Assessment of Lymphoid Molecular Clonality in Canine Thymoma

J. Comp. Path. 2018, Vol. 158, 66e70

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NEOPLASTIC DISEASE

Assessment of Lymphoid Molecular Clonality in Canine Thymoma F. Vessieres*,x, R. Rasotto*, I. Peters†, E. Villiers‡, D. Berlato* and F. Cian* * Animal Health Trust, Lanwades Park, Kentford, Newmarket, † TDDS Laboratory, Unit G, The Innovation Centre, University of Exeter, Rennes Drive, Exeter, ‡ Dick White Referrals, Station Farm, London Road, Six Mile Bottom, Cambridgeshire and x Anderson Moores Veterinary Specialists, The Granary, Bunstead Barns, Poles Lane, SO21 2LL Hursley, Hampshire, UK

Summary The aim of this study was to document the molecular clonality of lymphoid cells in canine thymoma using polymerase chain reaction for antigen receptor rearrangement (PARR). Fifteen formalin-fixed and paraffin wax-embedded samples of canine thymoma were analyzed for T- and B-cell receptor clonality. Two of these 15 cases were excluded from the study due to insufficient DNA concentration. Twelve of the 13 remaining samples (92.3%) showed a polyclonal lymphoid component and in one case the lymphoid component was monoclonal (T-cell clonality). PARR could therefore be a useful tool for differentiating canine thymoma from canine mediastinal lymphoma. Ó 2017 Elsevier Ltd. All rights reserved. Keywords: clonality; dog; polymerase chain reaction for antigen receptor rearrangement; thymoma

Thymoma and lymphoma are the most common causes of mediastinal masses in dogs (Yoon et al., 2004). Thymoma is a neoplasm originating from the epithelial cells of the thymus that are admixed with non-neoplastic lymphoid cells, while lymphoma is characterized by a neoplastic proliferation of lymphoid cells (De Mello Souza, 2013). In dogs, these two tumors have different prognoses and treatment options and being able to differentiate them has a major clinical relevance (De Mello Souza, 2013). Cytological samples from mediastinal masses may be difficult to interpret and the reported cytological accuracy for the diagnosis of mediastinal lesions in dogs varies from 40% to 80% (Atwater et al., 1994; Pintore et al., 2014). Histopathology is considered more reliable for the diagnosis of mediastinal lesions,

Correspondence to: andersonmoores.com).

F.

Vessieres

0021-9975/$ - see front matter https://doi.org/10.1016/j.jcpa.2017.12.001

(e-mail:

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although a definitive diagnosis may be challenging in cases of thymoma with few epithelial cells. Polymerase chain reaction for antigen receptor rearrangement (PARR) has recently become widely used for the diagnosis of lymphoma in dogs and cats (Burnett et al., 2003) and may also help in the distinction between mediastinal lymphoma and thymoma. This technique detects and amplifies via polymerase chain reaction (PCR) genes encoding particular chains of the immunoglobulin (for B cells) or T-cell receptor (for T cells) molecules in the lymphoid population of a sample. The resulting PCR products are analyzed by capillary gel electrophoresis. A clonal sample is reported if one or several discrete bands of appropriate DNA size are seen on the gel and distinct fluorescent peaks are seen on an electropheretogram. A polyclonal sample is reported if either no or multiple bands or peaks are seen. Lymphoid cells in lymphoma are typically monoclonal as they are derived from the same precursor cell (Burnett et al., 2003); Ó 2017 Elsevier Ltd. All rights reserved.

Lymphoid Clonality in Canine Thymoma

however, there is limited information about the clonality of the lymphoid component of canine thymoma. The aim of the present study was to evaluate the molecular clonality of the lymphoid component in canine thymoma, primarily assessing whether PARR performed on biopsy samples could be used routinely to distinguish this neoplasm from mediastinal lymphoma. Computerized databases from two referral centers (Animal Health Trust, Newmarket and Dick White Referrals, Six Mile Bottom, UK) were searched for confirmed cases of canine thymoma diagnosed by histopathology. A board-certified veterinary pathologist reviewed all of the samples, with immunohistochemistry for cytokeratin requested as needed (using monoclonal mouse anti-cytokeratin antibody; Dako, Glostrup, Denmark, catalogue number MNF116; dilution 1 in 200). DNA was isolated from sections taken from the paraffin wax-embedded tissue (DNeasy Blood and Tissue KitÒ, Qiagen, Manchester, UK) as per the manufacturer’s instructions, with minor modifications. The sections were added to a 2 ml tube (Safe-Lock Tube, Eppendorf, Stevenage, UK) and the wax was removed by heating the sections to 70 C for 10 min in 400 ml of ATL buffer (from the extraction kit). The samples were then centrifuged at 11,863 g for 2 min and the solidified wax ring was removed from the top of the ATL using a pipette tip. Proteinase K (40 ml) was added to the tube and the samples were incubated at 56 C with constant shaking (800 rotations per minute) in an incubator (Vortemp 56 Shaking Incubator, Labnet International, Edison, New Jersey, USA) before completion of the tissue extraction protocol. The resulting DNA was eluted in 100 ml of elution buffer before being stored at 20 C prior to analysis. The concentration of DNA was measured (Qubit dsDNA BR Assay Kit, Invitrogen, Paisley, UK). Samples showing insufficient DNA concentration to allow addition of 50 ng of DNA were excluded from the study, as the low quantity of DNA may have decreased the assay sensitivity. Samples with marginal, good and high DNA concentration were included in the study and the DNA concentration was recorded. The presence of amplifiable DNA was confirmed by performing a PCR for the succinate dehydrogenase (SDHA) gene (Peters et al., 2007) and for the presence of PCR inhibitors using an exogenous internal control (internal amplification control [IAC]) assay (Nolan et al., 2006) by real-time PCR as described previously, using a master mix (GoTaq G2 Hot Start Colorless Master Mix, Promega, Southampton, UK) in a specific instrument (Stratagene MX-3005P, Agilent Technologies, Stockport, UK). Samples that failed to have amplifi-

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able DNA with the SDHA assay were excluded from the study. Samples that had a negative canine DNA control (IAC), but for which the quantity of DNA extracted and measured was considered suitable for analysis, were not excluded, as this could have been due to presence of pigments acting as inhibitors. If PCR inhibitors were identified using the IAC assay, 1 mg of bovine serum albumin (BSA) was added to the PCR reaction mixes for the SDHA and IAC assays, and the analysis was repeated. In samples for which inhibition was not evident with reanalysis, an equivalent amount of BSA was added to the PCR reactions for the clonality analysis. PARR for the B-cell immunoglobulin heavy chain locus (IgH) and T-cell receptor gamma (TCRg) locus was performed using methods described previously (Vernau and Moore, 1999; Burnett et al., 2003; Keller and Moore, 2012). The resulting products were analyzed by capillary gel electrophoresis using specific software (QIAxcel Advanced System, QIAxcel ScreenGel Software, Version 1.4, Qiagen) and represented as electrophoretograms. Results were deemed clonal when there were identical, discrete electrophoretogram peaks, which were a minimum of twice the height of the other amplified products, present in both reaction repeats (Keller and Moore, 2012). As several primers were used, both polyclonal and clonal results were expected to express several peaks. The relative heights of the peaks was therefore the main criteria used to distinguish between clonal and polyclonal samples. Polyclonal controls were obtained from fresh thymic and lymph node tissue obtained post mortem from a juvenile dog without evidence of lymphoproliferative disease. The positive control material was obtained from a peripheral blood sample obtained from a leukemic dog that had previously yielded clonal results (Fig. 1). Fifteen samples were selected for the study, including four tru-cut and 11 intraoperative excisional biopsy samples. Breed, sex, age and type of sample are summarized in Table 1. The age of dogs with thymoma was 8.5  1.5 years (mean  SD). The represented breeds included Labrador retriever (n ¼ 4), golden retriever (n ¼ 2), flat coated retriever (n ¼ 2), German shepherd dog (n ¼ 2), Rhodesian ridgeback (n ¼ 1), Lhasa Apso (n ¼ 1), Jack Russell terrier (n ¼ 1) and crossbred (n ¼ 2). After reviewing the samples, the original diagnosis of thymoma was confirmed for all of the 15 samples. Cytokeratin IHC was performed for three samples. DNA was extracted and measured for 15 samples. Two of these samples (both obtained by tru-cut biopsy) showed insufficient DNA concentration and were therefore excluded from the study.

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Fig. 1. Electrophoretogram showing a monoclonal result in the PARR assay (positive control). The sample analyzed (peripheral blood) was obtained from a dog diagnosed with leukemia. The x-axis represents the size of the DNA molecules that migrated during electrophoresis (bp: base pairs), the y-axis represents the intensity of fluorescence, which correlates with the amount of DNA for each peak (RFU: relative fluorescence units). Note that the height of the tallest peak is more than twice the other amplified products, hence the monoclonal result.

Four of the remaining thirteen samples had marginal DNA concentration, indicating that they were suitable for analysis, but with a potential risk of lower sensitivity. Two of these were obtained by tru-cut biopsy and two by excisional biopsy. These four samples were included in the study.

Table 1 Breed, sex, age, type of biopsy, suitability and results of the PARR assay for the study population Dog

Breed

Sex

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Labrador retriever Labrador retriever Labrador retriever Labrador retriever Golden retriever Golden retriever Flat coated retriever Flat coated retriever German shepherd dog German shepherd dog Rhodesian ridgeback Lhasa Apso Jack Russell terrier Mixed breed Mixed breed

MN FN MN MN MN FN MN FN M FN M MN FN MN FN

Age Type of Samples PARR (years) biopsy suitable result sample for PARR analysis 7.7 11.5 9 10 9 7 7 8 10.8 9 8 10 7 10 7

E E E TC E E E TC E E E TC E E TC

+ + + + + + +  + + +  + + +

PC PC PC C PC PC PC PC PC PC PC PC PC

MN, male neutered; M, male; FN, female neutered; E, excisional biopsy; TC, tru-cut biopsy; +, sample suitable for PARR analysis (sufficient DNA quantity); , sample unsuitable for PARR analysis (insufficient DNA quantity); PC, polyclonal; C, clonal.

The remaining nine samples, all obtained by surgical biopsy, had good to high DNA concentration. Ten samples showed positive for the DNA control and therefore provided reliable results for PARR. Three of the 13 samples had a negative DNA control, although the quantity of DNA extracted and measured was considered suitable for analysis. As discussed earlier, the canine DNA control was repeated after addition of BSA and was found to be positive in all of these samples. In PARR analysis, 12 of the 13 thymoma samples were determined to have a polyclonal population of lymphoid cells (Fig. 2) and one was found to have a T-cell monoclonal lymphoid population (Fig. 3). None of the samples produced results consistent with B-cell receptor clonality. The only sample diagnosed as thymoma by histopathology, but with a clonal PARR result, was found to have relatively low peak heights on the PARR electrophoretogram, with the peak height being just twice the height of the background. This sample was obtained by trucut biopsy from a 10-year-old Labrador retriever who underwent thymectomy following histopathological diagnosis of thymoma. Two years after thymectomy the dog was still alive and showed no clinical signs suggestive of lymphoma. These results indicate that 92.3% (12/13) of the canine thymomas tested were polyclonal on PARR analysis in those samples considered suitable for analysis following assessment of DNA concentration and using an amplification control.

Lymphoid Clonality in Canine Thymoma

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Fig. 2. Electrophoretogram showing a polyclonal result in the PARR assay for one of the dogs diagnosed with thymoma. Note that the peaks are not discrete and the height of the tallest peak is less than twice the other amplified products (bp: base pairs, RFU: relative fluorescence units).

Fig. 3. Electrophoretogram of the dog diagnosed with thymoma, which exhibited a monoclonal pattern in the PARR assay. Note that the height of the tallest peaks is exactly twice the height of the other amplified products (bp: base pairs, RFU: relative fluorescence units).

The application of PARR for the diagnosis of mediastinal masses in dogs has previously only been evaluated on fine needle aspirate samples in a small number of cases (six dogs) (Lana et al., 2006). In that study, all of the aspirates showed a polyclonal population of lymphoid cells. In our study one confirmed case of thymoma (1/13) showed a monoclonal T-cell population. This dog was still alive at the time of publication (2 years following thymectomy) and did not develop any clinical signs consistent with lymphoma during the follow-up period.

Abdominal ultrasonography and exploratory laparotomy were performed 2 years following thymectomy for a gastric dilatation and volvulus, and were unremarkable. Different hypotheses could explain this monoclonal result, including pseudoclonality (although the results were comparable with the duplicate analysis), inadequate DNA concentration (unlikely based on satisfactory DNA concentration controls performed prior to clonality tests) and benign clonal lymphoid proliferation. Benign clonal lymphoid proliferation leading to false-positive results in clonality assays has

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been described in cases of Ehrlichia canis infection, nonneoplastic lymphocyte-rich thoracic effusion and other inflammatory processes such as inflammatory bowel disease (Burnett et al., 2003; Olivero et al., 2011; Qurollo et al., 2013). As a consequence, undiagnosed co-morbidities remain a possibility. Two of the original 15 samples included in the study were unsuitable for PARR analysis because of insufficient recovery of amplifiable DNA. These samples had been obtained by tru-cut biopsy. The remaining tru-cut biopsy samples in the study had only marginal DNA concentration after extraction, suggesting lower suitability of biopsies obtained by tru-cut compared with samples obtained by surgical excision. The authors therefore recommend retrieving several biopsy samples for each case to reduce the risk of having insufficient DNA for analysis. These results suggest that PARR may be a useful diagnostic test for differentiating between thymoma and lymphoma and could therefore be used in those cases where a definitive diagnosis based on histopathology is not possible, provided that pre-assay controls are performed to ensure sufficient amplifiable DNA is present.

Acknowledgments The authors thank S. Isaacson (Animal Health Trust, UK) for technical assistance and Bridge Pathology (Bristol, UK) for providing some of the case material. This work was supported by the Animal Health Trust, Newmarket, UK. The corresponding author’s current address is Anderson Moores Veterinary Specialists, Hursley, UK. An abstract of this study was presented as an oral communication at the ECVIM/ECVCP congress in Lisbon, Portugal, on 12th September 2015.

Conflict of Interest Statement The authors declare no conflict of interest with respect to the publication of this manuscript.

Supplementary data

Journal of the American Veterinary Medical Association, 205, 1007e1013. Burnett RC, Vernau W, Modiano JF, Olver CS, Moore PF et al. (2003) Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes. Veterinary Pathology, 40, 32e41. De Mello Souza CH (2013) Thymoma. In: Withrow and MacEwen’s Small Animal Clinical Oncology, 5th Edit., S Withrow, F Vail, R Page, Eds., Elsevier, St. Louis, pp. 686e691. Keller SM, Moore PF (2012) A novel clonality assay for the assessment of canine T cell proliferations. Veterinary Immunology and Immunopathology, 145, 410e419. Lana S, Plaza S, Hampe K, Burnett R (2006) Diagnosis of mediastinal masses in dogs by flow cytometry. Journal of Veterinary Internal Medicine, 20, 1161e1165. Nolan T, Hands RE, Ogunkolade W, Bustin SA (2006) SPUD: a quantitative PCR assay for the detection of inhibitors in nucleic acid preparations. Analytical Biochemistry, 351, 308e310. Olivero D, Turba ME, Gentilini F (2011) Reduced diversity of immunoglobulin and T-cell receptor gene rearrangements in chronic inflammatory gastrointestinal diseases in dogs. Veterinary Immunology and Immunopathology, 144, 337e345. Peters IR, Peeters D, Helps CR, Day MJ (2007) Development and application of multiple internal reference (housekeeper) gene assays for accurate normalisation of canine gene expression studies. Veterinary Immunology and Immunopathology, 117, 55e66. Pintore L, Bertazzolo W, Bonfanti U, Gelain ME, Bottero E (2014) Cytological and histological correlation in diagnosing feline and canine mediastinal masses. Journal of Small Animal Practice, 55, 28e32. Qurollo BA, Davenport AC, Sherbert BM, Grindem CB, Birkenheuer AJ (2013) Infection with Panola Mountain Ehrlichia sp. in a dog with atypical lymphocytes and clonal T-cell expansion. Journal of Veterinary Internal Medicine, 27, 1251e1255. Vernau W, Moore PF (1999) An immunophenotypic study of canine leukemias and preliminary assessment of clonality by polymerase chain reaction. Veterinary Immunology and Immunopathology, 69, 145e164. Yoon J, Feeney DA, Cronk DE (2004) Computed tomographic evaluation of canine and feline mediastinal masses in 14 patients. Veterinary Radiology & Ultrasound, 45, 542e546.

Supplementary data related to this article can be found at https://doi.org/10.1016/j.jcpa.2017.12.001.

References Atwater SW, Powers BE, Park RD, Straw RC, Ogilvie GK et al. (1994) Thymoma in dogs: 23 cases (1980e1991).

November 14th, 2017 ½ Received, Accepted, December 7th, 2017