Purpose: The success of immune checkpoint inhibitors strengthens the notion that tumor growth and regression are immune regulated. To determine whether distinct tissue immune microenvironments differentially affect clinical outcome in non–small cell lung cancer (NSCLC), an extended analysis of PD-L1 and tumor-infiltrating lymphocytes (TIL) was performed. Experimental Design: Samples from resected adenocarcinoma (ADC 42), squamous cell carcinoma (SCC 58), and 26 advanced diseases (13 ADC and 13 SCC) treated with nivolumab were analyzed. PD-L1 expression and the incidence of CD3, CD8, CD4, PD-1, CD57, FOXP3, CD25, and Granzyme B TILs were immunohistochemically assessed. Results: PD-L1 levels inversely correlated with N involvement, although they did not show a statistically significant prognostic value in resected patients. The incidence and phenotype of TILs differed in SCC versus ADC, in which EGFR and KRAS mutations conditioned a different frequency and tissue localization of lymphocytes. NSCLC resected patients with high CD8^pos lymphocytes lacking PD-1 inhibitory receptor had a longer overall survival (OS: HR = 2.268; 95% CI, 1.056–4.871, P = 0.03). PD-1-to-CD8 ratio resulted in a prognostic factor both on univariate (HR = 1.952; 95% CI, 1.34–3.12, P = 0.001) and multivariate (HR = 1.943; 95% CI, 1.38–2.86, P = 0.009) analysis. Moreover, low PD-1 incidence among CD8^pos cells was a distinctive feature of nivolumab-treated patients, showing clinical benefit with a prolonged progression-free survival (PFS: HR = 4.51; 95% CI, 1.45–13.94, P = 0.004). Conclusions: In the presence of intrinsic variability in PD-L1 expression, the reservoir of PD-1–negative effector T lymphocytes provides an immune-privileged microenvironment with a positive impact on survival of patients with resected disease and response to immunotherapy in advanced NSCLC. Clin Cancer Res; 1–13. ©2017 AACR.

OBJECTIVES: Tumour-infiltrating lymphocytes (TILs) are critically implicated in the clinical outcome and response to immunotherapy in non-small-cell lung cancer (NSCLC) patients. The functional competence of lymphocyte subpopulations is strongly conditioned by their spatial arrangement within the tumour immune microenvironment. The aim of this study was to determine whether the tissue localization of specific TIL subpopulations might have an impact on the risk of recurrence in surgically resected NSCLC. METHODS: High-speed scanning of whole slide images was performed on immunohistochemically stained tissue sections from 97 NSCLC patients to assess the number and ratio of CD3+, CD8+ and PD-1+ T-lymphocytes. TIL distribution was computed considering the intratumoural (proximal or distal) and peripheral (invasive margin) localization as well as their location within the fibrotic tissue (immune excluded). The tumour proliferative index was assessed by Ki67 labelling. The impact of TILs number and distribution on clinical-pathological characteristics and outcomes were statistically analysed. RESULTS: High density and percentage of proximal CD8+ TILs and low PD-1-to-CD8 ratio had a positive impact on disease-free-survival (P = 0.03) and overall survival (P = 0.003). An inverse correlation was observed between the abundance of intratumoural CD8+ TILs carrying PD-1 inhibitory receptor and cancer cell proliferation. Cases with high compared to low fraction of immune excluded CD8+ TILs had significantly reduced 5-year overall survival (n events: 22 vs 12; P = 0.04) and disease-free survival (n events: 24 vs 16; P = 0.03) rates while the amount of CD3+ and CD8+ TILs located at the invasive margin had a favourable effect on the clinical course. CONCLUSIONS: Mapping TIL subpopulations may implement the definition of prognostic parameters in surgically resected NSCLC.

Background: Pulmonary metastasectomy is considered a standard procedure in the treatment of metastatic colorectal cancer (CRC). Different prognostic factors including multiple metastatic nodules, the presence of extra-pulmonary metastases and BRAF mutation status have been associated with poor survival. The aim of this study was to evaluate which factors influenced survival in CRC patients undergoing pulmonary metastasectomy by studying primary tumors and pulmonary metastases. Methods: All patients treated for primary CRC who presented pulmonary metastases in a 10-year period were considered (group A). A control group treated for primary CRC who did not develop any pulmonary or extra-pulmonary metastases was taken for comparison (group B). Different prognostic factors including gender, age, tumor location, histological type, inflammatory infiltrate, BRAF, CDX2 and extra-pulmonary metastases were analyzed. Overall survival (OS) and patients’ survival after pulmonary metastasectomy were also considered. Results: Fifty-four patients were evaluated in group A and twenty-three in group B. In group A, BRAF immunohistochemistry did not significantly differ between primary tumors and pulmonary metastases; no difference of BRAF expression was found between group A and B. Even the expression of CDX2 was not significantly different in primary tumors and metastases. Similarly, in group B CDX2 did not significantly differ from primary CRC of group A. The most significant prognostic factor was the presence of extra-pulmonary metastases. Patients with extra-pulmonary metastases experienced a significant shorter survival compared to patients with pulmonary metastases alone (P=0.001 with log-rank test vs. P=0.003 with univariate Cox regression). Interestingly, patients with right pulmonary metastases presented a significant longer survival than those with left pulmonary metastases (P=0.027 with log-rank test vs. 0.04 with univariate Cox regression). Conclusions: The main prognostic factor associated with poor survival after lung resection of CRC metastases is a history of extra-pulmonary metastases. BRAF and CDX2 did not have a significant role in this small series of patients.

Lymphangiogenesis plays a critical role in the immune response, tumour progression and therapy effectiveness. The aim of this study was to determine whether the interplay between the lymphatic and the blood microvasculature, tumour-infiltrating lymphocytes and the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) immune checkpoint constitutes an immune microenvironment affecting the clinical outcome of patients with non-small-cell lung cancer. Samples from 50 squamous cell carcinomas and 42 adenocarcinomas were subjected to immunofluorescence to detect blood and lymphatic vessels. CD3^pos, CD8^pos and PD-1^pos tumour-infiltrating lymphocytes and tumour PD-L1 expression were assessed by immunohistochemical analysis. Quantification of vascular structures documented a peak of lymphatics at the invasive margin together with a decreasing gradient of blood and lymphatic vessels from the peritumour area throughout the neoplastic core. Nodal involvement and pathological stage were strongly associated with vascularization, and an increased density of vessels was detected in samples with a higher incidence of tumour-infiltrating lymphocytes and a lower expression of PD-L1. Patients with a high PD-L1 to PD-1 ratio and vascular rarefaction had a gain of 10 months in overall survival compared to those with a low ratio and prominent vascularity. Microvessels are an essential component of the cancer immune microenvironment. The clinical impact of the PD-1/PD-L1-based immune contexture may be implemented by the assessment of microvascular density to potentially identify patients with non-small-cell lung cancer who could benefit from immunotherapy and antiangiogenic treatment.

Black hairy tongue (BHT) is a self-limiting disorder characterized by abnormal hypertrophy and elongation of filiform papillae on the surface of the tongue. The exact mechanism of drug-induced BHT is unknown. Several factors have been implicated and included smoking or chewing tobacco, drinking alcohol, poor oral hygiene and antibiotics such as tetracyclines and penicillins. We report a quite uncommon case of Linezolid-induced BHT in a patient with a long-lasting history of chest wall infection.