Incidental pathogenic germline alterations detected through liquid biopsy in patients with solid tumors: prevalence, clinical utility and implications.

Liquid biopsy, a minimally invasive approach for detecting tumor biomarkers in blood, has emerged as a leading-edge technique in cancer precision medicine. New evidence has shown that liquid biopsies can incidentally detect pathogenic germline variants (PGVs) associated with cancer predisposition, including in patients with a cancer for which genetic testing is not recommended. The ability to detect these incidental PGV in cancer patients through liquid biopsy raises important questions regarding the management of this information and its clinical implications. This incidental identification of PGVs raises concerns about cancer predisposition and the potential impact on patient management, not only in terms of providing access to treatment based on the tumor molecular profiling, but also the management of revealing genetic predisposition in patients and families. Understanding how to interpret this information is essential to ensure proper decision-making and to optimize cancer treatment and prevention strategies. In this review we provide a comprehensive summary of current evidence of incidental PGVs in cancer predisposition genes identified by liquid biopsy in patients with cancer. We critically review the methodological considerations of liquid biopsy as a tool for germline diagnosis, clinical utility and potential implications for cancer prevention, treatment, and research.

Analysis of dermoscopic changes of blue nevi on digital follow-up: A 21-year retrospective cohort study.

BACKGROUND: Blue nevi are benign dermal melanocytic proliferations that are often easy to recognize clinically. Rarely, these lesions can display atypical features, suggesting the presence of a malignant blue nevus or mimicking cutaneous metastases of melanoma. OBJECTIVE: To describe the clinical evolution of blue nevi over time and to assess the need for monitoring these lesions. METHODS: We conducted a retrospective cohort study of 103 patients who were followed between December 1998 and November 2019. An artificial intelligence algorithm was used to identify blue nevi from the databases of two digital epiluminescence devices. Changes in the area of each lesion were calculated with a segmentation neural network. RESULTS: We included 123 blue nevi from 103 patients. Most of the lesions segmented, 99 (91.7%), were considered stable. Of the 9 (8.3%) growing blue nevi identified, 2 (1.85%) showed significant growth. The studied growing blue nevi turned out to be cellular blue nevi, presented with a low tumour mutation burden and GNAQ c.626A>T alteration was identified in both lesions. LIMITATIONS: Some clinical variants of blue nevi might not be included. CONCLUSIONS: Most blue nevi remain stable during their evolution. Rarely, they can show progressive growth, although histopathological or molecular signs of malignancy have not been identified.

Monitoring of Donor-Derived Cell-Free DNA by Short Tandem Repeats: Concentration of Total Cell-Free DNA and Fragment Size for Acute Rejection Risk Assessment in Liver Transplantation.

Monitoring of graft function is essential during the first months after liver transplantation (LT), but current liver function tests (LFTs) lack the specificity and sensitivity to ensure an efficient diagnosis of acute rejection (AR). Recently, donor-derived cell-free DNA (ddcfDNA) has emerged as a noninvasive biomarker to assess graft integrity. This study evaluated the feasibility of measuring the ddcfDNA through short tandem repeat (STR) analysis by quantitative fluorescent-polymerase chain reaction (QF-PCR) and to assess the role of the concentration and fragment size of total cfDNA as AR biomarkers. The total concentration and fragment size of cfDNA and the ddcfDNA percentage were monitored in plasma of 20 patients without rejection and 7 patients with T-cell-mediated AR during the first 3 months after LT. The median ddcfDNA percentage was 3-fold higher before AR diagnosis (34.8%; P < 0.001) and moderately higher at AR confirmatory diagnosis (23.8%; P = 0.049) compared with that of nonrejector patients (10.6%), showing a better performance (area under the curve = 84.6%) than conventional LFTs to predict the risk of rejection within the first 2 weeks following LT. The fraction of 100-250-bp cfDNA fragments was higher at AR diagnosis compared with that of nonrejector patients (68.0% versus 57.9%, P = 0.02). STR amplification by QF-PCR may be an alternative strategy for rapid ddcfDNA quantification, which is easily implementable in clinical laboratories. The results of this pilot study indicate that ddcfDNA increases very early, even 1-2 weeks before the diagnosis of AR, and so it could be useful as a prognostic biomarker in improving patient risk stratification.

Common genetic variants associated with melanoma risk or naevus count in patients with wildtype MC1R melanoma.

BACKGROUND: Hypomorphic MC1R variants are the most prevalent genetic determinants of melanoma risk in the white population. However, the genetic background of patients with wildtype (WT) MC1R melanoma is poorly studied. OBJECTIVES: To analyse the role of candidate common genetic variants on the melanoma risk and naevus count in Spanish patients with WT MC1R melanoma. METHODS: We examined 753 individuals with WT MC1R from Spain (497 patients and 256 controls). We used OpenArray reverse-transcriptase polymerase chain reaction to genotype a panel of 221 common genetic variants involved in melanoma, naevogenesis, hormonal pathways and proinflammatory pathways. Genetic models were tested using multivariate logistic regression models. Nonparametric multifactor dimensionality reduction (MDR) was used to detect gene-gene interactions within each biological subgroup of variants. RESULTS: We found that variant rs12913832 in the HERC2 gene, which is associated with blue eye colour, increased melanoma risk in individuals with WT MC1R [odds ratio (OR) 1·97, 95% confidence interval (CI) 1·48-2·63; adjusted P < 0·001; corrected P < 0·001]. We also observed a trend between the rs3798577 variant in the oestrogen receptor alpha gene (ESR1) and a lower naevus count, which was restricted to female patients with WT MC1R (OR 0·51, 95% CI 0·33-0·79; adjusted P = 0·002; corrected P = 0·11). This sex-dependent association was statistically significant in a larger cohort of patients with melanoma regardless of their MC1R status (n = 1497; OR 0·71, 95% CI 0·57-0·88; adjusted P = 0·002), reinforcing the hypothesis of an association between hormonal pathways and susceptibility to melanocytic proliferation. Last, the MDR analysis revealed four genetic combinations associated with melanoma risk or naevus count in patients with WT MC1R. CONCLUSIONS: Our data suggest that epistatic interaction among common variants related to melanocyte biology or proinflammatory pathways might influence melanocytic proliferation in individuals with WT MC1R. What is already known about this topic? Genetic variants in the MC1R gene are the most prevalent melanoma genetic risk factor in the white population. Still, 20-40% of cases of melanoma occur in individuals with wildtype MC1R. Multiple genetic variants have a pleiotropic effect in melanoma and naevogenesis. Additional variants in unexplored pathways might also have a role in melanocytic proliferation in these patients. Epidemiological evidence suggests an association of melanocytic proliferation with hormonal pathways and proinflammatory pathways. What does this study add? Variant rs12913832 in the HERC2 gene, which is associated with blue eye colour, increases the melanoma risk in individuals with wildtype MC1R. Variant rs3798577 in the oestrogen receptor gene is associated with naevus count regardless of the MC1R status in female patients with melanoma. We report epistatic interactions among common genetic variants with a role in modulating the risk of melanoma or the number of naevi in individuals with wildtype MC1R. What is the translational message? We report a potential role of hormonal signalling pathways in melanocytic proliferation, providing a basis for better understanding of sex-based differences observed at the epidemiological level. We show that gene-gene interactions among common genetic variants might be responsible for an increased risk for melanoma development in individuals with a low-risk phenotype, such as darkly pigmented hair and skin.

Inherited duplications of PPP2R3B predispose to nevi and melanoma via a C21orf91-driven proliferative phenotype.

PURPOSE: Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach. METHODS: Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays. RESULTS: We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70. CONCLUSION: This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching.

IRF4 rs12203592 functional variant and melanoma survival.

Inherited genetic factors may modulate clinical outcome in melanoma. Some low-to-medium risk genes in melanoma susceptibility play a role in melanoma outcome. Our aim was to assess the role of the functional IRF4 SNP rs12203592 in melanoma prognosis in two independent sets (Barcelona, N = 493 and Essen, N = 438). Genotype association analyses showed that the IRF4 rs12203592 T allele increased the risk of dying from melanoma in both sets (Barcelona: odds ratio [OR] = 6.53, 95% CI 1.38-30.87, Adj p = 0.032; Essen: OR = 1.68, 95% CI 1.04-2.72, Adj p = 0.035). Survival analyses only showed significance for the Barcelona set (hazard ratio = 4.58, 95% CI 1.11-18.92, Adj p = 0.036). This SNP was also associated with tumour localization, increasing the risk of developing melanoma in head or neck (OR = 1.79, 95% CI 1.07-2.98, Adj p = 0.032) and protecting from developing melanoma in the trunk (OR = 0.59, 95% CI 0.41-0.85, Adj p = 0.004). These findings suggest for the first time that IRF4 rs12203592 plays a role in the modulation of melanoma outcome and confirms its contribution to the localization of the primary tumour.

Inherited functional variants of the lymphocyte receptor CD5 influence melanoma survival.

Despite the recent progress in treatment options, malignant melanoma remains a deadly disease. Besides therapy, inherited factors might modulate clinical outcome, explaining in part widely varying survival rates. T-cell effector function regulators on antitumor immune responses could also influence survival. CD5, a T-cell receptor inhibitory molecule, contributes to the modulation of antimelanoma immune responses as deduced from genetically modified mouse models. The CD5 SNPs rs2241002 (NM_014207.3:c.671C > T, p.Pro224Leu) and rs2229177 (NM_014207.3:c.1412C > T, p.Ala471Val) constitute an ancestral haplotype (Pro224-Ala471) that confers T-cell hyper-responsiveness and worsens clinical autoimmune outcome. The assessment of these SNPs on survival impact from two melanoma patient cohorts (Barcelona, N = 493 and Essen, N = 215) reveals that p.Ala471 correlates with a better outcome (OR= 0.57, 95% CI = 0.33-0.99, Adj. p = 0.043, in Barcelona OR = 0.63, 95% CI = 0.40-1.01, Adj. p = 0.051, in Essen). While, p.Leu224 was associated with increased melanoma-associated mortality in both cohorts (OR = 1.87, 95% CI = 1.07-3.24, Adj. p = 0.030 in Barcelona and OR = 1.84, 95% CI = 1.04-3.26, Adj. p = 0.037, in Essen). Furthermore survival analyses showed that the Pro224-Ala471 haplotype in homozygosis improved melanoma survival in the entire set of patients (HR = 0.27, 95% CI 0.11-0.67, Adj. p = 0.005). These findings highlight the relevance of genetic variability in immune-related genes for clinical outcome in melanoma.

Characterization of individuals at high risk of developing melanoma in Latin America: bases for genetic counseling in melanoma.

PURPOSE: CDKN2A is the main high-risk melanoma-susceptibility gene, but it has been poorly assessed in Latin America. We sought to analyze CDKN2A and MC1R in patients from Latin America with familial and sporadic multiple primary melanoma (SMP) and compare the data with those for patients from Spain to establish bases for melanoma genetic counseling in Latin America. METHODS: CDKN2A and MC1R were sequenced in 186 Latin American patients from Argentina, Brazil, Chile, Mexico, and Uruguay, and in 904 Spanish patients. Clinical and phenotypic data were obtained. RESULTS: Overall, 24 and 14% of melanoma-prone families in Latin America and Spain, respectively, had mutations in CDKN2A. Latin American families had CDKN2A mutations more frequently (P = 0.014) than Spanish ones. Of patients with SMP, 10% of those from Latin America and 8.5% of those from Spain had mutations in CDKN2A (P = 0.623). The most recurrent CDKN2A mutations were c.-34G>T and p.G101W. Latin American patients had fairer hair (P = 0.016) and skin (P < 0.001) and a higher prevalence of MC1R variants (P = 0.003) compared with Spanish patients. CONCLUSION: The inclusion criteria for genetic counseling of melanoma in Latin America may be the same criteria used in Spain, as suggested in areas with low to medium incidence, SMP with at least two melanomas, or families with at least two cases among first- or second-degree relatives.Genet Med 18 7, 727-736.

The MC1R melanoma risk variant p.R160W is associated with Parkinson disease.

Epidemiological studies have reported the co-occurrence of Parkinson disease (PD) and melanoma. Common genetic variants in the MC1R (melanocortin 1 receptor) gene, which determines skin and hair color, are associated with melanoma. Here we investigated whether genetic variants in MC1R modulate the risk of PD by sequencing the entire gene in 870 PD patients and 736 controls ascertained from Spain. We found that the MC1R variant p.R160W (rs1805008) is marginally associated with PD (odds ratio = 2.10, gender- and age-adjusted p = 0.009, Bonferroni-corrected p = 0.063). Our results suggest that MC1R genetic variants modulate the risk of PD disease in the Spanish population.

Time and tumor type (primary or metastatic) do not influence the detection of BRAF/NRAS mutations in formalin fixed paraffin embedded samples from melanomas.

BACKGROUND: BRAF and NRAS mutation detection is crucial for advanced melanoma treatment. Our aim was to evaluate how different characteristics from formalin-fixed paraffin-embedded (FFPE) samples, age of the block or DNA concentration could influence the success of BRAF and NRAS mutational screening. METHODS: DNA was obtained from 144 FFPE samples (62 primary melanoma, 43 sentinel lymph nodes [SLN] and 39 metastasis). BRAF and NRAS were sequenced by Sanger sequencing. RESULTS: Complete sequencing results were obtained from 75% (108/144) of the samples, and at least one gene was sequenced in 89% (128/144) of them. BRAF was mutated in 55% (29/53) and NRAS in 11% (5/45) of the primary melanomas sequenced. DNA concentration correlated with the tumor area used for DNA extraction (mm2) (adj p-value<0.01, r=0.73). The age of the block did not affect sequencing success. In 60% of samples kept for more than 10 years, both BRAF and NRAS were successfully sequenced. CONCLUSIONS: Preserving sufficient tumor area in FFPE blocks is important. It is necessary to keep the FFPE blocks, no matter their age, as they are necessary to decide the best treatment for the melanoma patient.

Fine mapping of genetic susceptibility loci for melanoma reveals a mixture of single variant and multiple variant regions.

At least 17 genomic regions are established as harboring melanoma susceptibility variants, in most instances with genome-wide levels of significance and replication in independent samples. Based on genome-wide single nucleotide polymorphism (SNP) data augmented by imputation to the 1,000 Genomes reference panel, we have fine mapped these regions in over 5,000 individuals with melanoma (mainly from the GenoMEL consortium) and over 7,000 ethnically matched controls. A penalized regression approach was used to discover those SNP markers that most parsimoniously explain the observed association in each genomic region. For the majority of the regions, the signal is best explained by a single SNP, which sometimes, as in the tyrosinase region, is a known functional variant. However in five regions the explanation is more complex. At the CDKN2A locus, for example, there is strong evidence that not only multiple SNPs but also multiple genes are involved. Our results illustrate the variability in the biology underlying genome-wide susceptibility loci and make steps toward accounting for some of the "missing heritability."

Increased prevalence of lung, breast, and pancreatic cancers in addition to melanoma risk in families bearing the cyclin-dependent kinase inhibitor 2A mutation: implications for genetic counseling.

BACKGROUND: Cyclin-dependent kinase inhibitor 2A (CDKN2A) is the major high-risk susceptibility gene for melanoma. OBJECTIVE: We sought to evaluate the effect of CDKN2A mutations in Spanish patients with a high risk of developing melanoma and the association with clinical and family history features. METHODS: A cross-sectional study design was used to analyze the CDKN2A impact in 702 Spanish patients with a high risk of developing melanoma. RESULTS: The CDKN2A mutation prevalence was 8.5% in patients with sporadic multiple primary melanoma and 14.1% in familial melanoma. Number of cases in the family, number of primary melanomas, and age of onset were associated with the presence of CDKN2A mutation. Having a CDKN2A mutation in the family increased the prevalence of other cancers (prevalence ratio [PR] 2.99, P=.012) and prevalence of pancreatic (PR 2.97, P=.006), lung (PR 3.04, P<.001), and breast (PR 2.19, P=.018) cancers but not nephrourologic or colon cancer. LIMITATIONS: Smoking status was not assessed in the individuals with lung cancer. CONCLUSIONS: Melanoma-prone families with mutations in CDKN2A have an increased prevalence of a broad spectrum of cancers including lung, pancreatic, and breast cancer. This information should be included in genetic counseling and cancer prevention programs for CDKN2A mutation carriers.

Multiple primary acral melanomas in two young caucasian patients.

BACKGROUND: Acral melanoma (AM) is still one of the most poorly studied melanomas. It generally presents beyond the fifth decade of life and usually is a BRAF wild-type melanoma. OBJECTIVE: To report the first cases of multiple primary AM in Caucasians. METHODS: Clinical, dermoscopic, pathological and molecular profiles. RESULTS: A healthy 34- year-old male presented an in situ subungual melanoma on his finger, and 22 months later a fast-growing nodular melanoma appeared in an existing nevus on the sole. Both melanomas carried the V600E BRAF mutation. A 19-year-old female patient presented 2 in situ melanomas on different parts of her left foot within a 6-year period of time. The patients have neither familiar melanoma nor germline mutations in CDKN2A/CDK4 genes. CONCLUSION: Multiple AM in Caucasians is very rare. BRAF mutations are possible, especially in a high-risk set of patients with multiple nevi. Specific acral examination must be recommended since AM still suffers delayed detection.

Prevalence and predictors of germline CDKN2A mutations for melanoma cases from Australia, Spain and the United Kingdom.

BACKGROUND: Mutations in the CDKN2A and CDK4 genes predispose to melanoma. From three case-control studies of cutaneous melanoma, we estimated the prevalence and predictors of these mutations for people from regions with widely differing latitudes and melanoma incidence. METHODS: Population-based cases and controls from the United Kingdom (1586 cases, 499 controls) and Australia (596 early-onset cases, 476 controls), and a hospital-based series from Spain (747 cases, 109 controls), were screened for variants in all exons of CDKN2A and the p16INK4A binding domain of CDK4. RESULTS: The prevalence of mutations for people with melanoma was similar across regions: 2.3%, 2.5% and 2.0% for Australia, Spain and the United Kingdom respectively. The strongest predictors of carrying a mutation were having multiple primaries (odds ratio (OR) = 5.4, 95% confidence interval (CI: 2.5, 11.6) for 2 primaries and OR = 32.4 (95% CI: 14.7, 71.2) for 3 or more compared with 1 primary only); and family history (OR = 3.8; 95% CI:1.89, 7.5) for 1 affected first- or second-degree relative and OR = 23.2 (95% CI: 11.3, 47.6) for 2 or more compared with no affected relatives). Only 1.1% of melanoma cases with neither a family history nor multiple primaries had mutations. CONCLUSIONS: There is a low probability (<2%) of detecting a germline CDKN2A mutation in people with melanoma except for those with a strong family history of melanoma (≥2 affected relatives, 25%), three or more primary melanomas (29%), or more than one primary melanoma who also have other affected relatives (27%).

Serum 25-hydroxyvitamin D3 levels and vitamin D receptor variants in melanoma patients from the Mediterranean area of Barcelona.

BACKGROUND: Serum 25-hydroxyvitamin D3 (Vitamin D) insufficiency and single-nucleotide polymorphisms (SNPs) on its receptor, Vitamin D receptor (VDR), have been reported to be involved in melanoma susceptibility in populations mostly from northern countries. OBJECTIVE: To investigate 25-hydroxyvitamin D3 levels and VDR SNPs in melanoma patients from sunny area of Barcelona, two studies were carried out. The first study evaluated the levels of Vitamin D at time of melanoma diagnosis and the second one analyzed the association between VDR genetic variants and risk of having a high nevus number, the strongest phenotypic risk factor for melanoma. METHODS: The levels of 25-hydroxyvitamin D3 in 81 melanoma patients at diagnosis were measured. In a second group of melanoma patients, including 150 with low and 113 with high nevus number, 11 VDR SNPs were analyzed for their association with nevus number. RESULTS: In the first study, 68% of patients had insufficient levels of 25-hydroxyvitamin D3 (<25 ng/ml). Autumn-winter months and fair phototype were associated with 25-hydroxyvitamin D3 insufficiency; after multivariate analysis, season of sampling remained the only independent predictor of 25-hydroxyvitamin D3 levels. In the second study, VDR variant rs2189480 (P = 0.006) was associated with risk of high nevus number whereas rs2239179 (P = 0.044) and rs7975128 (P = 0.0005) were protective against high nevus number. After Bonferroni adjustment only rs7975128 remained significant. In stratified analysis, SNP rs7975128 was found protective against multiple melanomas (P = 0.021). CONCLUSION: This study showed that even in Barcelona, a sunny Mediterranean area, 25-hydroxyvitamin D3 levels were sub-optimal in the majority of melanoma patients at diagnosis. The involvement of VDR in nevi and, in turn, in melanoma susceptibility has also been suggested. Larger studies are needed to confirm our findings.

Role of CPI-17 in restoring skin homoeostasis in cutaneous field of cancerization: effects of topical application of a film-forming medical device containing photolyase and UV filters.

Cutaneous field of cancerization (CFC) is caused in part by the carcinogenic effect of the cyclobutane pyrimidine dimers CPD and 6-4 photoproducts (6-4PPs). Photoreactivation is carried out by photolyases which specifically recognize and repair both photoproducts. The study evaluates the molecular effects of topical application of a film-forming medical device containing photolyase and UV filters on the precancerous field in AK from seven patients. Skin improvement after treatment was confirmed in all patients by histopathological and molecular assessment. A gene set analysis showed that skin recovery was associated with biological processes involved in tissue homoeostasis and cell maintenance. The CFC response was associated with over-expression of the CPI-17 gene, and a dependence on the initial expression level was observed (P = 0.001). Low CPI-17 levels were directly associated with pro-inflammatory genes such as TNF (P = 0.012) and IL-1B (P = 0.07). Our results suggest a role for CPI-17 in restoring skin homoeostasis in CFC lesions.

Benefits of total body photography and digital dermatoscopy ("two-step method of digital follow-up") in the early diagnosis of melanoma in patients at high risk for melanoma.

BACKGROUND: Early detection of melanoma is the best way to improve prognosis. Digital follow-up (DFU) programs of populations at high risk could be an efficient strategy for detecting early melanomas with low morbidity. OBJECTIVE: We sought to report the added value of the use of the "two-step method" (digital total body photography and digital dermatoscopy). METHODS: This was an analysis of the surveillance of 618 patients at high risk for melanoma included in our DFU program from 1999 to 2008. RESULTS: A total of 11,396 lesions were monitored (mean 18.44/patient) during a median follow-up of 96 months (median 10 visits/patient). A total of 1152 lesions, 1.86 per patient, were excised. Almost 70% (798) were lesions previously registered at least twice, whereas 356 (30%) were detected and removed in the same visit. During follow-up, 98 melanomas (8.5% of excised lesions) were diagnosed in 78 patients (12.6%). In all, 53 melanomas were in situ (53.3%), whereas invasive (45) showed a Breslow index of less than 1 mm (median 0.5 mm) and none were ulcerated. LIMITATIONS: Because there are no control groups we cannot determine if the combined use of total body photography and digital dermatoscopy is more beneficial than these techniques used separately. CONCLUSION: DFU with total body photography and dermatoscopy in a selected population at high risk demonstrated the early detection of melanomas with a low rate of excisions. Long-term follow-up is required to allow the detection of slow-growing melanomas. Based on our 10-year experience, melanomas can be diagnosed at any time, suggesting that in a population at high risk for melanoma, DFU should be maintained over time.