Polyclonal B-cell lymphocytosis in English bulldogs

Abstract

Background: English bulldogs disproportionally develop an expansion of small B-cells, which has been interpreted as B-cell chronic lymphocytic leukemia (BCLL). However, clonality testing in these cases has often not been supportive of neoplasia.

Hypothesis: English bulldogs have a syndrome of nonneoplastic B-cell expansion.

Animals: Eighty-four English bulldogs with small-sized CD21+ B-cell lymphocytosis in the blood as determined by flow cytometry.

Methods: This is a retrospective study. We characterized this syndrome by assessing B-cell clonality, clinical presentation, flow cytometric features, and immunoglobulin gammopathy patterns. We identified 84 cases with CD21+ lymphocytosis among 195 English bulldogs with blood samples submitted to the Colorado State University-Clinical Immunology laboratory for immunophenotyping between 2010 and 2019. Flow cytometry features were compared to normal B-cells and BCLL cases. PCR for antigen receptor rearrangements (PARR) by multiple immunoglobulin primers was performed to assess B-cell clonality. A subset of cases with gammopathy were examined by protein electrophoresis, immunofixation, and immunoglobulin subclass ELISA quantification.

Results: Seventy percent (58/83) of cases had polyclonal or restricted polyclonal immunoglobulin gene rearrangements, suggesting nonmalignant B-cell expansion. The median age of all dogs in the study was 6.8 years and 74% were male. The median (range) lymphocyte count was 22 400/μL (2000-384 400/μL) and B-cells had low expression of class II MHC and CD25. Splenomegaly or splenic masses were detected in 57% (26/46) of cases and lymphadenopathy in 11% (7/61). Seventy-one percent (52/73) of cases had hyperglobulinemia and 77% (23/30) with globulin characterization had IgA ± IgM polyclonal or restricted polyclonal gammopathy patterns.

Conclusions and clinical importance: Polyclonal B-cell lymphocytosis in English bulldogs is characterized by low B-cell class II MHC and CD25 expression, splenomegaly and hyperglobulinemia consisting of increased IgA ± IgM. We hypothesize that this syndrome has a genetic basis.

Keywords: IgA gammopathy; PCR for antigen receptor rearrangements; canine; clinical pathology; clonality; flow cytometry; hyperglobulinemia.

FIGURE 1

PCR for antigen receptor rearrangements (PARR) results for English bulldogs with B‐cell lymphocytosis. GeneMarker tracings for complete immunoglobulin heavy (IGH) chain variable (V)‐diversity (D)‐joining (J) (IGH‐VDJ) rearrangements are presented for 4 polyclonal cases (A), 4 restricted polyclonal cases (B), and 4 clonal cases (C). The size of the PARR amplicons is plotted on the horizontal axis and the abundance of amplicons is on the vertical axis. (A) Polyclonal cases have multiple peaks forming a Gaussian distribution. (B) Restricted polyclonal cases have 1 to 5 peaks >2000 in amplitude and 2 times the height of the polyclonal peaks forming the base. (C) Clonal cases have a peak >5000 in amplitude and >3 times the base height

FIGURE 2

Protein electrophoresis (PE) and immunofixation (IF) results in control dogs and English bulldogs with B‐cell lymphocytosis. An agarose gel protein electrophoresis gel and tracing (top) and immunofixation gel (bottom) are provided. Immunofixation gels are labeled with anti‐whole serum (WS), anti‐canine IgG_FC heavy chain (G), anti‐canine IgA heavy chain (A), anti‐canine IgM heavy chain (M), and anti‐canine light chain (L) antibodies. (A) Serum PE and IF results from a healthy control English bulldog with no CD21+ B‐cell lymphocytosis reveal polyclonal immunoglobulin proteins that predominantly label with anti‐IgG_FC. (B) Serum PE and IF results from a non‐bulldog with polyclonal gammopathy. Immunoglobulin proteins form a broad smear and predominantly label with anti‐IgG_FC. (C) Serum PE and IF results from an English bulldog B‐cell lymphocytosis case diagnosed with polyclonal gammopathy characterized by broad peaks in beta and gamma regions on PE, with no evidence of restricted bands by PE or IF. There is heavy labeling with anti‐IgA by IF relative to IgG_FC, which is atypical as compared to the control (A), and a lack of a hypergammaglobulinemia, which is atypical for an IgG‐centric polyclonal gammopathy. (D) Serum PE and IF results from an English bulldog B‐cell lymphocytosis case diagnosed with restricted polyclonal gammopathy, characterized by multiple restricted peaks within a polyclonal background. By IF, the majority of protein labels with anti‐IgA. (E) Plasma PE and IF results from an English bulldog B‐cell lymphocytosis case with a clonal immunoglobulin gene rearrangement by PARR. There is a tall narrow peak in the beta 2 region on PE, which labels with anti‐IgM by IF, consistent with an IgM monoclonal gammopathy

FIGURE 3

Blood smear and histopathology and immunohistochemistry of spleen from English bulldog cases with polyclonal B‐cell lymphocytosis. (A) Peripheral blood film from an English bulldog with B‐cell lymphocytosis and polyclonal immunoglobulin gene rearrangements. Lymphocytes are small with condensed chromatin and scant basophilic cytoplasm (Wright Giemsa, ×60 objective, 10 μm scale bar). (B‐D) Histopathology of spleen from a different English bulldog with B‐cell lymphocytosis and polyclonal immunoglobulin gene rearrangements. There is lymphoid hyperplasia characterized by nodules of multifocal to rarely coalescing lymphoid follicular structures (B, H&E, ×2 objective, 500 μm scale bar). The follicular structures are composed of primarily small lymphocytes with condensed chromatin with fewer intermediate‐sized lymphocytes and scattered lymphocytes with marginal zone appearance with a single central prominent nucleolus (C, H&E, ×40 objective, 20 μm scale bar). Lymphocytes within the follicles are predominated by B‐cells with strong nuclear immunoreactivity for PAX5 (D, PAX5, Fast Red chromogen, ×4 objective, 200 μm scale bar)

FIGURE 4

B‐cell expression of CD25, class II MHC, and CD21 in English bulldog B‐cell lymphocytosis cases compared to clinically healthy non‐bulldog controls, English bulldog controls with normal B‐cell counts, and small breed B‐cell chronic lymphocytic leukemia (BCLL) cases. Expression of CD25 (A), class II MHC (B) and CD21 (C) by flow cytometry is plotted for individual cases. Lines depict the median and interquartile range for each group. English bulldog cases had significantly lower expression of CD25 and class II MHC and significantly higher expression of CD21 compared to peripheral blood B‐cells from healthy non‐bulldog and English bulldog controls and small breed BCLL cases

FIGURE 5

IgA, IgM, and IgG_FC protein quantification by ELISA in English bulldog cases with B‐cell lymphocytosis. Each English bulldog case is colored consistently across the 3 graphs. Data points plotted at 10 g/dL were above the limits of quantification for the assay. Dotted lines represent the mean (black line) and range (gray lines) of values for 7 control dogs. English bulldog cases had significantly greater quantities of serum IgA (P < .001), and a subset of cases had increased IgM, compared to control dogs. English bulldogs had significantly less IgG_FC than controls (P = .004)

FIGURE 6

Signalment and B‐cell CD25 expression in nonclonal and clonal English bulldog cases with B‐cell lymphocytosis. (A) The age at diagnosis for nonclonal cases with polyclonal or restricted polyclonal immunoglobulin PARR results and clonal cases with clonal immunoglobulin PARR results. Clonal cases (median, 8.2 years old) were significantly older than nonclonal cases (median, 6.3 years old) (P = .002). (B) The percentage of males and females within nonclonal and clonal groups is presented. There were significantly more males in the nonclonal group (P = .03). (C) The percentage of B‐cells expressing CD25 by flow cytometry in nonclonal and clonal cases. Nonclonal cases had significantly lower CD25 expression than clonal cases (P = .003)

FIGURE 7

Sequential PCR for antigen receptor rearrangements (PARR) results with immunoglobulin primers for 3 English bulldog cases with B‐cell lymphocytosis. For each case, immunoglobulin PARR results at initial presentation (top) and sequential PARR results (bottom) 3.4‐64.5 months after diagnosis are presented. The size of the PCR amplicons is indicated along the horizontal axis. Restricted peaks persist over time and are identical in size to those present at initial presentation. The PARR tracings maintain a similar pattern over time in 2 cases (A, B) and become more restricted in a case that has been monitored over 5 years (C)