Tertiary lymphoid structures (TLS) identification and density assessment on H&E-stained digital slides of lung cancer.

Tertiary lymphoid structures (TLS) are ectopic aggregates of lymphoid cells in inflamed, infected, or tumoral tissues that are easily recognized on an H&E histology slide as discrete entities, distinct from lymphocytes. TLS are associated with improved cancer prognosis but there is no standardised method available to quantify their presence. Previous studies have used immunohistochemistry to determine the presence of specific cells as a marker of the TLS. This has now been proven to be an underestimate of the true number of TLS. Thus, we propose a methodology for the automated identification and quantification of TLS, based on H&E slides. We subsequently determined the mathematical criteria defining a TLS. TLS regions were identified through a deep convolutional neural network and segmentation of lymphocytes was performed through an ellipsoidal model. This methodology had a 92.87% specificity at 95% sensitivity, 88.79% specificity at 98% sensitivity and 84.32% specificity at 99% sensitivity level based on 144 TLS annotated H&E slides implying that the automated approach was able to reproduce the histopathologists' assessment with great accuracy. We showed that the minimum number of lymphocytes within TLS is 45 and the minimum TLS area is 6,245µm2. Furthermore, we have shown that the density of the lymphocytes is more than 3 times those outside of the TLS. The mean density and standard deviation of lymphocytes within a TLS area are 0.0128/µm2 and 0.0026/µm2 respectively compared to 0.004/µm2 and 0.001/µm2 in non-TLS regions. The proposed methodology shows great potential for automated identification and quantification of the TLS density on digital H&E slides.

Positron emission tomography and single photon emission computed tomography imaging of tertiary lymphoid structures during the development of lupus nephritis.

Lymphoid neogenesis occurs in tissues targeted by chronic inflammatory processes, such as infection and autoimmunity. In systemic lupus erythematosus (SLE), such structures develop within the kidneys of lupus-prone mice ((NZBXNZW)F1) and are observed in kidney biopsies taken from SLE patients with lupus nephritis (LN). The purpose of this prospective longitudinal animal study was to detect early kidney changes and tertiary lymphoid structures (TLS) using in vivo imaging. Positron emission tomography (PET) by tail vein injection of 18-F-fluoro-2-deoxy-D-glucose (18F-FDG)(PET/FDG) combined with computed tomography (CT) for anatomical localization and single photon emission computed tomography (SPECT) by intraperitoneal injection of 99mTC labeled Albumin Nanocoll (99mTC-Nanocoll) were performed on different disease stages of NZB/W mice (n = 40) and on aged matched control mice (BALB/c) (n = 20). By using one-way ANOVA analyses, we compared two different compartmental models for the quantitative measure of 18F-FDG uptake within the kidneys. Using a new five-compartment model, we observed that glomerular filtration of 18FFDG in lupus-prone mice decreased significantly by disease progression measured by anti-dsDNA Ab production and before onset of proteinuria. We could not visualize TLS within the kidneys, but we were able to visualize pancreatic TLS using 99mTC Nanocoll SPECT. Based on our findings, we conclude that the five-compartment model can be used to measure changes of FDG uptake within the kidney. However, new optimal PET/SPECT tracer administration sites together with more specific tracers in combination with magnetic resonance imaging (MRI) may make it possible to detect formation of TLS and LN before clinical manifestations.

Tertiary Lymphoid Structures in Colorectal Cancer Liver Metastases: Association With Immunological and Clinical Parameters and Chemotherapy Response.

BACKGROUND/AIM: The presence of TLS (tertiary lymphoid structures) is detectable in the microenvironment of human cancers and linked to better patient outcomes. The role of TLS in colorectal cancer liver metastases (CRCLM) is unclear. PATIENTS AND METHODS: Medical records of patients with CRCLM were reviewed (n=33) and corresponding tissue samples (n=21) were obtained. Whole slide imaging analyses on immunohistochemical staining of immune cell infiltrates and TLS were correlated to clinical outcomes (including chemotherapy response) and other parameters. RESULTS: Neither the size (p=0.6) nor the quantity (p=0.47) of TLS was associated with the clinical course. When considering spatial differences, TLS in the invasive margin (IM) were associated with progression-free survival (p=0.03), while TLS in the LM (liver metastasis) were not. Also, TLS in the IM were associated with the presence of CD3+ T cells, which itself was prognostically favorable (p<0.001). CONCLUSION: TLS in the IM are associated with good prognosis in CRCLM, but not chemotherapy response.

B cell activation in the cecal patches during the development of an experimental colitis model.

Although previous studies have suggested that appendix seems to be involved in the colitis, the role of this in the pathogenesis remains unclear. In this study, we assessed the importance of appendiceal lymphoid follicles, specifically the cecal patches (CP) in mice, using an experimental colitis model. Treatment with oxazolone resulted in ulcerations particularly at CP with follicular expansion as well as colitis. The colitis was attenuated by either appendectomy or the absence of mature B cells. We therefore established an intravital imaging system accompanied by the fluorescence resonance energy transfer technology to analyze the dynamic immune response of CP B cells. Our observation revealed frequent Ca2+ signaling in CP B cells during the early phase of colitis development. These findings suggested that the CP B cells may be involved in the pathogenesis of colitis including inflammatory bowel diseases in humans.

Lymphoid Aggregates Remodel Lymphatic Collecting Vessels that Serve Mesenteric Lymph Nodes in Crohn Disease.

Early pathological descriptions of Crohn disease (CD) argued for a potential defect in lymph transport; however, this concept has not been thoroughly investigated. In mice, poor healing in response to infection-induced tissue damage can cause hyperpermeable lymphatic collecting vessels in mesenteric adipose tissue that impair antigen and immune cell access to mesenteric lymph nodes (LNs), which normally sustain appropriate immunity. To investigate whether analogous changes might occur in human intestinal disease, we established a three-dimensional imaging approach to characterize the lymphatic vasculature in mesenteric tissue from controls or patients with CD. In CD specimens, B-cell-rich aggregates resembling tertiary lymphoid organs (TLOs) impinged on lymphatic collecting vessels that enter and exit LNs. In areas of creeping fat, which characterizes inflammation-affected areas of the bowel in CD, we observed B cells and apparent innate lymphoid cells that had invaded the lymphatic vessel wall, suggesting these cells may be mediators of lymphatic remodeling. Although TLOs have been described in many chronic inflammatory states, their anatomical relationship to preestablished LNs has never been revealed. Our data indicate that, at least in the CD-affected mesentery, TLOs are positioned along collecting lymphatic vessels in a manner expected to affect delivery of lymph to LNs.