Considerations on diagnosis and surveillance measures of PTEN hamartoma tumor syndrome: clinical and genetic study in a series of Spanish patients.

BACKGROUND: The limited knowledge about the PTEN hamartoma tumor syndrome (PHTS) makes its diagnosis a challenging task. We aimed to define the clinical and genetic characteristics of this syndrome in the Spanish population and to identify new genes potentially associated with the disease. RESULTS: We reviewed the clinical data collected through a specific questionnaire in a series of 145 Spanish patients with a phenotypic features compatible with PHTS and performed molecular characterization through several approaches including next generation sequencing and whole exome sequencing (WES). Macrocephaly, mucocutaneous lesions, gastrointestinal polyposis and obesity are prevalent phenotypic features in PHTS and help predict the presence of a PTEN germline variant in our population. We also find that PHTS patients are at risk to develop cancer in childhood or adolescence. Furthermore, we observe a high frequency of variants in exon 1 of PTEN, which are associated with renal cancer and overexpression of KLLN and PTEN. Moreover, WES revealed variants in genes like NEDD4 that merit further research. CONCLUSIONS: This study expands previously reported findings in other PHTS population studies and makes new contributions regarding clinical and molecular aspects of PHTS, which are useful for translation to the clinic and for new research lines.

MicroRNAs Targeting HIF-2α, VEGFR1 and/or VEGFR2 as Potential Predictive Biomarkers for VEGFR Tyrosine Kinase and HIF-2α Inhibitors in Metastatic Clear-Cell Renal Cell Carcinoma.

Metastatic clear-cell renal cell carcinoma (m-ccRCC) is characterized by increased hypoxia-induced factor (HIF)-2α and vascular endothelial growth factor receptor (VEGFR)-dependent angiogenesis through loss of function of the von Hippel-Lindau protein. VEGFR tyrosine kinase inhibitors (VEGFR-TKIs) are a cornerstone of m-ccRCC treatment, and new treatments targeting HIF-2α are currently under investigation. However, predictive biomarkers for these treatments are lacking. In this retrospective cohort study including 109 patients treated with VEGFR-targeted therapies as first-line treatment, we aimed to study the possible predictive function of microRNAs (miRNAs) targeting HIF-2α, VEGFR1 and VEGFR2. We selected miRNAs inversely correlated with HIF-2α, VEGFR1 and/or VEGFR2 expression and with predicted target sites in the respective genes and subsequently studied their impact on therapeutic outcomes. We identified four miRNAs (miR-34c-5p, miR-221-3p, miR-222-3p and miR-3529-3p) inversely correlated with VEGFR1 and/or VEGFR2 expression and associated with tumor shrinkage and progression-free survival (PFS) upon treatment with VEGFR-TKIs, highlighting the potential predictive value of these miRNAs. Moreover, we identified three miRNAs (miR-185-5p, miR-223-3p and miR-3529-3p) inversely correlated with HIF-2α expression and associated with tumor shrinkage and PFS upon treatment with VEGFR-TKIs. These three miRNAs can have a predictive value not only upon treatment with VEGFR-TKIs but possibly also upon treatment with the upcoming HIF-2α inhibitor belzutifan.

MicroRNAs Possibly Involved in the Development of Bone Metastasis in Clear-Cell Renal Cell Carcinoma.

Bone metastasis in clear-cell renal cell carcinoma (ccRCC) leads to substantial morbidity through skeletal related adverse events and implicates worse clinical outcomes. MicroRNAs (miRNA) are small non-protein coding RNA molecules with important regulatory functions in cancer development and metastasis. In this retrospective analysis we present dysregulated miRNA in ccRCC, which are associated with bone metastasis. In particular, miR-23a-3p, miR-27a-3p, miR-20a-5p, and miR-335-3p specifically correlated with the earlier appearance of bone metastasis, compared to metastasis in other organs. In contrast, miR-30b-3p and miR-139-3p were correlated with less occurrence of bone metastasis. These miRNAs are potential biomarkers and attractive targets for miRNA inhibitors or mimics, which could lead to novel therapeutic possibilities for bone targeted treatment in metastatic ccRCC.

A Comprehensive Framework for Uncovering Non-Linearity and Chaos in Financial Markets: Empirical Evidence for Four Major Stock Market Indices.

The presence of chaos in the financial markets has been the subject of a great number of studies, but the results have been contradictory and inconclusive. This research tests for the existence of nonlinear patterns and chaotic nature in four major stock market indices: namely Dow Jones Industrial Average, Ibex 35, Nasdaq-100 and Nikkei 225. To this end, a comprehensive framework has been adopted encompassing a wide range of techniques and the most suitable methods for the analysis of noisy time series. By using daily closing values from January 1992 to July 2013, this study employs twelve techniques and tools of which five are specific to detecting chaos. The findings show no clear evidence of chaos, suggesting that the behavior of financial markets is nonlinear and stochastic.

Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/mTOR axis in metastatic pheochromocytoma/paraganglioma.

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors.

Clinical and Molecular Comparative Study of Colorectal Cancer Based on Age-of-onset and Tumor Location: Two Main Criteria for Subclassifying Colorectal Cancer.

Our aim was to characterize and validate that the location and age of onset of the tumor are both important criteria to classify colorectal cancer (CRC). We analyzed clinical and molecular characteristics of early-onset CRC (EOCRC) and late-onset CRC (LOCRC), and we compared each tumor location between both ages-of-onset. In right-sided colon tumors, early-onset cases showed extensive Lynch syndrome (LS) features, with a relatively low frequency of chromosomal instability (CIN), but a high CpG island methylation phenotype. Nevertheless, late-onset cases showed predominantly sporadic features and microsatellite instability cases due to BRAF mutations. In left colon cancers, the most reliable clinical features were the tendency to develop polyps as well as multiple primary CRC associated with the late-onset subset. Apart from the higher degree of CIN in left-sided early-onset cancers, differential copy number alterations were also observed. Differences among rectal cancers showed that early-onset rectal cancers were diagnosed at later stages, had less association with polyps, and more than half of them were associated with a familial LS component. Stratifying CRC according to both location and age-of-onset criteria is meaningful, not only because it correlates the resulting categories with certain molecular bases, but with the confirmation across larger studies, new therapeutical algorithms could be defined according to this subclassification.

Concomitant Medications and Risk of Chemotherapy-Induced Peripheral Neuropathy.

BACKGROUND: Peripheral neuropathy is the dose-limiting toxicity of many oncology drugs, including paclitaxel. There is large interindividual variability in the neuropathy, and several risk factors have been proposed; however, many have not been replicated. Here we present a comprehensive study aimed at identifying treatment and physiopathology-related paclitaxel-induced neuropathy risk factors in a large cohort of well-characterized patients. PATIENTS AND METHODS: Analyses included 503 patients with breast or ovarian cancer who received paclitaxel treatment. Paclitaxel dose modifications caused by the neuropathy were extracted from medical records and patients self-reported neuropathy symptoms were collected. Multivariate logistic regression analyses were performed to identify concomitant medications and comorbidities associated with paclitaxel-induced neuropathy. RESULTS: Older patients had higher neuropathy: for each increase of 1 year of age, the risk of dose modifications and grade 3 neuropathy increased 4% and 5%, respectively. Cardiovascular drugs increased the risk of paclitaxel dose reductions (odds ratio [OR], 2.51; p = .006), with a stronger association for beta-adrenergic antagonists. The total number of concomitant medications also showed an association with dose modifications (OR, 1.25; p = .012 for each concomitant drug increase). A dose modification predictive model that included the new identified factors gave an area under the curve of 0.74 (p = 1.07 × 10-10). Preexisting nerve compression syndromes seemed to increase neuropathy risk. CONCLUSION: Baseline characteristics of the patients, including age and concomitant medications, could be used to identify individuals at high risk of neuropathy, personalizing chemotherapy treatment and reducing the risk of severe neuropathy. IMPLICATIONS FOR PRACTICE: Peripheral neuropathy is a common adverse effect of many cancer drugs, including chemotherapeutics, targeted therapies, and immune checkpoint inhibitors. About 40% of survivors of cancer have functional deficits caused by this toxicity, some of them irreversible. Currently, there are no effective treatments to prevent or treat this neuropathy. This study, performed in a large cohort of well-characterized patients homogenously treated with paclitaxel, identified concomitant medications, comorbidities, and demographic factors associated with peripheral neuropathy. These factors could serve to identify patients at high risk of severe neuropathy for whom alternative non-neurotoxic alternatives may be considered.

Whole exome sequencing identifies PLEC, EXO5 and DNAH7 as novel susceptibility genes in testicular cancer.

Testicular germ cell tumors (TGCTs) are a clinically and pathologically heterogeneous disease, and little is known of its genetic basis. Only low susceptibility risk loci have been identified for both sporadic and familial cases. Therefore, we tried to identify new susceptibility genes responsible for familial testicular cancer that may contribute to increasing our knowledge about the genetic basis of the disease. Nineteen Spanish families with at least two affected individuals with TGCT were selected. WES was performed on those individuals using an Illumina Hiseq2000 sequencing platform. Data were analyzed under a monogenic and polygenic model of inheritance, and candidate variants were evaluated in a case-control association study performed on 391 Spanish sporadic cases and 1,170 healthy Spanish controls. Results were replicated in a second series consisting of 101 TGCTs from the Cancer Genome Atlas (TGCA) and 27,000 controls from the Exome Aggregation Consortium (ExAC) database. Logistic regression was carried out to analyze the association strength (risk) of candidate variants obtained among cases and controls in different populations. Despite the sample size, we detected a significant earlier age of onset in familial TGCT (28y) than sporadic cases (33y), using a Mann-Whitney U test. We identified significant variants in the comparative study of TGCT cases (391) versus controls (almost 1,170), and three of them [PLEC (OR = 6.28, p = 6.42 × 10-23 ) (p.Arg2016Trp), EXO5 (OR = 3.37, p = 4.82 × 10-09 ) (p.Arg344AlafsTer10) and DNAH7 (OR = 1.64, p = 0.048)] were replicated as potential candidates that may contribute to explaining the genetic basis of TGCT.

Polymorphisms associated with everolimus pharmacokinetics, toxicity and survival in metastatic breast cancer.

PURPOSE: Metastatic breast cancer (MBC) progressing after endocrine therapy frequently activates PI3K/AKT/mTOR pathway. The BOLERO-2 trial showed that everolimus-exemestane achieves increased progression free survival (PFS) compared with exemestane. However, there is great inter-patient variability in toxicity and response to exemestane-everolimus treatment. The objective of this study was to perform an exploratory study analyzing the implication of single nucleotide polymorphisms (SNPs) on outcomes from this treatment through a pharmacogenetic analysis. PATIENTS AND METHODS: Blood was collected from 90 postmenopausal women with hormone receptor-positive, HER2-negative MBC treated with exemestane-everolimus following progression after prior treatment with a non-steroidal aromatase inhibitor. Everolimus pharmacokinetics was measured in 37 patients. Twelve SNPs in genes involved in everolimus pharmacokinetics and pharmacodynamics were genotyped and associations assessed with drug plasma levels, clinically relevant toxicities (non-infectious pneumonitis, mucositis, hyperglycemia and hematological toxicities), dose reductions or treatment suspensions due to toxicity, progression free survival (PFS) and overall survival. RESULTS: We found that CYP3A4 rs35599367 variant (CYP3A4*22 allele) carriers had higher everolimus blood concentration compared to wild type patients (P = 0.019). ABCB1 rs1045642 was associated with risk of mucositis (P = 0.031), while PIK3R1 rs10515074 and RAPTOR rs9906827 were associated with hyperglycemia and non-infectious pneumonitis (P = 0.016 and 0.024, respectively). Furthermore, RAPTOR rs9906827 was associated with PFS (P = 0.006). CONCLUSIONS: CYP3A4*22 allele influenced plasma concentration of everolimus and several SNPs in PI3K/AKT/mTOR pathway genes were associated with treatment toxicities and prognosis. These results require replication, but suggest that germline variation could influence everolimus outcomes in MBC.

PheoSeq: A Targeted Next-Generation Sequencing Assay for Pheochromocytoma and Paraganglioma Diagnostics.

Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed.

Targeted Sequencing Reveals Low-Frequency Variants in EPHA Genes as Markers of Paclitaxel-Induced Peripheral Neuropathy.

Purpose: Neuropathy is the dose-limiting toxicity of paclitaxel and a major cause for decreased quality of life. Genetic factors have been shown to contribute to paclitaxel neuropathy susceptibility; however, the major causes for interindividual differences remain unexplained. In this study, we identified genetic markers associated with paclitaxel-induced neuropathy through massive sequencing of candidate genes.Experimental Design: We sequenced the coding region of 4 EPHA genes, 5 genes involved in paclitaxel pharmacokinetics, and 30 Charcot-Marie-Tooth genes, in 228 cancer patients with no/low neuropathy or high-grade neuropathy during paclitaxel treatment. An independent validation series included 202 paclitaxel-treated patients. Variation-/gene-based analyses were used to compare variant frequencies among neuropathy groups, and Cox regression models were used to analyze neuropathy along treatment.Results: Gene-based analysis identified EPHA6 as the gene most significantly associated with paclitaxel-induced neuropathy. Low-frequency nonsynonymous variants in EPHA6 were present exclusively in patients with high neuropathy, and all affected the ligand-binding domain of the protein. Accumulated dose analysis in the discovery series showed a significantly higher neuropathy risk for EPHA5/6/8 low-frequency nonsynonymous variant carriers [HR, 14.60; 95% confidence interval (CI), 2.33-91.62; P = 0.0042], and an independent cohort confirmed an increased neuropathy risk (HR, 2.07; 95% CI, 1.14-3.77; P = 0.017). Combining the series gave an estimated 2.5-fold higher risk of neuropathy (95% CI, 1.46-4.31; P = 9.1 × 10-4).Conclusions: This first study sequencing EPHA genes revealed that low-frequency variants in EPHA6, EPHA5, and EPHA8 contribute to the susceptibility to paclitaxel-induced neuropathy. Furthermore, EPHA's neuronal injury repair function suggests that these genes might constitute important neuropathy markers for many neurotoxic drugs. Clin Cancer Res; 23(5); 1227-35. ©2016 AACR.

Multilayer OMIC Data in Medullary Thyroid Carcinoma Identifies the STAT3 Pathway as a Potential Therapeutic Target in RETM918T Tumors.

Purpose: Medullary thyroid carcinoma (MTC) is a rare disease with few genetic drivers, and the etiology specific to each known susceptibility mutation remains unknown. Exploiting multilayer genomic data, we focused our interest on the role of aberrant DNA methylation in MTC development.Experimental Design: We performed genome-wide DNA methylation profiling assessing more than 27,000 CpGs in the largest MTC series reported to date, comprising 48 molecularly characterized tumors. mRNA and miRNA expression data were available for 33 and 31 tumors, respectively. Two human MTC cell lines and 101 paraffin-embedded MTCs were used for validation.Results: The most distinctive methylome was observed for RETM918T-related tumors. Integration of methylation data with mRNA and miRNA expression data identified genes negatively regulated by promoter methylation. These in silico findings were confirmed in vitro for PLCB2, DKK4, MMP20, and miR-10a, -30a, and -200c. The mutation-specific aberrant methylation of PLCB2, DKK4, and MMP20 was validated in 25 independent MTCs by bisulfite pyrosequencing. The methylome and transcriptome data underscored JAK/Stat pathway involvement in RETM918T MTCs. Immunostaining [immunohistochemistry (IHC)] for the active form of signaling effector STAT3 was performed in a series of 101 MTCs. As expected, positive IHC was associated with RETM918T-bearing tumors (P < 0.02). Pharmacologic inhibition of STAT3 activity increased the sensitivity to vandetanib of the RETM918T-positive MTC cell line, MZ-CRC-1.Conclusions: Multilayer OMIC data analysis uncovered methylation hallmarks in genetically defined MTCs and revealed JAK/Stat signaling effector STAT3 as a potential therapeutic target for the treatment of RETM918T MTCs. Clin Cancer Res; 23(5); 1334-45. ©2016 AACR.

Genetic variation in the NEIL2 DNA glycosylase gene is associated with oxidative DNA damage in BRCA2 mutation carriers.

In this report, we have tried to gain molecular insight into a single nucleotide polymorphism (SNP) in the NEIL2 gene previously identified as "cancer risk modifier" for BRCA2 mutation carriers. To that end, we studied the role of this SNP (rs804271) on NEIL2 transcriptional regulation, oxidative DNA damage and genome instability in two independent set of samples: The first one was a series of eighty-six BRCA1 and BRCA2 mutation carriers and eighty non-carrier controls in which we evaluated the effect of the SNP on NEIL2 gene expression and oxidative DNA damage accumulation. The second was a set of twenty lymphoblastoid cell lines (LCLs), thirteen BRCA1 mutation carriers and seven non-carriers control, that were used to analyze the correlation between NEIL2 mRNA and/or protein levels, the oxidative and the double stranded break (DSB) DNA damage levels. Our results suggest that an excessive production of NEIL2 enzyme, associated with the SNP, may have a deleterious effect modifying cancer risk susceptibility in BRCA2 mutation carriers. We hypothesize that due to the SNP impact on NEIL2 transcriptional upregulation, a cascade of events may converge in the accumulation of oxidative DNA damage and its posterior conversion into DSBs for this specific group of patients.

Deep sequencing reveals microRNAs predictive of antiangiogenic drug response.

The majority of metastatic renal cell carcinoma (RCC) patients are treated with tyrosine kinase inhibitors (TKI) in first-line treatment; however, a fraction are refractory to these antiangiogenic drugs. MicroRNAs (miRNAs) are regulatory molecules proven to be accurate biomarkers in cancer. Here, we identified miRNAs predictive of progressive disease under TKI treatment through deep sequencing of 74 metastatic clear cell RCC cases uniformly treated with these drugs. Twenty-nine miRNAs were differentially expressed in the tumors of patients who progressed under TKI therapy (P values from 6 × 10-9 to 3 × 10-3). Among 6 miRNAs selected for validation in an independent series, the most relevant associations corresponded to miR-1307-3p, miR-155-5p, and miR-221-3p (P = 4.6 × 10-3, 6.5 × 10-3, and 3.4 × 10-2, respectively). Furthermore, a 2 miRNA-based classifier discriminated individuals with progressive disease upon TKI treatment (AUC = 0.75, 95% CI, 0.64-0.85; P = 1.3 × 10-4) with better predictive value than clinicopathological risk factors commonly used. We also identified miRNAs significantly associated with progression-free survival and overall survival (P = 6.8 × 10-8 and 7.8 × 10-7 for top hits, respectively), and 7 overlapped with early progressive disease. In conclusion, this is the first miRNome comprehensive study, to our knowledge, that demonstrates a predictive value of miRNAs for TKI response and provides a new set of relevant markers that can help rationalize metastatic RCC treatment.

Molecular insights into the OGG1 gene, a cancer risk modifier in BRCA1 and BRCA2 mutations carriers.

We have recently shown that rs2304277 variant in the OGG1 glycosidase gene of the Base Excision Repair pathway can increase ovarian cancer risk in BRCA1 mutation carriers. In the present study, we aimed to explore the role of this genetic variant on different genome instability hallmarks to explain its association with cancer risk.We have evaluated the effect of this polymorphism on OGG1 transcriptional regulation and its contribution to telomere shortening and DNA damage accumulation. For that, we have used a series of 89 BRCA1 and BRCA2 mutation carriers, 74 BRCAX cases, 60 non-carrier controls and 23 lymphoblastoid cell lines (LCL) derived from BRCA1 mutation carriers and non-carriers.We have identified that this SNP is associated to a significant OGG1 transcriptional down regulation independently of the BRCA mutational status and that the variant may exert a synergistic effect together with BRCA1 or BRCA2 mutations on DNA damage and telomere shortening.These results suggest that this variant, could be associated to a higher cancer risk in BRCA1 mutation carriers, due to an OGG1 transcriptional down regulation and its effect on genome instability.

DNA copy number profiling reveals different patterns of chromosomal instability within colorectal cancer according to the age of onset.

Chromosomal instability resulting in copy number alterations is a hallmark of colorectal cancer (CRC). However, few studies have attempted to characterize the chromosomal changes occurring in early-onset CRC in order to compare them with those taking place within the more extensively studied late-onset CRC subset. Our aim was to characterize the genomic profiles of these two groups of colorectal tumors and to compare them to each other. Array comparative genomic hybridization profiling of 146 colorectal tumors (60 early-onset and 86 late-onset) in combination with an unsupervised analysis was used to define common and specific copy number alterations. We found a number of important differences between the chromosomal instability profiles of each age subset. Thus, losses at 1p36, 1p12, 1q21, 9p13, 14q11, 16p13, and 16p12 were significantly more frequent in younger patients, whereas gains at 7q11 and 7q22 were more frequent in older patients. Moreover, the unsupervised analysis stratified the tumors into two clusters, each one of which was enriched in patients from one of the age subsets. Our findings confirm the existence of substantial differences between the chromosomal instability profiles of the two groups which are more important from a qualitative point of view. Further studies are needed to understand the clinicopathological implications of these dissimilarities.

Recommendations for somatic and germline genetic testing of single pheochromocytoma and paraganglioma based on findings from a series of 329 patients.

BACKGROUND: Nowadays, 65-80% of pheochromocytoma and paraganglioma (PPGL) cases are explained by germline or somatic mutations in one of 22 genes. Several genetic testing algorithms have been proposed, but they usually exclude sporadic-PPGLs (S-PPGLs) and none include somatic testing. We aimed to genetically characterise S-PPGL cases and propose an evidence-based algorithm for genetic testing, prioritising DNA source. METHODS: The study included 329 probands fitting three criteria: single PPGL, no syndromic and no PPGL family history. Germline DNA was tested for point mutations in RET and for both point mutation and gross deletions in VHL, the SDH genes, TMEM127, MAX and FH. 99 tumours from patients negative for germline screening were available and tested for RET, VHL, HRAS, EPAS1, MAX and SDHB. RESULTS: Germline mutations were found in 46 (14.0%) patients, being more prevalent in paragangliomas (PGLs) (28.7%) than in pheochromocytomas (PCCs) (4.5%) (p=6.62×10(-10)). Somatic mutations were found in 43% of those tested, being more prevalent in PCCs (48.5%) than in PGLs (32.3%) (p=0.13). A quarter of S-PPGLs had a somatic mutation, regardless of age at presentation. Head and neck PGLs (HN-PGLs) and thoracic-PGLs (T-PGLs) more commonly had germline mutations (p=2.0×10(-4) and p=0.027, respectively). Five of the 29 metastatic cases harboured a somatic mutation, one in HRAS. CONCLUSIONS: We recommend prioritising testing for germline mutations in patients with HN-PGLs and T-PGLs, and for somatic mutations in those with PCC. Biochemical secretion and SDHB-immunohistochemistry should guide genetic screening in abdominal-PGLs. Paediatric and metastatic cases should not be excluded from somatic screening.

Functional and in silico assessment of MAX variants of unknown significance.

UNLABELLED: The presence of germline mutations affecting the MYC-associated protein X (MAX) gene has recently been identified as one of the now 11 major genetic predisposition factors for the development of hereditary pheochromocytoma and/or paraganglioma. Little is known regarding how missense variants of unknown significance (VUS) in MAX affect its pivotal role in the regulation of the MYC/MAX/MXD axis. In the present study, we propose a consensus computational prediction based on five "state-of-the-art" algorithms. We also describe a PC12-based functional assay to assess the effects that 12 MAX VUS may have on MYC's E-box transcriptional activation. For all but two of these 12 VUS, the functional assay and the consensus computational prediction gave consistent results; we classified seven variants as pathogenic and three as nonpathogenic. The introduction of wild-type MAX cDNA into PC12 cells significantly decreased MYC's ability to bind to canonical E-boxes, while pathogenic MAX proteins were not able to fully repress MYC activity. Further clinical and molecular evaluation of variant carriers corroborated the results obtained with our functional assessment. In the absence of clear heritability, clinical information, and molecular data, consensus computational predictions and functional models are able to correctly classify VUS affecting MAX. KEY MESSAGES: A functional assay assesses the effects of MAX VUS over MYC transcriptional activity. A consensus computational prediction and the functional assay show high concordance. Variant carriers' clinical and molecular data support the functional assessment.

Tumor microRNA expression profiling identifies circulating microRNAs for early breast cancer detection.

BACKGROUND: The identification of novel biomarkers for early breast cancer detection would be a great advance. Because of their role in tumorigenesis and stability in body fluids, microRNAs (miRNAs) are emerging as a promising diagnostic tool. Our aim was to identify miRNAs deregulated in breast tumors and evaluate the potential of circulating miRNAs in breast cancer detection. METHODS: We conducted miRNA expression profiling of 1919 human miRNAs in paraffin-embedded tissue from 122 breast tumors and 11 healthy breast tissue samples. Differential expression analysis was performed, and a microarray classifier was generated. The most relevant miRNAs were analyzed in plasma from 26 healthy individuals and 83 patients with breast cancer (36 before and 47 after treatment) and validated in 116 healthy individuals and 114 patients before treatment. RESULTS: We identified a large number of miRNAs deregulated in breast cancer and generated a 25-miRNA microarray classifier that discriminated breast tumors with high diagnostic sensitivity and specificity. Ten miRNAs were selected for further investigation, of which 4 (miR-505-5p, miR-125b-5p, miR-21-5p, and miR-96-5p) were significantly overexpressed in pretreated patients with breast cancer compared with healthy individuals in 2 different series of plasma. MiR-505-5p and miR-96-5p were the most valuable biomarkers (area under the curve 0.72). Moreover, the expression levels of miR-3656, miR-505-5p, and miR-21-5p were decreased in a group of treated patients. CONCLUSIONS: Circulating miRNAs reflect the presence of breast tumors. The identification of deregulated miRNAs in plasma of patients with breast cancer supports the use of circulating miRNAs as a method for early breast cancer detection.

DNA Methylation Profiling in Pheochromocytoma and Paraganglioma Reveals Diagnostic and Prognostic Markers.

PURPOSE: Pheochromocytoma and paraganglioma (PPGL) are rare neuroendocrine tumors, associated with highly variable postoperative evolution. The scarcity of reliable PPGL prognostic markers continues to complicate patient management. In this study, we explored genome-wide DNA methylation patterns in the context of PPGL malignancy to identify novel prognostic markers. EXPERIMENTAL DESIGN: We retrospectively investigated DNA methylation patterns in PPGL with and without metastases using high-throughput DNA methylation profiling data (Illumina 27K) from two large, well-characterized discovery (n = 123; 24 metastatic) and primary validation (n = 154; 24 metastatic) series. Additional validation of candidate CpGs was performed by bisulfite pyrosequencing in a second independent set of 33 paraffin-embedded PPGLs (19 metastatic). RESULTS: Of the initial 86 candidate CpGs, we successfully replicated 52 (47 genes), associated with metastatic PPGL. Of these, 48 CpGs showed significant associations with time to progression even after correcting for SDHB genotype, suggesting their value as prognostic markers independent of genetic background. Hypermethylation of RDBP (negative elongation factor complex member E) in metastatic tumors was further validated by bisulfite pyrosequencing [Δßmetastatic-benign = 0.29, P = 0.003; HR, 1.4; 95% confidence interval (CI), 1.1-2.0; P = 0.018] and may alter transcriptional networks involving (RERG, GPX3, and PDZK1) apoptosis, invasion, and maintenance of DNA integrity. CONCLUSIONS: This is the first large-scale study of DNA methylation in metastatic PPGL that identifies and validates prognostic markers, which could be used for stratifying patients according to risk of developing metastasis. Of the three CpGs selected for further validation, one (RDBP) was clearly confirmed and could be used for stratifying patients according to the risk of developing metastases.