Combined BIOMED-2 Clonality Assays for IGH and IGK and Assessment of t(14;18) on Formalin-Fixed, Paraffin Embedded Tissues Effectively Separates Primary Cutaneous B Cell Lymphomas from Reactive Cutaneous Lymphoid Hyperplasia.

Primary cutaneous B-cell lymphomas (PCBCL) are a diverse group of lymphomas that are limited to the skin at the time of diagnosis. Clonally rearranged immunoglobulin (Ig) genes are a key feature of B-cell malignancies. While PCR for Ig rearrangement is well-established and characterized in nodal malignancies, PCR investigations of Ig clonality in PCBCL have been unstandardized and extremely inconsistent. In this study, we analyzed 27 cases of PCBCL (22 cases of PCBCL diagnosed at initial presentation and 5 cases of atypical lymphoid infiltrates (ALI)) for Ig clonality using standardized BIOMED-2 PCR protocols (InVivoScribe Technologies, San Diego, CA). We defined lesions of ALI as those that were clinically suspicious for lymphoma, but could not be confirmed as lymphoma by morphologic and/or immunohistochemical methods. These patients were followed clinically where an eventual diagnosis of lymphoma was made by morphologic and/or immunohistochemical methods. Our primary objective was to evaluate the sensitivity and specificity of the BIOMED-2 method for detecting clonality in PCBCL, including cases initially diagnosed as ALI. Our secondary objective was to assess the role of clonality in the diagnosis of PCBCL. 47 biopsies from 27 cases of PCBCL (7 T1aN0M0, 4 T2aN0M0, 3 T2bN0M0, 3 T3aN0M0, and 10 T3bN0M0) were tested for IGH and IGK gene rearrangements and the (14;18) translocation using BIOMED-2 primers. Select cases from patients with multiple biopsies were further analyzed by sequencing to identify identical clones. We found a clone in 24/27 (89%) cases overall (10/11, 91% marginal zone lymphoma (MZL); 10/11, 91% follicle center lymphoma (FCL); and 4/5 ALI)). Among the 47 biopsies, a clone was detected in 35/47 (75%) cases (13/19, 68% MZL; 15/19, 79% FCL; and 7/9, 78% ALI). We found that clonal detection was improved in cases with multiple biopsies. The t(14;18) translocation was present in 3/11 FCL cases and 1/5 ALI cases, but not detected in any biopsy from MZL cases. Addition of IGK analysis to IGH analysis alone increased sensitivity of clonal detection from 57% to 75%. No clonality was detected in 9 cases of reactive cutaneous lymphoid hyperplasia. While our study is small, the initial results demonstrate that

1. the BIOMED-2 protocol shows high sensitivity and specificity in clonality detection in PCBCL

2. the (14;18) translocation is rare in PCBCL in general, but can be detected in a minority of cases of FCL

3. adding IGK primers to the analysis for clonality may increase its sensitivity over detection of IGH rearrangements alone

4. clonality detection may lead to earlier diagnosis of PCBCL in cases otherwise thought to be ALI

5. multiple biopsies can lead to a more definitive diagnosis of PCBCL with increased likelihood of clonal detection and

6. formalin-fixed paraffin embedded tissues can be used very effectively to establish clonality in PCBCL.

To our knowledge, this is the first report of a standardized approach for the detection of clonality in PCBCL. We propose a new algorithm in the diagnosis of PCBCL, where clonality assays are implemented when a definitive diagnosis cannot be made on clinical, morphologic, and/or immunophenotypic grounds. This novel characterization of PCBCL using archival tissue and the BIOMED-2 assay is a powerful strategy for clonality assessment and validation of the diagnosis of PCBCL.