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Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma
Published on: January 9, 2019
Immunoglobulin light chain transcript detection by ultrasensitive RNA in situ hybridization for B-cell lymphoma
Luisa Lorenzi1,2, Silvia Lonardi3, Michela Bonezzi3
1Pathology Unit, Department of Molecular and Translational Medicine-DMMT, University of Brescia, Brescia, Italy. lorenziluisa@gmail.com.
Insights
B-cell clonality testing using IgLC-RNAscope (RNAsc) shows superior performance for diagnosing lymphomas, especially in challenging cases with limited tumor cells. This method offers a powerful in situ diagnostic tool for B-cell lymphomas.
Area of Science:
- Hematopathology
- Molecular Pathology
- Oncology
Background:
- Assessing B-cell clonality in tissue sections is crucial but challenging for diagnosing lymphoid infiltrates.
- Current gold standard, IG gene rearrangements by PCR (IG-PCR), and flow cytometry (FC) have limitations in accessibility and sample requirements.
- Immunohistochemistry (IHC) and in situ hybridization (ISH) are alternatives but can be less sensitive, especially with limited tumor cells.
Purpose of the Study:
- To evaluate the diagnostic performance of B-cell clonality detection using IgLC-RNAscope (RNAsc) on formalin-fixed, paraffin-embedded (FFPE) tissue samples.
- To compare RNAsc performance against established methods like FC, IHC, and ISH, particularly in challenging cases.
Main Methods:
- Analyzed 216 FFPE samples: 185 non-Hodgkin lymphomas, 11 Hodgkin lymphomas (HL), and 20 reactive samples.
- Performed IgLC-RNAsc in parallel with FC on 53 cases.
- Compared RNAsc results with IHC, ISH, and IG-PCR where applicable, focusing on sensitivity in limited tumor cell content scenarios.
Main Results:
- IgLC-RNAsc demonstrated higher overall performance (93%) compared to FC (83%), with significant improvements in diffuse large B-cell lymphoma (98% vs 71%) and follicular lymphoma (93% vs 83%).
- RNAsc outperformed IHC and ISH in samples with limited tumor cells where IG-PCR was uninformative.
- First-time application on mediastinal lymphomas revealed monotypic IgLC transcripts in 69% of primary mediastinal large B-cell lymphoma (PMBCL) and 67% of mediastinal gray zone lymphomas (MGZL).
Conclusions:
- IgLC-RNAsc is a powerful and sensitive tool for B-cell lymphoma diagnosis, especially in difficult cases with scarce tumor cells.
- The method provides valuable in situ insights into immunoglobulin light chain regulation across various lymphoma entities.
- RNAsc offers a viable alternative to molecular and flow cytometry methods, enhancing diagnostic capabilities in routine pathology settings.
Abstract:
Evaluation of B-cell clonality can be challenging in the interpretation of lymphoid infiltrates on tissue sections. Clonality testing based on IG gene rearrangements analysis by PCR (IG-PCR) is the gold standard. Alternatively, B-cell clonality can be assessed by the recognition of immunoglobulin light chain (IgLC) restriction, by immunohistochemistry (IHC), chromogenic in situ hybridization (ISH) or flow cytometry (FC). IG-PCR requires molecular facilities, and FC requires cell suspensions, both not widely available in routine pathology units. This study evaluates the performance of B-cell clonality detection by IgLC-RNAscope® (RNAsc) in a group of 216 formalin-fixed, paraffin-embedded samples including 185 non-Hodgkin B-cell lymphomas, 11 Hodgkin lymphomas (HL) and 20 reactive samples. IgLC-RNAsc, performed in parallel with FC in 53 cases, demonstrated better performances (93% vs 83%), particularly in diffuse large B-cell lymphoma (98% vs 71%) and follicular lymphoma (93% vs 83%) diagnosis. IgLC-RNAsc was also superior to IHC and ISH especially in samples with limited tumor cell content, where IG-PCR was not informative. Performed for the first time on mediastinal lymphomas, IgLC-RNAsc identified monotypic IgLC transcripts in 69% of primary mediastinal large B-cell lymphoma (PMBCL) and 67% of mediastinal gray zone lymphomas (MGZL). IGK/L double-negative cells were detected in 1 PMBCL, 2 MGZL, and all classical HL, while monotypic IgLC expression appeared to be a hallmark in nodular lymphocyte-predominant HL. IgLC-RNAsc demonstrates to be a powerful tool in B-cell lymphoma diagnosis, above all in challenging cases with limited tumor cell content, ensuring in situ investigations on mechanisms of Ig regulation across lymphoma entities.
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