Publications

ABSTRACT Extramedullary multiple myeloma (EMM) has an overall survival of 6 months and occurs in 20% of multiple myeloma (MM) patients. Genetic and epigenetic mechanisms involved in EMM and the therapeutic role of new agents for MM are not well established. Besides, well-characterized preclinical models for EMM are not available. Herein, a patient-derived orthotopic xenograft (PDOX) was generated from a patient with an aggressive EMM to study in-depth genetic and epigenetic events, and drug responses related to extramedullary disease. A fresh punch of an extramedullary cutaneous lesion was orthotopically implanted in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ(NSG) mouse. The PDOX mimicked histologic and phenotypic features of the tumor of the patient. Cytogenetic studies revealed a hyperploid genome with multiple genetic poor-prognosis alterations. Copy number alterations (CNAs) were detected in all chromosomes. The IGH translocation t(14;16)(q32;q23)IGH/MAF was already observed at the medullary stage and a new one, t(10;14)(p?11-12;q32), was observed only with extramedullary disease and could be eventually related to EMM progression in this case. Exome sequencing showed 24 high impact single nucleotide variants and 180 indels. From the genes involved, only TP53 was previously described as a driver in MM. A rather balanced proportion of hyper/hypomethylated sites different to previously reported widespread hypomethylation in MM was also observed. Treatment with lenalidomide, dexamethasone and carfilzomib showed a tumor weight reduction of 90% versus non-treated tumors, whereas treatment with the anti-CD38 antibody daratumumab showed a reduction of 46%. The generation of PDOX from a small EMM biopsy allowed us to investigate in depth the molecular events associated with extramedullary disease in combination with drug testing.

Supplementary Figure 3 from Discovery of Epigenetically Silenced Genes by Methylated DNA Immunoprecipitation in Colon Cancer Cells

Rett Syndrome is a neurodevelopmental autism spectrum disorder caused by mutations in the gene coding for methyl CpG-binding protein (MeCP2). The disease is characterized by abnormal motor, respiratory, cognitive impairment, and autistic-like behaviors. No effective treatment of the disorder is available. Mecp2 knockout mice have a range of physiological and neurological abnormalities that resemble the human syndrome and can be used as a model to interrogate new therapies. Herein, we show that the combined administration of Levodopa and a Dopa-decarboxylase inhibitor in RTT mouse models is well tolerated, diminishes RTT-associated symptoms, and increases life span. The amelioration of RTT symptomatology is particularly significant in those features controlled by the dopaminergic pathway in the nigrostratium, such as mobility, tremor, and breathing. Most important, the improvement of the RTT phenotype upon use of the combined treatment is reflected at the cellular level by the development of neuronal dendritic growth. However, much work is required to extend the duration of the benefit of the described preclinical treatment.

<p>PDF file - 53K, Supplementary table 1 showing toxicities</p>

<p>Integration of three scRNA-seq datasets from adult murine mammary gland</p>

APOE-e4 is the major genetic risk factor for late-onset Alzheimer's Disease (LOAD) with e4 homozygotes showing earlier amyloid accumulation and onset of cognitive decline as compared to heterozygotes and e4 noncarriers [1]. The study of middle-aged healthy amyloid-free e4 homozygotes can shed light on the mechanisms through which APOE modulates the risk of AD. We aimed to determine whether APOE-e4 status has an effect on gray matter volume of healthy amyloid-negative individuals. The first 121 consecutive participants of the ALFA+ study [2] underwent 3D T1 MRI and [18F]-Flutemetamol PET brain imaging. Out of them, 87 turned out to be amyloid-negative, as assessed both by visual reading and by Centiloid quantification (<15 CL). Individuals were grouped according to the number of APOE-e4 alleles into homozygotes (N=14), heterozygotes (N=15) and non-carriers (N=58). Gray matter volume (GMV) was analyzed with Voxel Based Morphometry (VBM) using linear model that accounted for age, sex and educational level as confounders. The e4 homozygote group was compared to the heterozygote and non-carrier ones by means of one-sided t-tests (uncorrected p<0.001; k>100). Atrophy in the medial temporal lobe represents the most characteristic morphometric feature of AD. Our finding of lower GMV in middle-aged e4 homozygotes is in line with previous reports in children and adolescents [3] and add to existing literature of AD neuroimaging endopehnotypes observed, to a milder degree, in healthy APOE-e4 homozygotes [4]. Found in amyloid-negative individuals, lower entorhinal GMV can be regarded as to be a genetic trait that could contribute to their risk of developing Alzheimer's dementia. References [1] Liu et al. Nat Rev Neurol. 2013 Feb;9(2):106-18 [2] Molinuevo et al. Alzheimers Dement (N Y). 2016 Jun; 2(2): 82–92. [3] Shaw et al. Lancet Neurol. 2007 Jun;6(6):494-500. [4] Reiman et al. Proc Natl Acad Sci USA. 2005 Jun 7;102(23):8299-302.

In this Report, the Authors present the new Value Added Tax (VAT) Gap estimates for 2016, as well as updated estimates for 2012-2016. In addition to the analysis of the Compliance Gap, this Report examines the Policy Gap in 2016 as well as the contribution that reduced rates and exemptions made to the theoretical VAT revenue losses. Moreover, the Report contains an econometric analysis of VAT Gap determinants, which is a novelty introduced from this year’s Study.

// Esperanza Martín-Sánchez 1 , Saioa Mendaza 1 , Ane Ulazia-Garmendia 1 , Iñaki Monreal-Santesteban 1 , Idoia Blanco-Luquin 2 , Alicia Córdoba 3 , Francisco Vicente-García 4 , Noemí Pérez-Janices 1 , David Escors 2 , Diego Megías 5 , Paula López-Serra 6 , Manel Esteller 6, 7, 8 , José Juan Illarramendi 9 , David Guerrero-Setas 1, 3 1 Cancer Epigenetics Group, Navarrabiomed. Departmento de Salud-UPNA. IdiSNA, Pamplona, Spain 2 Immunomodulation Group, Navarrabiomed. Departmento de Salud-UPNA. IdiSNA, Pamplona, Spain 3 Department of Pathology, Complejo Hospitalario de Navarra, Servicio Navarro de Salud-Osasunbidea, Pamplona, Spain 4 Department of Surgery, Complejo Hospitalario de Navarra, Servicio Navarro de Salud-Osasunbidea, Pamplona, Spain 5 Confocal Microscopy Unit, Spanish National Cancer Research Centre, Madrid, Spain 6 Cancer Epigenetics Group, Cancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute - IDIBELL, Barcelona, Spain 7 Department of Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona, Spain 8 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain 9 Department of Oncology, Complejo Hospitalario de Navarra, Servicio Navarro de Salud-Osasunbidea, Pamplona, Spain Correspondence to: Esperanza Martín-Sánchez, email: emartisa@navarra.es David Guerrero-Setas, email: dguerres@navarra.es Keywords: CHL1, DNA methylation, breast cancer, prognostic biomarker Received: July 04, 2016 Accepted: January 03, 2017 Published: February 02, 2017 ABSTRACT The CHL1 gene encodes a cell-adhesion molecule proposed as being a putative tumour-suppressor gene in breast cancer (BC). However, neither the underlying molecular mechanisms nor the clinical value of CHL1 downregulation in BC has been explored. The methylation status of three CpG sites in the CHL1 promoter was analysed by pyrosequencing in neoplastic biopsies from 142 patients with invasive BC and compared with that of non-neoplastic tissues. We found higher CHL1 methylation levels in breast tumours than in non-neoplastic tissues, either from mammoplasties or adjacent-to-tumour, which correlated with lower levels of protein expression in tumours measured by immunohistochemistry. A panel of five BC cell lines was treated with two epigenetic drugs, and restoration of CHL1 expression was observed, indicating in vitro dynamic epigenetic regulation. CHL1 was silenced by shRNA in immortalized but non-neoplastic mammary cells, and enhanced cell proliferation and migration, but not invasion, were found by real-time cell analysis. The prognostic value of CHL1 hypermethylation was assessed by the log-rank test and fitted in a Cox regression model. Importantly, CHL1 hypermethylation was very significantly associated with shorter progression-free survival in our BC patient series, independent of age and stage ( p = 0.001). In conclusion, our results indicate that CHL1 is downregulated by hypermethylation and that this epigenetic alteration is an independent prognostic factor in BC.

The clinical relevance of the colorectal cancer serrated pathway is evident, but the screening of serrated lesions remains challenging. We aimed to characterize the serum methylome of the serrated pathway and to evaluate circulating cell‐free DNA (cfDNA) methylomes as a potential source of biomarkers for the non‐invasive detection of serrated lesions. We collected serum samples from individuals with serrated adenocarcinoma (SAC), traditional serrated adenomas, sessile serrated lesions, hyperplastic polyps and individuals with no colorectal findings. First, we quantified cfDNA methylation with the MethylationEPIC array. Then, we compared the methylation profiles with tissue and serum datasets. Finally, we evaluated the utility of serum cfDNA methylation biomarkers. We identified a differential methylation profile able to distinguish high‐risk serrated lesions from no serrated neoplasia, showing concordance with tissue methylation from SAC and sessile serrated lesions. Serum methylation profiles are pathway‐specific, clearly separating serrated lesions from conventional adenomas. The combination of ninjurin 2 ( NINJ2 ) and glutamate‐rich 1 ( ERICH1 ) methylation discriminated high‐risk serrated lesions and SAC with 91.4% sensitivity (64.4% specificity), while zinc finger protein 718 ( ZNF718 ) methylation reported 100% sensitivity for the detection of SAC (96% specificity). This is the first study exploring the serum methylome of serrated lesions. Differential methylation of cfDNA can be used for the non‐invasive detection of colorectal serrated lesions.

&lt;p&gt;Supplementary Table 2&lt;/p&gt;

Abstract Taxanes are standard therapy in clinical practice for metastatic breast cancer; however, primary or acquired chemoresistance are a common cause of mortality. Breast cancer patient-derived xenografts (PDX) are powerful tools for the study of cancer biology and drug treatment response. Specific DNA methylation patterns have been associated to different breast cancer subtypes but its association with chemoresistance remains unstudied. Aiming to elucidate docetaxel resistance mechanisms, we performed genome-wide DNA methylation in breast cancer PDX models, including luminal and triple-negative breast cancer (TNBC) models sensitive to docetaxel, their matched models after emergence of chemoresistance and residual disease after short-term docetaxel treatment. We found that DNA methylation profiles from breast cancer PDX models maintain the subtype-specific methylation patterns of clinical samples. Two main DNA methylation clusters were found in TNBC PDX and remain stable during the emergence of docetaxel resistance; however, some genes/pathways were differentially methylated according to docetaxel response. A DNA methylation signature of resistance able to segregate TNBC based on chemotherapy response was identified. Transcriptomic profiling of selected sensitive/resistant pairs and integrative analysis with methylation data demonstrated correlation between some differentially methylated and expressed genes in docetaxel-resistant TNBC PDX models. Multiple gene expression changes were found after the emergence of docetaxel resistance in TNBC. DNA methylation and transcriptional changes identified between docetaxel-sensitive and -resistant TNBC PDX models or residual disease may have predictive value for chemotherapy response in TNBC. Implications: Subtype-specific DNA methylation patterns are maintained in breast cancer PDX models. While no global methylation changes were found, we uncovered differentially DNA methylated and expressed genes/pathways associated with the emergence of docetaxel resistance in TNBC.

Abstract CpG island promoter hypermethylation of tumor suppressor genes is a common hallmark of human cancer, and new large-scale epigenomic technologies might be useful in our attempts to define the complete DNA hypermethylome of tumor cells. Here, we report a functional search for hypermethylated CpG islands using the colorectal cancer cell line HCT-116, in which two major DNA methyltransferases, DNMT1 and DNMT3b, have been genetically disrupted (DKO cells). Using methylated DNA immunoprecipitation methodology in conjunction with promoter microarray analyses, we found that DKO cells experience a significant loss of hypermethylated CpG islands. Further characterization of these candidate sequences shows CpG island promoter hypermethylation and silencing of genes with potentially important roles in tumorigenesis, such as the Ras guanine nucleotide-releasing factor (RASGRF2), the apoptosis-associated basic helix-loop transcription factor (BHLHB9), and the homeobox gene (HOXD1). Hypermethylation of these genes occurs in premalignant lesions and accumulates during tumorigenesis. Thus, our results show the usefulness of DNMT genetic disruption strategies combined with methylated DNA immunoprecipitation in searching for unknown hypermethylated candidate genes in human cancer that might aid our understanding of the biology of the disease and be of potential translational use. [Cancer Res 2007;67(24):11481–6]

&lt;p&gt;Supplementary Table 3. The twenty-eight HDAC inhibitors used in the study and their specificity&lt;/p&gt;

The study of genetic variants alone is not enough to explain a complex disease like cancer. Alterations in DNA methylation patterns have been associated with different types of tumor. In order to detect markers of susceptibility for the development of cutaneous melanoma and breast cancer in the Uruguayan population, we integrated genetic and epigenetic information of patients and controls.We performed two case-control studies that included 49 individuals with sporadic cutaneous melanoma and 73 unaffected controls, and 179 women with sporadic breast cancer and 209 women controls. We determined the level of global leukocyte DNA methylation using relative quantification of 5mdC by HPLC, and we compared methylation levels between cases and controls with nonparametric statistical tests. Since the Uruguayan population is admixed and both melanoma and breast cancer have very high incidences in Uruguay compared to other populations, we examined whether individual ancestry influences global leucocyte DNA methylation status. We carried out a correlation analysis between the percentage of African, European and Native American individual ancestries, determined using 59 ancestry informative markers, and global DNA methylation in all participants.We detected global DNA hypomethylation in leukocytes of melanoma and breast cancer patients compared with healthy controls (p < 0.001). Additionally, we found a negative correlation between African ancestry and global DNA methylation in cancer patients (p <0.005).These results support the potential use of global DNA methylation as a biomarker for cancer risk. In addition, our findings suggest that the ancestral genome structure generated by the admixture process influences DNA methylation patterns, and underscore the importance of considering genetic ancestry as a modifying factor in epigenetic association studies in admixed populations such as Latino ones.

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, EGFR, MUC1 and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.