Characterization of CD3+/CD20+ canine large-cell lymphoma

Abstract

Immunophenotyping of canine large-cell lymphoma (LCL) for B-cell and T-cell surface antigens is commonly performed to better predict the clinical outcome. Expression of surface antigen CD3 is associated with T-cell malignancies; surface antigen CD20 is expressed on B cells. However, a small subset of canine LCLs expresses both CD3 and CD20 (CD3+/CD20+); this form of lymphoma remains poorly defined at the molecular level. In a retrospective study, we aimed to better characterize immunophenotypic properties and antigen receptor clonality of CD3+/CD20+ LCL. We selected formalin-fixed, paraffin-embedded tissues from 10 cases of CD3+/CD20+ LCL and breed-matched controls of peripheral large T-cell lymphoma (PTCL) and diffuse large B-cell lymphoma (DLBCL). Using PCR for antigen receptor rearrangement (PARR), we identified monoclonal T-cell receptor gamma (TCRγ) rearrangements in all CD3+/CD20+ cases. Three of 10 cases had monoclonal rearrangements in the immunoglobulin heavy chain (IgH), supportive of cross-lineage rearrangement. There was no significant difference in the frequency of antigen receptor rearrangement between CD3+/CD20+ and PTCL cases. In comparison with DLBCL, CD3+/CD20+ LCL had TCRγ rearrangement more frequently and IgH rearrangement less frequently, respectively. Immunolabeling of the B-cell marker PAX5 occurred less frequently in all CD3+/CD20+ LCL cases compared to the DLBCL controls. Immunolabeling for BCL-2 was robust, regardless of immunophenotype. Nuclear Ki67 positivity was variable in CD3+/CD20+ cases, indicating a heterogeneity in proliferation. Overall, cases of canine CD3+/CD20+ LCL had properties similar to PTCL, suggesting a similar histogenesis of these 2 subsets.

Keywords

BCL-2 canine CD3 CD20 DLBCL large-cell lymphoma PARR PAX5 PTCL

Canine non-Hodgkin lymphoma refers to a diverse group of hematologic neoplasms, with aggressive, large-cell lymphomas (LCL) representing the most prevalent subset.³⁰ Diagnostic recommendations provided by the World Health Organization assume a specific cellular origin for these cancers.²⁸ Specifically, most lymphomas arise from either CD3+ T lymphocytes or CD20+ B lymphocytes. Diffuse large B-cell lymphoma (DLBCL) and peripheral large T-cell lymphoma (PTCL) remain the most common subtypes in dogs.³⁰ Immunophenotyping is clinically important because high-grade T-cell lymphomas have the shortest survival times, and T-cell neoplasms have been reported to have decreased response rates to chemotherapy.^14,27 Therefore, immunohistochemistry (IHC) for B- (CD20 and PAX5) and T-lymphocyte (CD3) antigens is often recommended in the diagnostic workup of lymphoma.

Although immunophenotyping allows for either a B- or T-cell classification for most LCL cases, a small subset of canine lymphomas expresses both CD3 and CD20. Conversely, null LCL has been reported in dogs and lacks expression of both CD3 and CD20.¹⁹ The CD3+/CD20+ phenotype has been described in a small number of case reports spanning a few different types of lymphoma, including enteropathy-associated T-cell lymphoma, anaplastic lymphoma, and cutaneous epitheliotropic T-cell lymphoma.^{2,9,15
–17,26} Despite these publications, understanding of CD3+/CD20+ LCL in dogs remains extremely limited.

We compared 10 CD3+/CD20+ LCLs, which affected mostly peripheral lymph nodes, to breed-matched cases of DLBCL and PTCL. More specifically, we determined clonality in all cases as well as provided immunolabeling properties for paired box gene 5 (PAX5), B-cell lymphoma 2 (BCL-2), and Ki67. We found that CD3+/CD20+ lymphomas are similar to PTCL with respect to antigen receptor rearrangement and PAX5 expression. Ki67 nuclear positivity was variable in our case series, indicating a difference in the proliferative activity of CD3+/CD20+ LCL.

Materials and methods

Case selection

We selected formalin-fixed, paraffin-embedded (FFPE) tissues retrospectively from the Michigan State University Veterinary Diagnostic Laboratory (IACUC exempt; MSU-VDL, Lansing, MI, USA). Cases of LCL, as determined by a board-certified anatomic pathologist (B.K. Harrington), that immunolabeled positive for both CD3 and CD20 met the criteria for inclusion in the study. DLBCL and PTCL case controls were selected based on breed similarity to the CD3+/CD20+ LCL group. Seven of 10 cases had exact breed matches. For the 3 unmatched patients, we selected Labrador Retriever controls for the Newfoundland and Shi Tzu controls for the Dachshund based on reports on the genetic similarities between these breeds,^3,18 and a mixed-breed control for the Beagle (Tables 1, 2; Fig. 1).

Table 1.

Demographics of biopsy and autopsy submissions of canine lymphoma cases at the Michigan State University Veterinary Diagnostic Laboratory.

Case	Breed	Sex	Age, y	Submission type
CD3+/CD20+ LCL cases
1	Labrador Retriever	CM	6.5	Biopsy (not specified)
2	Dachshund	F	10	Biopsy (not specified)
3	Mixed	F	5	Biopsy (punch)
4	American Pit Bull Terrier	CM	7	Biopsy (excisional)
5	Newfoundland	CM	6	Biopsy (not specified)
6	Belgian Shepherd	CM	12	Autopsy (whole mass)
7	Beagle	CM	8	Autopsy (whole mass)
8	American Pit Bull Terrier	SF	7	Biopsy (wedge)
9	Mixed	CM	4	Biopsy (punch)
10	Dachshund	CM	9	Biopsy (not specified)
CD3+/CD20– PTCL cases
11	Labrador Retriever	CM	12	Biopsy (not specified)
12	Shih Tzu	SF	8	Autopsy (whole mass)
13	Mixed	SF	7	Autopsy (whole mass)
14	American Pit Bull Terrier	CM	10	Autopsy (whole mass)
15	Labrador Retriever	F	11	Autopsy (whole mass)
16	Belgian Shepherd	SF	6	Biopsy (excisional)
17	Mixed	CM	11	Autopsy (whole mass)
18	American Pit Bull Terrier	M	5	Biopsy (not specified)
19	Mixed	CM	7	Biopsy (excisional)
20	Dachshund	CM	8	Biopsy (not specified)
CD3–/CD20+ DLBCL cases
21	Labrador Retriever	SF	7	Autopsy (whole mass)
22	Shih Tzu	SF	10	Autopsy (whole mass)
23	Mixed	F	6	Autopsy (whole mass)
24	American Pit Bull Terrier	SF	4	Biopsy (not specified)
25	Labrador Retriever	SF	11	Biopsy (excisional)
26	Belgian Shepherd	CM	8	Autopsy (whole mass)
27	Beagle	M	8	Biopsy (excisional)
28	American Pit Bull Terrier	SF	7	Biopsy (excisional)
29	Mixed	CM	5	Biopsy (incisional)
30	Dachshund	CM	2	Biopsy (excisional)

CM = castrated male; DLBCL = diffuse large B-cell lymphoma; F = female; LCL = large-cell lymphoma; M = male; PTCL = peripheral large T-cell lymphoma; SF = spayed female.

Table 2.

Nodal and/or extranodal involvement of biopsy and autopsy submissions of canine lymphoma cases at the Michigan State University Veterinary Diagnostic Laboratory.

Case	Location	Multicentric involvement	Additional details
CD3+/CD20+ LCL cases
1	LN (not specified)	+	Gen. lymphadenopathy
2	LN (not specified)	+	Gen. lymphadenopathy
3	Popliteal LN	+	Gen. lymphadenopathy
4	Submandibular LN	–	Submandibular LN only
5	Popliteal LN	+	Gen. lymphadenopathy
6	Liver mass	+	Liver, small intestines
7	Heart mass	–	Cardiac tissue only
8	Popliteal, prescapular LN	+	Gen. lymphadenopathy
9	Submandibular LN	+	Gen. lymphadenopathy
10	Submandibular LN	+	Multiple masses
CD3+/CD20– PTCL cases
11	Submandibular LN	–	Submandibular LN only
12	Mass near duodenum	+	Intestines, heart
13	Axillary LN	+	Spleen, LN
14	Mesenteric LN	+	Disseminated
15	Mediastinal LN	–	Mediastinal LN only
16	Ureter, vena cava	+	Ureter, intestines
17	LN (not specified)	+	Heart, liver, spleen
18	Mesenteric LN	+	LN, spleen
19	Mesenteric mass	+	Mesentery, liver
20	Submandibular, popliteal LN	+	Gen. lymphadenopathy
CD3–/CD20+ DLBCL cases
21	Submandibular LN	+	Gen. lymphadenopathy
22	Submandibular, popliteal LN	+	Gen. lymphadenopathy
23	LN (multiple)	+	Gen. lymphadenopathy
24	Multiple LNs	+	Gen. lymphadenopathy
25	Soft palate mass	–	Soft palate mass only
26	Submandibular, popliteal LN	+	Gen. lymphadenopathy
27	Submandibular LN	–	Submandibular LN only
28	Prescapular LN	+	Gen. lymphadenopathy
29	Cervical LN	+	Gen. lymphadenopathy
30	Rectal MALT	–	Rectal MALT only

DLBCL = diffuse large B-cell lymphoma; Gen. lymphadenopathy = generalized lymphadenopathy; LCL = large-cell lymphoma; LN = lymph node; MALT = mucosal-associated lymphoid tissue; PTCL = peripheral large T-cell lymphoma.

Figure 1.

Representative H&E stains and CD3 and CD20 immunolabeling images from CD3+/CD20+ large-cell lymphoma (LCL) and control cases. Case 10, CD3+/CD20+ LCL: A = H&E; B = CD3; C = CD20. Case 20, peripheral large T-cell lymphoma: D = H&E; E = CD3; F = CD20. Case 28, diffuse large B-cell lymphoma: G = H&E; H = CD3; I = CD20. Bars = 50 µm.

Parr

DNA was extracted from FFPE tissues (QIAamp DNA FFPE tissue kit; Qiagen) according to the manufacturer’s instructions. IgH and TCRγ primer sets were designed based on previous publications (Table 3).^4,11 The negative reaction control was performed by using the DNA FFPE extraction kit with nuclease-free water. In the positive control condition, previously documented DNA from a canine lymphoma sample unrelated to our study was used. PCR was performed (Type-it mutation PCR kit; Qiagen) with 20–100 ng of DNA from each case. For amplification of the TCRγ CDR3 region, a forward and reverse primer “cocktail” of the 6 listed sets in equal parts was made as described previously.¹¹ PCR was conducted in a T100 thermocycler (Bio-Rad). Denaturing, annealing, and extension were allowed to proceed for 35 cycles. The annealing temperature for the TCRγ region was 64°C for 90 s; B-cell amplification was at 60°C for 30 s. The subsequent PCR product was then detected, separated by capillary electrophoresis, and analyzed (QIAxcel advanced instrument; Qiagen) using a high-resolution pre-cast gel cartridge. DNA intensity peaks were obtained (QIAxcel ScreenGel software; Qiagen).

Table 3.

Sequences of primers used in PCR for antigen receptor rearrangements to determine clonality in either the IgH or TCRγ gene in canine lymphoma cases.

Target	Forward	Reverse
IgH Major CB1/CB3	5′-CAGCCTGAGAGCCGAGGACAC-3′	5′-TGAGGAGACGGTGACCAGGGT-3′
IgH Minor CB2/CB3	5′-CAGCCTGAGAGCCGAGGACAC-3′	5′-TGAGGACACAAAGAGTGAGG-3′
TCRγ
V2/6a/Ja	5′-GAAGGCGTGTACTACTGCGCTG-3′	5′-TTGTGCCAGGACCAAACACTTT-3′
V2/6b/Jb	5′-GAGGGCGTGTACTACTGTGCTG-3′	5′-GGGGAGTTACTATGAGCTTAGTTCCTT-3′
V3a/Jc	5′-TGTTAAGGAAACAAGATGAGGCCA-3′	5′-GAGGAGTTACTATAAGCCTAGTACCTTCTG-3′
V3b/J2-1	5′-TCTTAAGGAAACAACATGAGGCTGTG-3′	5′-GAGGAGTTACTATAAACCTGTTAACTTCTG-3′
V7a/J5-1	5′-AAGTAAAAATGCTCTTACTTCCACTTCAAC-3′	5′-GGGGAGTTACTATGAGATTAGTTCCTTCTG-3′
TV7b/J6-2	5′-GTAAAAATGCCGTTACTTCCACATCAACTT-3′	5′-GTGTGTCAGGACCCATCACTTTGTT-3′

IgH = immunoglobulin heavy chain; TCRγ = T-cell receptor gamma.

Immunohistochemistry

All tissues were fixed in 10% neutral-buffered formalin for 12–24 h and processed at the MSU-VDL. Slides were deparaffinized and re-hydrated using standard laboratory methods. IHC was conducted with the following parameters, as described previously^5,12,24,25 (Table 4). Antigen retrieval was performed with CC1 (Cell conditioning I, Discovery Ultra; Ventana) at 95°C for 4 min, or ER1 (Bond epitope retrieval solution I; Leica) at 95°C for 20 min. A Bond-Max automated IHC stainer was used for CD3, CD20, and Ki67 with the Bond polymer detection system (Vision Biosystems; Leica) and 3,3′-diaminobenzidine (DAB) as the chromogen. The Discovery Ultra automated staining system was used for BCL-2 and PAX5 with the Discovery UltraMap alkaline phosphatase system and Discovery Red (Roche). Sections of canine lymph node tissue were used as a positive control for all antibodies. The antibody used for BCL-2 has been established in primary colorectal follicular canine lymphoma, marginal zone lymphoma, and mantle cell lymphoma.^20,25

Table 4.

Primary antibody source, dilution, antigen retrieval method, and detection method employed in immunohistochemistry of canine lymphoma cases.

Primary antibody	Host	Source	Catalog	Dilution (primary)	Antigen retrieval	Detection
BCL-2	Mouse	Leica Biosystems	NCL-BCL-2-486	1:100	CC1	UltraMap Red
CD3	Rabbit	Agilent	A0452	1:200	ER1	DAB
CD20	Rabbit	Fisher Scientific	RB9013P	1:200	ER1	DAB
PAX5	Rabbit	Roche	760-4270	RTU	CC1	UltraMap Red
Ki67	Mouse	Agilent	GE020	1:50	CCI	DAB

CC1 = cell conditioning solution 1 (Tris-EDTA–based buffer, pH 7.8); ER1 = epitope retrieval solution 1; RTU = ready-to-use.

Ki67 quantitation

Nuclear Ki67 positivity was quantified using the QuPath (v.0.4.3) positive cell detection feature.¹ Positive cells were determined using the optical density sum and standard settings assuming default DAB and hematoxylin staining parameters.

Statistical analysis

Fisher exact tests were performed in Prism v.9.1.2 (GraphPad) between groups for comparisons involving positive or negative variables. P ≤ 0.0125 was considered statistically significant by using an alpha value of 0.05 and a Bonferroni adjustment with 4 comparisons (0.05/4). For analysis of Ki67 positivity, groups were examined using a one-way ANOVA and a Tukey honestly significant difference test, with p ≤ 0.05 being considered statistically significant.

Results

Antigen receptor clonality in CD3+/CD20+ LCL

To ascertain antigen receptor clonality for each case, we performed PARR using primers specific to TCRγ and IgH genes (Fig. 2). Monoclonal TCRγ rearrangements occurred in all CD3+/CD20+ cases. Three of 10 cases also had rearrangements in the IgH, supportive of cross-lineage rearrangement (Table 5). Interestingly, 5 cases of PTCL and 2 cases of DLBCL also had cross-lineage rearrangement. CD3+/CD20+ LCLs had TCRγ rearrangement more frequently and IgH rearrangement less frequently than DLBCL cases (p = 0.0007 and 0.003, respectively). We found no statistical difference comparing the CD3+/CD20+ LCL and PTCL groups (Table 6), suggesting that CD3+/CD20+ LCLs resemble PTCLs with respect to antigen receptor clonality.

Figure 2.

Antigen receptor clonality in CD3+/CD20+ canine large-cell lymphoma. A. Representative images from the amplification of immunoglobulin heavy chain (IgH). C1–8 and E10–12 correspond to the lane identification in the QIAxcel advanced capillary electrophoresis cartridge. B. Representative images from the amplification of T-cell receptor gamma (TCRγ). A1–A12 correspond to the lane identification in the QIAxcel advanced capillary electrophoresis cartridge.

Table 5.

Frequency of antigen receptor rearrangements in CD3+/CD20+ large-cell lymphoma (LCL), diffuse large B-cell lymphoma (DLBCL), and peripheral large T-cell lymphoma (PTCL) cases.

Group	TCRγ	IgH	Cross lineage	Total
CD3+/CD20+ LCL	7	0	3	10
PTCL*	2	2	5	10
DLBCL	0	8	2	10

Neither immunoglobulin heavy chain (IgH) nor T-cell receptor gamma (TCRγ) were amplified for one case of PTCL.

Table 6.

Summary of Fisher exact tests comparing CD3+/CD20+ large-cell lymphoma (LCL), diffuse large B-cell lymphoma (DLBCL ), and peripheral large T-cell lymphoma (PTCL) for TCRγ or IgH rearrangements (*p ≤0.0125). All samples were run in triplicate with the appropriate positive and negative controls.

Comparison	Variable	p
CD3+/CD20+ LCL vs. PTCL	TCRγ	p = 0.210
CD3+/CD20+ LCL vs. DLBCL	TCRγ	p = 0.007*
CD3+/CD20+ LCL vs. PTCL	IgH	p = 0.179
CD3+/CD20+ LCL vs. DLBCL	IgH	p = 0.003*

IgH = immunoglobulin heavy chain; TCRγ = T-cell receptor gamma.

Immunophenotyping of CD3+/CD20+ LCL

To further characterize our CD3+/CD20+ LCL cases, we performed IHC for PAX5 and BCL-2 (Fig. 3). PAX5 is a nuclear transcription factor that regulates the differentiation and development of B cells and is, therefore, used commonly as a B-cell marker.²⁹ In all CD3+/CD20+ cases, we observed negative PAX5 immunolabeling. This was unlike our DLBCL group, which immunolabeled for PAX5 in 90% of cases. PAX5 was only positive in 10% of PTCL cases. We observed no statistical difference in the PAX5 immunolabeling between CD3+/CD20+ LCLs and PTCLs (p = 0.999). PAX5 immunolabeling occurred more frequently in DLBCLs compared to our CD3+/CD20+ LCL group (p = 0.0001; Table 7). Our PAX5 immunolabeling data support the idea that CD3+/CD20+ LCLs have histogenesis similar to PTCL cases.

Figure 3.

Expression of PAX5 and BCL-2 in CD3+/CD20+ canine large-cell lymphoma (LCL). Representative images of immunolabeling of PAX5 and BCL-2. Case 10, CD3+/CD20+ LCL: A = PAX5; B = BCL-2. Case 28, peripheral large T-cell lymphoma: C = PAX5; D = BCL-2. Case 11, diffuse large B-cell lymphoma: E = PAX5; F = BCL-2. Bars = 50 µm.

Table 7.

Summary of Fisher exact tests comparing PAX5 or BCL-2 immunolabeling in CD3+/CD20+ large-cell lymphoma (LCL), diffuse large B-cell lymphoma (DLBCL), or peripheral large T-cell lymphoma (PTCL) where indicated (*p ≤0.0125).

Comparison	Variable	p
CD3+/CD20+ LCL vs. DLBCL	PAX5	p = 0.0001*
CD3+/CD20+ LCL vs. PTCL	PAX5	p = 0.999
CD3+/CD20+ LCL vs. DLBCL	BCL-2	p = 0.999
CD3+/CD20+ LCL vs. PTCL	BCL-2	p = 0.999

Next, we immunolabeled for BCL-2, an anti-apoptotic protein commonly overexpressed and therapeutically targeted in numerous cancer types.⁷ BCL-2 has also been reported to be expressed ubiquitously in canine DLBCLs.⁶ Moreover, a high BCL-2:BAX ratio has been associated more commonly with T-cell lymphomas, which may explain the poor clinical outcome in T-cell lymphomas.¹³ Robust immunolabeling for BCL-2 occurred in all cases, and no difference was observed among groups (p = 0.999; Table 7).

High Ki67 positivity has been associated with a shorter survival time in dogs with DLBCL treated with chemotherapy.²³ Differences in proliferation markers, such as Ki67, and the apoptotic activity of the neoplastic cells, can account for variations in the proliferative activity of tumors and subsequent clinical outcome.²¹ Therefore, we were interested in the Ki67 immunolabeling index of our CD3+/CD20+ LCL cases (Fig. 4A–D). Nuclear positivity was variable in both the PTCL and CD3+/CD20+ subsets (range: 4–99%), suggesting variability in the proliferation of these tumors. However, the average Ki67 immunolabeling index for the CD3+/CD20+ group was significantly different and higher than that of the DLBCL group (p = 0.04; Fig. 4E, Table 8).

Figure 4.

Ki67 immunolabeling index in CD3+/CD20+ canine large-cell lymphoma (LCL). Representative images of immunolabeling of Ki67 from CD3+/CD20+ lymphoma: A = case 1; CD3+/CD20+ LCL; B = case 4, CD3+/CD20+ LCL; C = case 11, peripheral large T-cell lymphoma; D = case 28, diffuse large B-cell lymphoma. Bars = 50 µm. E. Quantitation of % Ki67 positivity. Results were compared using a one-way ANOVA and a Tukey HSD (*p ≤ 0.05). The box-and-whisker plot indicates the minimum, first quartile, median, x– (“x”), third quartile, and maximum of each dataset.

Table 8.

Average Ki67 index and variation in CD3+/CD20+ large-cell lymphoma (LCL), diffuse large B-cell lymphoma (DLBCL), and peripheral large T-cell lymphoma (PTCL).

Group	Average Ki67 index, %	SD, %
CD3+/CD20+ LCL	53.4	27.1
PTCL	27.2	30.7
DLBCL	23.3	15.0

Discussion

The frequency with which the TCRγ and IgH were rearranged differed statistically between CD3+/CD20+ LCL cases and DLBCL cases. However, there was no statistical difference in antigen receptor rearrangement frequency between CD3+/CD20+ LCL cases and PTCL cases. Furthermore, PAX5 immunolabeling patterns were similar between PTCL and CD3+/CD20+ cases but differed statistically between DLBCL and CD3+/CD20+ cases. The Ki67 immunolabeling index was highly variable within all groups, but a significant difference was noted between the CD3+/CD20+ LCL and DLBCL groups. Overall, our data highlight similarities between PTCL and CD3+/CD20+ LCL, suggesting that CD3+/CD20+ LCL cases have histogenesis similar to PTCL.

Cross-lineage rearrangement occurred frequently in our study: in 7 of our control cases and 3 CD3+/CD20+ LCL cases. V(D)J recombination in both the T-cell receptor and IgH genes has been reported in dogs,¹⁶ although with a lower frequency than we observed. However, the true frequency with which cross-lineage rearrangement occurs in canine lymphoma is not known, given that a large-scale study has not yet been reported. Additionally, PARR is often assessed in only one antigen receptor for many diagnostic cases, further hindering our ability to retrospectively assess the frequency with which this phenomenon occurs. Other investigators have developed an improved primer set for canine PARR.⁸ The sensitivity of the IgH primer set that we used is reported to be ~70%, which should be considered when interpreting our results.⁸ Overall, the frequency of cross-lineage rearrangement, in addition to occasional aberrant expression of PAX5 in PTCL, bring into question the accuracy and adequacy of immunophenotyping with a single surface marker for canine lymphoma cases.

Inhibitors of the anti-apoptotic protein BCL-2 are under investigation in a wide variety of human cancers and have shown success in some forms of non-Hodgkin lymphoma.²² BCL-2 expression levels are not prognostic in determining a response to CHOP chemotherapy, but BH3 mimetics have had in vitro efficacy activity against canine T-cell neoplastic cells.^6,10 We observed no differences among groups in the immunolabeling of the therapeutic target BCL-2 and noted that BCL-2 had robust expression regardless of lymphoma subtype. Our data support the exploration of BCL-2 inhibitors in canine LCL patients regardless of immunophenotype.

Limitations of our study include the small sample size and retrospective approach. Despite these shortcomings, we performed a robust statistical analysis, identifying clear similarities and differences among lymphoma subtypes. Future studies will be directed at determining prognostic differences among these patients and evaluating additional prognostic markers and therapeutic targets.

Footnotes

Acknowledgements

We thank all of the individuals in the MSU-VDL histology laboratory for their assistance with our project. Our work was presented as a student poster at the 2021 American College of Veterinary Pathologists Annual Meeting (Virtual: Oct 30–Nov 2, 2021).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

Morris Animal Foundation Veterinary Student Scholar Award.

ORCID iD

Cory M. Howard

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