- Open Access
Kappa and lambda light chain mRNA in situ hybridization compared to flow cytometry and immunohistochemistry in B cell lymphomas
© Rimsza et al.; licensee BioMed Central Ltd. 2014
- Received: 18 March 2014
- Accepted: 27 June 2014
- Published: 21 July 2014
Detection 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.
The 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.
39/79 (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.
Optimized 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.
The virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1430491067123856
- B cell lymphoma
- In situ hybridization
- Light chains
Detection of B cell clonality has long been a useful tool for identification of monoclonal B cell populations, which are potentially malignant, from polyclonal B cell populations that result during the normal immune response. The copy number range of light chain mRNA and protein expressed by B cells is variable and depends on stage of differentiation. While naive and memory cells may have only 10-100 mRNA copies per cell, plasma cells may have approximately 100,000 mRNA copies per cell. Therefore detection methods need to have a wide dynamic range to be suitable for application in different types of B cell malignancies.
In diagnostic pathology, ISH on histologic sections commonly starts with haptenated probes (labeled with either DNP (2,4-dinitorphenol) or DIG (digoxigenin), followed by an anti-hapten antibody, and an indirect biotin streptavidin system with either alkaline phosphatase or silver deposition for visualization [8;9]. Clinical examples common in diagnostic hematopathology include detection of KAPPA and LAMBDA mRNA, as discussed above, as well as Epstein Barr Virus Encoded RNAs (EBER). New probe design approaches eliminating any repeat segments, as well as the development of novel haptens and chromogens have opened the possibility for more specific staining with greater sensitivity and the detection of multiple probes per slide[8, 9].
Given the advantages of ISH (suitability for fixed tissues, absence of background) as well as technological developments in the field, the current study sought to develop a new methodology known as dual color in situ hybridization (CISH) and to compare its performance in assessing B cell clonality to more commonly employed methods in a series of non-Hodgkin B cell lymphomas. Herein, we describe for the first time this new method and report initial promising results for future clinical application.
Benign tissues included 2 tonsils removed for tonsillitis and 1 with reactive paracortical hyperplasia. B cell non-Hodgkin lymphomas (B-NHL) cases were assembled as follows: Stanford University submitted 19 cases with flow cytometry results, Cleveland Clinic submitted 18 cases with flow cytometry results, the University of Arizona submitted 27 cases with flow cytometry results, and the University College of London submitted 15 cases with IHC results. The case criteria included a diagnosis of mature B-cell non-Hodgkin lymphoma, remaining tumor in the paraffin block, and documented monotypic light chain pattern by flow or IHC. Each site performed flow cytometry according to their own clinical laboratory standards and so was not standardized across the case series. Decalcified samples from bone marrow were not included since the method is not yet optimized for that setting. This project was conducted under a human subjects research ethical approval from the University of Arizona Institutional Review Board (protocol # 0500000226) according to the Declaration of Helsinki.
Probe synthesis and formulation
Novel KAPPA and LAMBDA anti-sense probes, complementary to mRNA encoding each light chain constant region, were chemically labeled with different haptens using Mirus linker arms as directed by the manufacturer (Mirus Bio LLC, Madison, WI). Twenty-five nanograms of each probe was suspended in one mL of Ribohybe™ (cat. 760-104, Ventana Medical Systems) solution and placed into a dispenser.
RNA in situ hybridization and tyramide-chromogen detection
The slides were interpreted by 3 pathologists (SM, TG, LR) without knowledge of the flow cytometry or IHC results. Each pathologist initially read all slides independently without access to each other’s interpretations. Results between pathologists were then compared, followed by discrepancy resolution by joint microscopic review to achieve consensus. After a consensus interpretation was achieved, the CISH results were unblinded and compared to the flow cytometry and IHC results.
Dual color CISH for kappa/lambda mRNA is a new technology with results not before visualized. Positive staining was therefore carefully defined as demonstrating a partial to full ring of punctuate cytoplasmic staining. The signals were small and delicate similar in size to iron particles identified in erythroid precursors using an Prussian Blue stain on bone marrow aspirate smears. In cells at the late stage of B cell differentiation such as plasma or plasmacytic cells, the staining was sometimes heavy enough to obscure the entire cell. Nucleolar staining was considered non-specific. Indeterminate staining was assigned when there was a lack of or minimal staining on the malignant B cells (no rings or partial rings) and the plasma cells in the sample were positively stained or absent. Failed staining was assigned when there was no or minimal staining on malignant B cells (no rings or partial rings) and the plasma cells, which are expected to be strongly stained, were present but unstained.
Total cases: concordant, discordant, indeterminate
Cases with minimum study criteria
Cases with interpretable CISH (excluding indeterminate cases)
Concordant with flow or IHC
Discordant with flow or IHC
Concordance of evaluable cases by lymphoma diagnosis
Diffuse Large B-cell
Small cell B-NHL, NOS
Indeterminate vs evaluable by lymphoma diagnosis
Diffuse Large B Cell
Small B Cell NHL, NOS
Interestingly, 2 cases which were negative for surface Ig by flow cytometry were submitted to the study. Surface Ig negativity is well recognized and considered a sign of monoclonality in the workup of B-NHL, and has been reported in a variety of B cell lymphomas including follicular lymphoma[10–12]. In this study, 2 FL were surface Ig negative by flow cytometry. Both of these cases had "indeterminate" CISH results. Of these, 1 case contained positively stained polyclonal plasma cells (a true SIg negative case by all methods), while the other case did not contain any discernable plasma cells to indicate that staining was or was not successful. This latter case could therefore be considered either a true negative (non-Ig expressing lymphoma) or perhaps a case with poorly preserved mRNA.
When considering the diagnosis of lymphoma in patient biopsies, the monoclonality or polyclonality of the B cell populations is a critical component of the decision making process. While many methods can be used, the most common (flow cytometry) requires fresh tissue, or does not take into account morphologic context (PCR), or is insufficiently sensitive for the majority of B cell NHL (current IHC and ISH methods)[5, 6]. In this study, we describe the feasibility and application of dual-color CISH technology for the evaluation of clonality of mature B cell populations in tissue sections. We demonstrate that with new, repeat-free probes and sensitive detection systems taking advantage of tyramide-induced deposition of chromogens, it is possible to detect not only KAPPA and LAMBDA mRNA levels typical of plasma cells and other post-germinal center B cells but even the very low level mRNA found in presumed naïve B cells in the mantle zones and germinal center B cells. This level of detection is then applicable to the lymphomas derived from most stages of B cell differentiation. The ability to automate the staining and then evaluate the results using light microscopy for slide evaluation holds the potential to easily integrate the CISH technique into routine diagnostic practice.
In this study we describe a preliminary accuracy of 98% as compared to reference methods including flow cytometry and immunohistochemistry. There was only 1 case called KAPPA by CISH and LAMBDA by IHC, which is difficult to explain biologically. However, the explanation could be as simple as a technical or transcriptional error in the original report. Nevertheless, biological complexity does exist in the interpretation of the lambda CISH results due to the presence of non-specific nucleolar staining with the LAMBDA probe seen in several cases. 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. 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. In contrast, those with pink nucleolar staining without cytoplasmic staining were scored as "indeterminate". In at least 2 cases, "indeterminate" is the most accurate interpretation since by flow cytometry these were both considered surface Ig-negative cases.
Dual-color CISH allowed for detection of both KAPPA and LAMBDA mRNA on a single slide under a light microscope, making interpretation of B-cell clonality in tissue sections reliable and highly optimized in the context of tissue architecture and cellular compartments. This application should be of particular interest to pathologists engaged in the field of lymphoma diagnostics. The availability of additional novel haptens and chromogens, which can be paired with highly specific probes, allows for the possibility of additional multi-color combinations and innumerable future applications.
- Segal GH, Shick HE, Tubbs RR, Fishleder AJ, Stoler MH: In situ hybridization analysis of lymphoproliferative disorders. Assessment of clonality by immunoglobulin light-chain messenger RNA expression. Diagn Mol Pathol. 1994, 3: 170-177.PubMedView ArticleGoogle Scholar
- de Tute RM: Flow cytometry and its use in the diagnosis and management of mature lymphoid malignancies. Histopathology. 2011, 58: 90-105.PubMedView ArticleGoogle Scholar
- Tembhare PR, Yuan CM, Venzon D, Braylan R, Korde N, Manasanch E, Zuchlinsky D, Calvo K, Kurlander R, Bhutani M, Tageja N, Maric I, Mulquin M, Roschewski M, Kwok M, Liewehr D, Landgren O, Stetler-Stevenson M: Flow cytometric differentiation of abnormal and normal plasma cells in the bone marrow in patients with multiple myeloma and its precursor diseases. Leuk Res. 2014, 38: 371-376.PubMedPubMed CentralView ArticleGoogle Scholar
- Beck RC, Tubbs RR, Hussein M, Pettay J, Hsi ED: Automated colorimetric in situ hybridization (CISH) detection of immunoglobulin (Ig) light chain mRNA expression in plasma cell (PC) dyscrasias and non-Hodgkin lymphoma. Diagn Mol Pathol. 2003, 12: 14-20.PubMedView ArticleGoogle Scholar
- Magro C, Crowson AN, Porcu P, Nuovo JG: Automated kappa and lambda light chain mRNA expression for the assessment of B-cell clonality in cutaneous B-cell infiltrates: its utility and diagnostic application. J Cutan Pathol. 2003, 30: 504-511.PubMedView ArticleGoogle Scholar
- Rimsza L, Vela E, Richter L, Bellamy W, Grogan T: Assessment of kappa and lambda light chain mRNA in paraffin embedded bone marrow core biopsies and lymph nodes using rapid automated colorimetric in situ hybridization. Lab Invest. 1997, 76: 775-Google Scholar
- van Dongen JJ, Langerak AW, Bruggemann M, Evans PA, Hummel M, Lavender FL, Delabesse E, Davi F, Schuuring E, García-Sanz R, van Krieken JH, Droese J, González D, Bastard C, White HE, Spaargaren M, González M, Parreira A, Smith JL, Morgan GJ, Kneba M, Macintyre EA: Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia. 2003, 17: 2257-2317.PubMedView ArticleGoogle Scholar
- Nitta H, Hauss-Wegrzyniak B, Lehrkamp M, Murillo AE, Gaire F, Farrell M, Walk E, Penault-Llorca F, Kurosumi M, Dietel M, Wang L, Loftus M, Pettay J, Tubbs RR, Grogan TM: Development of automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) for breast carcinomas and an assay performance comparison to manual dual color HER2 fluorescence in situ hybridization (FISH). Diagn Pathol. 2008, 3: 41-PubMedPubMed CentralView ArticleGoogle Scholar
- Nitta H, Zhang W, Kelly BD, Miller M, Pestic-Dragovich L, Bieniarz C, Vasicek TJ, Marafioti T, Rimsza L, Grogan TM: Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models. Methods. 2010, 52: 352-358.PubMedView ArticleGoogle Scholar
- de Martini RM, Turner RR, Boone DC, Lukes RJ, Parker JW: Lymphocyte immunophenotyping of B-cell lymphomas: a flow cytometric analysis of neoplastic and nonneoplastic cells in 271 cases. Clin Immunol Immunopathol. 1988, 49: 365-379.PubMedView ArticleGoogle Scholar
- Gregg EO, Al-Saffar N, Jones DB, Wright DH, Stevenson FK, Smith JL: Immunoglobulin negative follicle centre cell lymphoma. Br J Cancer. 1984, 50: 735-744.PubMedPubMed CentralView ArticleGoogle Scholar
- Ngan B, Warnke A, Cleary ML: Variability of immunoglobulin expression in follicular lymphoma. An immunohistologic and molecular genetic study. Am J Pathol. 1989, 135: 1139-1144.PubMedPubMed CentralGoogle Scholar
- Tubbs RR, Wang H, Wang Z, Minca EC, Portier BP, Gruver AM, Lanigan C, Luo Y, Cook JR, Ma XJ: 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. 2013, 140: 736-746.PubMedView ArticleGoogle Scholar
- Bossy D, Milili M, Zucman J, Thomas G, Fougereau M, Schiff C: Organization and expression of the lambda-like genes that contribute to the mu-psi light chain complex in human pre-B cells. Int Immunol. 1991, 3: 1081-1090.PubMedView ArticleGoogle Scholar
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