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The TNM classification is well established as a state-of-the-art prognostic and treatment-decision-making tool for non-small cell lung cancer (NSCLC) patients. However, incorporation of biological data may hone the TNM system. This article focuses on choosing and incorporating subsets of tissue-infiltrating lymphocyte (TIL), detected by specific immunohistochemistry and automatically quantified by open source software, into a TNM-Immune cell score (TNM-I) for NSCLC. We use common markers (CD3, CD4, CD8, CD20 and CD45RO) of TILs to identify TIL subsets in tissue micro-arrays comprising tumor tissue from 553 patients resected for primary NSCLC. The number of TILs is automatically quantified using open source software (QuPath). Their prognostic efficacy, alone and within a TNM-I model, is evaluated in all patients and histological subgroups. Compared with previous manual semi-quantitative scoring of TILs in the same cohort, the present digital quantification proved superior. As a proof-of-concept, we construct a TNM-I, using TNM categories and the CD8+ TIL density. The TNM-I is an independent prognosticator of favorable diagnosis in both the overall cohort and in the main histological subgroups. In conclusion, CD8+ TIL density is the most promising candidate marker for a TNM-I in NSCLC. The prognostic efficacy of the CD8+ TIL density is strongest in lung squamous cell carcinomas, whereas both CD8+ TILs and CD20+ TILs, or a combination of these, may be candidates for a TNM-I in lung adenocarcinoma. Furthermore, based on the presented results, digital quantification is the preferred method for scoring TILs in the future., (© The Author(s) 2020. Published by Oxford University Press.)

The presence of tumor-infiltrating lymphocytes (TILs) positively impacts the outcome of non-small cell lung cancer (NSCLC) patients. Most previous studies have assessed TILs using different immunohistochemical assays. The purpose of this study was to develop and validate a histopathological scoring model for the assessment of TILs in whole-tissue hematoxylin and eosin (H&E)-stained section slides of NSCLC patients and to evaluate the model in an immunoscore setting. Therefore, TIL was evaluated manually on H&E slides from 537 surgical specimens of primary resected stage I-III NSCLC patients. Using stromal TIL score as a stepwise discrete variable, increasing survival was seen with rising TIL level: disease-specific survival (DSS; P = .008), overall survival (P = .036) and disease-free survival (P = .006). Subgroup analysis revealed that high stromal TILs level was associated with superior DSS (P = .047) in patients with squamous cell carcinoma, but not in patients with adenocarcinoma. Multivariable analysis confirmed that high TIL levels independently predict improved prognosis for all endpoints in the overall cohort. In conclusion, high stromal TIL level is an independent favorable prognostic factor in stage I-III NSCLC patients. The comprehensive histological evaluation conducted in this study may be helpful in streamlining TIL quantification for routine clinical use in a future NSCLC immunoscore setting., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recent data indicate that tumor-associated neutrophils (TANs) serve a dual role in tumor progression and regression. CD66b is a neutrophil marker and has been associated with patient outcome in various cancers. However, its clinical impact in non-small cell lung cancer (NSCLC) remains controversial. 536 NSCLC patients, of which 172 harbored lymph node metastases, were included in this study. Tissue microarrays were constructed and multiplexed immunohistochemistry of CD66b, CD34 and pan-keratin was performed to evaluate the localization and quantity of CD66b+ TANs. High intratumoral CD66b+ TANs density in squamous cell carcinoma (SCC) subgroup was an independent positive prognosticator for disease-specific survival (P = 0.038). In contrast, high intratumoral TANs density was an independent negative prognostic factor in the adenocarcinoma (ADC) subgroup (P= 0.032). Likewise, in ADC patients with lymph node metastases, high level of intratumoral TANs was associated with poor prognosis (P = 0.003). Stromal CD66b+ TANs were not associated with outcome of NSCLC patients. In conclusion, CD66b+ TANs show diverging prognostic effect in NSCLC patients according to histological subgroups. The presence of CD66b+ TANs could prove pivotal for development of an immunoscore in ADC NSCLC patients.

Tumor-infiltrating lymphocytes (TILs) are vital in limiting cancer progression and may supplement the TNM classification. CD45RO(+) memory TILs show major prognostic impact in various malignancies but have not been extensively explored in non-small cell lung cancer (NSCLC). In this study, we aimed to evaluate their potential in a NSCLC TNM-Immunoscore. Tissue microarrays were constructed from tumor tissue samples from two cohorts including in total 536 patients (University Hospital of North Norway, n = 285; Nordland Hospital, n = 251) with primary resected stage I to IIIA NSCLC. The density of CD45RO(+) and CD8(+) TILs in tumor epithelial and stromal compartments of the tumors was evaluated by immunohistochemistry. In univariate analyses, intraepithelial CD45RO(+) TIL density (T-CD45RO) was a significant prognostic factor for disease-specific survival (P = .007), limited to the squamous cell carcinoma (SCC) histology subgroup (P < .001), where it was significant in both cohorts (University Hospital of North Norway, P = .003; Nordland Hospital, P = .022). Combining T-CD45RO and stromal CD8(+) TIL density (S-CD8) increased the prognostic impact in SCC (P < .001) and showed a significant impact within all pathological stages (I, P = .025; II, P < .001; III, P = .001). In the multivariate analysis, T-CD45RO was an independent positive prognostic factor for SCC (hazard ratio 2.65, 95% confidence interval 1.64-4.28, P < .001), and in combination with S-CD8, the prognostic impact increased vastly (high + high versus low + low: hazard ratio 6.50, 95% confidence interval 3.54-11.91, P < .001). In conclusion, T-CD45RO was an independent prognostic factor for SCC NSCLC. When combined with S-CD8, the prognostic impact increased and was significant within each pathological stage. We propose CD45RO as a candidate marker for TNM-Immunoscore in SCC NSCLC., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: To investigate if hypoxia induces vascular endothelial growth factor (VEGF)-A and VEGF-C secretion in non-small cell lung cancer (NSCLC) cells and if the secretion is cell type-dependent., Materials and Methods: Adenocarcinoma (AC) (H522, PAC) and squamous cell carcinoma (SCC) (H520) cell lines were exposed to hypoxia and normoxia. Supernatants were analysed with enzyme-linked immunosorbent assay (ELISA). Tissue microarrays, from 304 patients diagnosed with stage I-IIIA NSCLC, were immunohistochemically-stained and scored for VEGF-A and VEGF-C., Results: In vitro, VEGF-A expression in hypoxic AC cells was significantly higher than that in normoxic cells (H522: p=0.004, PAC; p=0.007). In contrast, hypoxia led to significantly reduced VEGF-A production in the SCC cell line compared to normoxic cells (p=0.005)., Conclusion: In vitro, AC and SCC exhibit different VEGF-A responses to hypoxia. Hypoxia mediates a pro-angiogenic response in AC, but apparently not in SCC.

Background: NOTCH and hypoxia pathways are both known to be highly involved in cancer. Because of the close interplay between both of these pathways, we investigated correlation and co-expression of molecules in these pathways., Materials and Methods: In 335 unselected stage I-IIIA NSCLC patients, protein expressions of hypoxia inducible factor 1α (HIF1α), hypoxia inducible factor 2α (HIF2α), glucose transporter 1 (GLUT1), lactate dehydrogenase 5 (LDH5), carbonic anhydrase IX (CAIX), delta like 4 (DLL4), JAGGED1, NOTCH1 and NOTCH4, evaluated by immunohistochemistry, were correlated and co-expressions tested in tumor and stromal cells., Results: HIF2α and LDH5 correlated moderately with DLL4, JAGGED1 and NOTCH4 in both tumor and stromal compartments (Spearman's r=0.16-0.33). The coexpression of HIF1α and NOTCH1 in tumor was significantly indicative of poor prognosis in univariate analysis. Hypoxia and NOTCH ligands and receptors were moderately correlated., Conclusion: The lack of appealing coexpression findings for HIF1α and NOTCH1 may be due to the way HIF1α directly influences NOTCH signalling without depending on an elevated NOTCH expression.

Background: Notch signaling plays a key role in embryonic vascular development and angiogenesis. The authors aimed to study the prognostic role of the angiogenesis-related Notch ligands and receptors and investigate the prognostic impact of the coexpression of vascular endothelial growth factor-A (VEGF-A) and Notch signaling., Methods: Tumor tissue samples from 335 resected patients with stage I to IIIA nonsmall cell lung cancer (NSCLC) were obtained, and tissue microarrays were constructed from duplicate cores of tumor cells and tumor-related stroma from each specimen. Immunohistochemistry was used to evaluate the expression of the molecular markers Notch-1, Notch-4, Delta-like ligand 4 (DLL4), and Jagged-1., Results: There were 191 squamous cell carcinomas (SCCs), 113 adenocarcinomas (ACs), and 31 large cell carcinomas. In AC, low tumor cell Delta-like ligand 4 expression was an independent negative prognostic factor (hazard ratio [HR], 2.9; 95% confidence interval [CI], 1.4-6.3 [P = .006]), whereas high tumor cell Notch-1 expression was an independent negative prognostic factor (HR, 2.2; 95% CI, 1.2-4.1 [P<.001]). In SCC, low stromal Delta-like ligand 4 expression was an independent indicator of poor prognosis (HR, 3.3; 95% CI, 1.8-6.1 [P<.001]). The coexpression of Notch-1 and VEGF-A had a significant prognostic impact (P<.001). For Notch-1 and VEGF-A, low/low (n = 142) versus high/high (n = 35) expression resulted in 5-year survival rates of 69% and 32%, respectively., Conclusions: Delta-like ligand 4 and Notch-1 are independent prognostic factors in NSCLC, but have diverse impacts in SCC and AC. The coexpression of tumor cell Notch-1/VEGF-A has a major impact on survival., (Copyright © 2010 American Cancer Society.)