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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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Ectopic follicular structures at the site of CRCLM.(A) When formed, ectopic follicles are attracted to the tumor – liver interface. Representative image of the large-scale metastatic area surrounded by ectopic follicles is shown; brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 2 mm. The higher-power views of follicles at border (a), proximal (b) and distant (c) to tumor portal veins are given. Scale bar: 50 µm. (B) Representative images of AID-positive ectopic follicular structures located at the tumor – liver border are shown (a, insert: the higher-power view); brown color, AID staining; blue color, nuclear counterstaining with haematoxylin. Scale bar 50 µm. (C) The characteristic structure of a mature, AID-positive lymphoid follicle (a-e) is shown. Consequent slides were stained for CD20 using immunofluorescent (a, merged, red color, CD20 staining; blue color, DAPI; CD20 gradient is visible with reduced intensity at the periphery of follicle) and immunohistochemical (b, brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin) procedure; AID (c), IgM (d), CD138 (e). Insert: the high-power view of CD138-positive cells around follicular structure. To visualize distribution of CD138-positive cells, the image (e) was reduced by 60% in comparison to those shown on a-d. The corresponding AID-positive core of the follicle is indicated by asterisk (c, e). Example of follicle which is predominantly composed of IgM-positive B-cell subset (f, brown color, IgM staining; blue color, nuclear counterstaining with haematoxylin). Scale bar: (a-d, f) 20 µm, (e) 50 µm. (D) CD138-positive plasma cells were not detected at portal vein areas distant to the border. Additional example of (a) follicular structure at border surrounded by CD138-positive plasma cells, (b) aggregate of infiltrating cells or (c) diffused cells around the portal veins distant to border which do not contain CD138-positive cells; brown color, CD138 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 50 µm.
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pone-0099008-g004: Ectopic follicular structures at the site of CRCLM.(A) When formed, ectopic follicles are attracted to the tumor – liver interface. Representative image of the large-scale metastatic area surrounded by ectopic follicles is shown; brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 2 mm. The higher-power views of follicles at border (a), proximal (b) and distant (c) to tumor portal veins are given. Scale bar: 50 µm. (B) Representative images of AID-positive ectopic follicular structures located at the tumor – liver border are shown (a, insert: the higher-power view); brown color, AID staining; blue color, nuclear counterstaining with haematoxylin. Scale bar 50 µm. (C) The characteristic structure of a mature, AID-positive lymphoid follicle (a-e) is shown. Consequent slides were stained for CD20 using immunofluorescent (a, merged, red color, CD20 staining; blue color, DAPI; CD20 gradient is visible with reduced intensity at the periphery of follicle) and immunohistochemical (b, brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin) procedure; AID (c), IgM (d), CD138 (e). Insert: the high-power view of CD138-positive cells around follicular structure. To visualize distribution of CD138-positive cells, the image (e) was reduced by 60% in comparison to those shown on a-d. The corresponding AID-positive core of the follicle is indicated by asterisk (c, e). Example of follicle which is predominantly composed of IgM-positive B-cell subset (f, brown color, IgM staining; blue color, nuclear counterstaining with haematoxylin). Scale bar: (a-d, f) 20 µm, (e) 50 µm. (D) CD138-positive plasma cells were not detected at portal vein areas distant to the border. Additional example of (a) follicular structure at border surrounded by CD138-positive plasma cells, (b) aggregate of infiltrating cells or (c) diffused cells around the portal veins distant to border which do not contain CD138-positive cells; brown color, CD138 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 50 µm.

Mentions: Important observation of the current study describes the presence of ectopic lymphoid follicles at site of the tumor – liver border. Such structures ranged from small aggregates (appr. 20 cells in diameter) to highly organized structures containing blood vessels, ductules, and bile ducts as determined by visual analysis of tissue morphology assessed on HE-stained images and staining of cytokeratin 19 (CK19) known to be expressed by bile duct epithelial cells (Figure S4). Ectopic follicles were detected in 12 out 14 patients of panel I and in 33 out of 51 patients of panel II. The presence of follicular structures was documented using a scoring system (0 – no follicular structures; 1 – low number of follicular structures with n ≤ 2, and 2 – high number of follicular structures with n>2). Comparison of panel I and panel II regarding the ectopic follicle score at the tumor – liver border revealed no statistically significant differences (Figure S5). Furthermore, similarly to the CD20-based results, no major differences were detected in the distribution of the ectopic follicle scores between the two chemotherapy groups of panel II (Table S3). Ectopic follicles could only be detected directly at the tumor – liver border or at the portal veins located in the close proximity, whereas typically no follicles were present within distant liver tissue (Figure 4, A).


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)

Ectopic follicular structures at the site of CRCLM.(A) When formed, ectopic follicles are attracted to the tumor – liver interface. Representative image of the large-scale metastatic area surrounded by ectopic follicles is shown; brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 2 mm. The higher-power views of follicles at border (a), proximal (b) and distant (c) to tumor portal veins are given. Scale bar: 50 µm. (B) Representative images of AID-positive ectopic follicular structures located at the tumor – liver border are shown (a, insert: the higher-power view); brown color, AID staining; blue color, nuclear counterstaining with haematoxylin. Scale bar 50 µm. (C) The characteristic structure of a mature, AID-positive lymphoid follicle (a-e) is shown. Consequent slides were stained for CD20 using immunofluorescent (a, merged, red color, CD20 staining; blue color, DAPI; CD20 gradient is visible with reduced intensity at the periphery of follicle) and immunohistochemical (b, brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin) procedure; AID (c), IgM (d), CD138 (e). Insert: the high-power view of CD138-positive cells around follicular structure. To visualize distribution of CD138-positive cells, the image (e) was reduced by 60% in comparison to those shown on a-d. The corresponding AID-positive core of the follicle is indicated by asterisk (c, e). Example of follicle which is predominantly composed of IgM-positive B-cell subset (f, brown color, IgM staining; blue color, nuclear counterstaining with haematoxylin). Scale bar: (a-d, f) 20 µm, (e) 50 µm. (D) CD138-positive plasma cells were not detected at portal vein areas distant to the border. Additional example of (a) follicular structure at border surrounded by CD138-positive plasma cells, (b) aggregate of infiltrating cells or (c) diffused cells around the portal veins distant to border which do not contain CD138-positive cells; brown color, CD138 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 50 µm.
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pone-0099008-g004: Ectopic follicular structures at the site of CRCLM.(A) When formed, ectopic follicles are attracted to the tumor – liver interface. Representative image of the large-scale metastatic area surrounded by ectopic follicles is shown; brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 2 mm. The higher-power views of follicles at border (a), proximal (b) and distant (c) to tumor portal veins are given. Scale bar: 50 µm. (B) Representative images of AID-positive ectopic follicular structures located at the tumor – liver border are shown (a, insert: the higher-power view); brown color, AID staining; blue color, nuclear counterstaining with haematoxylin. Scale bar 50 µm. (C) The characteristic structure of a mature, AID-positive lymphoid follicle (a-e) is shown. Consequent slides were stained for CD20 using immunofluorescent (a, merged, red color, CD20 staining; blue color, DAPI; CD20 gradient is visible with reduced intensity at the periphery of follicle) and immunohistochemical (b, brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin) procedure; AID (c), IgM (d), CD138 (e). Insert: the high-power view of CD138-positive cells around follicular structure. To visualize distribution of CD138-positive cells, the image (e) was reduced by 60% in comparison to those shown on a-d. The corresponding AID-positive core of the follicle is indicated by asterisk (c, e). Example of follicle which is predominantly composed of IgM-positive B-cell subset (f, brown color, IgM staining; blue color, nuclear counterstaining with haematoxylin). Scale bar: (a-d, f) 20 µm, (e) 50 µm. (D) CD138-positive plasma cells were not detected at portal vein areas distant to the border. Additional example of (a) follicular structure at border surrounded by CD138-positive plasma cells, (b) aggregate of infiltrating cells or (c) diffused cells around the portal veins distant to border which do not contain CD138-positive cells; brown color, CD138 staining; blue color, nuclear counterstaining with haematoxylin. Scale bar: 50 µm.
Mentions: Important observation of the current study describes the presence of ectopic lymphoid follicles at site of the tumor – liver border. Such structures ranged from small aggregates (appr. 20 cells in diameter) to highly organized structures containing blood vessels, ductules, and bile ducts as determined by visual analysis of tissue morphology assessed on HE-stained images and staining of cytokeratin 19 (CK19) known to be expressed by bile duct epithelial cells (Figure S4). Ectopic follicles were detected in 12 out 14 patients of panel I and in 33 out of 51 patients of panel II. The presence of follicular structures was documented using a scoring system (0 – no follicular structures; 1 – low number of follicular structures with n ≤ 2, and 2 – high number of follicular structures with n>2). Comparison of panel I and panel II regarding the ectopic follicle score at the tumor – liver border revealed no statistically significant differences (Figure S5). Furthermore, similarly to the CD20-based results, no major differences were detected in the distribution of the ectopic follicle scores between the two chemotherapy groups of panel II (Table S3). Ectopic follicles could only be detected directly at the tumor – liver border or at the portal veins located in the close proximity, whereas typically no follicles were present within distant liver tissue (Figure 4, A).

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