Applied Protein and Molecular Techniques for Characterization of B Cell Neoplasms in Horses

Abstract

Mature B cell neoplasms cover a spectrum of diseases involving lymphoid tissues (lymphoma) or blood (leukemia), with an overlap between these two presentations. Previous studies describing equine lymphoid neoplasias have not included analyses of clonality using molecular techniques. The objective of this study was to use molecular techniques to advance the classification of B cell lymphoproliferative diseases in five adult equine patients with a rare condition of monoclonal gammopathy, B cell leukemia, and concurrent lymphadenopathy (lymphoma/leukemia). The B cell neoplasms were phenotypically characterized by gene and cell surface molecule expression, secreted immunoglobulin (Ig) isotype concentrations, Ig heavy-chain variable (IGHV) region domain sequencing, and spectratyping. All five patients had hyperglobulinemia due to IgG1 or IgG4/7 monoclonal gammopathy. Peripheral blood leukocyte immunophenotyping revealed high proportions of IgG1-or IgG4/7-positive cells and relative T cell lymphopenia. Most leukemic cells lacked the surface B cell markers CD19 and CD21. IGHG1 or IGHG4/7 gene expression was consistent with surface protein expression, and secreted isotype and Ig spectratyping revealed one dominant monoclonal peak. The mRNA expression of the B cell-associated developmental genes EBF1, PAX5, and CD19 was high compared to that of the plasma cell-associated marker CD38. Sequence analysis of the IGHV domain of leukemic cells revealed mutated Igs. In conclusion, the protein and molecular techniques used in this study identified neoplastic cells compatible with a developmental transition between B cell and plasma cell stages, and they can be used for the classification of equine B cell lymphoproliferative disease.

PCRfor antigen receptor rearrangement (PARR) clonality testing in a horse with a solitary retropharyngeal lymphoma

“…Recently, PARR has been assessed using molecular techniques in horses with a rare condition of monoclonal gammopathy, B cell leukaemia and concurrent lymphadenopathy (lymphoma/leukaemia) (Badial et al . ). Additionally, PARR has been used to successfully identify diffuse intermediate to large B cell lymphoma with a robust haematogenous phase (TCRLBCL) in both blood and infiltrative tissue from a horse with multicentric disease (Meichner et al .…”

“…Recently, clonality has been assessed using molecular techniques in horses with leukaemia and concurrent lymphadenopathy (Badial et al . ). The current report represents one of only few reported cases of a solitary lymphoid tumour in a well‐fleshed horse with no evidence of multicentric disease, and the first documented use of polymerase chain reaction for analysis of antigen receptor rearrangement (PARR) in equine solitary extranodal lymphoma.…”

Developmental expression of B cell molecules in equine lymphoid tissues

“…Though murine B2 cells use lambda light chains in about 5% of Igs, peritoneal B1 cells have been shown to use lambda light chains in 20% of Igs (Hayakawa et al, 1986). Therefore, absolute quantification was performed for IGL and IGK, and RNA standard curves were prepared for each of these genes (Badial et al, 2015) to determine whether equine CD5 hi and CD5 lo B cells use similar ratios of IGL and IGK light chains. A region that included the SYBR primers was amplified from whole peripheral blood cDNA (Table 1) with iProof polymerase (iProof™ High-Fidelity PCR kit, Bio-Rad) for IGL or with 5′-rapid amplification of cDNA ends (RACE, SMARTer™ RACE cDNA Amplification Kit, Clontech, Mountain View, CA) for IGK according to manufacturer’s instructions (Badial et al, 2015) cloned into pJET1.2 vector (CloneJET™ PCR Cloning Kit, Thermo Fisher Scientific), and sequenced at the Cornell University Institute of Biotechnology (Ithaca, NY).…”

“… a (Tallmadge et al, 2012) b (Badial et al, 2015) c (Sanchez-Matamoros et al, 2013) d SMARTer™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA) …”

PCR for antigen receptor rearrangement (PARR) in equine veterinary medicine

“…; Badial et al . ). However, PARR has a major advantage over flow cytometry in that PARR can be performed on any cell, dead or alive; it can be performed on cytological samples, effusions and formalin‐fixed paraffin‐embedded tissue, and has a high specificity so may be an acceptable assay if fresh samples are not available (Thalheim et al .…”