Identification of prognostic collagen signatures and potential therapeutic stromal targets in canine mammary gland carcinoma

<div><p>Increasing evidence indicates that the tumor microenvironment plays a critical role in regulating the biologic behavior of breast cancer. In veterinary oncology, there is a need for improved prognostic markers to accurately identify dogs at risk for local and distant (metastatic) recurrence of mammary gland carcinoma and therefore would benefit from adjuvant therapy. Collagen density and fiber organization have been shown to regulate tumor progression in both mouse and human mammary tumors, with certain collagen signatures predicting poor outcomes in women with breast cancer. We hypothesized that collagen signatures in canine mammary tumor biopsies can serve as prognostic biomarkers and potential targets for treatment. We used second harmonic generation imaging to evaluate fibrillar collagen density, the presence of a tumor-stromal boundary, tumor associated collagen signatures (TACS) and individual collagen fiber characteristics (width, length and straightness) in grade I/II and grade III canine mammary tumors. Collagen density, as well as fiber width, length and straightness, were inversely correlated with patient overall survival time. Notably, grade III cases were less likely to have a tumor-stromal boundary and the lack of a boundary predicted poor outcome. Importantly, a lack of a defined tumor-stromal boundary and an increased collagen fiber width were associated with decreased survival even when tumor grade, patient stage, ovariohysterectomy status at the time of mammary tumor excision, and histologic evidence of lymphovascular invasion were considered in a multivariable model, indicating that these parameters could augment current methods to identify patients at high risk for local or metastatic progression/recurrence. Furthermore, these data, which identify for the first time, prognostic collagen biomarkers in naturally occurring mammary gland neoplasia in the dog, support the use of the dog as a translational model for tumor-stromal interactions in breast cancer.</p></div

Collagen density predicts poor outcome in canine mammary tumors.

<p>Integrated density of collagen signal from SHG images was quantified using Fiji (Image J) software. Graph represents averages from 5 images per tumor from 11 grade I/II and 9 grade III mammary tumors. *p<0.05 via an unpaired Mann-Whitney test (A). Kaplan-Meier survival curve for 18 dogs with collagen integrated density higher or lower than the mean integrated density value (B). The log-rank test was used to evaluate whether the collagen density significantly impacted survival (p = 0.013, HR 4.099, 95%CI 0.860–19.520).</p

Canine mammary tumor clinical parameters predict outcome.

<p>Kaplan-Meier survival curves and Cox regression univariate analysis was used to evaluate whether clinical parameters significantly impacted survival. A. Grade: grade I/II vs. grade III mammary tumors (p<0.001, hazard ratio (HR) 10.106, 95%CI 3.071–33.254). B. Stage: Stage 1/2 vs. 3/4 mammary tumors (p = 0.042, HR 2.778, 95%CI 1.039–7.420). C. Lymphovascular Invasion: Evidence vs. No Evidence (p<0.001, HR 7.462, 95%CI 2.359–23.603). D. Excision Completeness: Complete vs. Incomplete (p = 0.014, HR 4.255, 95%CI 1.335–13.558). E. OHE: At time of excision vs. prior to excision or not performed (p = 0.009, HR 0.133, 95%CI 0.029–0.604).</p

Collagen fiber characteristics in canine mammary carcinoma biopsies are associated with patient survival.

<p>Kaplan-Meier survival curve for 27 dogs, comparing lower and higher than the mean collagen fiber width (A; p = 0.009, HR 3.414, 95%CI 1.260–9.252) and length (B; p = 0.048, HR 2.501, 95%CI 0.895–6.990), as well as straightness (C; p = 0.025, HR 3.324, 95%CI 1.284–8.601), assessed by SHG analysis of histopathologic sections. The log-rank test was used to evaluate whether the collagen variable significantly impacted survival.</p

TACS in canine mammary tumor OS.

<p>TACS in canine mammary tumor OS.</p

Absence of a tumor-stromal boundary predicts poor outcome in canine mammary tumors.

<p>A panel of 7 observers graded SHG images as having (1) or lacking (0) a tumor-stromal boundary. The boundary score for each tumor represents the average of the 7 evaluators’ scores and the 5 images per tumor. Boundary scores were compared between 15 grade I/II and 19 grade III tumors. *p<0.05 via a Mann-Whitney test (A). Kaplan-Meier survival curve for 27 dogs with boundary scores higher or lower than the mean boundary score (B). The log-rank test was used to evaluate whether the boundary score significantly impacted survival (p = 0.012, HR 0.316, 95%CI 0.092–1.087).</p