Anne C. Avery

Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes.

Although the diagnosis of canine leukemia and lymphoma in advanced stages is usually uncomplicated, some presentations of the disease can be a diagnostic challenge. In certain situations, lymphoma and leukemia can be difficult to distinguish from a benign reactive proliferation of lymphocytes. Because clonality is the hallmark of malignancy, we have developed an assay that uses the polymerase chain reaction to amplify the variable regions of immunoglobulin genes and T-cell receptor genes to detect the presence of a clonal lymphocyte population. The assay detected clonally rearranged antigen receptor genes in 91% of the 77 dogs with lymphoid malignancy. Of the 24 dogs tested, that were either healthy or had clearly defined conditions not related to lymphoid malignancy, a clonally rearranged antigen receptor gene was found in one (a dog with Ehrlichia canis infection). Gene rearrangement was appropriate for the immunophenotype (immunoglobulin gene rearrangement in B-cell leukemias and T-cell receptor gene rearrangement in T-cell leukemias). Dilution analysis showed that the clonal rearrangement could be detected when 0.1–10% of the DNA was derived from neoplastic cells, depending on the source tissue. Potential applications of this assay include the diagnosis of lymphoma or leukemia in biopsy samples, cavity fluids, fine needle aspirates, bone marrow and peripheral blood; the determination of lineage (B or T cell); staging of lymphoma; and detection of residual disease after chemotherapy.

Distinct B-Cell and T-Cell Lymphoproliferative Disease Prevalence among Dog Breeds Indicates Heritable Risk

Immunophenotypes in lymphoproliferative diseases (LPD) are prognostically significant, yet causative factors for these conditions, and specifically those associated with heritable risk, remain elusive. The full spectrum of LPD seen in humans occurs in dogs, but the incidence and lifetime risk of naturally occurring LPD differs among dog breeds. Taking advantage of the limited genetic heterogeneity that exists within dog breeds, we tested the hypothesis that the prevalence of LPD immunophenotypes would differ among different breeds. The sample population included 1,263 dogs representing 87 breeds. Immunophenotype was determined by the presence of clonal rearrangements of immunoglobulin heavy chain or T-cell receptor gamma chain. The probability of observing the number of B-cell or T-cell tumors in a particular breed or breed group was compared with three reference populations. Significance was computed using chi2 test, and logistic regression was used to confirm binomial predictions. The data show that, among 87 breeds tested, 15 showed significant differences from the prevalence of LPD immunophenotypes seen across the dog population as a whole. More significantly, elevated risk for T-cell LPD seems to have arisen ancestrally and is retained in related breed groups, whereas increased risk for B-cell disease may stem from different risk factors, or combinations of risk factors, arising during the process of breed derivation and selection. The data show that domestic dogs provide a unique and valuable resource to define factors that mediate risk as well as genes involved in the initiation of B-cell and T-cell LPD.

Cutting Edge: The Acquisition of TLR Tolerance during Malaria Infection Impacts T Cell Activation

An effective immune response to infection requires control of pathogen growth while minimizing inflammation-associated pathology. During malaria infection, this balance is particularly important. Murine malaria is characterized by early production of proinflammatory cytokines, which declines in the face of continuing parasitemia. The mechanism by which this occurs remains poorly understood. In this study, we investigated the role of dendritic cells (DCs) in regulating pro- and anti-inflammatory cytokine responses. As malaria infection progresses, DCs become refractory to TLR-mediated IL-12 and TNF-α production, while increasing their ability to produce IL-10 and retaining the capacity for activation of naive T cells. IL-12-secreting DCs from early infection stimulate an IFN-γ-dominated T cell response, whereas IL-10-secreting DCs from later stages induce an IL-10-dominated T cell response. We suggest that phenotypic changes in DCs during Plasmodium yoelii infection represent a mechanism of controlling host inflammation while maintaining effective adaptive immunity.

A novel unstable duplication upstream of HAS2 predisposes to a breed-defining skin phenotype and a periodic fever syndrome in Chinese Shar-Pei dogs.

Hereditary periodic fever syndromes are characterized by recurrent episodes of fever and inflammation with no known pathogenic or autoimmune cause. In humans, several genes have been implicated in this group of diseases, but the majority of cases remain unexplained. A similar periodic fever syndrome is relatively frequent in the Chinese Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of a breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association (GWA) signal for susceptibility to the periodic fever syndrome (praw = 2.3×10−6, pgenome = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as the periodic fever syndrome (p<0.0001). When fragmented, HA can act as a trigger of the innate immune system and stimulate sterile fever and inflammation. The strong selection for the skin phenotype therefore appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a risk factor for human periodic fevers and as an important driver of chronic inflammation.

Dendritic Cells from Malaria-Infected Mice Are Fully Functional APC

Malaria infection has long been associated with diminished T cell responses in vitro and more recently in experimental studies in vivo. Suppression of T cell-proliferative responses during malaria has been attributed to macrophages in a variety of murine and human systems. More recently, however, attention has been directed at the role of dendritic cells in this phenomenon, with several studies suggesting that maturation of dendritic cells is inhibited in vitro by the presence of malaria-infected E. In the studies reported here, we have examined the function of dendritic cells taken directly from infected mice. We found that they express high levels of costimulatory proteins and class II MHC, can activate naive T cells to produce IL-2 as efficiently as dendritic cells from uninfected mice, and support high levels of IFN-γ production by naive T cells through an IL-12-dependent mechanism. Dendritic cells from infected mice also support higher levels of TNF-α production by naive T cells. These same dendritic cells present parasite Ag to a malaria-specific T cell hybridoma, a finding that demonstrates that dendritic cells participate in the generation of Ag-specific immunity during infection. Our findings challenge the contention that dendritic cell function is inhibited by malaria infection.

Canine Lymphoproliferative Disease Characterized by Lymphocytosis: Immunophenotypic Markers of Prognosis

BACKGROUND Canine lymphoproliferative disease often presents with lymphocytosis and is immunophenotypically diverse. HYPOTHESIS Immunophenotype predicts prognosis in canine lymphoproliferative disorders involving circulating lymphocytosis. ANIMALS Dogs that had peripheral blood evaluation performed by flow cytometry by the Clinical Immunology Service at Colorado State University between 2003 and 2005. METHODS Outcome data regarding treatment and survival were sought on patients with lymphocytosis comprising a single lymphocyte subset. Ninety-six patients that met the inclusion criteria had sufficient follow-up information to be included in the study. RESULTS Four main phenotypic classifications were found: CD8+ T-cell, CD21+ B-cell, CD4-8-5+ (aberrant T-cell phenotype), and CD34+ (undifferentiated progenitor). Expression of CD34 predicted poor outcome with median survival of 16 days (P < .0001) compared with other phenotypes. Within the CD8+ phenotype, dogs presenting with a lymphocytosis >30,000 lymphocytes/muL had significantly shorter median survival (131 days) than those presenting with <30,000 lymphocytes/muL (1098 days, P < .0008). Within the T-cell leukemias, there was no difference in outcome between dogs with CD4-8-5+ leukemia and dogs with the CD8+ T-cell phenotype nor was the loss of expression of the pan-leukocyte marker CD45 associated with decreased survival time. A CD21+ lymphocytosis composed of large cells was associated with shorter survival time (129 days) than those with smaller circulating cells (median survival not reached, P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE Immunophenotyping provides an objective method for determining prognosis in lymphoproliferative disorders characterized by lymphocytosis.

Diagnosis of Mediastinal Masses in Dogs by Flow Cytometry

BACKGROUND Biopsy of mediastinal masses can be invasive, but the procedure may be necessary if cytology of a mass aspirate is inconclusive. The 2 most common mediastinal masses, lymphoma and thymoma, may both be comprised of small lymphocytes. We investigated the ability of flow cytometry to distinguish between these 2 neoplasms. HYPOTHESIS Flow cytometry of mediastinal mass aspirates may provide a definitive diagnosis of thymoma or lymphoma, reducing the need for biopsy. ANIMALS Dogs with mediastinal masses presenting to the Veterinary Teaching Hospital/Animal Cancer Center were included in the study. METHODS Aspirates obtained over 2 years that met the inclusion criteria (i.e. sufficient viable cells and a definitive diagnosis by means other than flow cytometry) were analyzed by flow cytometry to determine the percentage of cells expressing B- and T-cell markers, and co-expressing CD4 and CD8. RESULTS All cases of thymoma (n = 6) consisted of > or = 10% lymphocytes coexpressing CD4 and CD8, a phenotype that is characteristic of thymocytes, whereas 6 of 7 lymphomas contained <2% CD4+CD8+ lymphocytes. The CD4+CD8+ lymphoma could be readily distinguished flow cytometrically from thymoma by light scatter properties. The phenotypes of the remaining lymphomas were CD4+ T cell (4), CD34+ (1) and B cell (1). CONCLUSIONS Our studies demonstrate that flow cytometry is a useful tool for discriminating mediastinal masses. Lymphocyte-rich mediastinal masses could be unambiguously identified by flow cytometry in 13/13 cases.

Class II Major Histocompatibility Complex Expression and Cell Size Independently Predict Survival in Canine B‐Cell Lymphoma

BACKGROUND Class II major histocompatibility complex (MHC) is an independent predictor of outcome in human B-cell lymphoma. We assessed class II expression together with other markers for their impact on prognosis in canine B-cell lymphoma. HYPOTHESIS Low class II MHC expression, large cell size, and expression of CD34 will predict a poorer outcome in canine B-cell lymphoma. Expression of CD5 and CD21 on tumor cells also may be associated with outcome. ANIMALS One hundred and sixty dogs with cytologically confirmed lymphoma. METHODS Patient signalment, treatment type, and flow cytometry characteristics were analyzed for their influence on outcome. A multivariable predictive model of survival was generated using 2/3 of the patients and validated on the remaining 1/3 of the dataset. RESULTS Class II MHC expression had a negative association with mortality and relapse. Treatment type also influenced relapse and mortality, whereas cell size and patient age was only associated with mortality. CD34, CD21, and CD5 expression was not associated with disease outcome. The constructed model performed variably in predicting the validation groups outcome at the 6-month time point. CONCLUSIONS AND CLINICAL IMPORTANCE Low levels of class II MHC expression on B-cell lymphoma predict a poor outcome, as in human B-cell lymphoma. This finding has implications for the use of dogs to model human lymphomas. Class II expression, cell size, treatment, and age can be combined to predict mortality with a high level of specificity.

Canine T‐Zone Lymphoma: Unique Immunophenotypic Features, Outcome, and Population Characteristics

Background Canine T‐cell lymphoma (TCL) is clinically and histologically heterogeneous with some forms, such as T‐zone lymphoma (TZL), having an indolent course. Immunophenotyping is an important tool in the classification of TCL in people, and can be equally useful in dogs. Hypothesis/Objectives We hypothesized that loss of expression of the CD45 antigen is a specific diagnostic feature of TZL. Animals Twenty dogs with concurrent histology and immunophenotyping by flow cytometry were studied in depth. An additional 494 dogs diagnosed by immunophenotyping were used to characterize the population of dogs with this disease. Methods Lymph node biopsies from 35 dogs with TCL were classified by 2 pathologists using WHO criteria. Twenty lymph nodes were from dogs with CD45− TCL and 15 were from CD45+ TCL. The pathologists were blinded to the flow cytometry findings. Outcome information was sought for the 20 dogs with CD45− lymphoma, and population characteristics of the additional 494 dogs were described. Results All 20 CD45− cases were classified as TZL. The 15 CD45+ cases were classified as aggressive TCL and are described in an accompanying paper. TZL cases had a median survival of 637 days. Examination of 494 additional dogs diagnosed with TZL by immunophenotyping demonstrated that 40% of cases are in Golden Retrievers, are diagnosed at a median age of 10 years, and the majority have lymphadenopathy and lymphocytosis. Conclusions TZL has unique immunophenotypic features that can be used for diagnosis.

Genome-wide Association Study Identifies Shared Risk Loci Common to Two Malignancies in Golden Retrievers

Dogs, with their breed-determined limited genetic background, are great models of human disease including cancer. Canine B-cell lymphoma and hemangiosarcoma are both malignancies of the hematologic system that are clinically and histologically similar to human B-cell non-Hodgkin lymphoma and angiosarcoma, respectively. Golden retrievers in the US show significantly elevated lifetime risk for both B-cell lymphoma (6%) and hemangiosarcoma (20%). We conducted genome-wide association studies for hemangiosarcoma and B-cell lymphoma, identifying two shared predisposing loci. The two associated loci are located on chromosome 5, and together contribute ~20% of the risk of developing these cancers. Genome-wide p-values for the top SNP of each locus are 4.6×10-7 and 2.7×10-6, respectively. Whole genome resequencing of nine cases and controls followed by genotyping and detailed analysis identified three shared and one B-cell lymphoma specific risk haplotypes within the two loci, but no coding changes were associated with the risk haplotypes. Gene expression analysis of B-cell lymphoma tumors revealed that carrying the risk haplotypes at the first locus is associated with down-regulation of several nearby genes including the proximal gene TRPC6, a transient receptor Ca2+-channel involved in T-cell activation, among other functions. The shared risk haplotype in the second locus overlaps the vesicle transport and release gene STX8. Carrying the shared risk haplotype is associated with gene expression changes of 100 genes enriched for pathways involved in immune cell activation. Thus, the predisposing germ-line mutations in B-cell lymphoma and hemangiosarcoma appear to be regulatory, and affect pathways involved in T-cell mediated immune response in the tumor. This suggests that the interaction between the immune system and malignant cells plays a common role in the tumorigenesis of these relatively different cancers.