Reflectance confocal microscopy-guided laser ablation of basal cell carcinomas: initial clinical experience

Abstract

Laser ablation offers a procedure for precise, fast, and minimally invasive removal of superficial and early nodular basal cell carcinomas (BCCs). However, the lack of histopathological confirmation has been a limitation toward widespread use in the clinic. A reflectance confocal microscopy (RCM) imaging-guided approach offers cellular-level histopathology-like feedback directly on the patient, which may then guide and help improve the efficacy of the ablation procedure. Following an ex vivo benchtop study (reported in our earlier papers), we performed an initial study on 44 BCCs on 21 patients in vivo, using a pulsed erbium:ytterbium aluminum garnet laser and a contrast agent (aluminum chloride). In 10 lesions on six patients, the RCM imaging-guided detection of either presence of residual tumor or complete clearance was immediately confirmed with histopathology. Additionally, 34 BCCs on 15 patients were treated with RCM imaging-guided laser ablation, with immediate confirmation for clearance of tumor (no histopathology), followed by longer-term monitoring, currently in progress, with follow-up imaging (again, no histopathology) at 3, 6, and 18 months. Thus far, the imaging resolution appears to be sufficient and consistent for monitoring efficacy of ablation in the wound, both immediately postablation and subsequently during recovery. The efficacy results appear to be promising, with observed clearance in 19 cases of 22 cases with follow-ups ranging from 6 to 21 months. An additional 12 cases with 1 to 3 months of follow-ups has shown clearance of tumor but a longer follow-up time is required to establish conclusive results. Further instrumentation development will be necessary to cover larger areas with a more automatically controlled instrument for more uniform, faster, and deeper imaging of margins.

Keywords: basal cell carcinoma; confocal microscopy; image-guided therapy; laser ablation; skin cancer.

Fig. 1

Detection and demarcation of preablation margins with RCM imaging. (a) Example of an $8\mathrm{mm}\times 8\mathrm{mm}$ mosaic captured at the dermal–epidermal junction. The red dashed lines from 12 to 6 and 3 to 9 o’clock indicate the quadrants utilized to keep localized record of the tumor location and estimated depth. The quadrants are defined in clockwise orientation. The tumor margins are delineated with the dashed yellow boundary, and the corresponding selected regions for control and ablation are shown (this is only for those 10 cases in which a thin excision was performed immediately postablation for histology correlation). (b) Images at different depths from a stack, captured at the location of the red square in (a), contain features of BCC used for estimation of tumor depth: at $\sim 60\mu \mathrm{m}$ (epidermal layer) enlarged blood vessels (yellow arrows); at $100\mu \mathrm{m}$ enlarged blood vessels (yellow arrows) and tumor islands (red arrows); and at $\sim 160\mu \mathrm{m}$ enlarged blood vessels (yellow arrow), tumor island (red arrow), and cell palisading (green arrow) surrounding the tumor island.

Fig. 2

Postlaser-ablated wound topography and RCM video-imaging approach. (a) The lines and arrows on the clinical photograph illustrate the approach of using a paper ring (orange ring) to isolate each quadrant of the wound for imaging and provide a more precise location of findings with respect to the clinical coordinates established by the surgeon. The wound edge (white boundary) is used as a reference for imaging along the epidermal margins, and imaging of the peripheral dermal margins below the edge (yellow dotted boundary) and at the center of the wound. The blue arrows illustrate that videos were acquired along the epidermal margin, starting from the 12 to 3, and proceeding from 3 to 6, 6 to 9, and 9 to 12 o’clock positions. (b) A cartoon of the side view of a postlaser-ablated wound showing the epidermal margins and deep dermal margins (base of wound).

Fig. 3

Laser ablation guided with perioperative feedback from RCM imaging of BCC tumor morphology with cellular-level resolution. (a) Preablation: mosaics and stacks are acquired to estimate BCC margins and tumor depth preablation. The use of Vivascope 1500 with a tissue contact ring to acquire mosaics and stacks preablation (top row, two panels on the right). (b) Laser ablation: number of passes is selected based on the estimated depth of tumor to obtain the sufficient depth of ablation for complete removal of tumor. (c) Postablation: videos are acquired with a Vivascope 3000 to verify clearance of margins and stacks to review suspicious areas as a function of depth after ablation (bottom). Video mosaics are generated to visualize larger fields of view postablation. The ablation guided by RCM imaging protocol mimics the standard procedure of Mohs surgery guided by frozen pathology.

Fig. 4

Video mosaic of a postablation wound that was produced from an RCM imaging video. (a) Clearance of tumor is seen after the removal of $\sim 130\mu \mathrm{m}$ of tissue with six passes of $25\mathrm{J}/{\mathrm{cm}}^2$; (b) enlarged view [of the red square in (a)] of the peripheral epidermal margin showing intact skin (left of yellow dotted line), bright nuclei of the basal cells, due to uptake of contrast agent (aluminum chloride), at the dermal–epidermal layer (shown with the yellow arrows); and (c) enlarge view of collagen fibers in the normal dermis at the deeper margin (inside of the white square, indicated by red arrows).

Fig. 5

RCM images showing morphologic structures that are typically seen in postablation wounds: (a) the wound edge (red line), with intact skin (top) showing the honeycomb pattern of dark nuclei and bright cytoplasm of the epidermal cells, at the wound edge bright nuclei (green arrows) of the exposed epidermis (enhanced by the aluminum chloride) and deeper in the dermis (lower side) collagen fibers at the dermis level (yellow arrows); (b) exposed of epidermis in the wound with cobblestone pattern of bright nuclei (yellow arrow); (c) inflammatory cells (green arrows), bright rounded small nuclei as seen in the deep dermal margin and hair follicles (lower yellow arrow); (d) bright fibrillar structures corresponding to fine collagen bundles; (e) thick collagen bundles seen in the deep dermis; and (f) eccrine glands (yellow arrow). Scale bar is $250\mu \mathrm{m}$ for all images.

Fig. 6

In vivo detection of clearance of tumor or presence of residual tumor postablation, for ablation with four passes at fluence of $25\mathrm{J}/{\mathrm{cm}}^2$. (a) A clinical photograph image of the lesion postablation, with a solid blue demarcation line, showing the intact area of the lesion that contains tumor (left, nonablated control area) and the ablated area (right); (b)–(c) RCM image of residual nodular BCC (white and black arrows) at the peripheral epidermal margin, located inside the solid red square in (a); (d) H&E-stained histopathology that confirms RCM findings, showing intact tumor [black arrow at the left of the solid blue line, corresponding to the nonablated control area in (a)] and the surgical margin (made by Mohs surgeon JC and labeled with light blue ink before treatment); and residual tumor in the ablated area (right of the solid blue line inside the solid red squares) corresponds to that in (b) (white arrows) and (c) black arrows. The vertically sectioned histology allows visualization of the residual tumor (green and black arrows) at the edge of the crater-shaped wound validating the continuity of the tumor from the nonablated control area to the ablated region.

Fig. 7

Follow-up RCM imaging of lesion number 3 (superficial BCC) at 1, 4, 7, and 12 months after ablation. In this case, ablation was with six passes at fluence of $25\mathrm{J}/{\mathrm{cm}}^2$. Presence of enlarged blood vessels (yellow arrows) and amorphous collagen fibers (red arrows) at 1 and 4 months (top) were observed as part of the wound healing process. At 7 months, presence of enlarged blood vessel (yellow arrows) was still observed with thickened collagen fibers (red arrows). Normal skin patterns without features of BCC were seen at 12 months of postablation. Dark nuclei patterns (yellow arrows, lower right) similar to that observed in normal skin epidermis.