BACKGROUND: Lymphocytic neoplasms with frequent reactive lymphocytes are uncommonly reported in dogs, and can pose a diagnostic challenge. Different diagnostic modalities such as cytology, flow cytometry, histopathology, immunohistochemistry, and clonality testing, are sometimes required for a diagnosis. This report illustrates the value of using a multi-modal diagnostic approach to decipher a complex lymphocytic tumor, and introduces immune repertoire sequencing as a diagnostic adjunct. CASE PRESENTATION: A 10-month-old Great Dane was referred for marked ascites. Cytologic analysis of abdominal fluid and hepatic aspirates revealed a mixed lymphocyte population including numerous large lymphocytes, yielding a diagnosis of lymphoma. Flow cytometrically, abdominal fluid lymphocytes were highly positive for CD4, CD5, CD18, CD45, and MHC II, consistent with T cell lymphoma. Due to a rapidly deteriorating clinical condition, the dog was euthanized. Post mortem histologic evaluation showed effacement of the liver by aggregates of B cells surrounded by T cells, suggestive of hepatic T cell-rich large B cell lymphoma. Immune repertoire sequencing confirmed the presence of clonal B cells in the liver but not the abdominal fluid, whereas reactive T cells with shared, polyclonal immune repertoires were found in both locations. CONCLUSIONS: T cell-rich large B cell lymphoma is a rare neoplasm in dogs that may be challenging to diagnose and classify due to mixed lymphocyte populations. In this case, the results of histopathology, immunohistochemistry and immune repertoire sequencing were most consistent with a hepatic B cell neoplasm and reactive T cells exfoliating into the abdominal fluid. Immune repertoire sequencing was helpful in delineating neoplastic from reactive lymphocytes and characterizing repertoire overlap in both compartments. The potential pitfalls of equating atypical cytomorphology and monotypic marker expression in neoplasia are highlighted.
- Klíčová slova
- Canine, Clonality, Flow cytometry, Immunocytochemistry, Immunohistochemistry, Liver, Neoplasia, Next-generation sequencing, PCR for antigen receptor gene rearrangements, TCRBL,
- MeSH
- ascites veterinární MeSH
- CD antigeny MeSH
- difúzní velkobuněčný B-lymfom diagnóza imunologie veterinární MeSH
- eutanazie u zvířat MeSH
- imunofenotypizace veterinární MeSH
- imunohistochemie veterinární MeSH
- nádory jater veterinární MeSH
- nemoci psů diagnóza patologie MeSH
- podskupiny lymfocytů patologie MeSH
- průtoková cytometrie veterinární MeSH
- psi MeSH
- T-lymfocyty patologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- Názvy látek
- CD antigeny MeSH
BACKGROUND: Evolution of indolent to aggressive lymphoma has been described in dogs but is difficult to distinguish from the de novo development of a second, clonally distinct lymphoma. Differentiation of these scenarios can be aided by next generation sequencing (NGS)-based assessment of clonality of lymphocyte antigen receptor genes. CASE PRESENTATION: An 8-year-old male intact Mastiff presented with generalized lymphadenomegaly was diagnosed with nodal T zone lymphoma (TZL) based on cytology, histopathology, immunohistochemistry and flow cytometry. Thirteen months later, the dog re-presented with progressive lymphadenomegaly, and based on cytology and flow cytometry, a large B cell lymphoma (LBCL) was diagnosed. Sequencing-based clonality testing confirmed the de novo development of a LBCL and the persistence of a TZL. CONCLUSIONS: The occurrence of two distinct lymphoid neoplasms should be considered if patient features and tumor cytomorphology or immunophenotype differ among sequential samples. Sequencing-based clonality testing may provide conclusive evidence of two concurrent and distinct clonal lymphocyte populations, termed most appropriately "composite lymphoma".
- Klíčová slova
- Antigen receptor gene rearrangement, Canine, Clonality, Composite lymphoma, Dog, Lymphoma, Lymphosarcoma, PARR,
- MeSH
- antitumorózní látky alkylující aplikace a dávkování terapeutické užití MeSH
- antitumorózní látky hormonální aplikace a dávkování terapeutické užití MeSH
- chlorambucil aplikace a dávkování terapeutické užití MeSH
- difúzní velkobuněčný B-lymfom komplikace patologie veterinární MeSH
- lymfom T-buněčný komplikace patologie veterinární MeSH
- nemoci psů patologie MeSH
- prednison aplikace a dávkování terapeutické užití MeSH
- psi MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- Názvy látek
- antitumorózní látky alkylující MeSH
- antitumorózní látky hormonální MeSH
- chlorambucil MeSH
- prednison MeSH
Several molecular clonality assays have been developed to assess canine B cell proliferations. These assays were based on different sequence data, utilized different assay designs and employed different testing strategies. This has resulted in a complex body of literature and complicates evidence-based selection of primer sets. In addition, further refinement of primer sets is difficult because it is unknown how well current primer sets cover the expressed sequence repertoire. The objectives of this study were 1) to provide an overview of published IGH clonality assays that highlights key differences in assay design and testing strategy and 2) to propose a novel method for optimizing primer sets that leverages large-scale sequencing data. A review of previously published assays highlighted confounding factors that hamper a direct comparison of performance metrics between studies. These findings illustrate the need for a multi-institutional effort to harmonize veterinary clonality testing. A novel in silico analysis of primer sequences using a large dataset of expressed sequences identified shortfalls of existing primer sets and was used to guide primer optimization. Three optimized primer sets were tested and yielded qualitative sensitivity values between 80-90%. The qualitative sensitivity ranged from 1% to over 50% and was dependent on the size of the neoplastic clone and the sample DNA used. These findings illustrate that inclusion of high-throughput sequencing data for primer design can be a useful tool to guide primer design and optimization. This strategy could be applied to other antigen receptor loci or species to further improve veterinary clonality assays.
- Klíčová slova
- B cell lymphoma, Clonality testing, Dog, Lymphoma, PARR, Sensitivity, Specificity,
- MeSH
- B-lymfocyty cytologie MeSH
- buněčné klony * MeSH
- DNA primery * MeSH
- psi genetika imunologie MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- zvířata MeSH
- Check Tag
- psi genetika imunologie MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- DNA primery * MeSH
- těžké řetězce imunoglobulinů MeSH
The ability to mount adaptive immune responses to a diverse array of pathogens is essential to maintaining the health of an individual. The outcome of adaptive immune responses is influenced by the pool of available lymphocyte antigen receptors. Understanding the composition and dynamics of immune repertoires is hence of relevance to characterizing physiologic immunological processes as well as understanding disease pathogenesis. The dog is increasingly recognized as a model for human disease. The objective of this study was to utilize NGS for comprehensive and unbiased analysis of the IGH repertoire in healthy dogs. First, the IGH locus was searched in silico for previously unidentified genes. Second, IGH transcripts from major lymphoid organs were amplified using a 5'RACE approach without V/J primer bias. Third, amplicons were sequenced on an Illumina MiSeq platform, and data were analyzed using the ARResT/Interrogate platform. Data analysis included V/J usage, V-J pairing biases, isotype frequency, CDR3 diversity, convergent recombination, and public repertoires. The results of this study provide a comprehensive IGH repertoire analysis for healthy dogs. These data will allow further improvement of V/J gene-specific primer sets and will serve as baseline for future studies investigating immune repertoires in health and disease.
- Klíčová slova
- Antigen receptor, CDR3, Dog, IGH, Rearrangement, Repertoire analysis,
- MeSH
- imunoglobulinové izotypy genetika MeSH
- psi MeSH
- receptory antigenů genetika MeSH
- sekvenční analýza DNA MeSH
- těžké řetězce imunoglobulinů genetika MeSH
- variabilní oblast imunoglobulinu genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- imunoglobulinové izotypy MeSH
- receptory antigenů MeSH
- těžké řetězce imunoglobulinů MeSH
- variabilní oblast imunoglobulinu MeSH
