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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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Patient-specific imprint of CD45-positive cells at the site of CRCLM.(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: (a) single cells; (b) large immune cell aggregates; (c) prominent ectopic follicle. In addition to the merged images (a–c, red channel for CD45 and blue channel for DAPI), pictures of individual channels are included (d–f for DAPI; g–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) (a–c) 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. (d) 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 (b). The median CD45 value, which was used for patient stratification into low and high risk groups on the Kaplan-Meier plot (b) is indicated by dashed line; p value is shown (t test).
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pone-0099008-g002: Patient-specific imprint of CD45-positive cells at the site of CRCLM.(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: (a) single cells; (b) large immune cell aggregates; (c) prominent ectopic follicle. In addition to the merged images (a–c, red channel for CD45 and blue channel for DAPI), pictures of individual channels are included (d–f for DAPI; g–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) (a–c) 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. (d) 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 (b). The median CD45 value, which was used for patient stratification into low and high risk groups on the Kaplan-Meier plot (b) is indicated by dashed line; p value is shown (t test).

Mentions: Gross examination revealed massive infiltration of CD45-positive cells confined to the tumor – liver border of the metastases. Representative images are shown in Figure 2, A. Different patterns of immune cell accumulations were observed among the patients and might coexist within the same specimen at the tumor – liver border: single cells, diffused cell aggregates, and ectopic follicles structures (Figure 2 B). The amount of CD45-positve cells at three regions of interest exhibited major inter-patient variability as determined by quantitative analysis: at the border ranging from 8% to 46% (median 19%), within the liver tissue at site of portal veins ranging from 9% to 56% (median 29%) and within the distant liver tissue ranging from 4% to 16% (median 9%); the analysis was performed for panel I and panel II together. Importantly, CD45-derived data sets showed no significant differences between panel I and panel II (Figure S2).


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)

Patient-specific imprint of CD45-positive cells at the site of CRCLM.(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: (a) single cells; (b) large immune cell aggregates; (c) prominent ectopic follicle. In addition to the merged images (a–c, red channel for CD45 and blue channel for DAPI), pictures of individual channels are included (d–f for DAPI; g–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) (a–c) 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. (d) 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 (b). The median CD45 value, which was used for patient stratification into low and high risk groups on the Kaplan-Meier plot (b) is indicated by dashed line; p value is shown (t test).
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Related In: Results  -  Collection

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pone-0099008-g002: Patient-specific imprint of CD45-positive cells at the site of CRCLM.(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: (a) single cells; (b) large immune cell aggregates; (c) prominent ectopic follicle. In addition to the merged images (a–c, red channel for CD45 and blue channel for DAPI), pictures of individual channels are included (d–f for DAPI; g–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) (a–c) 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. (d) 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 (b). The median CD45 value, which was used for patient stratification into low and high risk groups on the Kaplan-Meier plot (b) is indicated by dashed line; p value is shown (t test).
Mentions: Gross examination revealed massive infiltration of CD45-positive cells confined to the tumor – liver border of the metastases. Representative images are shown in Figure 2, A. Different patterns of immune cell accumulations were observed among the patients and might coexist within the same specimen at the tumor – liver border: single cells, diffused cell aggregates, and ectopic follicles structures (Figure 2 B). The amount of CD45-positve cells at three regions of interest exhibited major inter-patient variability as determined by quantitative analysis: at the border ranging from 8% to 46% (median 19%), within the liver tissue at site of portal veins ranging from 9% to 56% (median 29%) and within the distant liver tissue ranging from 4% to 16% (median 9%); the analysis was performed for panel I and panel II together. Importantly, CD45-derived data sets showed no significant differences between panel I and panel II (Figure S2).

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