Association of macrophage and lymphocyte infiltration with outcome in canine osteosarcoma

Abstract

Immunotherapeutic strategies have shown promise for the treatment of canine osteosarcoma (cOSA). Very little is known about the immune microenvironment within cOSA, however, limiting our ability to identify potential immune targets and biomarkers of therapeutic response. We therefore prospectively assessed the disease-free interval (DFI) and overall survival time (ST) of 30 dogs with cOSA treated with amputation and six doses of adjuvant carboplatin. We then quantified lymphocytic (CD3+, FOXP3+) and macrophage (CD204+) infiltrates within the primary tumours of this cohort using immunohistochemistry, and evaluated their association with outcome. Overall, the median DFI and ST were 392 and 455 days, respectively. The median number of CD3+ and FOXP3+ infiltrates were 45.8 cells/mm (4.6-607.6 cells/mm ) and 8.5 mm (0-163.1 cells/mm ), respectively. The median area of CD204+ macrophages was 4.7% (1.3%-23.3%), and dogs with tumours containing greater than 4.7% CD204+ macrophages experienced a significantly longer DFI (P = 0.016). Interestingly, a significantly lower percentage of CD204+ macrophages was detected in cOSA arising from the proximal humerus compared to other appendicular bone locations (P = 0.016). Lymphocytic infiltrates did not appear to correlate with outcome in cOSA. Overall, our findings suggest that macrophages may play a role in inhibiting cOSA progression, as has been suggested in human osteosarcoma.

Metastatic immune infiltrates correlate with those of the primary tumour in canine osteosarcoma

“…Broadly, host‐tumour interactions can vary from tumour‐promoting inflammation to effective anti‐tumour immunity . This may begin to explain the somewhat paradoxical increase in CD204+ macrophage infiltration in pulmonary metastases in this study given our previous findings identifying CD204+ macrophages in primary tumours as a positive prognostic factor in cOSA . This shift towards tumour‐promoting inflammation can involve alterations to the functionality of these macrophages between sites of disease .…”

“…For example, Biller et al identified a high CD8+/T‐regulatory cell (Treg) ratio through flow cytometry in the blood as a positive prognostic factor for survival although this did not reach significance within the tumour . In addition, Modiano et al reported that elevated T‐cell infiltrates in cOSA after treatment with Fas‐ligand gene therapy was associated with improved survival, while we identified elevated CD204+ macrophage tumour infiltration as being associated with a prolonged disease‐free interval (DFI) . To the authors' knowledge, the degree of leukocyte infiltration into pulmonary metastatic cOSA is currently unknown however.…”

“…The overall median densities of CD3+, FOXP3+, Pax‐5+, and CD204+ infiltrates in primary and metastatic lesions are listed in Table . Representative images of low and high Pax‐5+ infiltration are shown in Figure , while representative images of CD3+, FOXP3+ and CD204+ infiltrates in cOSA are presented in a previous manuscript by our group …”

“…Similar to a previous study by our group, we selected 3 cellular 100× fields on hematoxylin and eosin stained slides from each tumour to mimic the construction of tissue microarrays used in other studies to evaluate immune infiltrates in hOSA . Selected areas were marked on each IHC stained slide, enabling evaluation of each marker in similar areas on subsequent slides.…”

“…The number of cells/cellular area in pixels was then converted to the number of cells/mm 2 by dividing by 4 169 024 pixels 2 /mm 2 (2041.8 × 2041.8 pixels/mm 2 ). Macrophages were abundant and therefore measured as the percent area of CD204+ staining within each cellular (%CD204+) area using Image J, similar to that previously described by our group and others . The average density of CD3, FOXP3 and Pax‐5 infiltration (cells/mm 2 ), and %CD204+ (%) was then determined across the cellular fields evaluated for each sample.…”