Ultrastructural alteration of tape-stripped normal human skin after photodynamic therapy

The effect of photodynamic therapy on tape-stripped normal human skin was explored ultrastructurally. Back skin areas of 3 healthy Caucasian volunteers, 2 men and 1 woman, were tape-stripped 10 consecutive times followed by topical treatment with 5-aminolevulinic acid (20%, w/w) for 4 hours under occlusion (Tegaderm). Then the areas were irradiated for 30 minutes with Waldman PDT 1200 lamp (570-650 nm) with a total dose of 70 J/cm(2). Full-thickness punch biopsies were taken immediately after irradiation, and at 3 and 24 hours for electron microscopy examination. Photodynamic therapy caused morphological alterations mainly in the epidermis. Keratinocytes became oedematous and tonofilament bundles were split, but desmosomes remained normal. Many keratinocytes contained large intracellular vacuoles and extremely electronlucent cytoplasm implying cell damage. Although the majority of Langerhans cells were unaffected isolated Langerhans cells became apoptotic at 3 hours. The melanocytes preserved their normal morphology. The epidermal alterations recovered 24 hours after the irradiation. Inflammatory cell infiltrates were evident at 3 and 24 hours but no other dermal changes were observed. In conclusion, photodynamic therapy with 5-aminolevulinic acid affects mainly keratinocytes and can trigger apoptosis in Langerhans cells while melanocytes appear refractory, at least morphologically, to photodynamic therapy.     

Cutaneous field cancerization: clinical,histopathological and therapeutic aspects

The concept of "field cancerization" was first introduced by Slaughter in 1953 when studying the presence of histologically abnormal tissue surrounding oral squamous cell carcinoma. It was proposed to explain the development of multiple primary tumors and locally recurrent cancer. Organ systems in which field cancerization has been described since then are: head and neck (oral cavity, oropharynx, and larynx), lung, vulva, esophagus, cervix, breast, skin, colon, and bladder. Recent molecular studies support the carcinogenesis model in which the development of a field with genetically altered cells plays a central role. An important clinical implication is that fields often remain after the surgery for the primary tumor and may lead to new cancers, designated presently as "a second primary tumor" or "local recurrence," depending on the exact site and time interval. In conclusion, the development of an expanding pre-neoplastic field appears to be a critical step in epithelial carcinogenesis with important clinical consequences. Diagnosis and treatment of epithelial cancers should not only be focused on the tumor but also on the field from which it developed. The most important etiopathogenetic, clinical, histopathological and therapeutic aspects of field cancerization are reviewed in this article.     

Comparative study of histopathological and immunohistochemical findings in skin biopsies from patients with psoriasis before and after treatment with acitretin

Background:  Acitretin has been shown to be effective for psoriasis treatment. Its mechanism of action is not completely understood, and there are few studies focusing on histological and immunohistochemical differences before and after treatment of psoriasis with acitretin.
Methods:  This is a prospective study of 17 patients with plaque psoriasis treated with acitretin for 4 months with biopsies taken before and after therapy. Histological features and immunohistochemical reactions to cytokeratin (CK) 10, CK16, CK19, Ki67 and CD1a were evaluated and compared.
Results:  There were nine men and eight women with median age of 47 years. Epidermal thickness, CK16 positivity, Ki67 and CD1a-positive cell index reduced after treatment (p < 0.01). Suprapapillary plate thickness stayed the same (p > 0.05) although the epidermal/suprapapillary thickness ratio was significantly higher before treatment (p < 0.01). CK10 positivity was lower and a thicker basal cell layer was seen in the epidermis before treatment (p < 0.01). CK19 was negative in all cases.
Conclusions:  Acitretin therapy improved histological and immunohistochemical features typical of psoriasis. In psoriasis, suprapapillary plates are not thin, but the epidermal/suprapapillary thickness ratio is increased. Basal cell layer is expanded in psoriasis. Langerhans' cells were less frequent after treatment, and that finding has to be investigated further to determine its role in acitretin mechanism of action.     

Surgical scar remodelling after photodynamic therapy using aminolaevulinic acid or its methylester: a retrospective, blinded study of patients with field cancerization

Summary Background Photodynamic therapy (PDT) is a nonsurgical alternative to conventional tumour excision for nonmelanoma skin cancers (NMSCs). Objectives We evaluated whether patients with field cancerization (multiple NMSCs) treated with aminolaevulinic acid (ALA) or its methylester (MAL) for that indication had PDT‐induced changes in surgical scars in the treatment field. Methods Six adult patients with multiple NMSCs and a total of 21 scars from previous excisions were studied in a retrospective blinded evaluation from clinical photographs of scar response to ALA/MAL‐PDT. After a 3‐h application of topical 20% ALA or 16·8% MAL under occlusion, each field was irradiated with 635‐nm light‐emitting diode light at the fluence of 200 J cm^?2. Patients underwent one to three PDT sessions per field at ～1 month intervals, to fields that included scars on the back, thigh, arms and neck. Pre‐ and post‐treatment digital photographs of scars were combined into 92 pairs that were independently and blindly evaluated by three board‐certified dermatologists. This study was performed at our academic practice at the Massachusetts General Hospital. Results PDT produced a statistically significant improvement in scar appearance. The degree of improvement correlated with the number of treatment sessions (two or three treatments; P < 0·05). Improvement after a single treatment was not statistically different from baseline ratings (P = 0·99). Conclusions Surgical scar remodelling and clinical improvement may be accomplished via ALA/MAL‐PDT, but may require repeated treatment sessions. Larger, prospective studies are necessary to confirm the effectiveness of PDT for this indication.     

Nerve blocks enable adequate pain relief during topical photodynamic therapy of field cancerization on the forehead and scalp

Background  Topical photodynamic therapy (PDT) is an effective method when treating extensive areas of sun-damaged skin with multiple actinic keratoses (AKs) (field cancerization) on areas such as the forehead and scalp, and offers excellent cosmetic outcome. The major side-effect of PDT is the pain experienced during treatment.
Objectives  To investigate whether nerve blocks could provide adequate pain relief during PDT of AKs on the forehead and scalp.
Methods  Ten men with symmetrically distributed and extensive AKs on the forehead and scalp were included in the study. Prior to PDT one side of the forehead and scalp was anaesthetized by nerve blocks while the other side served as control.
Results  The mean visual analogue scale (VAS) score on the anaesthetized side was 1 compared with 6·4 on the nonanaesthetized side during PDT. This difference was significant ( P  <   0·0001), implying that nerve blocks reduce VAS scores during PDT.
Conclusions  The results of the study support the use of nerve blocks as pain relief during PDT of field cancerization on the forehead and scalp, although individual considerations must be taken into account to find the most adequate pain-relieving method for each patient.     

European guidelines for topical photodynamic therapy part 2: emerging indications – field cancerization,photorejuvenation and inflammatory/infective dermatoses

In addition to established indications in non‐melanoma skin cancer in immunocompetent patients, photodynamic therapy (PDT) has been studied for the treatment, and possible prevention, of superficial skin cancers in immunosuppressed patients. As a topical photosensitizer can be applied over large areas, PDT is also increasingly used for field cancerization in photodamaged skin, with evidence of potential to delay the development of actinic keratoses and basal cell carcinoma, although direct evidence of prevention of invasive squamous cell carcinoma remains limited. PDT has been studied in patch/plaque‐stage cutaneous T‐cell lymphoma, with efficacy more likely in unilesional disease. Accumulating evidence supports the use of PDT in acne and several other inflammatory/infective dermatoses including cutaneous leishmaniasis, although protocols are still to be refined. Despite proven efficacy, PDT is not widely used in viral/genital warts, where pain during treatment can be intense. PDT is a therapeutic option for photorejuvenation, with improvement in fine wrinkles, mottled hyperpigmentation, roughness and sallowness reported.     

European Dermatology Forum guidelines on topical photodynamic therapy 2019 Part 2: emerging indications – field cancerization,photorejuvenation and inflammatory/infective dermatoses

In addition to approved indications in non-melanoma skin cancer in immunocompetent patients, topical photodynamic therapy (PDT) has also been studied for its place in the treatment of, as well as its potential to prevent, superficial skin cancers in immune-suppressed patients, although sustained clearance rates are lower than for immune-competent individuals. PDT using a nanoemulsion of ALA in a daylight or conventional PDT protocol has been approved for use in field cancerization, although evidence of the potential of the treatment to prevent new SCC remained limited. High-quality evidence supports a strong recommendation for the use of topical PDT in photorejuvenation as well as for acne, refractory warts, cutaneous leishmaniasis and in onychomycosis, although these indications currently lack approvals for use and protocols remain to be optimized, with more comparative evidence with established therapies required to establish its place in practice. Adverse events across all indications for PDT can be minimized through the use of modified and low-irradiance regimens, with a low risk of contact allergy to photosensitizer prodrugs, and no other significant documented longer-term risks with no current evidence of cumulative toxicity or photocarcinogenic risk. The literature on the pharmacoeconomics for using PDT is also reviewed, although accurate comparisons are difficult to establish in different healthcare settings, comparing hospital/office-based therapies of PDT and surgery with topical ointments, requiring inclusion of number of visits, real-world efficacy as well as considering the value to be placed on cosmetic outcome and patient preference. This guideline, published over two parts, considers all current approved and emerging indications for the use of topical photodynamic therapy in Dermatology prepared by the PDT subgroup of the European Dermatology Forum guidelines committee. It presents consensual expert recommendations reflecting current published evidence.     

Clinical and immunohistochemical evaluation of psoriatic plaques treated with topical 5-aminolaevulinic acid photodynamic therapy

BACKGROUND/PURPOSE: The aims of this study were to investigate the clinical and immunohistochemical events of psoriatic plaques during photodynamic therapy (PDT) using topical application of 5-aminolaevulinic acid (ALA). METHODS: Twelve psoriatic patients were recruited for this study. Four of them dropped out because of pain during treatment. The effect of PDT was evaluated in the remaining eight. One plaque was selected in each patient and treated once weekly with PDT 10-30 J/cm(2) two to five times. It was evaluated by using the scale, erythema and induration (SEI) index (maximal score per patient=9). Pain during treatment was assessed by a visual analogue scale (VAS), ranging from 0 to 10. Skin biopsies were taken before treatment, after two treatments and after completion of treatment, and were evaluated by immunohistochemistry. RESULTS: Median SEI scores were significantly reduced from 7 (range 5-9) before to 1.5 (range 0-3) following treatment (P<0.0001). The median pain during PDT was 7. The number of vessels in the subpapillary dermis, identified by antibodies against Factor VIII and endoglin, increased during and/or after treatment in six of eight patients. Before treatment, the epidermal growth factor (EGF) receptor was displayed throughout the epidermis, keratin 16 suprabasally, involucrin from the stratum granulosum to the lower spinous layers and filaggrin in stratum granulosum with focal absence. There was a moderate dermal infiltrate of CD4(+) cells and a sparse one of CD8(+). Following treatment, the EGF receptor was still displayed throughout the epidermis in seven of eight specimens. Cytokeratin 16 expression decreased markedly. Involucrin was not seen as deep in the spinous layers as before PDT. Filaggrin was expressed throughout the stratum granulosum and often weakly in the upper stratum spinosum. The number of CD4(+) and CD8(+) dermal cells decreased. CONCLUSION: PDT improved psoriasis and induced dermal neovascularization. Although a good clinical response was seen in most of our patients, the high frequency of discomfort during treatment limits the usefulness of ALA-PDT for psoriasis. The mechanism of the neovascularization is unknown. It may be owing to an indirect effect of PDT on the microvasculature and immune system or recovery phenomena.     

Clinical, dermoscopic and immunohistochemical assessment of actinic keratoses and evaluation of the effectiveness of diclofenac therapy with immunohistochemical analysis

Actinic keratoses (AKs) is a keratinocytic neoplasm that typically develops on the face of elderly patients. Little is known regarding the clinical, dermatoscopic and immunohistochemical assessments of AK using topical diclofenac therapy. We sought to determine these assessments and evaluate the efficacy of topical diclofenac gel in AK. In this prospective, open-label study, 44 patients with 66 AKs were treated for 12 weeks with topically applied diclofenac (3 % gel in 2.5 % hyaluronic acid). Immunohistopathologic analyses were performed before and after diclofenac treatment using epidermal stem cell markers such as Cytokeratin 15 (CK15), Cytokeratin 19 (CK19) and p63, in addition to proliferation markers (Bcl-2, Ki-67). Diclofenac gel was found to be effective in AK, including the hyperkeratotic type. Surprisingly, complete remission was observed at a significantly higher rate in Grade 3 lesions (p = 0.017). However, imunohistochemical and histopathologic examinations revealed that 12-week treatment periods may not be sufficient to fully cure AK. The immunohistochemical analyses revealed no change in the expression levels of CK15, CK19 and Bcl-2 following diclofenac therapy. However, the expression of Ki-67 (p = 0.042) and p63 (p = 0.030) exhibited a significant decrease after therapy. Dermatoscopy is an effective method for diagnosis of AK, and topical diclofenac sodium gel was found as an effective additional treatment modality. Since positive histopathological findings were detected in some patients even with significant remission, a 12-week treatment period should be extended even in patients presenting with positive clinical response. Importantly, anti-proliferative effects of diclofenac were demonstrated by decreased Ki-67 and p63 expression levels.     

Intrinsic aging vs. photoaging: a comparative histopathological,immunohistochemical, and ultrastructural study of skin

Cutaneous aging is a complex biological phenomenon affecting the different constituents of the skin. To compare the effects of intrinsic and extrinsic aging processes, a total of 83 biopsies were collected from sun-exposed and protected skin of healthy volunteers representing decades from the 1st to the 9th (6-84 years of age). Routine histopathology coupled with computer-assisted image analysis was used to assess epidermal changes. Immunoperoxidase techniques with antibodies against type I and type III collagens and elastin were used to quantitatively evaluate changes in collagen and elastic fibers and their ultrastructure was examined by transmission electron microscopy. Epidermal thickness was found to be constant in different decades in both sun-exposed and protected skin; however, it was significantly greater in sun-exposed skin (P = 0.0001). In protected skin, type I and III collagen staining was altered only after the 8th decade, while in sun-exposed skin the relative staining intensity significantly decreased from 82.5% and 80.4% in the 1st decade to 53.2% and 44.1% in the 9th decade, respectively (P = 0.0004 and 0.0008). In facial skin the collagen fiber architecture appeared disorganized after the 4th decade. The staining intensity of elastin in protected skin significantly decreased from 49.2% in the 1st decade to 30.4% in the 9th decade (P = 0.05), whereas in sun-exposed skin the intensity gradually increased from 56.5% in the 1st decade to 75.2% in the 9th decade (P = 0.001). The accumulated elastin in facial skin was morphologically abnormal and appeared to occupy the areas of lost collagen. Collectively, the aging processes, whether intrinsic or extrinsic, have both quantitative and qualitative effects on collagen and elastic fibers in the skin.     

Photodynamic therapy for skin field cancerization: an international consensus. International Society for Photodynamic Therapy in Dermatology

Field cancerization is a term that describes the presence of genetic abnormalities in a tissue chronically exposed to a carcinogen. These abnormalities are responsible for the presence of multilocular clinical and sub-clinical cancerous lesions that explains the increased risks of multiple cancers in this area. With respect to the skin, this term is used to define the presence of multiple non-melanoma skin cancer, its precursors, actinic keratoses and dysplastic keratinocytes in sun exposed areas. The multiplicity of the lesions and the extent of the area influence the treatment decision. Providing at least equivalent efficacy and tolerability, field directed therapies are therefore often more worthwhile than lesion targeted approaches. Photodynamic therapy (PDT) with its selective sensitization and destruction of diseased tissue is one ideal form of therapy for this indication. In the following paper the use of PDT for the treatment of field cancerized skin is reviewed and recommendations are given for its use.     

Contribution to characterization of skin field cancerization activity: morphometric,chromatin texture,proliferation, and apoptosis aspects

BackgroundA skin field cancerization is a cutaneous area with subclinical changes resultant from chronic sun exposure, with a higher predisposition to development of pre-neoplastic and neoplastic lesions. So far, there are no well-defined objective parameters that can indicate their degree of activity.ObjectivesTo describe and compare morphometric aspects and expression of factors related to apoptosis and cell proliferation in actinic keratosis (AK), in both photoexposed and photoprotected epidermis.MethodsA cross-sectional study of patients with actinic keratosis in the forearms, biopsied at two points: the actinic keratosis and the axillary region. The biopsies of the actinic keratosis, perilesional area, and axilla were evaluated through keratinocyte intraepithelial neoplasia (KIN), and immunohistochemistry of p53, survivin, and Ki67. Nuclear morphometry of basal layer cells was performed through digital image analysis: entropy, area, perimeter, Ra, fractal dimension, circularity, color intensity, and largest diameter.ResultsThere were 13 patients included and 38 actinic keratosis biopsied. In morphometry, 1039 nuclei were analyzed, of which 228 represented axillary skin, 396 demonstrated actinic keratosis, and 415 represented the perilesional area to the actinic keratosis. There was a significant difference (p < 0.05) in all variables tested for the topographies evaluated. A significant correlation was identified between nucellar morphometric elements, KIN, proliferation markers, and apoptosis. Joint patterns of p53, Ki67, and KIN discriminated the topographies sampled.Study limitationsThis was a cross-sectional study with a small number of patients.ConclusionsThere are patterns of proliferation, resistance to apoptosis, and different cellular morphometrics between photoprotected skin and photoexposed skin. The joint expression of p53, Ki67, and KIN can characterize skin field cancerization activity.