Redox modulation of oxidatively-induced DNA damage by ascorbate enhances both in vitro and ex-vivo DNA damage formation and cell death in melanoma cells.

Elevated genomic instability in cancer cells suggests a possible model-scenario for their selective killing via the therapeutic delivery of well-defined levels of further DNA damage. To examine this scenario, this study investigated the potential for redox modulation of oxidatively-induced DNA damage by ascorbate in malignant melanoma (MM) cancer cells, to selectively enhance both DNA damage and MM cell killing. DNA damage was assessed by Comet and ɣH2AX assays, intracellular oxidising species by dichlorofluorescein fluorescence, a key antioxidant enzymatic defence by assessment of catalase activity and cell survival was determined by clonogenic assay. Comet revealed that MM cells had higher endogenous DNA damage levels than normal keratinocytes (HaCaT cells); this correlated MM cells having higher intracellular oxidising species and lower catalase activity, and ranked with MM cell melanin pigmentation. Comet also showed MM cells more sensitive towards the DNA damaging effects of exogenous H2O2, and that ascorbate further enhanced this H2O2-induced damage in MM cells; again, with MM cell sensitivity to induced damage ranking with degree of cell pigmentation. Furthermore, cell survival data indicated that ascorbate enhanced H2O2-induced clonogenic cell death selectively in MM cells whilst protecting HaCaT cells. Finally, we show that ascorbate serves to enhance the oxidising effects of the MM therapeutic drug Elesclomol in both established MM cells in vitro and primary cell cultures ex vivo. Together, these results suggest that ascorbate selectively enhances DNA damage and cell-killing in MM cells. This raises the option of incorporating ascorbate into clinical oxidative therapies to treat MM.

Comparison of the prognostic value of microscopically measured invasive width versus macroscopic width in cutaneous melanoma shows the superiority of microscopic invasive width measurement.

BACKGROUND: Invasive width, the distance between the most peripheral invasive melanoma cells on the section where Breslow thickness (BT) was measured, was recently identified as a prognostic feature. It is unclear whether a routine measurement is justified, given that macroscopic width is already included in many melanoma histopathology reports and may itself be a prognostic feature. This study sought to investigate this. METHODS: A retrospective cohort of 718 melanoma patients in which macroscopic width had been stated in the original histopathology report was used. Survival analysis was performed. RESULTS: Macroscopic and invasive widths were positively correlated (p < 0.001). Invasive width was typically smaller than the paired macroscopic width (median difference 3.7 mm, p < 0.001), a difference seen across all T groups. Both macroscopic and invasive widths were significantly associated with melanoma survival in Kaplan-Meier analysis, including overall survival, but invasive width survival curves were more widely separated. Both were significantly associated with outcome after correction for BT in Cox proportional hazards regression, but the models containing invasive width had a substantially better fit. CONCLUSIONS: This study shows that both macroscopic and invasive widths have prognostic values, but confirms that the latter is superior. It supports further investigation of this feature's prognostic value.

The Width of Invasion in Malignant Melanoma Is a Novel Prognostic Feature That Accounts for Outcome Better Than Breslow Thickness.

Breslow thickness (BT) is the cornerstone of malignant melanoma staging. However, to our knowledge no-one has ever assessed the horizontal width of invasion, measured microscopically, as a prognostic feature. This was investigated as a prognostic feature in this study. A retrospective cohort of 1329 melanoma cases was collected from patients presenting to a UK teaching hospital from January 1, 2004, to December 31, 2014. The main outcome was overall survival (OS). We found that width was associated with OS in multivariable analysis (hazard ratio=1.05, 95% confidence interval: 1.03-1.07, P<0.001) and was similarly significant for melanoma-specific survival and metastasis-free survival. Its presence rendered BT nonsignificant. The width was significantly associated with OS after adjustment for American Joint Committee on Cancer (AJCC), version 8 clinical tumor stage (hazard ratio=1.05, 95% confidence interval: 1.03-1.07, P<0.001), and bootstrap validation showed only slight model optimism. Similar associations were seen for melanoma-specific survival and metastasis-free survival. However, the combination of invasive width and BT did not account for the outcome as well as another novel histologic feature, tumor area, which was measured using the calculated tumor area method. In conclusion, this study is the first investigation of a novel histologic feature, invasive melanoma width, and demonstrates its strong independent association with outcome.

Global and mitosis-specific interobserver variation in mitotic count scoring and implications for malignant melanoma staging.

AIMS: Staging is the gold standard for predicting malignant melanoma outcome but changes in its criteria over time indicate ongoing evolution. One notable recent change from the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual was removal of mitotic count. We explore the extent to which this feature is limited by interobserver error in order to find ways to improve its fitness for use should it be revisited in future staging versions. METHODS AND RESULTS: In a cohort of 476 patients with melanoma ≤1.0 mm, a mitotic count of 0 versus 1 was significant for metastasis-free survival, but not melanoma-specific or overall survival. In 10 melanomas that were 0.9-1.0 mm thick, the mitotic count intraclass correlation coefficient for histopathologists was 0.58 (moderate agreement). Uniquely, we also assessed agreement for specific putative mitotic figures, identifying precise reasons why specific mitotic figures qualified for scoring or elimination. A kappa score was 0.54 (moderate agreement). We also gathered data on other staging features. Breslow thickness had an intraclass correlation coefficient of 0.41 (moderate agreement) and there was a systematic difference between histopathologists among cases (P = 0.04). Every case had a range that crossed the AJCC8 0.8-mm pT1a/pT1b staging boundary. Ulceration was only identified in two of the 10 cases. For ulceration, kappa agreement score was 0.31 (fair). CONCLUSION: This study supports the removal of mitotic count from staging, but shows that its scoring is substantially affected by interobserver variation, suggesting that more prescriptive guidelines might have a beneficial impact on its prognostic value.

Development and Initial Validation of Calculated Tumor Area as a Prognostic Tool in Cutaneous Malignant Melanoma.

IMPORTANCE: Breslow thickness is a 1-dimensional surrogate prognostic feature for tumor size, yet tissue sections have 2 dimensions. Therefore, a 2-dimensional feature, calculated tumor area (CTA), was devised. OBJECTIVE: To determine CTA precision and prognostic value. DESIGN, SETTING, AND PARTICIPANTS: This retrospective cohort of patients with cutaneous melanoma presented to the Leicester and Nottingham National Health Service hospital trusts in the United Kingdom. Eligible patients in the Leicester development sample had available primary tumor tissue; a diagnosis from January 1, 2004, through December 31, 2011; invasive disease; and Leicestershire residency. Patients in the Nottingham validation sample had an anonymized spreadsheet with primary melanoma diagnosed from January 1, 2003, through December 31, 2005, or from January 1, 2008, through December 31, 2010. From a starting population of 1463 patients in both data sets, a total of 224 (15.3%) were excluded to yield a study population of 1239. Data were analyzed from April 30, 2018, through January 10, 2019. INTERVENTION: An observational analysis of the prognostic value of CTA in patients with cutaneous melanoma. MAIN OUTCOMES AND MEASURES: Independent association of CTA with melanoma-specific survival and confounding effect of CTA on Breslow thickness in survival analysis. RESULTS: A total of 1239 patients with melanoma were assessed, including 649 (52.4%) women, with a median age of 60 years (interquartile range, 47-71 years). An intraclass correlation coefficient for CTA on 13 cases was 0.99. In 918 patients in the Leicester cohort, CTA was an independent prognostic factor in Cox proportional hazards regression models after adjusting for Breslow thickness, age, sex, ulcer, mitotic rate, and microsatellites (hazard ratio [HR], 1.87; 95% CI, 1.49-2.34; P < .001). Validation in 321 patients in the Nottingham cohort showed an HR of 1.55 (95% CI, 1.15-2.09; P = .005) and in the combined 1239 cases, an HR of 1.70 (95% CI, 1.43-2.03; P < .001). Breslow thickness was significant in multivariable analysis only when CTA was not in the model. The relative importance of CTA was shown by its retention in all 100 bootstrap multivariable models with backward selection, whereas Breslow thickness was retained in only 53. Melanomas stratified by CTA showed wider separation of survival curves than those stratified by Breslow thickness using the American Joint Committee on Cancer Staging Manual, 8th Edition (HRs, 1.00 to 41.46 vs 1.00 to 36.95, respectively), and the model with CTA categories had a Bayesian information criterion difference of 13.9 compared with T category, indicating substantially better fit. This model had a Harrell C index of 83.7%, and bootstrap analysis showed little evidence of model optimism, with a corrected calibration slope of 0.99. CONCLUSIONS AND RELEVANCE: This study provides a novel microscopic feature, CTA, with evidence of its independent prognostic value. This evidence suggests that CTA should be a priority for further study.

Compromised vitamin D receptor signalling in malignant melanoma is associated with tumour progression and mitogen-activated protein kinase activity.

The aims of this study were to investigate, in cutaneous malignant melanoma (MM), the integrity of nuclear vitamin D receptor (VDR) signalling, as implied by VDR subcellular location; to investigate the relationship between VDR and tumour progression and the inhibitory effect on VDR by mitogen-activated protein kinase (MAPK) overactivity. Archived tissue from 34 benign melanocytic naevi, 149 MMs and 44 matched metastases were stained by immunohistochemistry for VDR and a subset of primary MMs were stained for phosphorylated-extracellular signal-regulated kinase as a marker of MAPK activity. MM cell lines were investigated to show the subcellular location of VDR and cell viability in response to ligand±MAPK inhibitor. Benign melanocytic naevi showed mainly a strong nuclear VDR staining in contrast to MM where decreased nuclear and emergent cytoplasmic VDRs were associated with malignant progression in terms of dermal invasion and metastasis. MMs that retained exclusive nuclear VDR at the tumour base did not metastasize, a potentially important prognostic indicator. Decreased nuclear VDR correlated with increased cytoplasmic staining, suggesting the failure of nuclear entry as a primary cause of defective VDR signalling in MM. The histological subset analysis and MM cell line studies confirmed the inhibitory effect of MAPK activity on VDR signalling, but the pattern of VDR subcellular localization suggested failure of VDR nuclear entry as a primary effect of MAPK activity rather than direct inhibition of VDR-regulated transcription. Furthermore, high MAPK activity in tumours expressing cytoplasmic VDR was associated with worsened prognosis.

A case and literature review of orbital microcystic adnexal carcinoma.

Microcystic adnexal carcinoma (MAC) is a very rare and locally aggressive sweat gland tumour. MAC has been well reported presenting as a periocular cutaneous lesion, rarely with subsequent direct orbital invasion and only once as a primary orbital lesion. Local recurrence is frequent after primary surgical excision and the role of adjuvant radiotherapy is ill-defined. We describe a case of orbital MAC treated successfully with radiotherapy after incomplete margin clearance post exenteration surgery and review the associated literature.

Breslow Density Is a Novel Prognostic Feature That Adds Value to Melanoma Staging.

Histomorphologic prognostic biomarkers that can be measured using only an hematoxylin and eosin stain are very attractive because they are simple and cheap. We conceived an entirely novel biomarker of this type, the Breslow density (BD), which measures invasive melanoma cell density at the site where Breslow thickness (BT) is measured. This study assessed BD's prognostic value. In this study, BD was measured in 1329 melanoma patients. Measurement accuracy and precision was assessed using intraclass correlation coefficient (ICC). Survival was assessed with a primary end-point of melanoma-specific survival (MSS) and also overall survival and metastasis-free survival. We found that BD measurement was accurate compared with gold standard image analysis (ICC, 0.84). Precision was excellent for 3 observers with different experience (ICC, 0.93) and for an observer using only written instructions (ICC, 0.93). BD was a highly significant predictor in multivariable analysis for overall survival, MSS, and metastasis-free survival (each, P<0.001) and it explained MSS better than BT, but BT and BD together had best explanatory capability. A BD cut point of ≥65% was trained in 970 melanomas and validated in 359. This cut point showed promise as a novel way to upstage melanoma from T stage "a" to "b." BD was combined with BT to create a targeted burden score. This was a validated as an adjunct to American Joint Committee on Cancer stage. In summary, BD can be measured accurately and precisely. It demonstrated independent prognostic value and explained MSS better than BT alone. Notably, we demonstrated ways that BD could be used with American Joint Committee on Cancer version 8 staging.

A Novel Numerical Scoring System for Melanoma Tumor-infiltrating Lymphocytes Has Better Prognostic Value Than Standard Scoring.

The purpose of this study was to evaluate the prognostic value of tumor-infiltrating lymphocytes (TILs) in melanoma and to determine whether a simpler numerical scoring system would be more effective. In total, 655 patients presenting to a UK teaching hospital with primary invasive melanoma were analyzed. TILs were rescored using the standard Clark's method and univariable and multivariable analyses of the effect of TILs on overall survival (OS), disease-specific survival (DSS), and metastasis-free survival (MFS) was assessed using Cox regression. In total, 30 (5%) melanomas showed absent, 464 (71%) nonbrisk, and 161 (24%) brisk TILs. There was a statistically significant relationship between TILs and Breslow thickness, age, melanoma type, mitotic rate, and histologic regression. TIL grade was a significant predictor of MFS in multivariable analysis (hazard ratio [HR], 0.44; 95% confidence interval [CI], 0.25-0.77) but was not significant for OS or DSS. By contrast, when a simple numerical TIL percentage score was used this was a strong predictor of OS (HR, 0.55; 95% CI, 0.38-0.78), DSS (HR, 0.25; 95% CI, 0.14-0.44), and MFS (HR, 0.32; 95% CI, 0.21-0.51) in multivariable analysis. The percentage TIL score was also significant when adjusted for the prognostic gold standard, American Joint Committee on Cancer stage: OS (HR, 0.66; 95% CI, 0.46-0.95), DSS (HR, 0.33; 95% CI, 0.19-0.60), and MFS (HR, 0.41; 95% CI, 0.26-0.65). The TIL percentage score was subsequently validated in new cases. In summary, this study strongly confirms that higher amounts of TILs are associated with better prognosis and in addition demonstrates the value of a simplified numerical TIL scoring system.

A new cause of lacrimal gland calcification: Retained metallic foreign bodies.

A 49-year-old male presented with a 4 month history of dysaesthesia in the left periorbital region. A CT scan showed a lacrimal gland mass with areas of dense calcification. Biopsies of the left lacrimal gland revealed a silver material with associated chronic granulomatous inflammation and secondary calcification. The histological specimen was sent for X-ray microanalysis. This confirmed the silver material to be aluminium. The metal fragments were subsequently discovered to be from a car aerial, which caused an orbital impaling injury 20 years prior to presentation. This is the first reported case of lacrimal gland calcification secondary to chronic metal foreign body exposure. The case is presented alongside literature reviews of lacrimal gland calcification and associated radiological findings.

Breslow density is a novel prognostic feature in cutaneous malignant melanoma.

AIMS: In 1970, Breslow described his eponymously named thickness measurement. No-one has sought to enhance the Breslow thickness (BT). The aim of this study was to demonstrate a proof of concept that the density of melanoma cells at the position where the BT is measured is a morphological prognostic biomarker, which we name the Breslow density (BD). The hypothesis was that the BD has prognostic value for overall survival (OS) and is independent of the BT. METHODS AND RESULTS: We analysed 100 cutaneous melanomas, and followed REMARK guidelines. The BD was the estimated percentage dermal area occupied by melanoma cells in a specified location. The BT and BD had a strong correlation (P = 2.1 × 10-11 ) but, despite this, they were independent prognostic factors for OS in Cox regression [BD hazard ratio (HR) 1.03, P = 0.001849; and BT HR 1.09, P = 0.000146]. This was corroborated by an independent effect on melanoma-specific survival. We assessed whether the BT and BD could be combined into a Breslow score. A prognostic index based on Cox regression coefficients was used, and this showed a marginal improvement in predicted 5-year survival as compared with the BT alone (area under the curve of 94.8% versus 96.7%). CONCLUSIONS: We show a proof of concept that the BD represents a novel morphological prognostic biomarker that is independent of the BT, and that there is potential to combine these into a Breslow score. Larger studies are needed to validate the BD, but the simplicity of this biomarker makes it a strong candidate for translation to clinical practice.

5-Hydroxymethylcytosine is an independent predictor of survival in malignant melanoma.

Outcomes for melanoma patients vary within cancer stage. Prognostic biomarkers are potential adjuncts to provide more precise prognostic information. Simple, low-cost biomarker assays, such as those based on immunohistochemistry, have strong translational potential. 5-hydroxymethylcytosine (5 hmC) shows prognostic potential in melanoma but prior studies were small. We, therefore, analysed 5 hmC in a retrospective cohort to provide external validation of its prognostic value. Two hundred primary melanomas were evaluated for 5 hmC expression using immunohistochemistry. The primary objective was to assess the effect on overall survival while controlling for important confounders. Univariable and multivariable analyses were performed. REMARK guidelines were followed. The 5 hmC immunohistochemistry scoring showed very strong inter-observer agreement (ICC 0.88) and expression was significantly related to age, site, Breslow thickness, ulceration, mitotic rate, and stage. Kaplan-Meier analysis showed 5 hmC was associated with metastasis-free, melanoma-specific, and overall survival, P<0.0001 for each. In univariable Cox proportional hazards models, 5 hmC hazard ratios were significant and remained so in a multivariable model. A two-step cox model was created using stage and 5 hmC, as stage is the gold standard for clinical practice. The addition of 5 hmC produced significant improvement in the model and 5 hmC and stage were independent significant predictors. This is the largest study of the prognostic value of 5 hmC immunohistochemistry in melanoma. The 5 hmC scoring was easily and reproducibly performed and it was an independent predictor of metastasis-free survival, melanoma-specific survival, and overall survival. This work supports further development of 5 hmC as a prognostic biomarker and suggests that it could add more precision to American Joint Committee on Cancer staging.

A small multimarker panel using simple immunohistochemistry methods is an adjunct to stage for cutaneous melanoma prognosis.

Melanoma is an aggressive cancer. Outcomes can vary significantly for lesions within the same pathological stage - a problem of increasing relevance with the promise of adjuvant treatments on the basis of immune checkpoint modulators and targeted therapies. The use of a panel of prognostic molecular biomarkers as an adjunct to stage represents a possible solution. Immunohistochemistry-based biomarkers offer greater potential for translation into clinical practice than biomarkers utilizing more complex methods. Many immunohistochemistry-based biomarkers have been identified through discovery studies, but rigorous validation of these is scarce. We take the first steps towards validating a combination of three such biomarkers in a prognostic panel - 5hmC, ki-67 and p16. Immunohistochemistry was performed on a cohort of 50 melanomas to determine the expression of 5hmC, ki-67 and p16. 5hmC and p16 showed statistically significant differences in metastasis-free survival between low-score and high-score groups, whereas the use of all three biomarkers together with stage could predict the 5-year metastasis risk more accurately than stage alone. Our results suggest that the use of multimarker panels to improve the accuracy of prognostic predictions is feasible and worthy of further study. We have shown that a small immunohistochemistry-based panel utilizing simple, inexpensive, reproducible methods can be an effective adjunct to stage in prognostic prediction. A follow-up study consisting of a large cohort of melanomas is now indicated to continue the development of the prognostic panel.

microRNA-10b is a prognostic biomarker for melanoma.

Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disease has been revolutionized by new agents. More therapeutic options, coupled with the desire to extend treatment to the adjuvant setting mean that prognostic biomarkers that can be assayed from formalin-fixed paraffin-embedded clinical would be valuable. microRNAs have potential to fill this need. We analyzed 377 microRNAs in 79 primary melanomas and 32 metastases using a split sample discovery strategy. From a discovery analysis using 40 thick primary melanomas (20 cases with metastasis and 20 controls without metastasis at 5 years), microRNA expression was measured by quantitative RT-PCR (QRT-PCR). MiR-10b emerged as a candidate prognostic microRNA. This was confirmed in an independent validation set of thick primary melanomas (20 cases with metastasis and 19 controls without metastasis at 5 years). In the combined discovery and validation cohorts (n=79), miR-10b expression showed a 3.7-fold increase in expression between cases and controls (P=0.005) and showed a trend of increasing expression between primary melanomas and their matched metastases (P<0.001). In situ hybridization showed expression was in melanoma cells and correlated with expression measured by QRT-PCR (P=0.0005). We used the combined discovery and validation samples to verify the prognostic value of additional candidate microRNAs identified from other studies, and proceeded to analyze miR-200b. We demonstrated that miR-10b and miR-200b showed independent prognostic value (P=0.002 and 0.047, respectively) in multivariable analysis alongside known clinico-pathological prognostic features (eg, Breslow thickness) using a Cox proportional hazards regression model. Furthermore, the addition of these microRNAs to the clinico-pathological features led to an improved regression model with better identification of aggressive thick melanomas. Taken together, these data suggest that miR-10b is a new prognostic microRNA for melanoma and that there could be a place for microRNA analysis in stratifying melanoma for therapy.

MicroRNA-21 expression and its pathogenetic significance in cutaneous melanoma.

Identification of prognostic biomarkers is timely for melanoma as clinicians seek ways to stratify patients for molecular therapy. MicroRNAs are promising as tissue biomarkers because they can be assayed directly from formalin-fixed paraffin-embedded clinical samples. We previously reported that microRNA-21 (miR-21) was strongly expressed in melanoma relative to naevi and now sought to further assess the significance of this by assessing its relationship with its putative target, PTEN. Clinical melanoma samples were analysed by immunohistochemical analysis for PTEN, stem-loop qRT-PCR for miR-21 and PCR for BRAF/NRAS mutation status. Cell lines were investigated for the effect of anti-miR-21 on PTEN. A total of 81 clinical melanocytic tumour samples were investigated, with uniformly high PTEN expression in the nucleus and cytoplasm of naevi and with preferential loss of PTEN expression in the nucleus of melanoma cells. miR-21 expression was inversely associated with nuclear PTEN expression but not with cytoplasmic PTEN expression. An anti-miR-21 preferentially altered nuclear PTEN in melanoma cell lines. The presence of a BRAF or NRAS mutation had no significant effect on miR-21 expression. These data suggest miR-21 may exert an oncogenic effect in melanoma by favouring redistribution of PTEN to the nucleus.

Duplex Ratio Tests as Diagnostic Biomarkers in Malignant Melanoma.

Chromosomal instability is a well-described feature of malignant tumors. Melanomas have typical patterns of chromosomal instability compared with benign nevi, which have minimal DNA copy number change. A few malignant melanomas and their benign counterparts, nevi, prove difficult to diagnose on histopathologic analysis alone, which is currently the gold standard. Quantitative PCR-based assays called duplex ratio tests (DRTs) have been developed by our laboratory for application using DNA from FFPE samples of melanomas and nevi. The reproducibility and accuracy of the DRTs were demonstrated and appropriate correction factors for DNA quality calculated for each assay, based on the results of 108 diploid samples. As a panel, seven DRTs were able to differentiate unambiguous cases of melanoma and nevi with a sensitivity of 87% (95% CI, 83%-91%) and a specificity of 88% (95% CI, 84%-92%) in a series of 145 melanomas and 123 nevi. The DRT scores for 20 nonmetastasizing primary melanomas and 20 metastasizing primary melanomas revealed that DRTs had a marginal benefit as prognostic markers. DRTs have early potential to act as molecular biomarkers of melanoma on FFPE specimens pending validation, and DRTs may have applicability as prognostic markers in melanoma or other tumor types if new DRTs to relevant loci are developed.

A switch in the expression of embryonic EMT-inducers drives the development of malignant melanoma.

Aberrant expression of embryonic epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) in epithelial cells triggers EMT, neoplastic transformation, stemness, and metastatic dissemination. We found that regulation and functions of EMT-TFs are different in malignant melanoma. SNAIL2 and ZEB2 transcription factors are expressed in normal melanocytes and behave as tumor-suppressor proteins by activating an MITF-dependent melanocyte differentiation program. In response to NRAS/BRAF activation, EMT-TF network undergoes a profound reorganization in favor of TWIST1 and ZEB1. This reversible switch cooperates with BRAF in promoting dedifferentiation and neoplastic transformation of melanocytes. We detected EMT-TF reprogramming in late-stage melanoma in association with enhanced phospho-ERK levels. This switch results in E-cadherin loss, enhanced invasion, and constitutes an independent factor of poor prognosis in melanoma patients.

Accurate detection of copy number changes in DNA extracted from formalin-fixed, paraffin-embedded melanoma tissue using duplex ratio tests.

A minority of melanocytic lesions cannot confidently be classified as benign or malignant on histopathological examination, causing diagnostic uncertainty. DNA copy number changes can be used to distinguish nevi from melanoma, although the use of FFPE tissue can pose technical challenges. DNA copy number assays, called duplex ratio tests, have been developed with duplex real-time PCR, using a simple method with potential for high throughput. Five duplex ratio test assays targeting loci with common DNA copy number changes in melanoma were designed and tested using DNA extracted from FFPE samples microdissected from melanoma, common nevi, benign tonsil (10 each), and two melanoma cell lines. The assays proved accurate when DNA extracted from fresh and FFPE melanoma cell lines were compared (intraclass correlation coefficient, 0.99) and gave precise results when repeated on DNA from FFPE tissue (intraclass correlation coefficient range, 0.90 to 0.96). In combination, duplex ratio test values from three of the assays distinguished between the nevi and melanomas with 100% sensitivity (95% CI, 69.1% to 100%) and 100% specificity (95% CI, 69.1% to 100%). Duplex ratio test assays have been shown to be accurate and precise and can distinguish melanomas from common nevi using DNA from FFPE tissue. Appropriately designed assays could have value in assessment of other cancers.