The reliability of immunohistochemical analysis of the tumor microenvironment in follicular lymphoma: a validation study from the Lunenburg Lymphoma Biomarker Consortium

Abstract

The cellular microenvironment in follicular lymphoma is of biological and clinical importance. Studies on the clinical significance of non-malignant cell populations have generated conflicting results, which may partly be influenced by poor reproducibility in immunohistochemical marker quantification. In this study, the reproducibility of manual scoring and automated microscopy based on a tissue microarray of 25 follicular lymphomas as compared to flow cytometry is evaluated. The agreement between manual scoring and flow cytometry was moderate for CD3, low for CD4, and moderate to high for CD8, with some laboratories scoring closer to the flow cytometry results. Agreement in manual quantification across the 7 laboratories was low to moderate for CD3, CD4, CD8 and FOXP3 frequencies, moderate for CD21, low for MIB1 and CD68, and high for CD10. Manual scoring of the architectural distribution resulted in moderate agreement for CD3, CD4 and CD8, and low agreement for FOXP3 and CD68. Comparing manual scoring to automated microscopy demonstrated that manual scoring increased the variability in the low and high frequency interval with some laboratories showing a better agreement with automated scores. Manual scoring reliably identified rare architectural patterns of T-cell infiltrates. Automated microscopy analyses for T-cell markers by two different instruments were highly reproducible and provided acceptable agreement with flow cytometry. These validation results provide explanations for the heterogeneous findings on the prognostic value of the microenvironment in follicular lymphoma. We recommend a more objective measurement, such as computer-assisted scoring, in future studies of the prognostic impact of microenvironment in follicular lymphoma patients.

Revisiting the immune microenvironment of diffuse large B-cell lymphoma using a tissue microarray and immunohistochemistry: robust semi-automated analysis reveals CD3 and FoxP3 as potential predictors of response to R-CHOP

“…15 This study, conducted by highly experienced hematopathologists, found only low to moderate agreement in manual scoring of T cells and macrophage markers when four to five scoring categories were used. However, comparison of automated analyses set up by two different operators in two different instruments using the same methodology was highly reproducible for T-cell markers.…”

“…Large and interdigitating cells, such as macrophages, are difficult to quantify. 15 The image analysis training was challenging and focused on capturing the areas of dark brown IHC staining present in the cell bodies. CD68 staining was heterogeneous in this cohort.…”

Prognostic relevance of CD163 and CD8 combined with EZH2 and gain of chromosome 18 in follicular lymphoma: a study by the Lunenburg Lymphoma Biomarker Consortium

“…Previous IHC-based validation studies of microenvironment cell populations by the Lunenburg Lymphoma Biomarker Consortium (LLBC), in both diffuse large B-cell Lymphoma (DLBCL) and FL, have highlighted the poor reproducibility of manual scoring even by experienced pathologists, and advocated computer-assisted scoring as being the more reliable technique. 26,27 Recently, the mutational spectrum of FL tumor cells and their prognostic value have been reported. [28][29][30] Most notably, Pastore et al demonstrated the value of combining mutation status with clinical FLIPI and performance status to improve upon the prognostic value of FLIPI alone (45-55%) to 64-72% for M7-FLIPI, with a comparable negative prognostic value.…”

Catching up with solid tumor oncology: what is the evidence for a prognostic role of programmed cell death-ligand 1/programmed cell death-1 expression in B-cell lymphomas?

“…A validation study from a lymphoma consortium on the FL microenvironment reported considerable differences between manual scoring and automated microscopy systems and flow cytometry, which was also dependent on the investigating laboratory. 74 Within semi-automated image analysis systems, a high concordance seems to exist. 49 Among the included studies, expression was predominantly assessed by IHC.…”