Diversity of human papillomaviruses in skin lesions.

Pools of frozen biopsies from patients with squamous cell carcinoma (SCC) (n=29) actinic keratosis (AK) (n=31), keratoacanthoma (n=91) and swab samples from 84 SCCs and 91 AKs were analysed with an extended HPV general primer PCR and high-throughput sequencing of amplimers. We found 273 different HPV isolates (87 known HPV types, 139 previously known HPV sequences (putative types) and 47 sequences from novel putative HPV types). Among the new sequences, five clustered in genus Betapapillomavirus and 42 in genus Gammapapillomavirus. Resequencing of the three pools between 21 to 70 times resulted in the detection of 283 different known or putative HPV types, with 156 different sequences found in only one of the pools. Type-specific PCRs for 37 putative types from an additional 296 patients found only two of these putative types. In conclusion, skin lesions contain a large diversity of HPV types, but most appeared to be rare infections.

Aggressive curettage-cryosurgery for human papillomavirus-16 associated subungual squamous cell carcinoma in situ.

Squamous cell carcinoma (SCC) in situ is an uncommon tumour of the nail unit. Mohs micrographic surgery or wide surgical excision are often the preferred treatments. As an alternative therapy two patients with human papillomavirus (HPV)-16 associated SCC in situ were treated by curettage-cryosurgery. After a careful curettage with different-sized curettes freezing with liquid nitrogen in a double freeze-thaw was performed. Both patients were treated successfully and healed completely within 3 months. No adverse events were observed during a follow-up of at least 5 years and no recurrences were noted. Curettage-cryosurgery might be a safe and non-resource-demanding alternative treatment for patients with subungual SCC in situ.

Unbiased approach for virus detection in skin lesions.

To assess presence of virus DNA in skin lesions, swab samples from 82 squamous cell carcinomas of the skin (SCCs), 60 actinic keratoses (AKs), paraffin-embedded biopsies from 28 SCCs and 72 kerathoacanthomas (KAs) and fresh-frozen biopsies from 92 KAs, 85 SCCs and 92 AKs were analyzed by high throughput sequencing (HTS) using 454 or Ion Torrent technology. We found total of 4,284 viral reads, out of which 4,168 were Human Papillomavirus (HPV)-related, belonging to 15 known (HPV8, HPV12, HPV20, HPV36, HPV38, HPV45, HPV57, HPV59, HPV104, HPV105, HPV107, HPV109, HPV124, HPV138, HPV147), four previously described putative (HPV 915 F 06 007 FD1, FA73, FA101, SE42) and two putatively new HPV types (SE46, SE47). SE42 was cloned, sequenced, designated as HPV155 and found to have 76% similarity to the most closely related known HPV type. In conclusion, an unbiased approach for viral DNA detection in skin tumors has found that, although some new putative HPVs were found, known HPV types constituted most of the viral DNA.

5-year recurrence rates of Mohs micrographic surgery for aggressive and recurrent facial basal cell carcinoma.

Mohs micrographic surgery allows for complete microscopic examination of the surgical margin when treating aggressive and recurrent facial basal cell carcinomas. This leads to the highest cure rates and maximal preservation of healthy tissue. The 5-year recurrence rates of 587 aggressive and/or recurrent facial basal cell carcinomas treated during 1993 to 2003 at our centre were studied retrospectively. The resulting 5-year recurrence rates using Kaplan-Meier survival analysis were 2.1% for primary (previously untreated) tumours, 5.2% for recurrent basal cell carcinomas and 3.3% overall. In total, 87.9% of the tumours required at least two stages of Mohs micrographic surgery. The surgical defect's size after complete excision was, on average, approximately twice the size of the defect after excision of the clinically visible tumour with a 2-3 mm margin. Mohs micro-graphic surgery is underused in Scandinavia despite being the treatment of choice for aggressive and recurrent facial basal cell carcinomas.

Single low-dose red light is as efficacious as methyl-aminolevulinate--photodynamic therapy for treatment of acne: clinical assessment and fluorescence monitoring.

This controlled study investigated single low-dose red light photodynamic therapy and methyl-aminolevulinate (MAL) for treatment of moderate to severe facial acne in 19 patients. The right cheek was treated with MAL (160 mg/g) for 3 h prior to illumination. The left cheek received red light only. Both cheeks were illuminated with narrow-band red light (635 nm) at a light dose of 15 J/cm2. The global severity of acne was assessed at baseline and at follow-up, 10 and 20 weeks after treatment. Fluorescence images, clinical photographs and skin surface biopsies were obtained. Both MAL-photodynamic therapy and control areas showed a significant decrease in acne score at follow-up; no significant difference was found compared with control. MAL-photodynamic therapy was associated with adverse effects such as erythema and stinging. Fluorescence images revealed poor selectivity of MAL-induced fluorescence to the acne lesions, suggesting a general photoablating mechanism rather than selective destruction of sebaceous glands. No significant reduction in Propionibacterium acnes or sebum excretion was found.

Photodynamic therapy with methyl aminolevulinate for prevention of new skin lesions in transplant recipients: a randomized study.

BACKGROUND: Organ transplant recipients on long-term immunosuppressive therapy are at increased risk of non-melanoma skin lesions. Repeated field photodynamic therapy using topical methyl aminolevulinate (MAL) may have potential as a preventive treatment. METHODS: This open randomized, intrapatient, comparative, multicenter study included 81 transplant recipients with 889 lesions (90% actinic keratoses (AK)]. In each patient, the study treatment was initially administered to one 50 cm area on the face, scalp, neck, trunk, or extremities (n=476 lesions) twice (1 week apart), with additional single treatments at 3, 9, and 15 months. On each occasion, the area was debrided gently and MAL cream (160 mg/g) applied for 3 hr, before illumination with noncoherent red light (630 nm, 37 J/cm2). The control, 50 cm2 area (n=413 lesions) received lesion-specific treatment (83% cryotherapy) at baseline and 3, 9, and 15 months. Additionally, all visible lesions were given lesion-specific treatment 21 and 27 months in both treatment and control areas. RESULTS: At 3 months, MAL photodynamic therapy significantly reduced the occurrence of new lesions (65 vs. 103 lesions in the control area; P=0.01), mainly AK (46% reduction; 43 vs. 80; P=0.006). This effect was not significant at 27 months (253 vs. 312; P=0.06). Hypopigmentation, as assessed by the investigator, was less evident in the treatment than control areas (16% vs. 51% of patients; P<0.001) at 27 months. CONCLUSION: Our results suggest that repeated field photodynamic therapy using topical MAL may prevent new AK in transplant recipients although further studies are needed.

Cutaneous human papillomaviruses found in sun-exposed skin: Beta-papillomavirus species 2 predominates in squamous cell carcinoma.

BACKGROUND: A spectrum of cutaneous human papillomaviruses (HPVs) is detectable in nonmelanoma skin cancers, as well as in healthy skin, but the significance that the presence of these types of HPV DNA has for the pathogenesis of skin cancer remains unclear. METHODS: We studied 349 nonimmunosuppressed patients with skin lesions (82 with squamous cell carcinomas, 126 with basal cell carcinomas, 49 with actinic keratoses, and 92 with benign lesions). After superficial skin had been removed by tape, paired biopsy samples--from the lesion and from healthy skin from the same patient--were tested for HPV DNA. Risk factors for HPV DNA were analyzed in multivariate models. RESULTS: Overall, 12% of healthy skin samples were positive for HPV DNA, compared with 26% of benign lesions, 22% of actinic keratoses, 18% of basal cell carcinomas, and 26% of squamous cell carcinomas. HPV DNA was associated with sites extensively exposed to the sun, both for the lesions (odds ratio [OR], 4.45 [95% confidence interval {CI}, 2.44-8.11]) and for the healthy skin samples (OR, 3.65 [95% CI 1.79-7.44]). HPV types of Beta-papillomavirus species 2 predominate in squamous cell carcinomas (OR, 4.40 [95% CI, 1.92-10.06]), whereas HPV types of Beta-papillomavirus species 1 are primarily found in benign lesions (OR, 3.47 [95% CI, 1.72-6.99]). CONCLUSIONS: Cutaneous HPV types are primarily detected at sites extensively exposed to the sun. HPV types of Beta-papillomavirus species 2, but not of species 1, are associated with squamous cell carcinoma.

Photodynamic therapy for acne vulgaris: a pilot study of the dose-response and mechanism of action.

Acne vulgaris does not always respond to conventional therapy. Photodynamic therapy (PDT) has been proposed as a treatment option. The aim of this study was to determine the optimal light dose for effective PDT treatment of acne and to investigate whether PDT reduces sebum excretion and the amount of Propionibacterium acnes. Fifteen patients (9 men, 6 women, age range 16-44 years, mean age 25 years) with mild to severe acne were enrolled in an open, unblinded study. Aminolaevulinic acid cream (20% in Unguentum Merck) was applied on two circular areas 3 h before PDT. The areas of investigation were irradiated with red light (635 nm) from a Waldman PDT 1200 lamp. Ten patients with facial acne were treated with a light dose of 50 J/cm(2) on the right cheek and 30 J/cm(2) on the left cheek. Five patients with acne on their back were treated either with 50 J/cm(2) or with 70 J/cm(2). Clinical follow-up was performed for at least 10 weeks. In the patients with facial acne, sebum excretion was determined before PDT and at every follow-up visit. The amount of P. acnes was measured in a skin surface biopsy using a cyano-acrylate polymer to extract the content of the sebaceous follicles. In 9 patients with facial acne the improvement of lesions was the same for the two light doses. According to the patients' own assessment, 8 improved after PDT (p=0.02). No difference was found between the two doses in patients with acne on the back. Hyperpigmentation was more common at higher doses of light, and pain was experienced more often by the patients when higher doses were used. No significant reduction in P. acnes or sebum excretion was found at any time after PDT. It is concluded that PDT could be an alternative treatment of acne lesions. The lowest possible light dose should be used for minimal side-effects. Other mechanisms of action for PDT than eradication of P. acnes and sebosuppression should be considered.

Important factors for pain during photodynamic therapy for actinic keratosis.

Photodynamic therapy (PDT) is an efficient treatment for actinic keratosis. A common problem, however, is pain. The aim of this study was to investigate pain during PDT for actinic keratosis. The possibility of using capsaicin cream for pain relief was also assessed. Pain was investigated during aminolaevulinic acid PDT in 91 patients. Size, redness, scaling and induration of the lesions were recorded. Maximum pain during treatment was registered, using a visual analogue scale (0-10). The pain-reducing efficacy of capsaicin was tested in a pilot study in six patients (10 lesions). These patients were pre-treated with capsaicin cream for one week before commencing PDT. Pain was found to be normally distributed around a mean value of visual analogue scale 4.6. Larger lesions gave more pain (p=0.001). The redness of the actinic lesions was found to be related to PDT-induced pain (p=0.01), the reduction of actinic area (p=0.007), and the cure rate (p=0.01). The redder the actinic area, the better the treatment outcome and the more pain experienced. Patients with the largest reduction in the actinic area experienced more pain (p=0.053). The most important factors for presence of pain seem to be the size and the redness of the lesion. No significant pain relief was experienced after pre-treatment with capsaicin.

Penile intraepithelial neoplasia: results of photodynamic therapy.

Failure of response to treatment or recurrent disease is often noted in patients with penile intraepithelial neoplasia. Photodynamic therapy has recently been added to the list of treatment modalities used for this diagnosis. Our primary objective was to study the results of photodynamic therapy in the treatment of penile intraepithelial neoplasia in men over the age of 40 years. Ten patients aged 42-82 years with histopathologically confirmed lesions were studied. Lesions initially responded to photo-dynamic therapy in 7 out of 10 patients. Four of these patients presented no recurrences during a mean follow-up of 35 months, and were completely cleared after 2-8 treatments (mean 4.5 treatments). Three patients presented recurrences after treatment. No patient developed invasive penile cancer (mean follow-up 46.5 months). Photodynamic therapy is an alternative in the treatment of penile intraepithelial neoplasia, although prospective randomized trials are required to provide therapeutic guidelines.

Bispectral fluorescence imaging combined with texture analysis and linear discrimination for correlation with histopathologic extent of basal cell carcinoma.

Fluorescence imaging has been shown to be a potential complement to visual inspection for demarcation of basal cell carcinoma (BCC), which is the most common type of skin cancer. Earlier studies have shown promising results when combining autofluorescence with protoporphyrin IX (Pp IX) fluorescence, induced by application of delta-5-aminolaevulinic acid (ALA). In this work, we have tried to further improve the ability of this technique to discriminate between areas of tumor and normal skin by implementing texture analysis and Fisher linear discrimination (FLD) on bispectral fluorescence data of BCCs located on the face. Classification maps of the lesions have been obtained from histopathologic mapping of the excised tumors. The contrast feature obtained from co-occurrence matrices was found to provide useful information, particularly for the ALA-induced Pp IX fluorescence data. Moreover, the neighborhood average features of both autofluorescence and Pp IX fluorescence were preferentially included in the analysis. The algorithm was trained by using a training set of images with good agreement with histopathology, which improved the discriminability of the validation set. In addition, cross validation of the training set showed good discriminability. Our results imply that FLD and texture analysis are preferential for correlation between bispectral fluorescence images and the histopathologic extension of the tumors.

High prevalence of cutaneous human papillomavirus DNA on the top of skin tumors but not in "Stripped" biopsies from the same tumors.

Genomes of human papillomaviruses (HPV) are common in biopsies from non-melanoma skin cancers but are also found on healthy skin and it is possible that HPV positivity in tumor biopsies by PCR may merely reflect contamination of the lesion surface. To investigate this issue, 229 immunocompetent patients were tested for HPV DNA in swab samples collected on top of skin tumors and in biopsies of the same tumors, obtained after stripping with tape to remove superficial layers. HPV DNA was detected on top of 69% (159 of 229) of the lesions, and in 12% (28 of 229) of the stripped biopsies (p<0.001). The difference was seen for all four types of tumors studied. Seborrheic keratosis had 79% (34 of 43) HPV positivity on top of lesions versus 19% (eight of 43) in biopsies; actinic keratosis had 83% (38 of 46) HPV positivity on top versus 11% (five of 46) in biopsies; basal cell carcinoma had 63% (69 of 109) on top versus 8% (nine of 109) in biopsies and squamous cell carcinoma had 58% (18 of 31) on top versus 19% (six of 31) in biopsies. HPV DNA is common in superficial layers of lesions, but is not necessarily present throughout tumors.

Five-year results of curettage-cryosurgery for 100 consecutive auricular non-melanoma skin cancers.

Large excisions or Mohs' micrographic surgery (MMS) are often the suggested treatments for non-melanoma skin cancers (NMSCs) of the external ear. This five-year follow-up attempts to evaluate whether curettage-cryosurgery could be an alternative therapy for selected auricular NMSCs. One hundred auricular NMSCs, selected at a skin tumour clinic, were treated by a thorough curettage, with different-sized curettes, followed by cryosurgery in a double freeze-thaw cycle. Seventy-seven basal cell carcinomas (BCCs), 13 squamous cell carcinomas (SCCs), six SCCs in situ, and four basosquamous carcinomas were included. The mean diameter of the tumours was 18 mm (range 5-70). Morphoeiform BCCs, recurrent BCCs with fibrotic component, and most of the SCCs were selected for MMS. Seventy-one patients with 81 tumours were followed-up for at least five years with only one recurrence. Nineteen patients with 19 tumours, followed-up for two to four years, died from other causes with no sign of recurrence at their last visit. Patients followed-up for less than two years were excluded. No major problems were registered after treatment. The cosmetic result was good or acceptable in most patients. In carefully selected patients a thorough curettage followed by freezing with liquid nitrogen in a double freeze-thaw cycle could be a safe and inexpensive therapy even for large NMSCs of the external ear.

Computerized bipolar diathermy with scissors and forceps in cutaneous surgery.

BACKGROUND: Bipolar diathermy coagulates tissue as effectively as monopolar with less lateral tissue injury and no risk of interference with cardiac pacemakers or joint prostheses. OBJECTIVE: To test a novel computerized bipolar diathermy machine for combined cutting and coagulation in dermatologic surgery. METHODS: A divided cable was used to deliver current from a computerized bipolar diathermy unit to both scissors and forceps. The bipolar diathermy unit senses tissue contact with the instruments and starts automatically; a built-in microcomputer measures tissue impedance and automatically terminates the current when tissue coagulation is achieved. RESULTS: The equipment has been used successfully in more than 200 patients undergoing dermatologic surgery. The advantages were a reduced operating time and a more secure hemostasis. The microprocessor controlled bipolar diathermy unit minimized any tissue adherence to the instruments during use. CONCLUSION: We recommend the use of insulated scissors and computerized bipolar diathermy for safe and efficacious coagulation and cutting in dermatologic surgery.

A broad range of human papillomavirus types detected with a general PCR method suitable for analysis of cutaneous tumours and normal skin.

A pair of degenerate PCR primers (FAP59/64) was designed from two relatively conserved regions of the L1 open reading frame of most human papillomaviruses (HPV). The size of the generated amplicon was about 480 bp. PCR using these primers was found capable of amplifying DNA from 87% (65/75) of the HPV types tested, its sensitivity being 1-10 copies for HPV-5, -20 and -30 clones. HPV was found in 63% (5/8) of tumour samples and in 63% (5/8) of normal skin biopsies from patients with various cutaneous tumours. HPV-5, HPV-8, HPV-12, HPVvs20-4 and six putatively novel HPV types were identified. No correlation was found to exist between specific HPV and tumour types. Skin surface swab samples from one or more sites on three of four healthy volunteers were found to contain HPV, types 12 and 49 being identified, as well as eight novel HPV types, two of which were also found among the patients. In all, HPV was detected in 75% (9/12) of those tested, five HPV types and 12 novel candidate types being identified, and 37% (7/19) of HPV-positive samples were found to manifest more than one HPV type. All the HPV detected manifested high degrees of nucleotide sequence similarity with HPV types associated with skin lesions and epidermodysplasia verruciformis. The overall HPV finding in the skin samples was 50% (20/40) using the FAP primers as compared to 18% (7/40) using another PCR test designed for skin types. The results thus suggest the new method to be sensitive and generally applicable for detecting cutaneous HPV.