Clonality Testing in Veterinary Medicine

Abstract

The accurate distinction of reactive and neoplastic lymphoid proliferations can present challenges. Given the different prognoses and treatment strategies, a correct diagnosis is crucial. Molecular clonality assays assess rearranged lymphocyte antigen receptor gene diversity and can help differentiate reactive from neoplastic lymphoid proliferations. Molecular clonality assays are commonly used to assess atypical, mixed, or mature lymphoid proliferations; small tissue fragments that lack architecture; and fluid samples. In addition, clonality testing can be utilized to track neoplastic clones over time or across anatomic sites. Molecular clonality assays are not stand-alone tests but useful adjuncts that follow clinical, morphologic, and immunophenotypic assessment. Even though clonality testing provides valuable information in a variety of situations, the complexities and pitfalls of this method, as well as its dependency on the experience of the interpreter, are often understated. In addition, a lack of standardized terminology, laboratory practices, and interpretational guidelines hinders the reproducibility of clonality testing across laboratories in veterinary medicine. The objectives of this review are twofold. First, the review is intended to familiarize the diagnostic pathologist or interested clinician with the concepts, potential pitfalls, and limitations of clonality testing. Second, the review strives to provide a basis for future harmonization of clonality testing in veterinary medicine by providing diagnostic guidelines.

Optimisation and validation of a PCR for antigen receptor rearrangement (PARR) assay to detect clonality in canine lymphoid malignancies

“…Using this definition all ‘dominant peak only’ samples in this series would have been classified as clonal, increasing sensitivity to 97.6% but with a concomitant drop in specificity to 84.3%. These results lead us to concur with the recommendation made in a recent review of PARR (Keller et al, 2016), which concludes that mathematical algorithms such as this cannot be used to aid interpretation of PARR results.…”

“…Although rearrangement of IgH has been documented in human and canine acute myeloid leukaemia (Cheng et al, 1986, Kyoda et al, 1997), it is possible that the clonal products in this sample resulted from sampling error, since repeated testing of fresh samples gave polyclonal results (Elenitoba-Johnson et al, 2000, Bienzle and Vernau, 2011, Langerak et al, 2012). For cost reasons, we do not routinely perform testing of samples in duplicate as has been recommended (Keller et al, 2016) but where pseudoclonality is suspected or PARR gives unexpected results repeat testing of the same or additional samples should be performed.…”

Dogs with Acute Myeloid Leukemia Have Clonal Rearrangements in T and B Cell Receptors

“…This result indicates that clonality testing should not be relied upon for distinguishing between AML and lymphoid neoplasms, such as ALL or lymphoma with a secondary leukemia, as previously recommended (2). Recently, Keller and colleagues have also advocated against the use of clonality testing for distinguishing B from T cell neoplasms (1). However, in many cases of lymphoid neoplasia, the tumor shows fidelity between clonality and other phenotyping tests (IHC or flow cytometry) (2, 6, 20), and clonality assessment may still be useful, at the very least for confirming neoplasia, in sites where sufficient cells cannot be retrieved for other phenotyping techniques or invasive diagnostic procedures are contraindicated (12).…”

“…Testing is accomplished via polymerase chain reactions using primers designed to amplify the complementarity-determining region 3 (CDR3) of immunoglobulin chains (usually the heavy chain or IgH) of B cells (B cell receptor) and the T cell gamma receptor (1, 2). Thus, clonality testing is known colloquially as polymerase testing for antigen receptor rearrangements (2).…”

“…Amplified products have been traditionally detected using ethidium bromide and agarose gel electrophoresis (3, 4); however, higher resolution techniques, such as capillary gel electrophoresis, are supplanting this older method (1, 2, 5–7). Clonality testing is primarily used to distinguish between neoplastic and reactive lymphocyte expansions (1, 2, 6–8); however, this testing is also used as a means to phenotype lymphoid neoplasms as B or T in origin, particularly with tumors showing expression of more than one lineage with flow cytometry or immunohistochemical (IHC) staining (2, 5, 6, 8–13). The use of clonality as a phenotyping tool is being extended to myeloid neoplasms, where clonality testing has been used as a means to distinguish between acute myeloid leukemia (AML) and lymphoid neoplasms (lymphoma or leukemia) (6, 14, 15).…”