Localization and density of tertiary lymphoid structures associate with molecular subtype and clinical outcome in colorectal cancer liver metastases

Abstract

BackgroundTertiary lymphoid structures (TLSs) have been proposed to assess the prognosis of patients with cancer. Here, we investigated the prognostic value and relevant mechanisms of TLSs in colorectal cancer liver metastases (CRCLM).Methods603 patients with CRCLM treated by surgical resection from three cancer centers were included. The TLSs were categorized according to their anatomic subregions and quantified, and a TLS scoring system was established for intratumor region (T score) and peritumor region (P score). Differences in relapse-free survival (RFS) and overall survival (OS) between groups were determined. Multiplex immunohistochemical staining (mIHC) was used to determine the cellular composition of TLSs in 40 CRCLM patients.ResultsT score positively correlated with superior prognosis, while P score negatively associated with poor survival (all p<0.05). Meanwhile, T score was positively associated with specific mutation subtype of KRAS. Furthermore, TLSs enrichment gene expression was significantly associated with survival and transcriptomic subtypes of CRCLM. Subsequently, mIHC showed that the densities of Treg cells, M2 macrophages and Tfh cells were significantly higher in intratumor TLSs than in peritumor TLSs (p=0.029, p=0.047 and p=0.041, respectively), and the frequencies of Treg cells and M2 macrophages were positively correlated with P score, while the frequencies of Tfh cells were positively associated with T scores in intratumor TLSs (all p<0.05). Next, based on the distribution and abundance of TLSs, an Immune Score combining T score and P score was established which categorized CRCLM patients into four immune classes with different prognosis (all p<0.05). Among them, patients with higher immune class have more favorable prognoses. The C-index of Immune Class for RFS and OS was higher than Clinical Risk Score statistically. These results were also confirmed by the other two validation cohorts.ConclusionsThe distribution and abundance of TLSs is significantly associated with RFS and OS of CRCLM patients, and a novel immune class was proposed for predicting the prognosis of CRCLM patients.

Unveiling the impact of tertiary lymphoid structures on immunotherapeutic responses of clear cell renal cell carcinoma

“…Slides bound with primary and secondary antibodies but excluding fluorophores served as negative controls to gauge autofluorescence. These multiplex stained slides were scanned through the Vectra Polaris Quantitative Pathology Imaging System (Akoya Biosciences) at 20 nm wavelength intervals from 440 to 780 nm, producing a single image using inForm v.2.4.8 (Akoya Biosciences) to quantify the different biomarkers in each cell 60,61 . The quantity of various cell populations was expressed as both the number of stained cells per square millimeter and as the percentage of positively stained cells among all nucleated cells, following the manufacturer's instructions 59–61 …”

“…These multiplex stained slides were scanned through the Vectra Polaris Quantitative Pathology Imaging System (Akoya Biosciences) at 20 nm wavelength intervals from 440 to 780 nm, producing a single image using inForm v.2.4.8 (Akoya Biosciences) to quantify the different biomarkers in each cell. 60,61 The quantity of various cell populations was expressed as both the number of stained cells per square millimeter and as the percentage of positively stained cells among all nucleated cells, following the manufacturer's instructions. [59][60][61] For the evaluation of DCs in relation to TLS maturation, 75 ccRCC slides were co-stained for CD20, CD21, CD23, CD45, DC-LAMP, pan-CK, and DAPI.…”

“…60,61 The quantity of various cell populations was expressed as both the number of stained cells per square millimeter and as the percentage of positively stained cells among all nucleated cells, following the manufacturer's instructions. [59][60][61] For the evaluation of DCs in relation to TLS maturation, 75 ccRCC slides were co-stained for CD20, CD21, CD23, CD45, DC-LAMP, pan-CK, and DAPI. All dense lymphocytic aggregates and areas exhibiting DC-LAMP signal were imaged and analyzed using seven-color mIF.…”

“…First, a total of 63 tissue samples were initially deparaffinized in a BOND RX system (Leica Biosystems) and subjected to staining with specific primary antibodies. [59][60][61] The slides, following the primary antibody incubation, were then treated with secondary antibodies labeled by horseradish peroxidase (HRP) and corresponding reactive Opal fluorophores based on the tyramide signal amplification principle. 60,61 Slides bound with primary and secondary antibodies but excluding fluorophores served as negative controls to gauge autofluorescence.…”

Immunogenicity of mesenchymal stromal/stem cells

“…Tertiary lymphoid structures (TLSs) are aggregates of immune cells that occur in nonlymphoid tissues during chronic inflammation 77 . TLSs are similar in structure to secondary lymphoid organs (SLOs) and consist mainly of B cells, T cells, DCs, neutrophils, and macrophages, providing an important microenvironment for cellular immune responses 78,79 . MSCs may act as a lymphoid organizer (LTo) cell to activate T cells and induce the formation of TLSs, thus accelerating the early inflammatory process 54 …”