Non-small Cell Lung Carcinoma Specimens Research Articles

Spatial intratumor heterogeneity of programmed death-ligand 1 expression predicts poor prognosis in resected non-small cell lung cancer.

We quantified the pathological spatial intratumor heterogeneity (ITH) of programmed death-ligand 1 (PD-L1) expression and investigated its relevance to patient outcomes in surgically resected non-small cell lung carcinoma (NSCLC). This study enrolled 239 consecutive surgically resected NSCLC specimens of pathological stage IIA-IIIB. To characterize the spatial ITH of PD-L1 expression in NSCLC tissues, we developed a mathematical model based on texture image analysis and determined the spatial heterogeneity index of PD-L1 (SHIP) for each tumor. The correlation between the SHIP values and clinicopathological characteristics, including prognosis, was analyzed. Furthermore, an independent cohort of 70 cases was analyzed for model validation. Clinicopathological analysis showed correlations between high SHIP values and histological subtype (squamous cell carcinoma, p < .001) and vascular invasion (p = .004). Survival analysis revealed that patients with high SHIP values presented a significantly worse recurrence-free rate than those with low SHIP values (5-year RFS 26.3% vs 47.1%, p < .005). The impact of SHIP on cancer survival rates was verified through validation in an independent cohort. Moreover, high SHIP values were significantly associated with tumor recurrence in squamous cell carcinoma (5-year RFS 29.2% vs 52.8%, p < .05) and adenocarcinoma (5-year RFS 19.6% vs 43.0%, p < .01). Moreover, we demonstrated that a high SHIP value was an independent risk factor for tumor recurrence. We presented an image analysis model to quantify the spatial ITH of protein expression in tumor tissues. This model demonstrated that the spatial ITH of PD-L1 expression in surgically resected NSCLC predicts poor patient outcomes.

Progression of Non-Small-Cell Lung Carcinoma is Aggravated by LINC01410-Induced Upregulation of PGK1 via the Sponge Effect on miR-4523

This study investigates the role of LINC01410 in non-small-cell lung carcinoma (NSCLC) progression and the underlying mechanisms. Differential expressions of LINC01410, miR-4523, and PGK1 were examined in NSCLC and normal specimens using qRT-PCR. Prognostic and diagnostic potentials of LINC01410 were assessed via Kaplan-Meier and ROC analyses. Proliferation and migration rates in LINC01410-manipulated A549 and H1299 cells were evaluated using CCK-8 and transwell assays. Binding relationships between LINC01410/miR-4523 and miR-4523/PGK1 were determined through luciferase assays and correlation tests. Rescue experiments were conducted to elucidate the LINC01410/miR-4523/PGK1 axis’s role in NSCLC cell behavior regulation. LINC01410 and PGK1 were upregulated in NSCLC specimens, while miR-4523 was downregulated. High LINC01410 levels predicted poor NSCLC prognosis, with diagnostic potential confirmed by ROC curves. LINC01410 overexpression significantly enhanced A549 and H1299 cell proliferation and migration. LINC01410 acted as a miR-4523 sponge, and miR-4523 targeted PGK1. PGK1 overexpression partially attenuated LINC01410-induced NSCLC cell proliferation and migration. In conclusion, LINC01410 drives NSCLC cell proliferation and migration by upregulating PGK1 through miR-4523 sequestration.

Exploration of HER2 (ERBB2) immunohistochemistry in non-small cell lung cancer: correlation with ERBB2 mutational status: experimental research.

ERBB2 (HER2) is a gene in humans that encodes the ERBB2 protein, a member of the epidermal growth factor receptor family. Non-small cell lung carcinomas do not commonly harbour ERBB2 mutations, with clinical trials conducted to assess for targeted response and progression-free survival. We retrieved cases of lung adenocarcinoma with next-generation sequencing proven ERBB2 point mutations (n=8) or amplifications (n=11) and assessed the concordance of commercially available ERBB2 (HER2) immunohistochemical antibodies with the next-generation sequencing result. At present, no commercially available ERBB2 clone can accurately detect ERBB2 mutations consistently in non-small cell lung carcinoma specimens, but amplifications can be detected with reasonable diagnostic accuracy.

Abstract 5616: Optical metabolic imaging of treatment-naive human NSCLC to determine long-term outcome

Abstract Lung cancer remains the leading cause of cancer deaths in the US and worldwide.​ The five-year survival rate of patients with non-small cell lung carcinoma (NSCLC) remains significantly low given that over half present with locally advanced or metastatic disease at time of diagnosis, and experience tumor recurrence following therapeutic intervention.​Current evaluation techniques to assess treatment response are lacking, given they are implemented several weeks after treatment completion and are solely based on anatomical changes in tumor size, forgoing other criteria such as functional or metabolic changes.​ There is a critical need to identify markers early on following diagnosis that are indicative of patient long-term outcome. Two photon microscopy (TPM) techniques are well-suited to provide non-invasive high-resolution information on cell metabolism within three-dimensional tissue. Specifically, two-photon excited fluorescence imaging of autofluorescent cofactors, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), can be used to metabolically characterize cancerous tissue. The goal of this study is to utilize the optical redox ratio (ORR) of FAD/[NADH+FAD] autofluorescence and NADH fluorescence lifetime decay to identify measurable differences in optical metabolic endpoints of human NSCLC that are indicative of their long-term outcome. Twenty-nine NSCLC specimens were obtained from the Lung Cancer Biospecimen Resource Network. They were resected from the lung prior to the patient receiving therapy. Clinical detail reports were used to evaluate the follow-up data of each patient and classify them into groups that reflected their eventual outcomes: responder (N=21), non-responder (N=18), non-metastatic (N=25), and metastatic (N=14). TPM was used to determine the ORR for each sample. NADH lifetime images were collected and fit to a biexponential model to separate the short (A1) and long (A2) components of free and bound NADH. We observed a significantly higher optical redox ratio for tumors within the metastatic group compared to non-metastatic. ​This is consistent with previous findings from our lab that revealed increases in the ORR with increasing metastatic potential in cells, as well as other studies reporting a decrease in NADH concentration in metastatic cancers.​ Additionally, a significantly shorter mean NADH lifetime in the metastatic tumor group was revealed when compared to non-metastatic. This stands in agreement with other studies highlighting decreases in the average lifetime of NADH in metastatic cells compared to non-metastatic. These results demonstrate the feasibility of using optical imaging of autofluorescence and lifetimes of metabolic cofactors to characterize treatment-naïve primary NSCLC tumors and determine differences in their optical metabolic endpoints that are indicative of their long-term outcome. Citation Format: Paola Monterroso Diaz. Optical metabolic imaging of treatment-naive human NSCLC to determine long-term outcome. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5616.

Optimal Preservations of Cytological Materials Using Liquid-Based Cytology Fixatives for Next-Generation Sequencing Analysis

Introduction: Molecular targeted therapies have been established for various diseases, including cancers, and there is an increasing need for molecular testing on cytology specimens. The aim of this study was to determine the optimal preservation methods of liquid-based cytology (LBC) materials for molecular testing. Methods: Cytological samples from 35 surgical resected non-small cell lung carcinoma specimens were obtained between June 2016 and June 2021. The samples were fixed in CytoRich™ red Preservative and stored at 4°C. One week later, three tubes were prepared from each specimen sample and divided into the following groups: the SurePath™ group (continued storage at 4°C), Frozen (Fr) group (stored at −80°C after centrifugation), and LBC-Cell Block (LBC-CB) group (generation of paraffin-embedded CB and storage at 4°C). Samples from 5 patients were used for the time course analysis, and we performed evaluations on these samples at 1, 3, 6, 12, 24, and 36 months. The concentrations and purities of extracted DNA and RNA were measured. The double-stranded DNA (dsDNA) and RNA concentrations were also measured by a fluorometer. The DNA and RNA integrities were quantified by the DNA and RNA integrity number. Results: Evaluation of samples was performed at baseline and the six timepoints. In the LBC-CB group, DNA and dsDNA concentrations were higher rather than those in the other groups. The RNA concentration of the LBC-CB group was relatively high compared with those of the other groups at the 36-month timepoint. The Fr group maintained higher DNA quality compared with the other groups over 3 years. The LBC-CB group maintained a higher RNA quality than the other groups until 24 months. Conclusion: LBC-CB preparation is an effective method to maintain DNA/RNA quality and quantity in long-duration preservation for eventual molecular testing. Therefore, LBC-CB may have applications on preanalytical stage for molecular genomic testing such as next-generation sequencing.

Expression Level and Clinical Significance of AK021443 in Non-Small-Cell Lung Carcinoma.

To explore the prognostic potential of AK021443 in non-small-cell lung carcinoma (NSCLC), AK021443 levels in NSCLC specimens were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The correlation between the AK021443 level and pathological factors in NSCLC patients was analyzed. Kaplan–Meier curves were plotted for assessing the prognostic value of AK021443 in NSCLC patients. Potential factors influencing NSCLC prognosis were analyzed by multivariable Cox regression test. AK021443 was upregulated in NSCLC specimens than normal ones. Its level was correlated to histological type, tumor differentiation, TNM staging, and lymphatic metastasis in NSCLC patients. AK021443 was the independent risk factor for the overall survival of NSCLC. AK021443 is highly expressed in NSCLC specimens, which is correlated to histological type, tumor differentiation, TNM staging, and lymphatic metastasis in NSCLC patients. It is the independent prognostic factor for NSCLC.

454 Analysis of NY-ESO-1 expression in specimens from a Phase I/II NY-ESO-1 T-cell therapy clinical trial in non-small cell lung cancer and from exploratory studies in multiple tumor types

BackgroundThis analysis evaluates an NY-ESO-1 immunohistochemistry (IHC) clinical trial assay in multiple tumor types for the identification of patients who may be eligible for NY-ESO-1 TCR T-cell targeted therapy. We provide an analysis of NY-ESO-1 expression and prevalence in non-small cell lung carcinoma (NSCLC) tumor samples from a patient cohort of an early Phase I/II clinical trial assessing NY-ESO-1 TCR T-cell therapy. Furthermore, we describe exploratory analyses of NY-ESO-1 prevalence and expression in a preliminary set of multiple tumor types to identify new indications for NY-ESO-1 TCR T-cell therapy.MethodsAn IHC assay was developed to detect NY-ESO-1 expression in formalin-fixed paraffin-embedded (FFPE) specimens utilizing an anti-NY-ESO-1 monoclonal antibody, clone E978. NY-ESO-1 protein expression levels and diagnostic status were determined by pathological evaluation under light microscopy to capture the percentage of tumor cell staining across all tumor cells in specimens at staining intensities 0, 1+, 2+ and 3+. NY-ESO-1 expression data were assessed for: prevalence using a ≥10% cutoff at ≥ 1+ intensity to assign positivity, and prevalence across classification (primary and metastatic) and subtype (adenocarcinoma and squamous cell carcinoma) for the NSCLC specimens.ResultsThe overall prevalence for NSCLC specimens from the Phase I/II trial was 15% (49/325) for NY-ESO-1. A prevalence of 15% (29/191) for primary and 14% (19/132) for metastatic samples, 13% (20/159) for adenocarcinoma, and 14% (5/35) for squamous cell carcinoma was observed. No significant difference was observed between subtype or%Tumor at each intensity. The preliminary set of indications used in exploratory studies had an observed prevalence as follows: gastric adenocarcinoma, 14 (4/28)%; esophageal adenocarcinoma &amp; gastric esophageal junction, 9% (3/35); urothelial, 19% (6/31); head and neck squamous cell carcinoma, 10% (3/30); triple negative breast, 10% (3/30); hepatocellular carcinoma, 3%(1/30); and melanoma, 11% (3/27). NY-ESO-1 protein expression was localized in the cells’ nuclei and surrounding cytoplasm.ConclusionsMultiple indications assessed by the IHC clinical trial assay demonstrated similar NY-ESO-1 expression across the range of staining intensities and percentage of positive tumor observed as that in NSCLC, therefore warranting further development and validation of an IHC assay for NY-ESO-1 detection in these additional tumor types for use in clinical trials. These data support the use of IHC as a tool for the identification of patients whose tumors upregulate NY-ESO-1 in NSCLC and further encourage the investigation of multiple tumor types that may upregulate NY-ESO-1 as potential targets for NY-ESO-1 TCR T-cell therapies.AcknowledgementsThis study (NCT03709706) was funded by GlaxoSmithKline.Trial RegistrationNCT03709706ReferencesThomas R, et al. Front Immunol 2018;9:947Ethics ApprovalThis study was approved by the appropriate institutional review boards and independent ethics committees.

Prognostic Impact of PCK1 Protein Kinase Activity-Dependent Nuclear SREBP1 Activation in Non-Small-Cell Lung Carcinoma

Metabolic enzymes can perform non-metabolic functions and play critical roles in the regulation of a variety of important cellular activities. Phosphoenolpyruvate carboxykinase 1 (PCK1), a gluconeogenesis enzyme, was recently identified as an AKT-regulated protein kinase that phosphorylates INSIG1/2 to promote nuclear SREBP1-dependent lipogenesis. However, the relationship of this regulation with the progression of non-small-cell lung carcinoma (NSCLC) is unclear. Here, we demonstrate that epidermal growth factor receptor (EGFR) activation induces AKT-dependent PCK1 pS90, PCK1-mediated INSIG1 pS207/INSIG2 pS151, and nuclear SREBP1 accumulation in NSCLC cells. In addition, the expression levels of AKT pS473, PCK1 pS90, INSIG1 pS207/INSIG2 pS151, and nuclear SREBP1 are higher in 451 analyzed human NSCLC specimens than in their adjacent normal tissues and positively correlated with each other in the tumor specimens. Furthermore, the expression levels of PCK1 pS90, INSIG1 pS207/INSIG2 pS151, and nuclear SREBP1 are associated with TNM stage and progression in NSCLC. Importantly, levels of PCK1 pS90 or INSIG1 pS207/INSIG2 pS151 are positively correlated with poor prognosis in NSCLC patients, and the combined expression value of the PCK1 and INSIG1/2 phosphorylation has a better prognostic value than that of each individual protein phosphorylation value and is an independent prognostic marker for NSCLC. These findings reveal the role of PCK1-mediated nuclear SREBP1 activation in NSCLC progression and highlight the potential to target the protein kinase activity of PCK1 for the diagnosis and treatment of human NSCLC.

Prohibitin 2/PHB2 in Parkin-Mediated Mitophagy: A Potential Therapeutic Target for Non-Small Cell Lung Carcinoma.

BackgroundMitophagy, a selective autophagy process, plays various roles in tumors. Prohibitin 2 (PHB2) is an inner-mitochondrial membrane protein that participates in parkin-induced mitophagy. However, the role of PHB2 in non-small cell lung carcinoma (NSCLC) has not been previously reported.Material/MethodsPHB2 protein or PHB2-mRNA in NSCLC and paired normal tissues was determined by Western blot, qRT-PCR, and immunohistochemical staining. Cell proliferation was detected by CCK-8 assay. Cell migration was evaluated by wound healing and transwell migration assays. A 3D live-cell confocal system was used to monitor autophagic flux. Mitochondrial autolysosomes were observed by transmission electron microscopy (TEM). Finally, we performed JC-1 assay to measure mitochondrial membrane potential (MMP).ResultsThe level of PHB2 was significantly increased in human NSCLC specimens compared to paired adjacent specimens. Inhibition of PHB2 expression attenuated mitophagy in A549 and H1299 cells, as indicated by decreased levels of LC3 II/I and parkin markers and increased level of p62 protein. Furthermore, the inhibition caused reduction in mitochondrial autolysosomes and autophagic flux, as shown by TEM and live-cell imaging, respectively. In addition, PHB2 inhibition caused a remarkable increase in MMP and suppressed the proliferation and migration of A549 and H1299 cells.ConclusionsOur results suggest that downregulation of PHB2 reduced parkin-mediated mitophagy, which suppressed proliferation and migration of A549 and H1299 cells.

Autophagy inhibition of cancer stem cells promotes the efficacy of cisplatin against non-small cell lung carcinoma.

Background:Clinical treatment of non-small cell lung carcinoma (NSCLC) by cisplatin eventually results in drug resistance, which cancer stem cells and autophagy are believed to be involved in. In the present study, we aimed to explore the effect of autophagy-inhibited cancer stem cells in NSCLC.Methods:Cancer stem cells were identified by CD133 expression levels detected by immunochemistry, real-time polymerase chain reaction, western blot, and flow cytometry. Stemness was detected by sphere-forming assays of tumor cells. Autophagy was determined by LC3-II expression at mRNA and protein levels. The effect of chloroquine (CQ) on autophagy was detected by real-time polymerase chain reaction, western blot and sphere-forming assay in vitro, and tumor growth in male NOD/SCID mice.Results:Cisplatin (CDDP) treatment enhanced CD133+ cell ratios in clinical NSCLC specimens and NSCLC cell line A549. The CD133+ cells enriched by CDDP exhibited higher autophagy levels. Autophagy inhibition by CQ inhibited CD133+ stemness and promoted CDDP efficiency in A549 cells. In addition, the combination of CDDP and CQ treatment significantly inhibited autophagy levels and cancer stem cell proportions in vitro, and dramatically suppressed tumor growth compared with individual agents.Conclusion:Autophagy inhibition of cancer stem cells could promote the efficacy of cisplatin against NSCLC.

High CEP55 expression is associated with poor prognosis in non-small-cell lung cancer.

ObjectivesLung cancer is the most common and lethal malignancy worldwide. CEP55 was found to be overexpressed in multiple types of cancer. However, the expression pattern of CEP55 and its clinical significance in non-small-cell lung carcinoma (NSCLC) have not been investigated by immunohistochemistry.Materials and methodsIn this study, we analyzed 203 primary NSCLC specimens from Sun Yat-Sen University Cancer Center to investigate the clinical role of CEP55 in lung cancer. Tissue microarray was successfully generated for immunohistochemical evaluation. The correlation between CEP55 expression and clinical characteristics and survival was analyzed statistically. The predictive effect of CEP55 and APOBEC3B (AP3B) coexpression in lung cancer patients’ prognosis was evaluated.ResultsWe found that the CEP55 expression was commonly elevated in NSCLC tissues and overexpression of CEP55 was correlated with unfavorable prognosis in the patients with NSCLC. Furthermore, the combination of CEP55 and AP3B expression was significantly predictive of clinical outcome in all NSCLC patients.ConclusionCEP55 may act as a useful and novel prognostic biomarker for NSCLC. Further studies into the mechanism of CEP55 are warranted.

Estrogen receptor-alpha isoforms are the main estrogen receptors expressed in non-small cell lung carcinoma

The expression profile of estrogen receptors (ER) in Non-Small Cell Lung Carcinoma (NSCLC) remains contradictory. Here we investigated protein and transcriptome expression of ERα wild type and variants. Tissue Micro-Arrays of 200 cases of NSCLC (paired tumor/non-tumor) were assayed by immunohistochemistry using a panel of ERα antibodies targeting different epitopes (HC20, 6F11, 1D5, ERα36 and ERα17p). ERβ epitopes were also examined for comparison. In parallel we conducted a probe-set mapping (Affymetrix HGU133 plus 2 chip) meta-analysis of 12 NSCLC tumor public transcriptomic studies (1418 cases) and 39 NSCLC cell lines. Finally, we have investigated early transcriptional effects of 17β-estradiol, 17β-estradiol-BSA, tamoxifen and their combination in two NSCLC cell lines (A549, H520). ERα transcript and protein detection in NSCLC specimens and cell lines suggests that extranuclear ERα variants, like ERα36, prevail, while wild-type ERα66 is minimally expressed. In non-tumor lung, the wild-type ERα66 is quasi-absent. The combined evaluation of ERα isoform staining intensity and subcellular localization with sex, can discriminate NSCLC subtypes and normal lung. Overall ERα transcription decreases in NSCLC. ERα expression is sex-related in non-tumor tissue, but in NSCLC it is exclusively correlating with tumor histologic subtype. ERα isoform protein expression is higher than ERβ. ERα isoforms are functional and display specific early transcriptional effects following steroid treatment. In conclusion, our data show a wide extranuclear ERα-variant expression in normal lung and NSCLC that is not reported by routine pathology ER evaluation criteria, limited in the nuclear wild type receptor.

Prognostic Significance of EDIL3 Expression and Correlation with Mesenchymal Phenotype and Microvessel Density in Lung Adenocarcinoma

We examined the prognostic significance of Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3) expression and its correlations with mesenchymal phenotype and microvessel density in non-small cell lung carcinoma (NSCLC). A total of 268 NSCLC specimens were evaluated retrospectively by immunohistochemical staining for EDIL3, EMT markers (e-cadherin, β-catenin, and vimentin), and CD31 to measure microvessel density. EDIL3, e-cadherin, β-catenin, and vimentin were expressed in 16%, 22.8%, 3.7%, and 10.1% of the specimens, respectively. The mRNA level of EDIL3 in tumor was correlated with the level of EDIL3 protein expression using immunohistochemistry. In lung adenocarcinoma patients, EDIL3 expression was significantly correlated with low e-cadherin expression, high vimentin expression, and increased microvessel density (P < 0.001, P = 0.001, and P = 0.023, respectively). In lung squamous cell carcinoma patients, EDIL3 expression was significantly correlated with low e-cadherin expression and high vimentin expression (P = 0.021 and P = 0.002, respectively). In lung adenocarcinoma patients, EDIL3 was an independent prognostic factor for overall survival in a multivariate analysis (hazard ratio: 2.552, P = 0.004). EDIL3 is significantly correlated with mesenchymal phenotype, angiogenesis, and tumor progression in lung adenocarcinoma.

Recombinant EGFR/MMP-2 bi-targeted fusion protein markedly binding to non-small-cell lung carcinoma and exerting potent therapeutic efficacy

Overexpression of EGFR and MMP-2 plays an essential role in the initiation and progression of non-small-cell lung carcinoma (NSCLC). In this study, a novel format of EGFR/MMP-2 bi-targeted fusion protein Ec-LDP-TIMP2 and its enediyne-integrated analogue Ec-LDP(AE)-TIMP2 have been prepared by genetic engineering and molecular reconstitution. The Ec-LDP(AE)-TIMP2 comprises endogenous inhibitor of matrix metalloproteinase 2 (TIMP2), EGF-derived oligopeptide (Ec), lidamycin apoprotein (LDP), and the extremely potent cytotoxic enediyne (AE). By tissue microarray, Ec-LDP-TIMP2 showed high binding intensity and selectivity to human NSCLC specimens as compared with the matched non-cancerous tissues. By in vivo imaging, Ec-LDP-TIMP2 displayed prominent tumor-specific distribution in human NSCLC H460 xenograft. Particularly, Ec-LDP(AE)-TIMP2 inhibited tumor growth of H460 xenograft in athymic mice more striking. At doses of 0.2 and 0.4mg/kg, Ec-LDP(AE)-TIMP2 suppressed tumor growth by 74% and 89%, respectively. No histopathological changes were found in various organs of treated animals, suggesting that the effective dosage was tolerated. In summary, the ligand-based and enediyne-integrated fusion protein displaying extremely potent cytotoxicity might be highly effective for NSCLC therapy and useful as a carrier for drug delivery.

Heterogeneity of Anaplastic Lymphoma Kinase Gene Rearrangement in Non–Small-Cell Lung Carcinomas: A Comparative Study Between Small Biopsy and Excision Samples

The standard diagnostic method for echinoderm microtubule-associated protein-like 4-anaplastic lymphoma receptor tyrosine kinase translocation is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has been reported as a potential method in screening for anaplastic lymphoma kinase (ALK)-positive non-small-cell lung carcinomas (NSCLC), whereas several authors have reported a discordance between FISH and IHC results. We investigated the heterogeneity of ALK gene rearrangement in excision specimens by FISH and also examined whether the FISH score of ALK gene rearrangement corresponded in excision and biopsy samples from the same patient. Twenty ALK IHC-positive patients including six patients treated with crizotinib therapy were evaluated for the presence of ALK FISH. For evaluation of heterogeneity of ALK gene rearrangement in excision specimens, we defined six to 10 observation areas in each case, and the number of ALK FISH positive observation areas (≥15% rearrangement detected) was investigated. ALK FISH score in small biopsy samples was classified as positive (≥15% rearrangement detected), equivocal (5-14% rearrangement detected), or negative (<4% rearrangement detected). Of a total of 64 tumor observation areas from nine excision specimens, 50 areas were positive for ALK gene rearrangement (81.8%). In the comparison of excision and small biopsy samples, all excision specimens were ALK FISH-positive (100%; 6 of 6), whereas only three of the small biopsy samples in these patients were positive (50%; 3 of 6), two were equivocal (33%; 2 of 6), and one was negative (17%; 1 of 6). The two equivocal patients received crizotinib and showed a response. ALK gene rearrangement heterogeneity was observed in NSCLC specimens by FISH. Our findings suggested that IHC-positive/FISH-equivocal cases should not be considered true "false-negatives" when a small biopsy sample was used for ALK analysis.

CD8+CD103+ tumor-infiltrating lymphocytes are tumor-specific tissue-resident memory T cells and a prognostic factor for survival in lung cancer patients.

We had previously demonstrated the role of CD103 integrin on lung tumor-infiltrating lymphocyte (TIL) clones in promoting specific TCR-mediated epithelial tumor cell cytotoxicity. However, the contribution of CD103 on intratumoral T cell distribution and functions and the prognosis significance of TIL subpopulations in non-small cell lung carcinoma (NSCLC) have thus far not been systematically addressed. In this study, we show that an enhanced CD103(+) TIL subset correlates with improved early stage NSCLC patient survival and increased intraepithelial lymphocyte infiltration. Moreover, our results indicate that CD8(+)CD103(+) TIL, freshly isolated from NSCLC specimens, display transcriptomic and phenotypic signatures characteristic of tissue-resident memory T cells and frequently express PD-1 and Tim-3 checkpoint receptors. This TIL subset also displays increased activation-induced cell death and mediates specific cytolytic activity toward autologous tumor cells upon blockade of the PD-1-PD-L1 interaction. These findings emphasize the role of CD8(+)CD103(+) tissue-resident memory T cells in promoting intratumoral CTL responses and support the rationale for using anti-PD-1 blocking Ab to reverse tumor-induced T cell exhaustion in NSCLC patients.

Assessment of the prognostic value of methylation status and expression levels of FHIT, GSTP1 and p16 in non-small cell lung cancer in Egyptian patients.

Methylation of tumor suppressor genes has been investigated in all kinds of cancer. Tumor specific epigenetic alterations can be used as a molecular markers of malignancy, which can lead to better diagnosis, prognosis and therapy. Therefore, the aim of this study was to evaluate the association between gene hypermethylation and expression of fragile histidine triad (FHIT), glutathione S-transferase P1 (GSTP1) and p16 genes and various clinicopathologic characteristics in primary non-small cell lung carcinomas (NSCLC). The study included 28 primary non-small cell lung carcinomas, where an additional 28 tissue samples taken from apparently normal safety margin surrounding the tumors served as controls. Methylation-specific polymerase chain reaction (MSP) was performed to analyze the methylation status of FHIT, GSTP1 and p16 while their mRNA expression levels were measured using a real-time PCR assay with SYBR Green I. The methylation frequencies of the genes tested in NSCLC specimens were 53.6% for FHIT, 25% for GSTP1, and 0% for p16, and the risk of FHIT hypermethylation increased among patients with NSCLC by 2.88, while the risk of GSTP1 hypermethylation increased by 2.33. Hypermethylation of FHIT gene showed a highly significant correlation with pathologic stage (p<0.01) and a significant correlation with smoking habit and FHIT mRNA expression level (p<0.05). In contrast, no correlation was observed between the methylation of GSTP1 or p16 and smoking habit or any other parameter investigated (p>0.05). RESULTS of the present study suggest that methylation of FHIT is a useful biomarker of biologically aggressive disease in patients with NSCLC. FHIT methylation may play a role in lung cancer later metastatic stages while GSTP1 methylation may rather play a role in the early pathogenesis.

Regulation and novel action of thymidine phosphorylase in non-small cell lung cancer: crosstalk with Nrf2 and HO-1.

Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell proliferation and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1β and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1β and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC.

Evaluation of EGFR, KRAS, and TP53 mutations as predictive of disease recurrence in resected early non-small cell lung carcinomas (NSCLCs)

Purpose Identification of non-small cell lung carcinoma (NSCLC) patients at high-risk for recurrence after complete resection would potentially direct surveillance frequency and administration of adjuvant treatments. Genetic profiling of tumors could provide clinicians with information regarding recurrence risk. Conflicting evidence exists regarding EGFR, KRAS, and TP53 mutations as prognostic for recurrence. We aimed to test such mutations for prognostic significance in a cohort of early-resected NSCLC specimens.

A Single-Tube Multiplexed Assay for Detecting ALK, ROS1, and RET Fusions in Lung Cancer

Approximately 7% of non-small cell lung carcinomas (NSCLCs) harbor oncogenic fusions involving ALK, ROS1, and RET. Although tumors harboring ALK fusions are highly sensitive to crizotinib, emerging preclinical and clinical data demonstrate that patients with ROS1 or RET fusions may also benefit from inhibitors targeting these kinases. Using a transcript-based method, we designed a combination of 3' overexpression and fusion-specific detection strategies to detect ALK, ROS1 and RET fusion transcripts in NSCLC tumors. We validated the assay in 295 NSCLC specimens and showed that the assay is highly sensitive and specific. ALK results were 100% concordant with fluorescence in situ hybridization (FISH) (n = 52) and 97.8% concordant with IHC (n = 179) [sensitivity, 96.8% (95% CI 91.0%-98.9%); specificity, 98.8% (95% CI 93.6%-99.8%)]. For ROS1 and RET, we also observed 100% concordance with FISH (n = 46 and n = 15, respectively). We identified seven ROS1 and 14 RET fusion-positive tumors and confirmed the fusion status by RT-PCR and FISH. One RET fusion involved a novel partner, cutlike homeobox 1 gene (CUX1), yielding an in-frame CUX1-RET fusion. ROS1 and RET fusions were significantly enriched in tumors without KRAS/EGFR/ALK alterations. ALK/ROS1/RET/EGFR/KRAS alterations were mutually exclusive. As a single-tube assay, this test shows promise as a more practical and cost-effective screening modality for detecting rare but targetable fusions in NSCLC.