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Background: In breast cancer, gene signatures that predict the risk of metastasis after surgical tumor resection are mainly indicative of early events. The purpose of this study was to identify genes linked to metastatic recurrence more than three years after surgery., Methods: Affymetrix HG U133A and Plus 2.0 array datasets with information on metastasis-free, disease-free or overall survival were accessed via public repositories. Time restricted Cox regression models were used to identify genes associated with metastasis during or after the first three years post-surgery (early- and late-type genes). A sequential validation study design, with two non-adjuvantly treated discovery cohorts (n = 409) and one validation cohort (n = 169) was applied and identified genes were further evaluated in tamoxifen-treated breast cancer patients (n = 923), as well as in patients with non-small cell lung (n = 1779), colon (n = 893) and ovarian (n = 922) cancer., Results: Ten late- and 243 early-type genes were identified in adjuvantly untreated breast cancer. Adjustment to clinicopathological factors and an established proliferation-related signature markedly reduced the number of early-type genes to 16, whereas nine late-type genes still remained significant. These nine genes were associated with metastasis-free survival (MFS) also in a non-time restricted model, but not in the early period alone, stressing that their prognostic impact was primarily based on MFS more than three years after surgery. Four of the ten late-type genes, the ribosome-related factors EIF4B, RPL5, RPL3, and the tumor angiogenesis modifier EPN3 were significantly associated with MFS in the late period also in a meta-analysis of tamoxifen-treated breast cancer cohorts. In contrast, only one late-type gene (EPN3) showed consistent survival associations in more than one cohort in the other cancer types, being associated with worse outcome in two non-small cell lung cancer cohorts. No late-type gene was validated in ovarian and colon cancer., Conclusions: Ribosome-related genes were associated with decreased risk of late metastasis in both adjuvantly untreated and tamoxifen-treated breast cancer patients. In contrast, high expression of epsin (EPN3) was associated with increased risk of late metastasis. This is of clinical relevance considering the well-understood role of epsins in tumor angiogenesis and the ongoing development of epsin antagonizing therapies., Competing Interests: The authors have declared that no competing interests exist.

We analysed the whole-genome transcriptional profile of 6 cell lines of dark melanocytes (DM) and 6 of light melanocytes (LM) at basal conditions and after ultraviolet-B (UVB) radiation at different time points to investigate the mechanisms by which melanocytes protect human skin from the damaging effects of UVB. Further, we assessed the effect of different keratinocyte-conditioned media (KCM+ and KCM-) on melanocytes. Our results suggest that an interaction between ribosomal proteins and the P53 signaling pathway may occur in response to UVB in both DM and LM. We also observed that DM and LM show differentially expressed genes after irradiation, in particular at the first 6h after UVB. These are mainly associated with inflammatory reactions, cell survival or melanoma. Furthermore, the culture with KCM+ compared with KCM- had a noticeable effect on LM. This effect includes the activation of various signaling pathways such as the mTOR pathway, involved in the regulation of cell metabolism, growth, proliferation and survival. Finally, the comparison of the transcriptional profiles between LM and DM under basal conditions, and the application of natural selection tests in human populations allowed us to support the significant evolutionary role of MIF and ATP6V0B in the pigmentary phenotype.

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.

Single nucleotide-polymorphisms (SNPs) are a source of diversity among human population, which may be responsible for the different individual susceptibility to diseases and/or response to drugs, among other phenotypic traits. Several low penetrance susceptibility genes associated with malignant melanoma (MM) have been described, including genes related to pigmentation, DNA damage repair and oxidative stress pathways. In the present work, we conducted a candidate gene association study based on proteins and genes whose expression we had detected altered in melanoma cell lines as compared to normal melanocytes. The result was the selection of 88 loci and 384 SNPs, of which 314 fulfilled our quality criteria for a case-control association study. The SNP rs6854854 in ANXA5 was statistically significant after conservative Bonferroni correction when 464 melanoma patients and 400 controls were analyzed in a discovery Phase I. However, this finding could not be replicated in the validation phase, perhaps because the minor allele frequency of SNP rs6854854 varies depending on the geographical region considered. Additionally, a second SNP (rs6431588) located on ILKAP was found to be associated with melanoma after considering a combined set of 1,883 MM cases and 1,358 disease-free controls. The OR was 1.29 (95% CI 1.12-1.48; p-value = 4×10-4). Both SNPs, rs6854854 in ANXA5 and rs6431588 in ILKAP, show population structure, which, assuming that the Spanish population is not significantly structured, suggests a role of these loci on a specific genetic adaptation to different environmental conditions. Furthermore, the biological relevance of these genes in MM is supported by in vitro experiments, which show a decrease in the transcription levels of ANXA5 and ILKAP in melanoma cells compared to normal melanocytes.

Background: Biomarkers of the immune system are currently not used as prognostic factors in breast cancer. We analyzed the association of the B cell/plasma cell marker immunoglobulin kappa C (IGKC) and survival of untreated node-negative breast cancer patients., Material and Methods: IGKC expression was evaluated by immunostaining in a cohort of 335 node-negative breast cancer patients with a median follow-up of 152 months. The prognostic significance of IGKC for disease-free survival (DFS) and breast cancer-specific overall survival (OS) was evaluated with Kaplan-Meier survival analysis as well as univariate and multivariate Cox analysis adjusted for age at diagnosis, pT stage, histological grade, estrogen receptor (ER) status, progesterone receptor (PR) status, Ki-67 and human epidermal growth factor receptor 2 (HER-2) status., Results: 160 patients (47.7%) showed strong expression of IGKC. Univariate analysis showed that IGKC was significantly associated with DFS (P = 0.017, hazard ratio [HR] = 0.570, 95% confidence interval [CI] = 0.360-0.903) and OS (P = 0.011, HR = 0.438, 95% CI = 0.233-0.822) in the entire cohort. The significance of IGKC was especially strong in ER negative and in luminal B carcinomas. In multivariate analysis IGKC retained its significance independent of established clinical factors for DFS (P = 0.004, HR = 0.504, 95% CI = 0.315-0.804) as well as for OS (P = 0.002, HR = 0.371, 95% CI = 0.196-0.705)., Conclusion: Expression of IGKC has an independent protective impact on DFS and OS in node-negative breast cancer.

As the incidence of Malignant Melanoma (MM) reflects an interaction between skin colour and UV exposure, variations in genes implicated in pigmentation and tanning response to UV may be associated with susceptibility to MM. In this study, 363 SNPs in 65 gene regions belonging to the pigmentation pathway have been successfully genotyped using a SNP array. Five hundred and ninety MM cases and 507 controls were analyzed in a discovery phase I. Ten candidate SNPs based on a p-value threshold of 0.01 were identified. Two of them, rs35414 (SLC45A2) and rs2069398 (SILV/CKD2), were statistically significant after conservative Bonferroni correction. The best six SNPs were further tested in an independent Spanish series (624 MM cases and 789 controls). A novel SNP located on the SLC45A2 gene (rs35414) was found to be significantly associated with melanoma in both phase I and phase II (P<0.0001). None of the other five SNPs were replicated in this second phase of the study. However, three SNPs in TYR, SILV/CDK2 and ADAMTS20 genes (rs17793678, rs2069398 and rs1510521 respectively) had an overall p-value<0.05 when considering the whole DNA collection (1214 MM cases and 1296 controls). Both the SLC45A2 and the SILV/CDK2 variants behave as protective alleles, while the TYR and ADAMTS20 variants seem to function as risk alleles. Cumulative effects were detected when these four variants were considered together. Furthermore, individuals carrying two or more mutations in MC1R, a well-known low penetrance melanoma-predisposing gene, had a decreased MM risk if concurrently bearing the SLC45A2 protective variant. To our knowledge, this is the largest study on Spanish sporadic MM cases to date.