Histopathological analysis and in vivo optical spectroscopy were used to discriminate several histological stages of UV-irradiated mouse skin. At different times throughout the $30-week$ irradiation, autofluorescence (AF) and diffuse reflectance (DR) spectra were acquired in a bimodal approach. Then skin was sampled and processed to be classified, according to morphological criteria, into four histological categories: normal, and three types of hyperplasia (compensatory, atypical, and dysplastic). After extracting spectral characteristics, principal component analysis (data reduction) and the $k$-nearest neighbor classifying method were applied to compare diagnostic performances of monoexcitation AF (based on each of the seven excitation wavelengths: 360, 368, 390, 400, 410, 420, and $430nm$), multiexcitation AF (combining the seven excitation wavelengths), DR, and bimodal spectroscopies. Visible wavelengths are the most sensitive ones to discriminate compensatory from precancerous (atypical and dysplastic) states. Multiexcitation AF provides an average 6-percentage-point increased sensitivity compared to the best scores obtained with monoexcitation AF for all pairs of tissue categories. Bimodality results in a 4-percentage-point increase of specificity when discriminating the three types of hyperplasia. Thus, bimodal spectroscopy appears to be a promising tool to discriminate benign from precancerous stages; clinical investigations should be carried out to confirm these results.

This contribution presents the development of an optical spectroscopy device, called SpectroLive, that allows spatially-resolved multiply-excited autofluorescence and diffuse reflectance measurements. Besides describing the device, this study aims at presenting the metrological and safety regulation validations performed towards its aimed application to skin carcinoma in vivo diagnosis. This device is made of six light sources and four spectrometers for detection of the back-scattered intensity spectra collected through an optical probe (made of several optical fibers) featuring four source-to-detector separations (from 400 to 1000 µm). In order to be allowed by the French authorities to be evaluated in clinics, the SpectroLive device was successfully checked for electromagnetic compatibility and electrical and photobiological safety. In order to process spectra, spectral correction and metrological calibration were implemented in the post-processing software. Finally, we characterized the device’s sensitivity to autofluorescence detection: excitation light irradiance at the optical probe tip in contact with skin surface ranges from 2 to 11 W/m², depending on the light source. Such irradiances combined to sensitive detectors allow the device to acquire a full spectroscopic sequence within 6 s which is a short enough duration to be compatible with optical-guided surgery. All these results about sensitivity and safety make the SpectroLive device mature enough to be evaluated through a clinical trial that aims at evaluating its diagnostic accuracy for skin carcinoma diagnosis.

SignificanceThe clinical use of optical methods for in vivo skin imaging is limited by skin strong scattering properties, which reduce image contrast and probing depth. The efficiency of optical methods can be improved by optical clearing (OC). However, for the use of OC agents (OCAs) in a clinical setting, compliance with acceptable non-toxic concentrations is required.AimOC of in vivo human skin, combined with physical and chemical methods to enhance skin permeability to OCAs, was performed to determine the clearing-effectiveness of biocompatible OCAs using line-field confocal optical coherence tomography (LC-OCT) imaging.ApproachNine types of OCAs mixtures were used in association with dermabrasion and sonophoresis for OC protocol on three volunteers hand skin. From 3D images obtained every 5 min for 40 min, the intensity and contrast parameters were extracted to assess their changes during the clearing process and evaluate each OCAs mixture’s clearing efficacy.ResultsThe LC-OCT images average intensity and contrast increased over the entire skin depth with all OCAs. The best image contrast and intensity improvement was observed using the polyethylene glycol, oleic acid, and propylene glycol mixture.ConclusionsComplex OCAs featuring reduced component concentrations that meet drug regulation-established biocompatibility requirements were developed and proved to induce significant skin tissues clearing. By allowing deeper observations and higher contrast, such OCAs in combination with physical and chemical permeation enhancers may improve LC-OCT diagnostic efficacy.

In the context of cutaneous carcinoma diagnosis based on in vivo optical biopsy, Diffuse Reflectance (DR) spectra, acquired using a Spatially Resolved (SR) sensor configuration, can be analyzed to distinguish healthy from pathological tissues. The present contribution aims at studying the depth distribution of SR-DR-detected photons in skin from the perspective of analyzing how these photons contribute to acquired spectra carrying local physiological and morphological information. Simulations based on modified Cuda Monte Carlo Modeling of Light transport were performed on a five-layer human skin optical model with epidermal thickness, phototype and dermal blood content as variable parameters using (i) wavelength-resolved scattering and absorption properties and (ii) the geometrical configuration of a multi-optical fiber probe implemented on an SR-DR spectroscopic device currently used in clinics. Through histograms of the maximum probed depth and their exploitation, we provide numerical evidence linking the characteristic penetration depth of the detected photons to their wavelengths and four source–sensor distances, which made it possible to propose a decomposition of the DR signals related to skin layer contributions.

Optical spectroscopy is studied to contribute to skin cancer diagnosis. Indeed, optical spectra are modified along cancer progression and provide complementary information (e.g., on metabolism and tissue structure) to clinical examination for surgical guidance [1,2]. The current original dataset is made of autofluorescence and diffuse reflectance spectra acquired in vivo on 131 patients' skin with the SpectroLive device [3,4]. Spatially-resolved spectroscopy measurements were performed using a multi-fiber optic probe featuring 4 distances (0.4-1 mm) between excitation and collection optical fibers: spatial resolution allows spectra acquired at different distances to carry information from different depths in skin tissues. Five types of autofluorescence spectra were acquired using five different wavelength excitations (on the 365-415 nm spectral range) in order to collect information on several skin endogenous fluorophores (e.g., flavins, collagen). A sixth light source (white broadband) was used to acquire diffuse reflectance spectra carrying information about skin scattering properties and skin endogenous absorbers such as melanin and hemoglobin. Patients were proposed to be included into the clinical trial if they were suspected of suffering from actinic keratoses (precancerous skin lesions) or from basal or squamous cell carcinomas: in all cases, complete diagnostics is provided in the dataset. To increase the interest of the dataset and evaluate the dependence of optical spectra (intensity, shape) not only on pathological states but also on healthy skin features (civil age, skin age, gender, phototype, anatomical site), spectra were acquired for all 131 patients on two so-called "reference" skin sites known to rarely suffer from skin cancer: palm of the hand (featuring a thick skin type) and inner wrist (featuring thin skin). Spectra are available in .tab files: first column displays the spectral range on which intensity spectra were recorded (317-788 nm) and each following column provides an intensity spectrum acquired by each spectrometer for a given combination of light source excitation and distance. Each of the 131 folders corresponding to each of the 131 patients contains a .json file providing patients clinical features: gender, civil age, skin age, phototype score and class. All .tab files names include anatomical site and anatomopathological diagnostics of the skin site on which spectra were acquired: codes were defined to match a letter or an acronym to each diagnostic and anatomical site. To ensure quality control, a spectrum was acquired on the same calibration standard before starting spectra acquisition on each patient. It is therefore possible to follow the impact of the acquisition optical chain ageing during the 4.5 years that the patients were included. This dataset can be used by epidemiologists for the characterization of populations affected by skin cancers (gender ratio, mean age, anatomical sites typically affected, etc.); it may also be used by researchers in artificial intelligence to develop innovative methods to process such data and contribute to non-invasive diagnostics of skin cancers whose incidence is steadily increasing.

Actinic keratoses (AKs) are sun-damaged skin areas that affect 20% of the European adult population and more than 50% of people aged 70 years and over. There are currently no clinical or histological features allowing us to identify to which clinical class (i.e., regression or progression) an AK belongs. A transcriptomic approach seems to be a robust tool for AK characterization, but there is a need for additional studies, including more patients and elucidating the molecular signature of an AK. In this context, the present study, including the largest number of patients to date, is the first aiming at identifying biological features to objectively distinguish different AK signatures. We highlight two distinct molecular profiles: AKs featuring a molecular profile similar to squamous cell carcinomas (SCCs), which are called “lesional AKs” (AK_Ls), and AKs featuring a molecular profile similar to normal skin tissue, which are called “non-lesional AKs” (AK_NLs). The molecular profiles of both AK subclasses were studied, and 316 differentially expressed genes (DEGs) were identified between the two classes. The 103 upregulated genes in AK_L were related to the inflammatory response. Interestingly, downregulated genes were associated with keratinization. Finally, based on a connectivity map approach, our data highlight that the VEGF pathway could be a promising therapeutic target for high-risk lesions.