LT-RPA: An Isothermal DNA Amplification Approach for Improved Microsatellite Genotyping and Microsatellite Instability Detection.

Microsatellites are short tandem repeats of one to six nucleotides that are highly polymorphic and extensively used as genetic markers in numerous biomedical applications, including the detection of microsatellite instability (MSI) in cancer. The standard analytical method for microsatellite analysis relies on PCR amplification followed by capillary electrophoresis or, more recently, next-generation sequencing (NGS). However, their amplification during PCR generates undesirable frameshift products known as stutter peaks caused by polymerase slippage, complicating data analysis and interpretation, while very few alternative methods for microsatellite amplification have been developed to reduce the formation of these artifacts. In this context, the recently developed low-temperature recombinase polymerase amplification (LT-RPA) is an isothermal DNA amplification method at low temperature (32 °C) that drastically reduces and sometimes completely abolishes the formation of stutter peaks. LT-RPA greatly simplifies the genotyping of microsatellites and improves the detection of MSI in cancer. In this chapter, we describe in detail all the experimental steps necessary for the development of LT-RPA simplex and multiplex assays for microsatellite genotyping and MSI detection, including the design, optimization, and validation of the assays combined with capillary electrophoresis or NGS.

DNA methylome combined with chromosome cluster-oriented analysis provides an early signature for cutaneous melanoma aggressiveness.

Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients' outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations.

A high-throughput real-time PCR tissue-of-origin test to distinguish blood from lymphoblastoid cell line DNA for (epi)genomic studies.

Lymphoblastoid cell lines (LCLs) derive from blood infected in vitro by Epstein-Barr virus and were used in several genetic, transcriptomic and epigenomic studies. Although few changes were shown between LCL and blood genotypes (SNPs) validating their use in genetics, more were highlighted for other genomic features and/or in their transcriptome and epigenome. This could render them less appropriate for these studies, notably when blood DNA could still be available. Here we developed a simple, high-throughput and cost-effective real-time PCR approach allowing to distinguish blood from LCL DNA samples based on the presence of EBV relative load and rearranged T-cell receptors γ and ß. Our approach was able to achieve 98.5% sensitivity and 100% specificity on DNA of known origin (458 blood and 316 LCL DNA). It was further applied to 1957 DNA samples from the CEPH Aging cohort comprising DNA of uncertain origin, identifying 784 blood and 1016 LCL DNA. A subset of these DNA was further analyzed with an epigenetic clock indicating that DNA extracted from blood should be preferred to LCL for DNA methylation-based age prediction analysis. Our approach could thereby be a powerful tool to ascertain the origin of DNA in old collections prior to (epi)genomic studies.

Tumor DNA hypomethylation of LINE-1 is associated with low tumor grade of breast cancer in Tunisian patients.

DNA hypomethylation of long interspersed repetitive DNA retrotransposon (LINE-1) and Alu repeats elements of short interspersed elements family (SINEs) is an early event in carcinogenesis that causes transcriptional activation and leads to chromosomal instability. In the current study, DNA methylation levels of LINE-1 and Alu repeats were analyzed in tumoral tissues of invasive breast cancer in a Tunisian cohort and its association with the clinicopathological features of patients was defined. DNA methylation of LINE-1 and Alu repeats were analyzed using pyrosequencing in 61 invasive breast cancers. Median values observed for DNA methylation of LINE-1 and Alu repeats were considered as the cut-off (59.81 and 18.49%, respectively). The results of the current study demonstrated a positive correlation between DNA methylation levels of LINE-1 and Alu repeats (rho=0.284; P<0.03). DNA hypomethylation of LINE-1 was also indicated to be associated with low grade (P=0.023). To the best of our knowledge, the current study is the first study regarding DNA methylation of LINE-1 and Alu repeats element in breast cancer of the Tunisian population. The results of the current study suggest that, since hypomethylation of LINE-1 is associated with low grade, it could be used as a biomarker for prognosis for patients with breast cancer.

Low temperature isothermal amplification of microsatellites drastically reduces stutter artifact formation and improves microsatellite instability detection in cancer.

Microsatellites are polymorphic short tandem repeats of 1-6 nucleotides ubiquitously present in the genome that are extensively used in living organisms as genetic markers and in oncology to detect microsatellite instability (MSI). While the standard analysis method of microsatellites is based on PCR followed by capillary electrophoresis, it generates undesirable frameshift products known as 'stutter peaks' caused by the polymerase slippage that can greatly complicate the analysis and interpretation of the data. Here we present an easy multiplexable approach replacing PCR that is based on low temperature isothermal amplification using recombinase polymerase amplification (LT-RPA) that drastically reduces and sometimes completely abolishes the formation of stutter artifacts, thus greatly simplifying the calling of the alleles. Using HT17, a mononucleotide DNA repeat that was previously proposed as an optimal marker to detect MSI in tumor DNA, we showed that LT-RPA improves the limit of detection of MSI compared to PCR up to four times, notably for small deletions, and simplifies the identification of the mutant alleles. It was successfully applied to clinical colorectal cancer samples and enabled detection of MSI. This easy-to-handle, rapid and cost-effective approach may deeply improve the analysis of microsatellites in several biological and clinical applications.

Local Inhibition of MEK/Akt Prevents Cellular Growth in Human Congenital Melanocytic Nevi.

The management of large congenital melanocytic nevi (lCMN) is based exclusively on iterative surgical procedures in the absence of validated medical therapy. The aim of our study was to develop an intra-lesional medical treatment for lCMN. Seventeen patients harboring NRAS-mutated lCMN were included. Nevocytes obtained from lCMN displayed an overactivation of mitogen-activated protein kinase and phosphoinositide 3-kinase (Akt) pathways. Mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and Akt inhibitors reduced the nevosphere diameter in sphere-forming assays, as well as cell viability and proliferation in in vitro assays. Standardized lCMN explants were then cultured ex vivo with the same inhibitors, which induced a decrease in MelanA+ and Sox10+ cells in both epidermis and dermis. Finally, intradermal injections of these inhibitors were administered within standardized lCMN xenografts in Rag2-/- mice. They induced a dramatic decrease in nevocytes in treated xenografts, which persisted 30 days after the end of treatment. Using original nevus explant and xenograft preclinical models, we demonstrated that intradermal MEK/Akt inhibition might serve as neoadjuvant therapy for the treatment of NRAS-mutated congenital melanocytic nevi to avoid iterative surgeries.

Microenvironment tailors nTreg structure and function.

Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFß, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts.

Demethylation by low-dose 5-aza-2'-deoxycytidine impairs 3D melanoma invasion partially through miR-199a-3p expression revealing the role of this miR in melanoma.

BACKGROUND: Efficient treatments against metastatic melanoma dissemination are still lacking. Here, we report that low-cytotoxic concentrations of 5-aza-2'-deoxycytidine, a DNA demethylating agent, prevent in vitro 3D invasiveness of metastatic melanoma cells and reduce lung metastasis formation in vivo. RESULTS: We unravelled that this beneficial effect is in part due to MIR-199A2 re-expression by promoter demethylation. Alone, this miR showed an anti-invasive and anti-metastatic effect. Throughout integration of micro-RNA target prediction databases with transcriptomic analysis after 5-aza-2'-deoxycytidine treatments, we found that miR-199a-3p downregulates set of genes significantly involved in invasion/migration processes. In addition, analysis of data from melanoma patients showed a stage- and tissue type-dependent modulation of MIR-199A2 expression by DNA methylation. CONCLUSIONS: Thus, our data suggest that epigenetic- and/or miR-based therapeutic strategies can be relevant to limit metastatic dissemination of melanoma.

Major improvement in the detection of microsatellite instability in colorectal cancer using HSP110 T17 E-ice-COLD-PCR.

Every colorectal cancer (CRC) patient should be tested for microsatellite instability (MSI) to screen for Lynch syndrome. Evaluation of MSI status involves screening tumor DNA for the presence of somatic deletions in DNA repeats using PCR followed by fragment analysis. While this method may lack sensitivity due to the presence of a high level of germline DNA, which frequently contaminates the core of primary colon tumors, no other method developed to date is capable of modifying the standard PCR protocol to achieve improvement of MSI detection. Here, we describe a new approach developed for the ultra-sensitive detection of MSI in CRC based on E-ice-COLD-PCR, using HSP110 T17, a mononucleotide DNA repeat previously proposed as an optimal marker to detect MSI in tumor DNA, and an oligo(dT)16 LNA blocker probe complementary to wild-type genotypes. The HT17 E-ice-COLD-PCR assay improved MSI detection by 20-200-fold compared with standard PCR using HT17 alone. It presents an analytical sensitivity of 0.1%-0.05% of mutant alleles in wild-type background, thus greatly improving MSI detection in CRC samples highly contaminated with normal DNA. HT17 E-ice-COLD-PCR is a rapid, cost-effective, easy-to-implement, and highly sensitive method, which could significantly improve the detection of MSI in routine clinical testing.

Hypoxia inducible factor-1 mediates the expression of the immune checkpoint HLA-G in glioma cells through hypoxia response element located in exon 2.

HLA-G is an immune checkpoint molecule with specific relevance in cancer immunotherapy. It was first identified in cytotrophoblasts, protecting the fetus from maternal rejection. HLA-G tissue expression is very restricted but induced in numerous malignant tumors such as glioblastoma, contributing to their immune escape. Hypoxia occurs during placenta and tumor development and was shown to activate HLA-G. We aimed to elucidate the mechanisms of HLA-G activation under conditions combining hypoxia-mimicking treatment and 5-aza-2'deoxycytidine, a DNA demethylating agent used in anti-cancer therapy which also induces HLA-G. Both treatments enhanced the amount of HLA-G mRNA and protein in HLA-G negative U251MG glioma cells. Electrophoretic Mobility Shift Assays and luciferase reporter gene assays revealed that HLA-G upregulation depends on Hypoxia Inducible Factor-1 (HIF-1) and a hypoxia responsive element (HRE) located in exon 2. A polymorphic HRE at -966 bp in the 5'UT region may modulate the magnitude of the response mediated by the exon 2 HRE. We suggest that therapeutic strategies should take into account that HLA-G expression in response to hypoxic tumor environment is dependent on HLA-G gene polymorphism and DNA methylation state at the HLA-G locus.

Genome-wide DNA methylation analyses in lung adenocarcinomas: Association with EGFR, KRAS and TP53 mutation status, gene expression and prognosis.

BACKGROUND: DNA methylation alterations are early events in tumorigenesis and important in the regulation of gene expression in cancer cells. Lung cancer patients have in general a poor prognosis, and a deeper insight into the epigenetic landscape in lung adenocarcinoma tumors and its prognostic implications is needed. RESULTS: We determined whole-genome DNA methylation profiles of 164 fresh frozen lung adenocarcinoma samples and 19 samples of matched normal lung tissue using the Illumina Infinium 450K array. A large number of differentially methylated CpGs in lung adenocarcinoma tissue were identified, and specific methylation profiles were observed in tumors with mutations in the EGFR-, KRAS- or TP53 genes and according to the patients' smoking status. The methylation levels were correlated with gene expression and both positive and negative correlations were seen. Methylation profiles of the tumor samples identified subtypes of tumors with distinct prognosis, including one subtype enriched for TP53 mutant tumors. A prognostic index based on the methylation levels of 33 CpGs was established, and was significantly associated with prognosis in the univariate analysis using an independent cohort of lung adenocarcinoma patients from The Cancer Genome Atlas project. CpGs in the HOX B and HOX C gene clusters were represented in the prognostic signature. CONCLUSIONS: Methylation differences mirror biologically important features in the etiology of lung adenocarcinomas and influence prognosis.

Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors.

Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof.

DNA Methylation Analysis of ChIP Products at Single Nucleotide Resolution by Pyrosequencing®.

Interaction and co-occurrence of protein and DNA-based epigenetic modifications have become a topic of interest for many fundamental and biomedical questions. We describe within this chapter a protocol that combines two techniques in order to determine the methylation status of the DNA specifically associated with a protein of interest. First, DNA that directly interacts with the selected protein (such as a specific histone modification, a transcription factor, or any other DNA-associated protein) is purified by standard chromatin immunoprecipitation (ChIP). Second, the level of DNA methylation of this immunoprecipitated DNA is measured by bisulfite conversion and Pyrosequencing, a quantitative sequencing-by-synthesis method. This procedure allows determining the methylation status of genomic DNA associated to a specific protein at single nucleotide resolution.

A novel tumor suppressor function of Kindlin-3 in solid cancer.

Kindlin-3 (FERMT-3) is known to be central in hemostasis and thrombosis control and its deficiency disrupts platelet aggregation and causes Leukocyte Adhesion Deficiency disease. Here we report that Kindlin-3 has a tumor suppressive role in solid cancer. Our present genetic and functional data show that Kindlin-3 is downregulated in several solid tumors by a mechanism involving gene hypermethylation and deletions. In vivo experiments demonstrated that Kindlin-3 knockdown in 2 tumor cell models (breast cancer and melanoma) markedly increases metastasis formation, in accord with the in vitro increase of tumor cell malignant properties. The metastatic phenotype was supported by a mechanism involving alteration in ß3-integrin activation including decreased phosphorylation, interaction with talin and the internalization of its active form leading to less cell attachment and more migration/invasion. These data uncover a novel and unexpected tumor suppressor role of Kindlin-3 which can influence integrins targeted therapies development.

Variation in genomic landscape of clear cell renal cell carcinoma across Europe.

The incidence of renal cell carcinoma (RCC) is increasing worldwide, and its prevalence is particularly high in some parts of Central Europe. Here we undertake whole-genome and transcriptome sequencing of clear cell RCC (ccRCC), the most common form of the disease, in patients from four different European countries with contrasting disease incidence to explore the underlying genomic architecture of RCC. Our findings support previous reports on frequent aberrations in the epigenetic machinery and PI3K/mTOR signalling, and uncover novel pathways and genes affected by recurrent mutations and abnormal transcriptome patterns including focal adhesion, components of extracellular matrix (ECM) and genes encoding FAT cadherins. Furthermore, a large majority of patients from Romania have an unexpected high frequency of A:T>T:A transversions, consistent with exposure to aristolochic acid (AA). These results show that the processes underlying ccRCC tumorigenesis may vary in different populations and suggest that AA may be an important ccRCC carcinogen in Romania, a finding with major public health implications.

Ultrasensitive detection and identification of BRAF V600 mutations in fresh frozen, FFPE, and plasma samples of melanoma patients by E-ice-COLD-PCR.

A number of molecular diagnostic methods have been developed for the detection and identification of mutations in tumor samples, which are important for the choice of treatment in the context of personalized medicine. For the treatment of metastatic melanoma, Vemurafenib is recommended for patients with BRAF V600 activating mutations. However, the different assays developed to date for the detection of these mutations lack sensitivity or specificity or do not allow a sequencing-based identification or validation of the mutation. Recently, enhanced improved and complete enrichment co-amplification at lower denaturation temperature-polymerase chain reaction (E-ice-COLD-PCR) has been developed as a sensitive method for the detection and identification of mutations in KRAS codons 12/13. Here, we present the first E-ice-COLD-PCR assay for the detection and identification of BRAF codon 600 mutations, which has a large dynamic range, as 25 pg to 25 ng can be used as DNA input without any reduction in mutation enrichment efficiency, and which can detect down to 0.01 % of mutated alleles in a wild-type background. The assay has been validated on fresh frozen, formalin-fixed paraffin-embedded (FFPE), and plasma samples of melanoma patients and has allowed the detection and identification of BRAF mutations present in samples appearing as wild type using standard pyrosequencing, endpoint genotyping, or Sanger sequencing. Thus, the BRAF V600 E-ice-COLD-PCR assay is currently one of the most powerful molecular diagnostic tools for the ultrasensitive detection and identification of BRAF codon 600 mutations.

Synergistic chromatin repression of the tumor suppressor gene RARB in human prostate cancers.

DNA methylation and polycomb proteins are well-known mediators of epigenetic silencing in mammalian cells. Usually described as mutually exclusive, this statement is today controversial and recent in vitro studies suggest the co-existence of both repressor systems. We addressed this issue in the study of Retinoic Acid Receptor ß (RARß), a tumor suppressor gene frequently silenced in prostate cancer. We found that the RARß promoter is hypermethylated in all studied prostate tumors and methylation levels are positively correlated with H3K27me3 enrichments. Thus, by using bisulfite conversion and pyrosequencing of immunoprecipitated H3K27me3 chromatin, we demonstrated that DNA methylation and polycomb repression co-exist in vivo at this locus. We found this repressive association in 6/6 patient tumor samples of different Gleason score, suggesting a strong interplay of DNA methylation and EZH2 to silence RARß during prostate tumorigenesis.

Sensitive detection of KRAS mutations using enhanced-ice-COLD-PCR mutation enrichment and direct sequence identification.

A number of methods allowing the detection of low levels of KRAS mutations have been developed in the last years. However, although these methods have become increasingly sensitive, they can rarely identify the mutated base directly without prior knowledge on the mutated base and are often incompatible with a sequencing-based read-out desirable in clinical practice. Here, we present a modified version of the ice-COLD-PCR assay called Enhanced-ice-COLD-PCR (E-ice-COLD-PCR) for KRAS mutation detection and identification, which allows the enrichment of the six most frequent KRAS mutations. The method is based on a nonextendable chemically modified blocker sequence, complementary to the wild-type (WT) sequence leading to the enrichment of mutated sequences. This assay permits the reliable detection of down to 0.1% mutated sequences in a WT background. A single genotyping assay of the amplification product by pyrosequencing directly following the E-ice-COLD-PCR is performed to identify the mutated base. This developed two-step method is rapid and cost-effective, and requires only a small amount of starting material permitting the sensitive detection and sequence identification of KRAS mutations within 3 hr. This method is applied in the current study to clinical colorectal cancer samples and enables detection of mutations in samples, which appear as WT using standard detection technologies.