B Cells and Ectopic Follicular Structures: Novel Players in Anti-Tumor Programming with Prognostic Power for Patients with Metastatic Colorectal Cancer

<div><p>Remarkably limited information is available about biological mechanisms that determine the disease entity of metastatic colorectal cancer in the liver (CRCLM) with no good clinical parameters to estimate prognosis. For the last few years, understanding the relationship between tumor characteristics and local immune response has gained increasing attention. Given the multifaceted roles of B-cell-driven responses, we aimed to elucidate the immunological imprint of B lymphocytes at the metastatic site, the interrelation with macrophages, and their prognostic relevance. Here we present novel algorithm allowing to assess a link between the local patient-specific immunological capacity and clinical outcome. The microscopy-based imaging platform was used for automated scanning of large-scale tissue sections and subsequent qualitative and quantitative analyses of immune cell subtypes using lineage markers and single-cell recognition strategy. Results indicate massive infiltration of CD45-positive leukocytes confined to the metastatic border. We report for the first time the accumulation of CD20-positive B lymphocytes at the tumor – liver interface comprising the major population within the large CD45-positive aggregates. Strikingly, functionally active, activation-induced cytidine deaminase (AID)-positive ectopic lymphoid structures were found to be assembled within the metastatic margin. Furthermore, the CD20-based data set revealed a strong prognostic power: patients with high CD20 content and/or ectopic follicles had significantly lower risk for disease recurrence as revealed by univariate analysis (p<0.001 for both) and in models adjusted for clinicopathological variables (p<0.001 and p = 0.01, respectively), and showed prolonged overall survival. In contrast, CD68 staining-derived data set did not show an association with clinical outcome. Taken together, we nominate the magnitude of B lymphocytes, including those organized in ectopic follicles, as novel prognostic marker which is superior to clinicopathological parameters. Findings emphasize anti-tumoral role of B cell-driven mechanism(s) and thus indicate a new way of thinking about potential treatment strategies for CRCLM patients.</p></div

Patient-specific imprint of CD45-positive cells at the site of CRCLM.

<p>(A) Massive infiltration of CD45-positive cells confined to the tumor – liver border of the metastases. Representative parts of the metastatic areas for two CRCLM patients are shown (red channel for CD45, blue channel for nuclei/DAPI). T: tumor; L: liver. Scale bar: 200 µm. (B) Different patterns of CD45-positive cell accumulations at the metastatic border. Representative images of various types of immune cell infiltrates are shown: (<i>a</i>) single cells; (<i>b</i>) large immune cell aggregates; (<i>c</i>) prominent ectopic follicle. In addition to the merged images (<i>a–c</i>, red channel for CD45 and blue channel for DAPI), pictures of individual channels are included (<i>d–f</i> for DAPI; <i>g–i</i> for CD45); the individual channels are shown in black/white, whereas merged images are shown in color. T: tumor; L: liver. Scale bar: 50 µm. (C) (<i>a</i>–<i>c</i>) Kaplan-Meier estimates for patients stratification based on the CD45-derived values at the border. Kaplan-Meier curves for RFS based on CD45 values for panel I, panel II, and their combination are shown giving patients' stratification into low and high risk groups (higher than median indicates low risk); p value of the log-rank test is indicated. Panel I: median is equal to 20.95, below median n = 6, above median n = 7; panel II: median is equal to 17.66, below median n = 10, above median n = 9; panel I+II: median is equal to 19.13, below median n = 16, above median n = 16. (<i>d</i>) Boxplots of CD45 data sets for patients without recurrence (No) versus patients with recurrence (Yes) at the time point of 17 months. The cut off was set according to the latest event occurrence (16.3 months) where no censoring has occurred, thereby, providing clear separation from the censored subjects (≥32.2 months) as visualized on (<i>b</i>). The median CD45 value, which was used for patient stratification into low and high risk groups on the Kaplan-Meier plot (<i>b</i>) is indicated by dashed line; p value is shown (t test).</p

CD20-positive B lymphocytes at the site of CRCLM.

<p>(A) Subpopulation of CD20-positive B cells at metastatic border. Representative examples of the CD45-positive (<i>a</i>) ectopic follicle, (<i>c</i>) large cell aggregate and (<i>e</i>) non-organized cell population and the corresponding areas stained for CD20 are shown (<i>b</i>, <i>d</i>, <i>f</i>, respectively). Quantitative analysis was done using TissueQuest and HistoQuest software; percentage of positive cells is indicated. Scale bar: 100 µm. (B) Different organisation patterns of CD20-positive cells within the three regions of interest. Representative images (i) at the border: (<i>a</i>) single cells, (<i>b</i>) cell aggregates; insert: higher power view of CD20-positive cells in contact with the colon tumor epithelial cells, (<i>c</i>) ectopic follicular structures; (ii) around the portal veins (<i>d</i>) proximate to the border and (<i>e</i>) distant to the border as well as (iii) within liver tissue (<i>f</i>) are shown. T: tumor; L: liver. Scale bar: 50 µm. (C) Kaplan-Meier estimates for patients stratification based on the CD20-derived values at the border. Kaplan-Meier curves for RFS based on CD20 values for panel I, panel II and panel I+II are shown giving patient stratification into low and high risk groups (higher than median indicates low risk); p value of the log-rank test is indicated. Panel I: median is equal to 2.70, below median n = 5, above median n = 6; panel II: median is equal to 2.11, below median n = 25, above median n = 26; panel I+II: median is equal to 2.22, below median n = 31, above median n = 31. (<i>d</i>) Boxplots of CD20 data sets for patients without recurrence (No) versus patients with recurrence (Yes) at the time point of 24 months. The cut off was set according to the latest event occurrence (23.3 month) where no censoring has occurred, giving separation from the censored subjects (≥29.4 months) as visualized on (<i>b</i>). Dashed line: the median CD20 value, which was used for patient stratification into low and high risk groups on Kaplan-Meier plot (<i>b</i>); p value is shown (t test).</p

Prognostic effect of ectopic follicular structures allocated at the tumor – liver border.

<p>Kaplan-Meier estimates for patients stratification based on the ectopic follicle score at the border (<i>a</i>–<i>c</i>). Kaplan-Meier curves for RFS for panel I, panel II, and panel I+II show patient stratification into low (high number of follicular structures), intermediate (low number of follicular structures) and high risk (no follicular structures) groups; p value of the log-rank trend test is indicated. Panel I: low risk n = 5, intermediate risk n = 7, high risk n = 2; panel II: low risk n = 16, intermediate risk n = 17, high risk n = 18; panel I+II: low risk n = 21, intermediate risk n = 24, high risk n = 20. (<i>d</i>) Comparative assessment of contribution of <i>no</i>, <i>low</i>, and <i>high</i> ectopic follicle score to the total quantity for patient sub-groups without (No) and with (Yes) disease recurrence as estimated at the time point of 24 months where no censoring has occurred; p value of chi-square trend test is shown.</p

Localization patterns of CD68-positive macrophage populations across CRCLM specimens.

<p>(A) Representative images of three major distribution patterns are shown: (<i>a</i>) accumulation of CD68-positive macrophages lining the tumor body within the tumor – liver border sub-region; (<i>b</i>) CD68-positive rim bounding tumor parts and differentiating them from necrotic areas within the metastatic body; tumor – liver margin is indicated by dashed line; (<i>c</i>) no preferential accumulation either at the border or tumor. Brown color, CD68 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar 500 µm. T: tumor; L: liver. (B) Various subpopulations of CD68-positive lymphocytes (<i>a, b</i>) at the tumor – liver border and (<i>c</i>) within the portal vein area characterized by well-spread or round shaped morphology are shown; in addition, (<i>d</i>, <i>e</i>) resident CD68-positive Kupffer cells within distant liver shows inter-patient variability regarding intensity of staining and density; representative images of CD68-positive cells within (<i>f</i>) large immune cell aggregates and (<i>g</i>) ectopic follicles. Scale bar 50 µm. Inserts: the high-power views. T: tumor; L: liver. (C) Double immunofluorescent staining to visualize the co-localization of CD20-positive B cells and CD68-positive macrophages within the tumor – liver sub-region. The merged images are shown (green channel for CD20, red channel for CD68, and blue channel for DAPI): (<i>a</i>) direct cell-cell contact between CD20- and CD68-positive lymphocytes; (<i>b</i>) CD20-positive B cells might co-localize with the resident CD68-positive Kupffer cells within vascular sinusoids; example of such sinusoid is highlighted by a dashed line. Scale bar 50 µm. Inserts: the high-power views.</p