Second harmonic generation imaging of human breast cancer biopsies. A and B: Two representative examples of H&E-stained slices that demonstrated varying histopathological findings and tumor grade. C and D: The corresponding second harmonic generation (SHG) images illustrate the complexity and variability of collagen localization in these samples. E–H: Insets in A and B show that endogenous fluorescence intensity from cells and stroma was preserved in histopathology slides where the corresponding SHG image can either be overlaid with the fluorescence image (E, F) or examined by itself (G, H) to visualize the relationship of collagen to cells. Scale bars (A–D) = 200 μm, and (E–H) = 50 μm.

Region of interest (ROI) analysis of tumor-associated collagen signature-3 (TACS-3) presence. A: The second harmonic generation (SHG) image of each entire tissue sample was overlaid with 14 ROIs, which were positioned and numbered reproducibly. Image intensity statistics were measured from each individual ROI independently. B: For TACS-3 analysis, each ROI was rated by each of three panelists for TACS-3 presence at ×3 zoom. A typical example of an ROI that would be rated “yes” is shown in ROIs 7, 9, and 12 (A). In ROI 9, bundles of straightened, aligned collagen fibers in the SHG image were observed to terminate at a region of negative space, which was confirmed by examination of the H&E image to be epithelial cells. The definitions for the TACS-3 evaluation scores are: Score 1: For each ROI, TACS-3 was classified as “present” if at least two panelists rated “yes.” The percentages of ROI (across all 14 measurements) rated as “present” were computed for each patient. Therefore, this score is a percentage measure of the number of TACS-3 events in a slice. A receiver operating curve (ROC) analysis was performed to determine “optimal” threshold values for classifying patients as either TACS-3 positive or TACS-3 negative. Specifically, the threshold value was determined by maximizing the positive likelihood ratio [sensitivity/(1 – specificity)] for predicting the 5-year disease-specific survival status. The threshold used here for score 1 was 0.1. Score 2: For each ROI, panelists' ratings of “no” were scored as 0 and “yes” were scored as 1. An average of the total number of “yes” ratings per ROI was computed across all 14 ROIs. Because there were three panelists, score 2 ranged in value between 0 and 3 for each patient. As for score 1 previously, an ROC analysis was performed to determine a threshold for splitting data as TACS-3 positive or TACS-3 negative. This threshold was determined to be 0.5. Score 3: For each ROI, TACS-3 was classified as “present” if at least two panelists rated “yes.” The patient sample was classified as “positive” for TACS-3 if at least one ROI was rated as “present” (ie, at least two panelists rated “yes”). Therefore, this is the least stringent of the three definitions where we determine whether any ROI with TACS-3 is present in the entire sample.

Tumor-associated collagen signature-3 (TACS-3) presence is correlated with poor survival. A–C: The Kaplan-Meier curves for all three TACS-3 scores demonstrated that both the disease-free survival and the disease-specific survival rates of patients rated as TACS-3 positive were significantly worse with time than those patients rated as TACS-3 negative. For score 1, the number of patients rated as TACS-3 negative (black lines) was n = 98, TACS-3 positive (red lines) n = 97. Score 2 TACS-3 negative n = 168, TACS-3 positive n = 27; score 3 TACS-3 negative n = 72, TACS-3 positive n = 123.

Classification and regression tree analysis of tumor-associated collagen signature-3 (TACS-3) score in predicting breast cancer survival. Using the classification and regression tree method for partitioning patient survival based on three independent predictor variables (previously derived from the multivariate analysis), the following decision tree was created. Tumor size and estrogen receptor (ER) status were better predictors of survival and were the first criteria used for splitting the data, where a final result of “yes” or “no” refers to whether or not the patient survived to 10 years. The predicted 10-year disease-specific survival probabilities for the various marker profiles are shown in parentheses with 95% confidence intervals. The value for the TACS-3 score 1 was shown to be able to split patient survival based on the threshold value of 0.04.