The identification of new familial pheochromocytoma/paraganglioma genes using whole exome sequencing

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 30-04-2015Los feocromocitomas (PCC) son tumores neuroendocrinos, desarrollados a partir del tejido cromafín de la médula adrenal, que suelen causar hipertensión arterial por sobre-secreción de catecolaminas. Los paragangliomas (PGL) son PCCs, en su mayoría secretores y con un gran riesgo de malignizar, que se desarrollan a partir de paraganglios localizados fundamentalmente en la región intra-abdominal o torácica. Algunos PGLs pueden desarrollarse en la región de la cabeza y el cuello, y en esa localización suelen comportarse como masas benignas no secretoras. Los PCCs presentan una incidencia anual en población española de 2 casos por millón de habitantes y son por tanto una enfermedad rara. Tanto la secuenciación masiva del genoma completo, como la limitada a las regiones codificantes (secuenciación exómica, SE), se han convertido a lo largo de los últimos años en herramientas de gran utilidad para el descubrimiento de genes de susceptibilidad responsables de enfermedades mendelianas. De este modo, el objetivo principal de esta tesis doctoral fue la identificación de nuevos genes de susceptibilidad implicados en el desarrollo de PCC/PGL mediante el uso de la SE aplicada a 3 proyectos independientes. En el primer proyecto, se llevó a cabo la SE de tres pacientes no relacionados, con antecedentes familiares de la enfermedad y sin mutaciones en ninguno de los genes conocidos. Los pacientes fueron seleccionados como candidatos para el estudio debido a que sus correspondientes tumores presentaban perfiles de expresión muy homogéneos. El filtrado y posterior análisis de los datos de secuenciación permitió identificar mutaciones germinales patogénicas en el gen MAX en los tres pacientes. Además, la pérdida de heterocigosidad del alelo silvestre, la ausencia de proteína MAX en los tumores y el descubrimiento de otras 5 mutaciones en pacientes aquejados de la enfermedad permitió demostrar que MAX constituía un nuevo gen supresor de tumores asociado con el desarrollo de PCC hereditario. En un segundo trabajo, el estudio de una serie compuesta por más de 1500 pacientes no relacionados permitió establecer tanto la prevalencia de las mutaciones en MAX en pacientes con PCC/PGL (1,12%), como el fenotipo asociado a dichas mutaciones. Por último, con el objeto de determinar la patogenicidad de las variantes con significado desconocido halladas en el gen MAX, se llevó a cabo un estudio funcional de las mismas en células de PCC de rata (PC12) y se implementó una herramienta de predicción in silico basada en el consenso de 5 predictores. En el segundo proyecto, los candidatos a estudio mediante SE (3 tríos paciente/madre/padre) fueron seleccionados en base a la presencia de una característica fenotípica poco frecuente en pacientes con PCC/PGL: policitemia idiopática. Durante el análisis de los datos, se publicó el descubrimiento de mutaciones somáticas post-zigóticas en el gen EPAS1 en pacientes con PCC/PGL múltiple y policitemia idiopática. El análisis de EPAS1 en los correspondientes tumores de los pacientes seleccionados para la SE, reveló la presencia de mutaciones somáticas en mosaico en el gen EPAS1 en todos ellos. Además, el estudio de una serie adicional de tumores identificó mutaciones somáticas en tumores de pacientes sin policitemia. Finalmente, se identificó la ganancia de la región 2p como exclusiva de tumores con mutación en EPAS1. En el tercer proyecto, se llevó a cabo una selección de pacientes basada en la presencia de tumores múltiples (más de 5) como indicador de la existencia de una enfermedad hereditaria. Durante el filtrado de las variantes encontradas en uno de los pacientes seleccionados, se identificó una mutación en el gen MDH2, implicado en el ciclo de Krebs. La ausencia de RNAm, proteína y actividad enzimática malato deshidrogenasa, así como el diagnóstico de la enfermedad en un pariente portador de la variante permitió concluir que el gen MDH2 es un nuevo gen supresor tumoral responsable de susceptibilidad a desarrollar PCC/PGL. La identificación de este segundo gen de susceptibilidad a desarrollar PCC/PGL demuestra la eficacia de la SE en la identificación de nuevos genes responsables de enfermedades mendelianas.Pheochromocytomas (PCCs) are neuroendocrine tumors arising from the medulla of the adrenal gland that usually cause hypertension due to oversecretion of cathecolamines. On the other hand, paragangliomas (PGLs) are normally secretor tumors with high risk of malignancy that arise from the paraganglia of the intra-abdominal or thoracic regions. Some PGLs arise in the head and neck region, usually as benign, non-secretor tumors. PCC/PGL is a rare disease, with an incidence in the Spanish population of approximately 2 cases per million habitants. High-throughput techniques such as wholegenome sequencing (WGS) and whole-exome sequencing (WES) are nowadays widely applied to discover susceptibility genes involved in mendelian diseases. Thus, the main objective of this thesis was to identify new genes related to the susceptibility to develop PCC/PGL by applying WES in three independent projects. In the first project, we applied WES to three unrelated patients with a family history of disease testing negative for mutations in the major PCC/PGL susceptibility genes. The three patients were selected because they shared a common homogeneous transcriptional profile, suggesting a common underlying genetic alteration. The variant filtering process and the posterior analysis of the WES data allowed us to identify pathogenic germline mutations in the MAX (MYC associated factor X) gene in all three patients. Moreover, loss of heterozygosity of the wild type allele, loss of the protein in the tumors and the identification of MAX mutations in additional patients with the disease, together indicated that MAX constitutes a novel tumor suppressor gene. In a posterior international collaborative study, we recruited more than 1500 unrelated patients and established the prevalence of MAX mutations to be 1.12%; we also characterized the associated phenotype. Finally, we developed a functional model to determine the pathogenicity of variants of unknown significant (VUS) found in MAX. This model was implemented based on a consensus in silico prediction obtained from five algorithms available online. In the second project, the selection of three trios (patient/mother/father) was based on the presence of a very infrequent phenotypic characteristic in patients suffering for PCC/PGL: idiopathic polycythemia. While the analysis of the WES data was underway, a study was published reporting the presence of somatic post-zygotic mutations in the gene EPAS1 (HIF2A) in patients presenting multiple PCC/PGL and idiopathic polycythemia. Our posterior screening for EPAS1 mutations in the tumors from the index patients revealed the presence of somatic mutations in all of them. In addition, the analysis of an additional series of tumors identified somatic mutations in tumors from patients without polycythemia. Finally, we indentified a gain of the chromosomic region 2p exclusive to tumors harboring EPAS1 mutations. In the last WES Project, we selected patients based on the presence of multiple (more than five) tumors as phenotypic marker of hereditary disease. We identified a splice-site mutation affecting the MDH2 gene involved in the Krebs cycle. The observed absence of the messenger RNA, protein and malate dehydrogenase enzymatic activity, as well as the positive diagnosis for the disease of a family member, led us to conclude that MDH2 is a novel tumor suppressor gene implicated in susceptibility to develop PCC/PGL. The identification of this second susceptibility gene implicated in PCC/PGL development demonstrates the efficacy of WES in discovering susceptibility genes involved in Mendelian disease

Development of a Novel NGS Methodology for Ultrasensitive Circulating Tumor DNA Detection as a Tool for Early-Stage Breast Cancer Diagnosis

Breast cancer (BC) is the most prevalent cancer in women. While usually detected when localized, invasive procedures are still required for diagnosis. Herein, we developed a novel ultrasensitive pipeline to detect circulating tumor DNA (ctDNA) in a series of 75 plasma samples from localized BC patients prior to any medical intervention. We first performed a tumor-informed analysis to correlate the mutations found in tumor tissue and plasma. Disregarding the tumor data next, we developed an approach to detect tumor mutations in plasma. We observed a mutation concordance between the tumor and plasma of 29.50% with a sensitivity down to 0.03% in mutant variant allele frequency (VAF). We detected mutations in 33.78% of the samples, identifying eight patients with plasma-only mutations. Altogether, we determined a specificity of 86.36% and a positive predictive value of 88.46% for BC detection. We demonstrated an association between higher ctDNA median VAF and higher tumor grade, multiple plasma mutations with a likelihood of relapse and more frequent TP53 plasma mutations in hormone receptor-negative tumors. Overall, we have developed a unique ultra-sensitive sequencing workflow with a technology not previously employed in early BC, paving the way for its application in BC screening.Comino-Mendez’s contract is funded by the Spanish Association Against Cancer Scientific Foundation (AECC). This study was supported by the “Consejería de Salud y Familias—Junta de Andalucía” (PI-0291-2019), “Fundación Unicaja” is funding Alba-Bernal’s contract and the Andalusia-Roche Network in Precision Medical Oncology Quirós-Ortega’s contract. Carbajosa-Antona’s contract is funded by the “Ayudas María Zambrano para la atracción de talento internacional—Universidad de Málaga”. Partial funding for open access charge: Universidad de Málag

Diverse BRCA1 and BRCA2 Reversion Mutations in Circulating Cell-Free DNA of Therapy-Resistant Breast or Ovarian Cancer

Purpose:; Resistance to platinum-based chemotherapy or PARP inhibition in germline; BRCA1; or; BRCA2; mutation carriers may occur through somatic reversion mutations or intragenic deletions that restore BRCA1 or BRCA2 function. We assessed whether; BRCA1/2; reversion mutations could be identified in circulating cell-free DNA (cfDNA) of patients with ovarian or breast cancer previously treated with platinum and/or PARP inhibitors.; Experimental Design:; cfDNA from 24 prospectively accrued patients with germline; BRCA1; or; BRCA2; mutations, including 19 patients with platinum-resistant/refractory ovarian cancer and five patients with platinum and/or PARP inhibitor pretreated metastatic breast cancer, was subjected to massively parallel sequencing targeting all exons of 141 genes and all exons and introns of; BRCA1; and; BRCA2; Functional studies were performed to assess the impact of the putative; BRCA1/2; reversion mutations on BRCA1/2 function.; Results:; Diverse and often polyclonal putative; BRCA1; or; BRCA2; reversion mutations were identified in cfDNA from four patients with ovarian cancer (21%) and from two patients with breast cancer (40%).; BRCA2; reversion mutations were detected in cfDNA prior to PARP inhibitor treatment in a patient with breast cancer who did not respond to treatment and were enriched in plasma samples after PARP inhibitor therapy. Foci formation and immunoprecipitation assays suggest that a subset of the putative reversion mutations restored BRCA1/2 function.; Conclusions:; Putative; BRCA1/2; reversion mutations can be detected by cfDNA sequencing analysis in patients with ovarian and breast cancer. Our findings warrant further investigation of cfDNA sequencing to identify putative; BRCA1/2; reversion mutations and to aid the selection of patients for PARP inhibition therapy.; Clin Cancer Res; 23(21); 6708-20. ©2017 AACR;

Unveiling the Potential of Liquid Biopsy in HER2-Positive Breast Cancer Management

Invasive breast cancer (BC) is the most common cancer in women with a slightly increasing yearly incidence. BC immunohistochemical characterisation is a crucial tool to define the intrinsic nature of each tumour and personalise BC patients’ clinical management. In this regard, the characterisation of human epidermal growth factor receptor 2 (HER2) status guides physicians to treat with therapies tailored to this membrane receptor. Standardly, a tumour solid biopsy is therefore required, which is an invasive procedure and has difficulties to provide the complete molecular picture of the tumour. To complement these standard-of-care approaches, liquid biopsy is a validated methodology to obtain circulating tumour components such as circulating tumour DNA (ctDNA) and circulating tumour cells (CTCs) from body fluids in an easy-to-perform minimal-invasive manner. However, its clinical validity in cancer is still to be demonstrated. This review focusses on the utilisation of both ctDNA and CTCs in early and metastatic HER2-positive BC tumours. We discuss recently published studies deciphering the capacity of liquid biopsy to determine the response to neoadjuvant and adjuvant therapies as well as to predict patients’ outcomes

Challenges and achievements of liquid biopsy technologies employed in early breast cancer

Breast cancer is the most common cancer type in women worldwide and its early detection is crucial to curing the disease. Tissue biopsy, currently the method of choice to obtain tumour molecular information, is invasive and might be affected by tumour heterogeneity rendering it incapable to portray the complete molecular picture. Liquid biopsy permits to study disease features in a more comprehensive manner by sampling biofluids and extracting tumour components such as circulating-tumour DNA (ctDNA), circulating-tumour cells (CTCs), and/or circulating-tumour RNA (ctRNA) amongst others in a monitoring-compatible manner. In this review, we describe the recent progress in the utilization of the circulating tumour components using early breast cancer samples. We review the most important analytes and technologies employed for their study.ICM's contract is funded by the Spanish Association Against Cancer (AECC). AAB is contracted by the “Garantía Juvenil en I+D+i Subprograma Estatal de Incorporacion” by the Ministry of Science and Innovation (Spanish Government). MIQO contract is supported by the “Miguel Servet Type II” program (CPI13/00003), ISCIII, Spain and cofunded by the "Fondo Europeo de Desarrollo Regional-(FEDER)", and by the “Nicolas Monardes” research program from the "Consejería de Salud" (C-0030-2018), Andalusian Gobernment, Spain.Ye

Breast and Gut Microbiota Action Mechanisms in Breast Cancer Pathogenesis and Treatment

In breast cancer (BC) the employment of sequencing technologies for metagenomic analyses has allowed not only the description of the overall metagenomic landscape but also the specific microbial changes and their functional implications. Most of the available data suggest that BC is related to bacterial dysbiosis in both the gut microenvironment and breast tissue. It is hypothesized that changes in the composition and functions of several breast and gut bacterial taxa may contribute to BC development and progression through several pathways. One of the most prominent roles of gut microbiota is the regulation of steroid-hormone metabolism, such as estrogens, a component playing an important role as risk factor in BC development, especially in postmenopausal women. On the other hand, breast and gut resident microbiota are the link in the reciprocal interactions between cancer cells and their local environment, since microbiota are capable of modulating mucosal and systemic immune responses. Several in vivo and in vitro studies show remarkable evidence that diet, probiotics and prebiotics could exert important anticarcinogenic effects in BC. Moreover, gut microbiota have an important role in the metabolism of chemotherapeutic drugs and in the activity of immunogenic chemotherapies since they are a potential dominant mediator in the response to cancer therapy. Then, the microbiome impact in BC is multi-factorial, and the gut and breast tissue bacteria population could be important in regulating the local immune system, in tumor formation and progression and in therapy response and/or resistance.This work was supported in part by PE-0106-2019 from the Consejería de Salud de la Junta de Andalucía, C19047-2018 from Fundación Unicaja and UMA18-FEDERJA-042 from UMA-FEDER. Maria Isabel Queipo-Ortuño is recipient of a “Miguel Servet Type II” program (CPI13/00003) from ISCIII, co-funded by the Fondo Europeo de Desarrollo Regional-FEDER, Madrid, Spain and also belongs to the regional “Nicolas Monardes” research program of the Consejería de Salud (C-0030-2018, Junta de Andalucía, Spain. Aurora Laborda-Illanes was recipient of a predoctoral grant PFIS-ISCIII (FI19-00112) co-funded by the Fondo Europeo de Desarrollo Regional-FEDER, Madrid, Spain. Lidia Sanchez-Alcoholado was recipient of a predoctoral grant (PE-0106-2019) from the Consejería de Salud y Familia (co-funded by the Fondo Europeo de Desarrollo Regional-FEDER, Andalucia, Spain).Ye

Regulatory Polymorphisms in β-Tubulin IIa Are Associated with Paclitaxel-Induced Peripheral Neuropathy

PURPOSE: Peripheral neuropathy is the dose-limiting toxicity of paclitaxel, a chemotherapeutic drug widely used to treat several solid tumors such as breast, lung, and ovary. The cytotoxic effect of paclitaxel is mediated through β-tubulin binding in the cellular microtubules. In this study, we investigated the association between paclitaxel neurotoxicity risk and regulatory genetic variants in β-tubulin genes. EXPERIMENTAL DESIGN: We measured variation in gene expression of three β-tubulin isotypes (I, IVb, and IIa) in lymphocytes from 100 healthy volunteers, sequenced the promoter region to identify polymorphisms putatively influencing gene expression and assessed the transcription rate of the identified variants using luciferase assays. To determine whether the identified regulatory polymorphisms were associated with paclitaxel neurotoxicity, we genotyped them in 214 patients treated with paclitaxel. In addition, paclitaxel-induced cytotoxicity in lymphoblastoid cell lines was compared with β-tubulin expression as measured by Affymetrix exon array. RESULTS: We found a 63-fold variation in β-tubulin IIa gene (TUBB2A) mRNA content and three polymorphisms located at −101, −112, and −157 in TUBB2A promoter correlated with increased mRNA levels. The −101 and −112 variants, in total linkage disequilibrium, conferred TUBB2A increased transcription rate. Furthermore, these variants protected from paclitaxel-induced peripheral neuropathy [HR, 0.62; 95% confidence interval (CI), 0.42–0.93; P = 0.021, multivariable analysis]. In addition, an inverse correlation between TUBB2A and paclitaxel-induced apoptosis (P = 0.001) in lymphoblastoid cell lines further supported that higher TUBB2A gene expression conferred lower paclitaxel sensitivity. CONCLUSIONS: This is the first study showing that paclitaxel neuropathy risk is influenced by polymorphisms regulating the expression of a β-tubulin gene

Hematologic β-tubulin VI isoform exhibits genetic variability that influences paclitaxel toxicity.

Cellular microtubules composed of α-β-tubulin heterodimers that are essential for cell shape, division, and intracellular transport are valid targets for anticancer therapy. However, not all the conserved but differentially expressed members of the β-tubulin gene superfamily have been investigated for their role in these settings. In this study, we examined roles for the hematologic isoform β-tubulin VI and functional genetic variants in the gene. β-tubulin VI was highly expressed in blood cells with a substantial interindividual variability (seven-fold variation in mRNA). We characterized DNA missense variations leading to Q43P, T274M, and R307H, and a rare nonsense variant, Y55X. Because variations in the hematologic target of microtubule-binding drugs might alter their myelosuppressive action, we tested their effect in cell lines stably expressing the different β-tubulin VI full-length variants, finding that the T274M change significantly decreased sensitivity to paclitaxel-induced tubulin polymerization. Furthermore, patients treated with paclitaxel and carrying β-tubulin VI T274M exhibited a significantly lower thrombocytopenia than wild-type homozygous patients (P = 0.031). Together, our findings define β-tubulin VI as a hematologic isotype with significant genetic variation in humans that may affect the myelosuppresive action of microtubule-binding drugs. A polymorphism found in a tubulin isoform expressed only in hemapoietic cells may contribute to the patient variation in myelosuppression that occurs after treatment with microtubule-binding drugs.This article was supported by a project from the Spanish Ministry of Science and Innovation (SAF2009-08307). L. J. Leandro-Garcia was supported by a FIS fellowship (FI08/00375) and Susanna Leskela by a fellowship from the Spanish Ministry of Science and Innovation (AP2005-4514).S

Development of a Novel NGS Methodology for Ultrasensitive Circulating Tumor DNA Detection as a Tool for Early-Stage Breast Cancer Diagnosis

Breast cancer (BC) is the most prevalent cancer in women. While usually detected when localized, invasive procedures are still required for diagnosis. Herein, we developed a novel ultrasensitive pipeline to detect circulating tumor DNA (ctDNA) in a series of 75 plasma samples from localized BC patients prior to any medical intervention. We first performed a tumor-informed analysis to correlate the mutations found in tumor tissue and plasma. Disregarding the tumor data next, we developed an approach to detect tumor mutations in plasma. We observed a mutation concordance between the tumor and plasma of 29.50% with a sensitivity down to 0.03% in mutant variant allele frequency (VAF). We detected mutations in 33.78% of the samples, identifying eight patients with plasma-only mutations. Altogether, we determined a specificity of 86.36% and a positive predictive value of 88.46% for BC detection. We demonstrated an association between higher ctDNA median VAF and higher tumor grade, multiple plasma mutations with a likelihood of relapse and more frequent TP53 plasma mutations in hormone receptor-negative tumors. Overall, we have developed a unique ultra-sensitive sequencing workflow with a technology not previously employed in early BC, paving the way for its application in BC screening

Technical-methodological report : a nomogram for peak leg power output in the vertical jump.

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 [Deltabetametastatic-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. Clin Cancer Res; 21(13); 3020-30. (c)2015 AACR