Assessment of breast pathologies using nonlinear microscopy

Abstract

Rapid intraoperative assessment of breast excision specimens is clinically important because up to 40% of patients undergoing breast-conserving cancer surgery require reexcision for positive or close margins. We demonstrate nonlinear microscopy (NLM) for the assessment of benign and malignant breast pathologies in fresh surgical specimens. A total of 179 specimens from 50 patients was imaged with NLM using rapid extrinsic nuclear staining with acridine orange and intrinsic second harmonic contrast generation from collagen. Imaging was performed on fresh, intact specimens without the need for fixation, embedding, and sectioning required for conventional histopathology. A visualization method to aid pathological interpretation is presented that maps NLM contrast from two-photon fluorescence and second harmonic signals to features closely resembling histopathology using hematoxylin and eosin staining. Mosaicking is used to overcome trade-offs between resolution and field of view, enabling imaging of subcellular features over square-centimeter specimens. After NLM examination, specimens were processed for standard paraffin-embedded histology using a protocol that coregistered histological sections to NLM images for paired assessment. Blinded NLM reading by three pathologists achieved 95.4% sensitivity and 93.3% specificity, compared with paraffin-embedded histology, for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. Interobserver agreement was κ = 0.88 for NLM and κ = 0.89 for histology. These results show that NLM achieves high diagnostic accuracy, can be rapidly performed on unfixed specimens, and is a promising method for intraoperative margin assessment.

Keywords: breast cancer; imaging; nonlinear microscopy; pathology.

Fig. 1.

Schematic of extended FOV nonlinear microscopy. (A) Dual detection channels collect TPF and SHG signals for nuclear and stromal contrast, respectively. High-magnification fields were imaged using a 20×, 0.95 N.A. objective with a 480 × 480-µm field sampled with 1,024 × 1,024 pixels at 19 frames per s. (B) Overlapping fields are automatically acquired using a motorized stage translating the specimen in a serpentine pattern and serial depth sections obtained by motorized translation of the optics. (C) Field distortion correction, pseudo-H&E color remapping, and (D) image mosaicking are applied in postprocessing.

Fig. 2.

Normal human breast tissue imaged using NLM. (A) NLM and (B) corresponding paraffin-embedded H&E histology. (Scale bar: 1 mm.) Magnified regions show a TDLU (red box) (scale bar: 150 µm), adipocytes (green box) (scale bar: 150 µm), and fibrocollagenous stroma (blue box) (scale bar: 150 µm). Image shows maximum-intensity projection through eight depth sections acquired at 25-µm increments from the tissue surface. FOV: 6 × 6.25 mm (600 megapixels). NLM data: tinyurl.com/a3qgzco. H&E data: tinyurl.com/ajdwe8d.

Fig. 3.

Human breast specimen with DCIS in various histologic patterns. (A) NLM and (B) corresponding paraffin-embedded histology showing different DCIS subtypes. (Scale bar: 1 mm.) Finger-like projections of neoplastic cells invading the duct lumen are indicative of micropapillary DCIS (red box). (Scale bar: 250 µm.) Irregular-shaped cavities with cellular debris and comedo plug are indicative of comedo DCIS (green box). (Scale bar: 250 µm.) Individual cell nuclei at the periphery of the comedo DCIS exhibit prominent nucleoli consistent with high-grade DCIS (blue box). (Scale bar: 50 µm.) Image shows maximum-intensity projection through six depth sections acquired at 10-µm increments from the tissue surface. FOV: 8.25 × 5 mm (660 megapixels). NLM data: tinyurl.com/cwyly6s. H&E data: tinyurl.com/9teff9a.

Fig. 4.

Human breast specimen with regions of DCIS and IDC adjacent to normal tissue. (A) NLM and (B) corresponding paraffin-embedded histology showing DCIS and IDC (Right) vs. benign breast tissue (Left). (Scale bar: 2 mm.) Sieve-like appearance is associated with cribriform DCIS, consistent with low- to intermediate-grade DCIS (red box). (Scale bar: 500 µm.) IDC region shows neoplastic cells infiltrating breast stroma (green box). (Scale bar: 500 µm.) Magnified view of abnormal cellular regions show features consistent with columnar cell change (blue box). (Scale bar: 500 µm.) Image shows maximum-intensity projection through eight depth sections acquired at 15-µm increments from the tissue surface. FOV: 18.25 × 12 mm (3.5 gigapixels). NLM data: tinyurl.com/ccueff3. H&E data: tinyurl.com/cnvp8yy.