Ultrasensitive Detection of Unknown Colon Cancer-Initiating Mutations Using the Example of the <i>Adenomatous Polyposis Coli</i> Gene

Gerecke, Christian; Mascher, Conny; Gottschalk, Uwe; Kleuser, Burkhard; Scholtka, Bettina

doi:10.1158/1940-6207.capr-13-0145

Cited by 9 publications

(4 citation statements)

References 29 publications

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“…Although in the past PTT was considered as the most sensitive method for exon 15 APC mutation detection, 29,30 recently it has been shown that NGS and other new ultrasensitive methods are able to detect APC mutations previously unnoted by either PTT or Sanger sequencing. 31,32 In the three patients tested by NGS in the present series, a previously PTT undetected exon 15 mutation was found in one patient, which is in accordance with these data.…”

Section: Discussionsupporting

confidence: 92%

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Sporadic colorectal cancer: Studying ways to an end

Rosa

Fidalgo

Filipe³

et al. 2016

UEG Journal

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Introduction: Although colorectal cancer (CRC) has often been regarded as a single entity, different pathways may lead to macroscopically similar cancers. These pathways may evolve into a patchy colonic field defect that we aimed to study in consecutive CRC patients. Methods: In a single-center, observational, prospective study, consecutive CRC patients were included if surgery and a perioperative colonoscopy were planned. Personal and familial history data were collected. Tumors were studied for microsatellite instability (MSI) status, DNA repair protein expression (DRPE) and presence of BRAF and/or APC mutations.Macroscopically normal mucosa samples were tested for APC mutations. Presence and location of synchronous and metachronous adenomas and patient follow-up were analyzed. The association of two categorical variables was tested through the Fisher's exact test (SPSS 19). Results: Twenty-four patients (12 male, mean age 69 years) were studied. High-grade MSI (MSI-H) was found in eight tumors-these were significantly more common in the right colon (p ¼ 0.047) and more likely to have an altered DRPE (p ¼ 0.007). BRAF mutation was found in two of six tested MSI-H tumors. APC gene mutations were found in nine of 16 non-MSI-H tumors and absent in normal mucosa samples. There was a nonsignificant co-localization of CRC and synchronous adenomas and a significant co-localization (p ¼ 0.05) of synchronous and metachronous adenomas. Discussion: Sporadic CRCs evolve through distinct pathways, evidenced only by pathological and molecular analysis, but clinically relevant both for patients and their families. In non-MSI-H tumors, the expected APC gene mutations were not detected by the most commonly used techniques in a high number of cases. More studies are needed to fully characterize these tumors and to search for common early events in normal mucosa patches, which might explain the indirect evidence found here for a field defect in the colon.

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Section: Discussionsupporting

confidence: 92%

“…31,32 In the three patients tested by NGS in the present series, a previously PTT undetected exon 15 mutation was found in one patient, which is in accordance with these data.…”

Section: Discussionsupporting

confidence: 92%

Sporadic colorectal cancer: Studying ways to an end

Rosa

Fidalgo

Filipe³

et al. 2016

UEG Journal

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“…For the high-sensitivity detection of low-frequency mutations in tumor-derived cfDNA, a technology has been developed that uses a polymerase chain reaction xenonucleic acid clamping approach (XNA-PCR; QClamp™). This novel technology has been developed for the detection of low-frequency mutations in tumor-derived DNA [ 52 ] and the early detection of mutations in cfDNA from cancer patients [ 53 ] and from stool-derived DNA in colorectal cancer patients [ 54 ]. This technology allows for the selective primer-mediated DNA polymerase-based amplification of only mutant template in tumor-derived DNA because the wild-type templates are blocked by the highly specific XNA probes used during PCR (Fig.…”

Section: Qclamp™ Mutation Assays Using Cfdnamentioning

confidence: 99%

Genetic alteration and mutation profiling of circulating cell-free tumor DNA (cfDNA) for diagnosis and targeted therapy of gastrointestinal stromal tumors

Wang

Zhang²,

Powell³

2016

Chin J Cancer

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Gastrointestinal stromal tumors (GISTs) have been recognized as a biologically distinctive type of tumor, different from smooth muscle and neural tumors of the gastrointestinal tract. The identification of genetic aberrations in proto-oncogenes that drive the growth of GISTs is critical for improving the efficacy of cancer therapy by matching targeted drugs to specific mutations. Research into the oncogenic mechanisms of GISTs has found that these tumors frequently contain activating gene mutations in either platelet-derived growth factor receptor A (PDGFRA) or a receptor tyrosine protein associated with a mast cell growth factor receptor encoded by the KIT gene. Mutant cancer subpopulations have the potential to disrupt durable patient responses to molecularly targeted therapy for GISTs, yet the prevalence and size of subpopulations remain largely unexplored. Detection of the cancer subpopulations that harbor low-frequency mutant alleles of target proto-oncogenes through the use of molecular genetic methods, such as polymerase chain reaction (PCR) target amplification technology, is hampered by the high abundance of wild-type alleles, which limit the sensitivity of detection of these minor mutant alleles. This is especially true in the case of mutant tumor DNA derived “driver” and “drug-resistant” alleles that are present in the circulating cell-free tumor DNA (cfDNA) in the peripheral blood circulation of GIST patients. So-called “liquid biopsy” allows for the dynamic monitoring of the patients’ tumor status during treatment using minimally invasive sampling. New methodologies, such as a technology that employs a xenonucleic acid (XNA) clamping probe to block the PCR amplification of wild-type templates, have allowed improved molecular detection of these low-frequency alleles both in tissue biopsy samples and in cfDNA. These new methodologies could be widely applied for minimally invasive molecular testing in the therapeutic management of GISTs.

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“…Massive parallel sequencing has been proven successful in identifying mutations from cancerous tissues ( Gerecke et al, 2013 , Kinde et al, 2011 ) and specific cancer panels, such as the Ion AmpliSeq™Cancer Hotspot Panel v2 and the TruSeq Amplicon — Cancer Panel (TSACP) are now available for second generation platforms. However, despite covering a broad spectrum of genes, their sensitivity is bound at 5% ( Frampton et al, 2013 , Fang et al, 2013 ; http://www.edgebio.com/ion-ampliseq-fixed-panels-hct-15-colon-carcinoma-cell-line ; Singh et al, 2013 ), a constraint which makes them ineffective when screening for CRC using stool DNA.…”

Section: Introductionmentioning

confidence: 99%

Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing

Russo

Patrignani

Poveda

et al. 2015

Applied & Translational Genomics

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Colorectal cancer (CRC) represents one of the most prevalent and lethal malignant neoplasms and every individual of age 50 and above should undergo regular CRC screening. Currently, the most effective preventive screening procedure to detect adenomatous polyps, the precursors to CRC, is colonoscopy. Since every colorectal cancer starts as a polyp, detecting all polyps and removing them is crucial. By exactly doing that, colonoscopy reduces CRC incidence by 80%, however it is an invasive procedure that might have unpleasant and, in rare occasions, dangerous side effects. Despite numerous efforts over the past two decades, a non-invasive screening method for the general population with detection rates for adenomas and CRC similar to that of colonoscopy has not yet been established. Recent advances in next generation sequencing technologies have yet to be successfully applied to this problem, because the detection of rare mutations has been hindered by the systematic biases due to sequencing context and the base calling quality of NGS.We present the first study that applies the high read accuracy and depth of single molecule, real time, circular consensus sequencing (SMRT-CCS) to the detection of mutations in stool DNA in order to provide a non-invasive, sensitive and accurate test for CRC. In stool DNA isolated from patients diagnosed with adenocarcinoma, we are able to detect mutations at frequencies below 0.5% with no false positives. This approach establishes a foundation for a non-invasive, highly sensitive assay to screen the population for CRC and the early stage adenomas that lead to CRC.

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Ultrasensitive Detection of Unknown Colon Cancer-Initiating Mutations Using the Example of the Adenomatous Polyposis Coli Gene

Cited by 9 publications

References 29 publications

Sporadic colorectal cancer: Studying ways to an end

Sporadic colorectal cancer: Studying ways to an end

Genetic alteration and mutation profiling of circulating cell-free tumor DNA (cfDNA) for diagnosis and targeted therapy of gastrointestinal stromal tumors

Highly sensitive, non-invasive detection of colorectal cancer mutations using single molecule, third generation sequencing

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