Erna-Elise Paulsen

Stromal CD8+ T-cell Density—A 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. Clin Cancer Res; 21(11); 2635–43. ©2015 AACR.

The Role of Tumor-Infiltrating Lymphocytes in Development, Progression, and Prognosis of Non-Small Cell Lung Cancer.

ABSTRACT A malignant tumor is not merely an accumulation of neoplastic cells, but constitutes a microenvironment containing endothelial cells, fibroblasts, structural components, and infiltrating immune cells that impact tumor development, invasion, metastasis, and outcome. Hence, the evolution of cancers reflects intricate cellular and molecular interactions between tumor cells and constituents of the tumor microenvironment. Recent studies have shed new light on this complex interaction between tumor and host immune cells and the resulting immune response. The composition of the immune microenvironment differs across patients as well as in cancers of the same type, including various populations of T cells, B cells, dendritic cells, natural killer cells, myeloid‐derived suppressor cells, neutrophils, and macrophages. The type, density, location, and organization of immune cells within solid tumors define the immune contexture, which has proved to be a major determinant of tumor characteristics and patient outcome. Lung cancer consists mostly of non–small cell lung cancer (85%); it is our most deadly malignant disease, with the 5‐year survival rate being merely 15%. This review focuses on the immune contexture; the tumor‐suppressing roles of tumor‐infiltrating lymphocytes; and the relevance of this immune contexture for cancer diagnostics, prognostication, and treatment allocation, with an emphasis on non–small cell lung cancer.

Strategies for clinical implementation of TNM-Immunoscore in resected nonsmall-cell lung cancer.

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.

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. Micro‐Abstract Novel immune biomarkers could complement the TNM classification for non–small cell cancer (NSCLC), improving the prognostic accuracy. The present study evaluated the prognostic significance of the immune checkpoint molecules programmed cell death protein 1 (PD‐1) and PD‐1 ligand (PD‐L1) in 536 patients with stage I to IIIA NSCLC using an Immunoscore approach. Independently, and in combination, the infiltration of immune cells expressing PD‐L1 and PD‐1 predicted patient survival, supplementing the TNM classification in each stage.

Cancer associated fibroblasts in stage I-IIIA NSCLC: Prognostic impact and their correlations with tumor molecular markers

Background Cancer Associated Fibroblasts (CAFs) are thought to regulate tumor growth and metastasis. Fibroblast Activating Protein 1 (FAP-1) is a marker for fibroblast activation and by many recognized as the main marker of CAFs. Alpha Smooth Muscle Actin (α-SMA) is a general myofibroblast marker, and can be used to identify CAFs. This study investigates the prognostic impact of FAP-1 and α-SMA in non-small cell lung cancer (NSCLC) patients and correlates their expression to 105 proteins investigated in the same cohort. Methods Tumor specimens from 536 NSCLC patients were obtained and tissue micro-arrays were constructed. Immunohistochemistry was used to evaluate the expression of FAP-1 and α-SMA and explore their impact on survival and association with other tumor molecular markers in NSCLC patients. Results High expression of FAP-1, but not α-SMA, in squamous cell carcinoma (SCC, P = 0.043, HR = 0.63 95% CI 0.40–0.99) was significantly associated with increased disease-specific survival. FAP-1 and α-SMA were not significantly correlated to each other. Analyses of FAP-1 and α-SMA associated with other tumor-related proteins revealed histotype-specific correlation patterns. Conclusion The presence of FAP-1 expressing CAFs is an indicator of positive outcome for NSCLC-SCC patients. In addition, correlation analyses suggest FAP-1 and α-SMA to label different subsets of fibroblasts and their associations with other tumor-related proteins diverge according to histological subtype.

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.

The prognostic role of immune checkpoint markers programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) in a large, multicenter prostate cancer cohort

Programmed cell death protein 1 (PD-1) and its ligand Programmed death ligand 1 (PD-L1) have gained massive attention in cancer research due to recent availability and their targeted antitumor effects. Their role in prostate cancer is still undetermined. We constructed tissue microarrays from prostatectomy specimens from 535 prostate cancer patients. Following validation of antibodies, immunohistochemistry was used to evaluate the expression of PD-1 in lymphocytes and PD-L1 in epithelial and stromal cells of primary tumors. PD-L1 expression was commonly seen in tumor epithelial cells (92% of cases). Univariate survival analysis revealed a positive association between a high density of PD-1+ lymphocytes and worse clinical failure-free survival, limited to a trend (p = 0.084). In subgroups known to indicate unfavorable prostate cancer prognosis (Gleason grade 9, age < 65, preoperative PSA > 10, pT3) patients with high density of PD-1+ lymphocytes had a significantly higher risk of clinical failure (p = < 0.001, p = 0.025, p = 0.039 and p = 0.011, respectively). In the multivariate analysis, high density of PD-1+ lymphocytes was a significant negative independent prognostic factor for clinical failure-free survival (HR = 2.48, CI 95% 1.12–5.48, p = 0.025).

Prognostic effect of intratumoral neutrophils across histological subtypes of non-small cell lung cancer

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.

The presence of intraepithelial CD45RO+ cells in resected lymph nodes with metastases from NSCLC patients is an independent predictor of disease-specific survival.

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.

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.