Poor Prognosis In NSCLC Patients Research Articles

Machine learning and BP neural network revealed abnormal B cell infiltration predicts the survival of lung cancer patients.

FAM83A gene is related to the invasion and metastasis of various tumors. However, the abnormal immune cell infiltration associated with the gene is poorly understood in the pathogenesis and prognosis of NSCLC. Based on the TCGA and GEO databases, we used COX regression and machine learning algorithms (CIBERSORT, random forest, and back propagation neural network) to study the prognostic value of FAM83A and immune infiltration characteristics in NSCLC. High FAM83A expression was significantly associated with poor prognosis of NSCLC patients (p = 0.00016), and had excellent prognostic independence. At the same time, the expression level of FAM83A is significantly related to the T, N, and Stage. Subsequently, based on machine learing strategies, we found that the infiltration level of naive B cells was negatively correlated with the expression of FAM83A. The low infiltration of naive B cells was significantly related to the poor overall survival rate of NSCLC (p = 0.0072). In addition, Cox regression confirmed that FAM83A and naive B cells are risk factors for the prognosis of NSCLC patients. The nomogram combining FAM83A and naive B cells (C-index = 0.748) has a more accurate prognostic ability than the Stage (C-index = 0.651) system. Our analysis shows that abnormal infiltration of naive B cells associated with FAM83A is a key factor in the prognostic prediction of NSCLC patients.

The prognosis of EGFR complex mutation or co-mutation with tyrosine kinase inhibitor treatment in non-small cell lung cancer.

e21086 Background: To compare the prognoses of EGFR complex mutation, co-mutation and single mutation with first-line TKI treatment in NSCLC patients. Methods: A retrospective study of 215 patients with EGFR-mutated NSCLC who were admitted to the Department of Medical Oncology, the Fourth Hospital of Hebei Medical University from January 2015 to January 2021, all received first-line EGFR-TKI therapy. According to the type of mutation, they were divided into 48 cases (22.3%) of EGFR single mutation group, 48 cases (22.3%) of complex mutation group, and 142 cases (66.0%) of co-mutation group, of which 23 cases (10.6%) both had complex mutation and co-mutation. The prognoses of the latter two groups were compared with that of the single mutation group respectively. SPSS 26.0 was used to analyze the data, and P < 0.05 indicated the difference was statistically significant. Results: Progression-free survival (PFS): the co-mutation group was shorter than the single mutation group (11.0m vs 13.5m; HR = 1.492, 95%CI: 1.036-2.149; P= 0.030), especially the co-mutation was occurred in TP53 (10.5 m vs 13.0m; HR = 1.665, 95%CI: 1.234-2.248; P= 0.001) or HER family (9.0m vs 12.0m; HR = 2.435, 95%CI: 1.492-3.974; P< 0.001). Overall survival (OS): compared with the single mutation group, complex mutation group (36.5m vs 52.0m; HR = 1.480, 95%CI: 0.821-2.668; P= 0.193), co-mutation group (41.0m vs 52.0m; HR = 1.418, 95%CI: 0.901-2.231; P= 0.131) were not statistically different, but the co-mutation of PTEN pathway significantly reduced OS (24.0m vs 46.0m; HR = 1.884, 95%CI: 1.137 -3.120; P= 0.014). Objective response rate (ORR) and disease control rate (DCR) of first-line EGFR-TKI: single mutation group separately compared with complex mutation group and co-mutation group (ORR: 64.6% vs 6.2% vs 27.4%, P< 0.001; DCR: 100% vs 70.8% vs 91.5%, P < 0.001 and 0.007 respectively). After the first-generation EGFR-TKI treatment and secondary EGFR E20 T790M mutation, the presence or absence of the original sensitive mutation affects the PFS of the first-generation TKI (12.0m vs 11.0m; HR = 1.109, 95%CI:0.739-1.664 ; P= 0.616), PFS of the third-generation TKI (10.0m vs 8.0m; HR = 1.130, 95%CI: 0.717-1.781; P= 0.599) and OS (34.0m vs 44.5m; HR = 0.834, 95%CI: 0.505-1.377; P= 0.478) were not statistically different. Conclusions: EGFR co-mutation is a predictor of poor prognosis in NSCLC patients treated with EGFR-TKI; after first-generation EGFR-TKI resistance, the disappearance of the original sensitive mutation cannot predict the prognosis.

FP12.06 Solid Subtype Predicts Early Bone Metastases in Sensitive EGFR-Mutated Lung Adenocarcinoma Patients After Surgery

Bone metastasis (BM) is common and associated with poor prognosis in NSCLC patients. However, valid predictors of BM remain insufficient. Solid pattern has been reported as a predictive factor in disease progression in resected NSCLC patients. This study aimed to explore the relationship between solid subtype and BM in EGFR-mutated lung adenocarcinoma patients after surgery. Patients with completely resected lung adenocarcinoma and sensitive EGFR mutation were included in our study. Patients with solid pattern were divided into solid-present group while the rest were in solid-absent group. CT (for chest and brain) was performed every 2-3 months. Bone scan was done every 4-6 months. BM was confirmed if one of the following criteria were fulfilled: (1) clinical or pathological confirmation of lung cancer and diagnosis of BM according to bone biopsy; or (2) pathologically diagnosed as lung cancer and with BM images. All patients received EGFR-TKI as the first-line treatment for progression after operation. Bone disease-free survival (bDFS), DFS and OS were assessed in this study. bDFS was defined as the time span between surgery and BM diagnosis. From January 2007 to January 2017, 237 eligible patients were identified. Among them, 81 patients (34.2 %) were solid-present. BM was observed in 94 patients (39.7 %). Solid-present patients had significantly shorter bDFS than solid-absent patients (14 months vs. 27 months; P < 0.001). The difference was still significant in patients with BM as first-site (12.5 months vs. 16.5 months; P = 0.016) and non-first-site progression (16.5 months vs. 45.5 months; P < 0.001) after surgery (Figure 1). In survival analysis, solid-present group showed a significantly shortened DFS (15 months vs. 19 months; P < 0.001) and OS (47 months vs. 77 months; P = 0.001) (Figure 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT) These findings suggested that solid pattern predicted poor prognosis and early BM occurrence.

Long non-coding RNA DLX6-AS1 knockdown suppresses the tumorigenesis and progression of non-small cell lung cancer through microRNA-16-5p/BMI1 axis.

BackgroundNon-small cell lung cancer (NSCLC) is a huge threat to sufferers’ life and overall health. Long non-coding RNA (lncRNA) distal-less homeobox 6 antisense RNA 1 (DLX6-AS1) has been revealed to function as a carcinogenesis factor in some cancers. This research aimed to scrutinize the role and mechanism underlying DLX6-AS1 in NSCLC tumorigenesis and progression.MethodsThe levels of DLX6-AS1, microRNA-16-5p (miR-16-5p), and BMI1 mRNA were estimated via reverse transcription-quantitative PCR (RT-qPCR) assay. The protein levels were disclosed by western blot assay. Cell proliferative potential was estimated by colony formation and Cell Counting Kit-8 (CCK-8) assays. Cell migration was estimated by Transwell and wound healing assay. A Transwell assay was executed to estimate cell invasion. The relationships of DLX6-AS1, miR-16-5p, and BMI1 were forecasted by bioinformatics analysis, and confirmed by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. A xenograft mice model was employed to to inspect the function of DLX6-AS1 knockdown on NSCLC tumorigenesis in vivo.ResultsDLX6-AS1 was overexpressed in NSCLC tissues and cells, and was inextricably linked with the poor prognosis of NSCLC patients. Depletion of DLX6-AS1 oppressed cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) but promoted apoptosis in NSCLC. MiR-16-5p is a target of DLX6-AS1 and directly targets BMI1. Moreover, the anti-tumor impacts of miR-16-5p were overturned by overexpression of DLX6-AS1 or BMI1 in NSCLC cells. Additionally, DLX6-AS1 silencing inhibited tumor growth of NSCLC in vivo.ConclusionsIn conclusion, lncRNA DLX6-AS1 downregulation suppressed the tumorigenesis and progression of NSCLC via miR-16-5p/BMI1 axis in vitro and in vivo, elucidating the vital roles and downstream targets of DLX6-AS1 in NSCLC.

Abstract 1770: Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice

Abstract ULBP2 is one of the ligands for NKG2D (NKG2DLs). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Previously, we reported high concentrations of serum-soluble ULBP2 (sULBP2) were correlated with poor prognosis in NSCLC patients. As a possible mechanism, we demonstrated that sULBP2 directly suppressed the cytolytic activity of peripheral blood mononuclear cells (PBMCs). However, little is known about the in vivo function of human NKG2DLs on tumor cells. The present study investigates the function of human NKG2DLs in the syngeneic C57BL/6 mice. We generated ULBP2 stably expressing B16 melanoma cell lines (ULBP2-B16). As previously reported, 4-hour 51Cr releasing assay showed NK cell cytotoxicity against the tumor cells was enhanced by ectopic expression of ULBP2 on the tumor cells. IFN-gamma production in NK cells was decreased by non-contact co-culture with ULBP2-B16. To investigate the in vivo function of ULBP2, C57BL/6 mice were inoculated s.c. with mock-B16 cells or ULBP2-B16 cells. ULBP2-B16 tumors grew rapidly compared to mock-B16 tumors. NKG2D blockade by anti-NKG2D antibody injected i.p. suppressed tumor growth of ULBP2-B16 tumors. Spleen cells and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. NKG2D was expressed on splenic NK cells, while it was rarely expressed on splenic CD8+ T cells. NKG2D was downregulated on NK cells in TILs (NK-TILs) from ULBP2-B16. NKG2D was highly expressed on CD8+ T cells in TILs (CD8+-TILs) from mock-B16 tumors, while its expression was suppressed on these from ULBP2-B16 tumors. Degranulation analysis revealed that ULBP2-B16 reduced activity of NK-TILs and CD8+-TILs compared to mock-B16. In conclusion, ectopic expression of ULBP2 on tumor cells promotes tumor progression via suppression of the activity of NK cells and CD8+ T cells. These findings suggest that neutralization of NKG2DLs may be a promising approach for the treatment of NKG2DLs-expressing tumors. Citation Format: Kosuke Yamaguchi, Naomi Miyake, Yasuhiko Teruya, Kohei Yamane, Naoki Kinoshita, Tomohiro Sakamoto, Masahiro Kodani, Hiroki Chikumi, Akira Yamasaki. Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1770.

Prognostic impacts of extracranial metastasis on non-small cell lung cancer with brain metastasis: A retrospective study based on surveillance, epidemiology, and end results database.

This study was designed to investigate the prognostic value of the number and sites of extracranial metastasis (ECM) in NSCLC patients with BM. NSCLC patients with BM from the Surveillance, Epidemiology, and End Results (SEER) database from 2010 to 2015 were enrolled in analysis. Patients from 2010 to 2013 were included in the training set and those from 2014 to 2015 in the validation set. ECM sites among different subtypes of NSCLC were compared by Chi‐square tests. Kaplan–Meier methods and Cox regression models were performed to analyze survival data. Competing‐risks analysis was used to predict cumulative incidence rates for CSS and non‐CSS cause. We included 5974 patients in the training cohort and 3561 patients in the validation cohort. Most (nearly 80%) NSCLC patients with BM showed 0–1 involved extracranial organ, with the most and least common ECM organ being bone and distant lymph nodes (DLNs) among all subtypes of NSCLC, respectively. The number of involved extracranial organs was an independent prognostic factor for patients with BM from NSCLC (p < 0.001). Patients with 0–1 ECM had better survival than those with larger number of involved extracranial organs (p < 0.001). Cumulative incidence rates for CSS were increased with the number of ECM raising (p < 0.001). All involved extracranial organs were associated with worse survival (p < 0.05). In patients with single‐organ ECM, we observed a better prognosis in lung and bone metastasis, while liver metastasis showed worst survival. But the difference in survival in these patient groups was relatively small. Patients with liver metastasis had higher cumulative incidence rates for CSS than that in patients with lung and bone metastasis (p < 0.05). More extracranial metastases were associated with poor prognosis in NSCLC patients with BM and ECM sites showed limited effect on survival. Tailored treatments would be reasonable for BM patients from NSCLC with different metastasis patterns.

Primary resistance to first-generation EGFR-TKIs induced by MDM2 amplification in NSCLC

IntroductionTargeted therapy for NSCLC is rapidly evolving. EGFR-TKIs benefit NSCLC patients with sensitive EGFR mutations and significantly prolong survival. However, 20–30% of patients demonstrate primary resistance to EGFR-TKIs, which leads to the failure of EGFR-TKI treatment. The mechanisms of primary resistance to EGFR-TKIs require further study.MethodsTargeted sequencing was used for the detection of genomic alterations among patients in our center. Regular cell culture and transfection with plasmids were used to establish NSCLC cell lines over-expressing MDM2 and vector control. We used the MTT assays to calculate the inhibition rate after exposure to erlotinib. Available datasets were used to determine the role of MDM2 in the prognosis of NSCLC.ResultsFour patients harboring concurrent sensitive EGFR mutations and MDM2 amplifications demonstrated insensitivity to EGFR-TKIs in our center. In vitro experiments suggested that MDM2 amplification induces primary resistance to erlotinib. Over-expressed MDM2 elevated the IC50 value of erlotinib in HCC2279 line and reduced the inhibition rate. In addition, MDM2 amplification predicted a poor prognosis in NSCLC patients and was associated with a short PFS in those treated with EGFR-TKIs. The ERBB2 pathway was identified as a potential pathway activated by MDM2 amplification could be the focus of further research.ConclusionMDM2 amplification induces the primary resistance to EGFR-TKIs and predicts poor prognosis in NSCLC patients. MDM2 may serve as a novel biomarker and treatment target for NSCLC. Further studies are needed to confirm the mechanism by which amplified MDM2 leads to primary resistance to EGFR-TKIs.

Decreased Preoperative Estimated Glomerular Filtration Rate Was Related With Poor Prognosis of NSCLC Patients.

(1) Background:The coexistence of chronic kidney disease (CKD) and cancer is common due to the increased incidence of cancer in patients with CKD. Glomerular filtration rate is the optimal way to measure kidney function. To date, little is known about the role preoperative renal function plays in the prognosis of NSCLC patients.(2) Methods:The study enrolled 140 patients who had been newly diagnosed NSCLC and received potential radical surgery for treatment from 2009 January to 2012 December. The detailed characteristics were collected including gender, age, smoking and drinking habits, KPS score, hemoglobin levels, tumor size, pathology type, differentiation, pTNM stage and serum creatinine before surgery. Univariate and multivariate analyses of overall survival (OS) and disease-free survival (DFS) were performed using Kaplan-Meier method and Cox model.(3) Results:The univariate analysis identified that the pTNM stage (p < 0.001), eGFR level (p = 0.006), and adjuvant treatment (p = 0.007) were prognostic factors for OS, while drinking habit (p = 0.032), pTNM stage (p = 0.002) and eGFR level (p = 0.006) were the prognostic factors for DFS. Further multivariate analysis found that pTNM stage (HR = 2.091, 95% CI 1.424-3.071; p < 0.001) and eGFR level (HR = 1.890, 95% CI 1.424-3.071; p = 0.004) were independent factors associated with OS. The pTNM stage (HR = 1.735, CI 1.215-2.479; p = 0.002) and eGFR (HR = 1.793, CI 1.193-2.696; p = 0.005) were independent factors associated with DFS. Further subgroup analyses found that in female patients/ no smoking patients/ patients younger than 60 years, better eGFR level was significantly associated with better OS and DFS.(4) Conclusions:Decreased preoperative eGFR was associated with poor clinical outcome of NSCLC patients.

PD-L1+ aneuploid circulating tumor endothelial cells (CTECs) exhibit resistance to the checkpoint blockade immunotherapy in advanced NSCLC patients.

Sustained angiogenesis and increased PD-L1 expression on endothelial and carcinoma cells contribute toward fostering an immunosuppressive microenvironment suitable for tumor growth. PD-L1+ CTCs were reported to associate with poor prognosis in NSCLC patients. However, whether or not aneuploid circulating tumor endothelial cells (CTECs) express PD-L1, then serve as a surrogate biomarker to evaluate immunotherapy efficacy remains unknown. In this study, a novel SE-iFISH strategy was established to comprehensively quantify and characterize a full spectrum of aneuploid CTCs and CTECs in advanced NSCLC patients subjected to second-line anti-PD-1 (nivolumab) immunotherapy. In situ co-detection of diverse subtypes of aneuploid CTCs and CTECs expressing PD-L1 and Vimentin was performed. The present clinical study demonstrated that significant amounts of PD-L1+ aneuploid CTCs and CTECs could be detected in histopathologic hPD-L1- patients. In contrast to decreased PD-L1+ CTCs, the number of multiploid PD-L1+ CTECs (≥tetrasomy 8) undergoing post-therapeutic karyotype shifting increased in patients along with tumor progression following anti-PD-1 treatment. Progressive disease (PD) lung cancer patients possessing multiploid PD-L1+ CTECs had a significantly shorter PFS compared to those without PD-L1+ CTECs. In carcinoma patients, aneuploid CTCs and CTECs may exhibit a functional interplay with respect to tumor angiogenesis, progression, metastasis, and response to immunotherapy.

Abstract 1111: Fucosyltransferase 4-mediated aberrant glycosylation and cell signaling networks promote lung cancer metastasis

Abstract Rationale:Aberrant glycosylation has been known to regulate cancer cell trafficking via promoting intravascular adhesion during metastasis. Clinicopathological studies have also shown a strong correlation between aberrant glycosylation and invasive/metastatic potentials of human cancers. However, the major glycosylation enzymes that drive lung cancer metastases and the signaling networks involved in the process remain incompletely understood. Methods:We identified fucosyltransferase 4 (FUT4) as a strong predictor of patient prognosis from two independent cohorts of patients with NSCLC (TCGA and NTUH). We analyzed functional roles of FUT4 through over-expression and knock-down studies using lung cancer cell lines in vitro. Metastatic and homing potentials of FUT4-overexpressed and/or knockdown cells were evaluated in vivo in immunodeficient mice. Genome-wide RNA-seq and MS-based immune-precipitation proteomics were performed to study molecular signaling networks. Results: High expressions of FUT4 were associated with poor survivals in lung adenocarcinomas (p= 0.000681). FUT4-overexpressed lung cancer cells showed significantly increased migration, invasion, and adhesion abilities in vitro, as well as enhanced homing ability to the lungs in vivo. FUT4-overexpressedcells displayed typical characteristics of epithelial-to-mesenchymal transition (EMT) in a protein level-dependent manner. Lewis X and AAL antigens were significantly up-regulated in FUT4-overexpressedcells, which facilitated binding to E-, L- and P-selections. GSEA of RNA-seq data revealed the enrichment of metastasis-related signaling, especially in EGF and TGF-β signalings. Molecularly, FUT4 enhanced EGFR/TGFBR signaling through glycosylation of TGFBR receptors, which lead to increased binding affinity between ligands and its receptors. Conclusion: FUT4 promoted metastasis through enhancing mesenchymal phenotype mediated by glycosylated EGFR/TGFBR signaling, and through increasing binding affinity of cancer cells to selectins. High levels of FUT4 in tumor tissues significantly correlate with poor prognosis in NSCLC patients. Our study not only provided new insights into the role of FUT4 on molecular signaling as a potential therapeutic target, but also identified FUT4 as a prognostic marker in NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Hsuan-Hsuan Lu, Yi-Hsiu Juan, Rueyhung Roc Weng, Shu-Yung Lin, Yen-Wei Chen, Hsin-Han Hou, Zheng-Ci Hung, Yi-Jhen Huang, Tsai-Yu Yang, Yi-Chieh Wu, Giovanni Audrey Oswita, Jin-Yuan Shih, Hsing-Chen Tsai, Chong-Jen Yu. Fucosyltransferase 4-mediated aberrant glycosylation and cell signaling networks promote lung cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1111.

CD117 expression is correlated with poor survival of patients and progression of lung carcinoma:a meta-analysis with a panel of 2645 patients.

The aim of this research was to investigate the clinical role and prognostic value of CD117 expression assessed immunohistochemically in lung carcinoma through acomprehensive meta-analysis in which 27 publications were acquired and 2645patients were ultimately analysed. Statistical analysis and corresponding plots were performed using STATA version 12.0. Publication bias was assessed by Begg's funnel plots and Egger's test. Pooled HR and its 95% CI (HR=1.53, 95% CI: 1.13-2.07, p=0.007) for overall survival of patients indicated apoor prognostic value for CD117 expression in lung carcinoma, which was accompanied by heterogeneity and publication bias. In the subgroup analysis, there was strong evidence that could support an association between CD117 expression and poor prognosis in NSCLC patients (HR=2.03, 95% CI: 1.41-2.90, p<0.001; heterogeneity: I2= 41.9%, c2=15.49, p=0.078). Multivariate analysis also revealed consistent results in high-quality studies with reported HRs (HR=2.16, 95% CI: 1.67-2.79, p<0.001), and Asian patients (HR=2.12, 95% CI: 1.45-3.10, p<0.001). The correlations between CD117 expression and age, clinical stage, TNM stage, lymph node metastasis, or histology were not statistically significant. In conclusion, CD117 expression might be apotential marker for predicting poor prognosis, faster tumour growth, and early lymph node metastasis in NSCLC.

Blocking the FSTL1-DIP2A Axis Improves Anti-tumor Immunity

Immune dysfunction is a strong factor in the resistance of cancer to treatment. Blocking immune checkpoint pathways is a promising approach to improve anti-tumor immunity, but the clinical efficacies are still limited. We previously identified follistatin-like 1 (FSTL1) as a determinant of immune dysfunction mediated by mesenchymal stromal/stem cells (MSCs) and immunoregulatory cells. Here, we demonstrate that blocking FSTL1 but not immune checkpoint pathways significantly suppresses cancer progression and metastasis in several mouse tumor models with increased MSCs. Expression of DIP2A (the receptor of FSTL1) in tumor cells is critical for FSTL1-induced immunoresistance. FSTL1/DIP2A co-positivity in tumor tissues correlates with poor prognosis in NSCLC patients. Thus, breaking the FSTL1-DIP2A axis may be a useful strategy for successfully inducing anti-tumor immunity.

Abstract B02: Liquid biopsy for screening of driver mutations in EGFR gene: Personalized medicine for use tyrosine kinase inhibitors in non-small cell lung cancer patients

Abstract Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers worldwide. In Brazil there were an estimated 28.220 new cases in 2016. The 5-year survival rate in advanced NSCLC patients is less than 5%, making necessary the development of diagnostic strategies to improve the treatment and survival of these patients. Activating mutations of epidermal growth factor receptor (EGFR) gene occur in approximately 10–15 % in Caucasian and approximately 30% in Brazilian NSCLC cases. The first- and second-generation of EGFR tyrosine kinase inhibitors (TKIs) have been widely used for advanced NSCLC patients who are eligible for the treatment. Liquid biopsies using plasma-derived cell-free DNA (cfDNA) is an emerging noninvasive test allowing a better selection and monitoring of NSCLC. The aim of this study was to evaluate the frequency of EGFR mutations using liquid biopsy samples before and after TKIs treatment in a cohort of NSCLC Brazilian patients. All the patients were investigated for EGFR mutations by real-time qPCR Cobas© EGFR Mutation Test v2. Clinical data were obtained prior to blood collection from physician and/or pathologists involved in the original diagnoses. Blood samples were collected at Progenetica Laboratory or sent from other laboratories, centrifuged immediately for the plasma separation, and frozen. Fisher´s exact test was calculated to compare frequency distribution between two groups (GraphPad Prism version 6.00). This study enrolled 535 NSCLC Brazilian patients from Southeast+South (74.9%), North+Northeast (8.8%), and Midwest+Federal District (4.7%). Regional information was missing for 11.3% of cases. As expected, NSCLC was more frequent among women (56.4%) compared to men (43.6%). Four hundred cases (74.8%) did not show any EGFR mutations; however, 135 cases presented 19Del (57.8%), L858R (16.3%), double 19Del; T790M (11.1%), double T790M; L858R (8.15%), G719X (2.9%), L861Q (1.48%), 20Ins, triple L858R; T790M; 20Ins and S768I; G719X (0.74% each one) mutations spanning 18, 19, 20, and 21 EGFR exons. Previous results from liquid biopsy for EGFR mutation were obtained for 75/535 cases (14%). Among these patients, 10/75 cases (13.3%) presented nondetected mutations. Deletions at exon 19 (19Del) and mutations at exon 21 (L858R) were the most frequent, accounting for 61.5% and 23%, respectively. Since 19Del and L858R are drug-sensitive mutations, we compared the frequency of EGFR mutations in patients before and after TKIs treatment. Interestingly, patients harboring drug-sensitive mutations before the treatment were negative for the presence of 19Del (37.5%) and L858R (66.7%) after the treatment, respectively (p&amp;lt;0.0001). In addition, EGFR T790M mutation already associated with drug resistance was identified in 3/65 (4.6%) of cases in double with 19Del before the TKIs treatment. After treatment, one patient was negative and two patients remained positive for both mutations, respectively. These results showed that liquid biopsy based on cfDNA is a powerful tool for personalized medicine for early detection, stratification, and monitoring of EGFR druggable mutations in NSCLC patients. The presence of cfDNA has been related to aggressiveness and poor prognosis of NSCLC patients, and it is important to note that not all cases have the tumor cfDNA. To our knowledge this is the first report showing the application of liquid biopsies for management of NSCLC patients in Brazil. Clinical data collection is under way for better reliability results. High index of nondetected results was observed in this study; however, new protocols using more sensitive methodologies such as next-generation sequencing and digital PCR are under development in our institution to improve the sensitivity of this test. Citation Format: Priscila D. R. Cirillo, Natália P. Lopes, Carolina Bustamante, Agatha A. Morais, Natália L. Viana, Camila S. Portella, Elvis C. C. Mateo, Maíra C. M. Freire, Mariano G. Zalis. Liquid biopsy for screening of driver mutations in EGFR gene: Personalized medicine for use tyrosine kinase inhibitors in non-small cell lung cancer patients [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B02.

Downregulation of CPA4 to suppress NSCLC proliferation by inducing apoptosis and G1 arrest.

e20074 Background: Carboxypepidase A4 (CPA4) is a member of the metallocarboxypeptidase family. Previous study discovered that CPA4 may participate in cell growth and differentiation of prostate epithelial cells. Meanwhile, CPA4 is a printed gene and thought to be involved in prostate cancer aggressiveness. As is reported, CPA4 was increased in NSCLC tissues compared to normal lung tissues and high expression of CPA4 was correlated with poor prognosis of NSCLC patients. However, the role of CPA4 play in lung tumorigenesis is still unclear. Methods: We examined the mRNA and protein expression level of CPA4 via real-time PCR and immunohistochemistry in NSCLC tissues and adjacent tissues. Growth assays both in vitro and in vivo were performed to elucidate the role of CPA4 may play in lung cancer and Fluorescence Activated Cell Sorter was conducted to uncover the putative mechanism. Results: CPA4 expression was increased both in mRNA and protein levels in NSCLC tissues compared to adjacent tissues. MTT and colony formation assays showed that downregulation of CPA4 in H1299 and A549 cells inhibited lung cancer cells proliferation. We further confirmed this result by using cellomics and celligo. Depleting CPA4 also suppressed tumor growth in mice. Mechanically, we found that suppressing CPA4 expression in lung cancer cells could induce apoptosis and G1 arrest. We supposed that CPA4 expression may be associated with caspase family and it needs further studies. Conclusions: Collectively, we demonstrate that decreased CPA4 inhibits NSCLC proliferation via inducing apoptosis and G1 arrest.

The role of comorbidity in the management and prognosis in nonsmall cell lung cancer: a population-based study

Background: Coexisting disease constitutes a challenge for the provision of optimal cancer care. The influence of comorbidity on lung cancer management and prognosis remains incompletely understood. We assessed the influence of comorbidity on treatment intensity and prognosis in a population-based setting in patients with nonsmall cell lung cancer.Material and methods: Our study was based on information available in Lung Cancer Data Base Sweden (LcBaSe), a database generated by record linkage between the National Lung Cancer Register (NLCR) and several other population-based registers in Sweden. The NLCR includes data on clinical characteristics on 95% of all patients with lung cancer in Sweden since 2002. Comorbidity was assessed using the Charlson Comorbidity Index. Logistic regression and time to event analysis was used to address the association between comorbidity and treatment and prognosis.Results: In adjusted analyses encompassing 19,587 patients with a NSCLC diagnosis and WHO Performance Status 0–2 between 2002 and 2011, those with stage-IA–IIB disease and severe comorbidity were less likely to be offered surgery (OR: 0.45; 95% CI: 0.36–0.57). In late-stage disease (IIIB–IV), severe comorbidity was also associated with lower chemotherapy treatment intensity (OR: 0.76; 95% CI: 0.65–0.89). In patients with early, but not late-stage disease, severe comorbidity in adjusted analyses was associated with an increased all-cause mortality, while lung cancer-specific mortality was largely unaffected by comorbidity burden.Conclusions: Comorbidity contributes to the poor prognosis in NSCLC patients. Routinely published lung cancer survival statistics not considering coexisting disease conveys a too pessimistic picture of prognosis. Optimized management of comorbid conditions pre- and post-NSCLC-specific treatment is likely to improve outcomes.

PIG3 promotes NSCLC cell mitotic progression and is associated with poor prognosis of NSCLC patients

BackgroundNon-small cell lung cancer (NSCLC) is the most commonly diagnosed type of lung cancer that is associated with poor prognosis. In this study we explored the potential role of p53-induced gene 3 (PIG3) in the progression of NSCLC.MethodsImmunohistochemistry was used to determine the expression levels of PIG3 in 201 NSCLC patients. We performed in vitro studies and silenced endogenous PIG3 by using specific siRNAs that specific target PIG3. Immunofluorescent staining was performed to determine the effect of PIG3 on mitotic progression in NSCLC cells. The growth rates of microtubules were determined by microtubule nucleation analysis. Cell proliferation and chemosensitivity were analyzed by CCK8 assays. Annexin V staining and β-galactosidase activity analysis were used to evaluate PIG3 deficiency-related apoptosis and senescence, respectively.ResultsPIG3 expression levels negatively correlated with overall survival and disease-free survival of NSCLC patients. Knock down of PIG3 resulted in repressed proliferation of NSCLC cells and increased aberrant mitosis, which included misaligning and lagging chromosomes, and bi- or multi-nucleated giant cells. In addition, PIG3 contributed to mitotic spindle assembly by promoting microtubule growth. Furthermore, loss of PIG3 sensitized NSCLC cells to docetaxel by enhancing docetaxel-induced apoptosis and senescence.ConclusionsOur results indicate that PIG3 promotes NSCLC progression and therefore suggest that PIG3 may be a potential prognostic biomarker and novel therapeutic target for the treatment of NSCLC.

Knockdown of Lncrna PVT1 Enhances Radiosensitivity in Non-Small Cell Lung Cancer by Sponging Mir-195

Background/Aims: Plasmacytoma variant translocation 1 (PVT1) exerts an oncogenic role in many tumors, including lung cancer. However, the roles of PVT1 in regulating radiosensitivity of NSCLC and its underlying mechanism are still unclear. Methods: Expression levels of PVT1 and miR-195 in NSCLC tissues and cells were examined by qRT-PCR. Effects of PVT1 and miR-195 on cell proliferation, apoptosis and colony formation abilities were assessed by MTT assay, flow cytometry and colony formation assay. Luciferase reporter assay was performed to confirm the relationship between PVT1 and miR-195. Tumor xenograft experiments were conducted to observe the effect of PVT1 on radiosensitivity of NSCLC in vivo. Results: PVT1 was negatively correlated with miR-195 expression in NSCLC tissues and associated with poor prognosis of NSCLC patients. Expression of PVT1 and miR-195 varied inversely after irradiation in NSCLC cells. PVT1 knockdown or miR-195 overexpression enhanced radiosensitivity of NSCLC in vitro by inhibiting proliferation and inducing apoptosis. PVT1 directly interacted with miR-195 and regulated its expression. Moreover, PVT1 knockdown improved radiosensitivity of NSCLC cells in vitro and in vivo by sponging miR-195. Conclusion: Knockdown of PVT1 enhances radiosensitivity of NSCLC by sponging miR-195, providing a novel therapeutic target to improve radiotherapy efficiency in NSCLC.

Blockade of Notch3 inhibits the stem-like property and is associated with ALDH1A1 and CD44 via autophagy in non-small lung cancer.

Acquired resistance to standard chemotherapy causes treatment failure in patients with local advanced and advanced non-small lung cancer (NSCLC). Cancer stem cells (CSCs) are a small subpopulation within cancer that is thought to be resistant to conventional chemotherapy. The Notch pathway is one of the most intensively studied for putative therapeutic targets of CSCs in solid tumors. In our study, suppression of Notch3 decreased colony and sphere formation of stem-like property in lung cancer cells. In addition, Notch3 expression was demonstrated to be upregulated in the patients with chemoresistance and related to poor prognosis of NSCLC patients. Our results also showed that CSC markers ALDH1A1 and CD44 were highly expressed in NSCLC patients with chemoresistance and these two markers were positively correlated with Notch3 expression in lung cancer specimens from TCGA database. Furthermore, the lung cancer cells with drug resistance were shown to be associated with activation of autophagy. All the data support a crucial role of Notch3 in the increase of stem-like property in NSCLC cells that might be associated with upregulation of ALDH1A1 and CD44 and activation of autophagy.

Increased osteopontin protein expression may be correlated with poor prognosis in non-small-cell lung cancer: A meta analysis.

The present meta-nalysis investigates the prognostic value of osteopontin. (OPN) expression in patients with non-small-cell lung cancer. (NSCLC). The Web of Science (1945 ~ 2013), the Cochrane Library Database (Issue 12, 2013), PubMed (1966 ~ 2013), EMBASE (1980 ~ 2013), CINAHL (1982 ~ 2013), and the Chinese Biomedical Database (CBM) (1982 ~ 2013) were searched, without language restrictions, to retrieve studies related to OPN and NSCLC. We compiled carefully selected data and a meta-analysis was conducted using STATA software (Version 12.0, Stata Corporation, and College Station, Texas USA). Hazard ratios (HR) with corresponding 95% confidence interval (95%CI) were calculated. Ten clinical cohort studies were selected for statistical analysis, representing a total of 1,133 NSCLC patients. The main findings of our meta-analysis are that patients who were OPN-positive had significantly shorter overall survival than OPN-negative patients. (HR = 1.47, 95%CI = 1.15. ~ 1.79,P< 0.001). Ethnicity.stratified analysis revealed a significant correlation between expression levels of OPN and poor prognosis of NSCLC patients among both Caucasians and Asians. (Asians: HR = 1.53, 95%CI = 0.95. ~ 2.11, P < 0.001; Caucasians: HR = 1.56, 95%CI = 1.08. ~ 2.03, P < 0.001; respectively). The present meta-analysis is consistent with the hypothesis that increased expression of OPN protein may be significantly associated with poor prognosis in patients with NSCLC.

Abstract C93: RYBP predicts survival of patients with non-small cell lung cancer and sensitizes chemotherapy

Abstract Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers and associates with a poor prognosis. Despite several targeted therapies have been developed with promising results, the therapeutic options for NSCLC are limited and the 5-year survival rate remains poor (∼15%). Therefore, there is an urgent need for a better understanding of its pathogenesis and exploring novel therapeutic targets. Ring1 and YY1 binding protein (RYBP) is a member of polycomb group (PcG) proteins that typically act as transcriptional repressors via epigenetic modification of chromatin and participate in the establishment and maintenance of cell fates. Recent studies have shown that RYBP also possesses PcG-independent functions that promote apoptosis and cell cycle arrest in cancer cells. Our previous study has shown that RYBP stabilizes p53 through inhibiting MDM2 activity. Recent study indicates that RYBP is downregulated in human cancers, including NSCLC, but the underlying mechanisms are largely unknown. The present study was designed to demonstrate the molecular role of RYBP in NSCLC development and progression, chemotherapy, and patient survival. We systemically investigated the expression of RYBP in human NSCLC tissues and matched non-cancerous samples and evaluated the associations between the RYBP expression in NSCLCs and patient survial. We also generated a replication-deficient recombinant adenovirus driving the expression of RYBP (Adv-RYBP) and carried out in vitro and in vivo studies to explore the molecular basis for the tumor suppressing role of RYBP in NSCLC. We first demonstrated that the RYBP mRNA and protein expressions levels were significantly down-regulated in NSCLC tissues compared with matched noncancerous lung tissues. The low expression of RYBP was significantly associated with the poor prognosis in NSCLC patients. We then identified that enforced RYBP expression decreased cell viability, inhibited colony formation and induced apoptosis in NSCLC cells, while RYBP knockdown led to the opposite effects. RYBP affected the expression of several apoptosis-related proteins; and promoting apoptosis was one of the main mechanisms of RYBP-mediated cell growth inhibition in NSCLC. Moreover, AdRYBP led to a decreased NSCLC tumor xenograft growth. Additionally, clinically used chemotherapeutic agents induced the expression of RYBP, and RYBP sensitized NSCLC cells to chemotherapy in vitro and in vivo. In conclusion, our results reveal that RYBP is a potential biomarker for the diagnosis and prognosis of NSCLC and that reactivating RYBP in cancer cells may provide an effective and safe therapeutic approach to NSCLC therapy. (Supported by NIH/NCI grants R01 CA186662). Citation Format: wei wang, Sukesh Voruganti, Jiang-Jiang Qin, Biyun Qian, Ruiwen Zhang. RYBP predicts survival of patients with non-small cell lung cancer and sensitizes chemotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C93.