1.

Introduction

Incidences and mortality from skin cancer are still increasing.¹ Depending on the melanin concentration, skin tumors are broadly classified into two types—malignant melanomas (MM) and nonmelanoma skin cancers (NMSC). MM is the most aggressive skin cancer modality with death rate $\sim 80\%$ of all fatal skin cancer cases.¹ The most common NMSC are basal cell carcinoma (BCC, about 80% of new cases) and squamous cell carcinoma (SCC, about 20% of new cases) derived from the basal and squamous cells of the epidermis, respectively.² BCC is characterized by very slow growth tendency, low mortality rate, and high risk of recurrence, while SCC is more aggressive and associated with the risk of metastasis.^3,4

Early detection and removal of skin cancers can significantly increase the survival time. Noninvasive methods in primary oncological diagnostics of skin tumors are still topical for dermatologists and oncologists worldwide. One of those is skin autofluorescence (AF) imaging and spectroscopy, based on differences of AF specific information (intensity, spectral shape, and lifetime) in the tumor and surrounding normal skin.^5–8

The feasibility of AF spectroscopy for BCC diagnostics and differentiation has been studied extensively over this most recent decade. AF spectra from BCC lesions excited in UV/blue region (337 to 450 nm) were broadly characterized by decreased fluorescence intensity in comparison with surrounding healthy skin,⁸ most often attributed to the shift in the levels of NADH/NAD+ (reduced form and oxidized form of nicotinamide adenine dinucleotide) and reduced elastin and collagen, affected by malignant process. In some cases, especially in late tumor growing stages, a weak red fluorescence peak of the endogenous porphyrins has been observed.⁸

Tissue AF usually shows the photobleaching effect,⁹ which may be helpful in biomedical applications.^10–14 Under continuous wave (cw) excitation, skin AF intensity mainly drops during the first 15 to 20 s, followed by a slow decrease. Photobleaching kinetics can be well described by empirical double-exponential function with subsequent extraction of time constants ${\tau }_1$ and ${\tau }_2$ that characterize the rate of fast and slow phases of the AF decrease.⁹ Our previous research has demonstrated that each skin pathology as well as healthy skin has its own specific AF intensity decrease kinetics depending on excitation, localization, melanin content, and blood perfusion. Furthermore, the analysis of AF decrease kinetics during the first 15 to 20 s of cw laser excitation seems to be most suitable for clinical implementation.^15,16

Currently available smartphones equipped with high-resolution RGB cameras in combination with good light sensitivity and color representation mainly satisfy the required technical properties for adequate image acquisition.¹⁷ This technology may become a useful diagnostic tool for dermatologists and oncologists thanks to wide accessibility, convenient use, and low cost.^18,19 However, the ability to switch off the “embedded” automatized settings such as exposure time, white balance, and ISO is crucial for the skin parametric imaging. Our latest studies have shown that smartphones such as Galaxy and Nexus are suitable for mapping of skin chromophores.¹⁷

So far use of smartphones in skin pathology diagnostics has been mainly related to dermatoscopy—specifically magnified image acquisition under white or color illumination with subsequent analysis based on ABCD rules, fractal image analysis or other algorithms established in dermatoscopy.^20–24 In this paper, we present a smartphone-compatible technique for acquisition and analysis of 405 nm light-emitting diode (LED) excited skin autofluorescence images.

2.

Materials and Methods

3.

Results and Discussion

A total of 10 solid and 3 ulcerating BCCs were selected for the study. In all BCC cases (confirmed by cytological examination) the AF images showed lowered AF intensity in malignant tissue as compared with the healthy surrounding skin, which may be attributed to decreased levels of fluorophores and increased blood perfusion caused by the malignancy process.^2,5,7 AF spectra from in vivo BCC under 405 nm excitation are characterized by broad (450 to 750 nm) emission spectrum with maximum in green spectral region (510 to 530 nm). In comparison with surrounding healthy skin, the intensity of AF from malignant tissue usually are lower, while the shape of the spectrum remains unchanged. Moreover, the intensity of AF is strictly correlated with the tumor pigmentation, specifically, the higher the pigmentation, the lower is the intensity of fluorescence.^2,8 In all BCC cases, the G-band (corresponding to the AF maximum) AF intensity images in comparison with R-band images showed the more pronounced intensity contrast within the tumor tissue and surrounding healthy skin. Moreover, G-band AF decrease maps showed more structured compositions at tumor area in comparison with R-band AF decrease maps. In the cases of solid BCCs the AF images showed clearly bordered tumor areas with relatively low AF intensity in comparison with surrounding healthy skin. Whereas the ulcerating BCCs can be characterized by the high AF intensity in the ulcerating part surrounded by clearly bordered tissue emitting relatively low AF intensity. Furthermore, in all BCC cases the normalized AF intensity decrease maps showed high AF intensity decrease rates at the tumor areas with low AF intensity in comparison with the surrounding healthy skin and the internal ulcerating area.

Figure 1 represents images of ulcerating BCC: (a) filtered AF image at the excitation start moment, AF intensity G-band image (b), and parametric map of normalized AF intensity decrease rates in the green band (c). AF intensity image [Fig. 1(b)] shows high AF intensity in the ulcerating part (pathology center), surrounded by clearly bordered tissue emitting relatively low AF intensity. Furthermore, the AF intensity decreasing is more intensive at the external tumor area exposures in comparison with the surrounding healthy skin and the internal ulcerating area [Fig. 1(c)].

Fig. 1

Images of ulcerating basal cell carcinoma (BCC). Filtered AF color image (a) at excitation start moment, (b) the corresponding G-band image, and (c) normalized AF intensity decrease map. The pseudo color scale represents (b) the G-band intensity range and (c) the normalized AF intensity variation range.

Another case of solid BCC is presented in Fig. 2. G-band AF intensity image [Fig. 2(b)] shows relatively low intensity within the tumor area with clear margins between tumor and surrounding healthy skin. The tumor area also shows higher AF intensity decrease rate [Fig. 2(c)] in comparison with the surrounding healthy skin. Besides, the images presented in Fig. 2 reveal a small area with lowered fluorescence located at “five o’clock” from the main tumor. The low AF intensity and high AF intensity decrease rate in that skin area similarly indicates to cancerous process, which is probably determined by multicentric tumor growing process.

Fig. 2

Images of solid BCC. Filtered AF color image (a) at excitation start moment, (b) the corresponding G-band image, and (c) normalized AF intensity decrease map. The pseudo color scale represents (b) the G-band intensity range and (c) the normalized AF intensity variation range.

In addition, one atypical nevus was selected for the study (Fig. 3). The nevus before surgical excision was suspected as melanoma; histological analysis of the removed tissue samples had confirmed three different types of tissue cells within the lesion area. Specifically, the upper part of the pathology mostly prevailed by intradermal nevus, the middle part by dysplastic nevus, and the lower part by junctional nevus. Normalized AF decrease distribution map [Fig. 3(c)], on the other hand, showed the fastest intensity decrease in the lower (junctional nevus) and upper side (intradermal nevus), while the middle part (dysplastic nevus) of lesion photobleached slower. The observed different AF photobleaching rates most probably are associated with different tissue fluorophore concentration, melanin content, localization, and metabolic state.

Fig. 3

Color filtered AF image of skin atypical nevus (a) at the excitation start moment, (b) the corresponding G-band image and (c) normalized AF intensity decrease map. The color scale at (b) image represents G-band intensity range and at (c) image—range of normalized AF intensity variations.

4.

Conclusions

Smartphone AF imaging has shown potential for remote primary evaluation of cancerous or suspicious skin tissues. The proposed noninvasive technique and method adequately (with respect to the available literature data) represented planar distribution of AF intensities in malignant and healthy tissues. Moreover, the temporal analysis of AF intensity during the photobleaching showed a potential to be used as an additional indicator for demarcation of suspicious tissues. It may find clinical implementation, e.g., for primary evaluation of BCC, such as determination of precise excision margins prior to surgery, adequate selection of treatment method (in the case of multicentric growing process the nonsurgical methods are more desirable for the patients), as well as in selective application of immune response modulators for BCC therapy. The proposed excitation band around 405 nm covers the absorption maxima of several porphyrines and may find applications in photodynamic diagnostics of superficial nonmelanoma lesions. The most intriguing result of this research was the fact that AF photobleaching rate map showed quantitative correlation with the histology tests in the case of atypical dysplastic nevus. To explain, one can assume that specific tissue fluorophores might have individual bleaching kinetics features, which eventually could provide information on fluorophore concentration and environmental factors. The increased photobleaching rates in the tumor area most probably indicate different fluorophore content composition affected by the tumor growing process, e.g., destruction of collagen elastin cross-links along with decrease in NADH levels. Undoubtedly, this phenomenon requires additional studies to clarify the exact mechanism of uneven photobleaching of skin fluorophores under continuous optical excitation.