Detection of B-lymphocyte Clonality
Lymphoma is one of the most common cancers. Clonal expansion of B-lymphocytes harboring specific immunoglobulin gene rearrangements is a hallmark of B-cell lymphomas, resulting in expression of either immunoglobulin kappa (IgK) or lambda (IgL) light chains in combination with a specific immunoglobulin heavy chain.1 This selective expression of immunoglobulin kappa or lambda is known as “light chain restriction”. Evaluation of a population of lymphocytes for light chain restriction is an important component in the pathologic work-up. Currently, flow cytometric analysis serves as the gold standard for evaluation of kappa and lambda light chain expression.2 However, some B-cell lymphomas lack sufficient surface immunoglobulin expression for detection by flow cytometry. In addition, fresh tissue is not always set aside or available for flow-cytometry. In these cases, evaluation of light chain expression in formalin-fixed paraffin-embedded (FFPE) tissues are required. Several methods including Immunohistochemistry (IHC) and conventional bright field in situ hybridization are available for FFPE tissue evaluation but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells relative to plasma cells.
The RNAscope RNA ISH Platform, including chromogenic detection reagents and target specific probes for kappa and lambda light chain mRNA, provides assessment of light chain restriction in FFPE tissue (Figure 1).
Figure 1. RNAscope ISH Evaluation of kappa and lambda light chain mRNA expression. Case A. Neoplastic B cell population (A) demonstrates kappa light chain restriction with clear IgK ISH expression (B) and negative IgL ISH expression (C). Case B. Sheets of neoplastic B cells (D) demonstrate absence of kappa light chain expression (E) and clear lambda light chain restriction (F).
In some cases, positivity is seen with both Kappa and Lambda RNAscope ISH probes. This “polyclonal” pattern can be reflective of a non-clonal B cell proliferation. However, this pattern can also be seen in approximately 10-20% of B cell lymphomas. A third RNAscope ISH probe for immunoglobulin lambda like peptide 5 (IGLL5) can be employed to resolve the assessment. IGLL5 is expressed during B cell development and in a subset of B cell lymphomas, regardless of clonality. IGLL5 shares some but not all sequences with the IgL constant region gene segments.2,3 The RNAscope ISH probe IGLL5 targets those sequences unique to the IGLL5 gene, leading to no cross-hybridization with IgL mRNA. However, the Lambda probe targets a region of the IgL gene, the constant region, that overlaps with the IGLL5 gene and therefore has the potential to cross-hybridize with IGLL5 mRNA if present. As such, apparent co-expression of IgK and IgL with the RNAscope ISH Kappa and Lambda probes can actually be due to the detection of IGLL5 expression by the IgL probe rather than expression of IgL.
Combining Kappa and Lambda target probes with a third probe for IGLL5, an immunoglobulin-like protein that is expressed in lymphoid and non-lymphoid cells, creates additional clarity for potential confusion due to IGLL5 expression – something that is unavailable in IHC. The use of RNAscope ISH Probe Kappa, Lambda, and IGLL5 can help detect B-cell clonality in multiple subtypes of B-cell lymphoma.
Product Information
| Cat. No. | Product Name | Regulatory Status |
|---|---|---|
| 201428 | RNAscope ISH Probe Kappa | ASR |
| 201438 | RNAscope ISH Probe Lambda | ASR |
| 201448 | RNAscope ISH Probe IGLL5 | ASR |
Analyte Specific Reagents* (ASRs) are critical building blocks for Laboratory Developed Tests (LDTs) and play a key role in ensuring the accuracy and reliability of test results. By using ASRs, Clinical Laboratory Improvement Amendments (CLIA) certified labs can develop customized tests that are tailored to the specific needs of their patient population, while maintaining high standards of analytical and clinical performance.
In compliance with FDA regulations, ASRs may only be sold to in vitro diagnostic manufacturers, and clinical laboratories regulated under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), as qualified to perform high complexity testing under 42 CFR part 483 or under VHA directive 1106.
References:
Tubbs, R. R., H. Wang, et al. (2013). "Ultrasensitive RNA in situ hybridization for detection of restricted clonal expression of low-abundance immunoglobulin light chain mRNA in B-cell lymphoproliferative disorders." Am J Clin Pathol 140(5): 736-46.
Guo, L., Z. Wang, et al. (2018). "Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry." Mod Pathol 31(3): 385-394.
Warford, A., M. Rahman, et al. (2019) "Pushing the boundaries of in situ hybridisation for mRNA demonstration: demonstration of kappa and lambda light chain restriction in follicular lymphoma." Br J Biomed Sci 76(3): 143-146.
* Analyte Specific Reagent. Analytical and performance characteristics are not established.
