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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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Localization patterns of CD68-positive macrophage populations across CRCLM specimens.(A) Representative images of three major distribution patterns are shown: (a) accumulation of CD68-positive macrophages lining the tumor body within the tumor – liver border sub-region; (b) CD68-positive rim bounding tumor parts and differentiating them from necrotic areas within the metastatic body; tumor – liver margin is indicated by dashed line; (c) 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 (a, b) at the tumor – liver border and (c) within the portal vein area characterized by well-spread or round shaped morphology are shown; in addition, (d, e) 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 (f) large immune cell aggregates and (g) 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): (a) direct cell-cell contact between CD20- and CD68-positive lymphocytes; (b) 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.
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pone-0099008-g006: Localization patterns of CD68-positive macrophage populations across CRCLM specimens.(A) Representative images of three major distribution patterns are shown: (a) accumulation of CD68-positive macrophages lining the tumor body within the tumor – liver border sub-region; (b) CD68-positive rim bounding tumor parts and differentiating them from necrotic areas within the metastatic body; tumor – liver margin is indicated by dashed line; (c) 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 (a, b) at the tumor – liver border and (c) within the portal vein area characterized by well-spread or round shaped morphology are shown; in addition, (d, e) 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 (f) large immune cell aggregates and (g) 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): (a) direct cell-cell contact between CD20- and CD68-positive lymphocytes; (b) 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.

Mentions: Microscopic evaluation of whole tissue specimens revealed strong inter-patients variability regarding spatial distribution of CD68-positive cells. Three major distribution patterns could be observed as summarized in Figure 6, A: CD68-positive macrophages strongly attracted to the tumor – liver interface forming a clear rim at the metastatic border (32 out of 62 patients); no accumulation of CD68-positive cells at the tumor – liver border, however, apparent CD68-positive layer bounding the tumor islands within the partially necrotic metastatic body (15 out of 62 patients); low number and homogenous distribution of CD68-positive cells within border, liver, and tumor without any preferred place of accumulation throughout the tissue sample (15 out of 62 patients). Of note, in some patients mixed distribution patterns were observed; thus, the numbers should be considered as approximate estimations. Furthermore, detailed analysis of three regions of interest revealed heterogeneity in morphology and staining intensities among the patients. CD68-positive macrophages characterized by their elongated, well-spread morphology as well as round shaped phagocytes were detected at the tumor – liver border both from the side of the tumor body and the liver (Figure 6, B, a, b) and within the portal vein areas (Figure 6, B, c). Homogenously distributed CD68-positive resident Kupffer cells detectable within sinusoids in the liver showed inter-patients variability regarding morphology and staining intensity (Figure 6, B, d, e) likely reflecting their activation status. Additionally, large cellular aggregates and ectopic follicular structures were found to be intercalated and/or surrounded by CD68-positive cells (Figure 6, B, f, g). In line, as demonstrated by double staining, CD68-positive macrophages were observed in close proximity to and/or directly attached to CD20-positive B cells at the border (Figure 6, C). In line with visual observations, quantitative evaluations of three regions of interest revealed the CD68-positive cells at the tumor – liver border ranging from 2% to 24% (median 5%), within the liver tissue around the portal veins ranging from 1% to 18% (median 3%), and within distant liver tissue ranging from 1% to 10% (median 3%); analysis was performed for panel I and II in combination. Results revealed no significant differences for panel I and panel II within three regions of interest (Figure S6). Additionally, there are no indications for group differences between chemotherapies within panel II (Table S3). CD68-positive macrophages were significantly increased at portal fields and even stronger at the border compared to the distant liver (p<0.001 and p<0.001, respectively). However, despite strong inter-patient variation in the CD68-attributed immunological imprint, the univariate Cox regression analysis did not show significant prognostic effects for both RFS and OS (Table S6).


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)

Localization patterns of CD68-positive macrophage populations across CRCLM specimens.(A) Representative images of three major distribution patterns are shown: (a) accumulation of CD68-positive macrophages lining the tumor body within the tumor – liver border sub-region; (b) CD68-positive rim bounding tumor parts and differentiating them from necrotic areas within the metastatic body; tumor – liver margin is indicated by dashed line; (c) 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 (a, b) at the tumor – liver border and (c) within the portal vein area characterized by well-spread or round shaped morphology are shown; in addition, (d, e) 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 (f) large immune cell aggregates and (g) 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): (a) direct cell-cell contact between CD20- and CD68-positive lymphocytes; (b) 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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048213&req=5

pone-0099008-g006: Localization patterns of CD68-positive macrophage populations across CRCLM specimens.(A) Representative images of three major distribution patterns are shown: (a) accumulation of CD68-positive macrophages lining the tumor body within the tumor – liver border sub-region; (b) CD68-positive rim bounding tumor parts and differentiating them from necrotic areas within the metastatic body; tumor – liver margin is indicated by dashed line; (c) 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 (a, b) at the tumor – liver border and (c) within the portal vein area characterized by well-spread or round shaped morphology are shown; in addition, (d, e) 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 (f) large immune cell aggregates and (g) 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): (a) direct cell-cell contact between CD20- and CD68-positive lymphocytes; (b) 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.
Mentions: Microscopic evaluation of whole tissue specimens revealed strong inter-patients variability regarding spatial distribution of CD68-positive cells. Three major distribution patterns could be observed as summarized in Figure 6, A: CD68-positive macrophages strongly attracted to the tumor – liver interface forming a clear rim at the metastatic border (32 out of 62 patients); no accumulation of CD68-positive cells at the tumor – liver border, however, apparent CD68-positive layer bounding the tumor islands within the partially necrotic metastatic body (15 out of 62 patients); low number and homogenous distribution of CD68-positive cells within border, liver, and tumor without any preferred place of accumulation throughout the tissue sample (15 out of 62 patients). Of note, in some patients mixed distribution patterns were observed; thus, the numbers should be considered as approximate estimations. Furthermore, detailed analysis of three regions of interest revealed heterogeneity in morphology and staining intensities among the patients. CD68-positive macrophages characterized by their elongated, well-spread morphology as well as round shaped phagocytes were detected at the tumor – liver border both from the side of the tumor body and the liver (Figure 6, B, a, b) and within the portal vein areas (Figure 6, B, c). Homogenously distributed CD68-positive resident Kupffer cells detectable within sinusoids in the liver showed inter-patients variability regarding morphology and staining intensity (Figure 6, B, d, e) likely reflecting their activation status. Additionally, large cellular aggregates and ectopic follicular structures were found to be intercalated and/or surrounded by CD68-positive cells (Figure 6, B, f, g). In line, as demonstrated by double staining, CD68-positive macrophages were observed in close proximity to and/or directly attached to CD20-positive B cells at the border (Figure 6, C). In line with visual observations, quantitative evaluations of three regions of interest revealed the CD68-positive cells at the tumor – liver border ranging from 2% to 24% (median 5%), within the liver tissue around the portal veins ranging from 1% to 18% (median 3%), and within distant liver tissue ranging from 1% to 10% (median 3%); analysis was performed for panel I and II in combination. Results revealed no significant differences for panel I and panel II within three regions of interest (Figure S6). Additionally, there are no indications for group differences between chemotherapies within panel II (Table S3). CD68-positive macrophages were significantly increased at portal fields and even stronger at the border compared to the distant liver (p<0.001 and p<0.001, respectively). However, despite strong inter-patient variation in the CD68-attributed immunological imprint, the univariate Cox regression analysis did not show significant prognostic effects for both RFS and OS (Table S6).

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