Actinic Keratosis Pathogenesis Update and New ...

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Actinic Keratosis Pathogenesis Update and New Patents Carmen Cantisani1,*, Giovanni Paolino1, Marcello Melis2, Valentina Faina1, Federico Romaniello1, Dario Didona1, Michele Cardone1 and Stefano Calvieri1 1

UOC of Dermatology.Policlinico Umberto I, University “Sapienza” of Rome, Italy; 2Profilocolore Srl, Rome, Italy Received: August 8, 2015; Revised: May 3, 2016; Accepted: May 3, 2016

Abstract: Background: Actinic keratosis is a common premalignant skin lesion. Because of its increasing incidence, several efforts have been made to earlier detectection and to improve knowledge on photocarcinogenic pathways of keratinocytes. As a consequence, recently new discoveries have been done in this field.

Carmen Cantisani

Objective: Starting from our previous review on actinic keratosis, we reviewed the literature focusing on pathogenesis and new patents in order to highlight the most recent progresses in diagnosis and therapeutic approach. Conclusion: Although several efforts have been done in the field of photodamaged skin, new upgrades in diagnosis and therapy are needed to detect superficial actinic keratosis earlier, to improve the disease free survival of patient and to better treat the field cancerization.

Keywords: Actinic keratosis, aminolevulinic acidc, inflammation, patent, photochemotherapy, photodamage, sunlight, treatment outcome. INTRODUCTION Actinic keratoses (AKs) are one of the most common dermatological complaint of chronically sun- damaged skin [1], generally presenting in elderly people with fair skin as erythematous, scaly macules/papules or plaques, generally on sun damaged skin. Lesions can be single or more frequently multiple, across an area of sunlight exposure/damage (cancerization field) [24]. Described by Dubreuilh in 1826, AKs can be considered manifestations of cutaneous photo-ageing together with atrophy of skin with loss of elasticity, wrinkles, telangectasias and pigmentary changes [5]. Although AKs have been considered for years as pre-malignant lesions it is nowadays thought to be part of a disease continuum starting from normal sun-damaged skin to invasive squamous cell carcinoma (iSCC) [6, 7]. The number of people with AK is rapidly growing worldwide, especially in the UK, the US and Australia; in Italy the absence of registers determines the fact that the true incidence of this disease is not known. In the UK there is a prevalence of AK in patients over 70 years: 34% in men and 18% in women: the higher prevalence in men is explained, in part, by greater exposure to sun during outdoor work. In fact, it is estimated that outdoor workers are exposed to a UV-radiation dose 2-3 times higher than indoor workers. The risk for NMSC (non-melanoma skin *Address correspondence to this author at the Department of Dermatology and Plastic Surgery, Sapienza University, Rome, Italy, Policlinico Umberto I Hospital, Rome, Italy; Tel: +39-3479385719; Fax: +39-06490243; E-mails: [email protected]; [email protected]

1872-213X/16 $100.00+.00

cancer), including AK, among workers who have worked outdoor for more than 5-years, is 3-fold higher than the risk among those with no years of working outdoors, that’s why AK are recently considered an occupational disease [8]. From a histological point of view AKs are defined by dysplasia of keratinocytes in the epidermal basal layer manifesting atypical nuclei and irregular growth resulting in a thickened stratum corneum [9]. More than one histological subtype has been described, including lichenoid, hypertrophic, bowenoid, pagetoid, and pigmented [10]. Even though the evolution from AK to invasive SCC can be considered improbable, and spontaneous complete regression can be possible, it is currently impossible to predict the natural evolution of all lesions which must be prompted treated to avoid the risk of rapid dermal invasion and possible metastasis especially in immunosuppressed patients [11, 12]. AKs and SCC belong to the same kind of disease. It was also highlighted that the same genes that show an upregulation in AK and SCC are conversely downregulated in unaffected skin and more related to occupational UV exposure, compared to recreational exposure more related to BCC and melanoma incidence. EPIDEMIOLOGY AKs are extremely common. The incidence is rapidly increasing as reported by recent studies becoming a serious problem [13]. Countries with high sun exposure have a high prevalence, in particular the prevalence rate being highest in Australian people > 40%, affecting more than 40% of people in this group age [14]. AKs generally involve people with © 2016 Bentham Science Publishers

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skin phototype 1 and 2 (according to Fitzpatrick score) [15]. The prevalence ranges between 11 and 26% in United States while in Europe it is 15% for men and 6% for women. Usually patients with AKs show more than 6 lesions. Elderly, male sex, extreme UV damage, latitude, genetical syndromes, sunburns in childhood, wounds, chemical exposure, and immunosuppression, are predisposing conditions [16]. Pathogenesis UV radiation is the most important cause of development of AKs and skin cancer in general; both UVA and UVB are carcinogens, even if UVB has the most carcinogenic effect. UVA penetrating deeper in the skin cause predominantly photoaging with epidermal and dermal changes (atrophy of the skin, wrinkles) [9]. PATHOGENESIS AKs generally presents as recurrent lesions on chronically sun-exposed areas, also known as field cancerization, firstly reported by Slaughter et al. in 1953 [17]. In an interesting study Steven Padilla et al. showed the different genes expression profile in normal skin, AKs and SCC, revealing in AK and SCC a similar up-regulation of genes, down-regulated in normal skin and a similar downregulation of genes upregulated in normal skin, confirming the common origin of both lesion and in particular AK as a precursor lesion of SCC [18]. The most commonly mutated gene in AK is TP53. Other common mutations include Ras genes, c-myc protooncogenes, p16INK4 a tumor suppressor gene, and associated telomerase activity [19]. The main cause of AK is exposure to ultraviolet radiation (UVR). However, cutaneous HPVs may act as a co-carcinogen with UVR. Actually, prevalence and viral load is significantly higher in actinic keratosis than in SCC, suggesting that the virus may play a role in the early stages of carcinogenesis (known as "hit-andrun" hypothesis). Several studies have shown that E6 and E7 from certain cutaneous HPV types display transforming activities, further confirming their potential role in carcinogenesis [20]. AK are characterized by dysplasia of the basal epidermal layer with cyto-architectural keratinocytes abnormalities with a slight accumulation of mutated p53 in the nuclei, more aggressive AK even if KIN I (Keratinocytes Intraepithelial Neoplasia), may be due to higher accumulation of mutated p53 (they can evolve directly to more invasive SCC), while Bowen’s disease is characterized by abnormalities of all epidermal layers, SCC are characterized by infiltration of superficial and deep dermis of mutated cells with a great level of mutated p53, underlying a higher risk to give metastasis. Usually, progression from AK to cutaneous invasive SCC (iSCC) could be due to the growth of full thickness skin neoplasm in malignancies, in a multistep model, over many years, with a low aggressive potential, however sometimes iSCC may also straightly develop after a proliferation of atypical (complete dedifferentiated) basaloid cells restricted mainly to the basal layer of the epidermis (AK/KIN I) known as a differentiated pathway, not always related to the full thickness epidermal invasion but to the dermal infiltrating capacity to invade dermis and especially adnexal structures. The real risk of progression is unknown it was approximately estimated to vary from 0.6% at 1 year to 2.57% in 4 years and increase to 53% in patients with

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NMSC. There are no reliable clues to predict which lesion will become more aggressive. Conversion of AK into iSCC has been thought to occur as a result of progressive stages of keratinocyte intraepidermal neoplasia (KIN). Thus, lesions displaying-atypical keratinocytes with nuclear atypia and hyperchromatic nuclei high mitotic rate, and clusters and overlapping in the epidermis lower third (KIN I) would develop into lesions with the presence of atypical keratinocytes in the lower two-thirds of the epidermis (KIN II) and later to injuries with full thickness epidermal neoplasm (KIN III). This pathway, which needs complete dedifferentiation of the epidermis, is known as the classic pathway. Therefore, by analogy to CIN, usually KIN I/AK I features have been thought to be of low risk, because they would require progression to more advanced stage before acquiring infiltrating dermal phenotype. Although it is not unusual to find cases where iSCC seems to directly develop from proliferating atypical basaloid cells restricted to the lower third of the epidermis (KIN I/AK I), while the mild and upper epidermal layers remain virtually untouched [21]. Lastly, the assumption that iSCC may only develop from KIN III or at least KIN II, always needing previous evolution from KIN I to KIN III, is not universally accepted. They concluded that the direct transformation from AKI to iSCC (known as differentiated pathway), is the most common process [9, 10]. HISTOLOGY Histological findings in AKs include atypical keratinocytes showing polymorphism of nucleus, hyperchromasia and big nuclei surrounded by exiguous cytoplasm. Pathological keratinocytes are not uniformly disposed, displaying clusters that not cross the epidermal basal membrane even if these features can involve the above epidermis, showing a Bowenoid aspect. Sometimes, an increase in keratinocytes mitosis and is also found pathological mitoses in the suprabasal epidermis [4]. Another usual characteristic of AK is a thick cornified layer that shows either orthokeratosis or parakeratosis. However, hyperkeratosis is not present in lesions that show massive ulceration, trauma or topical therapy result. The capital features of AK is actinic elastosis, shown with different extent in every AK, even if it may sporadically be substituted by fibrotic collagen following repeated epidermal detachment, ulceration or topical therapies that lead to superficial dermal scar. The elastosis has been thought a side effect of sun exposure, even though latest studies have proposed the possibility that this kind of stromal atrophy may play a pivotal role in cancerization by promoting epidermal transformation, therefore contrasting photo-ageing with laser/IPL or other treatment that induce fibroblast proliferation and hyaluronic acid production may be interesting for aesthetic purposes but also prevent skin cancer development as pointed out by Tian et al. in the naked mole-rat fibroblasts. This rodent displays exceptional longevity, with a maximum lifespan exceeding 30 years and his fibroblasts secrete extremely high-molecular-mass hyaluronan (HA), which is over five times larger than human or mouse HA. This trait may provide cancer resistance and longevity to this species [22]. AK base usually exhibit chronic inflammation

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ranging between sparse lymphoid cells and a dense lichenoid lymphocytic infiltrates [5]. The existence of atypical keratinocytes going downwards along hair follicles and sweat ducts would delineate a subtype of AK with ‘proliferative’ features. The relevance of this subtype is linked to its resistance to the therapy due to the deep position of atypical cells, they are difficult to be identified [11, 23]. It was also supposed that the expression of MUC1 (a transmembrane glycoprotein that contributes to the progression of certain premalignant and malignant lesions) in AK would be induced by alteration of keratinocytes differentiation and associated to the degree of dysplasia rather than the thickness of the epidermis, in fact it is absent in normal keratinocytes while it appear on apical surface in the early stage, or on the entire surface in more advanced disease, therefore it can be considered a biomark in the evolution to invasive lesion with a diagnostic value and future therapeutic target. Also, recent studies have observed the expression of podoplanin changes during the neoplastic processes, we therefore aimed at assessing its expression in cancer and stromal cells of basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and actinic keratosis (AK), suggesting a potential role of podoplanin in the development and progression of this malignancy [24]. p300 and p300/CBP-associated factor (PCAF) are histone modifiers and transcriptional co-factors involved in a number of cell processes. Using tissue microarray and immunohistochemistry, is possible to evaluate the expression in relation to the type of the lesion (AK, Bowen’s disease and SCC) and prognostic parameters. In fact the increase in nuclear expression of p300, as well as the presence of cytoplasmic but loss of nuclear expression of PCAF, could play an important role in the development and progression of these cutaneous lesions [25]. Finally, the effectiveness of the fans in the treatment of AK explains how cyclooxygenase (COX) 1 and 2 enzyme up-regulation is involved in the pathogenetic process of these lesions. NSAID (non-steroidal anti-inflammatory drugs) acts by downregulating cyclooxygenase (COX) enzymes and then the production of prostaglandin E2 (PGE2) which is involved in the suppression of T and B cell proliferation and in the cytotoxic activity of natural killer cells. UV exposure of the skin induces COX-2 expression and prostaglandin production. COX-2 not only mediates dermal inflammation, but also promotes tumor growth by enhancing tumor cell proliferation, stimulating angiogenesis, and inhibiting apoptosis. Co-inhibition of COX-2 and COX-1 is needed in order to block Vascular Endothelian Growth Factor (VEGF) synthesis, which is involved in cancer development. These observations suggest that COX-1 plays a role in the regulation of angiogenesis by promoting VEGF synthesis. In comparison with diclofenac, piroxicam is characterized by higher COX-1 inhibition activity suggesting that this NSAID could be a good candidate for AK treatment [26]. DIAGNOSIS Although skin cancers are the only kind of tumors that is nearly always recognizable in its early-phases [13]. Early identification and eradication of skin cancer is necessary. Gold standard still remain the histology in the diagnostic

work-up of AK, while this invasive method may not be possible especially when many skin sites are affected given also to the low risk to progression [27]. In these cases, in vivo noninvasive diagnostic tools can be helpful for the diagnosis. Several technologies are currently available or under development to identify early skin cancers. Similarly, several molecular tests are increasingly being used by dermopathologist to classify lesions as benign vs malignant. Scale is histologically represented by orthokeratosis or parakeratosis and polygonal nucleated cells in the stratum corneum. AK shows uniform pink or brownish background and evident keratin. Specific dermoscopic patterns can improve the clinical diagnosis allowing the differentiation among AKs, intraepidermal carcinoma and invasive SCC [28]. Moreover, because there is currently more than one topical therapy choices available for AK, the comprehension of specific patterns helps in the selection of the best therapeutic option. The key clues of actinic keratosis are: erythematous pseudo-network, superficial scaling, linear-wavy vessels, yellow ovoid/white circles. Non-pigmented AK of the face is a mixed pattern called "strawberry pattern", marked by a background erythema/red pseudo-network of unfocused, large vessels situated among the hair follicles, in combination with evident follicular openings ringed by a white halo. Facial pigmented AK include several slate-gray to darkbrown dots and globules around the follicular ostia, annulargranular pattern and brown to gray pseudo-network. The classic dermoscopic features of SCC in situ have been described as a 6 scaly red surface with glomerular or dotted vessels corresponding to round vessels in the superficial dermis [29, 30]. Invasive squamous cell carcinoma (SCC) of the skin is highly protean in clinical presentation according to the rate of differentiation and localization. It looks like as a thick plaque or nodule. Ulceration may or may not be evident; extensive ulcerated lesions are not differentiable and may not show clear dermoscopic structures. Poorly differentiated neoplasms usually are pinker compared to whitish welldifferentiated neoplasms. Malignant tumors are destructive, causing the loss of normal tissue in comparison to noninvolved skin. The main characteristic of SCC is keratinization, so white structure less zones are frequent. In well differentiated tumors like keratoacanthoma, a central area of superficial scale, crust or horn (white, yellowish or brown) may be present, surrounded by dull-white structure less areas. There may be also irregular clusters of white perifollicular circles, blood spots, blood vessels arranged in different ways, with irregular round or coiled, looped, serpentine, branched or polymorphic morphology. A greater variation in vessels arrangement is associated with poor differentiation pathologically [31]. Reflectance Confocal Microscopy (RCM) is an optical imaging technique useful in the recognition of cellular and subcellular details of the skin. Reflectance confocal microscopy is a relatively new noninvasive imaging technique that has shown promise as a diagnostic aid in many dermatologic conditions although for this lesions, easily clinically diagnosed, it can be considered expensive and time consuming, except for differential diagnosis. It helps to bridge the gap between dermoscopy and histologic analysis, allowing hori-


zontal evaluation of a lesion (as with dermoscopy) while producing in vivo images of the epidermis and superficial dermis at a resolution that approximates that of histopathologic specimens. Furthermore, RCM is used to observe response to therapy and follow-up of skin cancer. RCM detects morphologic features of AK in both clinical and subclinical AK; furthermore, it identified subclinical AK by visualization of cellular and nuclear atypia within the spinous cell layer [32]. Atypical honeycomb and/or a disarranged epidermal pattern were seen in all the AKs and SCCs. While the SCCs showed extensive atypia and/or disarrangement of the 7 spinous-granular layer, most of the AKs revealed a focally disarranged or a mildly atypical honeycomb pattern. Lower aggressive AKs demonstrated sparse round vessels in the superficial dermis on RCM. Astner et al. described a superficial disruption of the stratum corneum, pleomorphic parakeratosis, severe atypical pleomorphism of the epidermis, severe architectural disarray of the epidermis, and atypical aggregates of keratinocytes in the dermis. Some of these features, including the changes at the stratum corneum and spinous-granular layers, dermal changes are associated to more aggressive forms. AKs demonstrated focal atypia of the epidermis on RCM and few vessels in the superficial dermis, findings that are similar to those of SCC. These findings reflect the concept that AK, in situ SCC, and invasive SCC are a continuous process on the spectrum of keratinocytic neoplasia and with the concept of “field cancerization,” which suggests that subclinical pre-neoplastic cells are present frequently in skin sites surrounding AKs and SCCs [33, 34]. Clinical diagnosis has intra and inter observer variability, and non-invasive and objective measure to diagnose them. With new devices dermatologists are trying to determine to automatically detect lesions not visible through naked eyes. Future research should focus on more typical features to enhance sensitivity for AKs without erythema and reduce false positives related to the anatomical structures of the face, furthermore it’d be better evaluated treatment efficacy. The best outcomes are critically dependent on careful and repeatable assessment. Diagnostic standardization is needed. Automatic image analysis is a promising tool to remove inter-observer variability and is represented as a first step towards standardizing the diagnosis and treatment of AKs. The automated identification through vascular pattern, made with non-invasive devices, as Antera-3D Miravex camera or similar, could be useful in everyday practice, especially for the identification of not-yet visible lesions already present in the cancerization field surrounding the visible one. Several technologies are currently available or under development to improve early diagnosis. Similarly, several molecular tests are increasingly being used by dermatopathologist for classifying lesions as benign vs malignant. Noninvasive imaging devices (dermoscopy, 3D digital cameras, total body microscopy, dermaspectra, digital derm, fotofinder, melanoscan, malesafe, canfield, ultrasound, confocal microscopy and optical coherence tomography) in clinical practice and research will help in better diagnosing and treat skin cancers [35].

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TREATMENT To date, a specific therapeutic flow-chart for first and second line therapies does not exist, also because it depends on lesions’ characteristic and number, anatomical site involved, patient’s comorbidities, devices availability and dermatologist experience. Since all lesions need to be treated, because it is not possible to predict which of them will develop metastatic type, it is important to consider some criteria before choose the better treatment for each patient. As described in our previous review, several therapies are available including lesion and field directed modes [36]. The guidelines so far, identify main active treatment options: cryosurgery, laser, curettage, DTC, laser, fluorouracil, diclofenac, imiquimod, ingenol mebutate, and photodynamic therapy (PDT) [31, 37]. Probably the combination of different alternatives available can be beneficial especially for long term results. Currently, the two main approaches to treating AK are in-office treatments, such as lesion directed therapy (such as cryotherapy) targeted at single isolated lesions and patient applied topical therapies directed at a more widespread field of lesions. Data on long-term, follow-up post treatment for AK are limited; however, there is much discussion about possible recurrence of existing lesions versus emergence of new AK lesions in a similar area [38]. PHOTODYNAMIC THERAPY Among treatment options for procedural field therapy the photodynamic therapy (with Methylaminolevulinate MAL under occlusion for 3 hours followed by 37 J/cm2 of red light or 9 without occlusion and with an incubation time of 90-120 minutes (depending on meteorological conditions play a special role) home-based daylight-PDT). Photodynamic therapy is a noninvasive technique with excellent cosmetic outcome and good curative results, when used in initial stages of skin cancers for superficial lesions. It allows treatment of large and multiple lesions with excellent cosmetic results, it’s efficacy is increased when combined with other topical immunomodulators for numerous and more deeper lesions. Its evolution, called daylight photodynamic therapy avoiding the use of the aklite lamp (Galderma®) overtake the limitation, strictly related to the devices, such as periocular regions, pain or other side effects during the procedure, and increase patient’s compliance, with self-limited (just one or due days durations) local skin reactions, well accepted by patients who can spend time outsite under visible light. Its efficacy, in our experience, is going to improve with time, in fact cosmetologic results become interesting from the second treatment, due to stimulation of memory T cell response and to fibroblast activation eradicating also not visible lesions [6]. Able to eradicate both the clinically visible and subclinical AK within the treatment area [4, 31]. The use of other topical agents, instead, is often limited by the protracted course of treatment required and local side effects, which are often not tolerated by patients and may make adherence difficult. With all topical treatment patients should avoid the orbit to prevent painful swelling, avoid UV radiation, start with one or not extensive body part at a time to avoid excessive systemic absorption or severe irritation, and wash hands after application. Patients should be told



about the course of therapy and shown pictures of the expected inflammation. It is impossible to forecast clinically which AK will invade the dermis and which will not, therefore, a target treatment is required. CLINICAL GUIDANCE AND TREATMENT OF AK RECOMMENDATIONS The modern guideline state that cryosurgery is a valid treatment for single lesions; it is effective especially for thicker lesions but may leave scars or pigmentary outcomes and has a high rate of recurrences. Curettage or excisional surgery are good for hypertrophic lesions, isolated lesions failing to respond to other therapies, lesions below the knee, or where histology is required. PDT is good for confluent scalp lesions, or lesions on sites of poor healing (such as the limbs), but is likely to be more expensive and not allowed in each hospitals. Ingenol mebutate can be considered an alternative options for multiple non hyperkeratotic AK on the face/scalp and/or on the body for a simultaneous or sequential treatment. According to its practical use and its short treatment duration, it is another new valid and efficacy treatment for AKs and cancerization field, with possible mild to moderate LRs self-resolving in a few weeks. Diclofenac gel shows a reasonable efficacy with few unwanted effects in mild lesions [39]. Fluorouracil is good for thin lesions (few or many), for the scalp (including confluent lesions) or face (except peri-orbital) or backs of hands but unwanted effects include soreness due to which it is not allowed in Italy [31]. Imiquimod 5%cream is good for multiple lesions of the face (including lips but except peri-orbital) scalp and trunk and for cancerization field the scalp with excellent long term and aesthetic results. Ingenolo Piroxicam Combination therapy could increase efficacy and patient satisfaction, but the risk of unwanted effects may be synergistic and need to be discussed fully with patients. All available treatments should be combined in different fashion according to the patient’s needs and history [6, 35, 40]. DISCUSSION Because of the rising incidence of AKs all over the world and the high rates of malignant evolution, it is indispensable to look for better treatment of each AK clinical or dermoscopy forms. Given also the chronic nature of AK, optimal management typically requires frequent retreatment to manage emerging lesions. Tolerability is an important consideration for any long-term therapeutic option. The goal of enhanced AK therapy is to simplify dosing frequency, expand treatment area, shortened treatment duration maintaining efficacy. Cryosurgery can be the first line treatment for single AK KIN II/III but according to the high recurrence rate due to its lack of specificity, it is important to focus attention to cancerization field, where mutated keratinocytes continue to be active, and probably the best approach can be a variable treatment combination with other immunomodulators in the market, which stimulate the immune system and are therefore able to clear mutated subclinical lesions over time. Close surveillance of elderly people with severe photodamage is extremely recommended for prompt diagnosis and

management of NMSC and new devices for early identifications are needed. CURRENT & FUTURE DEVELOPMENTS Except for new treatment developments, new therapy coming in the Italian market in the near future such as a new 5 fluoro-uracile formulation plus salicilyc acid,synthetic ingenol mebutate, or imiquimod 3.75%, and the worldwide diffusion of the daylight phodynamic therapy, or systemic agents such as an extract (PLE) from the fern polypodium leucotomos which decreases UV-induced immunosuppression and mutagenic activity improving efficacy of topical treatments research is focused on new devices for early diagnosis we will discuss them in this session. A new therapeutic patent is focusing on a solvent and powder, wherein the powder is prepared by mixing aminolevulinic acid and an effective traditional Chinese medicine component at a weight ratio of 2:1; when the medicament is in use, the solvent and the powder are mixed and compounded together. The medicament is simple and convenient to prepare, wide in medicine sources, low in cost, high in cure rate aiming at the skin cancer and wide in application prospect. Fifteen gram of crotalariasessiiflora, 13g of Dysosma versipellis, 8g of kirilow groundsel herb, 6g of nux vomica, 7g of Wikstroemia indica, 18g of edible tulip, 9g of 12 shorttube lycoris, 22g of Oldenlandia diffusa, 16g of Chinese lobelia, 3g of musk, 9g of rhizoma Smilacis glabrae, 13g of semen coicis and 15g of pinellia ternate.) The traditional Chinese medicine composition for treating the skin cancer has the beneficial effects such as good curative effects on the skin cancer and is fast acting [41, 42] Artoxanthochromane has a remarkable inhibitory effect on the growth of human skin cancer cell strains A431, HME1, A375 and SK23. Therefore, the Artoxanthochromane can be used for preparing antiskin cancer drugs and has a good prospect in development and application [43]. A technique for managing skin cancer applying an isothiocyanate functional surfactant to a zone involved in skin cancer, wherein the isothiocyanate functional surfactant is composed of one isothiocyanate functional group minimum, associated with an aliphatic and/or aromatic carbon atom of the isothiocyanate functional surfactant [44]. The present invention is related to a pulse photodynamic therapy (or pulse PDT) treatment of actinic keratosis (AK) or basal cell carcinoma (BCC). Photodynamic therapy (PDT), is a technique for the treatment of various abnormalities or disorders of the skin or other epithelial organs or mucosa, especially cancers or pre-cancerous lesions, as well as certain non-malignant lesions (e.g. skin complaints such as psoriasis, actinic keratosis (AK) and acne). PDT involves the application of photosensitizing (photochemotherapeutic) agents to the affected area of the body, followed by exposure to photoactivating light in order to activate the photosensitizing agents and convert them into cytotoxic form, whereby the affected cells are killed (necrosis, apoptosis). A range of photosensitizing agents is known, including the psoralens, the porphyrins (e.g. Photofrin (Registered trademark)), the chlorins and the phthalocyanins. Amongst the most clinically useful photosensitizing agents known in the art, however, are 5-aminolevulinic acid and its derivatives, for example esters such as 5-ALA esters.


The mechanism of action of PDT relies on intracellular porphyrins (including PplX) that are photoactive, fluorescing compounds and, upon light activation in the presence of oxygen, singlet oxygen is formed which causes damage to cellular compartments, in particular the mitochondria. Light activation of accumulated porphyrins leads to a photochemical reaction and thereby phototoxicity to the light-exposed target cells. Although PDT is clinically useful in the treatment of a wide range of diseases, a major drawback of such treatment is the concomitant side-effects, particularly at the treatment site. These often include inflammation such as erythema, swelling, edema, burning, itching, exfoliation, hyperpigmentation and prolonged irritation and hypersensitivity after treatment. Such side-effects are particularly undesirable when the treatment site is the face, scalp or neck. This is frequently the case when the PDT is for the treatment of lesions (e.g. acne, basal cell carcinoma, actinic keratosis, photodamage and Bowen's disease (BD)). Representative photosensitizers include preferably 5-aminolevulinicacid (5-ALA) and derivatives (e.g. an ester) of 5-ALA, more preferably 5-ALA methyl ester (or 5-MAL), or a pharmaceutically acceptable salt thereof. In the present uses and methods, photactivation is achieved by natural or artificial light. In a particular embodiment, the PDT comprises: (a) Optionally, preparing the area of skin to be treated with the appropriate pre-treatment, for example a curettage or micro perforation (b) Administering to said animal a composition comprising said photosensitizer for a short duration; and (c) photoactivating said photosensitizer. In a particular embodiment, the invention relates to a pulse-PDT treatment, comprising administering to a subject in need thereof a photosensitizer, in particular 5-MAL, for a short duration and then removing the photosensitizer from the skin surface. Photoactivation is then carried out as described throughout the present application. The pulse-PDT treatment of the invention ensures high intracellular PPIX and low extracellular PPIX. Excess amounts of PPIX formation during and after the end of the treatment are thus avoided. In particular, the inventors show that the pulse-PDT treatment of the invention shows less inflammation with unchanged efficacy. According to an embodiment, the pulse time during which the photosensitizer is left on the skin is between 5 and 120 minutes. According to a preferred embodiment, the pulse time during which the photosensitizer is left on the skin is between 15 and 60 minutes, in particular between 20 and 40 minutes. In a further particular embodiment, the photosensitizer is administered for about 30 minutes (e.g. for 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 minutes, more particularly during 30 minutes). With such a short photosensitizer treatment, pain level is not changed and PPIX concentration is clearly lower than for the conventional 3 hour exposure to 5-MAL for example, but the treatment is as efficient. Detailed description of the invention. Skin conditions treated according to the invention. By the term "animal" is meant

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herein any human or non-human being. Preferred animal s for treatment in accordance with the invention are humans. Any of the above particular or preferred embodiments may comprise a step of pretreating the skin as described above, before the step of applying the photosensitizer on the skin. Furthermore, in a particular embodiment, the PDT according to the invention al so comprises applying to the skin of the subject a glucocorticosteroid. According to this embodiment, the glucocorticosteroid is administered for further preventing or reducing the side effects associated with PDT. The glucocorticosteroid may be applied either simultaneousy, before, after or both before and after administration of the photosensitizer. In a particular embodiment, the glucocorti costeroid is selected from the group consisting of clobetasol propionate, betamethasone dipropionate, halobetasol proprionate, diflorasone diacetate, diflucortolone valerate, Hydrocortisone 17-butyrate, mometasone furoate, methyl prednisolone aceponate and halometasone. In a further particular embodiment, the glucocorticosteroid is clobetasol propionate or betamethasone. The purpose of this project is therefore to keep the PPIX formation within the mitochondria and avoid excess amounts of PPIX to be formed. Simultaneously PPIX should be allowed to be formed for such a long time that most abnormal cells will be affected. So the purpose of PDT is to kill abnormal , preferably by apoptosis. The ideal situation would be to keep PPIX inside the cell and to destroy the mitochondria only, thereby inhibiting the ATP formation necessary for cell functions. That should result in cell death by apoptosis. One possible way to achieve this would be to give a short 5-MAL pulse treatment to get a high concentration of 5-MAL in the cells initially and then diminish further access to 5-MAL by removing 5-MAL from the skin surface. This could be done by only exposing the skin to 5-MAL for a short time, after which all 5-MAL is removed from the skin surface. If the right "pulse time" can be found it might ensure high cellular PPIX and low extracellular PPIX. Excess amounts of PPIX formation during and after the end of the treatment would thus be avoided. The result shows less inflammation with unchanged efficacy and thus mitochondria destruction seems to be the most important factor in PDT [45]. The present invention relates to the use of a photoactivatable porphyrin-Derivative in extracorporeal photophoresis (ECP) treatment, in which a patient's blood or part of it containing said Porphyrin-derivative is/are exposed to light of a wavelength which activates said photoactivatable porphyrin-derivative. Extracorporeal photopheresis (ECP) is a form of apheresis and photodynamic therapy (PDT) where leucocytes (white blood cells) are separated from whole blood and exposed to photoactive 8-methoxypsoralen (8-MOP) as a photosensitiser and ultraviolet-A (UV-A) light before reintroduced back to the patient's circulation. Said extracorporeal photophoresis (ECP) treatment is mostly aimed at treating cancer, nlymphocyte-mediated malignant and non-malignant disorders, T-Cell-mediated diseases, autoimmune diseases and/or infections or at stimulat-



ing and/or modulating an immunological response against malignacies and immunological diseases. This especially includes lymphoma like cutaneous T-cell lymphoma or erythrodermic cutaneous T-cell lymphoma [46]. The invention relates to an alcohol-free cosmetic or pharmaceutical foam composition comprising water, a hydrophobic solvent, a surface-active agent, a gelling agent, an active component selected from the group of urea, hydroxylacid and a therapeutic enhancer and a propellant. The foam further comprises active agents and excipients with therapeutic properties having enhanced skin penetration. The foam composition is useful and advantageous for the treatment of skin disorders and for skin care and cosmetic care. The addition of an oil having refatting, protective and moisture-retaining properties in a spreadable foam form can substitute for currently available dermatological and cosmetic creams, lotions, gels, etc [47]. The present invention provides a method for topical delivery of pharmacologically active chemicals into local tissues (skin, subcutaneous, muscle and joint tissues) via the administration of their specially designed cationic prodrugs with anode iontophoresis. The pharmacologically active chemicals are either negatively charged or neutral under the physiologic pH, which are not suitable to be delivered with the anode iontophoresis. The cationic prodrugs of such pharmacologically active chemicals are suitable for anode iontophoresis for improving delivery efficiency of these drugs into the local tissues. The cationic prodrugs canal to be used in co-delivery with other cationic drugs such as vasoconstrictors or local anesthetic agents by iontophoresis for the treatment of disorders in the local tissues. The anode iontophoresis delivery of the specially designed cationic prodrugs can provide higher drug concentrations in the local tissues, which can be used for better topical treatment of musculoskeletal diseases or skin diseases. A lot of pharmacologically active chemical s are weak acids with carboxyl group in their molecules. Those chemicals include a lot of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), methotrexate, fusidic acid, aminolevulinic acid etc. Oral NSAIDs are commonly used for treating Musculoskeletal Disorders (MSD) such as rheumatoid arthritis, osteoarthritis, muscle pain and sprains, but treatments with oral NSAIDs are associated with gastric and cardiovascular risks and provide limited availability of the drug in the localized musculoskeletal tissue. Topical NSAIDs have also been used for treating MSD, and showed less systemic adverse effects because of limited systemic exposure of the topical Iontophoresis uses electric current to drive charged molecules into the skin. It can deliver drug much more quickly into the ski n and thus provide fast onset compared to passive topical delivery. Iontophoresis is generally well tolerated when the applied current density is below 0.5mA/cm. Negatively charged drug can be delivered by a cathode iontophoresis (drug placed in the cathode electrode side) and positively charged drug can be delivered by an anode iontophoresis (drug placed in the anode electrode side). Anode iontophoresis is usually more efficient than cathode iontophoresis in delivering drug into and across the skin. Under normal skin condition, electroosmotic flow induced by the applied electric current across the skin is in the same di recti on as

the anode iontophoresis drug delivery, but in the opposite direction of the cathode iontophoresis drug delivery, thus anode iontophoresis has a positive contribution from the electro-osmosis effect. In addition, for the commonly used silver/silver-chloride (Ag/AgCl) electrodes, anode electrode reaction does not generate competition ions into the anode chamber, but cathode electrode reaction generates chloride ions into the cathode chamber that compete with the delivery of negatively charged drug. Over time, as the chloride ion concentration increases in the cathode chamber, the delivery efficiency by the cathode iontophoresis decreases drugs. The major reason is because the skin blood flow cleared most of the drug in the skin from topical delivery, and thus, there is limited penetration of topical NSAIDs into the muscle or joint tissues underneath the skin. Iontophoresis uses electric current to drive charged molecules into the skin. It can deliver drug much more quickly into the skin and thus provide fast onset compared to passive topical delivery. Iontophoresis is generally well tolerated when the applied current density is below 0.5mA/cm. Negatively charged drug can be delivered by a cathode iontophoresis (drug placed in the cathode electrodeside) and positively charged drug can be delivered by an anode iontophoresis (drug placed in the anode electrodeside). Anode iontophoresis is usual l y more efficient than cathode iontophoresis in delivering drug into and across the skin. Under normal skin condition, electroosmotic flow induced by the applied electric current across the skin is in the same direction as the anode iontophoresis drug delivery, but in the opposite di recti on of the cathode iontophoresis drug delivery, thus anode iontophoresis has a positive contribution from the electro-osmosis effect. In addition, to the commonly used silver/silver-chloride (Ag/AgCl) electrodes, anode electrode reaction does not generate competition ions into the anode chamber, but cathode electrode reaction generates chloride ions into the cathode chamber that compete with the delivery of negatively charged drug. Over time, as the chloride ion concentration increases in the cathode chamber, the delivery efficiency by the cathode iontophoresis decreases. The present invention provides methods for achieving direct penetration of one or more pharmacologically active chemicals into local skin, muscle or joint tissue by iontophoretic delivery of their cationic prodrug salts across skin and methods of using the delivery to treat disease. Such diseases include musculoskeletal disorders and skin diseases, such as muscle strain, ankle sprain, artritis, tendinitis, bursitis, tenosynovitis, plantar fasciitis, patellar tendinitis and achilles tendinitis, carpal tunnel syndrome, temporomandibular disorder, gout, skin cancers, actinic keratosis, psoriasis, acne, warts, and sebaceous cyst, etc. In this method, higher concentrations of the drug is achieved in the local tissues compared to those from iontophoretic or passive delivery of the parent drugs [48]. A method for the treatment of an individual for a cellular proliferative disorder, which includes administering a therapeutically effective amount of the compound pterostilbene wherein UDP has been developed glucuronosyltransferase (UGT) activity is increased. In an embodiment, HETE levels can be reduced by administration of pterostilbene [49]. Or in terms of prevention: A method for treatment of skin cancer in tissue within the skin of a mammal requires the ingestion,


injection, infusion or application of an ingredient having a pharmaceutically active phenyl boric acid or salts of a phenyl boric acid to treat the affected tissue. The treatment may be with an amount of ingredient that inhibits the growth of at least one cancer cell line selected from other non-melanoma skin cancers and melanoma skin cell lines [50]. Focusing on diagnosis, more attention will be paid on the early detection of skin cancers and cancerization field. Such as skin cancer biomarker detection by infrared spectroscopy [51] or other devices for the diagnosis of skin cancer including laser, PC and optic system, consisting of optic fiber probe, that includes transmitting optic fiber with a transmission Bragg grating light filter on distal end, adjusted to laser wave length, and length, receiving optic fiber with formed in it notch filter in form of Bragg grating, blocking laser radiation, optic fiber splitter, to each outlet channel of which connected is its own Bragg grating, adjusted to transmission of a particular wavelength of spectrum of combination dispersion and connected optically to its own photo receiver or a series of photo receivers with parallel access, whose outlets are connected with card of PC data collection. Detachable metal figured washer, permitting disinfection or replacement in case if patients change, is installed on the butt end of fiber optic probe. This invention makes it possible to solve problems of diagnostics of skin malignant neoplasm boundaries in carrying out operations and medicinal impact, and endoscopic examinations [52]. Or for the evaluation of skin to detect the existence of neoplastic tissue, including a florescent probe that linking to a specific neoplasia marker is applied topically to the site involved. Following topical application, the probe preferentially binds to markers in neoplastic lesions in situ, the binding of which is recognized with a compact light unit that brings illumination at a wavelength appropriate for the capturing of the images. The light unit comprises a light source and fiber optic bundle to focus the light towards the examination zone. A detection unit serves to capture and record a picture of involved area. The collection unit may be a digital camera, film camera, etc. A mapping module can also be supplied to catalogue the site of examination [53].

ACKNOWLEDGEMENTS Declared none. REFERENCES [1] [2] [3]

[4] [5] [6]

[7]

[8]

[9]

[10] [11] [12]

[13] [14]

[15]

Another method for skin cancer detection is based on the exact measurement of the spectral reflectance using an imaging system (modified camera). The availability of a calibrated spectral reflectance of every pixel of the surface unleash a number of post-processing operation and differentiated analysis, and most importantly, allows to classify and store into the spectral database of the system the spectral signature of the different identified pathologies, with the use and the experience the database becomes the repository of a spectral based know how and a strong support to diagnosis [54]. Taking into account the dynamism of this area of interest new patents will appear in the near future helping physicians in the early diagnosis for a better treatment approach which would be applied also to the teledermatology, increasing patient’s compliance especially for patients that are able to move and to receive dermatological assistance.

[16]

CONFLICT OF INTEREST

[22]

The authors confirm that this article content has no conflict of interest.

Cantisani et al.

[17] [18]

[19]

[20] [21]

Goldberg LH, Mamelak AJ. Review of actinic keratosis. Part I: Etiology, epidemiology and clinical presentation. J Drugs Dermatol 2010; 9(9): 1125-32. Schmitt JV, Miot HA.Actinic keratosis: A clinical and epidemiological revision. A Bras Dermatol 2012; 87(3): 425-34. Hofbauer G, Anliker M, Boehncke WH, Brand C, Braun R, Gaide O, et al. Swiss clinical practice guidelines on field cancerization of the skin. Swiss Med Wkly 2014; 144: w14026. Philipp-Dormston WG. Field cancerization: From molecular basis to selective field-directed management of actinic keratosis. Curr Probl Dermatol 2015; 46: 115-21. Chia A, Moreno G, Lim A, Shumack S. Actinic keratoses. Aust Fam Physician 2007; 36(7): 539-43. Cantisani C, De Gado F, Ulrich M, Bottoni U, Iacobellis F, Richetta AG, et al. Actinic keratosis: Review of the literature and new patents. Recent Pat Inflamm Allergy Drug Discov 2013; 7(2):168-75. Kennedy C, Willemze R, de Gruijl FR, Bavinck JN, Bajdik CD. The influence of painful sunburns and lifetime sun exposure on the risk of actinic keratoses, seborrheic warts, melanocytic nevi, atypical nevi, and skin cancer. J Invest Dermatol 2003; 120: 1087-93. John SM, Trakatelli M, Gehring R, Finlay K, Fionda C, Wittlich M, et al. Consensus report: Recognizing non-melanoma skin cancer, including actinic keratosis, as an occupational disease- call to action. JEADV 2016; 30(S3):38-45. RoewertHuber J, Stockfleth E, Kerl H. Pathology and pathobiology of actinic (solar) keratosis - an update. Br J Dermatol 2007; 157(2):18-20. Rossi R, Mori M, Lotti T. Actinic keratosis. Int J Dermatol 2007; 46(9): 895-904. Werner RN, Sammain A, Erdmann R, Hartmann V, Stockfleth E, Nast A. The natural history of actinic keratosis: A systematic review. Br J Dermatol 2013; 169(3):502-18. Brougham ND, Dennett ER, Cameron R, Tan ST. The incidence of metastasis from cutaneous squamous cell carcinoma and the impact of its risk factors. J Surg Oncol 2012; 106(7):811-5. Rigel DS, Gold LF. The importance of early diagnosis and treatment of actinic keratosis. J Am Acad Dermatol 2013; 68(1 Suppl 1): S20-7. Marks, R. Epidemiology of non-melanoma skin cancer and solar keratoses in Australia: A tale of self-immolation in Elysian fields. Australas J Dermatol 1997; 38 Suppl 1:S26-9. Fitzpatrick TB. Sun and skin. Journal de Médecine Esthétique 1975; 2: 33-34. Commandeur S, De Gruijl FR, Willemze R, Tensen CP, El Ghalbzouri A. An in vitro three-dimensional model of primary human cutaneous squamous cell carcinoma. Exp Dermatol 2009; 18(10): 849-56. Slaughter, D.P, H.W. Southwick, W. Smejkal. Field cancerization in oral stratified squamous epithelium; Clinical implications of multicentric origin. Cancer 1953; 6(5): 963-8. Padilla RS, Sebastian S, Jiang Z, Nindl I, Larson R. Gene expression patterns of normal human skin, actinic keratosis, and squamous cell carcinoma: A spectrum of disease progression. Arch Dermatol 2010; 146: 288-93. Kubo Y, Matsudate Y, Fukui N, Nakasuka A, Sogawa M, Oshima M et al. Molecular tumorigenesis of the skin. J Med Invest 2014; 61:7-14. Accardi R, Gheit T. Cutaneous HPV and skin cancer. Presse Med 2014; 31; 43(12):435-43. Fernández-Figueras MT, Carrato C, Sáenz X, Puig L, Musulen E, Ferrándiz C, et al. Actinic keratosis with atypical basal cells (AK I) is the most common lesion associated with invasive squamous cell carcinoma of the skin. J Eur Acad Dermatol Venereol 2015; 29(5):991-7. Tian X, Azpurua J, Hine C, Vaidya A, Myakishev-Rempel M, Ablaeva J, et al. High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat. Nature 2013; 18; 499(7458):346-9.

Actinic Keratosis Update [23]

[24]

[25]

[26]

[27]

[28]

[29]

[30] [31]

[32]

[33]

[34]

[35]


Karia PS, Han J, Schmults CD. Cutaneous squamous cell carcinoma: Estimated incidence of disease, nodal metastasis, and deaths from disease in the United States 2012. J Am Acad Dermatol 2013; 68: 957. Wojciechowska-Zdrojowy M, Szepietowski JC, Matusiak , Dzigiel P, Pua B. Expression of podoplanin in non-melanoma skin cancers and actinic keratosis. Anticancer Res 2016; 36(4):1591-7. Bosic MM, Brasanac DC, Stojkovic-Filipovic JM, Zaletel IV, Gardner JM, Cirovic SL. Expression of p300 and p300/CBP associated factor (PCAF) in actinic keratosis and squamous cell carcinoma of the skin. Exp Mol Pathol 2016; 100(3): 378-85. doi: 10.1016/j.yexmp.2016.03.006. Babino G, Diluvio L, Bianchi L, Orlandi A, Di Prete M, Chimenti SM, et al. Long-term use of a new topical formulation containing piroxicam 0.8% and sunscreen: Efficacy and tolerability on actinic keratosis. A proof of concept study. Curr Med Res Opin 2016; DOI: 10.1080/03007995.2016.1174678 Ulrich M, Maltusch A, RiusDiaz fr, RöwertHuber JO, Gonzalez S.Sterry W, et al. Clinical applicability of in vivo reflectance confocal microscopy for the diagnosis of actinic keratoses. Dermatol Surg 2008; 34(5):610-9. Giacomel J, Lallas A, Argenziano G, Bombonato C, Zalaudek I. J Dermoscopic "signature" pattern of pigmented and nonpigmented facial actinic keratoses. Am Acad Dermatol 2015; 72(2): e57-9. doi: 10.1016/j.jaad.2014.10.043 Zalaudek, I.G. Argenziano. Dermoscopy of actinic keratosis, intraepidermal carcinoma and squamous cell carcinoma. Curr Probl Dermatol 2015; 46: 70-6. Rosendahl C, Cameron, Argenziano G, Zalaudek I, Tschandl P, Kittler H. Dermoscopy of squamous cell carcinoma and keratoacanthoma. Arch Dermatol 2012; 1-7. Stockfleth E, Terhorst D, Braathen L. Guideline subcommittee of the European dermatology forum. Guidelines for the management of actinic keratosis-update 2011. Available from: http://bestpractice.bmj.com/bestpractice/monograph/616/diagnosis/ guidelines.html. Ulrich M, Alarcon I, Malvehy J, Puig S. In vivo Reflectance Confocal Microscopy Characterization of Field-Directed 5-Fluorouracil 0.5%/Salicylic Acid 10% in Actinic Keratosis. Dermatology 2015; 230(3): 193-8. Rishpon A, Kim N, Scope A, Porges L, Oliviero MC, Braun RP, Marghoob AA, Fox CA, Rabinovitz HS. Reflectance confocal microscopy criteria for squamous cell carcinomas and actinic keratoses. Arch Dermatol 2009; 145(7):766-72. Ahlgrimm-Siess V, Cao T, Oliviero M, Hofmann-Wellenhof R, Rabinovitz HS, Scope A. The vasculature of nonmelanocytic skin tumors on reflectance confocal microscopy: Vascular features of squamous cell carcinoma in situ. Arch Dermatol 2011; 147(2): 264. Cantisani C, Paolino G, Corsetti P, Bottoni U, Didona D, Calvieri S. Evaluation of ingenol mebutate efficacy for the treatment of ac-

[36]

[37]

[38] [39] [40]

[41] [42] [43] [44] [45] [46] [47] [48]

[49] [50] [51] [52] [53] [54]

tinic keratosis with antera 3D camera. Eur Rev Med Pharmacol Sci 2015; 19(1):92-7. Cantisani C, De Gado F, Ulrich M, Bottoni U, Iacobellis F, Richetta AG. Actinic keratosis: Review of the literature and new patents. Recent Pat Inflamm Allergy Drug Discov 2013; 7(2):16875. Stockfleth E, Ferrandiz C, Grob JJ, Leigh I, Pehamberger H, Kerl H. Development of a treatment algorithm for actinic keratoses: A European Consensus. Eur J Dermatol 2008; 18(6): 651-9. Rosen T. Reexamination of field-directed therapy for actinic keratosis. Cutis 2012; 90(4):163-5. McEwan LE, Smith JG. Topical diclofenac/hyaluronic acid gel in the treatment of solar keratoses. Australas J Dermatol 1997; 38(4):187-9. Samrao A, CJ. Cockerell. Pharmacotherapeutic management of actinic keratosis: Focus on newer topical agents. Am J Clin Dermatol 2013; 14(4): 273-7. Lin, F., Li, G. Medicament for treating skin cancer and preparation method thereof. CN103816116 (2014). Xiao, Y. Traditional Chinese medicine composition for treating skin cancer. CN103520597 (2014). Chen, J. Application of Artoxanthochromane in skin cancer treatment drugs CN103638008 (2014). Silver, M.E. Method for treating skin cancer. US2014200275 (2014). Wulf, H.C. Pulse photodynamic treatment of skin conditions WO2015092052 (2015). Peng, Q. Modification of extracorporeal photopheresis technology with porphyrin precursors WO2015162279 (2015) & EP2015058986 (2015). Dov, T., Doron, F., Meir, E. Penetrating pharmaceutical foam. US20150157586 (2015). Yan, G. Iontophoresis delivery of cationic prodrugs for topical treatment of musculoskeletal or skin diseases US20150297718 (2015). Artos, J., Dellinger, R. Y. Method for treating non-melanoma skin cancer by inducing UDP-Glucuronosyltransferase activity using pterostilbene. US2015011650 (2015). Eacham, S., Carper, S. Espacenet treatment and reduction in incidence of skin cancer. US2014056831 (2014). Eikje, N. Skin cancer biomarker detection by infrared spectroscopy. AU2013236860 (2014). Bratchenko, I. A, Grishanov, V. N, Zakharov V P. Device for diagnosing skin cancer. RU2506049 (2014). Shachaf, C.M., Shachaf, A. Diagnostic system for the detection of skin cancer. US2014314661 (2014). Samadelli M, Melis M, Miccoli M, Egarter Vigl E, Zink A. R. Complete mapping of the tattoos of the 5300-year-old Tyrolean Iceman. J Cult Herit 2015; 31:16(5):753-8.