Abstract
BackgroundDetection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas. Clonality assessment can be accomplished through evaluation of KAPPA and LAMBDA light chain expression. Currently, only slide based methods are available for the majority of patient biopsies and do not detect light chain protein or mRNA in many B-cell lymphomas. Herein we evaluated a new method, known as colorimetric in situ hybridization (CISH), with improved sensitivity and multiplexing capacity, for its usefulness in clonality detection in mature B cell malignancies.MethodsThe KAPPA and LAMBDA ISH was performed on a Ventana Benchmark XT utilizing two color chromogenetic detection. The probes comprised 2 haptenated riboprobes each approximately 500 base pairs long directed against the conserved regions of either KAPPA or LAMBDA mRNA. The dual colors consisted of silver deposition (black) for KAPPA light chain and a novel (pink) chromogen for LAMBDA light chain. Following optimization, CISH allowed visualization of mRNA in benign B cells in reactive tissues including germinal center, mantle zone, and post-germinal center cells. We then identified 79 cases of B cell lymphoma with formalin-fixed paraffin-embedded (FFPE) biopsies including: follicular (36 cases), mantle cell (6 cases), marginal zone (12 cases), lymphoplasmacytic (6 cases), small lymphocytic (4 cases), and diffuse large B cell (15 cases), which were selected on the basis of either prior flow cytometry or immunohistochemistry (IHC) results to serve as the predicate, "gold standard," comparator.Results39/79 (49.4%) cases were classified as KAPPA and 29/79 (36.7%) as LAMBDA light chain restricted; while 9/79 (11.3%) cases were classified as indeterminate. Of the 70 cases with KAPPA or LAMBDA light chain restricted CISH, 69/70 (98.6%) were concordant with the reference method, while 1/70 (1.4%) was discordant.ConclusionsOptimized CISH detected lower levels of mRNA than can be visualized with current slide based methods, making clonality assessment in FFPE biopsies possible for mature B cell neoplasms. In this preliminary study, CISH was highly accurate compared to flow cytometry or IHC. CISH offers the possibility of wider applicability of light chain ISH and is likely to become a useful diagnostic tool.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1430491067123856
Highlights
Detection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas
After determining that the dual colorimetric in situ hybridization (CISH) assay was able to detect KAPPA and LAMBDA mRNA in mature B cells of increasing differentiation stages as encountered in peripheral lymphoid tissues, we collected cases of B cell nonHodgkin lymphomas (B-NHL) known to arise from the various lymph node
In this study we describe a preliminary accuracy of 98% as compared to reference methods including flow cytometry and immunohistochemistry
Summary
Detection of B cell clonality is useful for assisting in the diagnosis of B cell lymphomas. If lymphoma is not suspected ahead of time or only a limited amount of biopsy material is obtained, often only fixed tissues are available In this common situation, immunohistochemical (IHC) detection methods of kappa and lambda light chain protein in formalin fixed, paraffin embedded tissues (FFPET) are widely used. Current methods allow visualization of mRNA levels of B cells only in the later stages of differentiation and so do not have the dynamic range of flow cytometry (see Figure 1) [5,6] molecular methods aimed at amplification of KAPPA and LAMBDA light chain genes and examination for a predominant rearrangement motif are used These techniques usually performed in molecular laboratories, require longer time as nucleic acid extraction and amplification are needed and do not retain morphologic context i.e. exact visualization of the cell of interest [7]. Despite a variety of current methods, there are still many cases where assessment of B cell monoclonality is technically challenging, posing a limit to the diagnostic process
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