Evaluation of tumor-infiltrating lymphocytes using routine H&E slides predicts patient survival in resected non-small cell lung cancer.

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.

LAG-3 in Non-Small-cell Lung Cancer: Expression in Primary Tumors and Metastatic Lymph Nodes Is Associated With Improved Survival.

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.

CTLA-4 expression in the non-small cell lung cancer patient tumor microenvironment: diverging prognostic impact in primary tumors and lymph node metastases.

The immune checkpoint receptor CTLA-4 plays a crucial part in negatively regulating T cell activation and maintaining self-tolerance. It is frequently overexpressed in a variety of malignancies, yet its prognostic impact in non-small cell lung cancer (NSCLC) remains unclear. We constructed tissue microarrays from tumor tissue samples and evaluated the immunohistochemical expression of CTLA-4 in 536 patients with primary resected stage I-IIIA NSCLC. Expression of CTLA-4 was analyzed in tumor and stromal primary tumor tissue and in locoregional metastatic lymph nodes. CTLA-4 expression in neither tumor epithelial cells (T-CTLA-4) nor stromal cells (S-CTLA-4) of primary tumors was significantly associated with disease-specific survival (DSS) in all patients. However, high S-CTLA-4 expression independently predicted significantly improved DSS in the squamous cell carcinoma subgroup (HR 0.62, 95% CI 0.41-0.93, P = 0.021). In contrast, there was an independent negative prognostic impact of T-CTLA-4 expression in metastatic lymph nodes (HR 1.65, 95% CI 1.03-2.65, P = 0.039). Our results indicate that the expression of CTLA-4 has diverging prognostic impacts in metastatic NSCLC lymph nodes versus primary tumors. The presented results highlight important differences in the tumor microenvironments of primary and metastatic NSCLC tissues, and have potential to guide treatment and clinical sampling strategies.

Assessing PDL-1 and PD-1 in Non-Small Cell Lung Cancer: A Novel Immunoscore Approach.

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.

CD45RO(+) Memory T Lymphocytes--a Candidate Marker for TNM-Immunoscore in Squamous Non-Small Cell Lung Cancer.

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.

Lymphangiogenic Markers and Their Impact on Nodal Metastasis and Survival in Non-Small Cell Lung Cancer--A Structured Review with Meta-Analysis.

BACKGROUND: In non-small cell lung cancer (NSCLC), nodal metastasis is an adverse prognostic factor. Several mediating factors have been implied in the development of nodal metastases and investigated for predictive and prognostic properties in NSCLC. However, study results differ. In this structured review and meta-analysis we explore the published literature on commonly recognized pathways for molecular regulation of lymphatic metastasis in NSCLC. METHODS: A structured PubMed search was conducted for papers reporting on the expression of known markers of lymhangiogenesis in NSCLC patients. Papers of sufficient quality, presenting survival and/or correlation data were included. RESULTS: High levels of vascular endothelial growth factor C (VEGF-C, HR 1.57 95% CI 1.34-1.84) and high lymphatic vascular density (LVD, HR 1.84 95% CI 1.18-2.87) were significant prognostic markers of poor survival and high expression of VEGF-C, vascular endothelial growth factor receptor 3 (VEGFR3) and LVD was associated with lymph node metastasis in NSCLC. CONCLUSION: Lymphangiogenic markers are prognosticators of survival and correlate with lymph node metastasis in NSCLC. Their exact role and clinical implications should be further elucidated.

Prognostic impact of CXCL16 and CXCR6 in non-small cell lung cancer: combined high CXCL16 expression in tumor stroma and cancer cells yields improved survival.

BACKGROUND: The chemokine CXCL16 and its receptor CXCR6 are expressed by a variety of immune cells and have been shown to influence angiogenesis. The expression of CXCR6 and CXCL16 has been examined in numerous human cancers; however no studies have yet investigated their influence on prognosis in non-small cell lung cancer (NSCLC). We aimed to explore their prognostic significance in NSCLC, in addition to examining associations with previously investigated markers. METHODS: Resected tumor tissue from 335 consecutive unselected stage I-IIIA NSCLC patients (1990-2005) were collected. Immunohistochemistry was used to evaluate the expression of CXCR6 and CXCL16 on tissue microarrays. In vitro, NSCLC cells (NCI-H460, A549 cells) were transfected with CXCL16 siRNA to examine effects on proliferation. RESULTS: In univariate analysis, ↑ stromal cell CXCL16 expression was a significant positive prognostic factor (P = 0.016). CXCR6 was expressed in cancer cells, but did not show any prognostic impact. In the multivariate analysis, combined ↑cancer, and ↑stromal cell CXCL16 expression was an independent positive prognostic factor when compared to ↓stromal and ↓cancer cell expression (HR: 0.42; 95 % CI: 0.20-0.88; P = 0.022). Knockdown of CXCL16 by siRNA resulted in accelerated proliferation of NSCLC cell lines. CONCLUSION: We have shown that combined ↑cancer and ↑stromal cell CXCL16 expression is an independent positive prognostic factor in NSCLC. Further studies are warranted to elucidate the biological mechanism underlying this finding.

The prognostic role of progesterone receptor expression in non-small cell lung cancer patients: Gender-related impacts and correlation with disease-specific survival.

PURPOSE: Progesterone has been shown to impact the development of hormone-sensitive cancers, such as breast and ovarian cancers. Emerging evidence has revealed a possible role of progesterone in the tumorigenesis of other cancers, including lung cancer. Herein, we aimed to elucidate the prevalence and prognostic significance of progesterone receptor (PR) expression in non-small cell lung cancer (NSCLC) tissue. EXPERIMENTAL: Tumor tissue samples were collected from our patient cohort consisting of 335 NSCLC patients with stage I-IIIA disease. Tissue microarrays (TMAs) were constructed, and immunohistochemical (IHC) analyses were performed to evaluate the PR expression in the tumor epithelial and stromal compartments. RESULTS: In a univariate analysis, positive PR expression in the stromal tumor compartment (P=0.005) was significantly and independently associated with a favorable outcome for both genders. Furthermore, positive PR expression in tumor epithelial cells (P=0.003) correlated with a poor prognosis for female patients. In a multivariate analysis, positive PR expression in the tumor stroma (P=0.007) was an independent prognostic factor for improved disease-specific survival (DSS). Positive PR expression in tumor epithelial cells emerged as an independent prognostic factor in female patients (P=0.001) for poor DSS. CONCLUSIONS: We show that PR expression in tumor-surrounding stromal cells is associated with improved DSS for both male and female patients. Additionally, we reveal that positive PR expression in tumor epithelial cells is an independent, unfavorable prognosticator for DSS in female patients, making PR expression a potential marker for prognostic stratification in NSCLC.

Stromal CD8+ T-cell Density­A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.

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.

Positive prognostic impact of miR-210 in non-small cell lung cancer.

OBJECTIVES: miR-210 is an important regulator of the cellular response to hypoxia. Therefore, we aimed to explore the prognostic significance of miR-210 in non-small cell lung cancer (NSCLC) patients with stage I-IIIA disease. MATERIALS AND METHODS: In addition to clinicopathological and demograpic information, tumor tissues were collected and tissue micro arrays (TMAs) were constructed from 335 patients with stage I-IIIA NSCLC. Expression of miR-210 in cancer cells and stromal cells of the tumor was assessed by in situ hybridization. RESULTS: In univariate analyses, high cancer cell (p=0.039) and high stromal cell expression (p=0.008) of miR-210 were both significantly associated with an improved disease-spesific survival (DSS). High co-expression of miR-210 in cancer and stromal cells was also a positive prognostic factor for DSS (p=0.010). In multivariate analysis, miR-210 in stromal cells (p=0.011), and miR-210 co-expressed in cancer and stromal cells was an independent prognosticator for DSS (p=0.011). CONCLUSIONS: We show that miR-210 in stromal cells, and co-expressed in cancer cells and stromal cells mediates an independent prognostic impact. It is a candidate marker for prognostic stratification in NSCLC.

CD4/CD8 co-expression shows independent prognostic impact in resected non-small cell lung cancer patients treated with adjuvant radiotherapy.

BACKGROUND: Though traditionally regarded as immunosuppressive, radiotherapy may also stimulate immune cells and facilitate an anti-tumor immune response. We therefore aimed to explore the prognostic significance of immune cell markers in non-small cell lung cancer (NSCLC) patients treated with postoperative radiotherapy (PORT). METHODS: In addition to demographic and clinicopathological information, tumor tissue samples were collected and tissue microarrays (TMAs) were constructed from 55 patients with stage I-IIIA NSCLC who received PORT. Tumor and stromal expression of CD1a+, CD3+, CD4+, CD8+, CD20+, CD56+, CD68+, CD117+ and CD138+ cells, as well as M-CSF and CSF-1R, was assessed by immunohistochemistry. RESULTS: In univariate analysis, high co-expression of CD4+ and CD8+ T lymphocytes as well as high expression of CD1a+ dendritic cells in the tumor stroma correlated with improved disease-specific survival (DSS). In multivariate analysis patients with stromal ↓CD4/↓CD8 expression had a hazard ratio of 21.1 (CI95% 3.9-115.6, P<0.001) when compared to those with ↑CD4/↑CD8 expression. CONCLUSIONS: Stromal ↓CD4/↓CD8 expression was an independent negative prognostic factor for survival in NSCLC patients receiving PORT, indicating a highly detrimental prognosis.