Non-small Cell Lung Cancer Adenocarcinoma Research Articles

Prevalence of EGFR mutation testing in early-stage lung cancer: Implications of the ADAURA trial for clinical practice.

e20507 Background: It is reported that about 20% of patients with resected NSCLC adenocarcinoma harbor an EGFR driver mutation in the United States. Up to the recent approval of osimertinib in the adjuvant setting for resected EGFR + NSCLC based on the ADAURA trial, routine molecular profiling of early-stage lung cancer had not been standard of care. We hypothesize that there is a significant proportion of patients with resected adenocarcinoma with unknown EGFR status who could benefit from treatment that are missed with our current testing practices. Methods: We performed a retrospective analysis of Stage IB-IIIA lung adenocarcinomas resected at the University of Miami from 2014 to 2019. Eligible patients were identified from the Cancer Registry and information on EGFR mutation testing and treatment was obtained from chart review. We evaluated the prevalence of EGFR mutation testing in this population and outcomes based on EGFR mutation status. Disease free survival (DFS) and clinico-pathologic characteristics were evaluated. We estimated the number of patients that would have been eligible for EGFR testing and adjuvant osimertinib therapy in the pre-ADAURA era in our patient cohort. Results: A total of 120 patients had resected stage IB-IIIA adenocarcinoma during this five-year period (Stage IB 42.5%; Stage IIA 13.3%; Stage IIB 25%; Stage IIIA 19.2%) with a median age of 66 years. Most were females (59%), NHWs (51.5%), Hispanics (46.9%), and former smokers (66.7%). Out of patients with Stage IB-IIIA NSCLC with adenocarcinoma, 42.5% completed recommended adjuvant platinum-based chemotherapy. Only 40% of patients were referred for EGFR testing during this study period. The prevalence of EGFR mutations in this population was 10.8% (13 /120), but 59% of cases had no available EGFR testing. The most prevalent mutation was L858R (53.8%) followed by exon-19 deletions (30.8%). A total of 6 patients received an EGFR TKI therapy during the follow up period (2 in the adjuvant setting). With a median follow up of 12 mos, the rate of recurrence by stage was: Stage IB (3.9%); Stage IIB (10%); Stage IIIA (13%). Median time to disease progression or death was 13 months in this subgroup. There was no difference in disease free survival for patients with EGFR testing and those without results available in this short follow up period. Conclusions: Based on this retrospective review, up to 60% of patients with early-stage NSCLC with non-squamous histology have no available EGFR testing in the pre-ADAURA era. Of the anticipated 20% of patients with expected EGFR mutations based on historical controls, we have only identified half of patients that would have been eligible for adjuvant osimertinib. This study establishes the importance of upfront EGFR mutation testing in all NSCLC patients, not only to prognosticate, but also to identify the subset of patients who could benefit from adjuvant EGFR therapy.

Multiplex immune protein profiling of fine-needle aspirates from patients with non-small-cell lung cancer reveals signatures associated with PD-L1 expression and tumor stage.

Biomarker signatures identified through minimally invasive procedures already at diagnosis of non‐small‐cell lung cancer (NSCLC) could help to guide treatment with immune checkpoint inhibitors (ICI). Here, we performed multiplex profiling of immune‐related proteins in fine‐needle aspirate (FNA) samples of thoracic lesions from patients with NSCLC to assess PD‐L1 expression and identify related protein signatures. Transthoracic FNA samples from 14 patients were subjected to multiplex antibody‐based profiling by proximity extension assay (PEA). PEA profiling employed protein panels relevant to immune and tumor signaling and was followed by Qlucore® Omics Explorer analysis. All lesions analyzed were NSCLC adenocarcinomas, and PEA profiles could be used to monitor 163 proteins in all but one sample. Multiple key immune signaling components (including CD73, granzyme A, and chemokines CCL3 and CCL23) were identified and expression of several of these proteins (e.g., CCL3 and CCL23) correlated to PD‐L1 expression. We also found EphA2, a marker previously linked to inferior NSCLC prognosis, to correlate to PD‐L1 expression. Our identified protein signatures related to stage included, among others, CXCL10 and IL12RB1. We conclude that transthoracic FNA allows for extensive immune and tumor protein profiling with assessment of putative biomarkers of important for ICI treatment selection in NSCLC.

CD45RO⁺ Memory T Lymphocytes As A Candidate Marker for Non-small Cell Lung Cancer

背景与目的肺癌是世界范围内最常见的恶性肿瘤之一。肿瘤微环境中多种多样的免疫浸润细胞，是肿瘤免疫的重要组成，对患者预后具有临床意义。CD45RO+肿瘤浸润淋巴细胞（tumor infiltrating lymphocytes, TILs），即记忆T细胞，其表达与多种肿瘤预后相关。本研究旨在探讨非小细胞肺癌（non-small cell lung cancer, NSCLC）中评估肿瘤和基质区CD45RO+ TILs密度与患者临床特征和预后的关系，及其联合程序性死亡受体配体1（programmed cell death-ligand 1, PD-L1）作为预后标志物的临床价值。方法对167例NSCLC患者的组织微阵列进行多重荧光免疫组织化学染色，标记CD45RO、细胞角蛋白（cytokeratin, CK）和PD-L1。利用人工智能图像识别技术和肿瘤细胞特异性CK染色，划分组织中的肿瘤区和基质区，评估肿瘤区和基质区CD45RO+ TILs的密度以及肿瘤细胞的PD-L1表达水平。采用非参检验分析CD45RO+ TILs与患者临床特征的关系，使用Kaplan-Meier方法和Cox风险比例模型分析CD45RO+ TILs独立或与PD-L1联合与肿瘤预后的关系。结果CD45RO+ TILs的密度与患者年龄、吸烟、肿瘤分期和病理类型显著相关。在NSCLC和肺腺癌（lung adenocarcinoma, LUAD）患者中，基质区高密度CD45RO+ TILs具有更长的总生存期（overall survival, OS）（NSCLC: P=0.007; LUAD: P < 0.001），并且是OS的独立预后因素（NSCLC: HR=0.559, 95%CI: 0.377-0.829, P=0.004; LUAD: HR=0.352, 95%CI: 0.193-0.641, P=0.001）。联合肿瘤细胞的PD-L1评分以及所有区域CD45RO+ TILs的浸润评分将患者分为四组：其中PD-L1+/CD45RO+患者无病生存期（disease-free survival, DFS）最长，PD-L1+/CD45RO-的患者DFS时间最短，并可作为DFS预后的独立因素（HR=2.221, 95%CI: 1.258-3.919, P=0.006）。结论肿瘤组织中CD45RO+ TILs密度以及CD45RO+ TILs联合肿瘤区PD-L1，与NSCLC的临床病理特征及预后显著相关，可作为新的生存预后标志物。

High in vitro and in vivo synergistic activity between mTORC1 and PLK1 inhibition in adenocarcinoma NSCLC.

Significant rational is available for specific targeting of PI3K/AKT/mTOR pathway in the treatment of non-small cell lung cancer (NSCLC). However, almost all clinical trials that have evaluated Pi3K pathway-based monotherapies/combinations did not observe an improvement of patient’s outcome. The aim of our study was therefore to define combination of treatment based on the determination of predictive markers of resistance to the mTORC1 inhibitor RAD001/Everolimus. An in vivo study showed high efficacy of RAD001 in NSCLC Patient-Derived Xenografts (PDXs). When looking at biomarkers of resistance by RT-PCR study, three genes were found to be highly expressed in resistant tumors, i.e., PLK1, CXCR4, and AXL. We have then focused our study on the combination of RAD001 + Volasertib, a PLK1 inhibitor, and observed a high antitumor activity of the combination in comparison to each monotherapy; similarly, a clear synergistic effect between the two compounds was found in an in vitro study. Pharmacodynamics study demonstrated that this synergy was due to (1) tumor vascularization decrease, increase of the HIF1 protein expression and decrease of the intracellular pH, and (2) decrease of the Carbonic Anhydrase 9 (CAIX) protein that could not correct intracellular acidosis. In conclusion, all these preclinical data strongly suggest that the inhibition of mTORC1 and PLK1 proteins may be a promising therapeutic approach for NSCLC patients.

Abstract PO033: Regulation of immune checkpoint proteins in NSCLC adenocarcinoma

Abstract Lung cancer is the leading cause of cancer-related deaths in both men and women. Approximately 85% of lung cancers are non-small cell lung cancer (NSCLC), with adenocarcinoma (adenoCa) being the main subtype. Well-defined mutational signatures of various lung cancer subtypes have been exploited for targeted therapies, but cancer cells often acquire resistance to these treatments, which could be mediated by the inflammatory tumor microenvironment (TME). Inflammation within the TME is known to promote cancer progression and therapy resistance. The inflammatory cytokine, interleukin-1 beta (IL-1β) is elevated in the TME of several cancers and correlates with poor outcomes. In NSCLC patients, serum IL-1β is elevated relative to normal subjects; within the same patient, IL-1β is elevated in the tumor relative to adjacent non-tumor tissue. Significantly, the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) showed that blockade of IL-1 signaling could drastically reduce lung cancer incidence and mortality. Interestingly, we observed that NSCLC adenoCa cell lines H1838, H2228, H2347, HCC4150 and HCC827 have significantly higher basal expression of the Type 1 IL-1 receptor (IL-1R1), relative to immortalized normal Human Bronchial Epithelial Cell (HBEC-3KT) and Human Small Airway Epithelial Cell (HSAEC-1KT) lines, suggesting that adenoCa cells are primed to respond to IL-1 signaling. Cancer cells can promote immunosuppression through the expression of immune checkpoint proteins (ICPs). Previously, we identified several ICPs, including programmed death-ligand 1 (PD-L1), tryptophan 2,3-dioxygenase 2 (TDO2), indoleamine 2, 3-dioxygenase 1 (IDO1) and growth differentiation factor 15 (GDF15), that were developmentally upregulated by transcription factor nuclear factor erythroid 2-related factor 2 (NRF2/NFE2L2) in human fetal lung epithelial cells during differentiation in culture. Importantly, genomic studies have revealed that &amp;gt;20% of lung adenoCa harbor mutations in NRF2 or its inhibitor KEAP1, which cause NRF2 activation. Lung adenoCa with signatures of increased NRF2 activation manifest reduced immune cell infiltration, suggesting that increased expression of NRF2 might create a more permissive immune cell environment, resulting in evasion and tolerance to the host immune system. Given that IL-1β induces ROS which can activate NRF2, we hypothesized that IL-1β present in the TME may enhance expression of NRF2 and ICPs in NSCLC adenoCa. We found that increased expression of ICPs in adenoCa cell lines H1838, H2228, H2347, HCC4150 and HCC827 compared to HBEC-3KT and HSAEC-1KT cells was correlated with significantly increased expression of NRF2. When these adenoCa cell lines were treated with IL-1β, we observed a marked induction in NRF2 and ICP mRNA and protein levels. Taken together our studies indicate that NSCLC adenoCa cells are primed to respond to IL-1 signaling which induces NRF2 and ICPs to promote tumor cell survival through immunosuppression. Citation Format: Afshan Fathima Nawas, Ritu Mishra, Carole R. Mendelson. Regulation of immune checkpoint proteins in NSCLC adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO033.

P59.07 Molecular Profiling of Human Non-Small Cell Lung Cancer by Single-Cell RNA-Seq

Lung cancer is one of the malignant tumors with the highest morbidity and mortality worldwide. As the lung is a complex organ composed of more than 40 different types of cells, which contribute to the heterogeneity of lung cancers at tumor initiation, it is important to separate different cell types to profile their characteristics. In this study, we aim to comprehensively analyze the molecular heterogeneity of non-small-cell lung cancer (NSCLC). We performed single-cell RNA-seq analyses on 7,364 individual cells from tumor tissues and matched normal tissues from 19 primary lung cancer patients and 1 pulmonary chondroid hamartoma patient. We identified a significant proportion of cancer cells simultaneously expressing classical marker genes for two or three histologic subtypes of NSCLC—adenocarcinoma (ADC), squamous cell carcinoma (SCC), and large cell neuroendocrine carcinoma (LCNEC) in the same individual cell; this was verified by coimmunostaining and RNA in situ hybridization. These data suggest that mixed-lineage tumor cells are highly plastic with mixed features of different types of NSCLC. Both copy number variation (CNV) patterns and mitochondrial mutations clearly showed that the mixed-lineage and single-lineage tumor cells from the same patient had common tumor ancestors rather than different origins. Moreover, we revealed that patients with high mixed-lineage features of different cancer subtypes had worse survival than patients with low mixed-lineage features, indicating that mixed-lineage tumor features were associated with poorer prognosis. Based on the pseudotime trajectory of gene expression dynamics from normal epithelial cells to cancer cells, a cancer-specific gene set has been identified, which includes AKR1B1 and RAMP1. Knockdown experiments verified that both AKR1B1 and RAMP1 were necessary for tumor growth, suggesting that they can serve as candidate targets for tumor therapy. Our work provides novel insights into the heterogeneity of NSCLC and offers clues for the more refined classification, diagnosis and treatment of each subtype.

P35.09 Oncogenetic Differences in Never-Smokers versus Smokers with NSCLC Adenocarcinoma Treated at the Mt Sinai Tisch Cancer Institute

P35.09 Oncogenetic Differences in Never-Smokers versus Smokers with NSCLC Adenocarcinoma Treated at the Mt Sinai Tisch Cancer Institute

Detection of Tumor-Associated Membrane Receptors on Extracellular Vesicles from Non-Small Cell Lung Cancer Patients via Immuno-PCR.

Simple SummaryLung cancer is often detected at late stages when metastases are present and the genomic make-ups of the tumors are heterogeneous. Analyses of genomic alterations in non-small-cell lung cancer (NSCLC) have revealed mutated tumor-associated membrane receptors and fusion proteins, which can be targeted via tyrosine kinase inhibitors (TKIs). TKIs initially often have a good effect, but a fraction of the tumor lesions may develop resistance through additional mutations in the targeted kinases or by increased expression/function of other membrane receptors. Detection of TKI-bypassing mechanisms is difficult in tissue biopsies as these analyze only a subpart of tumors or lesions. Liquid biopsies based on tumor-secreted small extracellular vesicles (sEVs) into body fluids can assess tumor heterogeneity. We present an immuno-PCR method for the detection of the epidermal growth factor receptor (EGFR), the human epidermal growth factor receptor 2 (HER2), and the insulin-like growth factor 1 receptor (IGF-1R) on sEVs. Initial investigations of sEVs from EGFR-mutant NSCLC tumor cells or pleural effusion (PE) fluid from patients with NSCLC or benign diseases showed different protein profiles for individual sEV samples. Further development of the immuno-PCR could complement DNA/mRNA-based assays detecting kinase mutations to allow longitudinal treatment monitoring of diverse TKI-bypassing mechanisms.Precision cancer medicine for non-small-cell lung cancer (NSCLC) has increased patient survival. Nevertheless, targeted agents towards tumor-associated membrane receptors only result in partial remission for a limited time, calling for approaches which allow longitudinal treatment monitoring. Rebiopsy of tumors in the lung is challenging, and metastatic lesions may have heterogeneous signaling. One way ahead is to use liquid biopsies such as circulating tumor DNA or small extracellular vesicles (sEVs) secreted by the tumor into blood or other body fluids. Herein, an immuno-PCR-based detection of the tumor-associated membrane receptors EGFR, HER2, and IGF-1R on CD9-positive sEVs from NSCLC cells and pleural effusion fluid (PE) of NSCLC patients is developed utilizing DNA conjugates of antibody mimetics and affibodies, as detection agents. Results on sEVs purified from culture media of NSCLC cells treated with anti-EGFR siRNA, showed that the reduction of EGFR expression can be detected via immuno-PCR. Protein profiling of sEVs from NSCLC patient PE samples revealed the capacity to monitor EGFR, HER2, and IGF-1R with the immuno-PCR method. We detected a significantly higher EGFR level in sEVs derived from a PE sample of a patient with an EGFR-driven NSCLC adenocarcinoma than in sEVs from PE samples of non-EGFR driven adenocarcinoma patients or in samples from patients with benign lung disease. In summary, we have developed a diagnostic method for sEVs in liquid biopsies of cancer patients which may be used for longitudinal treatment monitoring to detect emerging bypassing resistance mechanisms in a noninvasive way.

Prognostic and predictive effect of KRAS gene copy number and mutation status in early stage non-small cell lung cancer patients.

BackgroundIn the current analysis, we characterize the prognostic significance of KRAS mutations with concomitant copy number aberrations (CNA) in early stage non-small cell lung cancer (NSCLC), and evaluate the ability to predict survival benefit from adjuvant chemotherapy.MethodsClinical and genomic data from the LACE (Lung Adjuvant Cisplatin Evaluation)-Bio consortium was utilized. CNAs were categorized as Gain (CN ≥2) or Neutral (Neut)/Loss; KRAS status was defined as wild type (WT) or mutant (MUT). The following groups were compared in all patients and the adenocarcinoma subgroup, and were correlated to survival endpoints using a Cox proportional hazards model: WT + Neut/Loss (reference), WT + Gain, MUT + Gain and MUT + Neut/Loss. A treatment-by-variable interaction was added to evaluate predictive effect.ResultsOf the 946 (399 adenocarcinoma) NSCLC patients, 41 [30] had MUT + Gain, 145 [99] MUT + Neut/Loss, 125 [16] WT + Gain, and 635 [254] WT + Neut/Loss. A non-significant trend towards worse lung cancer-specific survival (LCSS; HR =1.34; 95% CI, 0.83–2.17, P=0.232), DFS (HR =1.34; 95% CI, 0.86–2.09, P=0.202) and OS (HR =1.59; 95% CI, 0.99–2.54, P=0.055) was seen in KRAS MUT + Gain patients relative to KRAS WT + Neut/Loss patients. A negative prognostic effect of KRAS MUT + Neut/Loss was observed for LCSS (HR =1.32; 95% CI, 1.01–1.71, P=0.038) relative to KRAS WT + Neut/Loss on univariable analysis, but to a lesser extent after adjusting for covariates (HR =1.28; 95% CI, 0.97–1.68, P=0.078). KRAS MUT + Gain was associated with a greater beneficial effect of chemotherapy on DFS compared to KRAS WT + Neut/Loss patients (rHR =0.33; 95% CI, 0.11–0.99, P=0.048), with a non-significant trend also seen for LCSS (rHR =0.41; 95% CI, 0.13–1.33, P=0.138) and OS (rHR =0.40; 95% CI, 0.13–1.26, P=0.116) in the adenocarcinoma subgroup.ConclusionsA small prognostic effect of KRAS mutation was identified for LCSS, and a trend towards worse LCSS, DFS and OS was noted for KRAS MUT + Gain. A potential predictive effect of concomitant KRAS mutation and copy number gain was observed for DFS in adenocarcinoma patients. These results could be driven by the small number of patients and require validation.

Clinical research on the combined use of systemic chemotherapy and CT-guided radiofrequency ablation in treatment of lung cancer.

The purpose of this study is to describe the clinical efficacy and safety of the combined use of systemic chemotherapy and CT-guided radiofrequency ablation (RFA) in treatment of lung cancer. A panel of 256 patients with lung cancer who were admitted to our hospital from June 2017 to October 2019 were recruited. According to the treatment plan, the patients were divided into test group (n = 128) and control group (n = 128). Patients in the test group were treated by systemic chemotherapy combined with CT-guided RFA, while patients in the control group were given systemic chemotherapy only. After treatment, a comparative analysis was conducted in terms of clinical efficacy, level of tumor markers, and adverse reactions. Meanwhile, patients in the two groups were followed up for analysis of progression-free survival (PFS) and overall survival (OS). After treatment, the tumor objective response rate and disease control rate of patients in the test group were significantly higher than those in the control group. Besides, in patients of the test group, tumor markers, such as serum carcino-embryonic antigen, neuro-specific enolase, cytokeratin-19, and carbohydrate antigen-199, presented a remarkably lower level relative to those in the control group (p < 0.05). However, there was no significant difference observed with regard to the incidence of complications (p > 0.05). Additionally, patients in the test group were more likely to have better PFS and OS. Notably, we monitored that in the test group, superior clinical efficacy was achieved in patients with non-small cell lung cancer and lung adenocarcinoma relative to that in patients with small cell lung cancer and lung squamous cell carcinoma, respectively. The combined use of systemic chemotherapy and CT-guided RFA can produce good clinical efficacy in treatment of lung cancer. It is relatively safe and deserves promotion and application in clinic.

Roles of Lipid Profiles in Human Non-Small Cell Lung Cancer.

Aims: This review aims to identify lipid biomarkers of non-small cell lung cancer (NSCLC) in human tissue samples and discuss the roles of lipids in tissue molecular identification, the discovery of potential biomarkers, and surgical margin assessment. Methods: A review of the literature focused on lipid-related research using mass spectrometry (MS) techniques in human NSCLC tissues from January 1, 2015, to November 20, 2020, was conducted. The quality of included studies was assessed using the QUADAS-2 tool. Results: Twelve studies met the inclusion criteria and were included in the review. The risk of bias was unclear in the majority of the studies. The contents of lipids including fatty acids, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, cardiolipin, phosphatidyl serine, phosphatidyl glycerol, ceramide, lysophosphatidylethanolamine, lysophosphatidylcholine, and lysophosphatidylglycerol differed significantly between cancer and healthy tissues. The sensitivity or specificity of the discrimination model was reported in 8 studies, and the sensitivity and specificity varied among the reported methods. The lipid profiles differed between adenocarcinoma and squamous cell carcinoma NSCLC subtypes. Conclusion: In preclinical studies, MS analysis and multiple discrimination models can be combined to distinguish NSCLC tissues from healthy tissues based on lipid profiles, which provides a new opportunity to evaluate the surgical margin and cancer subtype intraoperatively. Future studies should provide guidance for selecting patients and discrimination models to develop an improved method for clinical application.

A meta-analysis of minimally invasive surgery versus thoracotomy for centrally located non-small cell lung cancer.

BackgroundSurgery is established as the most effective treatment for central lung cancer. Minimally invasive surgery (MIS) is gaining popularity. The decision of whether surgical treatment of central lung cancer should be minimally invasive or a conventional thoracotomy is a critical decision for the thoracic surgeon. However, whether MIS is more advantageous than other surgical treatments for central lung cancer. This study aimed to compare the short- and long-term results of MIS and conventional thoracotomy in patients with central lung cancer.MethodsThis meta-analysis was conducted using the PubMed, Embase, Wiley Online Library, Google Scholar, Wanfang, and China National Knowledge Infrastructure databases. Searches for relevant studies were conducted in strict accordance with research protocols detailed in the Cochrane handbook. The primary endpoints for comparison between the two surgical methods were perioperative and long-term survival. A 95% confidence interval (CI) for relative risk (RR)/mean difference (MD) was calculated to assess the strength of the correlation.ResultsNine studies that met the inclusion and exclusion criteria were eventually included in this meta-analysis. These studies involved a total of 5,869 patients [MIS, n =1,140 versus thoracotomy (TH), n =4,729]. The 3- and 5-year disease-free survival (DFS) and the 2-, 3-, and 5-year overall survival (OS) were similar for the MIS and TH groups [OR: 0.86 (95% CI: 0.51–1.43); P=0.55; OR: 1.01 (95% CI: 0.43–2.36); P=0.99, OR: 0.91 (95% CI: 0.78–1.05); P=0.18; OR: 0.90 (95% CI: 0.77–1.06); P=0.22; OR: 0.95 (95% CI: 0.25–1.53); P=0.30]. A subgroup analysis of 8 articles revealed no statistical difference in the pathological type of non-small cell lung cancer (squamous cell carcinoma or adenocarcinoma) between the two groups [OR: 0.99 (95% CI: 0.89–1.09); P =0.81]. However, the MIS group had a lower incidence of blood loss, duration of hospital stay, and fewer complications [MD: −46.25 (95% CI: −85.05 to −7.46); P=0.02; MD: −1.93 (95% CI: −3.15 to −0.7); P=0.002; MD: 0.73 (95% CI: 0.61–0.88); P=0.001].ConclusionsMIS was shown to be a more effective option to conventional thoracotomy for the treatment of central lung cancer.

The aqueous extract of brucea javanica reduces tumorigenicity of human lung cancer tumorspheres.

Aim: Therapy to overcome drug resistance by modulating epidermal growth factor receptor (EGFR) is a viable approach to suppress the proliferation of human non-small cell lung cancer (NSCLC) cells. A previous study demonstrated that the seeds of an aqueous Brucea javanica (BJ) (L.) Merr (Simaroubaceae) extract containing quassinoid mixtures effectively inhibited the growth and alleviated tumorigenesis in H1975 cells of NSCLC by targeting T790M/L858R EGFR. This study aimed to further determine whether the aqueous BJ extract affects the enriched H1975 spheroids in suspension culture and mouse xenograft tumor models. Methods: The spheroids of NSCLC adenocarcinoma H1975 cells were enriched in a serum-free media. The growth rate of sphere propagation by aqueous BJ extract was determined in suspended culture and in colony-formation assay. BJ extract was fed orally to nude mice bearing xenograft tumors. The resected tumors were analyzed by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and proliferating cell nuclear antigen assessment. Various markers were used to determine the pluripotency of tumors from mice treated with different concentrations of BJ extract. Results: BJ extract was demonstrated to be effective against the propagation of the enriched spheroids. In animal models, oral administration of the aqueous BJ extract reduced spheroid tumorigenicity. The alleviated growth of the established xenograft tumors can be attributed to the reduced drug resistance and induced apoptosis without distinct adverse effects. More evidence supports activated apoptotic death attenuated spheroid stemness of tumors. Conclusion: As an effective treatment regime to assuage lung cancer, the indigenous BJ extract promises to obliterate drug resistance and the growth of cancer stem cell tumors from NSCLC cells harboring T790M/L858R EGFR.

INSL4 as prognostic marker for proliferation and invasiveness in Non-Small-Cell Lung Cancer.

Non-small-cell-lung cancer accounts for 80-85% of all forms of lung cancer as leading cause of cancer-related death in human. Despite remarkable advances in the diagnosis and therapy of lung cancer, no significant improvements have thus far been achieved in terms of patients' prognosis. Here, we investigated the role of INSL4 - a member of the relaxin-family - in NSCLC. We overexpressed INSL4 in NSCLC cells to analyse in vitro the growth rate and the tumourigenic features. We investigated the signalling pathways engaged in INSL4 overexpressing cells and the tumour growth ability by studying the tumour development in a patient derived tumour xenograft mouse model. We found an INSL4 cell growth promoting effect in vitro in H1299 cells and in vivo in NOD/SCID mice. Surprisingly, in NSCLC-A549 cells, INSL4 overexpression has not similar effect, despite huge basal INSL4-mRNA expression respect to H1299. The INSL4-mRNA analysis of eight different NSCLC-derived cell lines, revealed highly difference in the INSL4-mRNA amount. Transfection of NSCLC lines with INSL4-Myc showed huge level of INSL4-mRNA with a very low amount of protein expressed. Notably, similar discrepancy has been observed in NSCLC patients. However, in a cohort of NSCLC patients analysing a database, we found a significant inverse correlation between INSL4 expression and Overall Survival. By combining the in vitro and in vivo results, suggest that in patients whose NSCLC adenocarcinoma spontaneously expressed high levels of INSL4 post-transcriptional modifications affecting INSL4 do not allow to assess precision therapy in selected patients without consider protein INSL4 amount.

Pilot Study of the Performance of 19-G Needle in Endobronchial Ultrasound-guided Transbronchial Aspiration for the Diagnosis and Testing of Molecular Markers in Lung Cancer.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has become the standard for diagnosis and staging of lung cancer. Historically, 21- and 22-G needles have been paired with EBUS. We evaluated the performance of EBUS-TBNA using a larger 19-G needle in the assessment of tumor tissue obtained and success of testing for molecular markers. We prospectively enrolled adult patients with lymphadenopathy concerning for metastatic lung cancer. Patients underwent diagnostic EBUS-TBNA utilizing 19-G needles. Cases of non-small cell lung cancer (NSCLC) were evaluated for programmed cell death receptor ligand (PD-L1) expression. Cases of adenocarcinoma or undifferentiated NSCLC were further evaluated for 3 molecular markers for driver mutations: epidermal growth factor receptor (EGFR), c-ros oncogene 1 (ROS-1), and anaplastic lymphoma kinase (ALK). Fifty patients were enrolled and underwent EBUS-TBNA using 19-G needles. PD-L1 assay was successfully performed in 90% of NSCLC cases. In adenocarcinoma or undifferentiated NSCLC cases, the success rate in testing was 90% for EGFR and 86% for ALK. ROS-1 testing had a success rate of 67%; 24% of these specimens had adequate tumor cells but there was technical difficulty with the assay. Block quality was judged by total number of tumor cells per hematoxylin and eosin-stained slide of each cell block (58% of specimens had >500 cells and 22% had 200 to 500 cells). There were no adverse events. EBUS-TBNA using 19-G needles can obtain a high number of tumor cells and has a high rate of success in performing assays for PD-L1, EGFR, and ALK in NSCLC patients without an increase in adverse events. The success rate of ROS-1 testing was lower.

Prognostic role of lipid phosphate phosphatases in non-smoker, lung adenocarcinoma patients

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid, plays a crucial role in tumorigenesis. It mediates its function through S1P receptors. A few components of the S1P signaling pathway, such as sphingosine kinase 1 (SphK1) and S1P receptor 1 (S1PR1), have been shown to contribute to lung carcinogenesis. In the present study, using web-based computational tools, we assessed the prognostic roles of eight S1P metabolizing enzymes and five S1P receptors in non-small-cell lung cancer (NSCLC) patients. Except for SPHK1, low expression of S1P metabolizing enzymes was correlated with worse overall survival (OS) in NSCLC patients. Moreover, lower expression of lipid phosphate phosphatase-1 and - 3 (PLPP1 and PLPP3) was significantly associated with worse OS in lung adenocarcinoma (LUAD) and non-smoker NSCLC patients. Furthermore, the UALCAN database analysis showed that mRNA and protein expression of PLPP3 and S1PR1 are significantly down regulated in primary tumors due to hypermethylation of their respective promoters. Expression of PLPP3, S1PR1, and S1PR4 was positively correlated with tumor-infiltrating immune cells in NSCLC patients. These results indicate that S1P signaling genes play a critical prognostic role in LUAD patients. Therefore, this gene signature could be used to predict their prognosis more accurately.

Clinical outcomes of immune checkpoint blockades and the underlying immune escape mechanisms in squamous and adenocarcinoma NSCLC.

Immune checkpoint blockades (ICBs) have changed the standard of care of squamous and adenocarcinoma non‐small cell lung cancer (NSCLC). Whereas detailed researches regarding ICBs in the two major histological subtypes are rare. In order to uncover the clinical efficacy differences between squamous and adenocarcinoma NSCLC and better understand the underlying immune‐regulatory mechanisms, we compared the survival benefits of ICBs between the two subtypes by revealing phase 3 randomized trials and attempted to uncover the immune‐regulatory discrepancy. Generally, compared with nonsquamous NSCLC, squamous NSCLC benefited more from ICBs in Keynote 024, CheckMate 026, CheckMate 227 and CheckMate 017 and similar in OAK, but less in Keynote 010 and PACIFIC. We revealed that the tumor mutation burden (TMB) level, the programmed cell death ligand 1 (PD‐L1) expression, tumor infiltrating lymphocytes (TILs) in the tumor microenvironment (TME), chemokines, and oncogenic driver alterations within the two subtypes may contributed to the clinical outcomes of ICBs. We prospected that the combinations of ICBs with chemotherapy, radiation therapy, and antiangiogenic therapy could be promising strategies to re‐immunize the less immunogenic tumors and further enhance the efficacy of ICBs.

Augmenting Anticancer Immunity Through Combined Targeting of Angiogenic and PD-1/PD-L1 Pathways: Challenges and Opportunities.

Cancer immunotherapy (CIT) with antibodies targeting the programmed cell death 1 protein (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis have changed the standard of care in multiple cancers. However, durable antitumor responses have been observed in only a minority of patients, indicating the presence of other inhibitory mechanisms that act to restrain anticancer immunity. Therefore, new therapeutic strategies targeted against other immune suppressive mechanisms are needed to enhance anticancer immunity and maximize the clinical benefit of CIT in patients who are resistant to immune checkpoint inhibition. Preclinical and clinical studies have identified abnormalities in the tumor microenvironment (TME) that can negatively impact the efficacy of PD-1/PD-L1 blockade. Angiogenic factors such as vascular endothelial growth factor (VEGF) drive immunosuppression in the TME by inducing vascular abnormalities, suppressing antigen presentation and immune effector cells, or augmenting the immune suppressive activity of regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages. In turn, immunosuppressive cells can drive angiogenesis, thereby creating a vicious cycle of suppressed antitumor immunity. VEGF-mediated immune suppression in the TME and its negative impact on the efficacy of CIT provide a therapeutic rationale to combine PD-1/PD-L1 antibodies with anti-VEGF drugs in order to normalize the TME. A multitude of clinical trials have been initiated to evaluate combinations of a PD-1/PD-L1 antibody with an anti-VEGF in a variety of cancers. Recently, the positive results from five Phase III studies in non-small cell lung cancer (adenocarcinoma), renal cell carcinoma, and hepatocellular carcinoma have shown that combinations of PD-1/PD-L1 antibodies and anti-VEGF agents significantly improved clinical outcomes compared with respective standards of care. Such combinations have been approved by health authorities and are now standard treatment options for renal cell carcinoma, non-small cell lung cancer, and hepatocellular carcinoma. A plethora of other randomized studies of similar combinations are currently ongoing. Here, we discuss the principle mechanisms of VEGF-mediated immunosuppression studied in preclinical models or as part of translational clinical studies. We also discuss data from recently reported randomized clinical trials. Finally, we discuss how these concepts and approaches can be further incorporated into clinical practice to improve immunotherapy outcomes for patients with cancer.

Canagliflozin Mediates Effective Blockade of the mTORC1 - HIF-1a Pathway, Inhibits Growth and Enhances Non-Small Cell Lung Cancer (NSCLC) Response to Radiotherapy (RT)

Alterations in NSCLC cell metabolism contribute to rapid tumor growth, resistance to RT and poor prognosis. The mammalian Target of Rapamycin Complex 1 (mTORC1) - Hypoxia-Inducible Factor-1α (HIF-1α) pathway mediates expression of the Warburg glycolytic phenotype in NSCLC and induces RT resistance. Earlier, we showed that the new diabetes agent Canagliflozin (CANA), developed to control hyperglycemia through inhibition of the renal Na+-glucose co-transporter 2 (SGLT2), also inhibits mitochondrial complex I respiration and blocks mTORC1 signaling. In this study, we analyzed CANA’s transcriptional and post-translational effects and its regulation on RT response in preclinical models of NSCLC. Cell proliferation and clonogenic survival assays were used to assess the efficacy of CANA (0–30 μM) and RT (0–8 Gy) in adenocarcinoma (A549, H1299, H1975) and squamous cell carcinoma (SK-MES-1) NSCLC cells. Protein immunoblotting, RT-qPCR and RNAseq of A549 NSCLC cells were performed to study regulation of pathway activation and gene expression in response to CANA and RT. In-vivo effects of the two agents were examined with ectopic A549 xenografts grown in immunodeficient nude mice treated with CANA (100 mg/kg/day in chow diet) and/or RT (5 Gy). RNAseq showed that clinically achievable CANA doses (10 μM) mediate profound regulation of NSCLC cell transcriptional activity with over 100 upregulated and 80 downregulated genes. CANA significantly downregulates HIF-1α pathway-related genes that mediate HIF-1α stability and nuclear transfer, glycolysis and lipogenesis, tyrosine kinase receptor signaling and inhibition of apoptosis. CANA treatment led to activation of AMP-activated protein kinase (AMPK) and blockade of the mammalian target of rapamycin complex 1 (mTORC1) pathway. It abolished phosphorylation of the mitosis marker, histone H3. This was consistent with long-term induction of the G1/S cyclin-dependent kinase inhibitor p27kip1. Clinically relevant doses of CANA suppressed proliferation and clonogenic survival in all NSCLC cell lines but this was enhanced in the K-Ras mutant/LKB1null A549 cells. Combination index analysis indicated additivity and sensitization interaction between the CANA and RT anti-proliferative effects. In the A549 xenograft model, CANA enhanced the cytotoxic effects of RT in an additive manner. CANA mediates effective suppression of the mTORC1-HIF-1α pathway, regulates gene expression and mediates potent anti-tumor activity in NSCLC models in-vitro and in-vivo. Given that CANA is a widely-used, well-tolerated and economical agent, this work indicates strong potential for this agent to be investigated rapidly in NSCLC clinical trials in combination with RT.

SHP-2 and PDL1 Inhibition Combined with Radiotherapy Enhances Systemic Antitumor Effects in an Anti-PD1-resistant Model of Non-small-cell-lung Cancer

Immune checkpoint inhibitors such as anti-PD1/PDL1 have emerged as promising therapies for advanced non-small-cell-lung cancer (NSCLC). However, approximately 80% of patients do not respond to immunotherapy given alone because of intrinsic or acquired resistance. Radiotherapy (XRT) can overcome PD1 resistance and improve treatment outcomes, but its efficacy remains suboptimal. The tyrosine phosphatase SHP-2, expressed in some cancers and in immune cells, has been shown to negatively affect antitumor immunity. Our hypothesis was that SHP-2 inhibition in combination with α-PDL1 would enhance immune-mediated responses to XRT and synergistically boost antitumor effects in an anti-PD1-resistant mouse model. We treated 129Sv/Ev mice with anti-PD1-resistant 344SQ NSCLC adenocarcinoma xenografts with oral SHP099 (an SHP-2 inhibitor) combined with XRT and intraperitoneal α-PDL1. Primary tumors were treated with XRT (3 fractions of 12 Gy each), whereas abscopal (out-of-field) tumors were observed but not treated. In vitro experiments include: qRT-PCR, western blot, flow-cytometry, single-cell-sequence and nano-string. Results: o The Results single-cell-sequence of biopsies from advanced NSCLC patients showed that SHP-2 was significantly higher expressed in to anti-PD-1 non-responders (n = 8) compared to responders (n = 8) (P = 0.041). Then, in vitro and in vivo experiments confirmed that SHP-2 was higher expressed in PD-1 resistant NSCLC model comparing to sensitive model in qRT-PCR, western blot and flow-cytometry. o XRT in combination with SHP099 and α-PDL1 promoted local and abscopal responses, reduced lung metastases, and improved mouse survival. o Expression analysis of 770 immune-related genes indicated that triple therapy significantly increased macrophage function (P = 0.008 vs XRT alone, P = 0.028 vs XRT+α-PDL1), T-cell function (P = 0.003 vs control, P = 0.009 vs XRT alone), and decreased cancer progression (P = 0.048 vs control) in abscopal tumor sites. o Flow-cytometry immune profiling indicated that addition of anti-SHP-2 was associated with higher M1/M2 ratio, greater numbers of CD8+ T cells, and fewer regulatory T cells, compared to iRT group (XRT+anti-PD-L1). o The depletion experiment showed that the anti-tumor effects of triple therapy took place mainly through M1 TAMs and CD8+ T cells. This novel triple-combination therapy had strong antitumor effects in this mouse model of anti-PD1-resistant NSCLC, and may be a novel therapeutic approach for anti-PD1- resistant NSCLC in patients.