Histopathologic diagnosis of thin, invasive cutaneous melanoma (CM) is only 34-62% accurate. Therefore, we sought to develop a transcriptomic biomarker to distinguish benign from malignant melanocytic neoplasms. We generated a targeted RNA-Sequencing dataset (TempO-Seq) of benign nevi (BN; n = 50) and CM (Breslow depth ≤ 1.0 mm; n = 51) and demonstrated enrichment of immune-related pathways among the 450 differentially expressed genes. Next, we trained a putative transcriptomic biomarker in two datasets, including BN and CM, and one dataset with CM in association with a nevus, macrodissected into CM and nevus regions. We refer to the nevus portion of CM in association with a nevus as progressing nevi (PN), since these nevi progressed to CM. Principal component analysis showed that PN samples clustered in a component intermediate to BN and CM. Ordinal regularized regression selected PYGL, AP000845.1, PHYHIP, WSCD1, FBXO7, TRPM1, SLC4A4, NALCN, FRMD4B, HHATL, COL1A1, CRYM, EPOP, RGS1, KRT6C, IGHG1, CNTN1, MMP11, GZMM, AP001880.1, TTYH3, TMEM132A, and PRAME; these genes were consistently selected in 1000 models using data from bootstrap resamples and had a single model predictive accuracy of at least 0.90 (area under the receiver operator characteristics curve). Linear regression models fit with these 23 genes in the TempO-Seq data, and publicly available microarray datasets from BN, dysplastic nevi, and CM, showed high consistency in the magnitude and directionality of gene expression differences between nevi and CM. Furthermore, immunohistochemical staining showed consistent protein-level changes in MMP11 and PYGL. These results illuminate the potential for a transcriptomic biomarker to differentiate benign from malignant melanocytic neoplasms and improve the accuracy of melanoma diagnosis.

Perspective on this Article from A Phase 2a Study of Topical Perillyl Alcohol Cream for Chemoprevention of Skin Cancer

Supplementary Figure S1 from Loss of Inositol Polyphosphate 5-Phosphatase Is an Early Event in Development of Cutaneous Squamous Cell Carcinoma

A 68-year-old man with rheumatoid arthritis presented for laser treatment of facial pigmentation (Fig 1, A). He had taken oral minocycline 100 mg 3 times weekly for 8 years (cumulative dose, 124.8 g) because of severe arthritic discomfort. Physical examination showed patchy slate blue/grey hyperpigmentation of the face, gums, teeth, and nail beds, consistent with type III minocycline-induced pigmentation (MP).1Mouton R.W. Jordaan H.F. Schneider J.W. A new type of minocycline-induced cutaneous hyperpigmentation.Clin Exp Dermatol. 2004; 29: 8-14Crossref PubMed Scopus (69) Google Scholar There was no pigmentation of sun-protected areas. Two different laser modalities were tested before treatment: Q-switched 755 nm (Qs755nm) alexandrite (3.4 J/cm2 with 4-mm spot size, 4.8 J/cm2 with 3-mm spot size) and Q-switched 1064 nm (Qs1064nm) neodymium:YAG (yttrium aluminum garnet) (4.8 J/cm2 with 3-mm spot size, 3 J/cm2 with 4-mm spot size). Qs755nm at 2.0 Hz/4-mm spot/3.4 J/cm2 showed the most improvement, with immediate pigment clearing, and was used for full facial treatment (3450 pulses). This setting was also selected because it had the largest spot size with lowest fluence, thus limiting the number of pulses and treatment time required. As observed with the test treatment, pigment resolution occurred immediately on the same day after therapy. There were no significant postoperative complications. Pigment resolution was maintained at 13 months despite continued minocycline use, and the patient was highly satisfied (Fig 1, B). The patient continued taking minocycline 100 mg 3 times weekly and re-presented 3 times over the next 13 years with facial hyperpigmentation (total cumulative dose of minocycline, 202.8 g). Each time, immediate pigment clearing was achieved with the Qs755nm alexandrite laser. Every visit concluded with a discussion that hyperpigmentation would recur with continued minocycline ingestion. At the time of this article's writing, he continues to take minocycline. The pretreatment biopsy showed intracellular and extracellular type III MP in the dermis1Mouton R.W. Jordaan H.F. Schneider J.W. A new type of minocycline-induced cutaneous hyperpigmentation.Clin Exp Dermatol. 2004; 29: 8-14Crossref PubMed Scopus (69) Google Scholar (Fig 2, A), with notably no detectable change in the posttreatment biopsy (Fig 2, B). However, in contrast to the pretreatment biopsy, the extracellular pigment in the posttreatment sample was no longer highlighted by Fontana-Masson (FM) stain, but intracellular pigment continued to stain with FM (Fig 2, C and D). The change in melanin staining is consistent with the clinically observed reduction in pigment intensity. Although minocycline is generally safe, cutaneous pigmentation is a common adverse effect.2La Placa M. Infusino S.D. Balestri R. Vincenzi C. Minocycline-induced blue-gray discoloration.Skin Appendage Disord. 2017; 3: 161-162Google Scholar Type III is the least common MP, involving sun-exposed areas and staining positively only for melanin. It is often unremitting or requires multiple treatments to achieve improvement.2La Placa M. Infusino S.D. Balestri R. Vincenzi C. Minocycline-induced blue-gray discoloration.Skin Appendage Disord. 2017; 3: 161-162Google Scholar Although we are not the first to document successful Qs laser treatment of MP,3Alster T.S. Gupta S.N. Minocycline-induced hyperpigmentation treated with a 755-nm Q-switched alexandrite laser.Dermatol Surg. 2004; 30: 1201-1204Crossref PubMed Scopus (31) Google Scholar, 4Green D. Friedman K.J. Treatment of minocycline-induced cutaneous pigmentation with the Q-switched alexandrite laser and a review of the literature.J Am Acad Dermatol. 2001; 44: 342-347Scopus (58) Google Scholar, 5Nisar M.S. Iyer K. Brodell R.T. Lloyd J.R. Shin T.M. Ahmad A. Minocycline-induced hyperpigmentation: comparison of 3 Q-switched lasers to reverse its effects.Clin Cosmet Investig Dermatol. 2013; 31: 159-162Google Scholar our case is notable for the rapidity of pigment resolution repeated on multiple occasions over 13 years. In contrast to previous cases requiring multiple treatments,3Alster T.S. Gupta S.N. Minocycline-induced hyperpigmentation treated with a 755-nm Q-switched alexandrite laser.Dermatol Surg. 2004; 30: 1201-1204Crossref PubMed Scopus (31) Google Scholar, 4Green D. Friedman K.J. Treatment of minocycline-induced cutaneous pigmentation with the Q-switched alexandrite laser and a review of the literature.J Am Acad Dermatol. 2001; 44: 342-347Scopus (58) Google Scholar our patient experienced immediate and sustained cosmetic improvement after only 1 treatment. Although successful treatment of minocycline-related pigmentation has also been reported with newer picosecond lasers,6Sasaki K. Ohshiro T. Ohshiro T. et al.Type 2 minocycline-induced hyperpigmentation successfully treated with the novel 755nm picosecond alexandrite laser – a case report.Laser Ther. 2017; 26: 137-144Google Scholar, 7Rodrigues M. Bekhor P. Treatment of minocycline-induced cutaneous pigmentation with the picosecond alexandrite (755-nm) laser.Dermatol Surg. 2015; : 1179-1182Google Scholar these devices are more expensive and less accessible than the Qs alexandrite laser. Other reports have also described the treatment of minocycline-associated pigmentation with combination laser modalities.8Vangipuram R.K. DeLozier W.L. Geddes E. Friedman P.M. Complete resolution of minocycline pigmentation following a single treatment wit non-ablative 1550-nm fractional resurfacing in combination with the 755-nm Q-switched alexandrite laser.Lasers Surg Med. 2016; 48: 234-237Google Scholar However, this is less convenient and more costly than using a single device such as the Qs alexandrite, as shown in our case. Because our patient continued to take minocycline, he did experience re-pigmentation; however, each recurrence resolved immediately with 1 treatment using the Qs755nm alexandrite laser, showing its effectiveness in achieving MP clearing. The findings of our case are reminiscent of immediate pigment darkening resulting from ferric oxide (red-brown) reduction to ferrous oxide (black).9Fitzpatrick R.E. Lupton J.R. Successful treatment of treatment-resistant laser-induced pigment darkening of a cosmetic tattoo.Lasers Surg Med. 2000; 27: 358-361Crossref PubMed Scopus (55) Google Scholar We term our outcome immediate pigment lightening, which may be another example of Q-switched laser alteration of dermal pigmentation through a physicochemical mechanism. The pigment in our case may be an insoluble minocycline-cation coordination complex, with calcium or magnesium as central metal ions. Because tetracyclines are susceptible to heat and photodecomposition,10Hassani M. Lazaro R. Perez C. Condon S. Pagan R. Thermostability of oxytetracycline, tetracycline, and doxycycline at ultrahigh temperatures.J Agric Food Chem. 2008; 56: 2676-2680Crossref PubMed Scopus (63) Google Scholar a physicochemical process that disrupts these complexes could explain our findings. Specifically, the discrepancy between H&E and FM stains suggests that extracellular pigment is still present after treatment but in an altered chemical state, such that it can no longer absorb visible light. To our knowledge, this is the first case to show immediate pigment lightening of type III MP repeated over multiple instances using only the Qs755nm alexandrite laser. We propose that an insoluble minocycline-cation coordination complex with selective sensitivity to the Qs755nm alexandrite laser may be deposited within the dermis. Our case shows that the Qs755nm alexandrite laser may be a preferable treatment modality for type III MP, especially because it is a single device and may be more convenient and cost effective compared with other laser modalities or combination laser treatments. Further studies investigating the mechanisms of MP deposition and resolution are warranted to clarify our findings.

Reduced melanoma risk has been reported with regular use of nonsteroidal anti‐inflammatory drugs (NSAIDs). However, the ability of NSAIDs to reach melanocytes in vivo and modulate key biomarkers in preneoplastic lesions such as atypical nevi has not been evaluated.

Abstract Reverse phase protein microarray analysis was used to identify cell signaling derangements in squamous cell carcinoma (SCC) compared with actinic keratosis (AK) and upper inner arm (UIA). We analyzed two independent tissue sets with isolation and enrichment of epithelial cells by laser capture microdissection. Set 1 served as a pilot and a means to identify protein pathway activation alterations that could be further validated in a second independent set. Set 1 was comprised of 4 AK, 13 SCC, and 20 UIA. Set 2 included 15 AK, 9 SCCs, and 20 UIAs. Activation of 51 signaling proteins, known to be involved in tumorigenesis, were assessed for set 1 and showed that the MEK–ERK [mitogen-activated protein (MAP)/extracellular signal-regulated (ERK; MEK)] pathway was activated in SCC compared with AK and UIA, and that epidermal growth factor receptor (EGFR) and mTOR pathways were aberrantly activated in SCC. Unsupervised two-way hierarchical clustering revealed that AK and UIA shared a common signaling network activation architecture while SCC was dramatically different. Statistical analysis found that prosurvival signaling through phosphorylation of ASK and 4EBP1 as well as increased Bax and Bak expression was higher in AK compared with UIA. We expanded pathway network activation mapping in set 2 to 101 key signaling proteins, which corroborated activation of MEK–ERK, EGFR, and mTOR pathways through discovery of a number of upstream and downstream signaling molecules within these pathways to conclude that SCC is indeed a pathway activation–driven disease. Pathway activation mapping of SCC compared with AK revealed several interconnected networks that could be targeted with drug therapy for potential chemoprevention and therapeutic applications. Cancer Prev Res; 5(3); 403–13. ©2012 AACR.

BACKGROUND:Reduced melanoma risk has been reported with regular use of nonsteroidal anti-inflammatory drugs (NSAIDs). However, the ability of NSAIDs to reach melanocytes in vivo and modulate key biomarkers in preneoplastic lesions such as atypical nevi has not been evaluated. METHODS:This randomized, double-blind, placebo-controlled trial of sulindac was conducted in individuals with atypical nevi (AN) to determine bioavailability of sulindac and metabolites in nevi and effect on apoptosis and vascular endothelial growth factor A (VEGFA) expression in AN. Fifty subjects with AN ≥ 4 mm in size and 1 benign nevus (BN) were randomized to sulindac (150 mg twice a day) or placebo for 8 weeks. Two AN were randomized for baseline excision, and 2 AN and BN were excised after intervention. RESULTS:Postintervention sulindac, sulindac sulfone, and sulindac sulfide concentrations were 0.31 ± 0.36, 1.56 ± 1.35, and 2.25 ± 2.24 μg/mL in plasma, and 0.51 ± 1.05, 1.38 ± 2.86, and 0.12 ± 0.12 μg/g in BN, respectively. Sulindac intervention did not significantly change VEGFA expression but did increase expression of the apoptotic marker cleaved caspase-3 in AN (increase of 3 ± 33 in sulindac vs decrease of 25 ± 45 in the placebo arm, P = .0056), although significance was attenuated (P = .1103) after adjusting for baseline expression. CONCLUSIONS:Eight weeks of sulindac intervention resulted in high concentrations of sulindac sulfone, a proapoptotic metabolite, in BN but did not effectively modulate VEGFA and cleaved caspase-3 expression. Study limitations included limited exposure time to sulindac and the need to optimize a panel of biomarkers for NSAID intervention studies.

Abstract Some epidemiological studies and several experimental publications suggest that regular use of non-steroidal anti-inflammatory drugs (NSAIDs) may reduce the risk of melanoma. However, specific information on the ability of NSAIDs to reach melanocytes in vivo and to modulate key biomarkers in pre-neoplastic lesions such as dysplastic nevi have not been evaluated to date. Methods: We conducted a randomized, double-blind, placebo controlled trial of sulindac in individuals at risk for melanoma to determine whether sulindac and related metabolites are bioavailable in the nevi and whether sulindac intervention has an effect on apoptosis and vascular endothelial growth factor (VEGF) expression in atypical nevi (AN). Fifty healthy subjects with ≥ 4 large (≥5 mm and < 15 mm) AN and one benign nevus (BN) were enrolled between 2/09 and 7/10. Subjects underwent clinical and dermoscopic evaluation of their melanocytic lesions with 2 AN randomized to be excised at baseline for biomarker evaluation and 2 AN to post-intervention evaluation. Subsequently, participants were randomized to receive sulindac (150 mg BID) or placebo for 8 weeks. Upon completion of the intervention the residual 2 AN and the BN were surgically removed. Results: The study intervention was well tolerated with some mild or moderate gastrointestinal adverse events that were possibly or probably related to the intervention. Sulindac, sulindac sulfone, sulindac sulfide concentrations were 0.31 ± 0.36, 1.56 ± 1.35, 2.25 ± 2.24 μg/ml, respectively, in post-intervention plasma samples from the sulindac group. Sulindac, sulindac sulfone, sulindac sulfide concentrations were 0.51 ± 1.05, 1.38 ± 2.86, 0.12 ± 0.12 μg/g tissue, respectively, in post-intervention nevi samples. Eight weeks of sulindac intervention did not result in significant changes in VEGF expression. Sulindac intervention increased the expression of a marker of apoptosis, cleaved caspase 3 (an increase of 3 ± 33 in the sulindac arm vs. a decrease of 25 ± 45 in the placebo arm, p = 0.0056) in dysplastic nevi. After adjusting for baseline expression, the significance of this finding is attenuated to a p-value of 0.1103 based on a linear regression model. Overall, these findings are consistent with the identification of high concentrations of sulindac sulfone, a pro-apoptotic metabolite in the nevi. Conclusion: The study showed that sulindac and sulindac metabolites can reach measurable levels in melanocytic nevi. Eight weeks of sulindac intervention induced the expression of a marker of apoptosis in dysplastic nevi but did not result in significant changes in VEGF expression. The study findings support the further evaluation of sulindac as a chemopreventive agent for melanoma. Citation Information: Cancer Prev Res 2011;4(10 Suppl):CN06-01.

Abstract Cutaneous squamous cell carcinoma (SCC) occurs commonly and can metastasize. Identification of specific molecular aberrations and mechanisms underlying the development and progression of cutaneous SCC may lead to better prognostic and therapeutic approaches and more effective chemoprevention strategies. To identify genetic changes associated with early stages of cutaneous SCC development, we analyzed a series of 40 archived skin tissues ranging from normal skin to invasive SCC. Using high-resolution array-based comparative genomic hybridization, we identified deletions of a region on chromosome 10q harboring the INPP5A gene in 24% of examined SCC tumors. Subsequent validation by immunohistochemistry on an independent sample set of 71 SCC tissues showed reduced INPP5A protein levels in 72% of primary SCC tumors. Decrease in INPP5A protein levels seems to be an early event in SCC development, as it also is observed in 9 of 26 (35%) examined actinic keratoses, the earliest stage in SCC development. Importantly, further reduction of INPP5A levels is seen in a subset of SCC patients as the tumor progresses from primary to metastatic stage. The observed frequency and pattern of loss indicate that INPP5A, a negative regulator of inositol signaling, may play a role in development and progression of cutaneous SCC tumors. Cancer Prev Res; 3(10); 1277–83. ©2010 AACR.

Abstract Some epidemiological studies and several experimental publications suggest that regular use of NSAIDs may reduce the risk of melanoma. Proposed mechanisms of action include: inhibition of MAP kinase activity and nuclear translocation, reduction in the concentration of prostaglandins associated with the inhibition of VEGF and bFGF production and HIF-1α stabilization, reduction of the activation state of the VEGF and Ftl-1 genes, and inhibition of PI-3 kinase/Akt signaling pathway. However, specific information on the ability of NSAIDs to reach melanocytes in vivo and to modulate key biomarkers in pre-neoplastic lesions, such as dysplastic nevi, have not been evaluated to date. Methods: A phase IIa placebo controlled clinical intervention trial was designed with the primary end point to determine whether oral sulindac and related metabolites were present in benign nevi (BN) after an 8 week course of oral sulindac; secondary end points were included to determine whether a short exposure to sulindac had an effect on apoptosis and VEGF expression in atypical nevi (AN), as well as measurement of sulindac and metabolite levels in plasma. Exploratory objectives will evaluate the feasibility of reverse phase protein microarray to identify intervention related changes in cell signaling proteins, focusing initially on the pAKT/mTOR pathway and COX expression. Fifty healthy subjects with ≥ 4 large (≥5 mm and < 15 mm) AN and one BN were enrolled between 2/09 and 7/10. Subjects underwent clinical and dermoscopic evaluation of their melanocytic lesions with 2 AN randomized to be excised at baseline for biomarker evaluation and 2 AN to post-intervention evaluation. Subsequently, participants were randomized to receive sulindac (150 mg BID) or placebo for 8 weeks. Upon completion of the intervention the residual 2 AN and the BN were surgically removed. Results: To date, accrual has been completed and study intervention in all subjects is expected to be concluded in 09/10. The study intervention was well tolerated with some mild or moderate gastrointestinal adverse events that are possibly or probably related to the intervention. Sulindac, sulindac sulfide, sulindac sulfone concentrations were 0 — 1.4, 0 −9.07, and 0 — 6.1 µg/ml, respectively, in 41 post-intervention plasma samples analyzed to date. Sulindac, sulindac sulfide, sulindac sulfone concentrations were 0 — 5.2, 0 — 0.25, and 0 — 14.27 µg/g, respectively, in 40 postintervention BN samples. Conclusion: Current results suggest that NSAIDs, in particular sulindac and the active sulindac metabolites, can reach measurable levels in the skin, and specifically in melanocytic nevi, further supporting the need to assess the effect of this group of drugs as chemopreventive agents for melanoma. Supported by N01CN35158 from the National Cancer Institute, Division of Cancer Prevention. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A66.