As an important component of tumor microenvironment, cancer-associated fibroblasts (CAFs) have lately gained prominence owing to their crucial role in the resistance to therapies. Photodynamic therapy (PDT) stands out as a successful therapeutic strategy to treat cutaneous squamous cell carcinoma. In this study, we demonstrate that the transforming growth factor β1 (TGFβ1) cytokine secreted by CAFs isolated from patients with SCC can drive resistance to PDT in epithelial SCC cells. To this end, CAFs obtained from patients with in situ cSCC were firstly characterized based on the expression levels of paramount markers as well as the levels of TGFβ1 secreted to the extracellular environment. On a step forward, two established human cSCC cell lines (A431 and SCC13) were pre-treated with conditioned medium obtained from the selected CAF cultures. The CAF-derived conditioned medium effectively induced resistance to PDT in A431 cells through a reduction in the cell proliferation rate. This resistance effect was recapitulated by treating with recombinant TGFβ1 and abolished by using the SB525334 TGFβ1 receptor inhibitor, providing robust evidence of the role of TGFβ1 secreted by CAFs in the development of resistance to PDT in this cell line. Conversely, higher levels of recombinant TGFβ1 were needed to reduce cell proliferation in SCC13 cells, and no induction of resistance to PDT was observed in this cell line in response to CAF-derived conditioned medium. Interestingly, we probed that the comparatively higher intrinsic resistance to PDT of SCC13 cells was mediated by the elevated levels of TGFβ1 secreted by this cell line. Our results point at this feature as a promising biomarker to predict both the suitability of PDT and the chances to optimize the treatment by targeting CAF-derived TGFβ1 in the road to a more personalized treatment of particular cSCC tumors. Full article

Image classification with convolutional neural networks (CNN) offers an unprecedented opportunity to medical imaging. Regulatory agencies in the USA and Europe have already cleared numerous deep learning/machine learning based medical devices and algorithms. While the field of radiology is on the forefront of artificial intelligence (AI) revolution, conventional pathology, which commonly relies on examination of tissue samples on a glass slide, is falling behind in leveraging this technology. On the other hand, ex vivo confocal laser scanning microscopy (ex vivo CLSM), owing to its digital workflow features, has a high potential to benefit from integrating AI tools into the assessment and decision-making process. Aim of this work was to explore a preliminary application of CNN in digitally stained ex vivo CLSM images of cutaneous squamous cell carcinoma (cSCC) for automated detection of tumor tissue. Thirty-four freshly excised tissue samples were prospectively collected and examined immediately after resection. After the histologically confirmed ex vivo CLSM diagnosis, the tumor tissue was annotated for segmentation by experts, in order to train the MobileNet CNN. The model was then trained and evaluated using cross validation. The overall sensitivity and specificity of the deep neural network for detecting cSCC and tumor free areas on ex vivo CLSM slides compared to expert evaluation were 0.76 and 0.91, respectively. The area under the ROC curve was equal to 0.90 and the area under the precision-recall curve was 0.85. The results demonstrate a high potential of deep learning models to detect cSCC regions on digitally stained ex vivo CLSM slides and to distinguish them from tumor-free skin. Full article

Reflectance confocal microscopy (RCM) presents a non-invasive method to image actinic keratosis (AK) at a cellular level. However, RCM criteria for AK response monitoring vary across studies and a universal, standardized approach is lacking. We aimed to identify reliable AK response criteria and to compare the clinical and RCM evaluation of responses across AK severity grades. Twenty patients were included and randomized to receive either cryotherapy (n = 10) or PDT (n = 10). Clinical assessment and RCM evaluation of 12 criteria were performed in AK lesions and photodamaged skin at baseline, 3 and 6 months. We identified the RCM criteria that reliably characterize AK at baseline and display significant reduction following treatment. Those with the highest baseline odds ratio (OR), good interobserver agreement, and most significant change over time were atypical honeycomb pattern (OR: 12.7, CI: 5.7–28.1), hyperkeratosis (OR: 13.6, CI: 5.3–34.9), stratum corneum disruption (OR: 7.8, CI: 3.5–17.3), and disarranged epidermal pattern (OR: 6.5, CI: 2.9–14.8). Clinical evaluation demonstrated a significant treatment response without relapse. However, in grade 2 AK, 10/12 RCM parameters increased from 3 to 6 months, which suggested early subclinical recurrence detection by RCM. Incorporating standardized RCM protocols for the assessment of AK may enable a more meaningful comparison across clinical trials, while allowing for the early detection of relapses and evaluation of biological responses to therapy over time. Full article

Line-field confocal optical coherence tomography (LC-OCT) is a novel, non-invasive technique for real-time skin imaging. Imiquimod (IQ) 5% cream is an immune response modifier currently approved for the treatment of small, superficial basal cell carcinoma (BCC). The aim of this study was to investigate if LC-OCT may be useful to enhance the treatment monitoring of BCC. Twenty superficial BCCs from 12 patients were treated with IQ 5% cream once daily, five days a week, for six weeks. Clinical and LC-OCT evaluations were performed at baseline and 4 weeks after the end of treatment. At the end of the study, 13 lesions showed a complete clinical and LC-OCT response, 4 lesions a partial clinical and LC-OCT response, and 3 lesions a complete clinical response but residual tumoral signs at LC-OCT. Our pilot study suggests that LC-OCT may represent a promising tool able to enhance the evaluation of the treatment response of BCCs to non-invasive treatments. In our case series, its use highlighted, through a detailed, fast, and complete examination of the treated area, three cases of residual BCC that otherwise would have gone undetected at clinical examination. Future studies on larger series of patients treated with different modalities and with a longer follow-up are advisable. Full article

Non-melanoma skin cancer (NMSC) is the most common malignant tumor affecting fair-skinned people. Increasing incidence rates of NMSC have been reported worldwide, which is an important challenge in terms of public health management. Surgical excision with pre-operatively identified margins is one of the most common and effective treatment strategies. Incomplete tumor removal is associated with a very high risk of recurrence and re-excision. Biological tissues can absorb and re-emit specific light wave-lengths, detectable through spectrophotometric devices. Such a phenomenon is known as autofluorescence (AF). AF spectroscopy has been widely explored for non-invasive, early detection of NMSC as well as for evaluation of surgical margins before excision. Fluorescence-aided diagnosis is based on differences in spectral characteristics between healthy and neoplastic skin. Understanding the biological basis of such differences and correlating AF intensity to histological features could improve the diagnostic accuracy of skin fluorescence spectroscopy. The primary objective of the present pre-clinical ex vivo study is to investigate the correlation between the intensity of cutaneous AF and the histopathological features of NMSC. Ninety-eight lesions suggestive for NMSCs were radically excised from 75 patients (46 M; 29 F; mean age: 79 years). After removal, 115 specific reference points on lesions (“cases”; 59 on BBC, 53 on SCC and 3 on other lesions) and on peri-lesional healthy skin (controls; 115 healthy skin) were identified and marked through suture stitches. Such reference points were irradiated at 400–430 nm wavelength, and resulting emission AF spectra were acquired through spectrophotometry. For each case, AFIR (autofluorescence intensity ratio) was measured as the ratio between the number of photons emitted at a wavelength ranging between 450 and 700 nm (peak: 500 nm) in the healthy skin and that was captured in the pathological tissue. At the histological level, hyperkeratosis, neoangiogenesis, cellular atypia, epithelial thickening, fibrosis and elastosis were quantified by light microscopy and were assessed through a previously validated grading system. Statistical correlation between histologic variables and AFIR was calculated through linear regression. Spectrometric evaluation was performed on 230 (115 cases + 115 controls) reference points. The mean AFIR for BCC group was 4.5, while the mean AFIR for SCC group was 4.4 and the fluorescence peaks at 500 nm were approximately 4 times lower (hypo-fluorescent) in BCCs and in SCCs than in healthy skin. Histological variables significantly associated with alteration of AFIR were fibrosis and elastosis (p < 0.05), neoangiogenesis, hyperkeratosis and epithelial thickening. Cellular atypia was not significantly associated with alteration of AFIR. The intensity of fluorescence emission in neoplastic tissues was approximately 4 times lower than that in healthy tissues. Histopathological features such as hyperkeratosis, neoangiogenesis, fibrosis and elastosis are statistically associated with the decrease in AFIR. We hypothesize that such tissue alterations are among the possible biophysical and biochemical bases of difference in emission AF between neoplastic and healthy tissue. The results of the present evaluation highlighted the possible usefulness of autofluorescence as diagnostic, non-invasive and real-time tool for NMSCs. Full article

The exact mechanisms of the imiquimod (IMQ)-induced antitumor effect have not been fully understood. Although both topical IMQ treatment and anti-PD-1 antibody may be used for primary skin lesions or skin metastases of various cancers, the efficacy of each monotherapy for these lesions is insufficient. Using a murine tumor model and human samples, we aimed to elucidate the detailed mechanisms of the IMQ-induced antitumor effect and analyzed the antitumor effect of combination therapy of topical IMQ plus anti-PD-1 antibody. Topical IMQ significantly suppressed the tumor growth of MC38 in wildtype mice. IMQ upregulated interferon γ (IFN-γ) expression in CD8⁺ T cells in both the lymph nodes and the tumor, and the antitumor effect was abolished in both Rag1-deficient mice and IFN-γ-deficient mice, indicating that IFN-γ produced by CD8⁺ T cells play a crucial role in the IMQ-induced antitumor effect. IMQ also upregulated PD-1 expression in T cells as well as PD-L1/PD-L2 expression in myeloid cells, suggesting that IMQ induces not only T-cell activation but also T-cell exhaustion by enhanced PD-1 inhibitory signaling. Combination therapy of topical IMQ plus anti-PD-1 antibody exerted a significantly potent antitumor effect when compared with each single therapy, indicating that the combination therapy is a promising therapy for the skin lesions of various cancers. Full article

Chronic ultraviolet B (UV-B) irradiation is known to be one of the most important hazards acting on the skin and poses a risk of developing photoaging, skin with cutaneous field cancerization (CFC), actinic keratosis (AKs), and squamous cell carcinomas (SCCs). Most of the UV-B light is absorbed in the epidermis, affecting the outermost cell layers, the stratum corneum, and the stratum granulosum, which protects against this radiation and tries to maintain the permeability barrier. In the present work, we show an impairment in the transepidermal water loss, stratum corneum hydration, and surface pH after chronic UV-B light exposure in an immunologically intact mouse model (SKH1 aged mice) of skin with CFC. Macroscopic lesions of AKs and SCCs may develop synchronically or over time on the same cutaneous surface due to both the presence of subclinical AKs and in situ SCC, but also the accumulation of different mutations in keratinocytes. Focusing on skin with CFC, yet without the pathological criteria of AKs or SCC, the presence of p53 immunopositive patches (PIPs) within the epidermis is associated with these UV-B-induced mutations. Reactive epidermis to chronic UV-B exposure correlated with a marked hyperkeratotic hyperplasia, hypergranulosis, and induction of keratinocyte hyperproliferation, while expressing an upregulation of filaggrin, loricrin, and involucrin immunostaining. However, incidental AKs and in situ SCC might show neither hypergranulosis nor upregulation of differentiation markers in the upper epidermis. Despite the overexpression of filaggrin, loricrin, involucrin, lipid enzymes, and ATP-binding cassette subfamily A member 12 (ABCA12) after chronic UV-B irradiation, the permeability barrier, stratum corneum hydration, and surface pH were severely compromised in the skin with CFC. We interpret these results as an attempt to restore the permeability barrier homeostasis by the reactive epidermis, which fails due to ultrastructural losses in stratum corneum integrity, higher pH on skin surface, abundant mast cells in the dermis, and the common presence of incidental AKs and in situ SCC. As far as we know, this is the first time that the permeability barrier has been studied in the skin with CFC in a murine model of SCC induced after chronic UV-B irradiation at high doses. The impairment in the permeability barrier and the consequent keratinocyte hyperproliferation in the skin of CFC might play a role in the physiopathology of AKs and SCCs. Full article

It is known that actinic keratoses (AKs) can progress to invasive squamous cell carcinoma (SCC). The histological PRO grading of AKs is based on the growth pattern of basal keratinocytes and relates to their progression risk. AKs can be non-invasively characterized by line-field confocal optical coherence tomography (LC-OCT). The aim of the study was to define criteria for an LC-OCT grading of AKs based on the PRO classification and to correlate it with its histological counterpart. To evaluate the interobserver agreement for the LC-OCT PRO classification, fifty AKs were imaged by LC-OCT and biopsied for histopathology. PRO histological grading was assessed by an expert consensus, while two evaluator groups separately performed LC-OCT grading on vertical sections. The agreement between LC-OCT and histological PRO grading was 75% for all lesions (weighted kappa 0.66, 95% CI 0.48–0.83, p ≤ 0.001) and 85.4% when comparing the subgroups PRO I vs. PRO II/III (weighted kappa 0.64, 95% CI 0.40–0.88, p ≤ 0.001). The interobserver agreement for LC-OCT was 90% (Cohen’s kappa 0.84, 95% CI 0.71–0.91, p ≤ 0.001). In this pilot study, we demonstrated that LC-OCT is potentially able to classify AKs based on the basal growth pattern of keratinocytes, in-vivo reproducing the PRO classification, with strong interobserver agreement and a good correlation with histopathology. Full article

Cutaneous squamous cell carcinomas (CSCC) account for about 20% of all keratinocyte carcinomas, which are the most common form of cancer. Heterogeneity of treatments and low mortality are a challenge in obtaining accurate incidence data and consistent registration in cancer registries. Indeed, CSCC mostly presents as an indolent, low-risk lesion, with five-year cure rates greater than 90% after surgical excision, and only few tumors are associated with a high-risk of local or distant relapse; therefore, it is particularly relevant to identify high-risk lesions among all other low-risk CSCCs for the proper diagnostic and therapeutic management. Chemotherapy achieves mostly short-lived responses that do not lead to a curative effect and are associated with severe toxicities. Due to an etiopathogenesis largely relying on chronic UV radiation exposure, CSCC is among the tumors with the highest rate of somatic mutations, which are associated with increased response rates to immunotherapy. Thanks to such strong pre-clinical rationale, clinical trials led to the approval of anti-PD-1 cemiplimab by the FDA (Food and Drug Administration) and EMA (European Medicines Agency), and anti-PD-1 pembrolizumab by the FDA only. Here, we provide a literature review and clinical recommendations by a panel of experts regarding the diagnosis, treatment, and follow-up of CSCC. Full article

A variety of well-characterized cutaneous paraneoplastic syndromes (PNS) are diagnosed during internal malignancies; however, the spectrum of keratinocyte skin neoplasms (KSC) related to PNS is still obscure. The aim of the present review is to compile and evaluate the literature data on PNS associated with a keratinocyte skin neoplasm (KSC). Employing Pubmed, MEDLINE was searched for KSC-associated PNS reports. Forty relevant entries were assembled, reporting a total of 41 PNS cases associated with a KSC (34 male). No review paper compiling this topic was found. Six distinct PNS entities were identified, and malignancy associated hypercalcemia (MAH; 78%), anemia (10%) and Bazex syndrome (5%) were the most frequently reported among them. 85% of the PNS were reported in association with SCC, 10% with BCC, and the rest with adnexal tumors. The median age of the patients at the time of PNS diagnosis was 58 years (range: five–83 years). In most cases the PNS was diagnosed either concurrently or after the KSC diagnosis. KSC predisposing conditions, as scars (22%) or hidradenitis suppurativa (20%), were reported in >70% of the PNS cases. Most PNS resolved after KSC treatment. In conclusion, PNS of a rather limited spectrum of entities are reported in association with KSC. They also seem to be rare, possibly reflecting a limited capacity of KSC to provoke overt PNS. Full article

The role of the immune system in cancer growth is well recognized and the development of immunotherapy represents a breakthrough in cancer treatment. Recently, the use of systemic immunotherapy was extended to keratinocyte carcinoma (KC), specifically locally advanced and metastasizing basal and squamous cell carcinoma. However, since most KC lesions are non-aggressive, systemic treatment with associated side effects is rarely justified. Conversely, topical immunotherapy with imiquimod remains restricted to premalignant and superficial lesions. Use of laser in the treatment of KC has evolved from physical tumor destruction and laser-assisted drug delivery to laser-mediated immune modulation. Evidence indicates that laser monotherapy can lead to immune cell infiltration, tumor reduction and resistance to tumor re-inoculation. Combining laser with immunotherapeutic agents, termed laser immunotherapy (LIT), may further potentiate immune activation and tumor response. Studies on LIT show not only direct anti-tumor effects but systemic adaptive immunity, illustrated by the prevention of tumor recurrence and regression in distant untreated tumors. These findings imply a therapeutic potential for both local and metastatic disease. This work provides rationales for immune-based treatment of KC and presents the current status of KC immunotherapy. Aiming to expand the field of KC immunotherapy, the review discusses the literature on immune activation following laser monotherapy and LIT. Full article

Ultraviolet radiation (UVR) arising from sun exposure represents a major risk factor in the development of keratinocyte carcinomas (KCs). UVR exposure induces dysregulated signal transduction, oxidative stress, inflammation, immunosuppression and DNA damage, all of which promote the induction and development of photocarcinogenesis. Because the incidence of KCs is increasing, better prevention strategies are necessary. In the concept of photoprevention, protective compounds are administered either topically or systemically to prevent the effects of UVR and the development of skin cancer. In this review, we provide descriptions of the pathways underlying photocarcinogenesis and an overview of selected photoprotective compounds, such as repurposed pharmaceuticals, plant-derived phytochemicals and vitamins. We discuss the protective potential of these compounds and their effects in pre-clinical and human trials, summarising the mechanisms of action involved in preventing photocarcinogenesis. Full article

Oral squamous cell carcinoma (OSCC) is the most prevalent form of cancer of lips and oral cavity, and its diagnostic delay, caused by misdiagnosis at the early stages, is responsible for high mortality ratios. Biopsy and histopathological assessment are the gold standards for OSCC diagnosis, but they are time-consuming, invasive, and do not always enable the patient’s compliance, mainly in cases of follow-up with the need for more biopsies. The use of adjunctive noninvasive imaging techniques improves the diagnostic approach, making it faster and better accepted by patients. The present review aims to focus on the most consolidated diagnostic techniques, such as vital staining and tissue autofluorescence, and to report the potential role of some of the most promising innovative techniques, such as narrow-band imaging, high-frequency ultrasounds, optical coherence tomography, and in vivo confocal microscopy. According to their contribution to OSCC diagnosis, an ideal three-step diagnostic procedure is proposed, to make the diagnostic path faster, better, and more accurate. Full article

We aimed to provide a comprehensive overview of the link between vitamin D and non-melanoma skin cancer (NMSC). For this purpose, we conducted a systematic literature review (updated to 3 February 2021) and meta-analysis of the studies reporting on the association between vitamin D intake (from diet and supplements) and blood concentration, polymorphisms of the vitamin D receptor (VDR) and vitamin D binding protein (VDBP) genes, and the risk of NMSC. Random effects meta-analysis models were fitted to merge study-specific risk estimates into summary relative risk (SRR) and corresponding 95% confidence intervals (CI). Twenty-four studies altogether were included. There was a suggestive association between increasing serum/plasma vitamin D concentration and NMSC risk (SRR for highest vs. lowest concentration 1.67, 95%CI 0.61–4.56), although with large heterogeneity across studies (I² = 91%). NMSC risk was associated with highest vitamin D intake in observational studies but not in clinical trials. Finally, there was no significant association between any polymorphism of the VDR and VDBP genes and NMSC risk. In conclusion, no strong relationship between vitamin D metabolism and NMSC risk appears to exist according to our systematic review and meta-analysis, although some findings are worthy of further investigation. Full article