Research Techniques Made Simple: Emerging Imaging Technologies for Noninvasive Optical Biopsy of Human Skin.

Over the past few years, high-resolution optical imaging technologies such as optical coherence tomography (OCT), reflectance confocal microscopy (RCM), and multiphoton microscopy (MPM) have advanced significantly as new methodologies for clinical research and for real-time detection, diagnosis, and therapy monitoring of skin diseases. Implementation of these technologies into clinical research and practice requires clinicians to have an understanding of their capabilities, benefits, and limitations. This concise review provides insights on the application of OCT, RCM, and MPM for clinical skin imaging through images acquired in vivo from the same lesions. The presented data are limited to pigmented lesions and basal cell carcinoma.

Inhibition of the hedgehog pathway in patients with basal-cell nevus syndrome: final results from the multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Aberrant hedgehog signalling underlies the development of basal-cell carcinomas. We previously reported the interim analysis of a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial in patients with the basal-cell nevus (Gorlin) syndrome indicating that the smoothened inhibitor vismodegib reduces basal-cell carcinoma tumour burden and prevents new basal-cell carcinoma growth in patients with basal-cell nevus syndrome. We report the final results of this 36 month trial. METHODS: In our multicentre, randomised, double-blind, placebo-controlled, phase 2 trial we enrolled patients aged 35-75 years with basal-cell nevus syndrome with at least ten surgically eligible basal-cell carcinomas at the Children's Hospital Oakland, Columbia University outpatient dermatology clinic (NY, USA) and a private practice outpatient dermatology office in Newport Beach (CA, USA). Patients were assigned to vismodegib or placebo (2:1) according to a randomisation sequence generated by computer code. The primary endpoint of the trial of 41 patients was to compare the effect of oral vismodegib (150 mg/day) versus placebo on the incidence of new surgically eligible basal-cell carcinomas after 3 months of treatment. In the subsequent, open-label phase (n=37) patients continued vismodegib at two sites for as long as month 36 (n=25) and at the third site were monitored up to month 36 (n=12). Additional endpoints for this phase were: whether continuous versus interrupted dosing differentially affected tumour burden; time to reach various levels of reduction in tumour burden; reduction in tumour size in patients who took less than 50% of the expected number of vismodegib tablets; reduction in the number of surgical excisions required per year before, during, and after treatment; and the effect of vismodegib on hedgehog target gene expression. We monitored patients at visits every 3 months for up to 36 months. The primary endpoint was analysed on a modified intention-to-treat basis. This trial is registered with ClinicalTrials.gov, number NCT00957229. FINDINGS: Between Sept 22, 2009, and Jan 24, 2011, 41 patients were monitored for a median of 36 months (IQR 36-36). Patients treated with vismodegib (n=26) had a mean reduced rate of new surgically eligible basal-cell carcinomas compared with patients randomly assigned to placebo (n=15; two [SD 0·12] new surgically eligible basal-cell carcinomas per patient per year vs 34 [1·32] new surgically eligible basal-cell carcinomas per patient per year, p<0·0001). In the 11 patients initially assigned to placebo, mean cross over to vismodegib reduced the development of new surgically eligible basal-cell carcinomas compared with placebo (0·4 [SD 0·2] new surgically eligible basal-cell carcinomas per patient per year vs 30·0 [7·8] new surgically eligible basal-cell carcinomas per patient per year, p<0·0001). Only three (17%) of 18 patients tolerated vismodegib continuously for the full 36 months. Fewer new surgically eligible basal-cell carcinomas developed in patients receiving vismodegib continuously than in those who interrupted dosing (mean 0·6 [0·72] new surgically eligible basal-cell carcinomas per patient per year vs 1·7 [1·8] new surgically eligible basal-cell carcinomas per patient per year, p<0·0001). Treatment-related grade 3-4 adverse events included weight loss of 20% or more (n=6) and muscle cramps (n=2). Two patients died during the course of the trial, one each from laryngeal and metastatic prostate cancer, deemed probably unrelated to drug. INTERPRETATION: Vismodegib reduces basal-cell carcinoma tumour burden in patients with basal-cell nevus syndrome. Adverse events associated with vismodegib frequently led to interruption of treatment, which is followed by basal-cell carcinoma recurrence. FUNDING: Genentech investigator-initiated trial funding, Clinical and Translational Science Award from the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Cancer Institute, Damon Runyon Cancer Research Foundation Clinical Investigator Award, Swim across America Foundation, and Michael J Rainen Family Foundation.

Tazarotene: randomized, double-blind, vehicle-controlled, and open-label concurrent trials for basal cell carcinoma prevention and therapy in patients with basal cell nevus syndrome.

Sporadic human basal cell carcinomas (BCC) are generally well managed with current surgical modalities. However, in the subset of high-risk patients predisposed to developing large numbers of BCCs, there is an unmet need for effective, low-morbidity chemoprevention. This population includes fair-skinned patients with extensive sun exposure and those with genodermatoses such as the basal cell nevus (Gorlin) syndrome (BCNS). Tazarotene (Tazorac, Allergan) is a topical retinoid with relative specificity for RAR-ß and RAR-γ receptors. We previously demonstrated tazarotene's robust anti-BCC efficacy in Ptch1(+/-) mice, a murine equivalent of BCNS, and others have found it to have some efficacy against sporadic human BCCs. We report here results of a randomized, double-blind, vehicle-controlled study in patients with BCNS evaluating the efficacy of topically applied tazarotene for BCC chemoprevention (N = 34 subjects), along with an open-label trial evaluating tazarotene's efficacy for chemotherapy of BCC lesions (N = 36 subjects) for a maximum follow-up period of 3 years. We found that only 6% of patients had a chemopreventive response and that only 6% of treated BCC target lesions were clinically cured. Our studies provide no evidence for either chemopreventive or chemotherapeutic effect of tazarotene against BCCs in patients with BCNS.

Inhibiting the hedgehog pathway in patients with the basal-cell nevus syndrome.

BACKGROUND: Dysregulated hedgehog signaling is the pivotal molecular abnormality underlying basal-cell carcinomas. Vismodegib is a new orally administered hedgehog-pathway inhibitor that produces objective responses in locally advanced and metastatic basal-cell carcinomas. METHODS: We tested the anti-basal-cell carcinoma efficacy of vismodegib in a randomized, double-blind, placebo-controlled trial in patients with the basal-cell nevus syndrome at three clinical centers from September 2009 through January 2011. The primary end point was reduction in the incidence of new basal-cell carcinomas that were eligible for surgical resection (surgically eligible) with vismodegib versus placebo after 3 months; secondary end points included reduction in the size of existing basal-cell carcinomas. RESULTS: In 41 patients followed for a mean of 8 months (range, 1 to 15) after enrollment, the per-patient rate of new surgically eligible basal-cell carcinomas was lower with vismodegib than with placebo (2 vs. 29 cases per group per year, P<0.001), as was the size (percent change from baseline in the sum of the longest diameter) of existing clinically significant basal-cell carcinomas (-65% vs. -11%, P=0.003). In some patients, all basal-cell carcinomas clinically regressed. No tumors progressed during treatment with vismodegib. Patients receiving vismodegib routinely had grade 1 or 2 adverse events of loss of taste, muscle cramps, hair loss, and weight loss. Overall, 54% of patients (14 of 26) receiving vismodegib discontinued drug treatment owing to adverse events. At 1 month, vismodegib use had reduced the hedgehog target-gene expression by basal-cell carcinoma by 90% (P<0.001) and diminished tumor-cell proliferation, but apoptosis was not affected. No residual basal-cell carcinoma was detectable in 83% of biopsy samples taken from sites of clinically regressed basal-cell carcinomas. CONCLUSIONS: Vismodegib reduces the basal-cell carcinoma tumor burden and blocks growth of new basal-cell carcinomas in patients with the basal-cell nevus syndrome. The adverse events associated with treatment led to discontinuation in over half of treated patients. (Funded by Genentech and others; ClinicalTrials.gov number, NCT00957229.).

High prevalence of vitamin D deficiency in patients with basal cell nevus syndrome.

OBJECTIVES: To evaluate vitamin D status in patients with basal cell nevus syndrome (BCNS) who practice photoprotection because of their genetic predisposition to skin cancer and to determine risk factors for deficiency. DESIGN: Retrospective cohort study. SETTING: Academic medical centers. PATIENTS: Forty-one ambulatory patients with BCNS who participated in a 2-year chemoprevention clinical trial. Population-based controls (n = 360) were selected and matched by age, sex, Fitzpatrick skin type, and season/geography. MAIN OUTCOME MEASURES: Levels of 25-hydroxyvitamin D (25[OH]D) and vitamin D deficiency (defined as a 25[OH]D level of ≤20 ng/mL). RESULTS: Twenty-three patients with BCNS (56%) were vitamin D deficient. Patients with BCNS had mean 25(OH)D levels below those of the general population (-3 ng/mL; P = .02) and were 3 times more likely to be vitamin D deficient (56% vs 18%; P < .001). Levels of 25(OH)D were lower in patients who were overweight (-3.0 ng/mL; P = .04) and who had blood collected in the winter compared with the summer (-7.1 ng/mL; P < .001). CONCLUSION: Patients with BCNS may be at increased risk for vitamin D deficiency, depending on their adherence to photoprotection practices.

Basal cell carcinoma chemoprevention with nonsteroidal anti-inflammatory drugs in genetically predisposed PTCH1+/- humans and mice.

In vitro and epidemiologic studies favor the efficacy of nonsteroidal anti-inflammatory drugs (NSAID) in preventing skin squamous photocarcinogenesis, but there has been relatively little study of their efficacy in preventing the more common skin basal cell carcinoma (BCC) carcinogenesis. We first compared the relative anti-BCC effects of genetic deletion and NSAID pharmacologic inhibition of cyclooxygenase (COX) enzymes in the skin of Ptch1(+/-) mice. We then assessed the effects of celecoxib on the development of BCCs in a 3-year, double-blinded, randomized clinical trial in 60 (PTCH1(+/-)) patients with the basal cell nevus syndrome. In Ptch1(+/-) mice, genetic deletion of COX1 or COX2 robustly decreased (75%; P < 0.05) microscopic BCC tumor burden, but pharmacologic inhibition with celecoxib reduced microscopic BCCs less efficaciously (35%; P < 0.05). In the human trial, we detected a trend for oral celecoxib reducing BCC burden in all subjects (P = 0.069). Considering only the 60% of patients with less severe disease (<15 BCCs at study entry), celecoxib significantly reduced BCC number and burden: subjects receiving placebo had a 50% increase in BCC burden per year, whereas subjects in the celecoxib group had a 20% increase (P(difference) = 0.024). Oral celecoxib treatment inhibited BCC carcinogenesis in PTCH1(+/-) mice and had a significant anti-BCC effect in humans with less severe disease.

Long-term establishment, characterization and manipulation of cell lines from mouse basal cell carcinoma tumors.

There have been few reports of successful long-term culture of cells established from cutaneous basal cell carcinoma (BCC) tumors. Here, we describe techniques that have enabled us to establish three long-term cultures of BCC cells isolated from BCC tumors that arose in irradiated Patched 1 (Ptch1)(+/-) mice. All three cell lines showed cellular morphology similar to that of BCC tumors and could be propagated for at least 20 passages. In addition, similar to BCC tumors, all cell lines had lost the wildtype Ptch1 allele, expressed BCC molecular markers, and responded similarly to cyclopamine, a small molecule inhibitor of Hedgehog signaling. Finally, we describe an efficient electroporation technique for DNA transfection into the BCC cell lines and show that they have activated Hedgehog signaling activity, albeit at a level lower than that of murine BCCs in vivo. These data indicate that the cell lines are bona fide long-term cultures of BCC cells and that DNA plasmids can be introduced into the BCC cell lines with relatively high transfection efficiency using a modified electroporation technique.

Topical tazarotene chemoprevention reduces Basal cell carcinoma number and size in Ptch1+/- mice exposed to ultraviolet or ionizing radiation.

Oral retinoids can reduce basal cell carcinoma (BCC) incidence in genetically susceptible patients, and one topical retinoid, tazarotene, has been reported to cure some sporadic BCCs. Therefore, we have tested whether this agent would affect BCCs in Ptch1+/- mice in a controlled chemoprevention trial. We found that topical tazarotene dramatically inhibits the formation of BCCs induced with either UV or ionizing radiation. The ability of tazarotene to inhibit BCC formation in this mouse model provides encouragement for the use of tazarotene in skin cancer chemoprevention trials in humans.

Ornithine decarboxylase is a target for chemoprevention of basal and squamous cell carcinomas in Ptch1+/- mice.

Solar ultraviolet B (UVB) radiation induces cutaneous ornithine decarboxylase (ODC), the first enzyme in the polyamine-biosynthesis pathway, which drives continued proliferation and clonal expansion of initiated (mutated) cells, leading to tumorigenesis. Therefore ODC is a potentially important target for chemoprevention of basal cell carcinomas (BCCs), the majority of which have mutations in the tumor-suppressor gene known as patched (PTCH). To assess this possibility, we first overexpressed ODC in the skin of Ptch1+/- mice using a keratin 6 (K6) promoter that directs constitutive ODC expression in the outer root sheath of the hair follicle. UVB irradiation of these mice accelerated induction of BCCs as compared with their Ptch1+/- littermates. To further verify the role of ODC in BCC tumorigenesis, we used an antizyme (AZ) approach to inhibit ODC activity in the Ptch1+/- mice. Ptch1+/- mice with AZ overexpression driven by the K6 promoter were resistant to the induction of BCCs by UVB. Furthermore, oral administration of the suicidal ODC inhibitor alpha-difluoromethylornithine reduced UVB-induced BCCs in Ptch1+/- mice. These results demonstrate the crucial importance of ODC for the induction of BCCs and indicate that chemopreventive strategies directed at inhibiting this enzyme may be useful in reducing BCCs in human populations.

Evidence for lack of enhanced hedgehog target gene expression in common extracutaneous tumors.

Abnormal hedgehog signaling, most commonly caused by loss of PTCH1 inhibitor activity,drives tumorigenesis of basal cell carcinomas (BCCs). To assess whether other tumors also have abnormal hedgehog signaling, we have assayed RNA from common cancers at nine different sites for levels of expression of hedgehog target genes that are up-regulated uniformly in BCCs. We report here that such dysregulation appears not to be common in the types of non-BCC cancers studied, indicating that the molecular pathogenesis of BCCs, like their frequency and behavior, differs markedly from that of most other cancers.

CUSP/p63 expression in basal cell carcinoma.

Chronic ulcerative stomatitis protein (CUSP), the most abundant cutaneous isoform of p63, is a p53-related gene essential for epithelial development. CUSP lacks the N-terminal transactivation domain found on other p53 family members and has been shown to inhibit p53 function in vitro. In this study, biopsies of normal skin (21 of 21), benign neoplasms [seborrheic keratosis (3 of 3), acrochordon (2 of 3), and verruca plana (3 of 3)], and squamous cell carcinomas (SCC) (4 of 4) displayed strong nuclear CUSP immuno-reactivity in epidermal cells. In contrast few basal cell carcinomas (BCC) (7 of 27) and sebaceous nevi (1 of 2) displayed this pattern of CUSP immunoreactivity. Thus, biopsies of cutaneous conditions characterized by sonic hedgehog (SHH) pathway dysregulation were more than 86 times as likely to lack CUSP/p63 immunofluorescence as were other cutaneous samples. Adjacent normal-appearing skin from patients with basal cell nevus syndrome (BCNS) (2 of 3) also lacked CUSP immuno-staining. Lastly, a BCC arising in a patched heterozygous mouse also lacked CUSP immuno-staining. Because CUSP mRNA and protein were detected via Northern and Western analysis in BCC samples lacking CUSP immuno-staining, we sequenced the coding region of CUSP from two non-staining BCCs but found no mutations. Therefore, CUSP appears to be present, unmutated, and yet frequently undetectable by immunofluorescence in cutaneous lesions in both humans and mice that are associated with SHH pathway dysregulation (BCCs, BCNS, and nevus sebaceous).

Cyclooxygenase-2 expression in murine and human nonmelanoma skin cancers: implications for therapeutic approaches.

Inflammatory stimuli result in the production of cutaneous eicosanoids, which are known to contribute to the process of tumor promotion. Cyclooxygenase (COX), the rate-limiting enzyme for the production of prostaglandins (PG) from arachidonic acid, exists in at least two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and plays various physiological roles, whereas increased COX-2 expression is known to occur in several types of epithelial neoplasms. Enhanced PG synthesis is a potential contributing factor in UVB-induced nonmelanoma skin cancers (NMSC). Increased COX-2 staining occurs in murine skin neoplasms after chronic exposure to carcinogenic doses of UVB. In this study, immunohistochemical and Western blot analyses were employed to assess longitudinally COX-2 expression in a standard mouse UVB complete carcinogenesis protocol and in human basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). During UVB irradiation of mice, COX-2 expression consistently increased in the hyperplastic skin, the benign papillomas and the SCC. COX-2 expression was also increased in human actinic keratoses, SCC and BCC as well as in murine SCC and BCC. The pattern of COX-2 expression was quite variable, occurring in a patchy distribution in some lesions with staining confined mainly to suprabasal cell layers. In general, COX-2 expression progressively became more extensive in benign papillomas and well-differentiated murine SCC. The staining was predominantly cytoplasmic and perinuclear in some focal areas in tissue stroma around both murine and human tumors. Western blot analysis confirmed negative COX-2 expression in normal skin, whereas acute UVB exposure resulted in increased enzyme expression, which continued to increase in developing papillomas and SCC. Because of the evidence indicating a pathogenic role for eicosanoids in murine and human skin neoplasms, we performed studies to assess the anti-inflammatory and anticarcinogenic effects of green tea extracts, which are potent antioxidants. Acute exposure of the human skin to UVB (minimum erythema dose x 4) caused a transient enhancement of the COX-2 expression, which reverted to baseline within hours; however, in murine skin the expression persisted for several days. Pretreatment with the topically applied green tea extract (1 mg/cm2) largely abrogated the acute COX-2 response to UVB in mice or humans. In summary, enhanced COX-2 expression serves as a marker of epidermal UVB exposure for murine and human NMSC. These results suggest that COX-2 inhibitors could have potent anticarcinogenic effects in UVB-induced skin cancer.