Enrichment of Mutant KRAS Alleles in Pancreatic Juice by Subtractive Iterative Polymerase Chain Reaction

Fischer, Carsten; Büthe, Julia; Nollau, Peter; Hollerbach, Stephan; Schulmann, Karsten; Schmiegel, Wolff; Wagener, Christoph; Tschentscher, Peter

doi:10.1038/labinvest.3780292

Cited by 10 publications

(7 citation statements)

References 19 publications

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“…Also, the testing for the presence of mutant KRAS in stool has been proposed for the detection of pancreatic and colorectal carcinoma (32,33). Some studies mentioned other samples like bile or pancreatic juice (34,13,29). Detection of KRAS mutation in pancreatic juice alone is considered insuffi cient for discriminating between pancreatic cancer and benign diseases (35).…”

Section: Discussionmentioning

confidence: 99%

“…The problem of using somatic mutations as markers of malignancy is minimal amounts of mutant DNA in a large excess of wild-type DNA containing in clinical samples. Variety of technologies based on allele discrimination strategies have been applied in the identifi cation of point mutations, such as oligonucleotide ligation (12,13), PCR enriched enzymatic cleavage (14,15,16), allele-specifi c oligonucleotide hybridization (17) and integration of allele-specifi c oligonucleotide ligation assay (OLA) with magnetic beads based on electrochemiluminescence (18). Pyrosequencing methods based on chemiluminescence (19,20) and primer extension (21) was also described.…”

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confidence: 99%

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Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

Ugorcakova

Hlavatý²,

Novotna³

et al. 2012

BLL

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Abstract:Objectives: The determination of gene mutations is important for the diagnosis and prognosis of various gastrointestinal cancers. The aim of our study was to develop a new procedure for the analysis of KRAS gene mutation by application of the real-time PCR method. Background: The detection process requires discriminate trace amount of mutant allele in a large excess of wild-type DNA in various samples. Methods:The real-time PCR based technique using hybridization probes for fi ve most frequently KRAS codon 12 mutations and WT specifi c peptide nucleic acid (PNA) was performed. Our multiplex detection system was tested in various DNA samples (tissue, bile, pancreatic juice) of patients with different diagnoses of gastrointestinal tract disease obtained by endoscopy and ERCP. Results: We designed and optimized the real-time PCR conditions and tested various amount of PNA in PCR reaction to suppress amplifi cation of the wild-type DNA. We determined the interassay variability of the melting temperatures and the results of mutation testing were confi rmed by DNA sequencing with the 100 % accuracy. Incidence of searched mutations was 67.5 % in cohort of 40 patients; for KRAS G12D it was in 44.4 %, KRAS G12V in 22.2 %, KRAS G12S in 14.8 %, KRAS G12A in 14.8 % and KRAS G12C in 3.8 %. The sensitivity of the assays is 1x10 -5 . Conclusions: Advantages of this technique are rapidity, accuracy and it is generally easy to perform. This method can be adapted for synchronic detection of multiple mutations and after readjustment by other type mutation of KRAS gene may serve as useful clinical tool for analyzing point mutations in various clinical samples (Tab. 3, Fig. 3, Ref. 42). Full Text in PDF www.elis.sk.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

Ugorcakova

Hlavatý²,

Novotna³

et al. 2012

BLL

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“…However, tumor-cell enrichment is not always achievable, particularly in samples with abundant desmoplastic stroma surrounding a minority of tumor cells and abundant inflammatory cells, or in scanty tumor cells after chemotherapy or radiation therapy in the follow-up setting. 20,21 As a result, conventional PCR-based assays are limited in their ability to identify low levels of mutation-bearing tumor cells and this may greatly effect on therapeutic decisions.…”

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confidence: 99%

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples

et al. 2009

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KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 801C (vs 941C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by 44.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffinembedded tissue.

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“…Because the X-chromosome inactivation analysis is based on comparing amplification of two alleles of a gene before and after digestion with a restriction enzyme, the results are dependent of the completeness of digestion and equal amplification of the alleles on both X-chromosomes. 4 Therefore, we used an internal control. Taking these considerations into account, the results of the X-chromosome analysis in the present study indicate that the ductules are polyclonal while the endocrine component is monoclonal.…”

Section: Discussionmentioning

confidence: 99%

Ductuloinsular Tumors of the Pancreas: Endocrine Tumors With Entrapped Nonneoplastic Ductules

Eeden

Leng

Morsink

et al. 2004

American Journal of Surgical Pathology

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Rare pancreatic neoplasms have been reported that show both endocrine and exocrine differentiation in the neoplastic components. In addition, pancreatic endocrine tumors may contain small, cytologically bland ductules intimately admixed with the endocrine component. It was recently suggested that these ductules represent an intrinsic part of the tumor, ie, that the ductules are neoplastic, and the term "ductulo-insular tumors of the pancreas" was proposed. In the present study, the nature of the ductular component of 16 cases of ductule-containing pancreatic endocrine tumors was investigated at the molecular level. Molecular genetic changes often present in ductal pancreatic neoplasms were not found by immunohistochemistry for DPC4, p53, and ERBB2 and by sequence analysis of KRAS codon 12. An X-chromosome inactivation clonality assay of one such tumor from a female patient indicated that the neuroendocrine component was monoclonal, contrasting with the ductular component that was polyclonal. The lymph node and liver metastases from three patients only contained the neuroendocrine component, and no ductules were observed. Although certain morphologic features of ductule-containing endocrine tumors are reminiscent of the embryonic development of the human pancreas, none of the tumors expressed PDX-1, a transcription factor essential in pancreatic organ development. Based on our results, it is suggested that the ductular component occasionally found in pancreatic endocrine tumors is the result of entrapment of preexisting nonneoplastic ductules and that the tumors are otherwise not distinctive from conventional pancreatic endocrine tumors. Although the phenomenon is rare, it is important to recognize and to distinguish these tumors from true mixed ductal-endocrine neoplasms, which are generally more clinically aggressive. "Pancreatic endocrine tumors with entrapped ductules" would be the preferred nomenclature since it better reflects the nonneoplastic nature of the ductules.

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Enrichment of Mutant KRAS Alleles in Pancreatic Juice by Subtractive Iterative Polymerase Chain Reaction

Cited by 10 publications

References 19 publications

Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

Detection of point mutations in KRAS oncogene by real-time PCR-based genotyping assay in GIT diseases

Application of COLD-PCR for improved detection of KRAS mutations in clinical samples

Ductuloinsular Tumors of the Pancreas: Endocrine Tumors With Entrapped Nonneoplastic Ductules

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