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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.)

Background: Lymphocyte activation gene-3 (LAG-3) is an immune checkpoint receptor and a putative therapeutic target in non-small-cell lung cancer (NSCLC). We explored the prognostic effect of LAG-3 + tumor-infiltrating lymphocytes (TILs) in primary tumors and metastatic lymph nodes in NSCLC and its potential for inclusion in an immunoscore, supplementing the TNM classification., Materials and Methods: Primary tumor tissue from 553 stage I-IIIB NSCLC patients and 143 corresponding metastatic lymph nodes were collected. The expression of LAG-3 was evaluated by immunohistochemistry on tissue microarrays., Results: On univariate analysis, LAG-3 + TILs in the intraepithelial and stromal compartments of primary tumors and in the intraepithelial and extraepithelial compartments of metastatic lymph nodes were associated with improved disease-specific survival (DSS). On multivariate analysis, stromal LAG-3 + TILs were a significant independent predictor of improved DSS (hazard ratio [HR], 0.59; 95% confidence interval [CI], 0.43-0.82; P = .002). Stromal LAG-3 + TILs did not have prognostic impact across all pathologic stages. In the metastatic lymph nodes, intraepithelial (HR, 0.61; 95% CI, 0.38-0.99; P = .049) and extraepithelial (HR, 0.54; 95% CI, 0.29-0.70; P < .001) LAG-3 + TILs were independently associated with favorable DSS., Conclusion: LAG-3 + TILs are an independent positive prognostic factor in stage I-IIIB NSCLC. LAG-3 in metastatic lymph nodes is a candidate marker for an immunoscore in NSCLC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction: Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) or its ligand, PD-L1, have gained momentum in the treatment of non-small cell lung cancer (NSCLC). However, their prognostic significance remains controversial. The present study evaluated the expression of PD-L1 and PD-1 and their potential role in an Immunoscore, supplementing the TNM classification of NSCLC., Materials and Methods: Tissue microarrays constructed from tumor tissue samples from 2 cohorts of a total of 536 patients (University Hospital of North Norway, n = 285; Nordland Hospital, n = 251) with primary resected stage I to IIIA NSCLC. PD-L1 and PD-1 were evaluated by immunohistochemistry in the primary tumor and metastatic lymph node tissue., Results: In univariate analysis, a high density of PD-L1 + immune cells in the stromal compartment (S-PD-L1) and PD-1 + intraepithelial tumor infiltrating lymphocytes (T-PD-1) was associated with favorable disease-specific survival (DSS; S-PD-L1, P = .004; T-PD-1, P = .012), both limited to the squamous cell carcinoma histologic subgroup (S-PD-L1, P = .002; T-PD-1, P = .034). A combined low S-PD-L1 and T-PD-1 was associated with poor survival in all patients (DSS: hazard ratio [HR], 1.81; 95% confidence interval [CI], 1.37-2.40; P < .001) at both centers and for all pathologic stages. In multivariate analysis, S-PD-L1 and T-PD-1 were independent positive prognostic factors, and combined low scores remained an independent prognosticator for poor survival (DSS: HR, 1.72; 95% CI, 1.29-2.28; P < .001; disease-free survival, P = .001; overall survival, P = .005)., Conclusion: Our study identified S-PD-L1 and T-PD-1 as independent positive prognostic factors for NSCLC patients. Their combination added significant prognostic impact within each pathologic stage and hence are feasible to include in a TNM Immunoscore., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Operable non-small cell lung cancer (NSCLC) patients whose tumours have spread to regional or central lymph nodes at the time of diagnosis have dismal prognoses compared with those who have limited disease. The current TNM staging system for NSCLC poorly distinguishes patients with lymph-node metastases who will succumb to, and those who will eventually be cured from, their disease. This novel study: (1) evaluates the presence of different subsets of intraepithelial tumour-infiltrating lymphocytes (TILs) in lymph nodes with metastases from NSCLC patients; (2) explores the impact of intraepithelial TILs in lymph nodes on survival; (3) correlates their presence with both intraepithelial and stromal TILs in their corresponding primary tumours., Methods: Metastatic lymph-node tissue from 143N+ NSCLC patients was collected and tissue microarrays were constructed. Immunohistochemistry was used to evaluate the presence of intraepithelial CD3+, CD4+, CD8+, CD20+ and CD45RO+ TILs and their impact on survival., Results: A high level of intraepithelial CD45RO+ TILs in lymph-node metastases from N+ NSCLC patients was an independent positive prognostic factor for disease-specific survival in all patients (HR=0.58, P=0.029) and in squamous cell carcinoma (HR=0.31, P=0.006), but not in adenocarcinoma patients., Conclusions: The presence of intraepithelial CD45RO+ cells in lymph-node metastases from N+ NSCLC patients predicts favourable disease-specific survival and outperforms the established TNM staging system in the SCC subgroup.

Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates and appears highly promising as a supplement to the tumor-node-metastasis (TNM) classification of various tumors. In colorectal cancer, an international task force has initiated prospective multicenter studies aiming to implement TNM-Immunoscore (TNM-I) in a routine clinical setting. In breast cancer, recommendations for the evaluation of tumor-infiltrating lymphocytes (TILs) have been proposed by an international working group. Regardless of promising results, there are potential obstacles related to implementing TNM-I into the clinic. Diverse methods may be needed for different malignancies and even within each cancer entity. Nevertheless, a uniform approach across malignancies would be advantageous. In nonsmall-cell lung cancer (NSCLC), there are several previous reports indicating an apparent prognostic importance of TILs, but studies on TILs in a TNM-I setting are sparse and no general recommendations are made. However, recently published data is promising, evoking a realistic hope of a clinical useful NSCLC TNM-I. This review will focus on the TNM-I potential in NSCLC and propose strategies for clinical implementation of a TNM-I in resected NSCLC., (© The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.)

Purpose: Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates, which appears to be superior to the tumor-node-metastasis (TNM) classification in colorectal cancer. In non-small cell lung cancer (NSCLC), no immunoscore has been established, but in situ tumor immunology is recognized as highly important. We have previously evaluated the prognostic impact of several immunological markers in NSCLC, yielding the density of stromal CD8(+) tumor-infiltrating lymphocytes (TIL) as the most promising candidate. Hence, we validate the impact of stromal CD8(+) TIL density as an immunoscore in NSCLC., Experimental Design: The prognostic impact of stromal CD8(+) TILs was evaluated in four different cohorts from Norway and Denmark consisting of 797 stage I-IIIA NSCLC patients. The Tromso cohort (n = 155) was used as training set, and the results were further validated in the cohorts from Bodo (n = 169), Oslo (n = 295), and Denmark (n = 178). Tissue microarrays and clinical routine CD8 staining were used for all cohorts., Results: Stromal CD8(+) TIL density was an independent prognostic factor in the total material (n = 797) regardless of the endpoint: disease-free survival (P < 0.001), disease-specific survival (P < 0.001), or overall survival (P < 0.001). Subgroup analyses revealed significant prognostic impact of stromal CD8(+) TIL density within each pathologic stage (pStage). In multivariate analysis, stromal CD8(+) TIL density and pStage were independent prognostic variables., Conclusions: Stromal CD8(+) TIL density has independent prognostic impact in resected NSCLC, adds prognostic impact within each pStage, and is a good candidate marker for establishing a TNM-Immunoscore., (©2015 American Association for Cancer Research.)

In addition to malignant neoplastic cells, cancer tissues also include immune cells, fibroblasts, and endothelial cells, including an abundant collection of growth factors, proangiogenic mediators, cytokines, chemokines, and components of the extracellular matrix. The main physiological function of the immune cells is to monitor tissue homeostasis, to protect against invading pathogens, and to eliminate transformed or damaged cells. Between immune cells and malignant cells in the tumor stroma, there is in fact a complex interaction which has significant prognostic relevance as the immune system has both tumor-promoting and -inhibiting roles. In non-small cell lung cancer (NSCLC), there is a marked infiltration of different types of immune cells, and the distribution, tissue localization, and cell types are significantly associated with progression and survival. Cancer immunotherapy has seen a significant progress during the last decade. An increased understanding of the mechanisms by which lung cancer cells escape the immune system, and the recognition of the key tumor antigens and immune system components in tumor ignorance have led to the development of several lung cancer vaccines. As the NSCLC prognosis in general is dismal, one may hope that future immunotherapy may be an effective adjunct to standard therapy, reversing immunologic tolerance in the tumor microenvironment. This review reports on the tumor stroma and in particular tumor-suppressing and -promoting roles of the immune system. Furthermore, it presents recent literature on relevant immune cell-related research in NSCLC.

Purpose: The purpose of this study was to clarify the prognostic significance of lymphocyte infiltration in soft tissue sarcomas (STS). Prognostic markers in potentially curable STS should guide therapy after surgical resection. The immune status at the time of resection may be important, but the prognostic significance of tumor infiltrating lymphocytes is controversial as the immune system has conflicting roles during cancer development., Experimental Design: Tissue microarrays from 249 patients with STS were constructed from duplicate cores of viable and representative neoplastic tumor areas. Immunohistochemistry was used to evaluate the CD3+, CD4+, CD8+, CD20+ and CD45+ lymphocytes in tumors., Results: In univariate analyses, increased numbers of CD4+ (P = 0.008) and CD20+ (P = 0.006) lymphocytes in tumor correlated significantly with an improved disease-specific survival (DSS) in patients with wide resection margins (n = 108). In patients with non-wide resection margins (n = 141) increased numbers of CD3+ (P = 0.028) lymphocytes in tumor correlated significantly with shorter DSS. In multivariate analyses, a high number of CD20+ lymphocytes (HR = 5.5, CI 95%  = 1.6-18.6, P = 0.006) in the tumor was an independent positive prognostic factor for DSS in patients with wide resections margins., Conclusions: High density of CD20+ lymphocytes in STS with wide resection margins is an independent positive prognostic indicator for these patients. Further research is needed to define if CD20+ cells can modify tumors in a way that reduces disease progression and metastatic potential.

Background: There seems to be a close interplay between angiogenesis and the immune system. The authors of this report investigated the prognostic role of angiogenic markers in coexpression with immune system markers in patients with nonsmall cell lung cancer (NSCLC)., Methods: Tumor resection samples from 335 patients with stage I to IIIA NSCLC were obtained, and tissue microarrays were constructed. Immunohistochemistry was used to evaluate the expression of vascular endothelial growth factor (VEGF) A (VEGF-A), VEGF receptor 2 (VEGFR-2), and lymphocytes that were positive for the cluster of differentiation 4 (CD4) and CD8 coreceptors., Results: In univariate analysis, 5-year survival rates were 87% for the combination of low tumor cell expression of VEGF-A and VEGFR-2 (↓VEGF-A/↓VEGFR-2) and high tumor cell expression of CD4 and CD8 (↑CD4/↑CD8) (n = 19), 58% for mixed combinations (n = 290), and 27% for the ↑VEGF-A/↑VEGFR-2 and ↓CD4/↓CD8 combination (n = 26). In multivariate analysis, the coexpression of ↑VEGF-A/↑VEGFR-2 and ↓CD4/↓CD8 was an independent negative prognostic factor (hazard ratio, 9.16; 95% confidence interval, 2.11-39.8; P = .003)., Conclusions: Low tumor cell VEGF-A and VEGFR-2 expression in combination with high adaptive immune cell expression in the tumor-related stroma had a strong and independent favorable prognostic impact in patients with NSCLC., (© 2010 American Cancer Society.)

Aims: The major value of prognostic markers in potentially curable non-small cell lung carcinoma (NSCLC) should be to guide therapy after surgical resection. The prognostic significance of tumour-infiltrating macrophages, their growth factor, macrophage colony-stimulating factor (M-CSF), and its receptor, colony-stimulating factor-1 receptor (CSF-1R), as well as natural killer cells and dendritic cells, is controversial. The aim of this study was to elucidate the prognostic significance of these markers in the epithelial and stromal compartments of NSCLC., Methods and Results: Tissue microarrays from 335 resected NSCLC, stage I-IIIA were constructed from duplicate cores of epithelial and stromal areas. Immunohistochemistry was used to evaluate epithelial and stromal areas for CD68, M-CSF, CSF-1R, CD56 and CD1a. On univariate analysis, increasing numbers of stromal CD1a+ (P = 0.011) and CD56+ cells (P = 0.014) correlated significantly with improved disease-specific survival (DSS). On multivariate analysis, stromal CD56+ cells were an independent prognostic factor for DSS (hazard ratio = 2.3, confidence interval = 1.1, 5.0, P = 0.031)., Conclusions: High density of stromal CD56+ cells is an independent factor associated with improved prognosis in resected NSCLC, suggesting that these cells mediate an antitumour immune response in the tumour stroma.

Purpose: The major value of prognostic markers in potentially curable non-small cell lung cancer (NSCLC) should be to guide therapy after surgical resection. In this regard, the patients' immune status at the time of resection may be important and also measurable. The immune system has paradoxical roles during cancer development. However, the prognostic significance of tumor-infiltrating lymphocytes is controversial. The aim of this study is to elucidate the prognostic significance of epithelial and stromal lymphocyte infiltration in NSCLC., Experimental Design: Tissue microarrays from 335 resected NSCLC, stage I to IIIA were constructed from duplicate cores of viable and representative neoplastic epithelial and stromal areas. Immunohistochemistry was used to evaluate the epithelial and stromal CD4+, CD8+, and CD20+ lymphocytes., Results: In univariate analyses, increasing numbers of epithelial CD8+ (P = 0.023), stromal CD8+ (P = 0.002), epithelial CD20+ (P = 0.023), stromal CD20+ (P < 0.001), and stromal CD4+ (P < 0.001) lymphocytes correlated significantly with an improved disease-specific survival. No such relation was noted for epithelial CD4+ cells. Furthermore, a low level of stromal CD8+ lymphocyte infiltration was associated with an increased incidence of angiolymphatic invasion (P = 0.032). In multivariate analyses, a high number of stromal CD8+ (P = 0.043) and CD4+ (P = 0.002) cells were independent positive prognostic factors for disease-specific survival., Conclusions: High densities of CD4+ and CD8+ lymphocytes in the stroma are independent positive prognostic indicators for resected NSCLC patients. This may suggest that these cells are mediating a strong antitumor immune response in NSCLC.