High Detection Rates of Colorectal Neoplasia by Stool DNA Testing With a Novel Digital Melt Curve Assay

Zou, Hongzhi; Taylor, William R.; Harrington, John P.; Hussain, Fareeda Taher Nazer; Cao, Xiaoming; Loprinzi, Charles L.; Levine, Theodore R.; Rex, Douglas K.; Ahnen, Dennis J.; Knigge, Kandice L.; Lance, Peter; Jiang, Xuan; Smith, Jeremy C.; Ahlquist, David A.

doi:10.1053/j.gastro.2008.10.023

Cited by 85 publications

(54 citation statements)

References 34 publications

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“…Regarding HRM, KRAS mutations at a ratio of 5% were necessary to be detectable (27,28). A related method, the Digital Melt Curve (DMC) assay, was conducted as a two-step method enriching target genes by sequence-specific capture before combined digital PCR and HRM (29). By DMC, 0.1% levels of KRAS mutations and 0.6% levels of APC mutations were detected when applying 1,000 copies of mutant DNA (29).…”

Section: Discussionmentioning

confidence: 99%

“…A related method, the Digital Melt Curve (DMC) assay, was conducted as a two-step method enriching target genes by sequence-specific capture before combined digital PCR and HRM (29). By DMC, 0.1% levels of KRAS mutations and 0.6% levels of APC mutations were detected when applying 1,000 copies of mutant DNA (29). Deep-sequencing technology was reported to achieve a detection limit of 0.2% mutant DNA (30).…”

Section: Discussionmentioning

confidence: 99%

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

Gerecke

Mascher²,

Gottschalk³

et al. 2013

Cancer Prevention Research

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Detection of cancer precursors contributes to cancer prevention, for example, in the case of colorectal cancer. To record more patients early, ultrasensitive methods are required for the purpose of noninvasive precursor detection in body fluids. Our aim was to develop a method for enrichment and detection of known as well as unknown driver mutations in the Adenomatous polyposis coli (APC) gene. By coupled wild-type blocking (WTB) PCR and high-resolution melting (HRM), referred to as WTB-HRM, a minimum detection limit of 0.01% mutant in excess wild-type was achieved according to as little as 1 pg mutated DNA in the assay. The technique was applied to 80 tissue samples from patients with colorectal cancer (n ¼ 17), adenomas (n ¼ 50), serrated lesions (n ¼ 8), and normal mucosa (n ¼ 5). Any kind of known and unknown APC mutations (deletions, insertions, and base exchanges) being situated inside the mutation cluster region was distinguishable from wild-type DNA. Furthermore, by WTB-HRM, nearly twice as many carcinomas and 1.5 times more precursor lesions were identified to be mutated in APC, as compared with direct sequencing. By analyzing 31 associated stool DNA specimens all but one of the APC mutations could be recovered. Transferability of the WTB-HRM method to other genes was proven using the example of KRAS mutation analysis. In summary, WTB-HRM is a new approach for ultrasensitive detection of cancer-initiating mutations. In this sense, it appears especially applicable for noninvasive detection of colon cancer precursors in body fluids with excess wild-type DNA like stool. Cancer Prev Res; 6(9); 898-907. Ó2013 AACR.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Gerecke

Mascher²,

Gottschalk³

et al. 2013

Cancer Prevention Research

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“…32,33 However, the former lacks enough specificity when used to detect gene mutations because the amplification of wild-type will proceed if there is a slight change in conditions; the latter has certain disadvantages of taking time for several consecutive amplifications and introducing mutations during these PCRs. 13,34 Digital melt curve (DMC), a newly developed screening method, showed a satisfactory scanning ability for low-abundance mutations (0.1%) in the informative regions of target genes, 12 whereas it was expensive at present for use in clinical molecular diagnostic purposes. Time-cost was another important aspect to evaluate the screening method.…”

Section: Discussionmentioning

confidence: 99%

“…As described in many papers, methods with high sensitivities could achieve high mutation detection rates. 12,32,33,37 Marchetti et al 37 compared direct sequencing with mutation-enriched sequencing in 90 colorectal carcinomas. The prevalence of tumors harboring K-ras mutations was 40% using direct sequencing and 55% using mutationenriched sequencing, which had a higher sensitivity.…”

Section: Discussionmentioning

confidence: 99%

“…[8][9][10] However, the proportion of genes derived from cancer cells in stools is as low as 1%. 11 At this concentration, mutations in stool DNA will not be readily detected using the most currently available gene analyzing methods, 12 such as restriction fragment length polymorphism (RFLP), 13 single-strand conformation polymorphism (SSCP), 14 and microarray chips. 15 The major drawbacks of these assays are that they lack sufficient sensitivity and high stability, and they are rather time and sample consuming and expensive for use in screening for molecular diagnostic purposes.…”

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confidence: 99%

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Rapid detection of low-abundance K-ras mutation in stools of colorectal cancer patients using chip-based temperature gradient capillary electrophoresis

Zhang

Wang

et al. 2011

Laboratory Investigation

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Mutant K-ras provides an independent negative predictive marker for epidermal growth factor receptor (EGFR)-targeted therapy in colorectal cancers (CRCs). Rapid, sensitive, and cost-effective screening for K-ras status will overarch rational personalized medicine. Stool-based DNA testing offers unique advantages for CRC screening such as noninvasiveness, high specificity, and patient compliance, whereas complicated procedures and the low sensitivity of the present approaches have hampered its application on a wide scale. In this study, a chip-based temperature gradient capillary electrophoresis (TGCE) technique was applied to detect low-abundance K-ras mutations under a pooled experiment and analyze K-ras mutations in 30 paired stool samples and cancer tissues of CRC patients and 15 stool samples of healthy volunteers. The chip-based TGCE results showed that the successful analysis of K-ras status could be achieved within 6 min with an extremely low sample consumption of 14 nl. Detection is sensitive enough to reliably report 0.2% mutant CRC cells in a wild-type background, and 0.5 ng of template DNA was sufficient for chip-based TGCE. Of the 30 stool samples of CRC patients analyzed, 17 (57%) harbored K-ras mutations, and the lowest percentage of the detectable mutant K-ras in stool samples was 2%. The coincidence rate for K-ras mutations between stools and tissues obtained by the chip-based method reached 97% (29/30). One of the 15 stool samples of normal controls carried K-ras mutations, producing a specificity of 93%. Clone sequencing data entirely confirmed the results obtained by chip-based TGCE. The study demonstrates that chip-based TGCE is capable of rapidly screening low-abundance K-ras mutations with high sensitivity, reproducibility, simplicity, and significant savings of time and sample. Application of this method to genotype the K-ras gene in stools would provide a potential means for predicting the effectiveness of EGFR-targeted therapy in CRC patients using noninvasive approaches.

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Stool DNA testing for the detection of pancreatic cancer

Kisiel

Yab

Taylor

et al. 2011

Cancer

Self Cite

107

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BACKGROUND Pancreatic cancer (PanC) presents at late stage with high mortality. Effective early detection methods are needed. Aberrantly methylated genes are unexplored as markers for noninvasive detection by stool testing. We aimed to select discriminant methylated genes and to assess accuracy of these and mutant KRAS in stool to detect PanC. METHODS Nine target genes were assayed by real-time methylation-specific PCR (MSP) in bisulfite-treated DNA from microdissected frozen specimens of 24 PanC cases and 30 normal colon controls. Archived stools from 58 PanC cases and 65 controls matched on sex, age, and smoking were analyzed. Target genes from fecal supernatants were enriched by hybrid capture, bisulfite-treated, and assayed by MSP. KRAS mutations were assayed using the QuARTS technique. RESULTS Areas under the receiver operating characteristics curves (AUCs) for tissue BMP3, NDRG4, EYA4, UCHL1, MDFI, Vimentin, CNTNAP2, SFRP2 and TFPI2 were 0.90, 0.79, 0.78, 0.78, 0.77, 0.77, 0.69, 0.67, and 0.66, respectively. The top 4 markers and mutant KRAS were evaluated in stool. BMP3 was the most discriminant methylation marker in stool. At 90% specificity: methylated BMP3 alone detected 51% of PanCs, mutant KRAS detected 50%, and combination detected 67%. AUCs for methylated BMP3, mutant KRAS, and combination in stool were 0.73, 0.75, and 0.85, respectively. CONCLUSIONS This study demonstrates that stool assay of a methylated gene marker can detect PanC. Among candidate methylated markers discriminant in tissue, BMP3 alone performed well in stool. Combining methylated BMP3 and mutant KRAS increased stool detection over either marker alone.

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High Detection Rates of Colorectal Neoplasia by Stool DNA Testing With a Novel Digital Melt Curve Assay

Cited by 85 publications

References 34 publications

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

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

Rapid detection of low-abundance K-ras mutation in stools of colorectal cancer patients using chip-based temperature gradient capillary electrophoresis

Stool DNA testing for the detection of pancreatic cancer

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