Fluorescence Confocal Microscopy of Basal Cell Carcinomas
Fluorescence Confocal Microscopy of Basal Cell Carcinomas
The introduction of FCM in the clinical setting for the margin assessment in BCC is quite recent and few studies have been done so far to provide the basic description of FCM images.
Beyond the initial studies, later studies on the possible application of ex vivo confocal microscopy for Mohs surgery were based on the use of either acetic acid and toluidine blue or citric acid to stain nuclear morphology in reflectance contrast (wavelength 830 nm). In reflectance mode, the use of acetowhitening or citric acid provides weak contrast of nuclei, compared with the background reflectance of collagen in the dermis. Thus, this modality enables the detection of only large nodular-type tumours that usually do not represent challenging surgical situations. Instead, the most difficult situation is represented by infiltrative and micronodular tumours characterized by tiny BCC nests that cannot be readily detected in reflectance mode. For such situations, ex vivo confocal microscopy can be used in fluorescence mode using the nuclear stain acridine orange. Nuclei appear brightly stained in fluorescence mode with acridine orange, whereas the cytoplasm and collagen appear darker. The capability of ex vivo FCM is much higher for optimal detection of all types of BCCs, including, especially, micronodular and infiltrative, in skin tissue.
The aim of our study was to provide the terminology of the FCM for both normal skin and adnexal structures and BCCs. To address this aim, 64 BCCs and the overlying skin were analysed morphologically. As a general point, we found excellent correspondence between FCM images and the related histopathology. Of note, the tissue subjected to acridine orange staining was not altered at all for the subsequent histopathological processing.
The skin analysed in our study was readily identifiable as well as the adnexal structures. However, normal skin coming from nonaffected tissue (non-BCC) needs to be analysed further to assess the variability of pattern according to distinct physiological and pathological conditions. A good correlation was found for fat tissue, muscle and adnexal structures.
Distinct BCC types appeared unique in terms of shape and size of tumour islands (bigger in nodular, smaller and rounded in micronodular and tiny cords for infiltrative ones) and for the presence of clefting, palisading and increased N/C ratio.
All BCCs showed the presence of higher fluorescence compared with the background. In superficial BCCs, the tumour islands were connected to the epidermis and were typified by the presence of peripheral palisading. However, the relatively small number of superficial BCCs analysed so far do not yet permit better investigation of other possible characteristic features.
Nodular BCCs were seen as well-demarcated nodular fluorescent islands with clefting and peripheral palisading; a high N/C ratio was typically observed. On FCM, tiny and angulated cords and strands of fluorescent cells were typically found in infiltrative BCCs. In this BCC type clefting and palisading were seen in seven of 30 and three of 30 cases, respectively. These aspects may render small areas of these tumours more difficult to recognize. Furthermore, we found that in four cases it was difficult to distinguish the infiltrative cords from the surrounding stroma although the latter showed no tendency to cluster.
Another possible pitfall was due to the presence of several sebaceous glands that may be confused with BCC islands. However, the former showed no palisading, less fluorescence and the presence of a centrally located nucleus compared with the tumours. Further prospective studies are needed to define the role of possible pitfalls and how to avoid them. Moreover, the combination of reflectance mode and FCM with digital staining to mimic conventional haematoxylin and eosin staining may impact the reading of the images.
Recently, Bennàssar et al. examined 69 BCCs on FCM to define the main diagnostic criteria. The description of BCC types is in line with our findings although the authors found the presence of nuclear pleomorphism to be a relevant finding. In our report, based on a similar number of cases, we did not explore this parameter as it was difficult to distinguish nuclear details in highly fluorescent tumours. However, this opens the question of whether multicentric studies should be performed, to develop a common terminology for this new and innovative technique.
In this report, we describe features of normal skin and BCC with ex vivo FCM of excised Mohs tissue compared with routine histopathology. The strengths of 'digital histopathology' in fluorescence mode, which distinguishes it from conventional histopathology, are as follows: direct observation of untreated fresh tissue, rapid acquisition of optical (nondestructive) serial sections in a few minutes, and the possibility to perform histopathology at the bedside in real time (12–15 min). The problems of FCM that remain to be solved in the future are as follows. Firstly, the possibility to scan larger tissue areas greater than 12 × 12 mm, which is the current limit, must be addressed. This would be beneficial in the case of large tumours with large margins that need to be checked for persistence. Secondly, faster scanning and an automated adjustment of laser power must be developed, to obtain reproducible and standardized controlled imaging. Thirdly, studies are needed to assess the diagnostic accuracy in clinical practice of Mohs surgery in diagnosing skin tissue belonging to nonmelanocytic tumours including not only BCC but also squamous cell carcinoma and others. Fourthly, beyond skin in the setting of Mohs surgery, FCM may be useful for the assessment of other solid tumours such as breast, colon, thyroid and other tissues in other settings, and must be tested for such applications.
Discussion
The introduction of FCM in the clinical setting for the margin assessment in BCC is quite recent and few studies have been done so far to provide the basic description of FCM images.
Beyond the initial studies, later studies on the possible application of ex vivo confocal microscopy for Mohs surgery were based on the use of either acetic acid and toluidine blue or citric acid to stain nuclear morphology in reflectance contrast (wavelength 830 nm). In reflectance mode, the use of acetowhitening or citric acid provides weak contrast of nuclei, compared with the background reflectance of collagen in the dermis. Thus, this modality enables the detection of only large nodular-type tumours that usually do not represent challenging surgical situations. Instead, the most difficult situation is represented by infiltrative and micronodular tumours characterized by tiny BCC nests that cannot be readily detected in reflectance mode. For such situations, ex vivo confocal microscopy can be used in fluorescence mode using the nuclear stain acridine orange. Nuclei appear brightly stained in fluorescence mode with acridine orange, whereas the cytoplasm and collagen appear darker. The capability of ex vivo FCM is much higher for optimal detection of all types of BCCs, including, especially, micronodular and infiltrative, in skin tissue.
The aim of our study was to provide the terminology of the FCM for both normal skin and adnexal structures and BCCs. To address this aim, 64 BCCs and the overlying skin were analysed morphologically. As a general point, we found excellent correspondence between FCM images and the related histopathology. Of note, the tissue subjected to acridine orange staining was not altered at all for the subsequent histopathological processing.
The skin analysed in our study was readily identifiable as well as the adnexal structures. However, normal skin coming from nonaffected tissue (non-BCC) needs to be analysed further to assess the variability of pattern according to distinct physiological and pathological conditions. A good correlation was found for fat tissue, muscle and adnexal structures.
Distinct BCC types appeared unique in terms of shape and size of tumour islands (bigger in nodular, smaller and rounded in micronodular and tiny cords for infiltrative ones) and for the presence of clefting, palisading and increased N/C ratio.
All BCCs showed the presence of higher fluorescence compared with the background. In superficial BCCs, the tumour islands were connected to the epidermis and were typified by the presence of peripheral palisading. However, the relatively small number of superficial BCCs analysed so far do not yet permit better investigation of other possible characteristic features.
Nodular BCCs were seen as well-demarcated nodular fluorescent islands with clefting and peripheral palisading; a high N/C ratio was typically observed. On FCM, tiny and angulated cords and strands of fluorescent cells were typically found in infiltrative BCCs. In this BCC type clefting and palisading were seen in seven of 30 and three of 30 cases, respectively. These aspects may render small areas of these tumours more difficult to recognize. Furthermore, we found that in four cases it was difficult to distinguish the infiltrative cords from the surrounding stroma although the latter showed no tendency to cluster.
Another possible pitfall was due to the presence of several sebaceous glands that may be confused with BCC islands. However, the former showed no palisading, less fluorescence and the presence of a centrally located nucleus compared with the tumours. Further prospective studies are needed to define the role of possible pitfalls and how to avoid them. Moreover, the combination of reflectance mode and FCM with digital staining to mimic conventional haematoxylin and eosin staining may impact the reading of the images.
Recently, Bennàssar et al. examined 69 BCCs on FCM to define the main diagnostic criteria. The description of BCC types is in line with our findings although the authors found the presence of nuclear pleomorphism to be a relevant finding. In our report, based on a similar number of cases, we did not explore this parameter as it was difficult to distinguish nuclear details in highly fluorescent tumours. However, this opens the question of whether multicentric studies should be performed, to develop a common terminology for this new and innovative technique.
In this report, we describe features of normal skin and BCC with ex vivo FCM of excised Mohs tissue compared with routine histopathology. The strengths of 'digital histopathology' in fluorescence mode, which distinguishes it from conventional histopathology, are as follows: direct observation of untreated fresh tissue, rapid acquisition of optical (nondestructive) serial sections in a few minutes, and the possibility to perform histopathology at the bedside in real time (12–15 min). The problems of FCM that remain to be solved in the future are as follows. Firstly, the possibility to scan larger tissue areas greater than 12 × 12 mm, which is the current limit, must be addressed. This would be beneficial in the case of large tumours with large margins that need to be checked for persistence. Secondly, faster scanning and an automated adjustment of laser power must be developed, to obtain reproducible and standardized controlled imaging. Thirdly, studies are needed to assess the diagnostic accuracy in clinical practice of Mohs surgery in diagnosing skin tissue belonging to nonmelanocytic tumours including not only BCC but also squamous cell carcinoma and others. Fourthly, beyond skin in the setting of Mohs surgery, FCM may be useful for the assessment of other solid tumours such as breast, colon, thyroid and other tissues in other settings, and must be tested for such applications.
