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B cells and ectopic follicular structures: novel players in anti-tumor programming with prognostic power for patients with metastatic colorectal cancer.

Meshcheryakova A, Tamandl D, Bajna E, Stift J, Mittlboeck M, Svoboda M, Heiden D, Stremitzer S, Jensen-Jarolim E, Grünberger T, Bergmann M, Mechtcheriakova D - PLoS ONE (2014)

Bottom Line: Strikingly, functionally active, activation-induced cytidine deaminase (AID)-positive ectopic lymphoid structures were found to be assembled within the metastatic margin.In contrast, CD68 staining-derived data set did not show an association with clinical outcome.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

ABSTRACT
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.

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CD20-positive B lymphocytes at the site of CRCLM.(A) Subpopulation of CD20-positive B cells at metastatic border. Representative examples of the CD45-positive (a) ectopic follicle, (c) large cell aggregate and (e) non-organized cell population and the corresponding areas stained for CD20 are shown (b, d, f, 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: (a) single cells, (b) cell aggregates; insert: higher power view of CD20-positive cells in contact with the colon tumor epithelial cells, (c) ectopic follicular structures; (ii) around the portal veins (d) proximate to the border and (e) distant to the border as well as (iii) within liver tissue (f) 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. (d) 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 (b). Dashed line: the median CD20 value, which was used for patient stratification into low and high risk groups on Kaplan-Meier plot (b); p value is shown (t test).
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pone-0099008-g003: CD20-positive B lymphocytes at the site of CRCLM.(A) Subpopulation of CD20-positive B cells at metastatic border. Representative examples of the CD45-positive (a) ectopic follicle, (c) large cell aggregate and (e) non-organized cell population and the corresponding areas stained for CD20 are shown (b, d, f, 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: (a) single cells, (b) cell aggregates; insert: higher power view of CD20-positive cells in contact with the colon tumor epithelial cells, (c) ectopic follicular structures; (ii) around the portal veins (d) proximate to the border and (e) distant to the border as well as (iii) within liver tissue (f) 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. (d) 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 (b). Dashed line: the median CD20 value, which was used for patient stratification into low and high risk groups on Kaplan-Meier plot (b); p value is shown (t test).

Mentions: Next, to characterize the B-cell lineage at CRCLM site, the tissue specimens were analysed for CD20-positive cells. Strikingly, the major fraction of cells within the large CD45-positive cellular aggregates and particularly within the ectopic follicles was found to be CD20-positive B cells; interestingly, only trace amounts of CD20-positive B cells were detected within the non-organized CD45-positive lymphocyte populations (Figure 3, A). Different organisation patterns of CD20-positive cells were observed within the three regions of interest. Representative images are provided in Figure 3, B. At the border, single cells (Figure 3, B, a) and/or cell aggregates (Figure 3, B, b), and/or ectopic follicles (Figure 3, B, c) were detected. Of note, CD20-positive B cells were found to accumulate around the tumor islands (Figure 3, B, b, insert). Large B-cell aggregates were as well detected around the portal veins in close proximity to the border area, whereas at more distant portal veins single CD20-positive cells were observed (Figure 3, B, d, e, respectively). Notably, within distant liver tissue, across the whole specimen only sparsely distributed, single events were found (Figure 3, B, f). In line, quantitative evaluations of three regions revealed the presence of B cells at the tumor – liver border ranging from 0.3% to 13% with a median value of 2% and within the liver tissue around the portal veins ranging from 0.1% to 36% with a median value of 4%; single CD20-positive cells were detected within distant liver tissue (median of 0.1%); analysis was performed for panel I and II in combination. Additionally, comparing the two chemotherapy groups within panel II we observed no major difference in the distribution of the staining-derived values and have no indication for group differences between chemotherapies (Table S3). Importantly, comparison of CD20 variables for panel I and panel II revealed no significant differences for three regions of interest (Figure S3).


B cells and ectopic follicular structures: novel players in anti-tumor programming with prognostic power for patients with metastatic colorectal cancer.

Meshcheryakova A, Tamandl D, Bajna E, Stift J, Mittlboeck M, Svoboda M, Heiden D, Stremitzer S, Jensen-Jarolim E, Grünberger T, Bergmann M, Mechtcheriakova D - PLoS ONE (2014)

CD20-positive B lymphocytes at the site of CRCLM.(A) Subpopulation of CD20-positive B cells at metastatic border. Representative examples of the CD45-positive (a) ectopic follicle, (c) large cell aggregate and (e) non-organized cell population and the corresponding areas stained for CD20 are shown (b, d, f, 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: (a) single cells, (b) cell aggregates; insert: higher power view of CD20-positive cells in contact with the colon tumor epithelial cells, (c) ectopic follicular structures; (ii) around the portal veins (d) proximate to the border and (e) distant to the border as well as (iii) within liver tissue (f) 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. (d) 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 (b). Dashed line: the median CD20 value, which was used for patient stratification into low and high risk groups on Kaplan-Meier plot (b); p value is shown (t test).
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Related In: Results  -  Collection

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Show All Figures
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pone-0099008-g003: CD20-positive B lymphocytes at the site of CRCLM.(A) Subpopulation of CD20-positive B cells at metastatic border. Representative examples of the CD45-positive (a) ectopic follicle, (c) large cell aggregate and (e) non-organized cell population and the corresponding areas stained for CD20 are shown (b, d, f, 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: (a) single cells, (b) cell aggregates; insert: higher power view of CD20-positive cells in contact with the colon tumor epithelial cells, (c) ectopic follicular structures; (ii) around the portal veins (d) proximate to the border and (e) distant to the border as well as (iii) within liver tissue (f) 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. (d) 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 (b). Dashed line: the median CD20 value, which was used for patient stratification into low and high risk groups on Kaplan-Meier plot (b); p value is shown (t test).
Mentions: Next, to characterize the B-cell lineage at CRCLM site, the tissue specimens were analysed for CD20-positive cells. Strikingly, the major fraction of cells within the large CD45-positive cellular aggregates and particularly within the ectopic follicles was found to be CD20-positive B cells; interestingly, only trace amounts of CD20-positive B cells were detected within the non-organized CD45-positive lymphocyte populations (Figure 3, A). Different organisation patterns of CD20-positive cells were observed within the three regions of interest. Representative images are provided in Figure 3, B. At the border, single cells (Figure 3, B, a) and/or cell aggregates (Figure 3, B, b), and/or ectopic follicles (Figure 3, B, c) were detected. Of note, CD20-positive B cells were found to accumulate around the tumor islands (Figure 3, B, b, insert). Large B-cell aggregates were as well detected around the portal veins in close proximity to the border area, whereas at more distant portal veins single CD20-positive cells were observed (Figure 3, B, d, e, respectively). Notably, within distant liver tissue, across the whole specimen only sparsely distributed, single events were found (Figure 3, B, f). In line, quantitative evaluations of three regions revealed the presence of B cells at the tumor – liver border ranging from 0.3% to 13% with a median value of 2% and within the liver tissue around the portal veins ranging from 0.1% to 36% with a median value of 4%; single CD20-positive cells were detected within distant liver tissue (median of 0.1%); analysis was performed for panel I and II in combination. Additionally, comparing the two chemotherapy groups within panel II we observed no major difference in the distribution of the staining-derived values and have no indication for group differences between chemotherapies (Table S3). Importantly, comparison of CD20 variables for panel I and panel II revealed no significant differences for three regions of interest (Figure S3).

Bottom Line: Strikingly, functionally active, activation-induced cytidine deaminase (AID)-positive ectopic lymphoid structures were found to be assembled within the metastatic margin.In contrast, CD68 staining-derived data set did not show an association with clinical outcome.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus