Gigapixel surface imaging of radical prostatectomy specimens for comprehensive detection of cancer-positive surgical margins using structured illumination microscopy

Abstract

Achieving cancer-free surgical margins in oncologic surgery is critical to reduce the need for additional adjuvant treatments and minimize tumor recurrence; however, there is a delicate balance between completeness of tumor removal and preservation of adjacent tissues critical for normal post-operative function. We sought to establish the feasibility of video-rate structured illumination microscopy (VR-SIM) of the intact removed tumor surface as a practical and non-destructive alternative to intra-operative frozen section pathology, using prostate cancer as an initial target. We present the first images of the intact human prostate surface obtained with pathologically-relevant contrast and subcellular detail, obtained in 24 radical prostatectomy specimens immediately after excision. We demonstrate that it is feasible to routinely image the full prostate circumference, generating gigapixel panorama images of the surface that are readily interpreted by pathologists. VR-SIM confirmed detection of positive surgical margins in 3 out of 4 prostates with pathology-confirmed adenocarcinoma at the circumferential surgical margin, and furthermore detected extensive residual cancer at the circumferential margin in a case post-operatively classified by histopathology as having negative surgical margins. Our results suggest that the increased surface coverage of VR-SIM could also provide added value for detection and characterization of positive surgical margins over traditional histopathology.

Figure 1. VR-SIM imaging system and prostate circumferential surface imaging procedure.

(a) The VR-SIM system is mounted on a movable cart. (b) Close-up of the VR-SIM imaging system with a prostate on the microscope stage. The imaging objective is below the prostate in an epi-illumination configuration. (c) Close-up (side view) of the posterior imaging position of the prostate. The posterior surface is contacting the slide; the imaging objective is seen below the prostate. The prostate is rotated about the urethral axis (yellow dashed line) to enable imaging of the prostate circumference. The prostate and the seminal vesicles/ductus deferens are indicated in the photograph. (d–g) Top views of the circumferential imaging positions (posterior, left lateral, anterior, right lateral). A wooden dowel rod is inserted through the urethra to demonstrate the rotation axis between imaging positions. In (d), the plane between the prostate and the attached seminal vesicles (s.v.) and ductus deferens (d.d.) is indicated by a dashed yellow line.

Figure 2. Circumferential VR-SIM surface images of the prostate of Case 6.

(a) Posterior prostate surface, with seminal vesicles and ductus deferens situated above the dashed yellow line, (b) left lateral prostate surface, (c) anterior prostate surface, with portions of the seminal vesicles and ductus deferens visible above the dashed yellow line and (d) right lateral prostate surface. The image area and image size in gigapixels (GP) is provided for each image, and totals 60.5 cm² and 15 GP in all. The orientation of each image with respect to the prostate apex (AP), base (B), anterior surface (AN), posterior surface (P), left lateral surface (L), and right lateral surface (R) is indicated by the direction arrows in each image.

Figure 3. Multi-scale visualization of prostate surface images from the macro-scale to the micro-scale.

(a) VR-SIM image of the 6.5 cm² right lateral surface of Case 6, (b) Zoom of the large dashed yellow box area indicated in (a). Features such as the neurovascular bundles (bright spindly features) and smooth prostatic pseudocapsule/fascia are apparent. (c) Zoom of the dashed yellow box in (b). The image is marked by a single neurovascular bundle extending across the field of view. A bright feature (d) is enclosed by a dashed yellow box, corresponding to a single prostate gland. Individual rounded nuclei are clearly resolved.

Figure 4. Clinical image atlas of VR-SIM versus histopathology images of benign and malignant prostate structures.

Arrows indicate corresponding features between VR-SIM and H&E-stained histology slides. (a,b) normal skeletal muscle, (c,d) nerves with ganglia, (e,f) neurovascular bundle, (g,h) smooth muscle bundle, (i,j) adipose tissue, (k,l) normal prostate glands with ‘cauliflower’ appearance, (m,n) benign prostate glands with elongated and rounded appearances, (o–r) examples of prostate adenocarcinoma, acinar type, Gleason grade 3 + 4 = 7.

Figure 5. VR-SIM image of the posterior surface of Case 14, with a pathologically-confirmed positive surgical margin with perineural invasion.

(a) Full surface image of the posterior side, with orientation provided by the directional arrows. Areas of interest on the right and central posterior surface are indicated by the dashed yellow boxes. (b,c) Areas of perineural invasion observed on the VR-SIM image. Small dense cancer glands (yellow arrows) are observed tracking along a prominent nerve (white arrows). (d) Histological cross-section from the right posterior quadrant, indicating the presence of malignant glands touching the inked surgical margin, as well as the presence of perineural invasion (glands wrapping around a nerve). (e) Large area of perineural invasion observed on VR-SIM, with small, dense, highly cellular glands (yellow arrows) tracking along nerve fibers (red asterisks). (f) Small, rounded and elliptical glands with malignant appearance (yellow arrows) on the VR-SIM image and (g) histological section of malignant glands at the inked surgical margin (black arrows), but not associated with nerve tissue.

Figure 6. VR-SIM image of the anterior surface of Case 22, depicting a positive surgical margin due to an intra-prostatic incision.

(a) Full VR-SIM image of the anterior surface with orientation provided by directional arrows. An area corresponding to exposed malignant glandular parenchyma is outlined in red. (b) Zoom of the area bounded by the dashed yellow box in (a). The area is characterized by extensive malignant glandular parenchyma, similar to that observed in the prostate cancer biopsy image from the atlas in Fig. 4o–p. (c,d) Histological confirmation of malignant glands similar to those in the VR-SIM image (yellow arrows) at the inked surgical margin, where the margin has breached the prostate capsule.

Figure 7. VR-SIM image of the posterior surface of Case 16 with a positive surgical margin missed by permanent histopathology.

(a) Full surface image of the posterior surface with orientation provided by the directional arrows. A nerve with infiltrating malignant glands is outlined in red in the left posterior area, with two regions of interest bounded by yellow dashed boxes. (b,c) Zooms of the VR-SIM image corresponding to the dashed yellow boxes in (a), showing the presence of small glands infiltrating the nerve structure (yellow arrows). (d) Histological section from the left posterior quadrant, showing a region of malignant glands (yellow arrows) invading nerves (yellow asterisks) ~500 μm below the surgical margin, consistent with the VR-SIM findings. Although the tumor does not touch the inked margin (black arrow) in this particular section, a prominent nerve is observed at the inked margin that may be associated with the perineural invasion observed in the VR-SIM image.