Evaluation of Breast Pathologies Using Multi-Photon Microscopy
Yury Sheykin, Yuankai K Tao, Osman O Ahsen, Dejun Shen, Daniel Schmolze, Nicole Johnson, James G Fujimoto, James L Connolly. Beth Israel Deaconess Medical Center, Boston, MA; Massachusetts Institute of Technology, Cambridge, MA
Background: Multi-photon microscopy (MPM) enables high-resolution imaging of intact tissue specimens using both endogenous and exogenous fluorescent and harmonic generation contrast. MPM is well-suited for visualizing cytologic and architectural markers used to differentiate between benign and neoplastic tissues including nuclear size and shape, cellular arrangement in tissue, and characteristics of intercellular stroma, all of which are assessed when examining hematoxylin and eosin (H&E)-stained histology slides. The advantage of MPM over standard histology is its ability to rapidly image freshly excised specimen surfaces without the need for histological processing, which could enable intraoperative margin assessment. Here, we investigate the utility of MPM for evaluating breast pathologies.
Design: In a prospective study, 179 fresh human breast tissue specimens from 50 patients were imaged using a 100 fs laser source at 740 nm wavelength. Specimens were rapid stained with a nuclear stain Acridine Orange (AO). Dual channels were used to detect exogenous AO fluorescence nuclear contrast and intrinsic second-harmonic generation stromal contrast. Multiple overlapping 480 μm2 fields were automatically imaged and mosaicked in post-processing, and MPM color channels were remapped to a colormap resembling H&E staining. Specimens were subsequently fixed in formalin and processed for corresponding H&E sections.
Results: Of 179 specimens, 138 MPM and corresponding H&E datasets were randomized and scored by 3 pathologists in a double-blinded study. The study included 59 normal (NBT), 5 fibroadenoma (FA), 11 usual ductal hyperplasia (UDH), 10 fibrocystic changes (FC), 20 ductal carcinoma in situ (DCIS), 5 lobular carcinoma in situ (LCIS), 38 invasive ductal carcinoma (IDC), and 9 invasive lobular carcinoma (ILC) specimens (figure). Analysis of the read showed that MPM had a sensitivity and specificity of 95.0% and 92.9%, respectively, for distinguishing between benign (NBT, FA, UDH, and FC) and malignant (DCIS, LCIS, IDC, and ILC) breast tissue as compared to standard H&E histology with an interobserver agreement (Fleiss' kappa) of 0.85.
Conclusions: MPM provides high sensitivity and specificity for distinguishing between benign and neoplastic breast tissue. The advantage of MPM imaging is that it potentially allows rapid nondestructive intraoperative margin assessment of resected breast specimens without the need for frozen section exam.
Monday, March 4, 2013 8:00 AM
Proffered Papers: Section B, Monday Morning