Ultrasensitive RNA In Situ Hybridization for Detection of Restricted Clonal Expression of Low-Abundance Immunoglobulin Light Chain mRNA in B-Cell Lymphoproliferative Disorders

Tubbs, Raymond R.; Wang, Hongwei; Wang, Zhen; Minca, Eugen C.; Portier, Bryce P.; Gruver, Aaron M.; Lanigan, Christopher; Luo, Yuling; Cook, James R.; Xiao, Jun

doi:10.1309/ajcpjtwk07fsabrj

Cited by 34 publications

(47 citation statements)

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“…Initially, this staining seemed to suggest conflicting results, particularly in the cases which had clear KAPPA mRNA cytoplasmic black colored staining while having apparent " LAMBDA " pink staining in the nucleoli of the same cells. However, a review of the literature revealed that this staining pattern may result from cross hybridization with the Ig lambda-like polypeptide 5 ( IGLL5 ) gene on chromosome 22, which is located within the immunoglobulin lambda locus but does not require somatic rearrangement for expression, meaning that it can be expressed in non-Ig expressing, non-B, cells [13]. As recently demonstrated by Tubbs et al .…”

Section: Discussionmentioning

confidence: 99%

“…As recently demonstrated by Tubbs et al . cross-hybridization with the LAMBDA CISH probe occurs because while the first exon of IGLL5 gene is unrelated to immunoglobulin variable genes, the second and third exons use the same loci as the Ig-lambda joining 1 and the Ig-lambda constant 1 gene segments [13,14]. Recognition of this non-specific pattern allowed interpretation of cases with pink nucleolar staining as well as black cytoplasmic staining as KAPPA restricted.…”

Section: Discussionmentioning

confidence: 99%

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Kappa and lambda light chain mRNA in situ hybridization compared to flow cytometry and immunohistochemistry in B cell lymphomas

et al. 2014

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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

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Kappa and lambda light chain mRNA in situ hybridization compared to flow cytometry and immunohistochemistry in B cell lymphomas

et al. 2014

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“…We then further used an ultrasensitive RNAscope in situ hybridization (ISH) method to detect and quantify low densities of CB 2 R mRNA expression in brain slices of rats and mice since the traditional ISH method may not be sensitive enough to detect the low CB 2 R mRNA in rodent brains (Tubbs et al, 2013;Wang et al, 2012). Figure 5A shows mouse and rat coronal atlas, illustrating PFC, VTA, DST and NAc.…”

Section: Rnascope In Situ Hybridization Of Cb 2 R In Rat and Mouse Brmentioning

confidence: 99%

Species Differences in Cannabinoid Receptor 2 and Receptor Responses to Cocaine Self-Administration in Mice and Rats

Zhang

et al. 2014

Neuropsychopharmacol

114

134

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The discovery of functional cannabinoid receptors 2 (CB2Rs) in brain suggests a potential new therapeutic target for neurological and psychiatric disorders. However, recent findings in experimental animals appear controversial. Here we report that there are significant species differences in CB2R mRNA splicing and expression, protein sequences, and receptor responses to CB2R ligands in mice and rats. Systemic administration of JWH133, a highly selective CB2R agonist, significantly and dose-dependently inhibited intravenous cocaine self-administration under a fixed ratio (FR) schedule of reinforcement in mice, but not in rats. However, under a progressive ratio (PR) schedule of reinforcement, JWH133 significantly increased breakpoint for cocaine self-administration in rats, but decreased it in mice. To explore the possible reasons for these conflicting findings, we examined CB2R gene expression and receptor structure in the brain. We found novel rat-specific CB2C and CB2D mRNA isoforms in addition to CB2A and CB2B mRNA isoforms. In situ hybridization RNAscope assays found higher levels of CB2R mRNA in different brain regions and cell types in mice than in rats. By comparing CB2R-encoding regions, we observed a premature stop codon in the mouse CB2R gene that truncated 13 amino-acid residues including a functional autophosphorylation site in the intracellular C-terminus. These findings suggest that species differences in the splicing and expression of CB2R genes and receptor structures may in part explain the different effects of CB2R-selective ligands on cocaine self-administration in mice and rats.

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“…Visualization of RNA transcripts for TNF-a, transforming growth factor (TGF)-b, IFN-g, IL-17A, IL-16, IL-10, CXCL9 and CXCL10 was done according to the manufacturer's instructions for RNAScope Ò 2.0 (Wang et al, 2012;Tubbs et al, 2013). A proprietary probe combination was used for each of the cytokine and chemokine mRNAs.…”

Section: Rna Chromogenic In-situ Hybridizationmentioning

confidence: 99%

Analysis of Cytokine Gene Expression using a Novel Chromogenic In-situ Hybridization Method in Pulmonary Granulomas of Cattle Infected Experimentally by Aerosolized Mycobacterium bovis

Palmer

Thacker

Waters

2015

Journal of Comparative Pathology

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Mycobacterium bovis is the cause of tuberculosis in most animal species including cattle and is a serious zoonotic pathogen. In man, M. bovis infection can result in disease clinically indistinguishable from that caused by Mycobacterium tuberculosis, the cause of most human tuberculosis. Regardless of host, the typical lesion induced by M. bovis or M. tuberculosis is the tuberculoid granuloma. Tuberculoid granulomas are dynamic structures reflecting the interface between host and pathogen and, therefore, pass through various morphological stages (I to IV). Using a novel in-situ hybridization assay, transcription of various cytokine and chemokine genes was examined qualitatively and quantitatively using image analysis. In experimentally infected cattle, pulmonary granulomas of all stages were examined 150 days after aerosol exposure to M. bovis. Expression of mRNA encoding tumour necrosis factor (TNF)-α, transforming growth factor-β, interferon (IFN)-γ, interleukin (IL)-17A, IL-16, IL-10, CXCL9 and CXCL10 did not differ significantly between granulomas of different stages. However, relative expression of the various cytokines was characteristic of a Th1 response, with high TNF-α and IFN-γ expression and low IL-10 expression. Expression of IL-16 and the chemokines CXCL9 and CXCL10 was high, suggestive of granulomas actively involved in T-cell chemotaxis.

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Ultrasensitive RNA In Situ Hybridization for Detection of Restricted Clonal Expression of Low-Abundance Immunoglobulin Light Chain mRNA in B-Cell Lymphoproliferative Disorders

Abstract: BRISH analysis of κ and λ immunoglobulin mRNA expression is a sensitive tool for establishing LCR in B-cell NHL when FCM results are not available.

Cited by 34 publications

References 31 publications

Kappa and lambda light chain mRNA in situ hybridization compared to flow cytometry and immunohistochemistry in B cell lymphomas

Kappa and lambda light chain mRNA in situ hybridization compared to flow cytometry and immunohistochemistry in B cell lymphomas

Species Differences in Cannabinoid Receptor 2 and Receptor Responses to Cocaine Self-Administration in Mice and Rats

Analysis of Cytokine Gene Expression using a Novel Chromogenic In-situ Hybridization Method in Pulmonary Granulomas of Cattle Infected Experimentally by Aerosolized Mycobacterium bovis

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