Intratumoral heterogeneity of second-harmonic generation scattering from tumor collagen and its effects on metastatic risk prediction

Metastases are the leading cause of breast cancer-related deaths. Tumor metastasis is affected by the microenvironment, including the extracellular matrix (ECM). Fibrillar collagen in the ECM produces an intrinsic optical signal called secondharmonic generation (SHG). The ratio of forward- to backward-scattered SHG photons (F/B) is sensitive to the collagen fiber internal structure and has been shown to be an independent prognostic indicator of metastasis-free survival time. We evaluated the effects of heterogeneity in the tumor ECM on F/B’s prognostic ability. Using SHG imaging we identified two distinct regions within 95 untreated primary tumor excisions: the tumor bulk and surrounding collagenous tumorstroma interface. We found that F/B measured in the tumor-stroma interface, but not tumor bulk, is prognostic of metastasis-free survival time (MFS) using both an intensity threshold selected by a blinded observer and adaptive thresholding. We calculated a 21-gene recurrence score (surrogate OncotypeDX®, or S-ODX) for each patient and using a Random Survival Forest method we found that F/B from the tumor-stroma interface, but not tumor bulk, and S-ODX contribute to predicting MFS in this patient cohort. These results suggest that F/B from the tumor-stroma interface of primary tumor excisions may provide information independent of genomic methods to stratify patients by metastatic risk and identify need for post-operative treatment.