Serum markers improve current prediction of metastasis development in early-stage melanoma patients: a machine learning-based study

Metastasis development represents an important threat for melanoma patients, even when diagnosed at early stages and upon removal of the primary tumor. In this scenario, determination of prognostic biomarkers would be of great interest. Serum contains information about the general status of the organism and therefore represents a valuable source for biomarkers. Thus, we aimed to define serological biomarkers that could be used along with clinical and histopathological features of the disease to predict metastatic events on the early-stage population of patients. We previously demonstrated that in stage II melanoma patients, serum levels of dermcidin (DCD) were associated with metastatic progression. Based on the relevance of the immune response on the cancer progression and the recent association of DCD with local and systemic immune response against cancer cells, serum DCD was analyzed in a new cohort of patients along with interleukin 4 (IL-4), IL-6, IL-10, IL-17A, interferon gamma (IFN-gamma), transforming growth factor-beta (TGF- beta), and granulocyte-macrophage colony-stimulating factor (GM-CSF). We initially recruited 448 melanoma patients, 323 of whom were diagnosed as stages I-II according to AJCC. Levels of selected cytokines were determined by ELISA and Luminex, and obtained data were analyzed employing machine learning and Kaplan-Meier techniques to define an algorithm capable of accurately classifying early-stage melanoma patients with a high and low risk of developing metastasis. The results show that in early-stage melanoma patients, serum levels of the cytokines IL-4, GM-CSF, and DCD together with the Breslow thickness are those that best predict melanoma metastasis. Moreover, resulting algorithm represents a new tool to discriminate subjects with good prognosis from those with high risk for a future metastasis.We are grateful to the Basque Biobank for providing the serum samples. We are also most grateful to Drs Arantza Arrieta and Natalia Maruri (Cruces University Hospital) for their technical support with the serum marker detection. This work was supported by grants from the Basque Government (KK2016-036 and KK2017-041 to MDB), UPV/EHU (GIU17/066 to MDB), H2020-ESCEL JTI (15/01 to MDB), and MINECO (PCIN-2015-241 to MDB

The Interplay between Natural Selection and Susceptibility to Melanoma on Allele 374F of SLC45A2 Gene in a South European Population

We aimed to study the selective pressures interacting on SLC45A2 to investigate the interplay between selection and susceptibility to disease. Thus, we enrolled 500 volunteers from a geographically limited population (Basques from the North of Spain) and by resequencing the whole coding region and intron 5 of the 34 most and the 34 least pigmented individuals according to the reflectance distribution, we observed that the polymorphism Leu374Phe (L374F, rs16891982) was statistically associated with skin color variability within this sample. In particular, allele 374F was significantly more frequent among the individuals with lighter skin. Further genotyping an independent set of 558 individuals of a geographically wider population with known ancestry in the Spanish population also revealed that the frequency of L374F was significantly correlated with the incident UV radiation intensity. Selection tests suggest that allele 374F is being positively selected in South Europeans, thus indicating that depigmentation is an adaptive process. Interestingly, by genotyping 119 melanoma samples, we show that this variant is also associated with an increased susceptibility to melanoma in our populations. The ultimate driving force for this adaptation is unknown, but it is compatible with the vitamin D hypothesis. This shows that molecular evolution analysis can be used as a useful technology to predict phenotypic and biomedical consequences in humans

Simultaneous purifying selection on the ancestral MC1R allele and positive selection on the melanoma-risk allele V60L in South Europeans

In humans, the geographical apportionment of the coding diversity of the pigmentary locus melanocortin-1 receptor (MC1R) is, unusually, higher in Eurasians than in Africans. This atypical observation has been interpreted as the result of purifying selection due to functional constraint on MC1R in high UV-B radiation environments. By analyzing 3,142 human MC1R alleles from different regions of Spain in the context of additional haplotypic information from the 1000 Genomes (1000G) Project data, we show that purifying selection is also strong in southern Europe, but not so in northern Europe. Furthermore, we show that purifying and positive selection act simultaneously on MC1R. Thus, at least in Spain, regions at opposite ends of the incident UV-B radiation distribution show significantly different frequencies for the melanoma-risk allele V60L (a mutation also associated to red hair and fair skin and even blonde hair), with higher frequency of V60L at those regions of lower incident UV-B radiation. Besides, using the 1000G south European data, we show that the V60L haplogroup is also characterized by an extended haplotype homozygosity (EHH) pattern indicative of positive selection. We, thus, provide evidence for an adaptive value of human skin depigmentation in Europe and illustrate how an adaptive process can simultaneously help to maintain a disease-risk allele. In addition, our data support the hypothesis proposed by Jablonski and Chaplin (Human skin pigmentation as an adaptation to UVB radiation. Proc Natl Acad Sci U S A. 2010;107:8962-8968), which posits that habitation of middle latitudes involved the evolution of partially depigmented phenotypes that are still capable of suitable tanning.This works was supported by the former Spanish Ministerio de Ciencia e Innovación, project CGL2008-04066/BOS to S.A.; by the Dpt. Educacion, Universidades e Investigación of the Basque Government, project IT542-10; by program UFI11/09 by the University of the Basque Country, by "Programa de Investigacion Cientifica de la Universidad de La Laguna" (boc-a- 2010-255-7177), and by grants from the Health Institute “Carlos III” (FIS PI08/1383, FIS PI11/00623) to C.F. and co-financed by the European Regional Development Funds, “A way of making Europe” from the European Union. M.P.Y. was supported by a postdoctoral fellowship from Fundación Ramón Areces. We thank the Spanish Banco Nacional de AND (BNADN) (http://www.bancoadn.org/) for providing us with DNA samples from all over Spain. We also thank the Spanish Agencia Estatal de Meteorología (AEMET) (http://www.aemet.es/) for kindly providing us with the UV-B radiation data

Small fiber neuropathy and phosphorylated alpha-synuclein in the skin of E46K- SNCA mutation carriers

Background and objective: In 2004 we described the E46K mutation in alpha-synuclein gene (E46K-SNCA), a rare point mutation causing an aggressive Lewy body disease with early prominent non-motor features and small fiber denervation of myocardium. Considering the potential interest of the skin as a target for the development of biomarkers in Parkinson's Disease (PD), in this work we aimed to evaluate structural and functional integrity of small autonomic nerve fibers and phosphorylated alpha-synuclein (p-synuclein) deposition in the skin of E46K- SNCA carriers as compared to those observed in parkin gene mutation (PARK2) carriers and healthy controls. Patients and methods: We studied 7 E46K-SNCA carriers (3 dementia with Lewy bodies, 2 pure autonomic failure, 1 PD and 1 asymptomatic), 2 PARK2 carriers and 2 healthy controls to quantify intraepidermal nerve fiber density and p-synuclein deposition with cervical skin punch biopsies (immunohistochemistry against anti PGP9.5/UCHL-1, TH and p-synuclein) and sudomotor function with electrochemical skin conductance (ESC) (SudoScan). Results: All E46K-SNCA carriers had moderate to severe p-synuclein deposits and small fiber neurodegeneration in different epidermal and dermal structures including nerve fascicles and glands, especially in carriers with Pure Autonomic Failure, while p-synuclein aggregates where absent in healthy controls and in one of two PARK2 carriers. The severity of the latter skin abnormalities in E46K-SNCA were correlated with sudomotor dysfunction (lower ESC) in hands (p = 0.035). Interpretation: These results together with our previous findings support the relevance of E46K-SNCA mutation as a suitable model to study small fiber neuropathy in Lewy body diseases

Pirin is a prognostic marker of human melanoma that dampens the proliferation of malignant cells by downregulating JARID1B/KDM5B expression

Originally considered to act as a transcriptional co‑factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient’s clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow‑up. Transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co‑transfection experiments and functional analysis. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow‑cycling JARID1B gene.This project was supported by grants from the Basque Government (KK2017-041 and KK2020-00069 to M.D.B.), the UPV/EHU (GIU17/066 to M.D.B.), H2020-ESCEL JTI (15/01 to M.D.B.) and MINECO (PCIN-2015-241 to M.D.B.). CP holds a predoctoral fellowship from the Basque Government. Part of this project is under European patent No. EP3051291 (EP14796149.4): “Method for diagnosis and prognosis of cutaneous melanoma”, Univer- sity of the Basque Country (UPV/EHU). The authors acknowledge the technical support SGIker resources at the UPV/EHU for the computational calculations, which were carried out in the Arina Informatics Cluster. The authors are grateful to the Basque Biobank for providing the biopsy samples and in particular, to María Jesús Fernández and Arantza Perez Dobaran for their technical support with the immunohistochemistry

Serum markers improve current prediction of metastasis development in early‐stage melanoma patients: a machine learning‐based study

Metastasis development represents an important threat for melanoma patients, even when diagnosed at early stages and upon removal of the primary tumor. In this scenario, determination of prognostic biomarkers would be of great interest. Serum contains information about the general status of the organism and therefore represents a valuable source for biomarkers. Thus, we aimed to define serological biomarkers that could be used along with clinical and histopathological features of the disease to predict metastatic events on the early-stage population of patients. We previously demonstrated that in stage II melanoma patients, serum levels of dermcidin (DCD) were associated with metastatic progression. Based on the relevance of the immune response on the cancer progression and the recent association of DCD with local and systemic immune response against cancer cells, serum DCD was analyzed in a new cohort of patients along with interleukin 4 (IL-4), IL-6, IL-10, IL-17A, interferon gamma (IFN-gamma), transforming growth factor-beta (TGF- beta), and granulocyte-macrophage colony-stimulating factor (GM-CSF). We initially recruited 448 melanoma patients, 323 of whom were diagnosed as stages I-II according to AJCC. Levels of selected cytokines were determined by ELISA and Luminex, and obtained data were analyzed employing machine learning and Kaplan-Meier techniques to define an algorithm capable of accurately classifying early-stage melanoma patients with a high and low risk of developing metastasis. The results show that in early-stage melanoma patients, serum levels of the cytokines IL-4, GM-CSF, and DCD together with the Breslow thickness are those that best predict melanoma metastasis. Moreover, resulting algorithm represents a new tool to discriminate subjects with good prognosis from those with high risk for a future metastasis.We are grateful to the Basque Biobank for providing the serum samples. We are also most grateful to Drs Arantza Arrieta and Natalia Maruri (Cruces University Hospital) for their technical support with the serum marker detection. This work was supported by grants from the Basque Government (KK2016-036 and KK2017-041 to MDB), UPV/EHU (GIU17/066 to MDB), H2020-ESCEL JTI (15/01 to MDB), and MINECO (PCIN-2015-241 to MDB

Frequency of 374F in Spain according to the intensity of annual UV irradiation.

<p>A colored map showing the frequency of 374F per UV intensity range and the correlation equations between UV intensity and frequency of 374F. UV ranges (J/m²) include: 23500–25500; 25501–27500; 27501–29500; 29501–31500; 31501–34500; and 43300. The green line corresponds to the correlation equation obtained when the Canary Islands were included. The red line corresponds to the correlation equation after excluding the Canary Islands.</p

Estimation of selection coefficient (s) of allele 314F using the haplotipic data of 60 unrelated CEU individuals from the HapMap.

<p>The highest likelihood was obtained for s = 0.0243 (95% CI, 0.0111–0.0375).</p

SNPs found in the resequencing of <i>SLC45A2</i>.

<p>We show the location of the 3 SNPs found in the coding region of the gene: rs16891982, rs2287949 and rs26722, plus the most frequent SNP in intron 5: rs35397.</p

Intermittent hypoxia is associated with high hypoxia inducible factor-1α but not high vascular endothelial growth factor cell expression in tumors of cutaneous melanoma patients

Epidemiological associations linking between obstructive sleep apnea and poorer solid malignant tumor outcomes have recently emerged. Putative pathways proposed to explain that these associations have included enhanced hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) cell expression in the tumor and altered immune functions via intermittent hypoxia (IH). Here, we examined relationships between HIF-1α and VEGF expression and nocturnal IH in cutaneous melanoma (CM) tumor samples. Prospectively recruited patients with CM tumor samples were included and underwent overnight polygraphy. General clinical features, apnea-hypopnea index (AHI), desaturation index (DI4%), and CM characteristics were recorded. Histochemical assessments of VEGF and HIF-1α were performed, and the percentage of positive cells (0, 75%) was blindly tabulated for VEGF expression, and as 0, 0-5.9, 6.0-10.0, > 10.0% for HIF-1α expression, respectively. Cases with HIF-1α expression > 6% (high expression) were compared with those 75% of cells was compared with those with < 75%. 376 patients were included. High expression of VEGF and HIF-1α were seen in 88.8 and 4.2% of samples, respectively. High expression of VEGF was only associated with increasing age. However, high expression of HIF-1α was significantly associated with age, Breslow index, AHI, and DI4%. Logistic regression showed that DI4% [OR 1.03 (95% CI: 1.01-1.06)] and Breslow index [OR 1.28 (95% CI: 1.18-1.46)], but not AHI, remained independently associated with the presence of high HIF-1α expression. Thus, IH emerges as an independent risk factor for higher HIF-1α expression in CM tumors and is inferentially linked to worse clinical CM prognostic indicators