Transposon mutagenesis identifies chromatin modifiers cooperating with Ras in thyroid tumorigenesis and detects ATXN7 as a cancer gene.

Oncogenic RAS mutations are present in 15-30% of thyroid carcinomas. Endogenous expression of mutant Ras is insufficient to initiate thyroid tumorigenesis in murine models, indicating that additional genetic alterations are required. We used Sleeping Beauty (SB) transposon mutagenesis to identify events that cooperate with HrasG12V in thyroid tumor development. Random genomic integration of SB transposons primarily generated loss-of-function events that significantly increased thyroid tumor penetrance in Tpo-Cre/homozygous FR-HrasG12V mice. The thyroid tumors closely phenocopied the histological features of human RAS-driven, poorly differentiated thyroid cancers. Characterization of transposon insertion sites in the SB-induced tumors identified 45 recurrently mutated candidate cancer genes. These mutation profiles were remarkably concordant with mutated cancer genes identified in a large series of human poorly differentiated and anaplastic thyroid cancers screened by next-generation sequencing using the MSK-IMPACT panel of cancer genes, which we modified to include all SB candidates. The disrupted genes primarily clustered in chromatin remodeling functional nodes and in the PI3K pathway. ATXN7, a component of a multiprotein complex with histone acetylase activity, scored as a significant SB hit. It was recurrently mutated in advanced human cancers and significantly co-occurred with RAS or NF1 mutations. Expression of ATXN7 mutants cooperated with oncogenic RAS to induce thyroid cell proliferation, pointing to ATXN7 as a previously unrecognized cancer gene.

Prospective study of the real impact of fusion centered genomic assays in patient management in a national collaborative group: the GETHI-XX-16 study.

PURPOSE: Precision medicine represents a paradigm shift in oncology. Access to genetic testing and targeted therapies is frequently limited. Assays based on DNA sequencing can miss druggable alterations. We aimed to determine the impact of a free access program to RNA tests in patient management. METHODS: We designed a multicenter prospective observational study within the Spanish National Group for Translational Oncology and Rare and Orphan Tumors (GETTHI). Eligible patients were adults with solid cancers that had progressed on standard therapies. Tumor samples were analyzed using two RNA sequencing assays (Trailblaze PharosTM and Archer FusionPlex Solid TumorTM). A central committee evaluated the actionability of genetic alterations and reported the findings to attending physicians, who made the final clinical management decisions. RESULTS: Between November 2016 and April 2019, 395 patients with 41 different tumors across 30 hospitals were included. Molecular analysis revealed actionable genetic alterations in 57 individuals (14.4%). Targeted therapies were advised for 23 and seven received a matched targeted therapy: two lung cancers (EML4-ALK and CD74-ROS1 fusion), three glioblastomas (EGFR point mutations), one oligodendroglioma (FGFR3-TACC3 fusion) and a prostate cancer (SND1-BRAF fusion). The outcomes included two tumor responses, one disease stabilization, one early withdrawal due to toxicity, one progression, and one unknown. CONCLUSION: Despite the growing knowledge of cancer biology and its translation to drug development, the overall impact of personalized treatments remains low. Access to comprehensive molecular tests covering properly all known actionable alterations and programs for a wide access to targeted therapies seem to be critical steps.

Proteomic profiles of peritoneal fluid-derived small extracellular vesicles correlate with patient outcome in ovarian cancer.

Cancer-derived small extracellular vesicles (sEVs) are capable of modifying the tumor microenvironment and promoting tumor progression. Ovarian cancer (OvCa) is a lethal malignancy that preferentially spreads through the abdominal cavity. Thus, the secretion of such vesicles into the peritoneal fluid could be a determinant factor in the dissemination and behavior of this disease. We designed a prospective observational study to assess the impact of peritoneal fluid-derived sEVs (PFD-sEVs) in OvCa clinical outcome. For this purpose, 2 patient cohorts were enrolled: patients with OvCa who underwent a diagnostic or cytoreductive surgery and nononcological patients, who underwent abdominal surgery for benign gynecological conditions and acted as the control group. Systematic extraction of PFD-sEVs from surgical samples enabled us to observe significant quantitative and qualitative differences associated with cancer diagnosis, disease stage, and platinum chemosensitivity. Proteomic profiling of PFD-sEVs led to the identification of molecular pathways and proteins of interest and to the biological validation of S100A4 and STX5. In addition, unsupervised analysis of PFD-sEV proteomic profiles in high-grade serous ovarian carcinomas (HGSOCs) revealed 2 clusters with different outcomes in terms of overall survival. In conclusion, comprehensive characterization of PFD-sEV content provided a prognostic value with potential implications in HGSOC clinical management.

Randomized phase II clinical trial of ruxolitinib plus simvastatin in COVID19 clinical outcome and cytokine evolution.

Background: Managing the inflammatory response to SARS-Cov-2 could prevent respiratory insufficiency. Cytokine profiles could identify cases at risk of severe disease. Methods: We designed a randomized phase II clinical trial to determine whether the combination of ruxolitinib (5 mg twice a day for 7 days followed by 10 mg BID for 7 days) plus simvastatin (40 mg once a day for 14 days), could reduce the incidence of respiratory insufficiency in COVID-19. 48 cytokines were correlated with clinical outcome. Participants: Patients admitted due to COVID-19 infection with mild disease. Results: Up to 92 were included. Mean age was 64 ± 17, and 28 (30%) were female. 11 (22%) patients in the control arm and 6 (12%) in the experimental arm reached an OSCI grade of 5 or higher (p = 0.29). Unsupervised analysis of cytokines detected two clusters (CL-1 and CL-2). CL-1 presented a higher risk of clinical deterioration vs CL-2 (13 [33%] vs 2 [6%] cases, p = 0.009) and death (5 [11%] vs 0 cases, p = 0.059). Supervised Machine Learning (ML) analysis led to a model that predicted patient deterioration 48h before occurrence with a 85% accuracy. Conclusions: Ruxolitinib plus simvastatin did not impact the outcome of COVID-19. Cytokine profiling identified patients at risk of severe COVID-19 and predicted clinical deterioration. Trial registration: https://clinicaltrials.gov/, identifier NCT04348695.

Genome-wide analysis of Pax8 binding provides new insights into thyroid functions.

BACKGROUND: The transcription factor Pax8 is essential for the differentiation of thyroid cells. However, there are few data on genes transcriptionally regulated by Pax8 other than thyroid-related genes. To better understand the role of Pax8 in the biology of thyroid cells, we obtained transcriptional profiles of Pax8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysis RESULTS: Exhaustive analysis of Pax8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of Pax8 in transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify Pax8 partners, including proteins involved in tertiary DNA structure (CTCF) and chromatin remodeling (Sp1), and these direct transcriptional interactions were confirmed in vivo. Moreover, both factors modulate Pax8-dependent transcriptional activation of the sodium iodide symporter (Nis) gene promoter. We ultimately combined putative and novel Pax8 binding sites with actual target gene expression regulation to define Pax8-dependent genes. Functional classification suggests that Pax8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes. CONCLUSION: Our study provides novel insights into the role of Pax8 in thyroid biology, exerted through transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new transcriptional partners of Pax8, which functionally cooperate with Pax8 in the regulation of thyroid gene transcription. Besides, our data demonstrate preferential location of Pax8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism that represents a novel role of Pax8 in the transcriptional output of the thyrocyte.

The variant rs1867277 in FOXE1 gene confers thyroid cancer susceptibility through the recruitment of USF1/USF2 transcription factors.

In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30-1.70; P = 5.9x10(-9)). Functional assays of rs1867277 (NM_004473.3:c.-283G>A) within the FOXE1 5' UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/alphaCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.

MEK inhibitor sensitivity in BRAF fusion-driven prostate cancer.

PURPOSE: The identification of subpopulations harboring druggable targets has become a major step forward in the subclassification of solid tumors into small groups suitable for specific therapies. BRAF fusions represent a paradigm of uncommon and targetable oncogenic events and have been widely correlated to the development of specific malignancies. However, they are only present in a limited frequency across most common tumor types. At this regard, we performed a genomic screening aimed to identifying rare variants associated to advanced prostate cancer development. METHODS: Tumoral tissue genomic screening of 41 patients developing advanced prostate cancer was performed at our center as part of the GETHI XX study. The project, sponsored by the Spanish Collaborative Group in Rare Cancers (GETHI), aims to analyze the molecular background of rare tumors and to discover unfrequent molecular variants in common tumors. RESULTS: Here we present the clinical outcome and an in-deep molecular analysis performed in a case harboring a SND1-BRAF fusion gene. The identification of such rearrangement in a patient refractory to standard therapies led to the administration of trametinib (MEK inhibitor). Despite unsensitive to standard therapies, the patient achieved a dramatic response to trametinib. A comprehensive study of the tumor demonstrated this event to be a trunk alteration with higher expression of MEK in areas of tumor invasion. CONCLUSIONS: Our study describes the patient-driven discovery of the first BRAF fusion-driven prostate cancer effectively treated with trametinib. Consequently, MAPK pathway activation could define a new subtype of prostate cancer susceptible to a tailored management.

Comprehensive molecular analysis of immortalization hallmarks in thyroid cancer reveals new prognostic markers.

BACKGROUND: Comprehensive molecular studies on tumours are needed to delineate immortalization process steps and identify sensitive prognostic biomarkers in thyroid cancer. METHODS AND RESULTS: In this study, we extensively characterize telomere-related alterations in a series of 106 thyroid tumours with heterogeneous clinical outcomes. Using a custom-designed RNA-seq panel, we identified five telomerase holoenzyme-complex genes upregulated in clinically aggressive tumours compared to tumours from long-term disease-free patients, being TERT and TERC denoted as independent prognostic markers by multivariate regression model analysis. Characterization of alterations related to TERT re-expression revealed that promoter mutations, methylation and/or copy gains exclusively co-occurred in clinically aggressive tumours. Quantitative-FISH (fluorescence in situ hybridization) analysis of telomere lengths showed a significant shortening in these carcinomas, which matched with a high proliferative rate measured by Ki-67 immunohistochemistry. RNA-seq data analysis indicated that short-telomere tumours exhibit an increased transcriptional activity in the 5-Mb-subtelomeric regions, site of several telomerase-complex genes. Gene upregulation enrichment was significant for specific chromosome-ends such as the 5p, where TERT is located. Co-FISH analysis of 5p-end and TERT loci showed a more relaxed chromatin configuration in short telomere-length tumours compared to normal telomere-length tumours. CONCLUSIONS: Overall, our findings support that telomere shortening leads to a 5p subtelomeric region reorganization, facilitating the transcription and accumulation of alterations at TERT-locus.

MCM3 is a novel proliferation marker associated with longer survival for patients with tubo-ovarian high-grade serous carcinoma.

Tubo-ovarian high-grade serous carcinomas (HGSC) are highly proliferative neoplasms that generally respond well to platinum/taxane chemotherapy. We recently identified minichromosome maintenance complex component 3 (MCM3), which is involved in the initiation of DNA replication and proliferation, as a favorable prognostic marker in HGSC. Our objective was to further validate whether MCM3 mRNA expression and possibly MCM3 protein levels are associated with survival in patients with HGSC. MCM3 mRNA expression was measured using NanoString expression profiling on formalin-fixed and paraffin-embedded tissue (N = 2355 HGSC) and MCM3 protein expression was assessed by immunohistochemistry (N = 522 HGSC) and compared with Ki-67. Kaplan-Meier curves and the Cox proportional hazards model were used to estimate associations with survival. Among chemotherapy-naïve HGSC, higher MCM3 mRNA expression (one standard deviation increase in the score) was associated with longer overall survival (HR = 0.87, 95% CI 0.81-0.92, p < 0.0001, N = 1840) in multivariable analysis. MCM3 mRNA expression was highest in the HGSC C5.PRO molecular subtype, although no interaction was observed between MCM3, survival and molecular subtypes. MCM3 and Ki-67 protein levels were significantly lower after exposure to neoadjuvant chemotherapy compared to chemotherapy-naïve tumors: 37.0% versus 46.4% and 22.9% versus 34.2%, respectively. Among chemotherapy-naïve HGSC, high MCM3 protein levels were also associated with significantly longer disease-specific survival (HR = 0.52, 95% CI 0.36-0.74, p = 0.0003, N = 392) compared to cases with low MCM3 protein levels in multivariable analysis. MCM3 immunohistochemistry is a promising surrogate marker of proliferation in HGSC.

Association study of 69 genes in the ret pathway identifies low-penetrance loci in sporadic medullary thyroid carcinoma.

To date, few association studies have been done to better understand the genetic basis for the development of sporadic medullary thyroid carcinoma (sMTC). To identify additional low-penetrance genes, we have done a two-stage case-control study in two European populations using high-throughput genotyping. We selected 417 single nucleotide polymorphisms (SNP) belonging to 69 genes either related to RET signaling pathway/functions or involved in key processes for cancer development. TagSNPs and functional variants were included where possible. These SNPs were initially studied in the largest known series of sMTC cases (n = 266) and controls (n = 422), all of Spanish origin. In stage II, an independent British series of 155 sMTC patients and 531 controls was included to validate the previous results. Associations were assessed by an exhaustive analysis of individual SNPs but also considering gene- and linkage disequilibrium-based haplotypes. This strategy allowed us to identify seven low-penetrance genes, six of them (STAT1, AURKA, BCL2, CDKN2B, CDK6, and COMT) consistently associated with sMTC risk in the two case-control series and a seventh (HRAS) with individual SNPs and haplotypes associated with sMTC in the Spanish data set. The potential role of CDKN2B was confirmed by a functional assay showing a role of a SNP (rs7044859) in the promoter region in altering the binding of the transcription factor HNF1. These results highlight the utility of association studies using homogeneous series of cases for better understanding complex diseases.

CREBBP/EP300 Bromodomain Inhibition Affects the Proliferation of AR-Positive Breast Cancer Cell Lines.

Inhibitors that prevent the binding of bromodomains to acetylated histones hold therapeutic potential. However, the effects of targeting most of the 60 different bromodomains found in the human proteome remain unexplored. Here, we investigate the molecular mechanisms responsible for the antiproliferative properties of CREBBP/EP300 bromodomain inhibition in ER-negative breast cancer cell lines. We show using genetic and chemical approaches that CREBBP/EP300 bromodomains are critical to support the proliferation of the triple-negative breast cancer cell line MDA-MB-453. Analysis of the transcriptional pathways affected by CREBBP/EP300 bromodomain inhibitors reveals that the expression of genes associated with super-enhancers is downregulated, which in turn are occupied by very high levels of androgen receptor (AR) in MDA-MB-453 cells. Treatment of MDA-MB-453 with CREBBP/EP300 bromodomain inhibitors downregulates the expression of an AR-dependent signature distinctive of breast cancer tumors that express AR and causes a decrease in H3K27ac levels at AR-binding sites. In accordance, in prostate cancer cell lines that express AR CREBBP/EP300 bromodomain inhibitors downregulate the expression of genes bound by AR and associated with super-enhancers. In summary, we report that triple-negative breast cancer cell lines that express AR are particularly sensitive to CREBBP/EP300 bromodomain inhibitors and consequently these inhibitors hold potential to treat this type of cancer. IMPLICATIONS: AR-dependent cancer cell lines are sensitive to CREBBP/EP300 bromodomain inhibitors.

CREBBP/EP300 bromodomains are critical to sustain the GATA1/MYC regulatory axis in proliferation.

BACKGROUND: The reported antitumor activity of the BET family bromodomain inhibitors has prompted the development of inhibitors against other bromodomains. However, the human genome encodes more than 60 different bromodomains and most of them remain unexplored. RESULTS: We report that the bromodomains of the histone acetyltransferases CREBBP/EP300 are critical to sustain the proliferation of human leukemia and lymphoma cell lines. EP300 is very abundant at super-enhancers in K562 and is coincident with sites of GATA1 and MYC occupancy. In accordance, CREBBP/EP300 bromodomain inhibitors interfere with GATA1- and MYC-driven transcription, causing the accumulation of cells in the G0/G1 phase of the cell cycle. The CREBBP/CBP30 bromodomain inhibitor CBP30 displaces CREBBP and EP300 from GATA1 and MYC binding sites at enhancers, resulting in a decrease in the levels of histone acetylation at these regulatory regions and consequently reduced gene expression of critical genes controlled by these transcription factors. CONCLUSIONS: Our data shows that inhibition of CREBBP/EP300 bromodomains can interfere with oncogene-driven transcriptional programs in cancer cells and consequently hold therapeutic potential.

Loss of the actin regulator HSPC300 results in clear cell renal cell carcinoma protection in Von Hippel-Lindau patients.

Clear cell renal cell carcinoma (ccRCC) is the most common malignant neoplasm of the kidney. The majority of hereditary and sporadic ccRCC cases are associated with germline and somatic mutations in the Von Hippel-Lindau gene (VHL), respectively. Gross deletions at the VHL locus can result either in ccRCC or in a mild clinical phenotype, with the absence of ccRCC development. Our goal in this study was to identify the molecular basis responsible for these differences in the clinical behavior in order to predict patients' phenotype. Using multiplex ligation-dependent amplification (MLPA), we identified and characterized gross VHL deletions in Spanish VHL families. A candidate gene related to this clinical association, HSPC300, was identified and depleted by RNA interference. It was possible to narrow the susceptibility region related to the mild clinical phenotype down to approximately 14 kb that included HSPC300 (C3orf10), a regulator of actin dynamics and cytoskeleton organization. Whereas 9 out of 10 families with ccRCC retained HSPC300 in the germline, loss of the HSPC300 locus was associated with mild clinical presentation of the disease in 6 out of 8 families. In fact, genetic depletion of HSPC300 resulted in cytoskeleton abnormalities and cytokinesis arrest in several tumor cell lines including ccRCC cells, suggesting that tumor cell proliferation was compromised in the absence of HSPC300. These clinical and functional data indicate a relevant function of HSPC300 in tumor cell progression, and suggest future therapeutic strategies based upon the inhibition of HSPC300 in renal cell carcinoma and possibly on other cancers.

PupaSNP Finder: a web tool for finding SNPs with putative effect at transcriptional level.

We have developed a web tool, PupaSNP Finder (PupaSNP for short), for high-throughput searching for single nucleotide polymorphisms (SNPs) with potential phenotypic effect. PupaSNP takes as its input lists of genes (or generates them from chromosomal coordinates) and retrieves SNPs that could affect the conserved regions that the cellular machinery uses for the correct processing of genes (intron/exon boundaries or exonic splicing enhancers), predicted transcription factor binding sites (TFBS) and changes in amino acids in the proteins. The program uses the mapping of SNPs in the genome provided by Ensembl. Additionally, user-defined SNPs (not yet mapped in the genome) can be easily provided to the program. Also, additional functional information from Gene Ontology, OMIM and homologies in other model organisms is provided. In contrast to other programs already available, which focus only on SNPs with possible effect in the protein, PupaSNP includes SNPs with possible transcriptional effect. PupaSNP will be of significant help in studies of multifactorial disorders, where the use of functional SNPs will increase the sensitivity of identification of the genes responsible for the disease. The PupaSNP web interface is accessible through http://pupasnp.bioinfo.cnio.es.

NSD2 contributes to oncogenic RAS-driven transcription in lung cancer cells through long-range epigenetic activation.

The histone methyltransferase NSD2/WHSC1/MMSET is overexpressed in a number of solid tumors but its contribution to the biology of these tumors is not well understood. Here, we describe that NSD2 contributes to the proliferation of a subset of lung cancer cell lines by supporting oncogenic RAS transcriptional responses. NSD2 knock down combined with MEK or BRD4 inhibitors causes co-operative inhibitory responses on cell growth. However, while MEK and BRD4 inhibitors converge in the downregulation of genes associated with cancer-acquired super-enhancers, NSD2 inhibition affects the expression of clusters of genes embedded in megabase-scale regions marked with H3K36me2 and that contribute to the RAS transcription program. Thus, combinatorial therapies using MEK or BRD4 inhibitors together with NSD2 inhibition are likely to be needed to ensure a more comprehensive inhibition of oncogenic RAS-driven transcription programs in lung cancers with NSD2 overexpression.

Succinate dehydrogenase D variants do not constitute a risk factor for developing C cell hyperplasia or sporadic medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a tumor that arises from parafollicular cells of the thyroid gland. MTC can occur sporadically (75%) or as part of inherited cancer syndromes (25%). In most cases, hereditary MTC evolves from preneoplastic C cell hyperplasia (CCH), so early detection of this pathology would evidently be critical. A recent study reports that alterations in succinate dehydrogenase (SDH) D are responsible for familial non-RET CCH. First, we studied SDHD in two families with hereditary non-RET CCH and found no alterations related to the inheritance of this disease. Then, we investigated whether the H50R variant could be a risk factor in the sporadic development of MTC in both Spanish and English patients. We found no evidence that the presence of the H50R is strongly associated with the risk of sporadic MTC, although we did observe an association with age at diagnosis of MTC in Spanish H50R carriers that we did not find in English patients. Finally, we looked for evidence of CCH or any other thyroid disease in a panel of germ-line SDH (B or D) mutation carriers and found none. We conclude that SDHD variants do not constitute a risk factor for developing CCH or sporadic MTC.

Genetic characterization and structural analysis of VHL Spanish families to define genotype-phenotype correlations.

Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations in the VHL gene. This gene, located in the 3p25-26 chromosome, is a tumor suppressor gene associated with the inhibition of angiogenesis and apoptosis, cell cycle exit, fibronectin matrix assembly, and proteolysis. To define the molecular basis of VHL in a Spanish population, we studied 33 patients suspected of suffering familial or de novo VHL disease and two familial pheochromocytoma cases. Sequence analysis of the coding regions of the VHL gene revealed germline sequence variants in 68.7% (24 out of 35) of the patients, and four of them presented with undescribed germline alterations: g.5429-5430insG, p.Leu128Arg, p.Tyr175Cys, and p.Tyr175Asn. For the remaining 11 patients who showed negative for point mutations, we performed Southern blot analysis and detected gross rearrangements in eight cases (22.8% of the index cases). Our results support the relevance of VHL gene analysis in familial pheochromocytoma cases and also in those with no familial history. In order to investigate the relevance of different amino acid changes in the VHL phenotype, we also analyzed the genotype-phenotype correlations using structural analysis to assess protein stability and complexes. The association of clear cell renal carcinoma (CCRC) development with a relatively high loss of structural stability in pVHL missense-mutants was consistent. Structural stability data in the genotype-phenotype correlations therefore provides us with a better understanding of VHL clinical implications. It is also a suitable approach to the evaluation of unknown significance changes.

Identification of novel SDHD mutations in patients with phaeochromocytoma and/or paraganglioma.

Familial paraganglioma is a dominantly inherited disorder characterised by the development of highly vascular tumours in the head and neck. Recently, a relationship between hereditary tumours derived from the autonomic nervous system and germline mutations in the gene encoding succinate dehydrogenase complex subunit D (SDHD) is increasingly a subject of study. Familial paraganglioma syndrome is embryologically related to phaeochromocytoma, another neuroendocrine tumour that shows great aetiological and genetic heterogeneity. Some hereditary phaeochromocytomas may be associated with germline mutations in VHL, RET and NF1 genes in genetic disorders such as von Hippel-Lindau disease (VHL), multiple endocrine neoplasia type 2 (MEN 2) and neurofibromatosis type 1 (NF 1), respectively. However, there are many cases that cannot be explained by mutations in these genes. In this report, we describe two previously unreported mutations in two patients from 25 unrelated kindreds with phaeochromocytoma and/or paraganglioma disorders and with or without familial antecedents: a mutation featuring the change of tryptophan to a termination codon in exon 2, and a 4-bp deletion in exon 4 that results in a truncated protein. We also describe one missense substitution of uncertain significance. The patients had previously tested negative for germline mutations in VHL and RET genes and had not been previously selected. The involvement of SDHD mutations in familial phaeochromocytoma and/or paraganglioma predisposition is of considerable interest since other studies have shown these alterations to be associated with highly expressed angiogenic factors.

Relief of feedback inhibition of HER3 transcription by RAF and MEK inhibitors attenuates their antitumor effects in BRAF-mutant thyroid carcinomas.

The RAF inhibitor vemurafenib (PLX4032) increases survival in patients with BRAF-mutant metastatic melanoma, but has limited efficacy in patients with colorectal cancers. Thyroid cancer cells are also comparatively refractory to RAF inhibitors. In contrast to melanomas, inhibition of mitogen-activated protein kinase (MAPK) signaling by PLX4032 is transient in thyroid and colorectal cancer cells. The rebound in extracellular signal-regulated kinase (ERK) in thyroid cells is accompanied by increased HER3 signaling caused by induction of ERBB3 (HER3) transcription through decreased promoter occupancy by the transcriptional repressors C-terminal binding protein 1 and 2 and by autocrine secretion of neuregulin-1 (NRG1). The HER kinase inhibitor lapatinib prevents MAPK rebound and sensitizes BRAF-mutant thyroid cancer cells to RAF or MAP-ERK kinase inhibitors. This provides a rationale for combining ERK pathway antagonists with inhibitors of feedback-reactivated HER signaling in this disease. The determinants of primary resistance to MAPK inhibitors vary between cancer types, due to preferential upregulation of specific receptor tyrosine kinases, and the abundance of their respective ligands.

An epistatic interaction between the PAX8 and STK17B genes in papillary thyroid cancer susceptibility.

Papillary Thyroid Cancer (PTC) is a heterogeneous and complex disease; susceptibility to PTC is influenced by the joint effects of multiple common, low-penetrance genes, although relatively few have been identified to date. Here we applied a rigorous combined approach to assess both the individual and epistatic contributions of genetic factors to PTC susceptibility, based on one of the largest series of thyroid cancer cases described to date. In addition to identifying the involvement of TSHR variation in classic PTC, our pioneer study of epistasis revealed a significant interaction between variants in STK17B and PAX8. The interaction was detected by MD-MBR (p = 0.00010) and confirmed by other methods, and then replicated in a second independent series of patients (MD-MBR p = 0.017). Furthermore, we demonstrated an inverse correlation between expression of PAX8 and STK17B in a set of cell lines derived from human thyroid carcinomas. Overall, our work sheds additional light on the genetic basis of thyroid cancer susceptibility, and suggests a new direction for the exploration of the inherited genetic contribution to disease using association studies.