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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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Algorithm for characterization, quantitative analysis, and estimation of the prognostic effect of immune cell populations.(A) Definition of regions of interest within CRCLM tissue samples to unify the quantification strategy between the specimens. Upon acquisition of a large-scale sample using the microscopy-based TissueFAXS system, the regions of interest were defined at the tumor – liver border (indicated by a red line), within liver tissue around the portal vein areas (indicated by dashed squares in green) and areas within the liver tissues distant to the portal veins (indicated by dashed squares in blue). Of note, the herein defined strategy for using 0.5×0.5 mm area for analysis of the tumor – liver border was established empirically based on the microscopic evaluation of CD45-stained sections showing the lymphocyte accumulation within this region; this was specified independent to but matching the quantification area defined by Halama et al [3]. Scale bar: 1 mm. (B) Study design. The study design is summarized by the flowchart including staining with established markers, immune cell organisation pattern, quantification, and assessment of clinical relevance by alignment with clinicopathological parameters.
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pone-0099008-g001: Algorithm for characterization, quantitative analysis, and estimation of the prognostic effect of immune cell populations.(A) Definition of regions of interest within CRCLM tissue samples to unify the quantification strategy between the specimens. Upon acquisition of a large-scale sample using the microscopy-based TissueFAXS system, the regions of interest were defined at the tumor – liver border (indicated by a red line), within liver tissue around the portal vein areas (indicated by dashed squares in green) and areas within the liver tissues distant to the portal veins (indicated by dashed squares in blue). Of note, the herein defined strategy for using 0.5×0.5 mm area for analysis of the tumor – liver border was established empirically based on the microscopic evaluation of CD45-stained sections showing the lymphocyte accumulation within this region; this was specified independent to but matching the quantification area defined by Halama et al [3]. Scale bar: 1 mm. (B) Study design. The study design is summarized by the flowchart including staining with established markers, immune cell organisation pattern, quantification, and assessment of clinical relevance by alignment with clinicopathological parameters.

Mentions: To analyse and quantify the presence of resident and infiltrating immune cells within the CRCLM tissue we established an algorithm using the TissueFAXS system. For specimen's comparison we applied uniform strategy by making sub-regions at the tumor – liver border (approximately 0.5×0.5 mm on each side of the border with a mean size of about 30 mm2) and within the liver tissue around the portal vein areas (five representative areas sized to 0.25 mm2) and areas within the liver tissues distant to the portal veins named as distant liver in the follow up descriptions (three representative areas sized to 0.25 mm2) (a representative picture is shown in Figure 1, A). Study design is summarized by the flowchart in Figure 1, B. Paraffin-embedded sections were first stained with anti-CD45 antibodies to detect all classes of resident and/or infiltrating immune cells across the patient specimen. Next, to specify the immune cell populations we discriminated between the B cell lineage and the monocyte/macrophage lineage (Figure 1, B). Quantitative analysis using the TissueQuest/HistoQuest software was applied to determine the amount of CD45-positive, CD20-positive, and CD68-positive cells found within the specified above locations of interest. The calculation algorithm is described in Figure S1. To determine the clinical relevance of the patient-specific immune response at site of metastasis, staining-derived data sets were used for alignment with clinicopathological parameters.


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)

Algorithm for characterization, quantitative analysis, and estimation of the prognostic effect of immune cell populations.(A) Definition of regions of interest within CRCLM tissue samples to unify the quantification strategy between the specimens. Upon acquisition of a large-scale sample using the microscopy-based TissueFAXS system, the regions of interest were defined at the tumor – liver border (indicated by a red line), within liver tissue around the portal vein areas (indicated by dashed squares in green) and areas within the liver tissues distant to the portal veins (indicated by dashed squares in blue). Of note, the herein defined strategy for using 0.5×0.5 mm area for analysis of the tumor – liver border was established empirically based on the microscopic evaluation of CD45-stained sections showing the lymphocyte accumulation within this region; this was specified independent to but matching the quantification area defined by Halama et al [3]. Scale bar: 1 mm. (B) Study design. The study design is summarized by the flowchart including staining with established markers, immune cell organisation pattern, quantification, and assessment of clinical relevance by alignment with clinicopathological parameters.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4048213&req=5

pone-0099008-g001: Algorithm for characterization, quantitative analysis, and estimation of the prognostic effect of immune cell populations.(A) Definition of regions of interest within CRCLM tissue samples to unify the quantification strategy between the specimens. Upon acquisition of a large-scale sample using the microscopy-based TissueFAXS system, the regions of interest were defined at the tumor – liver border (indicated by a red line), within liver tissue around the portal vein areas (indicated by dashed squares in green) and areas within the liver tissues distant to the portal veins (indicated by dashed squares in blue). Of note, the herein defined strategy for using 0.5×0.5 mm area for analysis of the tumor – liver border was established empirically based on the microscopic evaluation of CD45-stained sections showing the lymphocyte accumulation within this region; this was specified independent to but matching the quantification area defined by Halama et al [3]. Scale bar: 1 mm. (B) Study design. The study design is summarized by the flowchart including staining with established markers, immune cell organisation pattern, quantification, and assessment of clinical relevance by alignment with clinicopathological parameters.
Mentions: To analyse and quantify the presence of resident and infiltrating immune cells within the CRCLM tissue we established an algorithm using the TissueFAXS system. For specimen's comparison we applied uniform strategy by making sub-regions at the tumor – liver border (approximately 0.5×0.5 mm on each side of the border with a mean size of about 30 mm2) and within the liver tissue around the portal vein areas (five representative areas sized to 0.25 mm2) and areas within the liver tissues distant to the portal veins named as distant liver in the follow up descriptions (three representative areas sized to 0.25 mm2) (a representative picture is shown in Figure 1, A). Study design is summarized by the flowchart in Figure 1, B. Paraffin-embedded sections were first stained with anti-CD45 antibodies to detect all classes of resident and/or infiltrating immune cells across the patient specimen. Next, to specify the immune cell populations we discriminated between the B cell lineage and the monocyte/macrophage lineage (Figure 1, B). Quantitative analysis using the TissueQuest/HistoQuest software was applied to determine the amount of CD45-positive, CD20-positive, and CD68-positive cells found within the specified above locations of interest. The calculation algorithm is described in Figure S1. To determine the clinical relevance of the patient-specific immune response at site of metastasis, staining-derived data sets were used for alignment with clinicopathological parameters.

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