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.nonlinear microscopy | breast cancer | pathology | imaging B reast cancer is the most common cancer and cause of cancerrelated death in women worldwide (1). In the United States, one in eight women will develop breast cancer in their lifetimes with an estimated 226,870 new cases and 39,510 deaths in 2012 (2). Breast-conserving therapy (BCT), lumpectomy followed by adjuvant radiation and/or chemotherapy and hormonal therapy, is a standard of care for early breast cancer and achieves survival and local recurrence rates comparable to mastectomy while providing superior cosmetic outcomes and reduced morbidity (3-5). Local recurrence rates are correlated with surgical margin status, which is evaluated using intraoperative examination and postoperative histopathology. However, there is no consensus on surgical margin widths, and differing conventions for negative and close margins have shown inconsistent correlations with therapeutic outcomes (6-9). Despite these inconsistencies, margins initially positive for invasive cancer or ductal carcinoma in situ (DCIS) almost always require surgical reexcision (92.6% when positive for DCIS and 83.5% when positive for invasive cancer) (9), and up to 40% of all women undergoing BCT require repeat surgeries from positive or close margins (10-12). In addition to surgical complications and incr...

Multiscale nonlinear microscopy and widefield white light imaging enables rapid histological imaging of surgical specimen margins

“…Because physical sectioning accounts for the vast majority of processing delay for both FFPE and FSA histology, optical sectioning can dramatically reduce processing times and enable intraoperative histological examination in scenarios where FFPE and FSA histology are too time consuming. Various methods have been proposed for imaging breast, prostate and other surgical margins without physical sectioning, including optical coherence tomography (OCT) [18][19][20][21][22], reflectance confocal microscopy (RCM) [23,24], confocal fluorescence microscopy (CFM) [25][26][27][28][29], structured illumination microscopy (SIM) [30], light sheet microscopy [31], microscopy with ultraviolet surface excitation (MUSE) [32,33], and nonlinear microscopy (NLM) [34][35][36][37][38]. Stimulated Raman scattering (SRS) has also been demonstrated for surgical histology [39], but typically has appreciably lower imaging speed or signal to noise ratio when performed without physical sectioning in reflectance mode.…”

Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue

“…A water immersion objective was selected for 20× imaging in order to be consistent with our previous study. 24 Each image frame was acquired at 1024 × 1024 pixels. With the 20× and 10× objectives, the fields of view were 0.49 and 1.1 mm, with pixel sizes of 0.48 and 1.05 μm, respectively.…”

“…11 An alternative to time-consuming embedding and physical sectioning of fixed tissue is optical sectioning of unfixed tissue. Imaging unfixed tissue specimens has been demonstrated using techniques such as reflectance confocal microscopy (RCM), [12][13][14][15] confocal fluorescence microscopy (CFM), [16][17][18][19][20][21] multiphoton microscopy (MPM), [22][23][24] structured illumination microscopy, 25 spectrally encoded confocal microscopy, 26 and optical coherence tomography. 27,28 Furthermore, recently it has been demonstrated that fluorescent contrast agents can be rapidly diffused through viable human tissue, enabling fluorescent labeling without fixation.…”

“…Presently, only MPM has been comprehensively evaluated for breast pathology assessment. 24 This study performed a blinded reading of MPM mosaic images color remapped for an H&E-like appearance from 179 unfixed, discarded, and randomly selected breast tissue specimens from 50 patients. Specimens were mosaic imaged over large fields, fixed, and then corresponding paraffin-embedded H&E slides were obtained.…”

Methylene-blue aided rapid confocal laser endomicroscopy of breast cancer

“…6 One potential avenue for reducing reoperation rates following BCS is the use of high-resolution intraoperative imaging to determine if the radial margins are tumor-free during surgery. Optical imaging techniques, including optical coherence tomography, 7 confocal reflectance microscopy, 8 and nonlinear microscopy, 9 are currently being investigated for in-vivo visualization of breast morphology.…”