting that α-parvin is an ILC-specific invasion molecule that may have clinical utility as a biomarker for aggressive subsets of ILC.

Secondary Mutations of the EGFR Gene That Confer Resistance to Mobocertinib in EGFR Exon 20 Insertion.

INTRODUCTION: Approximately 10% of mutations in the EGFR gene in NSCLC are in-frame insertions in exon 20 (X20ins). These tumors usually do not respond to conventional EGFR tyrosine kinase inhibitors (TKIs). Several novel EGFR TKIs active for X20ins are in clinical development, including mobocertinib, which was recently approved by the U.S. Food and Drug Administration. However, acquired resistance during treatment with these TKIs still occurs as in the case of EGFR TKIs of earlier generations. METHODS: We chronically exposed murine pro-B-cell line cells transduced with the five most common X20ins (A763_Y764insFQEA, V769_D770insASV, D770_N771insSVD, H773_V774insNPH and H773_V774insH) to mobocertinib in the presence of N-ethyl-N-nitrosourea and searched for secondary EGFR mutations. We evaluated the efficacies of several EGFR X20ins inhibitors, including zipalertinib and sunvozertinib, against cells with acquired resistant mutations. RESULTS: All secondary mutations resulting in acquired resistance to mobocertinib were exclusively C797S in insFQEA and insSVD. However, in the case of other X20ins (insASV, insNPH, and insH), T790M or C797S secondary mutations contributed to acquired resistance to mobocertinib. The emergence of T790M was more frequent in cells treated with lower drug concentrations. Sunvozertinib exhibited good activity against resistant cells with T790M. Cells with C797S were refractory to all EGFR TKIs, except for erlotinib, which was active for insFQEA with C797S. CONCLUSIONS: T790M or C797S, depending on the original X20ins mutations, conferred acquired resistance to mobocertinib. Sunvozertinib may be the treatment of choice for patients with tumors resistant to mobocertinib because of T790M.

Patho-transcriptomic analysis of invasive mucinous adenocarcinoma of the lung (IMA): comparison with lung adenocarcinoma with signet ring cell features (SRCC).

Background: Invasive mucinous adenocarcinoma (IMA) comprises ∼5% of lung adenocarcinoma. There is no effective therapy for IMA when surgical resection is not possible. IMA is sometimes confused with adenocarcinoma with signet ring cell features (SRCC) pathologically since both adenocarcinomas feature tumor cells with abundant intracellular mucin. The molecular mechanisms by which such mucin-producing lung adenocarcinomas develop remain unknown. Methods: Using a Visium spatial transcriptomics approach, we analyzed IMA and compared it with SRCC patho-transcriptomically. Combining spatial transcriptomics data with in vitro studies using RNA-seq and ChIP-seq, we assessed downstream targets of transcription factors HNF4A and SPDEF that are highly expressed in IMA and/or SRCC. Results: Spatial transcriptomics analysis indicated that there are 6 distinct cell clusters in IMA and SRCC. Notably, two clusters (C1 and C3) of mucinous tumor cells exist in both adenocarcinomas albeit at a different ratio. Importantly, a portion of genes (e.g., NKX2-1 , GKN1 , HNF4A and FOXA3 ) are distinctly expressed while some mucous-related genes (e.g., SPDEF and FOXA2 ) are expressed in both adenocarcinomas. We determined that HNF4A induces MUC3A/B and TM4SF4 and that BI 6015, an HNF4A antagonist, suppressed the growth of IMA cells. Using mutant SPDEF that is associated with COVID-19, we also determined that an intact DNA-binding domain of SPDEF is required for SPDEF-mediated induction of mucin genes ( MUC5AC , MUC5B and AGR2 ). Additionally, we found that XMU-MP-1, a SPDEF inhibitor, suppressed the growth of IMA cells. Conclusion: These results revealed that IMA and SRCC contain heterogenous tumor cell types, some of which are targetable.

Current clinical trials with non-coding RNA-based therapeutics in malignant diseases: A systematic review.

This systematic review aimed to shed light on the trend of current clinical trials of non-coding RNA (ncRNA)-based therapeutics for malignant diseases. We conducted a database search for published literature and ongoing clinical trials using PubMed, clinicaltrials.gov, and University Medical Information Network (UMIN) clinical trial registry. To ensure that our review was based on up-to-date clinical trials, we limited our search to literature published within the last five years (January 2017-September 2022). Furthermore, due to the "clinical" nature of our review, we focused only on studies involving human participants. Among ncRNAs, microRNAs have been extensively explored in observational studies of malignant diseases as potential diagnostic markers and prognostic predictors, as well as for their therapeutic monitoring and profiling capabilities. As therapeutic agents, microRNA or siRNA were estimated in interventional human clinical trials and showed promising outcomes; however, the number of trials was small. Evidence and ongoing clinical trials in which ncRNAs other than microRNA or siRNA have been evaluated for their potential as therapeutic agents are limited. Here, we summarized microRNA as a potential therapeutic agent in malignant diseases, but most of the current evidence suggests that it is useful as a potential biomarker. siRNA is also a promising ncRNA technique in cancer, however more data from clinical trials are warranted for clinical use.

The role of biomarkers in stage III non-small cell lung cancer.

INTRODUCTION: Stage III non-small cell lung cancer (NSCLC) is a composite of the regional spread of lung cancer with different levels of potential lymph node involvement and tumor size that often deem the stage at time of diagnosis to be unresectable and suitable for chemoradiation plus consolidation immunotherapy with durvalumab for 12 months. Chemoradiation plus durvalumab consolidation yielded a landmark 49.2% 5-year overall survival in unresectable NSCLC. AREAS COVERED: Sub-optimal results lead us to focus on the mechanisms of resistance responsible for intractability in a significant proportion of cases that fail with chemoradiation and immunotherapy. In stage III NSCLC it is opportune to explore the accumulated evidence on ferroptosis resistance that can lead to cancer progression and metastasis. Strong data shows that three anti-ferroptosis pathways are principally involved in resistance to chemotherapy, radiation, and immunotherapy. EXPERT OPINION: Because a large part of stage III NSCLCs is resistant to chemoradiation and durvalumab consolidation, a ferroptosis-based therapeutic approach, combined with standard-of-care therapy, can lead to improved clinical outcomes in patients diagnosed with stage III and possibly stage IV NSCLCs.

Brief Report: circRUNX1 as Potential Biomarker for Cancer Recurrence in EGFR Mutation-Positive Surgically Resected NSCLC.

Introduction: As recently evidenced by the ADAURA trial, most patients with stages IB to IIIA of resected EGFR-mutant lung adenocarcinoma benefit from osimertinib as adjuvant therapy. Nevertheless, predictive markers of response and recurrence are still an unmet need for more than 10% of these patients. Some circular RNAs (circRNAs) have been reported to play a role in tumor growth and proliferation. In this project, we studied circRNA expression levels in formalin-fixed, paraffin-embedded lung tumor samples to explore their biomarker potential and develop a machine learning (ML)-based signature that could predict the benefit of adjuvant EGFR tyrosine kinase inhibitors in patients with EGFR-mutant NSCLC. Methods: Patients with surgically resected EGFR mutant-positive, stages I to IIIB NSCLC were recruited from February 2007 to December 2015. Formalin-fixed, paraffin-embedded tumor samples were retrospectively collected from those patients with a follow-up period of more than or equal to 36 months (N = 76). Clinicopathologic features were annotated. Total RNA was purified and quantified prior nCounter processing with our circRNA custom panel. Data analysis and ML were performed taking into consideration circRNA expression levels and recurrence-free survival (RFS). RFS was defined from the day of surgery to the day when recurrence was detected radiologically or the death owing to any cause. Results: Of the 76 patients with EGFR mutation-positive NSCLC included in the study, 34 relapsed within 3 years after resection. The median age of the relapsing cohort was 71.5 (range: 49-89) years. Most patients were nonsmokers (n = 21; 61.8%) and of female sex (n = 21; 61.8%). Most cases (n = 17; 50%) presented an exon 21 mutation, whereas 15 and four patients had an exon 19 and exon 18 mutation, respectively. Differential expression analysis revealed that circRUNX1, along with circFUT8 and circAASDH, was up-regulated in relapsing patients (p < 0.05 and >2 fold-change). A ML-based circRNA signature predictive of recurrence in patients with EGFR mutation-positive NSCLC, comprising circRUNX1, was developed. Our final model including selected 6-circRNA signature with random forest algorithm was able to classify relapsing patients with an accuracy of 83% and an area under the receiver operating characteristic curve of 0.91.RFS was significantly shorter not only for the subgroup of patients with high versus low circRUNX1 expression but also for the group classified as recurrent by the ML circRNA signature when compared with those classified as nonrecurrent. Conclusions: Our findings suggest that circRUNX1 and the presented ML-developed signature could be novel tools to predict the benefit of adjuvant EGFR tyrosine kinase inhibitors with regard to RFS in patients with EGFR-mutant NSCLC. The training and validation phases of our ML signature will be conducted including bigger independent cohorts.

FOXA2 Cooperates with Mutant KRAS to Drive Invasive Mucinous Adenocarcinoma of the Lung.

The endoderm-lineage transcription factor FOXA2 has been shown to inhibit lung tumorigenesis in in vitro and xenograft studies using lung cancer cell lines. However, FOXA2 expression in primary lung tumors does not correlate with an improved patient survival rate, and the functional role of FOXA2 in primary lung tumors remains elusive. To understand the role of FOXA2 in primary lung tumors in vivo, here, we conditionally induced the expression of FOXA2 along with either of the two major lung cancer oncogenes, EGFRL858R or KRASG12D, in the lung epithelium of transgenic mice. Notably, FOXA2 suppressed autochthonous lung tumor development driven by EGFRL858R, whereas FOXA2 promoted tumor growth driven by KRASG12D. Importantly, FOXA2 expression along with KRASG12D produced invasive mucinous adenocarcinoma (IMA) of the lung, a fatal mucus-producing lung cancer comprising approximately 5% of human lung cancer cases. In the mouse model in vivo and human lung cancer cells in vitro, FOXA2 activated a gene regulatory network involved in the key mucous transcription factor SPDEF and upregulated MUC5AC, whose expression is critical for inducing IMA. Coexpression of FOXA2 with mutant KRAS synergistically induced MUC5AC expression compared with that induced by FOXA2 alone. ChIP-seq combined with CRISPR interference indicated that FOXA2 bound directly to the enhancer region of MUC5AC and induced the H3K27ac enhancer mark. Furthermore, FOXA2 was found to be highly expressed in primary tumors of human IMA. Collectively, this study reveals that FOXA2 is not only a biomarker but also a driver for IMA in the presence of a KRAS mutation. SIGNIFICANCE: FOXA2 expression combined with mutant KRAS drives invasive mucinous adenocarcinoma of the lung by synergistically promoting a mucous transcriptional program, suggesting strategies for targeting this lung cancer type that lacks effective therapies.

Macrophage CD5L is a target for cancer immunotherapy.

BACKGROUND: Reprogramming of immunosuppressive tumor-associated macrophages (TAMs) presents an attractive therapeutic strategy in cancer. The aim of this study was to explore the role of macrophage CD5L protein in TAM activity and assess its potential as a therapeutic target. METHODS: Monoclonal antibodies (mAbs) against recombinant CD5L were raised by subcutaneous immunization of BALB/c mice. Peripheral blood monocytes were isolated from healthy donors and stimulated with IFN/LPS, IL4, IL10, and conditioned medium (CM) from different cancer cell lines in the presence of anti-CD5L mAb or controls. Subsequently, phenotypic markers, including CD5L, were quantified by flow cytometry, IF and RT-qPCR. Macrophage CD5L protein expression was studied in 55 human papillary lung adenocarcinoma (PAC) samples by IHC and IF. Anti-CD5L mAb and isotype control were administered intraperitoneally into a syngeneic Lewis Lung Carcinoma mouse model and tumor growth was measured. Tumor microenvironment (TME) changes were determined by flow cytometry, IHC, IF, Luminex, RNAseq and RT-qPCR. FINDINGS: Cancer cell lines CM induced an immunosuppressive phenotype (increase in CD163, CD206, MERTK, VEGF and CD5L) in cultured macrophages. Accordingly, high TAM expression of CD5L in PAC was associated with poor patient outcome (Log-rank (Mantel-Cox) test p = 0.02). We raised a new anti-CD5L mAb that blocked the immunosuppressive phenotype of macrophages in vitro. Its administration in vivo inhibited tumor progression of lung cancer by altering the intratumoral myeloid cell population profile and CD4+ T-cell exhaustion phenotype, thereby significantly modifying the TME and increasing the inflammatory milieu. INTERPRETATION: CD5L protein plays a key function in modulating the activity of macrophages and their interactions within the TME, which supports its role as a therapeutic target in cancer immunotherapy. FUNDING: For a full list of funding bodies, please see the Acknowledgements.

Novel application for pseudopodia proteomics using excimer laser ablation and two-dimensional difference gel electrophoresis.

We developed a novel application to conduct pseudopodia proteomics. Pseudopodia are ventral actin-rich protrusions and play functional roles in cell migrations. Identification of pseudopodia proteins leads to a further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies. In our application, tumor cells were placed on a fibronectin-coated porous membrane to form pseudopodia. According to the motile potentials of the cells, the cells formed pseudopodial microprocesses in the pores. An excimer laser, which was used for ophthalmic refractive surgeries, horizontally ablated cells at the membrane surface to remove the cell body. The microscopic observations and the protein expression studies suggested that the laser treatment caused no apparent damages to pseudopodia. Proteins in whole cells and pseudopodia fractions were individually solubilized, labeled with a highly sensitive fluorescent dye, and separated using two-dimensional difference gel electrophoresis. Among 2508 protein spots observed, 211 had different intensity between whole cells and pseudopodia fractions (more than fourfold differences and P-value of <0.05). The protein enrichment depended on the pore size. Mass spectrometric protein identification revealed 46 pseudopodia-localizing proteins. The localization of novel pseudopodia-localizing proteins such as RAB1A, HSP90B, TDRD7, and vimentin was confirmed using immunohistochemical examinations. The previous studies demonstrated that these four proteins may function in the cell migration process. This method will provide insights into the molecular details of pseudopodia and a further understanding of malignant phenotypes of tumor cells and novel therapeutic strategies.

The impact of epidermal growth factor receptor mutation status on adjuvant chemotherapy for patients with high-risk stage I lung adenocarcinoma.

OBJECTIVE: The aim of this study was to evaluate the role and effect of adjuvant chemotherapy based on epidermal growth factor receptor mutation status in patients with stage I lung adenocarcinoma. METHODS: Between 2010 and 2016, of 1901 patients with pathologic stage I (8th edition) non-small cell lung cancer, we identified 475 with high-risk (pT1c/T2a or positive for lymphovascular invasion) stage I lung adenocarcinoma who underwent lobectomy. We estimated propensity scores to adjust for confounding variables, including age, sex, Brinkman index, pulmonary functions, comorbidities, surgical approach, invasive component tumor size, visceral pleural, lymphatic, and vascular invasion, adenocarcinoma subtype, epidermal growth factor receptor mutation status, postoperative complications, and institution associated with the administration of adjuvant chemotherapy. The primary end point was recurrence-free survival. RESULTS: Of 292 patients without/unknown epidermal growth factor receptor mutation, 105 (36.0%) received adjuvant chemotherapy and 187 (64.0%) did not. In 69 pairs of patients who were propensity score matched, the 5-year recurrence-free survival was significantly better in those who underwent adjuvant chemotherapy (88.4%) than in those who did not (63.6%; P = .001). Of 183 patients with epidermal growth factor receptor mutation, 78 (42.6%) received adjuvant chemotherapy and 105 (57.4%) did not. In 49 pairs of propensity score-matched patients, there was no significant difference in the 5-year recurrence-free survival between those who underwent adjuvant chemotherapy (74.3%) and those who did not (80.5%; P = .573). CONCLUSIONS: The effect of adjuvant chemotherapy for high-risk stage I lung adenocarcinoma varied by epidermal growth factor receptor mutation status. Epidermal growth factor receptor mutation status may help to identify patients with high-risk stage I lung adenocarcinoma who may benefit from adjuvant chemotherapy.

Clinical Behavior of Combined Versus Pure High-Grade Neuroendocrine Carcinoma.

BACKGROUND: The aim of this study was to investigate and compare the clinical behaviors of combined and pure high-grade neuroendocrine carcinoma (large-cell neuroendocrine carcinoma [LCNEC] and small-cell lung carcinoma [SCLC]). PATIENTS AND METHODS: Data of 132 patients who underwent complete resection for combined or pure high-grade neuroendocrine carcinoma (combined group, 67; pure group, 65) between January 2001 and December 2015 were retrospectively reviewed. The clinicopathological features were analyzed and compared, and the prognoses were assessed by performing the Kaplan-Meier method and Cox regression analysis. RESULTS: The combined and pure groups had nearly equivalent clinicopathological characteristics, specifically, older males with smoking history, almost the same percentage of pleural/lymphatic/vascular invasion, and nearly the same recurrence rates and relapse patterns. The combined group had prognosis equivalent to that of the pure group (5-year overall survival [OS] rates: 61.8% vs. 52.2%, respectively; P = .82 and 5-year recurrence-free survival [RFS] rates: 42.4% vs. 43.9%, respectively; P = .96), and this trend was identified in sub-analyses only for patients with LCNEC, SCLC, and the same pathological stage. Multivariable Cox regression analysis in patients with high-grade neuroendocrine carcinoma revealed that vascular invasion and pathological stage were independent prognostic factors for OS; more importantly, combined and pure histologies were proven to have nearly equivalent associations with prognosis (hazard ratio, 0.96; 95% confidence interval, 0.22to 1.66; P = .96). RESULTS: Combined high-grade neuroendocrine carcinoma had clinical behavior equivalent to those of pure high-grade neuroendocrine carcinoma, with similar clinicopathological characteristics.

Sensitivity and optimal clinicopathological features for mutation-targeted liquid biopsy in pN0M0 EGFR-mutant lung adenocarcinoma.

PURPOSE: Liquid biopsy for early-stage lung cancer diagnosis is challenging, and optimal candidates' clinicopathological features are unknown. We investigated utility and clinicopathological features of optimal candidates in somatic mutation-targeted liquid biopsy using droplet digital polymerase chain reaction (ddPCR) in pN0M0 EGFR mutation-positive lung adenocarcinoma patients. METHODS: We performed EGFR mutation-targeted ddPCR liquid biopsy in 100 patients with resected pN0M0 invasive lung adenocarcinoma, whose tumor diameter in high-resolution computed tomography (HRCT) was ≤ 5 cm. Peripheral blood-derived serum was collected preoperatively. Two representative EGFR somatic variants (exon 19 [E746-A750 del (2235_2249 del)]; exon 21 (L858R)) were utilized as liquid biopsy targets. Clinicopathological features including radiological appearance, subhistology, and invasive status were compared between ddPCR-positive and ddPCR-negative patients. RESULTS: Among the 100 patients, 98 showed part-solid or pure-solid appearance in HRCT and 2 showed non-solid appearance; 98 were pathological stage IA1-IB. Of the 66 patients with EGFR mutation detection in ddPCR, 12 were significantly positive and 10 (83.3%, 10/12) exhibited pure-solid appearance in HRCT. Clinical invasive tumor ratio was significantly higher in ddPCR-positive than in ddPCR-negative patients (median: 100% vs. 85.4%, P = 0.0212), whereas other clinicopathological features were not significantly different. CONCLUSION: Mutation-targeted liquid biopsy using ddPCR detected lung cancer in 12.0% (12/100) of pN0M0 EGFR-mutant lung adenocarcinoma patients. In 83.3% of the ddPCR-positive patients, tumors showed pure-solid appearance in HRCT. The detection ratio increased to 21.3% (10/47) among patients with pure-solid appearance tumors. Tumor appearance might be useful for better selection of liquid biopsy candidates.

Impact of the amount of preoperative erector spinae muscle in stage I non-small-cell lung cancer.

OBJECTIVES: Erector spinae muscle (ESM) is an antigravity muscle group that can be evaluated as an index of muscle loss on chest computed tomography. The amount of ESM has been reported to be related to the prognosis of several respiratory diseases. However, few studies clarify the impact on postoperative non-small-cell lung cancer (NSCLC). We investigated the relationship between ESM and postoperative prognosis in patients with early-stage NSCLC. METHODS: We reviewed the medical records of 534 patients with stage I NSCLC who underwent lobectomy or segmentectomy. The ESM was identified by preoperative computed tomography, and the amount was normalized according to height and sex. Overall survival, lung cancer-related deaths and non-lung cancer-related deaths (NLCRD) were analysed using log-rank and Gray's tests. Multivariable analyses were conducted to identify factors that influenced overall survival (OS) and NLCRD. RESULTS: The amount of ESM normalized according to height and sex was significantly associated with age and body mass index. When the amount was low, OS (5-year OS, 79.6 vs 89.5%; P< 0.001) and NLCRD (5-year cumulative mortality rate, 14.7 vs 6.8%; P< 0.001) were significantly worse, although no difference was found in lung cancer-related deaths. CONCLUSIONS: The amount of preoperative ESM was strongly related to non-lung cancer-related death and was a significant prognostic factor for stage I NSCLC. Patients with a low amount of the muscle should be treated based on proper risk assessment.