Chemoprevention agents for melanoma: A path forward into phase 3 clinical trials.

Recent progress in the treatment of advanced melanoma has led to unprecedented improvements in overall survival and, as these new melanoma treatments have been developed and deployed in the clinic, much has been learned about the natural history of the disease. Now is the time to apply that knowledge toward the design and clinical evaluation of new chemoprevention agents. Melanoma chemoprevention has the potential to reduce dramatically both the morbidity and the high costs associated with treating patients who have metastatic disease. In this work, scientific and clinical melanoma experts from the national Melanoma Prevention Working Group, composed of National Cancer Trials Network investigators, discuss research aimed at discovering and developing (or repurposing) drugs and natural products for the prevention of melanoma and propose an updated pipeline for translating the most promising agents into the clinic. The mechanism of action, preclinical data, epidemiological evidence, and results from available clinical trials are discussed for each class of compounds. Selected keratinocyte carcinoma chemoprevention studies also are considered, and a rationale for their inclusion is presented. These data are summarized in a table that lists the type and level of evidence available for each class of agents. Also included in the discussion is an assessment of additional research necessary and the likelihood that a given compound may be a suitable candidate for a phase 3 clinical trial within the next 5 years.

A clinical instrument for combined raman spectroscopy-optical coherence tomography of skin cancers.

BACKGROUND AND OBJECTIVE: The current standard for diagnosis of skin cancers is visual inspection followed by biopsy and histopathology. This process can be invasive, subjective, time consuming, and costly. Optical techniques, including Optical Coherence Tomography (OCT) and Raman Spectroscopy (RS), have been developed to perform non-invasive characterization of skin lesions based on either morphological or biochemical features of disease. The objective of this work is to report a clinical instrument capable of both morphological and biochemical characterization of skin cancers with RS-OCT. MATERIALS AND METHODS: The portable instrument utilizes independent 785 nm RS and 1,310 nm OCT system backbones. The two modalities are integrated in a 4″ (H) × 5″(W) × 8″(L) clinical probe. The probe enables sequential acquisition of co-registered OCT and RS data sets. The axial response of the RS collection in the skin was estimated using scattering phantoms. In addition, RS-OCT data from patients with cancerous and non-cancerous lesions are reported. RESULTS: The RS-OCT instrument is capable of screening areas as large as 15 mm (transverse) by 2.4 mm (in depth) at up to 8 frames/second with OCT, and identifying locations to perform RS. RS signal is collected from a 44 µm transverse spot through a depth of approximately 530 µm. RS-OCT data sets from a superficial scar and a nodular BCC are reported to demonstrate the clinical potential of the instrument. CONCLUSION: The RS-OCT instrument reported here is capable of morphological and biochemical characterization of cancerous skin lesions in a clinical setting. OCT can visualize microstructural irregularities and perform an initial morphological analysis of the lesion. The images can be used to guide acquisition of biochemically specific Raman spectra. The two data sets can then be evaluated with respect to one another to take advantage of the mutually complimentary nature of RS and OCT.

Innovation interest within dermatology: a needs assessment for novel thought processes.

Medical innovation is crucial to advancing our field, and physicians have the potential to play a leading role due to their daily patient care experiences. The objective of this study was to evaluate the interest in, and barriers to participating in innovation. Two surveys were conducted; the first cross-sectional survey was conducted among attendees of the Advancing Innovation in Dermatology Forum in Feburary 2019. The second survey was conducted among trainees (resident/fellows) and faculty dermatologists at Brown, Emory, Iowa, Stanford, and Vanderbilt Universities between June and November 2019. Demographic data were collected, as well as factors involved with identifying problems, developing solutions, training in innovation, and perceived barriers to innovation. In the first survey, the greatest perceived benefits include bringing joy to one's work and increasing professional fulfillment with work. Innovation was also perceived to decrease burnout. In the second survey of academic centers, faculty more commonly expressed interest in identifying problems (p = 0.04), and was also more confident in their ability to generate solutions to these problems as compared to trainees (p < 0.01). Major barriers to participating in innovation processes included lack of time and lack of training or education in innovation. Both trainees and faculty groups noted a lack of knowledge in creating prototypes, understanding regulatory approval for medical products, and inexperience with pitching to investors or obtaining funding. These cross-sectional needs assessment surveys found a strong interest in innovation coupled with a lack of education in innovation processes. These findings suggest an urgent need and opportunity for providing formal training to empower dermatologists with the tools to lead innovation within our field.

Detecting temporal and spatial effects of epithelial cancers with Raman spectroscopy.

Epithelial cancers, including those of the skin and cervix, are the most common type of cancers in humans. Many recent studies have attempted to use Raman spectroscopy to diagnose these cancers. In this paper, Raman spectral markers related to the temporal and spatial effects of cervical and skin cancers are examined through four separate but related studies. Results from a clinical cervix study show that previous disease has a significant effect on the Raman signatures of the cervix, which allow for near 100% classification for discriminating previous disease versus a true normal. A Raman microspectroscopy study showed that Raman can detect changes due to adjacent regions of dysplasia or HPV that cannot be detected histologically, while a clinical skin study showed that Raman spectra may be detecting malignancy associated changes in tissues surrounding nonmelanoma skin cancers. Finally, results of an organotypic raft culture study provided support for both the skin and the in vitro cervix results. These studies add to the growing body of evidence that optical spectroscopy, in this case Raman spectral markers, can be used to detect subtle temporal and spatial effects in tissue near cancerous sites that go otherwise undetected by conventional histology.

Estrogen receptor beta expression in nevi during pregnancy.

Estrogen levels increase during pregnancy and clinical evidence has long suggested that melanocytes are estrogen-responsive. We hypothesized that nevi from pregnant patients would exhibit increased expression of estrogen receptor beta (ERbeta) and thus enhanced potential to respond to altered estrogen levels. Normal, dysplastic and congenital nevi (n = 212) were collected from pregnant and non-pregnant women ranging from 18 to 45 years of age. Immunohistochemical staining was performed on these nevi using antibodies specifically directed against estrogen receptor alpha (ERalpha) and ERbeta. ERalpha was not observed in any lesions; thus, ERbeta was the predominant estrogen receptor in melanocytic cells from all types of nevi. Enhanced positivity for ERbeta in normal nevi during pregnancy was noted, compared with non-pregnant controls including nevocytes residing in both the epidermal and dermal micro-environments (P = 0.005 and P = 0.001 respectively). Nevi with increasingly melanocytic atypia showed increased ERbeta in nevocytes nested within the epidermis. No additional increase in ERbeta in atypical nevi was observed during pregnancy. For normal and congenital nevi, regardless of pregnancy status, dermally associated nevocytes tended to have greater ERbeta immunoreactivity. Significant decreases in ERbeta immunoreactivity were observed in congenital nevi from pregnant women compared with normal and dysplastic nevi from pregnant women. Our data suggest that nevi possess the capacity to be estrogen-responsive. Factors such as pregnancy and degree of atypia are associated with enhanced ERbeta with the exception of congenital nevi where the melanocytes were unique in their response to pregnancy.

Raman microspectroscopy for skin cancer detection in vitro.

We investigate the potential of near-infrared Raman microspectroscopy to differentiate between normal and malignant skin lesions. Thirty-nine skin tissue samples consisting of normal, basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and melanoma from 39 patients were investigated. Raman spectra were recorded at the surface and at 20-microm intervals below the surface for each sample, down to a depth of at least 100 microm. Data reduction algorithms based on the nonlinear maximum representation and discrimination feature (MRDF) and discriminant algorithms using sparse multinomial logistic regression (SMLR) were developed for classification of the Raman spectra relative to histopathology. The tissue Raman spectra were classified into pathological states with a maximal overall sensitivity and specificity for disease of 100%. These results indicate the potential of using Raman microspectroscopy for skin cancer detection and provide a clear rationale for future clinical studies.

In vivo nonmelanoma skin cancer diagnosis using Raman microspectroscopy.

BACKGROUND AND OBJECTIVES: Nonmelanoma skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are the most common skin cancers, presenting nearly as many cases as all other cancers combined. The current gold-standard for clinical diagnosis of these lesions is histopathologic examination, an invasive, time-consuming procedure. There is thus considerable interest in developing a real-time, automated, noninvasive tool for nonmelanoma skin cancer diagnosis. In this study, we explored the capability of Raman microspectroscopy to provide differential diagnosis of BCC, SCC, inflamed scar tissue, and normal tissue in vivo. STUDY DESIGN: Based on the results of previous in vitro studies, we developed a portable confocal Raman system with a handheld probe for clinical study. Using this portable system, we measured Raman spectra of 21 suspected nonmelanoma skin cancers in 19 patients with matched normal skin spectra. These spectra were input into nonlinear diagnostic algorithms to predict pathological designation. RESULTS: All of the BCC (9/9), SCC (4/4), and inflamed scar tissues (8/8) were correctly predicted by the diagnostic algorithm, and 19 out of 21 normal tissues were correctly classified. This translates into a 100% (21/21) sensitivity and 91% (19/21) specificity for abnormality, with a 95% (40/42) overall classification accuracy. CONCLUSIONS: These findings reveal Raman microspectroscopy to be a viable tool for real-time diagnosis and guidance of nonmelanoma skin cancer resection.

Skin cancer screening: recommendations for data-driven screening guidelines and a review of the US Preventive Services Task Force controversy.

Melanoma is usually apparent on the skin and readily detected by trained medical providers using a routine total body skin examination, yet this malignancy is responsible for the majority of skin cancer-related deaths. Currently, there is no national consensus on skin cancer screening in the USA, but dermatologists and primary care providers are routinely confronted with making the decision about when to recommend total body skin examinations and at what interval. The objectives of this paper are: to propose rational, risk-based, data-driven guidelines commensurate with the US Preventive Services Task Force screening guidelines for other disorders; to compare our proposed guidelines to recommendations made by other national and international organizations; and to review the US Preventive Services Task Force's 2016 Draft Recommendation Statement on skin cancer screening.

Constitutive activation of Akt/protein kinase B in melanoma leads to up-regulation of nuclear factor-kappaB and tumor progression.

The serine/threonine kinase Akt/protein kinase B and the pleiotropic transcription factor nuclear factor-kappaB [NF-kappaB (p50/p65)] play important roles in the control of cell proliferation, apoptosis, and oncogenesis. Previous studies from our laboratory have shown the constitutive activation of NF-kappaB in melanoma cells. However, the mechanism of this activation is not clearly understood. The purpose of this study was to explore the role of Akt in the activation of NF-kappaB during melanoma tumor progression. Based on our observation that two of the five melanoma cell lines examined exhibit constitutive Akt activation, we evaluated Akt activation by immunohistochemistry in a series of pigmented skin lesions using an antibody specific for phospho-Akt Ser-473. Normal and slightly dysplastic nevi exhibited no significant Akt expression, in marked contrast to the dramatic Akt immunoreactivity seen in severely dysplastic nevi and melanomas (66.3% positive). When these same lesions were stained for nuclear p65, a similar expression pattern was observed. In addition, interruption of Akt activation resulted in increased apoptosis and decreased NF-kappaB promoter activity. These results indicate that activation of Akt kinase is linked to enhanced NF-kappaB nuclear localization and transactivation. We propose that activation of Akt may be an early marker for tumor progression in melanoma.

The effect of free-electron laser pulse structure on mid-infrared soft-tissue ablation: biological effects.

Previous studies have shown that changing the pulse structure of the free electron laser (FEL) from 1 to 200 ps and thus reducing the peak irradiance of the micropulse by 200 times had little or no effect on both the ablation threshold radiant exposure and the ablated crater depth for a defined radiant exposure. This study focuses on the ablation mechanism at 6.1 and 6.45 microm with an emphasis on the role of the FEL pulse structure. Three different experiments were performed to gain insight into this mechanism. The first was an analysis of the ablation plume dynamics observed for a 1 ps micropulse compared with a 200 ps micropulse as seen through bright-field analysis. Negligible differences are seen in the size, but not the dynamics of ablation, as a result of this imaging. The second experiment was a histological analysis of corneal and dermal tissue to determine whether there is less thermal damage associated with one micropulse duration versus another. No significant difference was seen in the extent of thermal damage on either canine cornea or mouse dermis for the micropulse durations studied at either wavelength. The final set of experiments involved the use of mass spectrometry to determine whether amide bond breakage could occur in the proteins present in tissue as a result of direct absorptions of mid-infrared light into the amide I and amide II absorption bands. This analysis showed that there was no amide bond breakage due to irradiation at 6.45 microm on protein.

Wound healing of 6.45-microm free electron laser skin incisions with heat-conducting templates.

We have previously shown a reduction in lateral thermal damage with acute studies of skin incisions made in vitro using heat-conducting templates. Here we examined the wound-healing response to laser incisions with heat-conducting templates and explored the use of an optically transparent template with the free electron laser (FEL) at 6.45 microm. First we evaluated the effects of a sapphire heat-conducting template on the lateral thermal damage of FEL incisions using in vitro human skin samples. Next we compared wound tensile strength and histological scoring of the healing of incisions created on the dorsal pelts of live rats with the FEL utilizing metal and sapphire heat-conducting templates and scalpel incisions. The animals were euthanized and the wounds were analyzed at postoperative days 7, 14, and 21. The depth and lateral thermal damage of FEL incisions on in vitro human skin were significantly reduced with the sapphire heat-conducting template. Nonstatistically significant differences in wound tensile strengths and histological scoring of wound healing were noted at days 7 and 14. By day 21, all of the incisions appeared similar. When the data from days 7 and 14 were combined, statistically significant differences were found for each of the templates (except the histological evaluation with the aluminum template) and the scalpel compared with laser incisions made without using a template. The use of metal or sapphire heat-conducting templates reduced the wound-healing delay of laser incisions seen at postoperative days 7 and 14.

Pregnancy and estrogen receptor beta expression in a large congenital nevus.

BACKGROUND: Large congenital nevi carry a slightly increased risk of melanoma. Pregnancy poses an additional challenge in the monitoring of these patients because little is known regarding the effects of increased estrogen levels on congenital nevi. OBSERVATIONS: A young woman was observed to have clinical lightening of her garment nevus and satellite nevi during 2 sequential pregnancies. Postpartum, the patient experienced darkening and repigmentation in her large garment nevus, with continued lightening of nearby satellite lesions. In addition to photographic documentation of these changes, biopsy samples taken during pregnant and nonpregnant periods underwent immunohistochemical evaluation for estrogen receptor beta (ERbeta), the predominant estrogen receptor in nevi and melanomas. Biopsy samples collected during pregnancy showed a decrease in nuclear staining for ERbeta compared with samples collected after pregnancy. These changes in ERbeta expression were not associated with histologic atypia during pregnancy or after delivery. CONCLUSIONS: Congenital nevi may be unique in their response to altered estrogen levels. Given the slightly increased risk of melanoma in giant congenital nevi and the dearth of information available regarding the effects of pregnancy on congenital nevi, this case illustrates the need for further study of these pigmented lesions.

The lymphotoxin-beta receptor is an upstream activator of NF-kappaB-mediated transcription in melanoma cells.

The pleiotropic transcription factor nuclear factor-kappaB (NF-kappaB (p50/p65)) regulates the transcription of genes involved in the modulation of cell proliferation, apoptosis, and oncogenesis. Furthermore, a host of solid and hematopoietic tumor types exhibit constitutive activation of NF-kappaB (Basseres, D. S., and Baldwin, A. S. (2006) 25, 6817-6830). However, the mechanism for this constitutive activation of NF-kappaB has not been elucidated in the tumors. We have previously shown that NF-kappaB-inducing kinase (NIK) protein and its association with Inhibitor of kappaB kinase alphabeta are elevated in melanoma cells compared with their normal counterpart, leading to constitutive activation of NF-kappaB. Moreover, expression of dominant negative NIK blocked this base-line NF-kappaB activity in melanoma cells. Of the three receptors that require NIK for activation of NF-kappaB, only the lymphotoxin-beta receptor (LTbeta-R) is expressed in melanoma. We show in this manuscript that for melanoma there is a strong relationship between expression of the LTbeta-R and constitutive NF-kappaB transcriptional activity. Moreover, we show that activation of the LTbeta-R can drive NF-kappaB activity to regulate gene expression that leads to enhanced cell growth. The inhibition by LTbeta-R shRNA resulted in decreased NF-kappaB promoter activity, decreased growth, and decreased invasiveness as compared with control. These results indicate that the LTbeta-R constitutively induces NF-kappaB activation, and this event may be associated with autonomous growth of melanoma cells.

Expression of activated Akt in benign nevi, Spitz nevi and melanomas.

BACKGROUND: Activated Akt expression (p-Akt) is reportedly increased in many melanomas as compared with benign nevi. The purpose of this study was to evaluate and compare p-Akt immunohistological staining in benign nevi, Spitz nevi and primary melanomas. METHODS: Immunostaining for phosphorylated Akt was performed in 41 melanocytic lesions previously classified as benign intradermal nevus (14 lesions), Spitz nevus (9 lesions) or melanoma (18 lesions). Lesions were graded for intensity of p-Akt staining by two independent observers (0, no staining; 1, slightly positive; 2, moderately positive; 3, highly positive). Scores were averaged, and statistical analyses were performed. RESULTS: Benign nevi showed less staining (mean score 1.18) compared with Spitz nevi (mean score 2.11) and melanomas (mean score 2.19). This difference was statistically significant between benign nevi and melanomas (p = 0.0047) and benign nevi and Spitz nevi (p = 0.0271). No statistical difference was detected in staining between Spitz nevi and melanomas (p = 0.8309). CONCLUSIONS: Activated Akt expression is increased in Spitz nevi and melanomas as compared with benign intradermal nevi, but is unlikely to prove useful in differentiating between the former.

Controlling thermal damage of incisions using diamond, copper, and sapphire heat-conducting templates with and without cooling.

INTRODUCTION: We investigated the reduction of thermal damage to the surrounding tissue when laser incisions were made with and without using thermal conducting templates at room temperature and cooled to 5 degrees C. STUDY DESIGN/MATERIALS AND METHODS: We used the Vanderbilt free-electron laser (FEL) at 5.4, 6.1, 6.45, and 7.7 microns. We also used a conventional continuous wave (CW) carbon dioxide laser at 10.6 microns. Incisions were made on 5x10 mm pieces of human breast skin (in vitro) and analyzed with histology. Computer morphometrics were used to measure the amount of thermal damage. RESULTS: All templates produced a statistically significant reduction in the thermal damage. Additionally, we showed that cooling the templates made a statistically significant greater reduction in the thermal damage. The cooled diamond template reduced the thermal damage from the FEL to 28% of the damage observed without a template. The same cooled template reduced the thermal damage from the CO(2) laser to 56% of the damage observed without a template. Lesser reductions were observed with the copper template and even less with the sapphire template. The sapphire template reduced the thermal damage to 39 and 67% of the damage observed without a template for the FEL and the CO(2) laser, respectively. CONCLUSION: These results indicate that unwanted lateral thermal damage from laser incisions can be reduced with cooled thermally conductive templates with the best results obtained with the diamond template, which is also the best thermal conductor.

Oestrogen receptor-beta expression in melanocytic lesions.

Melanomas rarely occur before puberty, have a higher death rate for males, and tend to be more invasive during pregnancy. Prior to the discovery of a second oestrogen receptor (ERbeta), studies with the initial oestrogen receptor, ERalpha, showed no obvious role for oestrogen in the pathophysiology of benign or malignant melanocytic lesions. To investigate the specific immunostaining patterns of ERalpha and ERbeta, benign nevocytic nevi, dysplastic nevi with mild, moderate and severe cytological atypia, lentigo malignas and melanomas of varying depth (Clark) and thickness (Breslow) were studied. ERbeta but not ERalpha was the predominant oestrogen receptor we found in all types of benign and malignant melanocytic lesions. The most intense ERbeta immunostaining was seen in melanocytes in dysplastic nevi with severe cytological atypia and in lentigo malignas. ERbeta expression levels also correlated with the malignant tumor microenvironment; i.e., melanocytes in proximity with keratinocytes>deeper dermal melanocytes in contact with stroma>minimally invasive melanomas>Clark Level III/IV or thick melanomas (Breslow). Discovery that ERbeta expression varies in relation to the tumor microenvironment and increasing depth of invasion suggests its possible usefulness as a surrogate marker for neoplasia and prognosis in malignant melanoma.

Free-electron laser and heat-conducting templates: a study of reducing cutaneous lateral thermal damage.

BACKGROUND AND OBJECTIVES: We developed novel heat-conducting templates, and tested whether they could effectively remove damaging heat from the tissue during laser ablation. The reduction of lateral thermal damage during cutaneous incisional laser procedures should decrease the time in wound healing. In addition, we selected various infrared wavelengths to determine whether the template effects would be influenced by the laser penetration depth and the particular chromophore absorbing the laser light. STUDY DESIGN/MATERIALS AND METHODS: This study utilized the Free-Electron Laser at wavelengths of 3.0, 5.5, 6.45, 7.5, and 7.7 microm to produce 1.0 cm incisions on in vitro lightly pigmented human skin. At each of these wavelengths, copper, aluminum, glass, and Plexiglas heat conducting templates were tested. At wavelength 5.5 microm, the study was duplicated using in vitro darkly pigmented skin. Histological samples were evaluated using computerized morphometric analysis. RESULTS: The adjunct use of both the copper and aluminum templates provided a decrease in thermal damage at each wavelength. Using the copper template reduced lateral thermal damage an average of 67% with no apparent wavelength dependence. The aluminum template reduced thermal damage an average of 54% with no apparent wavelength dependence. The glass and Plexiglas templates did not reduce the lateral thermal damage. At 5.5 microm, no statistically significant difference in lateral thermal damage was observed between darkly and lightly pigmented tissues. CONCLUSIONS: Heat-conducting templates are an effective new method to reduce lateral thermal damage from thermal laser incisions.

Reduction in lateral thermal damage using heat-conducting templates: a comparison of continuous wave and pulsed CO2 lasers.

BACKGROUND AND OBJECTIVES: The advantages of the continuous wave (c.w.) CO(2) laser are offset by the delay in laser wound healing secondary to thermal damage. We have developed novel heat-conducting templates to reduce laser thermal damage. Because shortened pulse durations also decrease thermal damage, we tested the effectiveness of heat-conducting templates with a c.w. CO(2) clinical laser and a short-pulsed CO(2) laser to determine the best method and mechanism to minimize thermal damage. STUDY DESIGN/MATERIALS AND METHODS: Comparison of 0.2-second shuttered c.w. and 5-microsecond pulsed CO(2) lasers were made by doing incisions on 150 tissue samples from reduction mammoplasties and abdominoplasties. Copper, aluminum, glass, and Plexiglass heat-conducting templates were tested against no template (air) with both lasers. Histological samples were evaluated using computerized morphometrics analysis. RESULTS: Statistically significant reductions in lateral thermal damage were seen with the copper (50%) and aluminum (39%) templates used with the c.w. CO(2) laser. Only the copper template (39%) significantly reduced thermal damage when used with the pulsed CO(2) laser. Less thermal damage was seen using the pulsed CO(2) laser compared to the c.w. CO(2) laser with each template. CONCLUSIONS: Heat-conducting templates significantly reduced the amount of lateral thermal damage when used with the c.w. CO(2) laser (copper and aluminum) and short-pulsed CO(2) laser (copper). The c.w. CO(2) laser with the copper template compared favorably to the short-pulsed CO(2) laser without a template. Therefore, both heat conductive templates and short-pulse structure provide successful methods for reducing lateral thermal damage, and a combination of the two appears to provide optimal results.