Tertiary lymphoid structures (TLS) form in chronically inflamed tissues and contain organized compartments of T cells, B cells and follicular dendritic cells (FDCs). TLS were first noticed in autoimmune diseases where they support ectopic germinal center (GC) reactions and facilitate the activation and infiltration of immune cells. In cancer B cell and mature DC markers have been used for TLS detection because of their frequent localisation in TLS, and show a significant association with improved survival in several tumor types. Together these studies have driven the interest in TLS as a potential site of anti-tumor immune response activation and/or facilitation of lymphocyte infiltration. Here we aimed to characterise TLS as a microanatomical structure excluding non-organised lymphocytic infiltrates and to determine their prognostic value and association with clinico-pathologic parameters in non-small cell lung cancer patients.
We characterised TLS density and presence of GCs in tumor center and periphery using H&E stained whole sections from 4 different tumor regions of 127 surgically resected lung squamous cell carcinoma (SCC) patients with an annotated 5-year follow-up. Intratumoral TLS where observed in 30% of patients, while >95% of cases showed TLS in the tumor periphery. Patients with TLS-rich (n=65) and TLS-poor (n=62) tumour microenvironments could be distinguished. High density and GC positivity of TLS correlated with increased lymphocyte infiltration (Pearson correlation 0.34, p<0.001), but not other clinical parameters.
In neo-adjuvant chemotherapy-naïve patients, high density of GC-containing TLS was a significant positive prognosticator for disease free survival independent of pTNM, tumor grade, vascular and pleural invasion, age, gender, or pack years (n=87, multivariate Cox regression p=0.006, hazard ratio=0.365, 95% CI 0.18-0.75). In contrast, prognostic significance was abrogated in patients treated by neo-adjuvant chemotherapy using a doublet of platinum-containing drug with either gemcitabine, taxanes or vinorelbine. We observed that the density of TLS in tumor periphery was not altered in this cohort compared to untreated patients, but the proportion of TLS with active GCs was decreased (Pearson correlation -0.32, p<0.001). Further, when we stratified the neo-adjuvant chemotherapy-naïve patients along adjuvant chemotherapy post-surgery, the prognostic significance was retained only in the adjuvant untreated group (n=55, multivariate Cox regression p=0.001, hazard ratio=0.17, 95% CI 0.06-0.5). This suggests that chemotherapy might affect the quality rather than formation of TLS and disturb their immunostimulatory function.
We further investigated, which factors might affect the density of TLS in tumors. Because the chemokines CXCL13, CXCL12 and CCL21 have been shown to induce TLS formation in the lung under inflammatory conditions, we analyzed their expression in tumor samples by immunohistochemistry. Positive pixel algorithm (ImageJ software) was used for the quantification of immunostained tissue sections in central and peripheral tumor areas. We saw that the overexpression of CXCL13 in both, stromal and cancer cells, was associated with increased density of TLS (Student's T test p<0.05), while the expression of CXCL12 and CCL21 did not differ between the TLS-rich and TLS-poor tumor microenvironments.
In conclusion, we propose that the deliberate induction of TLS in lung cancer, possibly by CXCL13, may represent a novel immunotherapeutic approach. Thereto, the identification of molecular changes that chemotherapy exerts on the functionality of TLS, and whether this can be reversed, will provide novel ideas for the combination of immunotherapy with chemotherapy and thereby increasing the efficiency of standard treatments.
Citation Format: Karina Silina, Alex Soltermann, Holger Moch, Aija Line, Maries van den Broek. Tertiary lymphoid structures in chemotherapy-treated and untreated lung squamous cell carcinoma patients. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B85.
- ©2015 American Association for Cancer Research.