Genomic and genetic dissection of thyroid cancer

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 17-06-2015Thyroid tumors can have two cellular origins and a variety of genetic drivers. Thus, thyroid cancer (TC) is a complex and heterogeneous disease. As much of its etiology remains poorly explored, TC represents an attractive model to study cancer disease process. Herein, we have coupled exhaustive genomic dissection of an exceptional collection of human samples to their genetic characterization, and comprehensive data analysis in order to address several aspects of clinical interest. In the first part of the study focused on follicular cell-derived cancer, by performing a two-step association study involving 1,820 cases and 2,410 controls, we provide novel insights into the genetic susceptibility of this disease. Apart from underscoring the importance of 9q22.33 locus in disease risk, we identify novel associations at 10q26.12 and 6q14.1 and highlight that genetic heterogeneity between populations could be a part of this disease’s hidden heritability. Moreover, we describe the genomic landscape of a total of 165 follicular cellderived tumors including both papillary and follicular cases. We identify distinct molecular subgroups closely related to oncogenic drivers, and explore the diagnostic and prognostic utility of several genomic features. According to our results, the methylome and miRNome of benign and malignant disease is largely overlapping, which prevents from diagnostic markers’ identification. Of note, elevated promoter methylation of WT1 and EI24, and aberrant expression of let-7a and miR-192 could serve as potential novel molecular markers of shorter time to progression. The second part of this study is focused on the less frequent tumors of the gland arising from C-cells, named medullary thyroid carcinomas (MTC). By characterizing the MTC methylome, we have complemented the genomic dissection of an outstanding collection of 64 frozen and confirmed that this disease comprises of several molecular entities closely related to the underlying mutations. Moreover, taking advantage of a series composed of 103 paraffin embedded tumors we show that even tyrosine kinase inhibitors’ (TKI) targets expression differs according to these mutations, and a priori genetic screening of MTC patients appears advisable to guide the selection of the most suitable TKI treatment. To summarize, we have performed genetic and genomic characterization of almost the whole spectrum of thyroid tumors. Our studies have revealed much of molecular mechanisms behind these tumors and shown that they tend to be tightly linked to the causal driver mutations. On the whole, these results are yet another example of the great potential that lies in highthroughput techniques to decipher disease etiology, and discover disease markers.Los tumores tiroideos pueden tener dos orígenes celulares y estar causados por una gran variedad de mutaciones genéticas, siendo entonces el cáncer de tiroides (CT) una enfermedad compleja y heterogénea. Gran parte de su etiología sigue siendo poco explorada, y el CT representa un modelo atractivo de estudio. En este trabajo, se ha llevado a cabo una disección exhaustiva de aspectos genómicos de una colección sobresaliente de muestras humanas caracterizadas genéticamente, con el fin de abordar varios aspectos de interés clínico. La primera parte del estudio se centra en el cáncer derivado de célula folicular. Mediante un estudio de asociación en dos etapas que incluía 1,820 casos y 2,410 controles, pudimos confirmar la importancia del locus 9q22.33 en el riesgo de la enfermedad, e identificamos nuevas asociaciones en 10q26.12 y 6q14.1. Nuestros datos sugieren que la heterogeneidad genética entre poblaciones podría en parte explicar la falta de replicación en distintos estudios. Además, describimos las características genómicas de 165 tumores derivados de célula folicular incluyendo tanto casos con patrón de crecimiento papilar como folicular. Identificamos subgrupos moleculares específicamente relacionados con mutaciones concretas, y exploramos la utilidad diagnóstica y pronostica de varias de las características genómicas estudiadas. Así, el metiloma y el miRNoma de enfermedades benignas y malignas se solapan en gran medida, lo cual impide la identificación de marcadores diagnósticos. Sin embargo, la hipermetilación del promotor de WT1 y EI24, y la expresión aberrante de let-7a y miR-192 podrían servir como nuevos marcadores moleculares para predecir el tiempo hasta la progresión. La segunda parte del estudio se centra en carcinomas tiroideos derivados de las células C, llamados carcinoma medular de tiroides (CMT). Se caracterizó el metiloma de 48 CMT congelados, complementando resultados previos de transcriptoma y miRNoma. Esta enfermedad se compone de varias entidades moleculares estrechamente relacionadas con las mutaciones subyacentes. Utilizando una colección de 103 tumores embebidos en parafina demostramos que la expresión de dianas de inhibidores tirosina quinasas difiere de acuerdo a la mutación. Por tanto, una caracterización genética previa de los pacientes con CMT parece aconsejable para guiar la selección del tratamiento más adecuado. En resumen, hemos caracterizado genética y genómicamente casi todo el espectro de tumores tiroideos. Nuestros estudios han revelado mecanismos moleculares involucrados en su desarrollo y han demostrado que los perfiles genómicos tienden a estar estrechamente vinculados a mutaciones causales. En conjunto, estos resultados son una muestra del gran potencial de las técnicas de alto rendimiento para descifrar la etiología de la enfermedad, y descubrir marcadores de la enfermedad.This work was supported by the following grants and fellowships: La Caixa/CNIO International PhD Fellowship, 2011-2015; Veronika Mančíková. Project PI11/01359 from Fondo de Investigaciones Sanitarias (FIS), Institute of Health.Carlos III.Project AP2775/2008 from Mutua Madrileña Foundation. Project PI14/00240 from Fondo de Investigaciones Sanitarias (FIS), Institute of Health Carlos III. Project S2011/BMD-2328 from the Community of Madri

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

El artículo original ha sido publicado: Landa I, Boullosa C, Inglada-Pe´rez L, Sastre-Perona A, Pastor S, et al. (2013) An Epistatic Interaction between the PAX8 and STK17B Genes in Papillary Thyroid Cancer Susceptibility. PLoS ONE 8(9): e74765. doi:10.1371/journal.pone.0074765

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

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 models, and define specific therapeutic options according to tumor genomic features. : 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 . : 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, =4.67·10), and was found associated 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 a repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. : 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

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

MicroRNA deep-sequencing reveals master regulators of follicular and papillary thyroid tumors

et al.MicroRNA deregulation could be a crucial event in thyroid carcinogenesis. However, current knowledge is based on studies that have used inherently biased methods. Thus, we aimed to define in an unbiased way a list of deregulated microRNAs in well-differentiated thyroid cancer in order to identify diagnostic and prognostic markers. We performed a microRNA deep-sequencing study using the largest well-differentiated thyroid tumor collection reported to date, comprising 127 molecularly characterized tumors with follicular or papillary patterns of growth and available clinical follow-up data, and 17 normal tissue samples. Furthermore, we integrated microRNA and gene expression data for the same tumors to propose targets for the novel molecules identified. Two main microRNA expression profiles were identified: one common for follicular-pattern tumors, and a second for papillary tumors. Follicular tumors showed a notable overexpression of several members of miR-515 family, and downregulation of the novel microRNA miR-1247. Among papillary tumors, top upregulated microRNAs were miR-146b and the miR-221∼222 cluster, while miR-1179 was downregulated. BRAF-positive samples displayed extreme downregulation of miR-7 and -204. The identification of the predicted targets for the novel molecules gave insights into the proliferative potential of the transformed follicular cell. Finally, by integrating clinical follow-up information with microRNA expression, we propose a prediction model for disease relapse based on expression of two miRNAs (miR-192 and let-7a) and several other clinicopathological features. This comprehensive study complements the existing knowledge about deregulated microRNAs in the development of well-differentiated thyroid cancer and identifies novel markers associated with recurrence-free survival.This work was supported in part by the Fondo de Investigaciones Sanitarias (projects PI11/01359, PI14/00240, PI11/01354, RD12/0036/0030 and RD12/0036/0013), the Fundacion Mutua Madrilena (project AP2775/2008), the Comunidad de Madrid S2011/BMD-2328 TIRONET, and the Spanish Ministry of Economy and Competitiveness (projects SAF2011/23638 and SAF2013/44709R). VM and AAdC are predoctoral fellows of ‘la Caixa’/CNIO international PhD programme. LI-P is a predoctoral fellow of the CIBERER.Peer Reviewe

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

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.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. © 2013 Landa et al.This work was supported by Grants from the Fondo de Investigaciones Sanitarias (FIS) project PI11/01359 (to MR), the Red Temática de Investigación Cooperativa en Cáncer, the Instituto de Salud Carlos III, RD12/0030/0060 (to PS), RD12/0036/0050 (to NM) and S2011/BMD-2328 TIRONET from the Comunidad de Madrid (to MR and PS), and European Regional Development Fund. IL is supported by FIS grant FI07/00326; CB is supported by the FPI grant BES-2008-006332; LI-P is supported by Centro de Investigación Biomédica en Red de Enfermedades Raras, and AS-P is a predoctoral fellows of the FPU program (MICINN) respectively. SR-LL is a postdoctoral fellow of the FIS (contract # CD05-0055). RDU is supported by project BIO2009-12458 from the Spanish Ministry of Economy and Innovation. RM, SP and AV are supported by the Generalitat de Catalunya, CIRIT (2009SGR-725).Peer Reviewe

Thyroid cancer GWAS identifies 10q26.12 and 6q14.1 as novel susceptibility loci and reveals genetic heterogeneity among populations

et al.Thyroid cancer is the most heritable cancer of all those not displaying typical Mendelian inheritance. However, most of the genetic factors that would explain the high heritability remain unknown. Our aim was to identify additional common genetic variants associated with susceptibility to this disease. In order to do so, we performed a genome-wide association study in a series of 398 cases and 502 controls from Spain, followed by a replication in four well-defined Southern European case-control collections contributing a total of 1,422 cases and 1,908 controls. The association between the variation at the 9q22 locus near FOXE1 and thyroid cancer risk was consistent across all series, with several SNPs identified (rs7028661: OR = 1.64, p = 1.0 × 10−22, rs7037324: OR = 1.54, p = 1.2 × 10−17). Moreover, the rare alleles of three SNPs (rs2997312, rs10788123 and rs1254167) at 10q26.12 showed suggestive evidence of association with higher risk of the disease (OR = 1.35, p = 1.2 × 10−04, OR = 1.26, p = 5.2 × 10−04 and OR = 1.38, p = 5.9 × 10−05, respectively). Finally, the rare allele of rs4075570 at 6q14.1 conferred protection in the series studied (OR = 0.82, p = 2.0 × 10−04). This study suggests that heterogeneity in genetic susceptibility between populations is a key feature to take into account when exploring genetic risk factors related to this disease.The authors thank the Spanish National Genotyping Center (CEGEN) for its support in this study. Grant sponsor: Fondo de Investigaciones Sanitarias (FIS); Grant numbers: PI11/01359 (to M.R.), PI13/01136 (to A.C.), PI12/00749 (to J.C.- T.); Grant sponsor: Comunidad de Madrid (to P.S., M.R. and A.C.); Grant number: S2011/BMD-2328 TIRONET; Grant sponsor: “La Caixa”/CNIO International PhD Program (to V.M.) and Centro de Investigación Biomédica en Red de Enfermedades Raras (to L.I.-P. and R.C.).Peer reviewe

Performance of anti-CD19 chimeric antigen receptor T cells in genetically defined classes of chronic lymphocytic leukemia

BackgroundWhile achieving prolonged remissions in other B cell-derived malignancies, chimeric antigen receptor (CAR) T cells still underperform when injected into patients with chronic lymphocytic leukemia (CLL). We studied the influence of genetics on CLL response to anti-CD19 CAR T-cell therapy.MethodsFirst, we studied 32 primary CLL samples composed of 26 immunoglobulin heavy-chain gene variable (IGHV)-unmutated (9 ATM-mutated, 8 TP53-mutated, and 9 without mutations in ATM, TP53, NOTCH1 or SF3B1) and 6 IGHV-mutated samples without mutations in the above-mentioned genes. Then, we mimicked the leukemic microenvironment in the primary cells by ‘2S stimulation’ through interleukin-2 and nuclear factor kappa B. Finally, CRISPR/Cas9-generated ATM-knockout and TP53-knockout clones (four and seven, respectively) from CLL-derived cell lines MEC1 and HG3 were used. All these samples were exposed to CAR T cells. In vivo survival study in NSG mice using HG3 wild-type (WT), ATM-knockout or TP53-knockout cells was also performed.ResultsPrimary unstimulated CLL cells were specifically eliminated after >24 hours of coculture with CAR T cells. ‘2S’ stimulated cells showed increased survival when exposed to CAR T cells compared with unstimulated ones, confirming the positive effect of this stimulation on CLL cells’ in vitro fitness. After 96 hours of coculture, there was no difference in survival among the genetic classes. Finally, CAR T cells were specifically activated in vitro in the presence of target knockout cell lines as shown by the production of interferon-γ when compared with control (CTRL) T cells (p=0.0020), but there was no difference in knockout cells’ survival. In vivo, CAR T cells prolonged the survival of mice injected with WT, TP53-knockout and ATM-knockout HG3 tumor cells as compared with CTRL T cells (p=0.0485, 0.0204 and <0.0001, respectively). When compared with ATM-knockout, TP53-knockout disease was associated with an earlier time of onset (p<0.0001), higher tumor burden (p=0.0002) and inefficient T-cell engraftment (p=0.0012).ConclusionsWhile in vitro no differences in survival of CLL cells of various genetic backgrounds were observed, CAR T cells showed a different effectiveness at eradicating tumor cells in vivo depending on the driver mutation. Early disease onset, high-tumor burden and inefficient T-cell engraftment, associated with TP53-knockout tumors in our experimental setting, ultimately led to inferior performance of CAR T cells

Distinct p53 phosphorylation patterns in chronic lymphocytic leukemia patients are reflected in the activation of circumjacent pathways upon DNA damage

TP53 gene abnormalities represent the most important biomarker in chronic lymphocytic leukemia (CLL). Altered protein modifications could also influence p53 function, even in the wild‐type protein. We assessed the impact of p53 protein phosphorylations on p53 functions as an alternative inactivation mechanism. We studied p53 phospho‐profiles induced by DNA‐damaging agents (fludarabine, doxorubicin) in 71 TP53‐intact primary CLL samples. Doxorubicin induced two distinct phospho‐profiles: profile I (heavily phosphorylated) and profile II (hypophosphorylated). Profile II samples were less capable of activating p53 target genes upon doxorubicin exposure, resembling TP53‐mutant samples at the transcriptomic level, whereas standard p53 signaling was triggered in profile I. ATM locus defects were more common in profile II. The samples also differed in the basal activity of the hypoxia pathway: the highest level was detected in TP53‐mutant samples, followed by profile II and profile I. Our study suggests that wild‐type TP53 CLL cells with less phosphorylated p53 show TP53‐mutant‐like behavior after DNA damage. p53 hypophosphorylation and the related lower ability to respond to DNA damage are linked to ATM locus defects and the higher basal activity of the hypoxia pathway