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Confocal Imaging–Guided Laser Ablation of Basal Cell Carcinomas: An Ex Vivo Study

      Abbreviations

      BCC
      basal cell carcinoma
      RCM
      reflectance confocal microscopy
      TO THE EDITOR
      Laser ablation is a promising approach for minimally invasive removal of superficial and early nodular basal cell carcinomas (BCCs;
      • Smucler R.
      • Vlk M.
      Combination of Er:YAG laser and photodynamic therapy in the treatment of nodular basal cell carcinoma.
      ;
      • Smucler R.
      • Kriz M.
      • Lippert J.
      • et al.
      Ultrasound guided ablative-laser assisted photodynamic therapy of basal cell carcinoma (US-aL-PDT).
      ). The skin can be removed in μm-thin layers in a well-controlled manner, increasing preservation of the surrounding normal tissue. However, tissue is vaporized such that there is none available for immediate histopathological confirmation. The efficacy tends to be variable and recurrence rate (8.25%) low (
      • Smucler R.
      • Vlk M.
      Combination of Er:YAG laser and photodynamic therapy in the treatment of nodular basal cell carcinoma.
      ), compared with that reported for Mohs surgery (2.1–3.5%;
      • Chren M.M.
      • Torres J.S.
      • Stuart S.E.
      • et al.
      Recurrence after treatment of nonmelanoma skin cancer: a prospective cohort study.
      ;
      • Chren M.M.
      • Linos E.
      • Torres J.S.
      • et al.
      Tumor recurrence 5 years after treatment of cutaneous basal cell carcinoma and squamous cell carcinoma.
      ), excision (3.5–4.2%), and electrodessication and curettage (1.6–4.9%). Thus, the implementation of laser ablation for BCCs in a clinical setting is still limited.
      A high-resolution nuclear-level optical imaging approach such as reflectance confocal microscopy (RCM;
      • Nori S.
      • Rius-Díaz F.
      • Cuevas J.
      • et al.
      Sensitivity and specificity of reflectance-mode confocal microscopy for in vivo diagnosis of basal cell carcinoma: a multicenter study.
      ;
      • Guitera P.
      • Menzies S.W.
      • Longo C.
      • et al.
      In vivo confocal microscopy for diagnosis of melanoma and basal cell carcinoma using a two-step method: analysis of 710 consecutive clinically equivocal cases.
      ) may guide laser ablation by detecting the presence or clearance of residual BCCs directly on the patient, to provide immediate histopathology-like feedback to improve the efficacy. Preliminary studies in human skin ex vivo with acetic acid for nuclear contrast (
      • Sierra H.
      • Larson B.A.
      • Chen C.
      • et al.
      Confocal microscopy to guide erbium:yttrium aluminum garnet laser ablation of basal cell carcinoma: an ex vivo feasibility study.
      ) and in vivo with aluminum chloride (
      • Chen Ch J.
      • Sierra H.
      • Cordova M.
      • et al.
      Confocal microscopy guided laser ablation for superficial and early nodular basal cell carcinoma.
      ) revealed feasibility of imaging in post-ablated tissue. However, the imaging was of variable quality. Ablation was performed with a pulsed Erbium-doped Yttrium Aluminum Garnet (Er:YAG) laser, with pulse duration of ∼250 μs that produces an underlying zone of thermal coagulation of ∼20 μm (
      • Hohenleutner U.
      • Hohenleutner S.
      • Bäumler W.
      • et al.
      Fast and effective skin ablation with an Er:YAG laser: determination of ablation rates and thermal damage zones.
      ).
      We therefore hypothesized that loss of tissue viability due to thermal coagulation must affect the repeatability and consistency of uptake of contrast agent and imaging of residual tumor. We may control thermal coagulation with optimal choice of ablation parameters (pulse duration, fluence, number of pulses, and wavelength). This may subsequently allow repeatable and consistent uptake of contrast agent and imaging of residual nuclear morphology and detection of residual BCC tumors. In this Letter, we report the results of an extended study for determining optimal fluence and number of pulses for a given wavelength and pulse duration.
      Fifty-eight discarded frozen-thawed BCC specimens from Mohs surgery were obtained under an Institutional review board-approved protocol. The tissue was immersed in acetic acid (5%, 30 s) for brightening nuclear morphology (“acetowhitening”) using a previously described protocol (
      • Patel Y.G.
      • Nehal K.S.
      • Aranda Y.I.
      • et al.
      Confocal reflectance mosaicing of basal cell carcinomas in Mohs surgical skin excisions.
      ). A reflectance confocal microscope (Vivascope 1500, Caliber Imaging and Diagnostics, Rochester, NY) was used to capture mosaics, displaying areas of 8 mm × 8 mm, of the skin and to determine regions containing BCCs. A selected region containing BCCs was ablated with our Er:YAG laser (Sciton Profile, Palo Alto, CA; wavelength 2.94 μm, spot diameter 4 mm), using fluences of 6.3, 12.5, 17.5, and 25.0 J cm−2 and number of passes 1–8. Each pass is a set of four independent pulses, separated by ∼40 ms. All specimens were imaged, ablated, once again immersed in acetic acid, then imaged and finally processed for frozen histopathology. En face sections were prepared of the ablated surface that was imaged.
      RCM mosaics were qualitatively evaluated against the corresponding histopathology for the appearance of nuclear, residual BCC tumor, and surrounding dermal morphology. The evaluation showed that a total delivered fluence of up to 150 J cm−2 (maximum fluence 25 J cm−2 and 6 consecutive passes) allows repeatable and consistent uptake of contrast agent and RCM imaging. For higher total fluences, delivered in more than 6 consecutive number of passes without any tissue cooling in-between, the nuclear morphology appears amorphous, and the residual tumor cannot be distinguished from the surrounding dermis. This must be due to the increase in thermal coagulation with increased number of passes (
      • Walsh J.T.
      • Flotte T.J.
      • Anderson R.
      • et al.
      Er:YAG laser ablation of tissue: effect of pulse duration and tissue type on thermal damage.
      ;
      • Hohenleutner U.
      • Hohenleutner S.
      • Bäumler W.
      • et al.
      Fast and effective skin ablation with an Er:YAG laser: determination of ablation rates and thermal damage zones.
      ). However, in specimens in which ablation was performed with time interval of at least 1–2 s between multiple treatments (each consisting of maximum 6 consecutive passes), for passive cooling of the tissue, we can control thermal coagulation to allow the subsequent uptake of contrast agent and imaging. These observations were confirmed in the histopathology. (The limit of 6 warrants further investigation. Possibly, 6 may not be an intrinsic limit, and active cooling of the skin may allow more number of consecutive passes.)
      To quantify the accuracy for detecting the clearance or the presence of tumor after ablation, 15 specimens were selected. We selected specimens with reasonably consistent initial conditions: (a) contained large tumors and (b) treated with highest available fluence of 25 J cm−2 and a total of 1–10 passes, with no more than 6 consecutive passes per treatment. The presence or absence of tumor was evaluated in 30 half-mosaics (approximately × 5 magnification) against histopathology. The clearance rate, sensitivity, and specificity were estimated. RCM imaging found an overall clearance rate of 40% compared with 23% by inspection of histopathology. Agreement between the confocal assessment for the presence of residual tumor with histopathology was 77%. The preliminary measures of accuracy are 74% sensitivity and 86% specificity.
      To mimic in vivo conditions, 10 additional specimens with intact stratum corneum were imaged and ablated with the intention of completely clearing tumor, using fluence of 25 J cm−2 and one treatment, each of 1–6 passes. The number of passes were determined on the basis of the depth of the tumor, as estimated with pre-ablation imaging. (We have previously characterized depth of ablation per pass with fluence for this laser (
      • Sierra H.
      • Larson B.A.
      • Chen C.
      • et al.
      Confocal microscopy to guide erbium:yttrium aluminum garnet laser ablation of basal cell carcinoma: an ex vivo feasibility study.
      ).) After ablation, a RCM mosaic of the ablated surface was captured. Vertical frozen sections were then prepared from the superficial and deep margins of the ablated regions.
      Figure 1 demonstrates the ability of RCM imaging to detect the presence and clearance of residual BCC tumor. Mosaics and images are shown of a specimen that was ablated through intact stratum corneum, with fluence of 25 J cm−2 and 6 passes. Vertical histopathology sections through the ablated region confirmed the observations at the superficial and deep margins of the post-ablated wounds.
      Figure thumbnail gr1
      Figure 1Reflectance confocal microscopy (RCM) imaging detects the clearance or the presence of residual basal cell carcinoma (BCC) tumor in skin specimens after ablation with 6 passes at fluence of 25 J cm−2. (a) RCM image through stratum of a skin specimen showing BCCs. (b) Enlarged view of region within solid yellow square in a shows details of nodular BCCs. (c) Enlarged view of the region within dashed yellow square in a shows details of BCCs. (d) RCM image of ablated specimen showing exposed dermis. (e) Enlarged view of region within solid yellow square in d shows clearance of tumor after ablation. (f) Enlarged view of ablated region within dashed yellow square in d shows residual nodular BCC. (g) Vertical hematology and eosin histology from dashed orange line in d. (h) Enlarged view of solid black rectangle in g shows normal dermis confirming clearance of tumor after ablation. (i) Enlarged view of dashed black rectangle in g shows residual BCC after ablation. Size bars=500 μm.
      In Figure 1a, a pre-ablation mosaic at the dermal–epidermal junction (∼130 μm depth) shows nodular BCCs (region inside both solid and dotted yellow squares). Enlarged views of the two regions within these solid and dotted squares (Figure 1b and c, respectively) show more clearly clusters of bright densely distributed nuclei and the nodular morphology of the tumors. Figure 1d shows a post-ablation mosaic. An enlarged view (Figure 1e) of the area in the solid yellow square shows only dermal collagen and confirms clearance of tumor. By comparison, an enlarged view (Figure 1f) of the region within the dashed yellow square shows clusters of densely distributed bright nuclei closer to the edge of the wound and indicates the presence of residual tumor. Figure 1g shows a vertical frozen histopathology section through the wound, at the location of the dashed orange line in Figure 1d. The section confirms the clearance of tumor in the center of the wound (solid black rectangle, which corresponds to the location of the dashed orange line within the solid yellow square in Figure 1d) and the presence of residual tumor closer to the edge (dashed black rectangle, which corresponds to the location of the dashed orange line within the dashed yellow square in Figure 1d). The pathology indicates the maximum depth of ablation to be ∼160 μm, and a thin layer of darker stained amorphous tissue (not obvious at low magnification) indicates a thermal coagulation zone of approximately 20–30 μm. Figure 1h and i show magnified views of the histopathology (corresponding to the location of the dashed orange line in Figure 1e and f), which further confirms, respectively, the clearance and the presence of tumor.
      For all 10 specimens, the histopathology sections confirm the observations in RCM mosaics regarding clearance of tumor or presence. The clearance, as intended, was seen in 9 specimens (true negatives) and the (unintended) presence in 1 (“false negative”). These initial results suggest that imaging may enable less invasive treatment via localized control on the depth of ablation, with potentially high negative predictive value. Furthermore, the estimation of lateral margins (not performed here, but feasible on patients (
      • Pan Z.Y.
      • Lin J.R.
      • Cheng T.T.
      • et al.
      In vivo reflectance confocal microscopy of Basal cell carcinoma: feasibility of preoperative mapping of cancer margins.
      )), in addition to depth, may improve the accuracy of ablation. However, the results highlight the current limitation of the imaging, which is mainly contrast (while resolution appears to be sufficient) for detectability of residual tumors. Further investigation and optimization of this approach for enhancement of tumor-to-dermis contrast is necessary.
      Our work, together with other studies in vivo (
      • Tannous Z.
      • Torres A.
      • González S.
      In vivo real-time confocal reflectance microscopy: a noninvasive guide for Mohs micrographic surgery facilitated by aluminum chloride, an excellent contrast enhancer.
      ;
      • Nori S.
      • Rius-Díaz F.
      • Cuevas J.
      • et al.
      Sensitivity and specificity of reflectance-mode confocal microscopy for in vivo diagnosis of basal cell carcinoma: a multicenter study.
      ;
      • Scope A.
      • Mahmood U.
      • Gareau D.S.
      • et al.
      In vivo reflectance confocal microscopy of shave biopsy wounds: feasibility of intra-operative mapping of cancer margins.
      ;
      • Guitera P.
      • Menzies S.W.
      • Longo C.
      • et al.
      In vivo confocal microscopy for diagnosis of melanoma and basal cell carcinoma using a two-step method: analysis of 710 consecutive clinically equivocal cases.
      ;
      • Pan Z.Y.
      • Lin J.R.
      • Cheng T.T.
      • et al.
      In vivo reflectance confocal microscopy of Basal cell carcinoma: feasibility of preoperative mapping of cancer margins.
      ;
      • Chen Ch J.
      • Sierra H.
      • Cordova M.
      • et al.
      Confocal microscopy guided laser ablation for superficial and early nodular basal cell carcinoma.
      ), suggests the potential possibility of peri-operative RCM imaging of superficial and early nodular BCCs to guide noninvasive diagnosis, pretreatment detection of tumor margins, less invasive (ablative) treatment, and post-treatment monitoring, directly on the patient. The ablation may be combined with other approaches such as debulking of tumor for enhancing the efficacy of treatment. Further clinical studies must be performed to rigorously test for accuracy (particularly, negative predictive value and recurrence rate for different subtypes of BCCs), combined with training for reading and interpretation of images.

      ACKNOWLEDGMENTS

      We thank the National Institutes of Health for funding support (grant R01EB012466 from NIBIB’s Image-guided Interventions program).

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