Applied Protein and Molecular Techniques for Characterization of B Cell Neoplasms in Horses Peres R. Badial,a,b Rebecca L. Tallmadge,a Steven Miller,a Tracy Stokol,c Kristy Richards,d Alexandre S. Borges,b M. Julia B. Felippea Department of Clinical Sciences, Cornell University College of Veterinary Medicine, Ithaca, New York, USAa; Department of Veterinary Clinical Sciences, School of Veterinary Medicine and Animal Sciences, Universidade Estadual Paulista, Botucatu, São Paulo, Brazilb; Department of Population Medicine and Diagnostic Sciences, Cornell University College of Veterinary Medicine, Ithaca, New York, USAc; Department of Biomedical Sciences, Cornell University College of Veterinary Medicine, Ithaca, New York, USAd Mature B cell neoplasms cover a spectrum of diseases involving lymphoid tissues (lymphoma) or blood (leukemia), with an over- lap between these two presentations. Previous studies describing equine lymphoid neoplasias have not included analyses of clon- ality using molecular techniques. The objective of this study was to use molecular techniques to advance the classification of B cell lymphoproliferative diseases in five adult equine patients with a rare condition of monoclonal gammopathy, B cell leukemia, and concurrent lymphadenopathy (lymphoma/leukemia). The B cell neoplasms were phenotypically characterized by gene and cell surface molecule expression, secreted immunoglobulin (Ig) isotype concentrations, Ig heavy-chain variable (IGHV) region domain sequencing, and spectratyping. All five patients had hyperglobulinemia due to IgG1 or IgG4/7 monoclonal gammopathy. Peripheral blood leukocyte immunophenotyping revealed high proportions of IgG1- or IgG4/7-positive cells and relative T cell lymphopenia. Most leukemic cells lacked the surface B cell markers CD19 and CD21. IGHG1 or IGHG4/7 gene expression was consistent with surface protein expression, and secreted isotype and Ig spectratyping revealed one dominant monoclonal peak. The mRNA expression of the B cell-associated developmental genes EBF1, PAX5, and CD19 was high compared to that of the plasma cell-associated marker CD38. Sequence analysis of the IGHV domain of leukemic cells revealed mutated Igs. In conclu- sion, the protein and molecular techniques used in this study identified neoplastic cells compatible with a developmental transi- tion between B cell and plasma cell stages, and they can be used for the classification of equine B cell lymphoproliferative disease. Lymphoproliferative disorders include a spectrum of neo- plasms, ranging from those that manifest with primary ex- tramedullary tissue involvement (lymphoma) to those with pri- mary blood or bone marrow involvement (leukemia). Lymphoid neoplasms can be further characterized by phenotype (B or T cell) and stage of maturity (precursor or mature cell). Some patients present with concurrent lymphoid tissue tumors and leukemia, complicating classification as lymphoma or leukemia, and these are frequently grouped as precursor lymphoma/leukemia or ma- ture lymphoma/leukemia (1). In the case of mature B cell neo- plasms (i.e., chronic lymphocytic leukemia [CLL] or small lym- phocytic lymphoma [SLL]), the two neoplasms are distinguished by the main site of involvement. In CLL, most tumor cells are located in the blood (�5,000 cells/�l), although some may infil- trate and develop masses in lymphoid tissues (1). In contrast, SLL presents with lymphadenopathy due to neoplastic small lympho- cytes but lacks the peripheral blood lymphocytosis characteristic of CLL (2, 3). In human patients, mature B cell neoplasms ex- press cell surface markers distinct from those of normal B cells and either somatically mutated or unmutated immunoglobu- lin heavy-chain variable (IGHV) genes (4). The mutational sta- tus of IGHV genes has been used as a prognostic indicator of B cell CLL (B-CLL) and for speculation of the developmental origin of neoplastic cells in human patients (5). B cell neoplasms that arise from differentiated mature cells may have undergone somatic hypermutation and productive Ig heavy-chain class switching in germinal centers (6). These cells may produce excessive monoclonal Ig detectable in serum and/or urine (7). The most common form of lymphoid neoplasia in horses is T cell-rich large B cell lymphoma, which is composed of large neoplastic B cells, with an accompanying infiltrate of non- neoplastic small mature T cells (8, 9). Other variants of neoplasms diagnosed in horses include peripheral T zone lymphoma, diffuse large B cell lymphoma, and lymphoma of granular lymphocytes (8). Acute or chronic lymphoid leukemias are reported less fre- quently in horses than in dogs and cats (10–13). In a recent case series of lymphoma in 203 horses, only 2% presented with leuke- mia (8). There have been some reports of concurrent B cell lym- phoma and leukemia in horses; however, expanded cell marker expression and IGHV sequencing were not pursued in those stud- ies (3, 9–11, 14–16). Horses have an IGHV domain repertoire as diverse as that in humans and sheep, with a similar profile of gene usage during developmental progression (17, 18). The classification of lymphoproliferative disorders is essential for the diagnosis and improvement of therapeutic regimens, both of which are in great need in equine medicine. The World Health Organization (WHO) classification is based on a combination of Received 25 June 2015 Returned for modification 31 July 2015 Accepted 19 August 2015 Accepted manuscript posted online 26 August 2015 Citation Badial PR, Tallmadge RL, Miller S, Stokol T, Richards K, Borges AS, Felippe MJB. 2015. Applied protein and molecular techniques for characterization of B cell neoplasms in horses. Clin Vaccine Immunol 22:1133–1145. doi:10.1128/CVI.00374-15. Editor: R. S. Abraham Address correspondence to M. Julia B. Felippe, mbf6@cornell.edu. P.R.B. and R.L.T. contributed equally to this work. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /CVI.00374-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved. November 2015 Volume 22 Number 11 cvi.asm.org 1133Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://dx.doi.org/10.1128/CVI.00374-15 http://dx.doi.org/10.1128/CVI.00374-15 http://dx.doi.org/10.1128/CVI.00374-15 http://cvi.asm.org http://cvi.asm.org/ clinical data, course of disease, cell morphology, immunopheno- typing (immunocytochemistry or histochemistry and flow cy- tometry), and genetic features (1). The WHO system has been adapted for the classification of canine and equine lymphomas based on cell morphology and immunohistochemistry for a few immunologic markers (8, 19). The aim of this study was to use the expression of genes and cell surface molecules, secreted Ig isotypes, IGHV domain sequencing, and Ig spectratyping to characterize a rare clinical condition of mature B cell neoplasms characterized by leukemia and lymph- adenopathy (mature lymphoma/leukemia) in 5 equine patients. This study offers an advancement in the diagnosis and classifica- tion of equine lymphoid neoplasms, with potential applications in future studies that evaluate treatment protocols in this species. MATERIALS AND METHODS Experimental design. The experimental studies were approved by the Cornell University Center for Animal Resources and Education and In- stitutional Animal Care and Use Committee for the use of vertebrates in research. This study performed assays using blood samples from 5 equine patients diagnosed with mature B cell leukemia, hyperglobulinemia, monoclonal gammopathy, and concomitant lymphadenopathy (Table 1) submitted to the Equine Immunology Laboratory at the Cornell Univer- sity College of Veterinary Medicine for lymphocyte immunophenotyping and serum Ig isotype quantification between 2000 and 2009. Clinical cases were managed in their respective referral teaching hospitals (horse 1 at the Ohio State University Veterinary Medical Center, horses 2, 3, and 5 at the Cornell University College of Veterinary Medicine, and horse 4 at the University of Pennsylvania School of Veterinary Medicine). Clinical ex- amination, history, clinical pathological, and postmortem findings (when available) were provided by the respective institutions; complete clinical records were not available for horse 1. Blood samples from �1 healthy research adult horse (of various breeds) were tested side by side with each patient sample as assay controls for immunophenotyping using flow cy- tometry. RNA archived from blood samples from 3 healthy research mares (2 Thoroughbred and 1 pony; age range, 10 to 15 years) and from bone marrow and mesenteric lymph node samples from 1 healthy adult Thoroughbred research gelding (unknown age) were processed side by side with leukemic samples as assay controls in the standard and quanti- tative reverse transcriptase PCR (RT-PCR) assays. Protein electrophoresis and immunoglobulin isotype concentra- tions. Agarose gel protein electrophoresis analyses with respective elec- trophoretograms were performed using serum or plasma samples at the Cornell University Clinical Pathology Laboratory, which provided an in- house established reference interval for serum but not plasma electropho- resis. Plasma was the only available sample type from horse 3 for protein electrophoresis. Serum IgM, IgG, and IgA concentrations were deter- mined using commercially available radial immunodiffusion (RID) kits for horses, as per the manufacturer’s instructions (VMRD, Pullman, WA). A standard curve was generated with the known concentrations of purified equine Igs provided in the kit and their respective precipitate diameters. The concentrations of IgM, IgG, and IgA in each serum sample were determined by comparing the individual precipitate diameters to those of the standard curve. Serum samples were diluted 2-fold when values were greater than the upper limit of the standard curve. Reference intervals for serum Igs were published previously (20). In addition, serum (or plasma from horse 3) IgG concentrations were measured using an immunoturbidimetric assay on an automated chemistry analyzer at the Cornell University Clinical Pathology Laboratory (Midland Bioproducts Corporation, Boone, IA, and Hitachi P-Modular; Roche Diagnostics, In- dianapolis, IN). Horses have 11 immunoglobulin heavy-chain constant genes in the IGH locus named and ordered as IGHM, IGHD, IGHG1, IGHG2, IGHG3, IGHG7, IGHG4, IGHG6, IGHG5, IGHE, and IGHA (20, 21). The IGHG7 T A B LE 1 C lin ic al da ta of af fe ct ed h or se s A ff ec te d h or se / re fe re n ce in te rv al Si gn al m en t C lin ic al si gn sa H C T (% )b H b (g /d l) c C ou n t (� 10 3 /� l) of : T ot al pr ot ei n (g /d l) P la te le ts N eu tr op h ils (n o. of ba n ds ) L ym ph oc yt es 1 17 -y r- ol d H afl in ge r m ar e Ly m ph ad en op at h y, re sp ir at or y di se as e N A N A N A N A 37 .0 10 .8 2 7- yr -o ld T h or ou gh br ed ge ld in g W t lo ss ,d er m at it is ,m u lt ip le n od u le s pa lp ab le in ab do m en (r ec ta le xa m in at io n ) 41 15 .6 79 .0 4. 1 (0 ) 5. 3 12 .1 3 14 -y r- ol d C ly de sd al e m ar e M an di bu la r, pr es ca pu la r, ce rv ic al ly m ph ad en op at h y, w t lo ss ,l et h ar gy ,f ev er ,v en tr al ed em a, ta ch yc ar di a, di ar rh ea ,b ila te ra le pi st ax is ,i n sp ir at or y st ri do r, pl eu ra lfl u id 24 9. 3 16 .0 2. 2 (2 .2 ) 10 3. 8 9. 0 4 28 -y r- ol d Q u ar te r H or se ge ld in g M an di bu la r ly m ph ad en op at h y, fe ve r, ve n tr al ed em a, re sp ir at or y st ri do r an d di st re ss ,t ac h yc ar di a, P P ID 37 13 .8 N A (a de qu at e on sm ea r) 6. 9 (0 .8 ) 68 .0 8. 8 5 11 -y r- ol d C ly de sd al e m ar e B ila te ra le ye lid ed em a, lim b ed em a, de cr ea se d ap pe ti te ,l et h ar gy ,c h ro n ic w t lo ss ,i n tr ap er it on ea l m as s (U S) 29 10 .5 15 1. 0 6. 2 (0 ) 17 .0 10 .9 R ef er en ce in te rv al 34 –4 6 11 .8 –1 5. 9 94 .0 –2 32 .0 2. 7– 6. 6 1. 2– 4. 9 5. 7– 7. 7 a P P ID ,p it u it ar y pa rs in te rm ed ia dy sf u n ct io n ;U S, u lt ra so u n d ex am . b H C T ,h em at oc ri t; N A ,n ot av ai la bl e. c H b, h em og lo bi n . Badial et al. 1134 cvi.asm.org November 2015 Volume 22 Number 11Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://cvi.asm.org http://cvi.asm.org/ gene has a high homology to the equine IGHG4 gene and the IGHG3 to IGHG5 genes; the description of the equine immunoglobulin heavy-chain genes substituted the formerly designated IgGa (now IgG1), IgGb (IgG4/ 7), IgGc (IgG6), and IgGT (IgG3/5) (20, 22). Serum IgG1 (IgGa, clone CVS48; AbD Serotec, Raleigh, NC) and IgG4/7 (IgGb, clone CVS39; AbD Serotec) isotype concentrations were determined at the Cornell Univer- sity Equine Immunology Laboratory using an enzyme-linked immu- nosorbent assay (ELISA) (23, 24). Briefly, goat anti-horse IgG(H�L) was coated onto ELISA plates as a capture antibody for the serum Igs (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Serial dilutions (1:102 to 1:108) of serum samples and horse Ig isotype reference serum were tested in triplicate (Bethyl Laboratories, Inc., Montgomery, TX). Murine monoclonal antibodies against equine IgG1 (IgGa) and IgG4/7 (IgGb) were used as the detection antibody. Peroxidase-conjugated goat anti-mouse IgG(H�L) antibodies detected bound mouse monoclonal antibodies (Jackson ImmunoResearch Laboratories, Inc.). A standard curve was generated from the known reference serum dilutions and their respective optical density (OD) values (Thermo Fisher Scientific, Wal- tham, MA). The concentrations of serum Ig isotypes in each testing sam- ple were determined from the standard curve. The reference intervals were published previously (23, 24). Peripheral blood leukocyte immunophenotyping. Peripheral blood leukocyte immunophenotyping was performed using monoclonal anti- bodies and flow cytometric analysis at the Cornell University Equine Im- munology Laboratory (23). Briefly, peripheral blood mononuclear cells (PBMC) (106) were isolated from heparinized blood using Ficoll density centrifugation. The cell surface molecules tested with monoclonal anti- bodies included CD2 (clone HB88a), CD4 (clone HB61A), CD5 (clone HT23A), and CD8 (clone HT14A) from the Washington State University Monoclonal Antibody Center, Pullman, WA; CD3 (F6G.3G12) from M. Blanchard, University of California—Davis, CA; CD19-like (CZ2.1), ma- jor histocompatibility complex (MHC) class I and class II (CZ3 and CZ11, respectively), and lymphocyte function-associated antigen (LFA-1 or CD11a/CD18, CZ3.2) from D. Antczak, Cornell University, Ithaca, NY; CD21 (B-ly4) from BD Biosciences, San Jose, CA); IgM (CM7), IgA (K1292G5), IgGa (CVS48), and IgGb (CVS39) from AbD Serotec, Raleigh, NC); IgGc (CVS53) and IgGT (CVS40) from P. Lunn, North Carolina State University, Raleigh, NC; and an irrelevant molecule (negative-control against canine parvovirus, C. Parrish, Cornell University) (25–27). The secondary- stage antibody was a fluorescein isothiocyanate (FITC)-conjugated goat anti- mouse IgG(H�L) antibody (Jackson ImmunoResearch Laboratories, Inc.). Samples were analyzed on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). Leukocyte subpopulations (lym- phocytes, monocytes, and neutrophils) were identified and gated based on their characteristic size and complexity in a forward-scatter (FSC) and side- scatter (SSC) dot plot. The percentage of cells in the lymphocyte gate positive for each marker was measured using histogram plots of fluorescence intensity (i.e., stronger fluorescence than the irrelevant control). The reference inter- vals were published previously (23, 24). Reverse transcriptase PCR. The expression of 31 genes associated with lymphoid origin and early or late stages of B cell development, in- cluding plasma cells, was qualitatively assayed by standard RT-PCR. Total RNA was isolated from leukocytes and assay control tissue samples using the RNeasy minikit with on-column digestion for genomic DNA, accord- ing to the manufacturer’s instructions, as described previously (Qiagen, Inc., Valencia, CA) (27). The relative purity and quantity of the isolated RNA were determined by spectrophotometry using NanoDrop 1000 (Thermo Fisher Scientific). The RevertAid reverse transcriptase kit was used to synthesize oligo(dT)-primed cDNA (Thermo Fisher Scientific). The amplification reaction mixtures contained 1� DreamTaq buffer, 0.2 mM deoxynucleoside triphosphates (dNTPs), 0.5 �M forward and re- verse primers specific for each gene, 1.25 U DreamTaq DNA polymerase (Thermo Fisher Scientific), and 25 ng of cDNA template. The primers used are listed in Table 2 or were previously published (23, 28, 29). The thermal cycling parameters were 95°C for 5 min; 35 cycles of 95°C for 1 min, 58°C for 1 min, and 72°C for 30 s; and a final extension of 72°C for 10 min. Amplification products were run on 1% agarose gels and stained with GelGreen nucleic acid stain (Phenix Research Products, Candler, NC) for visualization with the Gel Doc EZ system (Bio-Rad Laboratories, Hercules, CA). Quantitative real-time RT-PCR. Quantitative RT-PCR (qRT-PCR) was used to measure the expression of nine essential genes for B cell de- velopment in RNA isolated from leukocytes and control tissue samples (28). Reactions were performed in triplicate with 10 ng of RNA (leuko- cyte, bone marrow, or lymph node), primers (Table 2), and the iScript one-step RT-PCR kit with SYBR green mix in a CFX96 real-time PCR detection system (Bio-Rad Laboratories). In addition, a no-template con- trol was included on each plate in triplicate. The cycling parameters were 1 cycle of 50°C for 10 min, 1 cycle of 95°C for 5 min, 40 cycles of 95°C for 10 s, and then annealing for 30 s, followed by melt curve analysis (see Table 2 for annealing temperatures). The amplification of specific transcripts was confirmed by melting curve profiles generated at the end of each run. SYBR primers for ACTB, CD19, EBF1, IGHM, and PAX5 were validated previously (28). For the present study, SYBR primers spanning intron/ exon boundaries for CD38, IGHG1, IGHG4/7, IGKC, and IGLC were de- signed with Beacon Designer 7.91 software (PREMIER Biosoft Interna- tional, Palo Alto, CA) and validated on RNA standard curves. RNA standard curves were prepared for each gene by in vitro transcription with the TranscriptAid T7 high-yield transcription kit (Thermo Fisher Scientific) from linearized clones harboring the respective gene. In TABLE 2 RT-PCR and qRT-PCR SYBR primer sets Gene by primer type GenBank accession no. Forward (5=-3=) sequence Reverse (5=-3=) sequence Product size (bp) Annealing temp (°C) Primer concn (nM) RT-PCR primers CD34 XM_001491596 CTAGGGTGTGCTCCTTGCTC GACCAGTGCAATCAGGGTCT 209 58 500 RAG2 AF447533 CACCAAACAATGAGCTTTCG TTTGGGTGGAAGGGATGTAG 207 58 500 CD11b XM_001495590 GGGCAGCCCTGACAGTA GCTGATGCCCAGTCCTGA 131 58 500 CD38 XM_001498785 ATGGCCAACCACAGATTCAG CCAGATGTGCAAGATGGATG 923 58 500 ELA-DRA JQ254080 CTCCAGAGGTGACTGTGCTC TGTCTCTGAGAGGGGAGTTG 311 58 500 qRT-PCR primers CD38 XM_001498785 GGCAGATGCTACACCTACA TATGAGCGGTTGATAGTCTTGT 141 60 500 IGHG1 XM_001496465 CCAGCGAGACCTACATCT TGGTGGTTGTTGGGAATG 88 60 500 IGHG4/7 ECA302057, ECA302058 GCTCACTGTGGAGACTAA GAGACTTGGAGACGGATT 107 57 500 IGKC KJ741386 TTCAGTGGCAGTGGATCTGG CAGAAGACGGTGGGAAGATG 184 63 500 IGLC KF985132 TCCAGGCTGAGGACGAGGC GGAAGAGAGAGACCGAGGGT 135 63 300 Molecular Characterization of Equine B Cell Neoplasms November 2015 Volume 22 Number 11 cvi.asm.org 1135Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://www.ncbi.nlm.nih.gov/nuccore?term=XM_001491596 http://www.ncbi.nlm.nih.gov/nuccore?term=AF447533 http://www.ncbi.nlm.nih.gov/nuccore?term=XM_001495590 http://www.ncbi.nlm.nih.gov/nuccore?term=XM_001498785 http://www.ncbi.nlm.nih.gov/nuccore?term=JQ254080 http://www.ncbi.nlm.nih.gov/nuccore?term=XM_001498785 http://www.ncbi.nlm.nih.gov/nuccore?term=XM_001496465 http://www.ncbi.nlm.nih.gov/nuccore?term=KJ741386 http://www.ncbi.nlm.nih.gov/nuccore?term=KF985132 http://cvi.asm.org http://cvi.asm.org/ vitro-transcribed RNA was purified with RNA Clean & Concentrator columns (Zymo Research, Irvine, CA) and quantified with a Nano- Drop 1000 (Thermo Fisher Scientific). Ten-fold serial dilutions were made for the standard curve. The reaction efficiency ranged between 92.8 and 102.8% (the slope of the curve ranged between �3.256 and �3.506), and no primer dimers were observed on the melt curve anal- ysis. Absolute quantification of mRNA transcript numbers was deter- mined from the RNA standard curve with the CFX Manager software (Bio-Rad Laboratories). Amplification of the equine IGHV domain. Immunoglobulin heavy- chain variable region sequences were amplified from leukocyte cDNA of all leukemic samples, with conserved primers spanning 89% of the VDJ region (5=-GTGGTTCTTCCTCTTTCTGGTG-3= and 5=-CCTGAGGAG ACGGTGACCAG-3=) and a proofreading polymerase (Bio-Rad Labora- tories) (18). PCR products were visualized on a 1% agarose gel, directly purified using the GeneJET PCR purification kit, and ligated into CloneJET PCR cloning kit (Thermo Fisher Scientific). 5-Alpha com- petent Escherichia coli strains were transformed with ligation products, and single colonies were grown in liquid broth with ampicillin (New Eng- land BioLabs, Inc., Ipswich, MA). Plasmid DNA was isolated from �10 clones per sample with the GeneJET plasmid miniprep kit (Thermo Fisher Scientific) and sequenced at the Cornell University Institute of Biotech- nology Genomics Facility. Sequences were analyzed with the Geneious 6.1.5 software (Biomatters Ltd., Auckland, New Zealand). Percent nucle- otide identity to the germ line IGHV gene was determined by comparing the leukemic IGHV sequence against the equine reference genome EquCab2.0 with the NCBI BLAST tool (18). Immunoglobulin spectratyping. Fluorescent Ig heavy-chain variable region amplicons were obtained as described above in conjunction with the M13 tail fluorescent labeling method (30). The amplification reaction mixtures were composed of leukocyte cDNA, 1� DreamTaq buffer, 0.2 mM dNTPs, 1.25 U of DreamTaq DNA polymerase, 1 �M M13 forward primer labeled with 6-carboxyfluorescein (FAM) (5=-FAM-TGTAAAAC GACGGCCAGT-3=), 0.07 �M conserved Ig forward primer with an M13 tail (5=-TGTAAAACGACGGCCAGTGTGGTTCTTCCTCTTTCTGGT G-3=; M13 tail underlined), and 1 �M conserved Ig reverse primer (5=-C CTGAGGAGACGGTGACCAG-3=) (Thermo Fisher Scientific). The primers were commercially synthesized (Eurofins MWG Operon, Huntsville, AL). Touchdown thermal cycling was performed with an initial 5 min of denatur- ation at 95°C, 4 cycles in which the annealing temperature decreased 2 degrees each cycle from 68°C to 60°C (95°C for 30 s, annealing for 30 s, and 72°C for 1 min), which continued for 36 cycles at 60°C, followed by a final extension at 72°C for 10 min. PCR products were visualized on a 1% agarose gel with GelGreen nucleic acid stain before fragment analysis submission (Phenix Re- search Products, Candler, NC). To obtain the spectratype, 1 �l of PCR prod- uct was premixed with 15 �l of Hi-Di formamide and 0.5 �l of GeneScan 500 LIZ dye size standard (Life Technologies, Grand Island, NY). Samples were denatured at 95°C for 5 min. Fragment analysis was performed on the ABI 3730xl DNA analyzer at the Cornell University Institute of Biotechnology Genomics Facility, Ithaca, NY. Electropherograms were analyzed with the Applied Biosystems Peak Scanner software version 1.0 (Life Technologies). Statistical analysis. A Shapiro-Wilk normality test using the Graph- Pad software (GraphPad, San Diego, CA) revealed that the gene expres- sion data (quantitative RT-PCR for EBF1, PAX5, CD19, IGHM, CD38, IGKC, and IGLC) was not normally distributed, and the nonparametric Mann-Whitney-Wilcoxon rank sum test was performed for two-way comparisons between the leukemic (n � 5) and control healthy (n � 3) horse samples. The alpha value was 0.05. Other data were presented de- scriptively. Nucleotide sequence accession numbers. The equine IGHV sequences determined in this study are available in GenBank with the accession numbers KJ741369 to KJ741385. Other accession numbers for the prim- ers used are listed in Table 2. RESULTS The aim of this study was to use the expression of genes and cell surface molecules, secreted Ig isotypes, IGHV domain sequencing, and Ig spectratyping to characterize mature B lymphoma/leuke- mia in 5 equine patients. The application of molecular techniques to equine lymphoid neoplasms advances the classification of neo- plastic cells, in anticipation of better informing diagnosis and gaining insight into the underlying mechanisms. Clinical and histopathological findings. The 5 horses with concurrent mature B cell lymphoma/leukemia were of both sexes and different breeds, with an age range of 7 to 28 years old (Table 1). Three horses presented with peripheral lymphadenopathy, whereas intracavitary masses were detected by rectal examination or transabdominal ultrasound examination in the remaining two horses. Two of 4 horses with complete hemogram results had mild anemia, and 2 were also thrombocytopenic. Only 1 horse was neutropenic, and this horse also had a left shift and toxic changes evident in neutrophils on blood smear examination. All horses had lymphocytosis, but this was mild in 1 horse. Circulating tu- mor cells were a mixed population of small and intermediate cells (8 to 12 �m) with indented nuclei, containing clumped chroma- tin, no nucleoli, and a small amount of light blue cytoplasm. Low numbers of large lymphocytes (15 to 20 �m) with fine chromatin and indistinct nucleoli were seen in 3 horses, comprising �6% of the total lymphocytes (Fig. 1). In two horses, some of the tumor FIG 1 Representative photomicrograph of venous blood from a clinically healthy horse and a horse with leukemia. (A) In the healthy horse, a neutrophil and small lymphocyte (8 to 10 �m, arrow) are present, and erythrocytes are uniformly dispersed with no rouleaux formation. (B [low power]) In horse 3 with leukemia, there is a marked lymphocytosis, consisting of small cells (8 to 10 �m) with a few large lymphocytes (arrow). The small lymphocytes have clumped chromatin and small amounts of light blue cytoplasm. Some have lobulated or irregular nuclei. There is a blue hue to the background, and excessive rouleaux formation is evident in erythrocytes (arrowheads), indicating increased plasma protein and globulins, respectively. (B= [high power]) The small size of the lymphocytes is apparent in relation to the toxic segmented neutrophil. One of the lymphocytes has a lobulated nucleus (arrow). Scale � 10 �m, Wright’s stain. Badial et al. 1136 cvi.asm.org November 2015 Volume 22 Number 11Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://www.ncbi.nlm.nih.gov/nuccore?term=KJ741369 http://www.ncbi.nlm.nih.gov/nuccore?term=KJ741385 http://cvi.asm.org http://cvi.asm.org/ T A B LE 3 C ytologic an d/or h istopath ologic fi n din gs A ffected h orse B lood sm ear cytology B on e m arrow cytology Lym ph n ode cytology G ross path ology H istopath ology 1 N ot available N ot perform ed N ot perform ed N ot perform ed N ot perform ed 2 Lym ph ocytes w ith excess cytoplasm an d cleaved m atu re n u clei N ot perform ed N ot perform ed M u ltifocalfi rm n odu les on in testin al serosa;diffu sely en larged m esen teric lym ph n odes V ariou s sizes ofproliferatin g lym ph oblasts in m esen teric lym ph n odes,in testin al su bm u cosa,kidn eys (perivascu lar), h eart,an d liver (periportal); m u lticen tric lym ph oblastic lym ph om a 3 99% sm allto in term ediate lym ph ocytes w ith 1% blasts; bilobed,lobu lated,in den ted,or cleaved n u clei N ot perform ed N ot perform ed D iffu se lym ph aden opath ic tu m or n odu les in lu n gs,h eart,liver, kidn eys,spleen ,an d G I tract; diffu se petech iation s;m an dibu lar, prescapu lar,su perfi cialcervical, an d m esen teric lym ph aden opath y a P leom orph ic n eoplastic cells (7–15 � m ); scan t eosin oph ilic cytoplasm ;cen trally to periph erally located n u cleu s;coarse ch rom atin w ith m u ltiple n u cleoli; m oderate popu lation ofplasm a cells presen t diffu sely in th e lam in a propria; C D 79a- an d B LA 36-positive cells; m u lticen tric T cell-rich large B cell lym ph om a b 4 68% leu kocytosis du e to 12- to 15- � m atypicallym ph ocytes;rou n d to in den ted n u cleu s;gran u lar to stippled ch rom atin ;in distin ct n u cleoli;scan t dark cytoplasm H em odilu ted sam ple n ot diagn ostic Sm allm atu re lym ph ocytes w ith m oderate n o.ofm ediu m -sized lym ph ocytes an d few lym ph oblasts;n o eviden ce of n eoplasia D iffu se lym ph aden opath y;tu m or n odu les in lu n gs,liver,kidn eys, an d in testin es N eoplastic lym ph ocytes presen t in abdom in alparietalsu bperiton ealm ass; aortic w all,lym ph n odes,pitu itary glan d,lu n gs,liver,kidn eys,in testin es, an d lym ph n odes sligh tly in creased; n eoplastic cells in vesselm u lticen tric lym ph osarcom a 5 In term ediate to large m on on u clear cells;deeply in den ted to cleaved n u clei;sm allam t ofligh t blu e cytoplasm N ot perform ed N ot perform ed N ot perform ed N ot perform ed a G I,gastroin testin al. b C D ,clu ster ofdifferen tiation ;B LA 36,B lym ph ocyte an tigen 36. Molecular Characterization of Equine B Cell Neoplasms November 2015 Volume 22 Number 11 cvi.asm.org 1137Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://cvi.asm.org http://cvi.asm.org/ cells had abnormal morphological features, including clover-leaf- shaped, flower-like, and monocytoid nuclei. Excessive rouleaux formation was evident in the erythrocytes in 3 horses on blood smear examination (Fig. 1). Biochemical profiles were determined in 3 horses. Horse 3 had abnormalities attributable to concurrent liver injury (aspartate ami- notransferase [AST] level, 534 U/liter; reference interval, 212 to 426 U/liter; alkaline phosphatase [ALP] level, 412 U/liter; reference inter- val 75 to 220 U/liter) and diarrhea (decreased electrolytes and high anion gap titration metabolic acidosis). When horses 3 to 5 were tested, the results for creatinine and total calcium levels were within reference intervals (creatinine, 1.5 to 1.8 mg/dl; reference interval, 0.9 to 1.8 mg/dl; total calcium, 11.3 to 12.0 mg/dl; reference interval, 11.2 to 13.0 mg/dl). Mild hyperphosphatemia was present in horses 3 and 5 (4.9 and 5.9 mg/dl, respectively; reference interval, 2.1 to 4.7 mg/dl). Bone marrow aspirates from horse 4 were uninformative due to poor sample quality from hemodilution; in the same horse, lymph node biopsy imprint smear cytology resulted in no evidence of neoplasia but reactive lymphoid hyperplasia. Necropsy was performed in 3 horses (Table 3). Gross patho- logical examination revealed diffuse lymphadenopathy involving mesenteric (horses 2 and 3) or cervical and mediastinal (horse 4) lymph nodes and variably sized tumor nodules in multiple inter- nal organs, including the gastrointestinal tract (all 3 horses) and thoracic organs (horses 3 and 4). Histopathologic examination of tissue samples collected during necropsy revealed neoplastic lym- phocytes in the lymph nodes of all 3 horses and perivascular infil- trates in the intestinal tract, kidney, heart, and liver. In addition, multifocal to coalescing or nodular infiltrates of neoplastic lym- phocytes were found in various tissues of all 3 horses, including the intestine, heart, liver, lungs, spleen, pancreas, mammary gland, aortic and abdominal wall, and pituitary gland. Bone mar- row involvement was evident as a single lobulated mass in the proximal femur of horse 3. Immunohistochemical staining for CD3, CD79a, and B lymphocyte antigen 36 (BLA36) was per- formed on the large intestine and bone marrow of horse 3, and the results showed a mixed population of lymphocytes, with large B cells and small T cells, consistent with a diagnosis of a T cell-rich B cell lymphoma. Protein electrophoresis. Protein electrophoresis revealed hy- perglobulinemia (range, 7.50 to 13.60 g/dl) in all cases and hy- poalbuminemia in 3 horses (Table 4). The hyperglobulinemia was attributed to a monoclonal peak in the gamma region in 4 cases (gammaglobulin range, 2.13 to 6.93 g/dl) and 1 case in the beta 2-globulin region (horse 1, 6.52 g/dl) (Fig. 2). Horse 3 also pre- sented with an increased beta 2-globulin (small peak) value attrib- uted to fibrinogen (plasma sample). Serum immunoglobulin isotype concentration. Serum Ig isotype-specific quantitation using radial immunodiffusion re- vealed IgG hyperglobulinemia in �2 horses (Table 5). However, when using the immunoturbidimetric assay, all horses had mark- edly increased serum IgG concentrations. The serum IgM concen- tration was low (range, �25 to 70 mg/dl) in all affected horses. When tested, the serum IgA concentration was normal and low in 2 horses and 1 horse, respectively. The IgG serotype ELISA re- vealed high levels of IgG1 (IgGa; range, 2,862 to 11,025 mg/dl) and IgG4/7 (IgGb; 5,835 and 7,817 mg/dl) in 3 and 2 horses, respec- tively. Serum IgG3/5 was also elevated for horses 3 and 4 (IgGT; 687 mg/dl and 862 mg/dl, respectively). Peripheral blood leukocyte immunophenotyping. Peripheral blood leukocyte immunophenotyping revealed markedly in- creased percentages of leukemic IgG-positive cells and concomi- tant T cell lymphopenia (CD3 range, 1.1 to 11.0% positive cells; Table 6) in all horses. The majority of leukemic cells did not ex- press the B cell marker CD19 (range, 0.2 to 14.1%) or CD21 (range, 1.5 to 8.6%) (Fig. 3). For horse 3, the percentage of IgM- positive, CD19�, or CD21� cells suggested the presence of a re- sidual population of normal B cells. Three horses had a high per- centage of IgG1 (IgGa; range, 75.9 to 98.1%)-positive cells, whereas 2 horses had a high percentage of IgG4/7 (IgGb; 87.7 and 93.4%) cells. The surface IgG expression matched the secreted IgG isotype on ELISA (Tables 5 and 6). In all cases, almost all cells in the lymphocyte-gated area were positive for MHC classes I (range, 99.9 to 100%) and II (range, 96.4 to 99.5%) and the integrin CD11a/CD18 or LFA-1 (range, 98.3 to 100%). Gene expression. The isolated peripheral blood leukocytes from all horses expressed all leukocyte signature genes using the qualitative RT-PCR test, albeit with some variability of band in- tensity (see Fig. S1 in the supplemental material). Therefore, the expression of selected genes was measured using quantitative analysis. The mRNA copy numbers for EBF1, PAX5, and CD19 in affected horses were greater (P � 0.02) than those in the control healthy horses but were similar to those of the control horses for IGHM (P � 0.3) and CD38 (P � 0.07) (Fig. 4). The mRNA copy numbers for IGHG1 were relatively high in horses 1, 3, and 4; a similar result was observed for IGHG4/7 in horses 2 and 5 (statis- tical analyses were not performed because of the low power when dividing the leukemic horses in 2 groups). The IGLC copy number was higher (P � 0.02) in all affected horses than that in the con- trols. Horses 2 and 5 had IGKC copy number expression compa- rable to that of the control healthy horses (P � 0.13) (Fig. 4). The IGLC/IGKC copy number ratios for horses 1 through 5 were 21,063, 164, 9,898, 22,542, and 439, respectively, and the values for the 3 control healthy horses were 85, 68, and 142. Overall, the IGLC/IGKC copy number ratio was greater (P � 0.03) in affected horses than that in the control horses. The bone marrow and TABLE 4 Serum protein electrophoresis results (g/dl) Affected horse/ reference interval Albumin 1-Globulin 2-Globulin 1-Globulin 2-Globulin �-Globulin Total 1 1.64 0.51 1.12 0.82 6.52 0.50 11.10 2 2.96 0.73 1.41 1.11 0.47 6.93 13.60 3a 2.13 0.26 1.11 0.88 0.99 3.13 8.50 4 2.44 0.35 0.99 0.90 0.68 2.13 7.50 5 2.10 0.41 0.91 0.93 0.45 6.10 10.90 Reference interval 2.3–3.5 0.3–0.8 0.7–1.3 0.2–1.1 0.3–0.8 0.7–1.8 5.7–7.9 a Electrophoresis was performed on heparinized plasma from this horse; reference intervals for plasma were not determined. Badial et al. 1138 cvi.asm.org November 2015 Volume 22 Number 11Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://cvi.asm.org http://cvi.asm.org/ mesenteric lymph node assay control samples from a healthy horse were positive for all genes in both RT-PCR and quantitative RT-PCR analyses (not shown). Immunoglobulin spectratyping. Immunoglobulin spectratyp- ing revealed one dominant peak for each affected horse, with ampli- con lengths ranging from 420 to 439 bases in the individual horses (Fig. 5). For control healthy horses, �4 peaks were observed for each horse, ranging from 424 to 442 bases in 3-base intervals, and the FIG 2 Electrophoretic profile of serum (or plasma) proteins of 5 equine patients with B cell neoplasm, characterized by leukemia and hyperproteinemia. Note the monoclonal peaks of globulins in the gamma region, except for horse 1, which is seen in the beta 2 region. Electrophoresis was performed with a plasma sample from horse 3 and shows a small peak (fibrinogen) in the beta 2 region. A serum sample from a control healthy horse is included for comparison. TABLE 5 Serum immunoglobulin concentration results Affected horse or reference interval Radial immunodiffusion results (mg/dl) for: Turbidimetric assay results (mg/dl) for IgG ELISA results (mg/dl) for: IgA IgM IgG IgG1 (IgGa) IgG4/7 (IgGb) IgG3/5 (IgGT) 1 NAa 25 800 5,084 11,025 189 115 2 NA �25 �1,600 7,391 66 5,835 22 3 225 25 2,400 5,043 4,130 526 687 4 350 70 3,450 3,569 2,862 1,070 862 5 31 �25 �5,000 7,833 290 7,817 314 Reference interval 150–360 100–110 960–3,200 960–3,200 207–479 531–1,697 263–462 a NA, not available. Molecular Characterization of Equine B Cell Neoplasms November 2015 Volume 22 Number 11 cvi.asm.org 1139Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://cvi.asm.org http://cvi.asm.org/ amplicons were of approximately equivalent magnitude within a horse. Equine IGHV domain sequencing. Immunoglobulin heavy- chain variable region sequences were amplified from peripheral blood leukocytes for all 5 affected horses, and �10 clones were sequenced per horse. For some horses, the sequences obtained were 99 to 100% identical (horses 2, 3, and 4), and for other horses (1 and 5), more than one sequence was obtained (Fig. 6). Using the 98% germ line identity criterion, sequence analysis of the IGHV domain indicated a high frequency of mutated genes, with the exception of clones P1.12 to P1.15, (horse 1) and P5.1, P5.4, P5.5, and P5.7 to P5.11 (horse 5). DISCUSSION The lymphoid neoplasms presented in this study manifested in middle-age to elderly horses as concurrent lymphadenopathy, mild to marked leukemia, and hyperglobulinemia due to mono- clonal gammopathy of the IgG1 or IgG4/7 isotype. Peripheral blood leukocyte immunophenotyping revealed a markedly in- creased percentage of cells expressing surface IgG1 or IgG4/7 and concomitant T cell lymphopenia. The leukemic cells did not ex- press the classic B cell markers CD19 and CD21, although two horses had low numbers of presumably normal CD19� or CD21� cells. At the mRNA level, the expression of the B cell markers EBF1, PAX5, and CD19 was greater in leukocytes of affected horses than that in the control healthy horses, in contrast to the mRNA expression of the plasma cell marker CD38. In addition, high mRNA expression for IGHG1 or IGHG4/7 in leukocytes was con- sistent with their respective cell surface protein expression, se- creted monoclonal gammopathy, and single dominant peak on Ig spectratyping. The IGLC/IGKC copy number ratio was greater in affected horses than that in the control horses, but 2 affected horses still expressed detectable levels of mRNA for IGKC. Se- quence analysis of the IGHV domain indicated a high frequency of mutated Igs in comparison to the germ line. The expression of IGHM in horses 3 and 5 was not consistent with serum IgM con- centrations, which were low overall. Lymphocytosis of small to intermediate lymphocytes was the most consistent change in the hemogram. Lymphocytosis was mild in one horse and moderate to marked in the others. The lymphocytosis observed in these cases was attributed to leukemia due to aberrant phenotypes of the lymphocytes in blood. Mild anemia was attributed, in general, to the suppression of erythro- poiesis from chronic disease. Although myelophthisis was not re- ported in the postmortem evaluation, horse 3 had infiltrates in the bone marrow, which may have contributed to decreased red blood cell production and the concurrent severe thrombocytopenia, with associated bleeding signs. Thrombocytopenia might also have been due to concurrent consumption due to severe infection based on an inflammatory leukogram or immune-mediated mechanisms not investigated antemortem (31, 32). Monoclonal gammopathy was suspected in these equine pa- tients due to excessive rouleaux formation in erythrocytes, mod- erate to severe hyperglobulinemia, and associated lymphocytosis. Monoclonal gammopathy was confirmed by protein electropho- resis (serum or plasma) and Ig spectratyping (blood cell) in all cases. In one patient, the monoclonal protein was observed in the beta 2 region; although most IgG proteins migrate to the gamma region, they can be found throughout the electrophoretic spec- trum, such as in the beta region. Since urine samples were notT A B LE 6 P er ip h er al bl oo d le u ko cy te im m u n op h en ot yp in g re su lt s A ff ec te d h or se or re fe re n ce in te rv al % po si ti ve ce lls in th e ly m ph oc yt e- ga te d ar ea fo r: C D 2 C D 3 C D 4 C D 5 C D 8 C D 19 -l ik e C D 21 Ig M Ig G 1 (I gG a) Ig G 4/ 7 (I gG b) Ig G 3/ 5 (I gG T ) Ig G 6 (I gG c) Ig A M H C -I M H C -I I LF A -1 1 16 .9 6. 8 2. 6 3. 2 1. 4 10 .4 N D a 1. 7 92 .4 0. 4 0. 8 0. 3 3. 8 99 .9 96 .4 99 .5 2 N D 11 5. 3 6. 4 9. 6 2. 6 N D N D 2. 1 87 .7 1. 2 N D 0. 2 10 0 99 .1 98 .3 3 5. 5 6. 9 1. 3 5. 5 3. 6 14 .1 7 20 .1 75 .9 0. 2 0. 3 0. 1 N D 10 0 99 .1 99 .9 4 2. 7 1. 1 2. 3 3. 3 0. 7 0. 2 1. 5 0. 3 98 .1 0. 2 0. 7 2. 4 N D 10 0 98 .8 10 0 5 10 .1 9. 6 5. 2 10 .8 5. 4 2. 1 8. 6 4. 7 1. 4 93 .4 2. 5 N D N D 10 0 99 .5 98 .9 R ef er en ce in te rv al 80 .7 –8 9. 9 86 .3 –9 3. 0 57 .2 –7 2. 1 86 .2 –9 5. 6 14 .1 –2 2. 7 6. 3– 12 .6 3. 4– 17 .9 5. 6– 12 .9 � 2. 0 � 2. 0 � 2. 0 � 2. 0 � 2. 0 99 .9 –9 9. 9 84 .6 –9 8. 9 99 .7 –9 9. 9 a N D ,n ot de te rm in ed . Badial et al. 1140 cvi.asm.org November 2015 Volume 22 Number 11Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://cvi.asm.org http://cvi.asm.org/ available for these horses, tests for the presence of monoclonal light chain in urine (i.e., urine electrophoresis and heat precipita- tion methods) were not performed. None of the horses submitted to necropsy had evidence of renal azotemia, despite having mono- clonal gammopathy and neoplastic infiltrates in the kidneys. It is notable that the RID assay for the measurement of serum IgG was the least accurate method for detecting the monoclonal protein, yielding normal values in two horses despite markedly high con- centrations of IgG measured by both ELISA and the immunotur- bidimetric assay; these two assays were in closer agreement. This might be due to the insensitivity of the antibody in the RID kit to a potentially misfolded or mutated monoclonal IgG. For this study, we performed mRNA analysis in leukocytes that contained a mixture of normal and leukemic cells. Despite the mixed nature of the cells in the preparation, the expression of signature B cell genes (EBF1, PAX5, and CD19) was greater in affected horses, likely reflecting the increased proportion of neo- plastic B cells in the peripheral blood. Surprisingly, the increased CD19 mRNA expression in the leukemic cells was not associated with expression of the protein on the cell surface. The magnitude of gene expression of IGHG1 and IGHG4/7 agreed with the mag- nitude of correspondent distribution of circulating cells (immu- nophenotyping) and serum IgG1 and IgG4/7 concentrations, re- spectively. With the marked expression of IgG on the cell surface, this might be explained by a tumor arising from a B cell in transi- tion phase between a postgerminal center B cell and plasma cell, or perhaps an aberrant plasmablast or immature plasma cell. The high serum concentrations of secreted IgG antibodies indicated that the neoplastic B cell clone had undergone isotype switching after encountering antigen and moved on to the antibody-secret- ing phase. The CD19 molecule is expressed during B cell differen- tiation commitment through antigen-independent and -depen- dent development but not after differentiation into plasma cells (33). Along with CD21 and CD81 coreceptors, CD19 assembles the B cell Ig receptor and regulates B cell activation upon antigen binding. In support of our findings, previous studies of CD19 in horses affected with monoclonal gammopathy have shown that leukemic B cells frequently lose surface expression of this marker, despite the persistence of IgG expression (7, 34). These B cell monoclonal antibodies also detected a small population of normal (not leukemic) cells in the samples of these equine patients, indi- cating their potential to detect the equine molecule when present. Due to the lack of appropriate equine conjugated-antibody re- agents, confirmation of negative or low expression of CD19 and CD21 molecules in the IgG1 and IgG4/7 leukemic cells was not possible. The modest CD38 mRNA expression, along with lym- phoid features of the leukemic cells, suggests that the neoplastic clone had not fully differentiated into plasma cells. In human pa- tients, surface expression of CD38 in B-CLL carries an unfavor- able prognosis, since this protein is required for cell proliferation and survival (35). The lack of equine reagents for other markers used for the diagnosis of B and plasma cell neoplasms in human patients (e.g., CD20, CD23, CD27, CD138, and MUM-1) impairs further classification of the leukemic cells in horses at the protein level (36). In human patients with B-CLL, there is usually only a single FIG 3 Flow cytometric dot plot analysis of peripheral blood immunophenotyping of a horse with leukemia. Peripheral blood immunophenotyping of horse 5 shows the distribution of leukocytes (SSC-H, cell granularity side scatter height; FSC-H, cell size forward scatter height) with the prevalence of lymphocytes (R1) in comparison to neutrophils (R2) and monocytes (R3). Dot plots of SSC versus fluorescence show the distribution (%) of positive cells within the lymphocyte- gated area (R1) for each of the markers tested. Neg, negative. Molecular Characterization of Equine B Cell Neoplasms November 2015 Volume 22 Number 11 cvi.asm.org 1141Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://cvi.asm.org http://cvi.asm.org/ clone of cells that expresses one class of Ig light chain (kappa or lambda), which results in an altered kappa/lambda ratio when measured by flow cytometry (37). However, measurement of the kappa-lambda ratio by flow cytometry may fail to identify smaller clonal populations admixed with reactive polyclonal B cells, and mRNA expression may increase the sensitivity for detecting the neoplastic clone. As there are no reliable monoclonal antibodies available to measure equine kappa and lambda light chains using flow cytometry, we measured gene expression in leukocytes. Over- all, affected horses had a greater IGLC/IGKC copy number ratio than that of the control healthy horses, indicating the predomi- nance of the lambda chain in leukemic cells; this result was some- what expected, because horses produce Ig with a relative abun- dance of lambda (96%) in comparison to kappa (4%) light chains under normal conditions (38). Nevertheless, 2 horses had compa- rable IGKC expression to that of the control horse and relatively higher expression than that of the other affected horses, suggesting either expression of both light chains by leukemic cells or reflect- ing the presence of a high number of normal B cells expressing the kappa light chain. The combination of flow cytometric and molecular techniques has expanded our ability to diagnose and better understand the pathophysiology of lymphoid neoplasms. Furthermore, these techniques can readily be performed antemortem on blood sam- ples or tissue aspirates versus relying on necropsy and histologic evaluation of formalin-fixed tissue for a definitive diagnosis. Here, we provide the first report of two molecular techniques that con- firm the diagnosis of mature B cell lymphoma/leukemia in horses. Assessments of B cell IGHV clonality and sequencing have widely been used in human patients and infrequently in dogs with lym- phoid neoplasia (leukemia and lymphoma) (5, 39, 40). Rearrange- ment of the IGHV region and expression of a functional B cell receptor occur during B cell development in the bone marrow, and mutations may follow during antigen-dependent develop- ment (41). Human patients with unmutated IGHV genes (�98% homology with the germ line) have a poorer prognosis than that of patients with mutated IGHV genes (�98% homology) (5). The lack of chromosomal translocations in samples from human pa- tients suggests that B cell leukemia originates from the oncogenic transformation of a germinal center-derived B cell, i.e., after a T cell-dependent immune response, somatic IGHV hypermutation, and Ig isotype switching (42). Alternatively, marginal-zone B cells involved in T cell-independent immune responses become trans- formed with unmutated IGHV receptors, or transformation oc- curs before B cells enter the germinal centers, although mutated cases have also been reported (5). The expression of CD5 has been used to identify marginal-zone B cells (43). Our data showed that the equine leukemic cells were negative for the CD5 marker, and a majority had a mutated IGHV domain, suggesting a B cell neo- plasm of postgerminal center cells that had undergone somatic FIG 4 Quantitative gene expression (mean value � standard error) of B cell signature genes in peripheral blood leukocytes isolated from horses with B cell neoplasm and leukemia and in healthy horses. The mRNA copy number of B cell signature genes was measured in triplicate using quantitative real-time RT-PCR and RNA extracted from isolated leukocytes of affected horses (horses P1 to P5; filled bars) and healthy horses (controls C6 to C8; open bars). The results were compared to serial dilutions of respective in vitro-transcribed RNA used for the generation of a quantitative standard curve. The expression of the housekeeping gene -actin (ACTB) in the respective samples is shown as an inset. Bone marrow and mesenteric lymphoid tissues from a healthy horse were tested side by side as positive controls (not shown). Badial et al. 1142 cvi.asm.org November 2015 Volume 22 Number 11Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://cvi.asm.org http://cvi.asm.org/ hypermutation and isotype switching in the peripheral lymphoid tissues. Immunoglobulin spectratyping was a novel approach to measure monoclonality; it provided a graphic representation of the overall Ig repertoire based on the distribution of CDR3 lengths and revealed one dominant peak for affected horses in contrast to control horses (44). Ig gene rearrangement is im- precise; therefore, CDR3 lengths are variable in nucleotide length. Spectratyping assesses CDR3 length distribution, be- cause length heterogeneity is expected to represent overall se- quence diversity. In the assay presented here, Ig transcripts are amplified by conserved primers in fixed positions, and the re- sulting products directly reflect the CDR3 length distribution of the template. The number and distribution of peaks ob- served, or spectratyping, were consistent with the IGHV se- quences presented here and with previous IGHV sequence analyses (18). Although the IGHV sequence analysis provided additional detail, spectratyping was sufficient to discriminate between leukemic and healthy samples. In this study, spectra- typing had 100% agreement with protein and molecular tests performed in leukocytes, bringing the potential for antemor- tem diagnostic application of clinical cases, including nonse- cretory B cell leukemias (not diagnosed using protein electro- phoresis), cavitary fluids, or solid-tumor aspirates. The horses in this report had both extramedullary infiltrates (such as lymphadenopathy) and lymphocytosis, indicating con- current leukemia. In human patients, CLL is a lymphoprolifera- tive disorder characterized by monoclonal B lymphocytosis; the clinical signs are heterogeneous, ranging from asymptomatic and prolonged survival to the presence of lymphadenopathy, hepato- splenomegaly, autoimmune cytopenias, weight loss, fatigue, and poor outcome (2). SLL is characterized by lymphadenopathy, hepatosplenomegaly, and tumoral cell invasions; monoclonal B cells can be in circulation but in lower number than in CLL. SLL is considered the tissue equivalent (nonleukemic) of CLL. The mild differences in clinical manifestations can be associated with their distinct expression of chemokine receptors, integrins, and genetic abnormalities, although CLL and SLL in humans do not seem to differ morphologically and at the level of cell surface molecules: both types of tumors can express CD2, CD3, CD5, CD19, CD21, and CD23. Another unsolved observation in this study was the fact that patient 3 was diagnosed postmortem with a T-cell-rich large B cell lymphoma, which is characterized by large neoplastic B cells with a background population of presumably polyclonal small T cells. However, the majority of circulating neoplastic cells in this horse were small to intermediate in size and lacked T or B cell markers, similar to the other cases reported here. In human patients, CLL may show an increase over time in cell size and proliferative activ- ity in lymph nodes and bone marrow. Indeed, 2 to 8% of human patients with CLL/SLL develop diffuse large B cell lymphoma, with an aggressive clinical course and poor prognosis; the molec- ular mechanisms associated with neoplasia transformation are under investigation (45). In addition, human patients with follic- ular lymphoma may have lymphocytosis of the small B cells, while the tumor in the lymph node can be composed of larger cells (46). The lymph node tissues of these patients were not available for spectratyping and sequencing, which potentially might have helped solve the origin of and relationship with the leukemic cells. In addition to genetic and epigenetic abnormalities, lym- phomagenesis of mature B cell neoplasms involves the sus- tained activation of self- or nonself-antigen-driven B cell re- ceptor (BCR) clonal proliferation and impaired apoptosis (47). The active proliferation state creates an opportunity for the use of therapeutic agents that target the BCR signaling pathway, including those that inhibit the function of spleen tyrosine kinase (SYK), Bruton’s tyrosine kinase (BTK), phosphatidyl- inositol 3-kinases (PI3K), and B-cell lymphoma 2 (BCL-2) (48). In human patients, current treatment options include chemotherapy (e.g., fludarabine and cyclophosphamide), radi- ation therapy, immunotoxins (e.g., rituximab), and bone mar- row transplantation. In dogs, successful treatment has been reported with melphalan and prednisolone (49). In horses, treatment of lymphoid neoplasms is rarely performed due to poor characterization of neoplastic cells, high costs per body weight, and poor results, and it was not an economic possibility in these patients. In summary, the B cell neoplasms presented here were charac- terized by small to intermediate lymphocytes with concurrent tis- sue and blood involvement and monoclonal gammopathy of IgG isotypes. The leukemic cells expressed markers compatible with a developmental transition between a B cell and a plasma cell stage, including IgG� CD19�, and predominant mutated IGHV. Our study brings a systematic and comprehensive approach that uses molecular testing to complement previously used cytologic and histologic protein testing, enabling advanced diagnosis and clas- sification of B cell neoplasia in these patients. Future studies using this approach may help with identifying new therapeutics and prognostic markers for these tumors in horses. FIG 5 Immunoglobulin spectratyping of peripheral blood leukocytes isolated from horses with B cell neoplasm and leukemia and from healthy horses. Immunoglobulin heavy-chain transcripts were amplified from isolated leuko- cytes of affected horses (P1 to P5) and healthy horses (controls C6 to C8). Spectratypes (black peaks) of the fluorescently labeled amplicons were visual- ized on a genetic analyzer with a size standard (white peaks at 400 and 450 bases shown). Amplicon lengths are shown. Leukemic samples generated one am- plicon, indicating monoclonality, and samples from control healthy horses generated multiple amplicons, indicating a polyclonal population. Molecular Characterization of Equine B Cell Neoplasms November 2015 Volume 22 Number 11 cvi.asm.org 1143Clinical and Vaccine Immunology on M ay 31, 2019 by guest http://cvi.asm .org/ D ow nloaded from http://cvi.asm.org http://cvi.asm.org/ ACKNOWLEDGMENTS We thank Catherine Kohn at the Ohio State University and Brett Dolente and Amy Johnson at the University of Pennsylvania for sending samples and providing clinical data for affected horses 1 and 3, respectively; Midori Asakawa for her additional comments for the histopathologic da- ta; Mary Beth Matychak and Brendan Kraft for technical assistance; and Douglas Antczak and Donald Miller for generously providing ELA-DRA primer sequences. A graduate student scholarship (to P. Badial) was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (grant 2010/08774-7), Brazil. REFERENCES 1. Campo E, Swerdlow SH, Harris NL, Pileri S, Stein H, Jaffe ES. 2011. 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Protein electrophoresis and immunoglobulin isotype concentrations. Peripheral blood leukocyte immunophenotyping. Reverse transcriptase PCR. Quantitative real-time RT-PCR. Amplification of the equine IGHV domain. Immunoglobulin spectratyping. Statistical analysis. Nucleotide sequence accession numbers. RESULTS Clinical and histopathological findings. Protein electrophoresis. Serum immunoglobulin isotype concentration. Peripheral blood leukocyte immunophenotyping. Gene expression. Immunoglobulin spectratyping. Equine IGHV domain sequencing. DISCUSSION ACKNOWLEDGMENTS REFERENCES