Use of immune repertoire sequencing to resolve discordant microscopic and immunochemical findings in a case of T cell-rich large B cell lymphoma in a young dog

Abstract

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.

Research progress on application of single-cell TCR/BCR sequencing technology to the tumor immune microenvironment, autoimmune diseases, and infectious diseases

“…IR sequencing (IR-seq) targets T and B lymphocytes. Multiplex PCR or 5’-rapid amplification of cDNA (complementary Deoxyribonucleic acid) ends was used to amplify the complementarity-determining region (CDR) that determines the diversity of TCR or BCR, combined with high-throughput sequencing technology, to comprehensively assess the diversity of the immune system and explore the relationship between the IR and disease ( 3 ). IR-seq technology mainly includes single-cell TCR and BCR sequencing; a schematic illustration is shown in Figure 1 .…”

Doing more with less: multiple uses of a single slide in veterinary cytology. A practical approach

“…More recent technical developments in molecular biology , such as next generation sequencing, are already being applied in the veterinary field (Lee et al 2021 ). These advances may increase the precision of existing molecular based methods, such as PARR, or may allow the development of novel diagnostic procedures, like microRNAs collected by FNA.…”

Development and application of a next-generation sequencing protocol and bioinformatics pipeline for the comprehensive analysis of the canine immunoglobulin repertoire

“…Another study combined a 5' RACE protocol with traditional Sanger sequencing to characterize the IGH repertoire in three healthy dogs [24]. A more recent study used a targeted PCR approach coupled with NGS to evaluate the IGH, TRB, and TRG loci in a single dog with T cell-rich large B cell lymphoma [25]. However, to the best of our knowledge, there have been no published studies to date examining the entire BCR repertoire including IGH, IGK, and IGL chains using an unbiased high-throughput sequencing approach in populations of dogs.…”