MicroRNA and transcription factor co-regulatory networks and subtype classification of seminoma and non-seminoma in testicular germ cell tumors

Recent studies have revealed that feed-forward loops (FFLs) as regulatory motifs have synergistic roles in cellular systems and their disruption may cause diseases including cancer. FFLs may include two regulators such as transcription factors (TFs) and microRNAs (miRNAs). In this study, we extensively investigated TF and miRNA regulation pairs, their FFLs, and TF-miRNA mediated regulatory networks in two major types of testicular germ cell tumors (TGCT): seminoma (SE) and non-seminoma (NSE). Specifically, we identified differentially expressed mRNA genes and miRNAs in 103 tumors using the transcriptomic data from The Cancer Genome Atlas. Next, we determined significantly correlated TF-gene/miRNA and miRNA-gene/TF pairs with regulation direction. Subsequently, we determined 288 and 664 dysregulated TF-miRNA-gene FFLs in SE and NSE, respectively. By constructing dysregulated FFL networks, we found that many hub nodes (12 out of 30 for SE and 8 out of 32 for NSE) in the top ranked FFLs could predict subtype-classification (Random Forest classifier, average accuracy ≥90%). These hub molecules were validated by an independent dataset. Our network analysis pinpointed several SE-specific dysregulated miRNAs (miR-200c-3p, miR-25-3p, and miR-302a-3p) and genes (EPHA2, JUN, KLF4, PLXDC2, RND3, SPI1, and TIMP3) and NSE-specific dysregulated miRNAs (miR-367-3p, miR-519d-3p, and miR-96-5p) and genes (NR2F1 and NR2F2). This study is the first systematic investigation of TF and miRNA regulation and their co-regulation in two major TGCT subtypes

Genomic landscape of platinum resistant and sensitive testicular cancers

While most testicular germ cell tumours (TGCTs) exhibit exquisite sensitivity to platinum chemotherapy, ~10% are platinum resistant. To gain insight into the underlying mechanisms, we undertook undertake whole exome sequencing and copy number analysis in 40 tumours from 26 cases with platinum-resistant TGCT, and combined this with published genomic data on an additional 624 TGCTs. We integrated analyses for driver mutations, mutational burden, global, arm-level and focal copy number (CN) events, and SNV and CN signatures. Albeit preliminary and observational in nature, these analyses provide the first support for a possible mechanistic link between early driver mutations in RAS and KIT and the widespread copy number events by which TGCT is characterised

Germ Cell Tumor

The book aims to provide an overview of current knowledge regarding germ cell tumors. It deals with the clinical presentations, treatment modalities, the biology and genetics of germ cell tumors in children and adults. Most chapters are focused on testicular germ cell tumors whose incidence has been increasing in young males. Included are reviews on the pathogenesis, risk factors, diagnosis and treatment regimens applied to precursor, pre-invasive lesions as well as to seminomatous and non-seminomatous germ cell tumors of the testes. In addition, a review is included on the diagnosis and current management options for intracranial germ cell tumors in children. Authors have also contributed articles on the genetics and epigenetics of germ cell tumor development in humans and in the mouse model system. This book will be of interest to scientists, physicians and lay readers wishing to review recent developments in the field of germ cell cancers

Genomic landscape of platinum resistant and sensitive testicular cancers

Abstract: While most testicular germ cell tumours (TGCTs) exhibit exquisite sensitivity to platinum chemotherapy, ~10% are platinum resistant. To gain insight into the underlying mechanisms, we undertake whole exome sequencing and copy number analysis in 40 tumours from 26 cases with platinum-resistant TGCT, and combine this with published genomic data on an additional 624 TGCTs. We integrate analyses for driver mutations, mutational burden, global, arm-level and focal copy number (CN) events, and SNV and CN signatures. Albeit preliminary and observational in nature, these analyses provide support for a possible mechanistic link between early driver mutations in RAS and KIT and the widespread copy number events by which TGCT is characterised

Testicular germ cells tumors in adolescents and young adults: Management and outcomes from a single-center experience.

Objective: To investigate and compare the effectiveness of active surveillance versus post-surgical active treatment, in patients with testicular germ cells tumor (TGCT). Materials and methods: We retrospectively analyzed 52 patients who underwent surgery for TGCT from January 2009 to December 2014. All the patients were divided into two age groups: the Group A included children-adolescents from 18 months to 21 years old, while the Group B comprised young adults from 22 to 39 years old. Clinical, histopathological, therapeutic and follow-up data were collected. Results: Overall, 22 patients (42,3%) were enrolled in the Group A and 30 patients (57.7%) were categorized in the Group B. Inguinal orchiectomy was performed in all patients. Retroperitoneal lymphadenectomy was performed in 4 patients (7.7%). Post-surgical management differed based on clinical stage, resulting in active surveillance or adjuvant therapy. After an average 7 years follow-up period (range: 3.5-9.0 years), the overall survival rate is 100%. The relapse risk is significantly higher for the patients in the Group B, displaying a recurrence free-survival rate of 72% versus 95% (Group A); 11 relapses (21.1%) were recorded 2 years after surgery. Of these, 3 recurrences (12.0%) occurred in patients undergoing an active surveillance approach, while 8 (29.6%) in patients subjected to an active treatment. Conclusions: The excellent prognosis in both age groups confirms the high curability of this neoplasia. The active surveillance could represent an optimal option for low recurrence risk tumors. However, post-surgical treatments should be taken into consideration for TGCT with high risk factors, including tumor size, lymphovascular and rete testis invasion

Analysis of TERT isoforms across TCGA, GTEx and CCLE datasets

Reactivation of the multi-subunit ribonucleoprotein telomerase is the primary telomere maintenance mechanism in cancer, but it is rate-limited by the enzymatic component, telomerase reverse transcriptase (TERT). While regulatory in nature, TERT alternative splice variant/isoform regulation and functions are not fully elucidated and are further complicated by their highly diverse expression and nature. Our primary objective was to characterize TERT isoform expression across 7887 neoplastic and 2099 normal tissue samples using The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression Project (GTEx), respectively. We confirmed the global overexpression and splicing shift towards full-length TERT in neoplastic tissue. Stratifying by tissue type we found uncharacteristic TERT expression in normal brain tissue subtypes. Stratifying by tumor-specific subtypes, we detailed TERT expression differences potentially regulated by subtype-specific molecular characteristics. Focusing on β-deletion splicing regulation, we found the NOVA1 trans-acting factor to mediate alternative splicing in a cancer-dependent manner. Of relevance to future tissue-specific studies, we clustered cancer cell lines with tumors from related origin based on TERT isoform expression patterns. Taken together, our work has reinforced the need for tissue and tumour-specific TERT investigations, provided avenues to do so, and brought to light the current technical limitations of bioinformatic analyses of TERT isoform expression

Deciphering the genetic basis of Spanish familial testicular cancer

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 26-09-2017Esta tesis tiene embargado el acceso al texto completo hasta el 26-03-2019Testicular cancer is a frequently occurring disease among adult males, and it accounts for 1-2% of all male tumors. It can be classified into different types of cancer: germ cell tumors (GCT), which represent 98% of all cases, and tumors involving the gonadal stromal tumors and secondary tumors of the testes. The incidence of Testicular Germ Cell Tumors (TGCTs) has been increasing for the past decades, although the etiology of the disease and the reasons of its increased incidence remain unknown. Environmental factors, in particular exposure to endocrine disruptors during embryogenesis and perinatal life are suspected culprits. It is likely that genetic factors also play an important role in TGCT formation, as the estimated heritability, 48.9%, is the third highest among all cancers, and the Familial Testicular Germ Cell Tumor (FTGCT) risk is 2-fold higher than what is typical for more common cancers such as breast, colorectal and prostate cancer. Several candidate gene approaches failed to identify high susceptibility genes. In fact, in the last years scientists have come to believe that a polygenic model fits better with the genetic landscape of the disease, although the idea of polygenic susceptibility does not fit in with a history of familial aggregations in a disease. Due to the absence of information about the genetic basis of this disease, our objective was to identify high/moderate or low susceptibility genes using whole exome sequencing (WES) and case-control studies considering both monogenic and polygenic models of inheritance. These studies will help to increase our knowledge about the genetic basis of the disease and may have a significant impact on its prevention, early diagnosis and a possible treatment. The hypothesis of the monogenic model was tested using a pipeline previously described by our group, while the polygenic model was studied by performing family-based association tests in which we evaluated the level of additive and cumulative effects our variants could have in the familial aggregation of the disease. DNA of a group of 19 families (71 individuals) was sequenced with an Illumina HiSeq 2000 sequencer. Based on the analysis assuming both patterns of inheritance, a total of 120 candidate variants were evaluated in the case-control study performed in 391 sporadic cases and 382 healthy Spanish controls. In order to increase the size sample, we used data from the public database of the Spanish Center for Biomedical Research on Rare Diseases (CIBERER), which contains WES data of 788 unaffected individuals, and to perform statistical analysis. In this discovery analysis, 27 variants gave significant results and two of them (located in the VNN1 and SLC22A16 genes, which are both involved in spermatogenesis) were later on replicated in a large series studied in the English population. Moreover, the variant of the SLC22A16 gene appears to be specifically associated with the development of Seminoma germ cell tumors. In summary, our results present two new susceptibility risk genes whose variants are potential candidates for being associated with the development of familial testicular cancer.La presente Tesis Doctoral se realizó en el Grupo de Genética Humana en el Centro Nacional de Investigaciones Oncológicas (CNIO) de Madrid durante los años 2013 y 2017 bajo la supervisión del Dr. Javier Benítez Las siguientes becas, ayudas y proyectos han permitido la realización de esta Tesis Doctoral: “La Caixa”- Severo Ochoa International Phd Programmme at CNIO Proyecto FIS PI12/0070 BRIDGES Project (H2020

Pan-Cancer Analysis of Telomerase Reverse Transcriptase (TERT) Isoforms

Reactivation of the multi-subunit ribonucleoprotein telomerase is the primary telomere maintenance mechanism in cancer, but it is rate-limited by the enzymatic component, telomerase reverse transcriptase (TERT). While regulatory in nature, TERT alternative splice variant/isoform regulation and functions are not fully elucidated and are further complicated by their highly diverse expression. In this thesis, I characterized TERT expression across normal and neoplastic tissues using TCGA and GTEx RNA-sequencing data. In doing so, I demonstrated the global overexpression and splicing shift towards full-length TERT in neoplastic tissue. Furthermore, my studies identified tumour subtype expression differences possibly regulated by subtype-specific characteristics, detailed heterogeneity in both isoform function and prognostic potential and determined cancer cell lines with representative tumour specific TERT transcriptomes. Taken together, my work reinforced the need for tissue specific TERT investigations, provided avenues to do so, and brought to light the current technical limitations of bioinformatically analyzing TERT isoform expression

Characterization of the DNA methylation patterns of chemosensitive and chemoresistant human cancer cells: Biological and clinical impact

[spa] La quimioresistencia es el principal limitante para el tratamiento del paciente oncológico. La comprensión de los mecanismos celulares que conducen a la quimioresistencia puede tener un impacto drástico en la utilización de los fármacos. Estos agentes destruyen las células sensibles por inducción de programas de muerte celular. La desregulación de genes implicados en estos mecanismos puede ser una manera de quimioresistencia. Genes supresores tumorales y de reparación del ADN son importantes en estos procesos. Mientras que la inactivación de los primeros lleva a la quimioresistencia, la inactivación de los segundos puede conducir a quimosensibilidad. Mecanismos como la epigenética puede ser responsables por la alteración de estos genes. Por ejemplo, la hipermetilación del ADN en los promotores puede causar el silenciamiento de genes, y/o la hipometilación global producir la activación de otros genes. Pretendemos determinar si los cambios del perfil de metilación del ADN son importantes en la adquisición de resistencia a la quimioterapia. 1º estudio: 68 pacientes con cáncer colorectal (CCR) metastásico recibieron tratamiento con dacarbazina. 3% alcanzó una respuesta parcial y 12% la estabilización de la enfermedad. El mejor resultado se asoció a la hipermetilación del gen MGMT. 2º estudio: La resistencia al oxaliplatino depende en parte de la hipermetilación del gen SRBC. Este hecho predice una supervivencia libre de progresión más corta en pacientes tratados con oxaliplatino para los que no estaba indicada la cirugía de las metástasis. 3º Estudio: Descubrimos que el gen UGCG tiene un papel en resistencia de tumores testiculares germinales al cisplatino (CDDP). Sugerimos un agente químico que podría ser utilizado re-sensibilizar este tipo de tumores refractarios al CDDP. 4º estudio: MGMT se presenta hipermetilado en los modelos de cáncer testicular sensibles al cisplatino. En pacientes la hipermetilación de este gen encontrase asociada a una mayor supervivencia global. Se demostró que la inactivación de esto gen en tumores resistentes podría re-sensibilizarlos al CDDP. El estado de metilación de los promotores génicos influye en la sensibilidad tumoral a diferentes agentes quimioterapéuticos. Ponemos en evidencia que la inhibición de MGMT y UGCG pueden ser opciones terapéuticas para la rescatar pacientes refractarios al CDDP.[eng] Although chemotherapeutic drugs are widely used in order to improve the cancer outcome, drug resistance remain the most unpredictable factor affecting chemotherapy and a major impediment to successful patient’s treatment. Understanding the cellular mechanisms leading to chemoresistance may dramatically impact on the way chemotherapeutic drugs are used. Then, it would allow selecting the most suitable personalized therapy. It has become increasingly clear that many chemotherapeutic agents kill susceptible cells through the induction of the physiological cell death program. Accordingly, deregulation of any gene involved in the activation or execution of the death processes may be a major mechanism of chemoresistance. Tumor suppressor and DNA repair genes were classified as important mediators of chemotherapeutic respons. While inactivation of tumor suppressor genes could lead to drug resistance, inactivation of DNA repair genes, drug metabolisms, and detoxification genes might lead to drug sensitivity. This can be due to different mechanisms like regional hypermethylation and/or global hypomethylation. The possibility that some genes conferring chemoresistance are reversibly switched on/off by DNA methylation is particularly important and may have relevant clinical implications. A very potent specific inhibitor of DNA methylation, 5-AZA, has been widely used as a demethylating agent in vitro, and is used clinically in the treatment of acute leukemia and myelodysplasia. The present Doctoral Thesis has been devoted to provide further knowledge about the cross-talk between genes promoters DNA methylation status and tumors chemosensitivity, on a more detailed understanding of the influence of its changes in resistance to dacarbazine and to platinum agents, such oxaliplatin and cisplatin. We also pretend to explore alternative therapies as an attempt for reverse tumors chemoresistance the main cause of patients dead. In order to address these goals, we studied two cancer models, colorectal and testicular germ cell tumors. 1º Study: 68 patients were enrolled in this study and treated with dacarbazine. Overall, 2% achieved partial response and 12% had stable disease. Better outcome was significantly associated with MGMT promoter hypermethylation. 2º Study: We found that oxaliplatin resistance in colon cancer cells depends on the DNA methylation-associated inactivation of SRBC gene. SRBC over-expression or depletion gives rise to sensitivity or resistance to oxaliplatin, respectively. SRBC epigenetic inactivation occurred in primary tumors from a discovery cohort of colorectal cancer patients, predicting shorter progression free survival, particularly in oxaliplatin­treated cases for which metastasis surgery was not indicated. 3º Study: We report the perpetuation of serially cisplatin-refractory orthotopic transplantable patient-derived nonseminomatous tumor grafts in mice, named orthoxenografts, as a system to investigate cisplatin refractoriness from a genetic perspective and for the preclinical development of novel targeted therapies based on overcoming cisplatin-resistance. Here we found UGCG implicated in cisplatin resistance. By quimical inactivation we were able to revert cisplatin resistance in ortoxenograt tumors. 4º Study: To explore if MGMT promoter methylation changes have a role in cisplatin chemoresistance, we study it methylation status in cisplatin sensitive and paired resistant human non-seminoma cancer cell lines, xenograft tumors and in clinical samples from metastasic patients treated with cisplatin-based chemotherapy. We found that cisplatin sensitive samples are related with MGMT promoter hypermethylation associated with its loss of expression. Clinically, the presence of MGMT promoter methylation is related with better overall survival in metastasic patients with testicular germ cell cancer. Inhibition of MGMT with O6-benzylguanine in vitro or in vivo increases the sensitivity to cisplatin and temozolomide, being this a possible chemotherapeutic approach to re-sensibilize human non-seminoma refractory tumors. We conclude that the methylation status of the gene promoters influences tumor sensitivity to different chemotherapeutic agents. We demonstrated that inhibition of MGMT and UGCG can be therapeutic ways for rescue patient’s refractory to cisplatin

SALL4 Expression in Hepatocellular Carcinomas Is Associated with EpCAM-Positivity and a Poor Prognosis

BACKGROUND: There is increasing interest in hepatocellular carcinomas (HCC) expressing "stemness"-related markers, as they have been associated with aggressive behavior and poor prognosis. In this study, we investigated the usefulness of Sal-like protein 4 (SALL4), a recently proposed candidate marker of "stemness." METHODS: Immunohistochemical stains were performed for SALL4, K19, and epithelial cellular adhesion molecule (EpCAM) on tissue microarrays constructed from 190 surgically resected HCCs, and the results were correlated with the clinicopathological features and patient survival data. RESULTS: Nuclear SALL4 expression was observed in 39/190 HCCs (20.5%), while K19 and EpCAM were expressed in 30 (15.9%) and 92 (48.7%) HCCs, respectively. The nuclear expression was generally weak, punctate or clumped. SALL4 expression was significantly associated with a poor overall survival compared to SALL4-negative HCCs (p = .014) compared to SALL4-negative HCCs. On multivariate analysis adjusted for tumor size, multiplicity, vascular invasion, and pathological tumor stage, SALL4 remained as a significant independent predictor of decreased overall survival (p= .004). SALL4 expression was positively correlated with EpCAM expression (p = .013) but not with K19 expression. HCCs that expressed both SALL4 and EpCAM were associated with significantly decreased overall survival, compared to those cases which were negative for both of these markers (p = .031). CONCLUSIONS: Although SALL4 expression was not significantly correlated with other clinicopathological parameters suggestive of tumor aggressiveness, SALL4 expression was an independent predictor of poor overall survival in human HCCs, and was also positively correlated with EpCAM expression.ope