Metabolomic profiling and accurate diagnosis of basal cell carcinoma by MALDI imaging and machine learning.

Basal cell carcinoma (BCC), the most common keratinocyte cancer, presents a substantial public health challenge due to its high prevalence. Traditional diagnostic methods, which rely on visual examination and histopathological analysis, do not include metabolomic data. This exploratory study aims to molecularly characterize BCC and diagnose tumour tissue by applying matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and machine learning (ML). BCC tumour development was induced in a mouse model and tissue sections containing BCC (n = 12) were analysed. The study design involved three phases: (i) Model training, (ii) Model validation and (iii) Metabolomic analysis. The ML algorithm was trained on MS data extracted and labelled in accordance with histopathology. An overall classification accuracy of 99.0% was reached for the labelled data. Classification of unlabelled tissue areas aligned with the evaluation of a certified Mohs surgeon for 99.9% of the total tissue area, underscoring the model's high sensitivity and specificity in identifying BCC. Tentative metabolite identifications were assigned to 189 signals of importance for the recognition of BCC, each indicating a potential tumour marker of diagnostic value. These findings demonstrate the potential for MALDI-MSI coupled with ML to characterize the metabolomic profile of BCC and to diagnose tumour tissue with high sensitivity and specificity. Further studies are needed to explore the potential of implementing integrated MS and automated analyses in the clinical setting.

Oral intake of bucillamine, carvedilol, metformin, or phenformin does not protect against UVR-induced squamous cell carcinomas in hairless mice.

Squamous cell carcinoma represents the second most common type of keratinocyte carcinoma with ultraviolet radiation (UVR) making up the primary risk factor. Oral photoprotection aims to reduce incidence rates through oral intake of photoprotective compounds. Recently, drug repurposing has gained traction as an interesting source of chemoprevention. Because of their reported photoprotective properties, we investigated the potential of bucillamine, carvedilol, metformin, and phenformin as photoprotective compounds following oral intake in UVR-exposed hairless mice. Tumour development was observed in all groups in response to UVR, with only the positive control (Nicotinamide) demonstrating a reduction in tumour incidence (23.8%). No change in tumour development was observed in the four repurposed drug groups compared to the UV control group, whereas nicotinamide significantly reduced carcinogenesis (P = 0.00012). Metformin treatment significantly reduced UVR-induced erythema (P = 0.012), bucillamine and phenformin increased dorsal pigmentation (P = 0.0013, and P = 0.0005), but no other photoprotective effect was observed across the repurposed groups. This study demonstrates that oral supplementation with bucillamine, carvedilol, metformin, or phenformin does not affect UVR-induced carcinogenesis in hairless mice.

Does systemic hydrochlorothiazide increase the risk of developing ultraviolet radiation-induced skin tumours in hairless mice?

Hydrochlorothiazide (HCTZ) is a frequently prescribed diuretic that exhibits photosensitizing properties. It is used to treat hypertension and edema. Dermato-epidemiological studies in various populations have linked HCTZ treatment with increased risk of particular types of skin cancer, including malignant melanoma (lentigo subtype), and both basal cell carcinoma and squamous cell carcinoma (SCC). This study investigated whether either of two different doses of HCTZ increased the risk of SCC development in mice exposed to ultraviolet radiation (UVR). A total of three groups of hairless mice were used in this study (total, N = 71). One group received a low dose (0.26 mg/mouse/day) and another group received a high dose (0.52 mg/mouse/day) of HCTZ in their drinking water; a third UVR control group received only tap water. All three groups were irradiated with UVR until the mice developed three tumours that were 4 mm in size. The times to SCC tumour development were recorded. In the low-dose group, the median time to develop an SCC tumour was 170 days; in both the high-dose group and the control group, the median time to develop anexd SCC tumour was 163 days (p ≥ 0.331). In our hairless mouse model, we found that mice treated with UVR plus HCTZ did not develop SCCs more rapidly than mice treated with UVR but not HCTZ.

Phototesting in erythropoietic protoporphyria trials: A systematic review.

Severe skin pain when exposed to long wave ultraviolet radiation or visible light is the main symptom of erythropoietic protoporphyria (EPP). Treatment options for EPP are inadequate and new treatments are needed but hampered by the lack of valid efficacy outcomes. Phototesting with well-defined illumination of the skin can be performed reliably. We aimed to provide an overview of phototest procedures used to evaluate EPP treatments. Systematic searches of Embase, MEDLINE and the Cochrane Library were performed. Searches identified 11 studies using photosensitivity as efficacy outcome. The studies used eight different phototest protocols. Illuminations were performed with a filtered high-pressure mercury arc, or a xenon arc lamp equipped with monochromator or filters. Some used broadband, others narrowband illumination. In all protocols phototests were performed on the hands or the back. Endpoints were minimal dose required to induce either first symptom of discomfort, erythema, urticaria or intolerable pain. Other endpoints were change in erythema intensity or diameter of any type of flare after exposure compared to before. In conclusion, protocols displayed extensive variability in illumination set-up and evaluation of phototest reactions. Implementation of a standardized phototest method will allow more consistent and reliable outcome evaluation in future therapeutic research of protoporphyric photosensitivity.

Repeated exposure to fractional CO2 laser delays squamous cell carcinoma formation and prevents clinical and subclinical photodamage visualized by line-field confocal optical coherence tomography and histology.

OBJECTIVES: Ablative fractional laser (AFL) is a well-established modality for treating ultraviolet radiation (UVR)-induced skin photodamage. We aimed to investigate the potential of AFL to delay squamous cell carcinoma (SCC) formation and prevent photodamage in a preclinical UVR-induced SCC model. MATERIALS AND METHODS: Hairless C3.Cg-Hrhr /TifBomTac mice (n = 50) were exposed to UVR three times weekly throughout the study. UV-exposed mice were randomized to two groups that received dorsal CO2 AFL (10 mJ/mb, 10% density) or no treatment. AFL was performed every other week for a total of 16 weeks (nine treatments in total). The primary outcome was time to tumor occurrence. In a subset of mice on Day 150, prevention of clinical photodamage was assessed by examination of skin tightness and dyspigmentation. Concomitantly, assessment of subclinical photoprevention based on normalization of keratinocyte dysplasia, dermo-fiber morphology (collagen and elastin fibers), and skin thickness, was performed using line-field confocal optical coherence tomography (LC-OCT) and histology. RESULTS: Repeated AFL treatments delayed SCC tumor development compared to UVR control mice by 12, 19, and 30 days for first, second, and third tumors, respectively (p ≤ 0.0017). Compared to UVR controls, AFL prevented photodamage both clinically and subclinically, based on LC-OCT and histology. In the epidermal layer, AFL imparted photopreventative effects including reduced dyspigmentation and keratinocyte dysplasia (1 vs. 2.5, p = 0.0079) and partial normalization of the epidermal thickness (p < 0.0001). In the dermis, AFL led to twofold greater skin tightness (p = 0.0079), improved dermo-fiber structure, and dermal thickness (p = 0.0011). CONCLUSION: In conclusion, repeated AFL treatments of UVR-exposed skin significantly delayed SCC tumor formation and prevented clinical and imaging-assessed subclinical signs of photodamage, indicating a potential for AFL in prevention strategies for SCC and photodamage in high-risk populations.

A one-time pneumatic jet-injection of 5-fluorouracil and triamcinolone acetonide for treatment of hypertrophic scars-A blinded randomized controlled trial.

BACKGROUND: Patients with hypertrophic scars (HTS) risk reduced quality of life due to itching, pain, poor cosmesis, and restriction of movement. Despite good clinical efficacy, patients are often reluctant to undergo repeated needle injections due to pain or needle phobia. OBJECTIVES: To evaluate the applicability of needle-free pneumatic jet injection (PJI) and assess changes in hypertrophic scars following a single PJI treatment with 5-fluorouracil (5-FU) and triamcinolone acetonide (TAC). METHODS: Twenty patients completed this blinded, randomized, controlled, split-scar trial. The intervention side of the HTS received a one-time treatment with PJIs containing a mixture of TAC + 5-FU injected at 5 mm intervals (mean 7 PJI per HTS); the control side received no treatment. Assessments were made at baseline and 4 weeks posttreatment. Outcome measures included change in (1) Vancouver Scar Scale (VSS) total score and subscores, (2) scar volume and surface area assessed by three-dimensional imaging, (3) skin microarchitecture measured by optical-coherence tomography (OCT), (4) photo-assessed scar cosmesis (0-100), (5) patient-reported pain and satisfaction (0-10), and (6) depiction of drug biodistribution after PJI. RESULTS: PJI with TAC + 5-FU significantly decreased both HTS height (-1 VSS; p = 0.01) and pliability (-1 VSS; p < 0.01) with a nonstatistically significant reduction of -1 in total VSS score (0 in control; p = 0.09). On 3D imaging, a 33% decrease in scar volume (p = 0.016) and a 37% decrease in surface area (p = 0.008) was observed. OCT indicated trends towards smoother scar surface (Ra 11.1-10.3; p = 0.61), normalized dermal microarchitecture (attenuation coefficient: 1.52-1.68; p = 0.44), and a reduction in blood flow between 9% and 17% (p = 0.50-0.79). Despite advances in VSS subscores and OCT, no improved photo-assessed cosmesis was found (-3.2 treatment vs. -1.4 control; p = 0.265). Patient-reported pain was low (2/10) and 90% of the patients that had previously received needle injections preferred PJI to needle injection. Depositions of TAC + FU were imaged reaching deep into the scar at levels corresponding to the reticular dermis. CONCLUSION: A single PJI injection containing 5-FU and TAC can significantly improve the height and pliability of HTS. PJI is favored by the patients and may serve as a complement to conventional needle injections, especially for patients with needle phobia.

Few X-ray and PUVA treatments accelerate photocarcinogenesis in hairless mice.

PUVA is a treatment that combines oral methoxypsoralen (8-MOP) with ultraviolet radiation A (UVA). It is used for severe psoriasis and the early stages of T-cell lymphoma. X-rays are an effective treatment for skin cancers. Both treatments are in higher doses used to treat skin malignancies and simultaneously increase the risk of keratinocyte cancer. The main objective of this study was to test whether a few PUVA or X-ray treatments could delay the development of ultraviolet radiation (UVR)-induced skin tumors in a well-established hairless mouse model. Three groups of immunocompetent mice (total, N = 75) were included in the study. All groups were UVR-exposed during the study period. In addition, one group was treated with PUVA and another group was treated with X-rays at days 45, 52, 90 and 97. A control group was treated with UVR only. We recorded when the first, second and third skin tumors were induced in each mouse. Skin tumors developed significantly earlier in both the PUVA and X-ray groups (median, 188 days) than in the control mice (median, 215 days; p < 0.001). Therefore, a few X-ray and PUVA treatments both significantly accelerated the development of skin tumors in hairless mice, compared to UVR controls. Neither treatment showed a delay of UVR-induced skin tumors and caution should be exercised before applying these treatments to sun-damaged skin.

Tumor Clearance and Immune Cell Recruitment in UV-Induced Murine Squamous Cell Carcinoma Exposed to Ablative Fractional Laser and Imiquimod Treatment.

BACKGROUND AND OBJECTIVES: Keratinocyte carcinoma (KC) is the most common cancer worldwide, and squamous cell carcinoma (SCC) is the second most frequent subtype. Ablative fractional laser (AFL)-assisted drug delivery significantly enhances the uptake of topically applied drugs. The objective of this study was to assess tumor response and perform a descriptive characterization of the local recruitment of immune cells and systemic immune mediator levels in an ultraviolet radiation (UVR)-induced murine SCC model after AFL treatment alone and combined with topical imiquimod. STUDY DESIGN/MATERIALS AND METHODS: Immunocompetent hairless mice (C3·Cg/TifBomTac, n = 74) were irradiated with solar-simulated UVR until 3-mm SCCs developed. The mice were divided into four interventional groups: AFL alone, AFL + imiquimod, imiquimod alone, and untreated SCC controls. AFL was given as a single treatment, whereas imiquimod was applied daily until the mice were euthanized on Days 0, 2, 7, or 14. SCCs were photographed and measured (mm) to assess the therapeutic response. Skin samples were processed for histopathological and immunohistochemical analyses, as well as for flow cytometry. Cytokine expression changes in sera were analyzed using ELISpot cytokine arrays. RESULTS: Treatment of mouse SCCs with AFL + imiquimod induced the most robust immune cell infiltration and the greatest proportion of tumor clearance compared to other interventions. Early innate immune cell infiltration was induced by AFL + imiquimod treatment as the number of neutrophils and macrophages had increased fourfold within 2 days of treatment initiation compared with untreated SCC control mice (P < 0.05). AFL treatment alone had a more limited effect, with a fourfold increase in neutrophils (P < 0.05) but no significant increase in the number of macrophages. Correspondingly, treatment with AFL + imiquimod had the greatest effects on the adaptive immune cell recruitment: CD4+ T-helper cells increased threefold at Day 7 compared with untreated SCCs (P = 0.0001) and, notably, cytotoxic CD8+ T cells increased 14-fold at Day 14 (P = 0.0112). In addition, FOXP3+ regulatory T cells (Tregs) increased 14-fold at Day 7 (P = 0.0026), suggesting the resolution of the inflammatory infiltration. AFL treatment alone induced a moderate immune cell infiltration (a twofold increase in CD4+ T-helper cells, P = 0.0200; a threefold increase in CD8+ T cells, P = 0.0100; and a 14-fold increase in FOXP3+ Tregs at Day 14, P = 0.0021), whereas imiquimod alone did not significantly increase cell counts. AFL + imiquimod treatment increased CXCL12 serum levels threefold at Day 14 (P = 0.0200). CONCLUSION: AFL treatment alone and in combination with imiquimod induces substantial tumor clearance associated with local recruitment of innate and adaptive immune cells in UVR-induced murine SCCs. These results may provide a basis for new immunotherapeutic approaches to KC treatment.

A Comparison of Human and Porcine Skin in Laser-Assisted Drug Delivery of Chemotherapeutics.

BACKGROUND AND OBJECTIVES: Porcine skin is a widely used model in diffusion studies, but its usefulness for laser-assisted drug delivery (LADD) has not been evaluated in comparison with human skin. This study compared porcine and human skin in ex vivo LADD diffusion studies. STUDY DESIGN/MATERIALS AND METHODS: Ex vivo ablative fractional laser (AFL) treatments (5, 20, and 80 mJ/mb) were applied to skin samples from three sources: human, normal pig (Duroc × Landrace × Yorkshire breed), and a hyperkeratotic pig phenotype. Samples were stained using hematoxylin and eosin, photo-documented, and measured digitally. Samples (20 mJ/mb) were exposed to bleomycin or 5-fluorouracil (5-FU) for 19 hours in Franz diffusion cells. Drug uptake was quantified at three skin depths (100, 500, and 1,500 µm) by high-performance liquid chromatography-mass spectrometry. Drug biodistribution and endogenous lipids were visualized by matrix-assisted laser desorption/ionization-mass spectrometry imaging. RESULTS: Epidermal and dermal thicknesses of human and normal pig skin were similar (76-87 µm and 1,668-1,886 µm, respectively; P = 0.082-0.494). Endogenous lipids were investigated, and 116 compounds were identified. Of these compounds, 100 were found in all three skin types, while six were present exclusively in human skin. Laser channel depths (20 mJ/mb) in human and normal pig skin were similar (1,081 vs. 1,126 µm; P = 0.588). Bleomycin uptake was similar in all skin types at all depths (101.4-175.6 µg/cm3 ; P = 0.132-0.699). 5-FU uptake in human and normal pig skin was similar at 100 and 500 µm (80.5 vs. 140.3 µg/cm3 and 131.2 vs. 208.1 µg/cm3 , respectively; P = 0.065-0.093). At 1500 µm, 5-FU concentrations in the porcine skin types differed from those in human skin (104.7 vs. 196.7-344.8 µg/cm3 ; P = 0.002-0.026). Drug biodistribution was similar among skin types, but differences between bleomycin and 5-FU biodistribution were observed. CONCLUSIONS: Normal porcine and human skin showed similar morphology, the composition of endogenous lipids, and AFL-assisted cutaneous uptake, and biodistribution of chemotherapeutics. Therefore, normal porcine skin, but not hyperkeratotic pig phenotype skin, is a practical and reliable model for healthy human skin in ex vivo LADD diffusion studies. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Efficacy and Safety of Laser-Assisted Combination Chemotherapy: An Explorative Imaging-Guided Treatment With 5-Fluorouracil and Cisplatin for Basal Cell Carcinoma.

BACKGROUND AND OBJECTIVES: Rising incidences of basal cell carcinoma (BCC) have increased the need for effective topical therapies. By enhancing cutaneous uptake of the chemotherapeutic agents, cisplatin and 5-fluorouracil (5-FU), laser-assisted delivery may provide a new combination treatment for BCC. Accordingly, this study aimed to evaluate tumor response, safety, and drug biodistribution in tumors and blood after topical laser-assisted 5-FU + CIS treatment in BCC patients. STUDY DESIGN/MATERIALS AND METHODS: This open-label, proof-of-concept trial investigated laser-assisted combination cisplatin + 5-FU treatment in 20 patients with histologically verified, low-risk superficial or nodular BCCs on the face (<20 mm) or trunk/extremities (<50 mm). After tumor demarcation guided by optical coherence tomography (OCT), BCCs were exposed to ablative fractional CO2 laser followed by 60 minutes topical cisplatin solution and 7-day exposure to 5% 5-FU cream under occlusion. After 30 days, treatment was repeated if any tumor residual was identified. Tumor response at day 30 and month 3 was assessed clinically as well as by OCT, reflectance confocal microscopy, and ultrasound, supplemented by histological verification at 3 months. Local skin reactions (LSRs) and side effects were evaluated on days 1, 3-5, 14, 30, and month 3. Drug detection in tumors and blood was performed in a subset of patients 1- and 24 hours after treatment. RESULTS: Nineteen patients completed the trial, with 32% (6/19) receiving a single treatment and 68% (13/19) treated twice. At 3 months, clinical clearance was seen in 18/19 patients with a corresponding 94% (17/18) achieving histological clearance. Baseline tumor thickness and subtype did not influence treatment number or clearance rate (P ≥ 0.61). LSRs were well-tolerated and consisted of erythema, edema, and erosion, followed by crusting by day 14. Erythema declined gradually by month 3, with 94% of patients and 79% of physicians rating cosmesis as "good" or "excellent." Scarring or hyperpigmentation was noted in 50% and 56%, respectively, while pain and infection were not observed during the follow-up period. Although chemotherapy uptake was visualized extending to deep skin layers, no systemic exposure to cisplatin or 5-FU was detected in patient blood. CONCLUSION: Laser-assisted cisplatin + 5-FU shows potential as an effective and tolerable treatment option for low-risk BCC, particularly in instances where self-application is not possible or where in-office, non-surgical therapy is preferred. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

Topical delivery of vismodegib using ablative fractional laser and micro-emulsion formulation in vitro.

BACKGROUND AND OBJECTIVE: Hedgehog inhibitors such as vismodegib are targeted drugs widely used for the treatment of basal cell carcinomas; however, their use is significantly limited by frequent systemic side effects due to oral administration route. We aim to use ablative fractional laser (AFL) to enable the topical delivery of vismodegib to relevant dermal depths. MATERIALS AND METHODS: Pig skin was treated in vitro with a fractional 10,600 nm CO2 laser at 0 or 80 mJ/microbeam and exposed to vismodegib (6.4 mmol/L) in Franz-diffusion cells for 0.5, 4, and 24 hours (n = 54 samples), either formulated in a micro-emulsion composed of soybean oil and Tween 80 or dissolved in ethanol as vehicle control. Vismodegib biodistribution was studied at specific skin depths from 0 to 1,800 µm (incremental steps of 300 µm) by mass spectrometry. RESULTS: Combination of AFL and vismodegib emulsion substantially enhanced the delivery of drug into the skin. Emulsion formulation alone yielded higher vismodegib skin concentrations compared to vehicle control in superficial and mid-dermis (0-900 µm, P = 0.002-0.015). The over-all highest concentration found (554.5 µmol/L) was reached at 24 hours in superficial (0-300 µm) AFL exposed skin, 7.6-fold higher than vehicle control (P = 0.002) and 9.7-101.6 fold higher than previously reported steady-state plasma concentrations in patients treated with oral vismodegib (5.5-56.9 µmol/L). Compared to intact skin, AFL exposure significantly increased skin concentrations of vismodegib even in deep skin layers (24 h, 900-1,800 µm, emulsion: 8.7-74.3 µmol/L vs. 0.0-0.0 µmol/L, P = 0.004-0.048; vehicle control: 23.7-50.6 µmol/L vs. 0.0-1.6 µmol/L, P = 0.002). The total delivery of vismodegib-emulsion into mid-deep dermal skin layers from 600 to 1,800 µm was for AFL exposed skin 8.2 fold higher than intact skin. Also, delivery of emulsion vismodegib by AFL was time-dependent as seen by the continuous increase in concentrations found over time, with highest uptake detected after 24 hours (4-24 hours, 0-900 µm, P = 0.002-0.004). CONCLUSION: AFL enhances topical delivery of micro-emulsion formulated vismodegib, reaching concentrations similar to or above plasma concentrations previously reported in patients receiving oral vismodegib. Lasers Surg. Med. 51:79-87, 2019. © 2018 Wiley Periodicals, Inc.

Red tattoos, ultraviolet radiation and skin cancer in mice.

Ultraviolet radiation (UVR) induces skin cancer. The combination of UVR and red tattoos may be associated with increased risk of skin cancer due to potential carcinogens in tattoo inks. This combination has not been studied previously. Immunocompetent C3.Cg/TifBomTac hairless mice (n=99) were tattooed on their back with a popular red tattoo ink. This often used ink is banned for use on humans because of high content of the potential carcinogen 2-anisidine. Half of the mice were irradiated with three standard erythema doses UVR thrice weekly. Time to induction of first, second and third squamous cell carcinoma (SCC) was measured. All UV-irradiated mice developed SCCs. The time to the onset of the first and second tumor was identical in the red-tattooed group compared with the control group (182 vs 186 days and 196 vs 203 days, P=ns). Statistically, the third tumor appeared slightly faster in the red-tattooed group than in the controls (214 vs 224 days, P=.043). For the second and third tumor, the growth rate was faster in the red-tattooed group compared with the control (31 vs 49 days, P=.009 and 30 vs 38 days, P=.036). In conclusion, no spontaneous cancers were observed in skin tattooed with a red ink containing 2-anisidine. However, red tattoos exposed to UVR showed faster tumor onset regarding the third tumor, and faster growth rate of the second and third tumor indicating red ink acts as a cocarcinogen with UVR. The cocarcinogenic effect was weak and may not be clinically relevant.

Search for Internal Cancers in Mice Tattooed with Inks of High Contents of Potential Carcinogens: A One-Year Autopsy Study of Red and Black Tattoo Inks Banned in the Market.

BACKGROUND AND OBJECTIVE: Tattoo ink stock products often contain potential carcinogens, which on large-scale population exposure may be clinically relevant. The aim of this autopsy study in mice was to screen major organs for clinical and subclinical cancers. METHODS: Mice were tattooed on their backs. In total, 48 mice were included and divided into 4 groups; 11 mice tattooed black, 10 tattooed red, and 5 mice serving as untreated controls. A group of 22 mice with black tattoos and exposed to ultraviolet radiation (UVR) were also studied. The black and red inks were both stock products banned on the Danish market due to the measured contents of potential carcinogens; benzo(a)pyrene and 2-anisidine, respectively. The mice were housed for 1 year after tattooing, and autopsy study on internal organs was performed. Tissue samples were systematically taken from major organs for screening of subclinical changes, not detected by visual examination. Any observed deviation from normal structure was subject to biopsy and light microscopy. RESULTS: All mice survived the 1-year observation period. Autopsy revealed no macroscopic signs of cancer. Microscopic search of internal organs showed no subclinical or clinical cancer. CONCLUSION: Despite extensive tattoos with 2 banned inks, the long-term observation in mice showed no internal cancers nor was the combination of carcinogen and UVR associated with cancer. Lack of observed malignancy might be explained by the fact that tattooing is only a single dose exposure. Registered data on carcinogens relies on repeated or chronic exposures. The study does not support the hypothesis that tattooing causes cancer.

Tattoo Pigments Are Observed in the Kupffer Cells of the Liver Indicating Blood-Borne Distribution of Tattoo Ink.

AIM: Tattoo pigments are deposited in the skin and known to distribute to regional lymph nodes. Tattoo pigments are small particles and may be hypothesized to reach the blood stream and become distributed to peripheral organs. This has not been studied in the past. The aim of the study was to trace tattoo pigments in internal organs in mice extensively tattooed with 2 different tattoo ink products. MATERIAL/METHODS: Three groups of mice were studied, i.e., 10 tattooed black, 10 tattooed red, and 5 untreated controls. They were tattooed on the entire back with commercial tattoo inks, black and red. Mice were sacrificed after 1 year. Samples were isolated from tattooed skin, lymph nodes, liver, spleen, kidney, and lung. Samples were examined for deposits of tattoo pigments by light microscopy and transmission electron microscopy (TEM). RESULTS: TEM identified intracellular tattoo pigments in the skin and in lymph nodes. TEM in both groups of tattooed mice showed tattoo pigment deposits in the Kupffer cells in the liver, which is a new observation. TEM detected no pigment in other internal organs. Light microscopy showed dense pigment in the skin and in lymph nodes but not in internal organs. CONCLUSION: The study demonstrated black and red tattoo pigment deposits in the liver; thus, tattoo pigment distributed from the tattooed skin via the blood stream to this important organ of detoxification. The finding adds a new dimension to tattoo pigment distribution in the body, i.e., as observed via the blood in addition to the lymphatic pathway.

Impact of UVR Exposure Pattern on Squamous Cell Carcinoma-A Dose-Delivery and Dose-Response Study in Pigmented Hairless Mice.

Cumulative lifetime ultraviolet radiation (UVR) is an important factor in the development of squamous cell carcinoma. This study examines the impact of UVR exposure pattern on tumor development. Hairless C3.Cg/TifBomTac immunocompetent pigmented mice (n = 351) were irradiated with 12 standard erythema doses (SED)/week, given as 2 SED ×6, 3 SED ×4, 4 SED ×3, or 6 SED ×2 (dose-delivery study) or 0, 0.6, 1.2, 2, 3 or 4 SED ×3/week (dose-response study). All mice were irradiated until development of 3 tumors of 4 mm each. Pigmentation was measured once monthly. In the dose-delivery study, the median time until tumor development was independent of dose fractions. In the dose-response study, higher UVR doses resulted in faster tumor appearance. When the weekly UVR dose was decreased from 12 to 6 SED, the cumulative UVR dose needed for tumor development was reduced by 40%. In conclusion, delivery schedules of a fixed weekly UVR dose did not affect tumor development. When using different weekly UVR doses, longer time to tumor development was observed using lower UVR doses. Lower weekly UVR doses however resulted in lower cumulative UVR doses to induce tumors in hairless pigmented mice.

Is the thin layer of methyl aminolevulinate used during photodynamic therapy sufficient?

BACKGROUND: If the recommended 1.0 mm layer of methyl aminolevulinate (MAL) is used during photodynamic therapy (PDT) of large areas with multiple actinic keratoses (AK) huge amounts of cream are needed. OBJECTIVES: To report the amount of MAL used for PDT of AK and basal cell carcinomas (BCC) in daily routine. The association of protoporphyrin IX (PpIX) fluorescence and thickness of MAL was investigated in a randomized paired study in healthy volunteers. METHODS: Amount of cream used per cm(2) during conventional and daylight PDT was recorded. In 16 healthy volunteers, 0.1 mm, 0.2 mm, 0.5 mm and 1.0 mm MAL cream were applied for 3 h on tape-stripped areas on each forearm randomized to light-permeable or light-impermeable occlusion. PpIX fluorescence was measured. RESULTS: Less than 0.4 mm MAL was used during PDT of BCC and 0.2 mm for AK. No difference in PpIX fluorescence was found between the different thicknesses of MAL using light-impermeable occlusion. CONCLUSION: In daily routine <0.4 mm MAL was used during PDT of BCC and 0.1-0.2 mm MAL during PDT of AK. The recommended 1.0 mm MAL did not result in a higher accumulation of PpIX compared to thinner MAL layers after light-impermeable occlusion for 3 h.

Alternatives to Outdoor Daylight Illumination for Photodynamic Therapy--Use of Greenhouses and Artificial Light Sources.

Daylight-mediated photodynamic therapy (daylight PDT) is a simple and pain free treatment of actinic keratoses. Weather conditions may not always allow daylight PDT outdoors. We compared the spectrum of five different lamp candidates for indoor "daylight PDT" and investigated their ability to photobleach protoporphyrin IX (PpIX). Furthermore, we measured the amount of PpIX activating daylight available in a glass greenhouse, which can be an alternative when it is uncomfortable for patients to be outdoors. The lamps investigated were: halogen lamps (overhead and slide projector), white light-emitting diode (LED) lamp, red LED panel and lamps used for conventional PDT. Four of the five light sources were able to photobleach PpIX completely. For halogen light and the red LED lamp, 5000 lux could photobleach PpIX whereas 12,000 lux were needed for the white LED lamp. Furthermore, the greenhouse was suitable for daylight PDT since the effect of solar light is lowered only by 25%. In conclusion, we found four of the five light sources and the greenhouse usable for indoor daylight PDT. The greenhouse is beneficial when the weather outside is rainy or windy. Only insignificant ultraviolet B radiation (UVB) radiation passes through the greenhouse glass, so sun protection is not needed.

Correlation between treatment time, photobleaching, inflammation and pain after photodynamic therapy with methyl aminolevulinate on tape-stripped skin in healthy volunteers.

Photodynamic therapy (PDT) is an attractive treatment option for skin diseases such as actinic keratosis, since large skin areas can be treated with high response rates and good cosmetic outcomes. Nevertheless inflammation and pain are still major side effects. The aim of this study was to investigate the extent to which less time-consuming PDT treatment regimens using methyl aminolevulinate (MAL) decrease protoporphyrin IX (PpIX) photobleaching, inflammation and pain. Twenty-four healthy volunteers were treated with 4 different interventions on each forearm. All 8 fields were tape-stripped 10 times. On the right arm MAL was applied for 20, 40, 60 or 180 min, followed by further incubation after wiping off MAL until 180 min after start and then illuminating with red light 180 min after start. On the left arm MAL or vehicle was applied for 30, 60, or 90 min and illuminated immediately after MAL removal. PpIX fluorescence, photobleaching, objective and subjective erythema (as a measure for inflammation), pigmentation and pain were measured. The results showed a significant correlation between incubation time, time until illumination and photobleaching. Furthermore, there was a significant correlation between photobleaching and erythema and also between photobleaching and pain. In conclusion, shorter PDT regimens result in decreased photobleaching and also less inflammation and pain. We hypothesize that a shorter incubation time is important for the optimal specific subcellular distribution of PpIX and to avoid unspecific distribution. We propose a shorter PDT regimen, "Pulse PDT", comprising, for example 30 min incubation with MAL and illumination after 180 min, and we have planned a study of actinic keratosis and "Pulse PDT".

Black tattoos protect against UVR-induced skin cancer in mice.

BACKGROUND: Black tattoos may involve risk of cancer owing to polycyclic aromatic hydrocarbons including benzo(a)pyrene (BaP) in inks. Ultraviolet radiation (UVR) induces skin cancer. The combination of UVR and black tattoo may therefore potentially be very problematic, but has not been previously studied. METHODS: Immunocompetent C3.Cg/TifBomTac mice (n = 99) were tattooed on the back with Starbrite Tribal Black(™) . This ink has a high content of the carcinogen BaP. Half of the mice were irradiated with three standard erythema doses UVR thrice weekly. Time to induction of first, second and third squamous cell carcinoma (SCC) was measured. Controls were 'tattooed' without ink. RESULTS: All irradiated mice developed SCCs while no malignant tumours were found in the nonirradiated group. In the tattooed and irradiated group, the development of the first, second and third SCC was significantly delayed in comparison with the irradiated controls without black tattoos (212, 232, 247 days vs. 163, 183, 191 days, P < 0.001). CONCLUSION: In UVR-irradiated black tattoos, remarkably, the development of UVR-induced skin cancer was delayed by the tattoos. Skin reflectance measurement indicated that the protective effect of black pigment in the dermis might be attributed to UVR absorption by black pigment below the epidermis and thereby reduction of backscattered radiation.

miR-125b induces cellular senescence in malignant melanoma.

BACKGROUND: Micro RNAs (miRs) have emerged as key regulators during oncogenesis. They have been found to regulate cell proliferation, differentiation, and apoptosis. Mir-125b has been identified as an oncomir in various forms of tumours, but we have previously proposed that miR-125b is a suppressor of lymph node metastasis in cutaneous malignant melanoma. Our goal was therefore to further examine this theory. METHODS: We used in-situ-hybridization to visualise miR-125b expression in primary tumours and in lymph node metastasis. Then using a miRVector plasmid containing a miR-125b-1 insert we transfected melanoma cell line Mel-Juso and then investigated the effect of the presence of a stable overexpression of miR-125b on growth by western blotting, flow cytometry and ß-galactosidase staining. The tumourogenicity of the transfected cells was tested using a murine model and the tumours were further examined with in-situ-hybridization. RESULTS: In primary human tumours and in lymph node metastases increased expression of miR-125b was found in single, large tumour cells with abundant cytoplasm. A stable overexpression of miR-125b in human melanoma cell line Mel-Juso resulted in a G0/G1 cell cycle block and emergence of large cells expressing senescence markers: senescence-associated beta-galactosidase, p21, p27 and p53. Mel-Juso cells overexpressing miR-125b were tumourigenic in mice, but the tumours exhibited higher level of cell senescence and decreased expression of proliferation markers, cyclin D1 and Ki67 than the control tumours. CONCLUSIONS: Our results confirm the theory that miR-125b functions as a tumour supressor in cutaneous malignant melanoma by regulating cellular senescence, which is one of the central mechanisms protecting against the development and progression of malignant melanoma.