Biometrical Journal / Contribution to the discussion of ‘When should meta-analysis avoid making hidden normality assumptions?

(VLID)342423

A 5year update of patients with HPV positive versus negative oropharyngeal cancer after radiochemotherapy in Austria

Background After publishing promising results for the treatment of patients with human papilloma virus (HPV) positive oropharyngeal cancer with radiochemotherapy regarding 2year survival, we present an update of the disease-specific and disease-free survival after 5 years. Patients and methods A total of 29 patients of which 18 were HPV negative and 11 HPV positive with squamous cell carcinoma of the oropharynx received radiation therapy with or without chemotherapy (cisplatin) or immunotherapy (cetuximab) between 2007 and 2009. At time of the present analysis, six patients are still alive including four with HPV positive and two with HPV negative oropharyngeal carcinoma, while 15 out of 16 patients with HPV negative tumors died and 1 died of another cause with evidence of disease. Results Since the 2year disease-specific survival of patients with HPV positive cancer of the oropharynx was published with 100% versus 30.4% in HPV negative tumors, we now present the 5year disease-specific survival after treatment, which was 85.7% in HPV positive versus 11.1% in HPV negative patients. Conclusion We present the results of patients receiving radiochemo(immuno)therapy for oropharyngeal cancer regarding the HPV status, which is still promising.(VLID)354526

The Immune Phenotype of Isolated Lymphoid Structures in Non-Tumorous Colon Mucosa Encrypts the Information on Pathobiology of Metastatic Colorectal Cancer

The gut-associated lymphoid tissue represents an integral part of the immune system. Among the powerful players of the mucosa-associated lymphoid tissue are isolated lymphoid structures (ILSs), which as information centers, drive the local (and systemic) adaptive immune responses. Germinal center reactions, taking place within ILSs, involve the coordinated action of various immune cell types with a central role given to B cells. In the current study, we aimed at dissecting the impact of ILSs within non-tumorous colon tissue (NT) on the pathobiology of colorectal cancer (CRC) with metastasis in the liver (CRCLM). In particular, we focused on the immune phenotypes of ILSs and ectopic lymphoid structures (ELSs), built up at matching primary and metastatic tumor sites. We implemented an integrative analysis strategy on the basis of tissue image cytometry and clonality assessment to explore the immune phenotype of ILS/ELS at three tissue entities: NT, CRC, and CRCLM (69 specimens in total). Applying a panel of lineage markers used for immunostaining, we characterized and compared the anatomical features, the cellular composition, the activation, and proliferation status of ILSs and ELSs, and assessed the clinical relevance of staining-derived data sets. Our major discovery was that ILS characteristics at the NT site predefine the immune phenotype of ELSs at CRC and CRCLM. Thereby, B-cell-enriched (CD20) and highly proliferative (Ki67) ILSs and ELSs were found to be associated with improved clinical outcome in terms of survival and enabled patient stratification into risk groups. Moreover, the data revealed a linkage between B-cell clonality at the NT site and the metastatic characteristics of the tumor in the distant liver tissue. Consolidation of immunostaining-based findings with the results of compendium-wide transcriptomic analysis furthermore proposed CD27 as a novel marker of T follicular helper cells within lymphoid structures. Overall, the study nominates the ILS immune phenotype as a novel prognostic marker for patients with metastatic CRC

B Cells and Ectopic Follicular Structures: Novel Players in Anti-Tumor Programming with Prognostic Power for Patients with Metastatic Colorectal Cancer

<div><p>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.</p></div

Patient-specific imprint of CD45-positive cells at the site of CRCLM.

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

Ectopic follicular structures at the site of CRCLM.

<p>(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 (<i>a</i>), proximal (<i>b</i>) and distant (<i>c</i>) 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 (<i>a</i>, 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 (<i>a-e</i>) is shown. Consequent slides were stained for CD20 using immunofluorescent (<i>a</i>, merged, red color, CD20 staining; blue color, DAPI; CD20 gradient is visible with reduced intensity at the periphery of follicle) and immunohistochemical (<i>b</i>, brown color, CD20 staining; blue color, nuclear counterstaining with haematoxylin) procedure; AID (<i>c</i>), IgM (<i>d</i>), CD138 (<i>e</i>). Insert: the high-power view of CD138-positive cells around follicular structure. To visualize distribution of CD138-positive cells, the image (<i>e</i>) was reduced by 60% in comparison to those shown on <i>a-d</i>. The corresponding AID-positive core of the follicle is indicated by asterisk (<i>c</i>, <i>e</i>). Example of follicle which is predominantly composed of IgM-positive B-cell subset (<i>f</i>, brown color, IgM staining; blue color, nuclear counterstaining with haematoxylin). Scale bar: (<i>a-d, f</i>) 20 µm, (<i>e</i>) 50 µm. (D) CD138-positive plasma cells were not detected at portal vein areas distant to the border. Additional example of (<i>a</i>) follicular structure at border surrounded by CD138-positive plasma cells, (<i>b</i>) aggregate of infiltrating cells or (<i>c</i>) 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.</p

CD20-positive B lymphocytes at the site of CRCLM.

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

Prognostic effect of ectopic follicular structures allocated at the tumor – liver border.

<p>Kaplan-Meier estimates for patients stratification based on the ectopic follicle score at the border (<i>a</i>–<i>c</i>). Kaplan-Meier curves for RFS for panel I, panel II, and panel I+II show patient stratification into low (high number of follicular structures), intermediate (low number of follicular structures) and high risk (no follicular structures) groups; p value of the log-rank trend test is indicated. Panel I: low risk n = 5, intermediate risk n = 7, high risk n = 2; panel II: low risk n = 16, intermediate risk n = 17, high risk n = 18; panel I+II: low risk n = 21, intermediate risk n = 24, high risk n = 20. (<i>d</i>) Comparative assessment of contribution of <i>no</i>, <i>low</i>, and <i>high</i> ectopic follicle score to the total quantity for patient sub-groups without (No) and with (Yes) disease recurrence as estimated at the time point of 24 months where no censoring has occurred; p value of chi-square trend test is shown.</p