Confocal imaging of carbon dioxide laser-ablated basal cell carcinomas: An ex-vivo study on the uptake of contrast agent and ablation parameters.

Approximately 3.5 million new cases of non-melanoma skin cancers are diagnosed yearly in the United States, of which approximately 80% are basal cell carcinomas (BCCs) [1–2]. Mohs micrographic surgery, which relies on complete histopathological margin assessment, is the most common treatment for high-risk BCCs and provides highest cure rates (five-year, 97–99%) [3–4]. However, for less aggressive and low-risk subtypes of BCCs (i.e. superficial and early nodular), alternative treatment modalities can be less invasive, faster and lower-cost. These include electrodessication and curettage, topical chemotherapy (e.g. Imiquimod), cryotherapy, and coagulative and ablative laser therapies [5–8]. Laser ablation offers highly localized and precisely controlled removal of tissue, and can be particularly effective for minimally invasive treatment of superficial and early nodular BCCs [9–14]. To select the fluence and number of passes (sets of pulses), clinicians usually rely on visual examination and/or the diagnosis of tumor subtype and depth provided by a biopsy and histology [12–14]. However, visual examination may miss subclinical extension of lateral margins, while histology does not often allow for estimation of complete lateral and deep margins across the entire tumor. Furthermore, clinicians heavily rely on the visual appearance of the tissue between passes to estimate whether the tumor has been totally removed. During clinical follow up, monitoring the efficacy of the treatment is also typically based on visual observation. For all these reasons, there is high variability of recurrence rates, ranging from 3.23% to 50% [12–14]. Given the absence of definitive histological confirmation, there is a need for a non-invasive method to guide laser ablation, particularly for accurate determination of lateral and deep tumor margins, selection of laser parameters, and assessment of ablated tissue to confirm clearance of the tumor. Optical imaging is being investigated for its ability to noninvasively guide the detection of lateral and deep margins of tumor, pre-, during and post-ablation [15–17]. By using a contrast agent, high-resolution microscopic-level imaging of cellular and sub-cellular detail can be obtained in post-ablated tissue in vivo. This allows for the presence (or absence) of residual tumor to be detected in post-ablated tissue, providing histopathology-like control directly on the patient. However, this is possible only with a specific set of parameters (number of passes and delivered fluence) that allow for simultaneous removal of BCCs, while controlling of the amount of underlying thermal coagulation, such that cellular structures can be subsequently labeled with a contrast agent and imaged [15–16]. We previously reported the feasibility of reflectance confocal microscopy (RCM) imaging to guide Erbium-doped yttrium aluminum garnet (Er: YAG) laser ablation, in both bench-top studies and on a small number of patients [15–17]. The Er: YAG laser (wavelength 2900nm) allows for vaporization of very thin layers of tissue while producing only a thin underlying zone of thermal coagulation. Two contrast agents, acetic acid and aluminum chloride, were shown to label nuclei and enhance contrast of nuclear morphology [18–21]. From the results, RCM imaging appears to be capable of detecting residual BCCs in post-ablated tissue [16–17]. Our studies have now expanded to include the carbon dioxide (CO2) laser. The CO2 laser (wavelength 10600nm) offers relatively more aggressive vaporization, along with relatively deeper thermal coagulation compared to the Er: YAG laser, affording greater tissue destruction with fewer passes and producing hemostasis in the underlying microvasculature [22–24]. In this paper, we report an ex vivo bench-top study to determine a set of effective ablation parameters (fluence, number of passes) for a CO2 laser. We further report the results of an assessment of the uptake of acetic acid for contrast and our ability to utilize RCM imaging in post-ablated tissue to evaluate for the presence or absence of residual BCC tumors.