Accurate detection of copy number aberrations in FFPE samples using the mFAST-SeqS approach.

Background: Shallow whole genome sequencing (Shallow-seq) is used to determine the copy number aberrations (CNA) in tissue samples and circulating tumor DNA. However, costs of NGS and challenges of small biopsies ask for an alternative to the untargeted NGS approaches. The mFAST-SeqS approach, relying on LINE-1 repeat amplification, showed a good correlation with Shallow-seq to detect CNA in blood samples.

PCR-Free Shallow Whole Genome Sequencing for Chromosomal Copy Number Detection from Plasma of Cancer Patients Is an Efficient Alternative to the Conventional PCR-Based Approach.

Somatic copy number alterations can be detected in cell-free DNA (cfDNA) by shallow whole genome sequencing (sWGS). PCR is typically included in library preparations, but a PCR-free method could serve as a high-throughput alternative. To evaluate a PCR-free method for research and diagnostics, archival peripheral blood or bone marrow plasma samples, collected in EDTA- or lithium-heparin-containing tubes, were collected from patients with non-small-cell lung cancer (n = 10 longitudinal samples; 4 patients), B-cell lymphoma (n = 31), and acute myeloid leukemia (n = 15), or from healthy donors (n = 14).

Engineering large-scale chromosomal deletions by CRISPR-Cas9.

Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage.

Expression of let-7i and miR-192 is associated with resistance to cisplatin-based chemoradiotherapy in patients with larynx and hypopharynx cancer.

Objectives: The majority of patients with locally advanced larynx or hypopharynx squamous cell carcinoma are treated with organ-preserving chemoradiotherapy (CRT). Clinical outcome following CRT varies greatly. We hypothesized that tumor microRNA (miRNA) expression is predictive for outcome following CRT.

Circulating Tumor DNA as a Preoperative Marker of Recurrence in Patients with Peritoneal Metastases of Colorectal Cancer: A Clinical Feasibility Study.

Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy (CRS-HIPEC) may be curative for colorectal cancer patients with peritoneal metastases (PMs) but it has a high rate of morbidity. Accurate preoperative patient selection is therefore imperative, but is constrained by the limitations of current imaging techniques. In this pilot study, we explored the feasibility of circulating tumor (ct) DNA analysis to select patients for CRS-HIPEC.

Genomic imbalances defining novel intellectual disability associated loci.

Background: High resolution genome-wide copy number analysis, routinely used in clinical diagnosis for several years, retrieves new and extremely rare copy number variations (CNVs) that provide novel candidate genes contributing to disease etiology. The aim of this work was to identify novel genetic causes of neurodevelopmental disease, inferred from CNVs detected by array comparative hybridization (aCGH), in a cohort of 325 Portuguese patients with intellectual disability (ID).

Results: We have detected CNVs in 30.

Combination of a six microRNA expression profile with four clinicopathological factors for response prediction of systemic treatment in patients with advanced colorectal cancer.

Background: First line chemotherapy is effective in 75 to 80% of patients with metastatic colorectal cancer (mCRC). We studied whether microRNA (miR) expression profiles can predict treatment outcome for first line fluoropyrimidine containing systemic therapy in patients with mCRC.

Methods: MiR expression levels were determined by next generation sequencing from snap frozen tumor samples of 88 patients with mCRC.

Loss of Chromosome 18q11.2-q12.1 Is Predictive for Survival in Patients With Metastatic Colorectal Cancer Treated With Bevacizumab.

Purpose Patients with metastatic colorectal cancer (mCRC) have limited benefit from the addition of bevacizumab to standard chemotherapy. However, a subset probably benefits substantially, highlighting an unmet clinical need for a biomarker of response to bevacizumab. Previously, we demonstrated that losses of chromosomes 5q34, 17q12, and 18q11.

DNA copy number analysis of fresh and formalin-fixed specimens by shallow whole-genome sequencing with identification and exclusion of problematic regions in the genome assembly.

Detection of DNA copy number aberrations by shallow whole-genome sequencing (WGS) faces many challenges, including lack of completion and errors in the human reference genome, repetitive sequences, polymorphisms, variable sample quality, and biases in the sequencing procedures. Formalin-fixed paraffin-embedded (FFPE) archival material, the analysis of which is important for studies of cancer, presents particular analytical difficulties due to degradation of the DNA and frequent lack of matched reference samples. We present a robust, cost-effective WGS method for DNA copy number analysis that addresses these challenges more successfully than currently available procedures.

Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology.

Purpose: Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling.

Methods: TSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (n = 58/n = 45 FFPE-tumor DNA samples) against 'gold standard' high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA.

Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping.

Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA.

An exploration of pathways involved in lung carcinoid progression using gene expression profiling.

Pulmonary carcinoids comprise a well-differentiated subset of neuroendocrine tumors usually associated with a favorable prognosis, but mechanisms underlying disease progression are poorly understood. In an explorative approach to identify pathways associated with progression, we compared gene expression profiles of tumors from five patients with a favorable and five with a poor disease outcome. Differentially expressed genes were validated using quantitative real-time PCR on 65 carcinoid tumors, in combination with survival analysis.

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures.

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling, we used biotinylated trimethylpsoralen as a DNA structure probe to show that the human genome is organized into supercoiling domains.

Detection limits of DNA copy number alterations in heterogeneous cell populations.

Background: Array Comparative Genomic Hybridization (aCGH) is a widely used technique to assess chromosomal copy number alterations. Chromosomal content, however, is often not uniform throughout cell populations. Here we evaluated to what extent aCGH can detect DNA copy number alterations in heterogeneous cell populations.

CGH arrays compared for DNA isolated from formalin-fixed, paraffin-embedded material.

Formalin-fixed, paraffin-embedded (FFPE) archival tissue is an important source of DNA material. The most commonly used technique to identify copy number aberrations from chromosomal DNA in tumorigenesis is array comparative genomic hybridization (aCGH). Although copy number analysis using DNA from FFPE archival tissue is challenging, several research groups have reported high quality and reproducible DNA copy number results using aCGH.

Trifluorothymidine resistance is associated with decreased thymidine kinase and equilibrative nucleoside transporter expression or increased secretory phospholipase A2.

Trifluorothymidine (TFT) is part of the novel oral formulation TAS-102, which is currently evaluated in phase II studies. Drug resistance is an important limitation of cancer therapy. The aim of the present study was to induce resistance to TFT in H630 colon cancer cells using two different schedules and to analyze the resistance mechanism.

Investigation of a patient with a partial trisomy 16q including the fat mass and obesity associated gene (FTO): fine mapping and FTO gene expression study.

A female patient with a partial trisomy 16q was described previously. Her clinical characteristics included obesity, severe anisomastia, moderate to severe mental retardation, attention deficit hyperactivity disorder, dysmorphic facies, and contractions of the small joints. In this article, we describe a more detailed analysis of the genetic anomaly in this patient.

Anti-proliferative action of vitamin D in MCF7 is still active after siRNA-VDR knock-down.

Background: The active form of Vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), has strong anti-proliferative effects, yet the molecular mechanisms underneath this effect remain unclear. In contrast, the molecular mechanism of 1,25D for the regulation of calcium homeostasis has principally been resolved, demonstrating a pivotal role for the vitamin D receptor (VDR).

Results: We first addressed the question whether the anti-proliferative effects of 1,25D are influenced by VDR.

Genomic profiles associated with early micrometastasis in lung cancer: relevance of 4q deletion.

Purpose: Bone marrow is a common homing organ for early disseminated tumor cells (DTC) and their presence can predict the subsequent occurrence of overt metastasis and survival in lung cancer. It is still unclear whether the shedding of DTC from the primary tumor is a random process or a selective release driven by a specific genomic pattern.

Experimental Design: DTCs were identified in bone marrow from lung cancer patients by an immunocytochemical cytokeratin assay.

A novel method to identify syncytiotrophoblast-derived RNA products representative of trisomy 21 placental RNA in maternal plasma.

A novel in vitro method is described wherein gene expression profiling is reflective and informative for the way how syncytiotrophoblast cells shed RNA products in vivo in maternal plasma. After controlled denudation, RNA is obtained selectively from the syncytiotrophoblast cells of trisomy 21 placentae. cDNA copies are subsequently analyzed by microarray profiling and cDNA cloning with sequencing.

Human and mouse oligonucleotide-based array CGH.

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution.

In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant.

Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase.