Research letter: A new fluorescent three-dimensional and deep-imaging technique for histological identification of individual tumor cells in extramammary Paget's disease

In extramammary Paget's disease (EMPD), Paget cells are sometimes detected outside the clinical border (subclinical extension) with a skipping pattern. Therefore, “mapping biopsy” is performed to evaluate the clinical border before complete resection. To increase the proportion of Paget cells in biopsy specimens, it is necessary to increase the number of biopsies. However, this is associated with increased pain and discomfort to the patient. The tumor border decision is also a critical factor for prognosis. If we could detect the EMPD tumor cells from the skin surface directly without cutting the patient skin, we could check numerous points where the mapping biopsy is necessary for detecting the tumor border, and it may bring a good prognosis with a skin surgery. Recently, we have established a new fluorescent three-dimensional deep-imaging technique using two-photon excitation fluorescence microscopy for skin lesions.1, 2 The new technique is very good at describing the skin tissue structure in the epidermis and dermis, for example, acrosyringium, individual keratinocytes, and nerve fibre.1 Hence, to resolve the abovementioned issue, we evaluated whether a new fluorescent three-dimensional deep-imaging technique using two-photon excitation fluorescence microscopy for skin lesions is applicable for mapping biopsy in EMPD. EMPD samples resected during surgery were used in this study. Briefly, skin tissue samples were collected via 2-mm disposal biopsy punches from six areas around the main EMPD lesion. After fixation with 4% paraformaldehyde in PBS for 30 min, the samples were processed for propidium-iodide staining, and optically cleared using the transparency-enhancing ilLUmination of Cleared organs to IDentify target molecules method (LUCID),3 and newly established push-pull pyrene probe bearing a ketone acceptor group (PK), which is a solvatochromic dye.4 The samples were evaluated by two-photon excitation fluorescent microscopy (TPM), as described previously.1 All procedures were performed with prior approval from the Ethics Committees of Ehime University Graduate School of Medicine. This study was conducted in accordance with the principles of the Declaration of Helsinki. As shown in Figure 1A, typical histopathological findings of EMPD tumor cells include clear cytoplasm and a “shotgun” pattern on haematoxylin and eosin staining (HE) in the epidermis. In the same sagittal slice view, TPM confirmed similar LUCID/PK staining of the EMPD tumor cells (Figure 1B). The tumor cells had a dark, clear cytoplasm and nucleus, and there were tumor nests in the epidermis (Figure 1B, Video S1). On horizontal slices, EMPD tumor cells could be detected in the epidermis (Figure 1C) and dermis (Figure 1D, Video S2), suggesting that this tumor was already an invasive carcinoma as opposed to an in situ lesion. The major advantage of this new method is the ability of deep imaging to show the whole area from the surface to the deep lesion. This new technique has several advantages over the routine pathological procedure with HE staining. First, although we could obtain cell and tissue information with slices only 5 μm thick per slide via HE staining, our method can determine histological information over a thickness of 100–500 μm, which allowed us to obtain a three-dimensional image of each tissue slice. In addition, the tissue slices do not require processing; the thin tissue can be directly observed after LUCID/PK staining. Second, preparing the HE pathological specimen usually takes at least 1 week because a number of procedures are required, including making a paraffin block, cutting slices, staining with HE, dehydration, and mounting. However, the new technique only requires that the sample be placed in the LUCID/PK staining cocktail for several hours (overnight if needed), with no requirement for histopathological preparation. Two-photon microscopy is a very useful application of multiphoton microscopy in dermatological studies.5 Conventional skin tissue slices have autoimmunofluorescence and the basic structure of the epidermis, which can reveal the dermis and skin appendages. However, the resolution of this procedure is inferior to conventional HE staining. Therefore, a new technique using TPM with immunohistochemistry was developed that can reveal the detailed structure of skin appendages.6 Two-photon microscopy with immunohistochemical analysis has already been applied to EMPD,7 and the results revealed the EMPD tumor border histologically. Compared to their method, our new technique requires only a LUCID/PK cocktail and TPM; there is no requirement for immunohistochemical staining or sample processing, such as making paraffin blocks or slices. Our procedure also has s higher resolution and is very easy to perform in any laboratory where TPM is available. Furthermore, confocal laser microscopy can detect the signal in tissue (<100 μm thick). Further experiments are underway to establish the non-invasive “mapping biopsy” technique. MM, RK, and YN involved in conceptualization; MM, RK, and TT involved in data curation; MM and RK involved in methodology and formal analysis, writing—original draft preparation, writing—review and editing, and funding acquisition; MM, RK, and TT involved in validation, investigation, and visualization; RK, TI involved in technical supervision and advisory. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/RycH7j. The authors have no conflict of interest to declare. This study was supported by JSPS KAKENHI Grant number 20 K08691, Grant-in-Aid for Scientific Research on Innovative Areas_Platforms for Advanced Technologies and Research Resources “Advanced Bioimaging Support,” and AMED under Grant Number JP22ym0126810. Research data are not shared. Video S1. EMPD lesion in the epidermis with three-dimensional observation. Video S2. EMPD lesion scanned from the surface to the dermis. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.