2.1.

Laser Ablation Instrumentation

A pulsed erbium-doped yttrium aluminum garnet (Er:YAG, $2.94\mu \mathrm{m}$) laser (Sciton Profile, Palo Alto, California), equipped with a pulse duration of $\sim 250\mu \mathrm{s}$ (fixed), spot diameter of 4 mm, and fluence up to $25.0\mathrm{J}/{\mathrm{cm}}^2$ was used. More details can be found in our earlier papers.^19,20 One “pass” of $\sim 161\mathrm{ms}$ duration consists of a set of four individual pulses, each separated by 40 ms. [Note that shorter pulse durations, relative to the thermal relaxation time in skin, are usually preferable, such as femtosecond or picosecond lasers, which would result in highly selective “pure” ablation of thin cellular-level layers of tissue without any underlying thermal coagulation. Our particular choice of laser and pulse duration was mainly due to the routine use of Er:YAG lasers in dermatologic clinical settings, and thus the easy availability and access to the laser used by our collaborating Mohs surgeon (coauthor CSJC). For ablating superficial or nodular BCCs of $\sim 150$- to $200\text{-}\mu \mathrm{m}$ depth, very high selectivity is not necessary, and longer pulse durations on the order of 10 to 100 microseconds are clinically sufficient.] In this study, the fluence was fixed at $25.0\mathrm{J}/{\mathrm{cm}}^2$ for a single pass and, when necessary, a higher fluence was obtained by performing several passes, to produce a depth of ablation that reached the tumor’s deepest margins. The selection of the number of passes was based on an ex vivo study in human skin specimens, previously conducted, to characterize the depth of ablation versus number of passes. Increasing the number of passes to produce higher fluence values also increases the thermal residual damage to surrounding intact tissue postablation.²² As reported in our previous benchtop studies, the thermal damage depth for these laser parameters is 5 to $20\mu \mathrm{m}$, confirming the literature.²² Our study provided a set of parameters (number of passes at fixed fluence) to ablate BCCs while controlling thermal coagulation in the underlying wound and surrounding intact skin, to subsequently allow the uptake of contrast agent and enhance the appearance of nuclear patterns and tumor morphology for the detection of presence (or absence) of tumor postablation with RCM imaging. Thus, the uptake of aluminum chloride (seen in this in vivo study as well as in the previous benchtop studies), along with the wound healing and recovery process, indicates that healthy tissue remains undamaged. Further details may be gleaned from our earlier papers.^19,20

2.2.

Patients and Basal Cell Carcinomas Lesions

Twenty-one patients, with forty-four lesions, each with a diagnosis of superficial and/or nodular BCC, participated in this study. Prior to enrollment, patients gave consent to a research protocol, approved by Memorial Sloan Kettering Cancer Center’s (MSKCC’s) Institutional Review Board, for imaging and ablation. An initial group of six patients, with 10 BCCs that were confirmed with diagnostic biopsy, consented for RCM imaging-guided laser ablation. On these patients, immediately postablation, a thin excision was performed and the resulting frozen histology used for confirmation. A second set of 15 patients, on whom 34 superficial and early nodular BCCs had been clinically diagnosed [i.e., with only clinical (visual) examination and dermoscopy but without diagnostic biopsy], was treated with RCM imaging-guided laser ablation only (i.e., with no subsequent excision or histopathology), and all of these lesions are currently being followed up and noninvasively monitored with imaging.

2.3.

Reflectance Confocal Microscopy Imaging

Imaging on patients was performed with two reflectance confocal microscopes [Vivascope 1500 and Vivascope 3000, Caliber Imaging and Diagnostics (formerly, Lucid Inc.), Rochester, New York]. In both, the illumination is with the near-infrared wavelength of 830 nm, and imaging was done with a gel-immersion objective lens of magnification $30\times$ and 0.9 numerical aperture, optical sectioning of $\sim 3\mu \mathrm{m}$ and lateral resolution of $\sim 1\mu \mathrm{m}$. High-resolution RCM images were collected to a depth of $\sim 150$ to $200\mu \mathrm{m}$.

2.4.

Image-Guided Laser Ablation Protocol

Figures 1Fig. 2–3 show the complete perioperative imaging-guided protocol, with histopathology-like feedback obtained directly on the patient before, during, and after the ablation treatment.

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.

2.5.

Imaging-Guided Laser Ablation with Immediate Histology Confirmation

Ten BCCs lesions were treated and the imaging results immediately confirmed with histology. An $8\mathrm{mm}\times 8\mathrm{mm}$ area, centered within the lesion containing tumor, was selected for RCM imaging and ablation. The tumor in the surrounding area served as control when the lesion was greater in size. If the lesion was smaller than $8\mathrm{mm}\times 8\mathrm{mm}$, half of the lesion was selected for ablation with the remaining portion serving as control. RCM imaging-guided laser ablation protocol was executed and the postablation imaging detected the presence of residual tumor or complete clearance. Immediately afterward, our Mohs surgeon (coauthor CSJC) performed a thin excision and sent it to the laboratory for histology preparation to validate the imaging findings.

2.6.

Imaging-Guided Laser Ablation with Immediate Reflectance Confocal Microscopy Confirmation and Longer-Term Follow-Up (No Histopathology)

Thirty four superficial and early nodular BCCs were treated with RCM imaging-guided laser ablation for complete tumor removal. All lesions were diagnosed clinically and dermoscopically, and also confirmed with BCC features under RCM before the ablation. The number of passes was guided by the imaging and imaging-based measurement of depth until clearance of tumor was confirmed with immediate imaging. No histology was performed. This method was limited to relatively small lesions of diameter $<10\mathrm{mm}$.

Follow-up imaging is being currently conducted to measure the accuracy of the imaging-guided approach to detect clearance of tumor postablation, and to monitor the longer-term efficacy of the ablation treatment and achieve follow-up periods of at least 12 months for all treated lesions. Video imaging and mosaicking of the peripheral epidermal and dermal margins and the deeper dermal margins at the center region (base of wound) of the treated lesions is being conducted at 1 to 3, 6, and 18 months after treatment.

2.7.

Assessment Depth of Ablation, Uptake of Contrast Agent, Reflectance Confocal Microscopy Images, Video-Mosaics

Image stacks were collected (where presence of tumor was detected) to estimate tumor depth, which then allowed us to select the appropriate number of passes to provide the depth of ablation required for complete removal of the tumor.

The qualitative evaluation of postablation imaging was focused on the uptake of contrast agent and detectability of tumor and normal features as a function of depth of ablation and number of passes. Three clinicians (coauthors CSJC, MC, and OY) with experience in reading and analyzing confocal images of BCCs evaluated the quality of RCM images, stacks, videos, and video mosaics. The overall quality was qualitatively assessed as acceptable or not for resolution, contrast, and visualization of nuclear and morphological detail. The visualization of bright nuclear morphology in the exposed peripheral epidermal margin, dermal structures, such as collagen bundles, hair follicles, eccrine glands, and inflammatory cells in the deeper dermal margin (base of wound), as well as the presence or absence of tumor in each quadrant of the wound were analyzed.

3.1.

Uptake of Contrast Agent in the Superficial Epidermal and Deep Dermal Margins of the Ablated Wounds

In Fig. 4, a video mosaic generated from a video captured from the surface of a laser ablation wound is shown.

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).

Figure 4(a) shows an example video-mosaic postablation, in this case with seven passes of $25\mathrm{J}/{\mathrm{cm}}^2$. In Fig. 4(b), details of the superficial margins are shown. From left to right, we see intact skin, bright nuclei of basal cells at the epidermal margin, suggesting the uptake of contrast agent (yellow arrows) and in Fig. 4(c), collagen fibers in normal dermis in the deeper dermal margin (red arrows).

Overall, aluminum chloride produced repeatable and consistent brightening of nuclear morphology and enhanced the contrast and detectability of residual BCC and normal structures after laser ablation with four to nine passes of $25\mathrm{J}/{\mathrm{cm}}^2$ fluence. However, improper (such as excess or no uniform) application or tissue heterogeneity was found to sometimes produce saturation artifacts. In three postablation imaging cases, we observed saturation due to the contrast agent, preventing the distinction of normal from abnormal cellular patterns. However, as seen in Fig. 5, the nuclei, cellular detail, and dermal morphology, such as inflammatory cells, hair follicles, collagen fibers, eccrine, and sebaceous glands, could be differentiated.

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.

Thirty one out of thirty four laser-ablated wounds could be visually graded as being of clinically acceptable imaging quality, in terms of resolution, contrast, and detectability of features. Those graded as being of unacceptable image and/or video quality were due to artifacts, such as bubbles (created by an excess amount of gel and/or smearing of the gel during imaging), which resulted in completely or partially blocking the field of view, and saturation of brightness and loss of contrast, which compromised the distinction of cellular structures. Other reasons for low-quality videos included loss of information (dark images) due to sudden changes in wound topography, variability in operator movement and speed causing motion blur, and variability of pressure against skin, all of which produced discontinuities in the videos during the imaging.

3.2.

Depth of Ablation

The depth of ablation values range from 80 to $122\mu \mathrm{m}$ for 4 to 5 passes, 140 to $170\mu \mathrm{m}$ for 6 to 7 passes and 170 to $200\mu \mathrm{m}$ for 8 to 9 passes as a function of passes. In Table 1, these values are shown as a function of the number of passes. The depth of ablation measurements from our previous ex vivo study is also shown. In general, there is reasonable agreement between the measurements in vivo and those ex vivo. Variations between in vivo and ex vivo measurements are attributed to variations in skin sites and hydration conditions on patients, the presence of stratum corneum, and motion artifact due to the laser beam being slightly noncollimated and slightly diverging (the ablation was performed manually, such that slight variations in the distance between the laser scanner and the patient could result in slight variations in fluence). Nonetheless, the results indicate that the depth of ablation versus number of passes measurements ex vivo can be a reasonable initial “look up table” guide for choosing ablation parameters (fluence, number of passes) based on RCM-guided measurements of depth of tumor in vivo.

3.3.

Correlation to Histology in the First Six Patients (10 Basal Cell Carcinomas) for Presence or Absence of Tumor

Table 2 shows the selected laser ablation parameters (number of passes for fluence of $25\mathrm{J}/{\mathrm{cm}}^2$) based on depth of tumor that was estimated by preablation RCM imaging. For all lesions, a thin excision was taken for histology postablation. In 3 out of 10 lesions, residual BCCs were detected by RCM postablation. For those cases, the residual tumors were not further removed with laser ablation, but were surgically excised, and then confirmed and correlated to histology. The correlation of imaging findings to histology resulted in three true positives, six true negatives, and one false positive. For the false positive case, the presence of saturation artifacts in the imaging prevented our clinical imaging reading expert (coauthor MC) from reaching conclusive identification of the residual tumor.

Table 2

Imaging guided laser ablation with correlation to histology in 10 BCC lesions on six patients.

An example of detection of residual tumor postablation with four passes of $25\mathrm{J}/{\mathrm{cm}}^2$ is shown in Fig. 6. In Fig. 6(a), a clinical photograph (routinely taken in the clinic) of the lesion is shown, along with a solid blue line on the surgical scoring (performed by the Mohs surgeon) demarcating the ablated region (right side) and intact region as control (left). Postablation imaging detected residual tumor at the peripheral margins. Figures 6(b)–6(c) show RCM images of typical features of BCCs indicating the presence of residual tumor, corresponding to the region enclosed in the solid red rectangle in Fig. 6(a). In (b), the high density nuclei enhanced in contrast by the aluminum chloride form a flower shaped structure that is typical of BCCs surrounded by a palisading pattern (white arrows). Figure 6(c) shows small island of tumor formed by bright nuclei (black arrows). In Fig. 6(d), an H&E section taken at the location of the white dashed line in Fig. 6(a) is shown. At the left side, the intact tumor (black arrow) intentionally left for control and the surgical margin (the crater with the edge labeled with light blue ink) serves as a reference to identify the ablated region. At the right side, the green and black arrows indicate details of the presence of residual BCCs at the epidermal margins confirming the findings shown in Figs. 6(b) and 6(c), respectively.

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.

3.4.

Image-Guided Laser Ablation with Imaging Follow-Up on Remaining 15 Patients (34 Basal Cell Carcinomas)

Initial follow-up results are available for 26 superficial, six superficial and early nodular, and two early nodular BCCs that were treated with only the RCM imaging-guided laser ablation approach (i.e., no histopathology). The number of passes varied from 5 to 11 as shown in Table 1 (row 2). After the first ablation treatment RCM imaging detected clearance of tumor in 27 lesions and residual tumor in seven lesions (two treated with eight passes and five treated with 5 to 6 passes) for which a second treatment (of 2 to 4 passes) was applied to attain clearance of tumor. The results for 31 lesions show that the uptake of ${\mathrm{AlCl}}_3$ as contrast agent to label nuclei appears to be effective and consistent in wounds, with estimated depths of ablation from 80 to $200\mu \mathrm{m}$. However, in three cases (ablated with 6 to 8 passes), saturation was observed in the images that challenged the detection of nuclear structures. All lesions are currently being followed up and the ablation treatment monitored with imaging at 1 to 3 (7 lesions), 6 to 7 (12 lesions), and 10 to 21 (15 lesions) months. The monthly time points tend to vary a bit due to patients’ schedule for the follow-up appointments.

To date, 31 lesions have shown good cosmetic outcome and clearance of tumor under RCM images. Only two of the lesions (superficial and superficial/nodular types of BCCs at 7 and 17 months of follow-up, respectively) have shown recurrence after 6 months, respectively, where imaging showed features indicating presence of BCC. In one lesion (superficial type of BCC at 7 months follow-up), persisting large blood vessels and elongated nuclei patterns at the epidermis layer were observed as suspicious features of presence of BCC.

At early stages, most of the lesions were more raised and slightly erythematous. After 12 months, most of the followed lesions were the same color as surrounding skin or pink. Hypopigmented macules in five lesions ($>16$ months of follow-up), pink macules in two lesions ($>16$ months of follow-up), pink papules (raised) in three lesions ($>6$ months of follow-up), erythematous macules in three lesions ($>6$ months of follow-up), and hypertrophic scarring in one lesion (at 2 months of follow-up) were observed.

An example of follow-up imaging, collected at 1, 4, 7, and 12 months, is shown in Fig. 7, for one of the lesions (with 20 months of follow-up) representing the evolution of treated superficial type of BCC. The regrowth skin layers are seen at the first follow-up. However, significant presence of enlarged blood vessels (yellow arrows) and amorphous and thin collagen fibers (yellow arrows) are seen at 1 and 4 months. At 7 months, more normal dermis is visualized still presence of enlarged blood vessels with thickened collagen fibers (red arrows) were observed (Fig. 7-left bottom). The appearance and morphology of the epidermal layers were seen normal compared to untreated surrounding normal skin at 12 months (Fig. 7-right bottom).

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.