Gender-specific profiling in SCN1A polymorphisms and time-to-recurrence in patients with stage II/III colorectal cancer treated with adjuvant 5-fluoruracil chemotherapy

This study was designed to analyze the gender-related association between SCN1A polymorphisms (voltage-gated sodium channels; α-subunit) and time-to-recurrence (TTR) in patients with colorectal cancer (CRC) treated with 5-fluoruracil (5-FU)-based adjuvant chemotherapy. We enrolled from a prospective database patients with stage II and III CRC treated with adjuvant 5-FU-based chemotherapy. Genotypes for SCN1A rs3812718 and rs229877 were determined by direct DNA sequencing. One hundred twenty-seven males and 107 females were included in the study. In the univariate and multivariate analysis, the shortest TTR was associated with female patients carrying the rs3812718-TT genotype (hazard ratio (HR): 2.26 (95% confidence interval (CI): 0.89, 5.70), P=0.039) but with male patients carrying the rs3812718-CC genotype (HR: 0.49 (95% CI: 0.18, 1.38), P=0.048). For rs229877 the CT genotype was associated with a trend for shorter TTR in both gender populations. The study validated gender-dependent association between genomic SCN1A rs3812718 polymorphism and TTR in CRC patients treated with adjuvant 5-FU-based chemotherapy. This study confirms that voltage-gated Na+ channels may be a potential therapeutic target and a useful predictive biomarker before 5-FU infusion.

Common cancer stem cell gene variants predict colon cancer recurrence

PURPOSE: Recent evidence suggests that cancer stem cells (CSC) are responsible for key elements of colon cancer progression and recurrence. Germline variants in CSC genes may result in altered gene function and/or activity, thereby causing interindividual differences in a patient's tumor recurrence capacity and chemoresistance. We investigated germline polymorphisms in a comprehensive panel of CSC genes to predict time to tumor recurrence (TTR) in patients with stage III and high-risk stage II colon cancer.

EXPERIMENTAL DESIGN: A total of 234 patients treated with 5-fluorouracil-based chemotherapy at the University of Southern California were included in this study. Whole blood samples were analyzed for germline polymorphisms in genes that have been previously associated with colon CSC (CD44, Prominin-1, DPP4, EpCAM, ALCAM, Msi-1, ITGB1, CD24, LGR5, and ALDH1A1) by PCR-RFLP or direct DNA-sequencing.

RESULTS: The minor alleles of CD44 rs8193 C>T, ALCAM rs1157 G>A, and LGR5 rs17109924 T>C were significantly associated with increased TTR (9.4 vs. 5.4 years; HR, 0.51; 95% CI: 0.35-0.93; P = 0.022; 11.3 vs. 5.7 years; HR, 0.56; 95% CI: 0.33-0.94; P = 0.024, and 10.7 vs. 5.7 years; HR, 0.33; 95% CI: 0.12-0.90; P = 0.023, respectively) and remained significant in the multivariate analysis stratified by ethnicity. In recursive partitioning, a specific gene variant profile including LGR5 rs17109924, CD44 rs8193, and ALDH1A1 rs1342024 represented a high-risk subgroup with a median TTR of 1.7 years (HR, 6.71, 95% CI: 2.71-16.63, P < 0.001).

CONCLUSION: This is the first study identifying common germline variants in colon CSC genes as independent prognostic markers for stage III and high-risk stage II colon cancer patients.

Pharmacogenetic angiogenesis profiling for first-line Bevacizumab plus oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer

PURPOSE: There is substantial germline genetic variability within angiogenesis pathway genes, thereby causing interindividual differences in angiogenic capacity and resistance to antiangiogenesis therapy. We investigated germline polymorphisms in genes involved in VEGF-dependent and -independent angiogenesis pathways to predict clinical outcome and tumor response in metastatic colorectal cancer (mCRC) patients treated with bevacizumab and oxaliplatin-based chemotherapy.

EXPERIMENTAL DESIGN: A total of 132 patients treated with first-line bevacizumab and FOLFOX or XELOX were included in this study. Genomic DNA was isolated from whole-blood samples by PCR-RFLP or direct DNA sequencing. The endpoints of the study were progression-free survival (PFS), overall survival (OS), and response rate (RR).

RESULTS: The minor alleles of EGF rs444903 A>G and IGF-1 rs6220 A>G were associated with increased OS and remained significant in multivariate Cox regression analysis (HR: 0.52; 95% CI: 0.31-0.87; adjusted P = 0.012 and HR: 0.60; 95% CI: 0.36-0.99; adjusted P = 0.046, respectively). The minor allele of HIF1α rs11549465 C>T was significantly associated with increased PFS but lost its significance in multivariate analysis. CXCR1 rs2234671 G>C, CXCR2 rs2230054 T>C, EGFR rs2227983 G>A, and VEGFR-2 rs2305948 C>T predicted tumor response, with CXCR1 rs2234671 G>C remaining significant in multiple testing (P(act) = 0.003).

CONCLUSION: In this study, we identified common germline variants in VEGF-dependent and -independent angiogenesis genes predicting clinical outcome and tumor response in patients with mCRC receiving first-line bevacizumab and oxaliplatin-based chemotherapy.

Identification of novel Factor XI mutations in a patient requiring dental extractions

Factor XI is a serine protease that participates in the intrinsic pathway of blood coagulation. Patients deficient in factor XI exhibit varying degrees of post operative bleeding following invasive surgical procedures such as dental extractions. Objectives: The aim of the study was to identify the specific mutations in a patient from a family with known factor XI deficiency. Methods: Samples were obtained from the patient, his mother and his father and subjected to DNA sequencing. Each protein coding exon 2-15 of the factor XI gene was amplified by polymerase chain reaction (PCR) followed by bidirectional sequencing utilizing di-deoxy chain termination chemistry. Results: The patient had a factor XI level of 20% of normal. Initial sequencing of factor XI from the patient identified a point mutation (646G>A) and a putative splice site mutation (1567+4A>T) in intron 13. These are novel previously unreported mutations. DNA sequence analysis of the mother revealed the 1567+4A>T mutation and the father exhibited the 646G>A mutation. As a consequence the treatment proceeded without serious bleeding complication and required administration only of transexamic acid though factor XI was available as haemostatic cover. Conclusion: The two mutations identified in this family are novel; further laboratory investigation of the functional consequences of those mutations is currently underway. Although factor XI deficiency is rare in the Northern Irish population this study highlights the techniques available to sequence and analyse this and similar haematological disorders.

Mapping protein-DNA interactions using ChIP-sequencing

Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method.Chromatin immunoprecipitation (ChIP) allows enrichment of genomic regions which are associated with specific transcription factors, histone modifications, and indeed any other epitopes which are present on chromatin. The original ChIP methods used site-specific PCR and Southern blotting to confirm which regions of the genome were enriched, on a candidate basis. The combination of ChIP with genomic tiling arrays (ChIP-chip) allowed a more unbiased approach to map ChIP-enriched sites. However, limitations of microarray probe design and probe number have a detrimental impact on the coverage, resolution, sensitivity, and cost of whole-genome tiling microarray sets for higher eukaryotes with large genomes. The combination of ChIP with high-throughput sequencing technology has allowed more comprehensive surveys of genome occupancy, greater resolution, and lower cost for whole genome coverage. Herein, we provide a comparison of high-throughput sequencing platforms and a survey of ChIP-seq analysis tools, discuss experimental design, and describe a detailed ChIP-seq method.

Mapping Protein-DNA Interactions Using ChIP-exo and Illumina-Based Sequencing

Chromatin immunoprecipitation (ChIP) provides a means of enriching DNA associated with transcription factors, histone modifications, and indeed any other proteins for which suitably characterized antibodies are available. Over the years, sequence detection has progressed from quantitative real-time PCR and Southern blotting to microarrays (ChIP-chip) and now high-throughput sequencing (ChIP-seq). This progression has vastly increased the sequence coverage and data volumes generated. This in turn has enabled informaticians to predict the identity of multi-protein complexes on DNA based on the overrepresentation of sequence motifs in DNA enriched by ChIP with a single antibody against a single protein. In the course of the development of high-throughput sequencing, little has changed in the ChIP methodology until recently. In the last three years, a number of modifications have been made to the ChIP protocol with the goal of enhancing the sensitivity of the method and further reducing the levels of nonspecific background sequences in ChIPped samples. In this chapter, we provide a brief commentary on these methodological changes and describe a detailed ChIP-exo method able to generate narrower peaks and greater peak coverage from ChIPped material.

Partial structure of the phylloxin gene from the giant monkey frog, Phyllomedusa bicolor: parallel cloning of precursor cDNA and genomic DNA from lyophilized skin secretion

Phylloxin is a novel prototype antimicrobial peptide from the skin of Phyllomedusa bicolor. Here, we describe parallel identification and sequencing of phylloxin precursor transcript (mRNA) and partial gene structure (genomic DNA) from the same sample of lyophilized skin secretion using our recently-described cloning technique. The open-reading frame of the phylloxin precursor was identical in nucleotide sequence to that previously reported and alignment with the nucleotide sequence derived from genomic DNA indicated the presence of a 175 bp intron located in a near identical position to that found in the dermaseptins. The highly-conserved structural organization of skin secretion peptide genes in P. bicolor can thus be extended to include that encoding phylloxin (plx). These data further reinforce our assertion that application of the described methodology can provide robust genomic/transcriptomic/peptidomic data without the need for specimen sacrifice.

Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell adhesion and chromatin remodelling genes

Gastric cancer is a major cause of global cancer mortality. We surveyed the spectrum of somatic alterations in gastric cancer by sequencing the exomes of 15 gastric adenocarcinomas and their matched normal DNAs. Frequently mutated genes in the adenocarcinomas included TP53 (11/15 tumors), PIK3CA (3/15) and ARID1A (3/15). Cell adhesion was the most enriched biological pathway among the frequently mutated genes. A prevalence screening confirmed mutations in FAT4, a cadherin family gene, in 5% of gastric cancers (6/110) and FAT4 genomic deletions in 4% (3/83) of gastric tumors. Frequent mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) also occurred in 47% of the gastric cancers. We detected ARID1A mutations in 8% of tumors (9/110), which were associated with concurrent PIK3CA mutations and microsatellite instability. In functional assays, we observed both FAT4 and ARID1A to exert tumor-suppressor activity. Somatic inactivation of FAT4 and ARID1A may thus be key tumorigenic events in a subset of gastric cancers.

A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor-negative and -positive breast cancers

BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4).

Identifying trophic variation in a marine suspension feeder:DNA- and stable isotope-based dietary analysis in Mytilus spp.

Accurate field data on trophic interactions for suspension feeders are lacking, and new approaches to dietary analysis are necessary. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was integrated with stable isotope analysis to examine dietary patterns in suspension-feeding Mytilus spp. from seven spatially discrete locations within a semi-enclosed marine bay (Strangford Lough, Northern Ireland) during June 2009. Results of the two methods were highly correlated, reflecting dietary variation in a similar manner. Variation in PCR-DGGE data was more strongly correlated with the principal environmental gradient (distance from the opening to the Irish Sea), while values of dC and dN became progressively enriched, suggesting a greater dependence on animal tissue and benthic microalgae. Diatoms and crustaceans were the most frequently observed phylotypes identified by sequencing, but specific DNA results provided little support for the trophic trends observed in the stable isotope data. This combined approach offers an increased level of trophic insight for suspension feeders and could be applied to other organisms. © 2012 Springer-Verlag Berlin Heidelberg.

The Interlaboratory Robustness Of Next-Generation Sequencing (IRON) Study Phase II: Deep-Sequencing Analyses Of Hematological Malignancies Performed In 8,867 Cases By An International Network Involving 27 Laboratories

Introduction: Amplicon deep-sequencing using second-generation sequencing technology is an innovative molecular diagnostic technique and enables a highly-sensitive detection of mutations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Leukemia, 2011;25:1840-8).

Aims: In Phase II of the study, we established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and multiple myeloma. Currently, 27 laboratories from 13 countries are part of this research consortium. In total, 74 gene targets were selected by the working groups and amplicons were developed for a NGS deep-sequencing assay (454 Life Sciences, Branford, CT). A data analysis pipeline was developed to standardize mutation interpretation both for accessing raw data (Roche Amplicon Variant Analyzer, 454 Life Sciences) and variant interpretation (Sequence Pilot, JSI Medical Systems, Kippenheim, Germany).

Results: We will report on the design, standardization, quality control aspects, landscape of mutations, as well as the prognostic and predictive utility of this assay in a cohort of 8,867 cases. Overall, 1,146 primer sequences were designed and tested. In detail, for example in AML, 924 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 1,888 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and their utility as a biomarker to detect residual disease. Analyses of DNMT3A (n=1,041) were focused to perform landscape investigations and to address the prognostic relevance. Additionally, this working group is focusing on TET2, ASXL1, and TP53 analyses. A novel prognostic model is being developed allowing stratification of AML into prognostic subgroups based on molecular markers only. In ALL, 1,124 pediatric and adult cases have been screened, including 763 assays for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed additional content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, EP300, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R, NOTCH1, CREBBP, or FBXW7. Further, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B1, MYD88, XPO1, FBXW7 and BIRC3. Currently, 922 cases were screened to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance. In MDS, RUNX1 mutation analyses were performed in 977 cases. The prognostic relevance of TP53 mutations in MDS was assessed in additional 327 cases, including isolated deletions of chromosome 5q. Next, content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, U2AF1, and ZRSR2. In BCR-ABL1-negative MPN, nine genes of interest (JAK2, MPL, TET2, CBL, KRAS, EZH2, IDH1, IDH2, ASXL1) have been analyzed in a cohort of 155 primary myelofibrosis cases searching for novel somatic mutations and addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing run using just 20 ng of starting DNA. Finally, nine laboratories are studying CML, applying ultra-deep sequencing of the BCR-ABL1 tyrosine kinase domain. Analyses were performed on 615 cases investigating the dynamics of expansion of mutated clones under various tyrosine kinase inhibitor therapies.

Conclusion: Molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories analyzed a variety of disease entities applying amplicon-based NGS assays. Importantly, the consortium not only standardized assay design for disease-specific panels, but also achieved consensus on a common data analysis pipeline for mutation interpretation. Distinct working groups have been forged to address scientific tasks and in total 8,867 cases had been analyzed thus far.

Validation of Next Generation Sequencing Technologies in Comparison to Current Diagnostic Gold Standards for BRAF, EGFR and KRAS Mutational Analysis

Next Generation Sequencing (NGS) has the potential of becoming an important tool in clinical diagnosis and therapeutic decision-making in oncology owing to its enhanced sensitivity in DNA mutation detection, fast-turnaround of samples in comparison to current gold standard methods and the potential to sequence a large number of cancer-driving genes at the one time. We aim to test the diagnostic accuracy of current NGS technology in the analysis of mutations that represent current standard-of-care, and its reliability to generate concomitant information on other key genes in human oncogenesis. Thirteen clinical samples (8 lung adenocarcinomas, 3 colon carcinomas and 2 malignant melanomas) already genotyped for EGFR, KRAS and BRAF mutations by current standard-of-care methods (Sanger Sequencing and q-PCR), were analysed for detection of mutations in the same three genes using two NGS platforms and an additional 43 genes with one of these platforms. The results were analysed using closed platform-specific proprietary bioinformatics software as well as open third party applications. Our results indicate that the existing format of the NGS technology performed well in detecting the clinically relevant mutations stated above but may not be reliable for a broader unsupervised analysis of the wider genome in its current design. Our study represents a diagnostically lead validation of the major strengths and weaknesses of this technology before consideration for diagnostic use.

Whole-mitochondrial genome sequencing in primary open-angle glaucoma using massively parallel sequencing identifies novel and known pathogenic variants

Purpose:The aim of this study was to determine whether mutations in mitochondrial DNA play a role in high-pressure primary open-angle glaucoma (OMIM 137760) by analyzing new data from massively parallel sequencing of mitochondrial DNA.
Methods:Glaucoma patients with high-tension primary open-angle glaucoma and ethnically matched and age-matched control subjects without glaucoma were recruited. The entire human mitochondrial genome was amplified in two overlapping fragments by long-range polymerase chain reaction and used as a template for massively parallel sequencing on an Ion Torrent Personal Genome Machine. All variants were confirmed by conventional Sanger sequencing.
Results:Whole-mitochondrial genome sequencing was performed in 32 patients with primary open-angle glaucoma from India (n = 16) and Ireland (n = 16). In 16 of the 32 patients with primary open-angle glaucoma (50% of cases), there were 22 mitochondrial DNA mutations consisting of 7 novel mutations and 8 previously reported disease-associated sequence variants. Eight of 22 (36.4%) of the mitochondrial DNA mutations were in complex I mitochondrial genes.
Conclusion:Massively parallel sequencing using the Ion Torrent Personal Genome Machine with confirmation by Sanger sequencing detected a pathogenic mitochondrial DNA mutation in 50% of the primary open-angle glaucoma cohort. Our findings support the emerging concept that mitochondrial dysfunction results in the development of glaucoma and, more specifically, that complex I defects play a significant role in primary open-angle glaucoma pathogenesis.

Next-generation sequencing: a change of paradigm in molecular diagnostic validation

Next-generation sequencing (NGS) is beginning to show its full potential for diagnostic and therapeutic applications. In particular, it is enunciating its capacity to contribute to a molecular taxonomy of cancer, to be used as a standard approach for diagnostic mutation detection, and to open new treatment options that are not exclusively organ-specific. If this is the case, how much validation is necessary and what should be the validation strategy, when bringing NGS into the diagnostic/clinical practice? This validation strategy should address key issues such as: what is the overall extent of the validation? Should essential indicators of test performance such as sensitivity of specificity be calculated for every target or sample type? Should bioinformatic interpretation approaches be validated with the same rigour? What is a competitive clinical turnaround time for a NGS-based test, and when does it become a cost-effective testing proposition? While we address these and other related topics in this commentary, we also suggest that a single set of international guidelines for the validation and use of NGS technology in routine diagnostics may allow us all to make a much more effective use of resources.

Characterisation of a novel panel of polymorphic microsatellite loci for the liver fluke, Fasciola hepatica, using a next generation sequencing approach

The liver fluke, Fasciola hepatica is an economically important pathogen of sheep and cattle and has been described by the WHO as a re-emerging zoonosis. Control is heavily reliant on the use of drugs, particularly triclabendazole and as a result resistance has now emerged. The population structure of F. hepatica is not well known, yet it can impact on host-parasite interactions and parasite control with drugs, particularly regarding the spread of triclabendazole resistance. We have identified 2448 potential microsatellites from 83Mb of F. hepatica genome sequence using msatfinder. Thirty-five loci were developed and optimised for microsatellite PCR, resulting in a panel of 15 polymorphic loci, with a range of three to 15 alleles. This panel was validated on genomic DNA from 46 adult F. hepatica; 38 liver flukes sourced from a Northwest abattoir, UK and 8 liver flukes from an established isolate (Shrewsbury; Ridgeway Research). Evidence for null alleles was found at four loci (Fh_1, Fh_8, Fh_13 and Fh_14), which showed markedly higher levels of homozygosity than the remaining 11 loci. Of the 38 liver flukes isolated from cattle livers (n=10) at the abattoir, 37 genotypes were identified. Using a multiplex approach all 15 loci could be amplified from several life cycle stages that typically yield low amounts of DNA, including metacercariae, the infective life cycle stage present on pasture, highlighting the utility of this multiplex microsatellite panel. This study reports the largest panel of microsatellite markers available to date for population studies of F. hepatica and the first multiplex panel of microsatellite markers that can be used for several life cycle stages.