<div>Abstract<p><b>Purpose:</b> Current staging methods are imprecise for predicting prognosis of early-stage non–small-cell lung cancer (NSCLC). We aimed to develop a gene expression profile for stage I and stage II NSCLC, allowing identification of patients with a high risk of disease recurrence within 2 to 3 years after initial diagnosis.</p><p><b>Experimental Design:</b> We used whole-genome gene expression microarrays to analyze frozen tumor samples from 172 NSCLC patients (pT1-2, N0-1, M0) from five European institutions, who had undergone complete surgical resection. Median follow-up was 89 months (range, 1.2-389) and 64 patients developed a recurrence. A random two thirds of the samples were assigned as the training cohort with the remaining samples set aside for independent validation. Cox proportional hazards models were used to evaluate the association between expression levels of individual genes and patient recurrence-free survival. A nearest mean analysis was used to develop a gene-expression classifier for disease recurrence.</p><p><b>Results:</b> We have developed a 72-gene expression prognostic NSCLC classifier. Based on the classifier score, patients were classified as either high or low risk of disease recurrence. Patients classified as low risk showed a significantly better recurrence-free survival both in the training set (<i>P</i> < 0.001; <i>n</i> = 103) and in the independent validation set (<i>P</i> < 0.01; <i>n</i> = 69). Genes in our prognostic signature were strongly enriched for genes associated with immune response.</p><p><b>Conclusions:</b> Our 72-gene signature is closely associated with recurrence-free and overall survival in early-stage NSCLC patients and may become a tool for patient selection for adjuvant therapy.</p></div>
Abstract Background: Immune checkpoint inhibitors showed impressive activity in advanced NSCLC and are expected to be effective in postoperative treatment of early disease. New therapies aimed at T and NK cell activation are in development to expand treatments options. It is conceivable that markers of ‘inflamed’, ‘immune system ignorant’ and ‘immune system excluding’ tumor phenotypes may be predictive for specific immune-targeting therapies. Aim: We assessed prognostic value of expression of CD8(+) T and NK cell markers: CD96 and NKG2D in early stage NSCLC. In addition, we analyzed expression of 11 transcripts of APOBEC genes with hypothesis that APOBEC induced mutation burden may affect tumor immunogenicity or host adaptive immunity responsiveness. Methods: mRNA levels were measured by RT-PCR in frozen tumor tissue from 65 NSCLC stage I-IIIA patients who underwent pulmonary resection (75% lung adenocarcinoma, 33% never-smokers, 44% with subsequent dissemination), and in 11 NSCLC cell-lines. The relative gene expression (vs. 5 normalization genes) was compared between groups that did and did not disseminate, and in relation to clinico-pathological features. Results: After Bonferroni correction for multiple testing, NKG2D and CD96 mRNA expression was significantly lower in tumors with subsequent dissemination (p.adj.=0.045). Out of 11 APOBEC genes, expression of AICDA, APOBEC3A and APOBEC3G was lower in tumors with subsequent dissemination (p.unadj.=0.025). The NSCLC lines did not express NKG2D, CD96, AICDA or APOBEC3A. The Spearman corr. coeff. between the expression of NKG2D, CD96 and APOBEC3G were ≥0.8. AICDA expression was correlated with CD96, NKG2D and APOBEC3G with Spearman corr. coeff. in the range of 0.5-0.62. Expression of CD96 and NKG2D was significantly lower in tumors with vs. without subsequent brain metastasis (p.unadj.=0.048). The negative prognostic impact of low NKG2D and CD96 expression was independent from patient smoking status (FC <1, irrespective of smoking status). In the subset of never-smokers, the fold difference of NKG2D expression between tumors with vs. without subsequent dissemination was particularly pronounced (HR=0.37; p.unadj.=0.023). In multivariate Cox analysis including stage, CD96 and NKG2D expression was significantly correlated with distant metastasis free survival (p=0.028). The expression of CD96, NKG2D and APOBEC genes was not related to gender or NSCLC histological type. IHC co-localization and mRNA expression experiments at the background of other more established markers are planned. Conclusions: Low expression of CD96 and NKG2D in early stage NSCLC may be predictive for high propensity to dissemination, including metastasis to the brain. The expression of APOBEC transcripts does not seem to correlate with metastatic potential. Citation Format: Marcin Skrzypski, Michał Marczyk, Amelia Szymanowska, Anna Kowalczyk, Agnieszka Maciejewska, Ryszard Pawlowski, Wojciech Biernat, Joanna Polańska, Jacek Jassem. Prognostic value of the expression of NKG2D and CD96 in early stage non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3700. doi:10.1158/1538-7445.AM2017-3700
7612 Background: 5-year survival for surgically resected SCCL patients (p) is still limited. cDNA microarray studies have identified gene expression patterns that correlate with survival. We have examined the expression pattern of 29 genes selected by these studies to test their clinical prognostic value in early-stage SCCL. Methods: From 2000 to 2004, freshly frozen primary tumor specimens were obtained at the time of surgery from 66 Polish SCCL p. Sections were taken from blocks of tumor tissue for RNA extraction, and gene expression of the 29 genes was assessed by RT-QPCR (AB7900HT) using low density arrays (LDAs, Applied Biosystems). Expression values were dichotomized using the median value as the cut-off. Results: The univariate analysis showed 10 genes with prognostic value: PH4 (P=0.01); macrophage- colony stimulating factor (CSF1) (P=0.002); EGFR (P=0.05); KIAA0974 (P=0.02); ANLN (P=0.02); carbonic anhydrase IX (CA IX) (P=0.007); VEGFC (P=0.03); neurotrophic tyrosine receptor kinase 1 (P=0.04); fibronectin (P=0.002); insulin receptor (P=0.03). In the multivariate analysis of survival, CSF1, EGFR, CA IX and tumor size emerged as significant variables ( Table ). Conclusions: Tumor cells secrete CSF1, which attracts macrophages and induce them to express EGF, which in turn binds to EGFR in tumor cells. CA IX is regulated by hypoxia and plays a role in chemoresistance. Our findings highlight the relevance of tumor size and these markers in selecting p to receive adjuvant chemo- and targeted therapy. No significant financial relationships to disclose. [Table: see text]
Background: About 50% of NSCLC patients (pts) will develop distant metastases following pulmonary resection. Currently, apart from clinical stage at diagnosis, there are no reliable factors to select the high risk pts for adjuvant chemotherapy. We previously demonstrated prognostic value of 22 miRs in frozen tissue samples of early stage SqCLC, and the feasibility of their expression assessment in formalin fixed paraffin embedded (FFPE) samples (Skrzypski et. al. J Clin Oncol 2010; 28;15s). In this study, we validated the prognostic value of these miRs in an independent cohort of early AC pts. Methods: FFPE tumor samples were obtained from 82 stage I-II AC pts who underwent radical pulmonary resection, 44% of whom developed distant metastases. Median follow-up of pts who did not develop metastases was 5.53 years (range, 3,01-8.9 years). miRs were isolated from tumor tissue with RecoverAll kit (Ambion). Expression of 22 miRs previously found to be related to the risk of metastases was analyzed by RT-PCR assay (Appliedbiosystems). Raw data were normalized vs. the expression of U6. Individual miRs were correlated with distant metastases-free survival (MFS). Results: MiR-222* (p=0,0003) and miR-222 (p=0,002) were significantly related to MFS. Using the median of the miR-222* expression as a cut-off value, the median MFS was 2.12 years in the high risk group, and not reached in the low risk group (HR=1.95). Using the median of the miR-222 expression as a cut-off value, the median MFS was 2.56 years in the high risk group, and not reached in the low risk group (HR=1.78). Conclusions: MiR-222* and miR-222 are strong predictors of distant relapse in operable early AC.
Abstract Murine tissues harbor signature γδ T cell compartments with profound yet differential impacts on carcinogenesis. Conversely, human tissue-resident γδ cells are less well defined. In the present study, we show that human lung tissues harbor a resident Vδ1 γδ T cell population. Moreover, we demonstrate that Vδ1 T cells with resident memory and effector memory phenotypes were enriched in lung tumors compared with nontumor lung tissues. Intratumoral Vδ1 T cells possessed stem-like features and were skewed toward cytolysis and helper T cell type 1 function, akin to intratumoral natural killer and CD8 + T cells considered beneficial to the patient. Indeed, ongoing remission post-surgery was significantly associated with the numbers of CD45RA − CD27 − effector memory Vδ1 T cells in tumors and, most strikingly, with the numbers of CD103 + tissue-resident Vδ1 T cells in nonmalignant lung tissues. Our findings offer basic insights into human body surface immunology that collectively support integrating Vδ1 T cell biology into immunotherapeutic strategies for nonsmall cell lung cancer.
11556 Background: Therapies aimed at activation of T and NK cells are developed to expand NSCLC treatments options. It is conceivable that markers of ‘immune ignorant’, ‘immune excluding’ or ‘inflamed’ tumor phenotypes could be prognostic or predictive of benefit from specific immune-targeting therapies. Aim: To assess the prognostic value of expression of T and NK cells mRNA markers and immune-related genes in early stage NSCLC. Methods: qRT-PCR was used to assess 48 mRNAs levels in frozen cancer tissue sections and matched normal lung parenchyma from 56 surgically treated stage I-IIIA NSCLC patients. The mRNA expression (normalized vs. 4 reference genes) was compared between the groups that did (44%) or did not relapse, as well as clinicopathological features (33% never-smokers, 75% lung adenocarcinoma). Results: Low expression of FAS-L (p.adj. = 0.048) , TIGIT and LAG3 was correlated with shorter distant metastasis free survival (DMFS) (p < 0.04). Expression of PD-1 (p = 0.024) and CTLA4 (p = 0.04) was significantly lower in relapsed vs. non-relapsed NSCLCs, whereas there was no difference for PDL-1 and PDL-2. Expression of NK activation markers: NCR3 and NCR1, but not NCR3-ligand 1 was significantly lower in relapsed vs. not relapsed NSCLCs. Other NK cell markers: CD96 and NKG2D were expressed at lower levels (p = 0.02) in relapsed vs. not relapsed NSCLCs, whereas there was no difference for NKG2C and NKG2A. Expression of CXCR3 was lower in relapsed NSCLCs (p = 0.03), however, the expression of its ligands (chemoattractants for lymphocytes) - CXCL9, CXCL10 or CXCL11 or endothelin receptor type B was not different according to metastatic status. GITR and FOXP3 expression was significantly higher in cancers vs. normal lung parenchyma (p.adj. < 0.003). There were no differences in expression according to gender, smoking or NSCLC histological types. Conclusions: Non-inflamed NSCLC phenotype is associated with higher risk of dissemination after primary resection. Neoplastic tissue is characterized by higher level of immune tolerance in comparison to normal lung tissue.
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