Ramón Bosch

Outcome in Hodgkin's lymphoma can be predicted from the presence of accompanying cytotoxic and regulatory T cells.

Purpose: Recent studies of Hodgkins lymphoma (HL) have suggested that the presence of regulatory T cells in the reactive background may explain the inhibition of the antitumoral host immune response observed in these patients. This study aimed to assess the relevance of regulatory T cells and CTLs present in the background of HL samples in the prognosis of a series of classic HL (cHL) patients. Experimental Design: Expression of granzyme B and TIA-1 (markers for CTL) and FOXP3 (a marker for regulatory T cells) were evaluated independently by immunohistochemistry in tissue microarrays of 257 cHL patients and correlated with patient outcome. Results: The combined influence of the presence of FOXP3+ and TIA-1+ cells distinguished three risk groups of patients with 5-year overall survival of 100%, 88%, and 73%. The presence of a small number of FOXP3+ cells and a high proportion of TIA-1+ cells in the infiltrate represent an independent prognostic factor that negatively influenced event-free survival and disease-free survival in cHL. Compared with the features at diagnosis, relapsed samples tended to have more TIA-1+ cells and a lower proportion of FOXP3+ cells in the reactive background. Conclusions: These data suggest that low infiltration of FOXP3+ cells in conjunction with high infiltration of TIA-1+ cells in cHL may represent biological markers predicting an unfavorable outcome. Moreover, the variation of these markers over the course of the disease implies a possible role for them in the progression of HL cases.

Quantification of diverse subcellular immunohistochemical markers with clinicobiological relevancies: validation of a new computer-assisted image analysis procedure

Tissue microarray technology and immunohistochemical techniques have become a routine and indispensable tool for current anatomical pathology diagnosis. However, manual quantification by eye is relatively slow and subjective, and the use of digital image analysis software to extract information of immunostained specimens is an area of ongoing research, especially when the immunohistochemical signals have different localization in the cells (nuclear, membrane, cytoplasm). To minimize critical aspects of manual quantitative data acquisition, we generated semi‐automated image‐processing steps for the quantification of individual stained cells with immunohistochemical staining of different subcellular location. The precision of these macros was evaluated in 196 digital colour images of different Hodgkin lymphoma biopsies stained for different nuclear (Ki67, p53), cytoplasmic (TIA‐1, CD68) and membrane markers (CD4, CD8, CD56, HLA‐Dr). Semi‐automated counts were compared to those obtained manually by three separate observers. Paired t‐tests demonstrated significant differences between intra‐ and inter‐observer measurements, with more substantial variability when the cellular density of the digital images was > 100 positive cells/image. Overall, variability was more pronounced for intra‐observer than for inter‐observer comparisons, especially for cytoplasmic and membrane staining patterns (P < 0.0001 and P = 0.050). The comparison between the semi‐automated and manual microscopic measurement methods indicates significantly lower variability in the results yielded by the former method. Our semi‐automated computerized method eliminates the major causes of observer variability and may be considered a valid alternative to manual microscopic quantification for diagnostic, prognostic and therapeutic purposes.

Tumor-Infiltrated Immune Response Correlates with Alterations in the Apoptotic and Cell Cycle Pathways in Hodgkin and Reed-Sternberg Cells

Purpose: To analyze tumor-microenvironment relationships in Hodgkin lymphoma (HL) as potential determinants in the decision-making process related to the alterations in cell cycle and apoptotic pathways of Hodgkin/Reed-Sternberg (H/RS) cells. Experimental Design: Based on a cohort of 257 classic HL patients, we carried out a global descriptive correlational analysis and logistic regression study to identify tumor-infiltrated immune cell rate in HL that could be interconnected with genes involved in the regulation of apoptotic/proliferative pathways in H/RS cells. Results: Our results reveal the existence of a connection between the reactive microenvironment and molecular changes in apoptotic/proliferative pathways in H/RS cells. A lesser incidence of infiltrated cytotoxic cells in the tumor (CD8+ T lymphocytes, CD57+ natural killer, and granzyme B+ cells) was associated with overexpression of antiapoptotic proteins (Bcl-XL, survivin, caspase-3, and nuclear factor-κB) in tumoral cells. Increased incidence of general infiltrated immune cells, such as CD4+ T lymphocytes, CD57+ natural killer cells, activated CTL, and dendritic cells, in the microenvironment of the tumor was associated with increased growth fraction of tumoral cells, including G1-S checkpoint (cyclin D and cyclin E) and tumor suppressor pathways (p16 and SKP2), and with the presence of EBV (signal transducers and activators of transcription 1 and 3 expression; STAT1/STAT3). Conclusions: A lower level of cytotoxic cells correlated with an increase of antiapoptotic mechanisms in H/RS cells, whereas the global infiltrated immune population correlated with the growth fraction of the tumor. Our collective data suggest a causal relationship between infiltrated immune response and concurrent changes of the different proliferative checkpoints, tumor suppressor, and apoptotic pathways of H/RS cells in HL.

Validation of various adaptive threshold methods of segmentation applied to follicular lymphoma digital images stained with 3,3’-Diaminobenzidine&Haematoxylin

AbstractThe comparative study of the results of various segmentation methods for the digital images of the follicular lymphoma cancer tissue section is described in this paper. The sensitivity and specificity and some other parameters of the following adaptive threshold methods of segmentation: the Niblack method, the Sauvola method, the White method, the Bernsen method, the Yasuda method and the Palumbo method, are calculated. Methods are applied to three types of images constructed by extraction of the brown colour information from the artificial images synthesized based on counterpart experimentally captured images. This paper presents usefulness of the microscopic image synthesis method in evaluation as well as comparison of the image processing results. The results of thoughtful analysis of broad range of adaptive threshold methods applied to: (1) the blue channel of RGB, (2) the brown colour extracted by deconvolution and (3) the ’brown component’ extracted from RGB allows to select some pairs: method and type of image for which this method is most efficient considering various criteria e.g. accuracy and precision in area detection or accuracy in number of objects detection and so on. The comparison shows that the White, the Bernsen and the Sauvola methods results are better than the results of the rest of the methods for all types of monochromatic images. All three methods segments the immunopositive nuclei with the mean accuracy of 0.9952, 0.9942 and 0.9944 respectively, when treated totally. However the best results are achieved for monochromatic image in which intensity shows brown colour map constructed by colour deconvolution algorithm. The specificity in the cases of the Bernsen and the White methods is 1 and sensitivities are: 0.74 for White and 0.91 for Bernsen methods while the Sauvola method achieves sensitivity value of 0.74 and the specificity value of 0.99. According to Bland-Altman plot the Sauvola method selected objects are segmented without undercutting the area for true positive objects but with extra false positive objects. The Sauvola and the Bernsen methods gives complementary results what will be exploited when the new method of virtual tissue slides segmentation be develop.Virtual SlidesThe virtual slides for this article can be found here: slide 1: http://diagnosticpathology.slidepath.com/dih/webViewer.php?snapshotId=13617947952577 and slide 2: http://diagnosticpathology.slidepath.com/dih/webViewer.php?snapshotId=13617948230017.

Impact of interleukin-10 polymorphisms (-1082 and -3575) on the survival of patients with lymphoid neoplasms.

Background and Objectives Single-nucleotide polymorphisms (SNP) in interleukin-10 (IL-10) genes can influence immune responses, which may affect the outcome of patients with lymphoid neoplasms. The aim of this study was to explore the association between polymorphisms of IL-10-1082A>G and IL-10-3575T>A with the overall survival in patients with lymphoid neoplasms. Design and Methods We analyzed two IL-10 SNP (−1082 and −3575) in 472 consecutive cases with lymphoid neoplasms. Genotypes were tested for association with overall survival and classical prognostic factors by multivariate analysis. Haplotype analysis was carried out using the haplostats package implemented in R software. The implications for survival of patients with lymphoma were evaluated using multivariate analysis. Results Lymphoma patients with the IL-10-3575T>A genotype had a better overall survival (p= 0.002), as did the subgroup with non-Hodgkin’s lymphoma (NHL) (p=0.05). Patients with the IL10-1082GG genotype had a better median overall survival (p=0.05). When both genotypes were included in a multivariate analysis, IL-10-3575AA genotype was the only independent prognostic factor for survival (HR=0.20, 95%CI 0.05–0.92). Patients with the IL-10-1082 and -3575 G-A/G-A diplotype had a longer overall survival (p=0.003) and this combination appeared to be an independent prognostic factor for survival (HR:0.26; 95%CI 0.08–0.83). Interpretation and Conclusions The IL-10-3575A/A genotype was identified as a marker of favorable survival. Because the IL-10-1082 and -3575 G-A/G-A diplotype was also identified as an indicator of longer survival, we cannot exclude the potential additive role of the IL-10-1082GG genotype. These results need to be replicated in larger series and examined in different NHL subtypes.

Effects of Image Compression on Automatic Count of Immunohistochemically Stained Nuclei in Digital Images

This study investigates the effects of digital image compression on automatic quantification of immunohistochemical nuclear markers. We examined 188 images with a previously validated computer-assisted analysis system. A first group was composed of 47 images captured in TIFF format, and other three contained the same images converted from TIFF to JPEG format with 3x, 23x and 46x compression. Counts of TIFF format images were compared with the other three groups. Overall, differences in the count of the images increased with the percentage of compression. Low-complexity images (< or =100 cells/field, without clusters or with small-area clusters) had small differences (<5 cells/field in 95-100% of cases) and high-complexity images showed substantial differences (<35-50 cells/field in 95-100% of cases). Compression does not compromise the accuracy of immunohistochemical nuclear marker counts obtained by computer-assisted analysis systems for digital images with low complexity and could be an efficient method for storing these images.

Equalisation of archival microscopic images from immunohistochemically stained tissue sections

A method of image equalisation that reduces non-uniformity of light distribution caused by optical devices and dust on camera sensors is presented. The method explores non-uniformity which occurs in archival images captured by a typical optical set which consists of a light microscope and a digital camera. A sufficient number of images with low density of foreground objects has been used to extract a global map of non-uniformity of the particular microscope and camera. The proposed method consists of two steps: – (1) extraction of the map of non-uniformity based upon a set of chosen images and – (2) correction of images acquired by the optical set. The global map is created based upon a modified value layer, the third layer of HSV colour space. The proposed method has been tested on images of immunohistochemically (IHC) stained samples of a biopsy tissue, and it has been validated using an image segmentation method developed earlier. The results of the light distribution equalization, as well as the equalized images segmentation turn out to be more similar to the reference method results (namely the manual counting results), than the results of the original images segmentation. The equalization method can be used for other types of images, but all of them should be acquired by the same optical set.

Antibody Response to Merkel Cell Polyomavirus Associated with Incident Lymphoma in the Epilymph Case–Control Study in Spain

Background: Merkel cell polyomavirus (MCV) has been identified as the cause of Merkel cell carcinoma. The increased incidence of chronic lymphocytic leukemia in Merkel cell cancer cohorts and the lymphotropic properties of the virus suggest a possible viral association with lymphomagenesis. To investigate this potential role, we explored seroreactivity against MCV VP1 capsids within the Epilymph case–control study in Spain. Methods: Serum samples from 468 incident lymphomas, categorized into up to 11 entities, and 522 controls frequency matched by age, sex, and recruitment center were tested for MCV antibodies by enzyme immunoassay using Virus-Like-Particles. Adjusted multinomial logistic regression was used to estimate the OR and 95% confidence interval (CI) associated to MCV seroprevalence. Immunosuppressed subjects were excluded. Results: MCV seroprevalence was 82% in controls and 85% in lymphoma cases. Among 11 lymphoma categories, MCV seropositivity was significantly higher in diffuse large B-cell lymphomas (DLBCL; 96.4%; OR = 6.1, 95%CI = 1.9–19.8), as compared with controls. MCV prevalences were also higher in follicular lymphoma, lymphoplasmacytic lymphoma, chronic lymphocytic leukemia, Hodgkin lymphoma, and mature T-cell lymphoma but differences did not reach statistical significance. Lower prevalences were observed for multiple myeloma and other B-cell lymphoma. Exclusion of samples collected after start of treatment did not change the results. In a subset analysis, no significant association was observed between BKV and JCV seroprevalence and DLBCL. Conclusion: The association observed between serologic evidence of MCV exposure and DLBCL warrants further research. Impact: MCV might be involved in the pathway of DLBCL and other lymphomas. Cancer Epidemiol Biomarkers Prev; 21(9); 1592–8. ©2012 AACR.

The Method of Immunohistochemical Images Standardization

The standardization method of immunohistchemically staining tissue section images prior to the image processing and analysis is described in this paper. The effectiveness of the proposed standardization method is examined on thin tissue slices of breast cancer stained with DAB & H. The image analysis results after the initial image standardization are more closer to the results of traditional methods of cells nuclei quantification than for original images.

Evaluation of cytokeratin-19 in breast cancer tissue samples: a comparison of automatic and manual evaluations of scanned tissue microarray cylinders

BackgroundDigital image (DI) analysis avoids visual subjectivity in interpreting immunohistochemical stains and provides more reproducible results. An automated procedure consisting of two variant methods for quantifying the cytokeratin-19 (CK19) marker in breast cancer tissues is presented.MethodsThe first method (A) excludes the holes inside selected CK19 stained areas, and the second (B) includes them. 93 DIs scanned from complete cylinders of tissue microarrays were evaluated visually by two pathologists and by the automated procedures.Results and conclusionsThere was good concordance between the two automated methods, both of which tended to identify a smaller CK19-positive area than did the pathologists. The results obtained with method B were more similar to those of the pathologists; probably because it takes into account the entire positive tumoural area, including the holes. However, the pathologists overestimated the positive area of CK19. Further studies are needed to confirm the utility of this automated procedure in prognostic studies.