SBIR-STTR Award

Immunoglobulin (Ig) light chain restriction (LCR) detection is an important molecular diagnostic tool in the differential diagnosis that includes lymphoid hyperplasia, atypical lymphoid hyperplasia, chronic inflammation, and B cell neoplasms. Current methods of LCR identification in formalin fixed paraffin embedded (FFPE) tissue include immunohistochemistry (IHC) and conventional chromogenic in situ hybridization (CISH), which measure kappa or lambda protein and mRNA, respectively. However, both IHC and conventional CISH lack sufficient sensitivity to detect LCR in many non-Hodgkin lymphoma (NHL), including the most common NHL variants - follicular lymphoma, diffuse large B cell lymphoma, small B lymphocytic lymphoma, and extranodal marginal zone lymphoma of MALT type. RNAscope is novel RNA in situ hybridization technology that has single-molecule detection sensitivity and is fully compatible with FFPE tissue using either chromogenic or fluorescent detection. We hypothesize that RNAscope will accurately and reproducibly delineate LCR in all non-Hodgkin lymphoma types. We propose to develop an RNAscope-based CISH assay for kappa and lambda mRNAs for LCR detection in FFPE tissue.

Public Health Relevance:
The diagnosis of lymphoma can be difficult based on morphology alone. Detection of restricted immunoglobulin light chain expression is a molecular ancillary tool to resolve ambiguous lymphoproliferative disorders. The proposed product based on RNAscope will provide superior sensitivity to current methods.

Public Health Relevance Statement:
The diagnosis of lymphoma can be difficult based on morphology alone. Detection of restricted immunoglobulin light chain expression is a molecular ancillary tool to resolve ambiguous lymphoproliferative disorders. The proposed product based on RNAscope will provide superior sensitivity to current methods.

NIH Spending Category:
Biotechnology; Cancer; Genetics; Hematology; Lymphoma; Rare Diseases

Project Terms:
Address; Atypical Lymphoproliferative Disorder; B-Cell Neoplasm; B-Cell NonHodgkins Lymphoma; B-Lymphocytes; base; Benign; Biological Assay; Cell Line; Cell surface; Cells; Chromogenic in situ Hybridization; Chronic; Clinical; Clonality; Color; Detection; Development; Diagnosis; Diagnostic; Diagnostics Research; Differential Diagnosis; Evaluable Disease; Evaluation; Extranodal; Flow Cytometry; Follicular Lymphoma; Formalin; Goals; Immunohistochemistry; In Situ Hybridization; Inflammation; kappa-Chain Immunoglobulins; large cell Diffuse non-Hodgkin's lymphoma; Light; Light-Chain Immunoglobulins; lymphoid hyperplasia; Lymphoma; Lymphoproliferative Disorders; Measures; Medicine; meetings; Messenger RNA; Methods; Molecular; Morphology; mucosa-associated lymphoid tissue lymphoma; Non-Hodgkin's Lymphoma; novel; Paraffin Embedding; Pathologic; Phase; Plasma Cell Neoplasm; Prospective Studies; Proteins; Pseudolymphoma; RNA; single molecule; Slide; Small Business Innovation Research Grant; Small-Cell Lymphoma; Specificity; Staining method; Stains; success; Technology; Time; Tissues; tool; Translating; Validation; Variant

Immunoglobulin (Ig) light chain restriction (LCR) detection is an important molecular diagnostic tool in the differential diagnosis that includes lymphoid hyperplasia, atypical lymphoid hyperplasia, chronic inflammation, and B cell neoplasms. LCR can be detected by flow cytometry (FCM) as an abnormal kappa:lambda ratio on the cell surface. However, FCM requires fresh (unfixed) tissue. Some B cell lymphomas lack surface Ig expression detectable by FCM, some demonstrate nonspecific antibody binding. Often only formalin-fixed paraffin-embedded (FFPE) tissue is available for pathologic evaluation. Current methods of LCR identification in FFPE tissue include immunohistochemistry (IHC) and conventional chromogenic in situ hybridization (CISH), which measure kappa or lambda protein and mRNA, respectively. However, both IHC and conventional CISH lack sufficient sensitivity to detect LCR in many non-Hodgkin lymphoma (NHL), including the most common NHL variants - follicular lymphoma, diffuse large B cell lymphoma, small B lymphocytic lymphoma, and extranodal marginal zone lymphoma of MALT type. RNAscope is a novel RNA in situ hybridization technology which has single-molecule detection sensitivity and is fully compatible with FFPE tissue using either chromogenic or fluorescent detection. We have developed a manual bright-field kappa and lambda duplex RNA ISH assay for LCR, which has demonstrated broad applicability to all NHL subtypes. In this project, we will develop a fully automated duplex RNAscope LCR assay with digital pathology interpretation, which should greatly facilitate clinical adoption of this novel assay.

Public Health Relevance Statement:


Public Health Relevance:
The diagnosis of lymphoma can be difficult based on morphology alone. Detection of restricted immunoglobulin light chain expression is a molecular ancillary tool to resolve ambiguous lymphoproliferative disorders. The proposed product is a fully automated RNA in situ hybridization assay for light chain mRNAs based on the RNAscope technology, which will provide superior sensitivity to current methods.

Project Terms:
Address; Adoption; Antibodies; Aspirate substance; Atypical Lymphoproliferative Disorder; Automation; B lymphoid malignancy; B-Cell Lymphomas; B-Cell Neoplasm; B-Cell NonHodgkins Lymphoma; B-Lymphocytes; base; Benign; Binding (Molecular Function); Biological Assay; Biological Markers; Biopsy; Biopsy Specimen; Bone Marrow; Bone marrow biopsy; Cell surface; Cells; Chromogenic in situ Hybridization; Chronic; Clinical; Clonality; cohort; Color; Computer software; cost; Detection; Development; Diagnosis; Diagnostic; Differential Diagnosis; digital; Disease; Evaluable Disease; Evaluation; Extranodal; Flow Cytometry; Follicular Lymphoma; Formalin; Fresh Tissue; Goals; Hyperplasia; Image Analysis; Immunohistochemistry; In Situ Hybridization; Inflammation; instrument; kappa-Chain Immunoglobulins; Laboratories; large cell Diffuse non-Hodgkin's lymphoma; Light; Light-Chain Immunoglobulins; Lymphoid; lymphoid hyperplasia; Lymphoma; Lymphoproliferative Disorders; Manuals; Measures; Medicine; Messenger RNA; Methods; Molecular; Molecular Cytogenetics; Molecular Diagnostic Testing; Morphology; mucosa-associated lymphoid tissue lymphoma; Non-Hodgkin's Lymphoma; novel; novel diagnostics; Paraffin Embedding; Pathologic; Pathologist; Pathology; Performance; Phase; Probability; Procedures; prospective; Proteins; Protocols documentation; public health relevance; Reagent; RNA; Savings; Series; single molecule; Slide; Small Business Innovation Research Grant; Small-Cell Lymphoma; Specimen; Staining method; Stains; success; Surface; Surface Immunoglobulins; Technology; Testing; Time; Tissues; tool; Translational Research; Validation; Variant