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Chubarov AS, Baranovskaya EE, Oscorbin IP, Yushin II, Filipenko ML, Pyshnyi DV, Vasilyeva SV, Lomzov AA. Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR. Int J Mol Sci 2024; 25:617. [PMID: 38203788 PMCID: PMC10778797 DOI: 10.3390/ijms25010617] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Revised: 12/29/2023] [Accepted: 12/30/2023] [Indexed: 01/12/2024] Open
Abstract
Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.
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Affiliation(s)
- Alexey S. Chubarov
- Correspondence: or (A.S.C.); (A.A.L.); Tel.: +7-913-763-1420 (A.S.C.); +7-(383)363-51-51 (A.A.L.)
| | | | | | | | | | | | | | - Alexander A. Lomzov
- Institute of Chemical Biology and Fundamental Medicine, SB RAS, 8 Lavrentiev Avenue, 630090 Novosibirsk, Russia; (E.E.B.); (I.P.O.); (I.I.Y.); (M.L.F.); (D.V.P.); (S.V.V.)
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Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers. Diagnostics (Basel) 2020; 10:diagnostics10110872. [PMID: 33114622 PMCID: PMC7692470 DOI: 10.3390/diagnostics10110872] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Revised: 10/21/2020] [Accepted: 10/22/2020] [Indexed: 12/20/2022] Open
Abstract
Establishing the Kirsten rat sarcoma (KRAS) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common KRAS mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a KRAS mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.
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Plagnol V, Woodhouse S, Howarth K, Lensing S, Smith M, Epstein M, Madi M, Smalley S, Leroy C, Hinton J, de Kievit F, Musgrave-Brown E, Herd C, Baker-Neblett K, Brennan W, Dimitrov P, Campbell N, Morris C, Rosenfeld N, Clark J, Gale D, Platt J, Calaway J, Jones G, Forshew T. Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling. PLoS One 2018; 13:e0193802. [PMID: 29543828 PMCID: PMC5854321 DOI: 10.1371/journal.pone.0193802] [Citation(s) in RCA: 80] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2017] [Accepted: 02/20/2018] [Indexed: 12/21/2022] Open
Abstract
Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.
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Affiliation(s)
- Vincent Plagnol
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Samuel Woodhouse
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Karen Howarth
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Stefanie Lensing
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Matt Smith
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Michael Epstein
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Mikidache Madi
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Sarah Smalley
- Product Development, Inivata Inc, Research Triangle Park, North Carolina, United States of America
| | - Catherine Leroy
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Jonathan Hinton
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Frank de Kievit
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | | | - Colin Herd
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Katherine Baker-Neblett
- Clinical Development, Inivata Inc, Research Triangle Park, North Carolina, United States of America
| | - Will Brennan
- Product Development, Inivata Inc, Research Triangle Park, North Carolina, United States of America
| | - Peter Dimitrov
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Nathan Campbell
- Clinical Laboratory Operations, Inivata Inc, Research Triangle Park, North Carolina, United States of America
| | - Clive Morris
- Clinical Development, Inivata Inc, Research Triangle Park, North Carolina, United States of America
| | - Nitzan Rosenfeld
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - James Clark
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Davina Gale
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
| | - Jamie Platt
- Product Development, Inivata Inc, Research Triangle Park, North Carolina, United States of America
| | - John Calaway
- Product Development, Inivata Inc, Research Triangle Park, North Carolina, United States of America
| | - Greg Jones
- Product Development, Inivata Inc, Research Triangle Park, North Carolina, United States of America
| | - Tim Forshew
- Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom
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Salbe C, Trevisiol C, Ferruzzi E, Mancuso T, Nascimbeni R, Di Fabio F, Salerni B, Dittadi R. Molecular Detection of Codon 12 K-RAS Mutations in Circulating DNA from Serum of Colorectal Cancer Patients. Int J Biol Markers 2018; 15:300-7. [PMID: 11192825 DOI: 10.1177/172460080001500404] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Point mutations of the K-RAS gene at codon 12 are found in about 40% of cases with colorectal cancer. The diagnostic implications of the detection of these mutations and their clinical utility are still unclear. The aim of this study was to test both the feasibility of the detection of the mutated K-RAS gene in serum and its potential role in colorectal cancer detection and monitoring. Codon 12 K-RAS mutations were examined in DNA extracted from the serum of 35 patients with colorectal cancer and were compared with the K-RAS status in the corresponding primary tumor. Molecular detection was performed by the mutant-enriched PCR (ME-PCR) assay, a sensitive method capable of distinguishing a small quantity of mutated DNA in the presence of abundant wild-type DNA. The occurrence of mutations was compared with clinicopathological parameters as well as CEA and CA19.9 serum levels. We found codon 12 K-RAS mutations in the tissue of 13/35 (37%) patients. Serum mutations were detected in 5/13 (38.5%) patients with mutated K-RAS in the tissue. 26/35 (74%) patients showed an identical K-RAS pattern in tissue and serum. No codon 12 K-RAS alterations were found in serum samples of 22 patients with benign gastrointestinal diseases. Elevated serum CEA levels were detected in 16 patients, four of whom also presented serum RAS mutations. Our results confirm that K-RAS mutations can be found in circulating DNA extracted from serum samples of patients with colorectal cancer and show that there is a correspondence between serum and tissue K-RAS patterns.
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Affiliation(s)
- C Salbe
- Center for Biological Markers of Malignancy, Regional Hospital ULSS 12, Venice, Italy.
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5
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Wan JCM, Massie C, Garcia-Corbacho J, Mouliere F, Brenton JD, Caldas C, Pacey S, Baird R, Rosenfeld N. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer 2017; 17:223-238. [PMID: 28233803 DOI: 10.1038/nrc.2017.7] [Citation(s) in RCA: 1657] [Impact Index Per Article: 207.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Improvements in genomic and molecular methods are expanding the range of potential applications for circulating tumour DNA (ctDNA), both in a research setting and as a 'liquid biopsy' for cancer management. Proof-of-principle studies have demonstrated the translational potential of ctDNA for prognostication, molecular profiling and monitoring. The field is now in an exciting transitional period in which ctDNA analysis is beginning to be applied clinically, although there is still much to learn about the biology of cell-free DNA. This is an opportune time to appraise potential approaches to ctDNA analysis, and to consider their applications in personalized oncology and in cancer research.
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Affiliation(s)
- Jonathan C M Wan
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
- Cancer Research UK Cambridge Centre, Cambridge CB2 0RE, UK
| | - Charles Massie
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
- Cancer Research UK Cambridge Centre, Cambridge CB2 0RE, UK
| | - Javier Garcia-Corbacho
- Clinical Trials Unit, Clinic Institute of Haematological and Oncological Diseases, Hospital Clinic de Barcelona, IDIBAPs, Carrer de Villarroel, 170 Barcelona 08036, Spain
| | - Florent Mouliere
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
- Cancer Research UK Cambridge Centre, Cambridge CB2 0RE, UK
| | - James D Brenton
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
- Cancer Research UK Cambridge Centre, Cambridge CB2 0RE, UK
| | - Carlos Caldas
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
- Cancer Research UK Cambridge Centre, Cambridge CB2 0RE, UK
- Department of Oncology, University of Cambridge Hutchison-MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK
| | - Simon Pacey
- Cancer Research UK Cambridge Centre, Cambridge CB2 0RE, UK
- Department of Oncology, University of Cambridge Hutchison-MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK
| | - Richard Baird
- Cancer Research UK Cambridge Centre, Cambridge CB2 0RE, UK
- Department of Oncology, University of Cambridge Hutchison-MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK
| | - Nitzan Rosenfeld
- Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK
- Cancer Research UK Cambridge Centre, Cambridge CB2 0RE, UK
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Cushman-Vokoun AM, Stover DG, Zhao Z, Koehler EA, Berlin JD, Vnencak-Jones CL. Clinical utility of KRAS and BRAF mutations in a cohort of patients with colorectal neoplasms submitted for microsatellite instability testing. Clin Colorectal Cancer 2013; 12:168-78. [PMID: 23773459 DOI: 10.1016/j.clcc.2013.04.005] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2012] [Revised: 02/19/2013] [Accepted: 04/15/2013] [Indexed: 01/12/2023]
Abstract
BACKGROUND Molecular analysis has become important in colorectal carcinoma (CRC) evaluation. Alterations in KRAS, BRAF, or mismatch repair (MMR) genes may determine therapeutic response or define a hereditary cancer syndrome. Correlation of DNA studies with clinical findings will further clarify the clinical utility of these markers. PATIENTS AND METHODS A retrospective study was performed on 111 paraffin-embedded tumor specimens submitted for microsatellite instability (MSI) testing based on clinical history or histologic examination, or both. DNA samples were screened for 7 KRAS mutations and the BRAF p.V600E mutation using fluorescent allele-specific polymerase-chain reaction (PCR) and capillary electrophoresis. Clinical data were collected through chart review. RESULTS Fifty-eight male and 53 female patients were studied. The incidence of KRAS and BRAF mutations was 49.5% and 7.2%, respectively. Dideoxy sequencing verified KRAS mutation status in 46 of 49 specimens tested. There was a trend toward significance of individual KRAS mutations on survival (P = .003). Dually positive KRAS and MSI tumors exclusively demonstrated p.G12D and p.G13D mutations (G>A transitions). BRAF-mutated tumors were predominantly right-sided and associated with a borderline worse prognosis. Forty-eight percent of tumors with MSI were present in the left colon or rectum. CONCLUSION Allele-specific PCR is an accurate and convenient method to assess KRAS and BRAF mutations and may detect mutations not identified by dideoxy sequencing. KRAS mutation status, in conjunction with morphologic or clinical parameters, may be useful in determining whether a tumor should be tested for MSI. MSI testing should not be considered exclusively in right-sided lesions. BRAF analysis may not be useful in rectal adenocarcinomas and should be evaluated in larger studies.
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Affiliation(s)
- Allison M Cushman-Vokoun
- Department of Pathology and Microbiology, 985454 Nebraska Medical Center, Omaha, NE 68105-5454, USA.
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8
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Takei F, Igarashi M, Oka Y, Koga Y, Nakatani K. Competitive allele-specific hairpin primer PCR for extremely high allele discrimination in typing of single nucleotide polymorphisms. Chembiochem 2012; 13:1409-12. [PMID: 22689446 DOI: 10.1002/cbic.201200266] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2012] [Indexed: 11/08/2022]
Affiliation(s)
- Fumie Takei
- The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan
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Wentz SC, Vnencak-Jones C, Chopp WV. Neuroendocrine and squamous colonic composite carcinoma: Case report with molecular analysis. World J Gastroenterol 2011; 17:4729-33. [PMID: 22180717 PMCID: PMC3233680 DOI: 10.3748/wjg.v17.i42.4729] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/18/2010] [Revised: 01/24/2011] [Accepted: 01/31/2011] [Indexed: 02/06/2023] Open
Abstract
Composite colorectal carcinomas are rare. There are a modest number of cases in the medical literature, with even fewer cases describing composite carcinoma with neuroendocrine and squamous components. There are to our knowledge no reports of composite carcinoma molecular alterations. We present a case of composite carcinoma of the splenic flexure in a 33 year-old Caucasian male to investigate the presence and prognostic significance of molecular alterations in rare colonic carcinoma subtypes. Formalin-fixed paraffin-embedded (FFPE) tissue was hematoxylin and eosin- and mucicarmine-stained according to protocol, and immuno-stained with cytokeratin (CK)7, CK20, CDX2, AE1/AE3, chromogranin-A and synaptophysin. DNA was extracted from FFPE tissues and molecular analyses were performed according to lab-developed methods, followed by capillary electrophoresis. Hematoxylin and eosin staining showed admixed neuroendocrine and keratinized squamous cells. Positive nuclear CDX2 expression confirmed intestinal derivation. CK7 and CK20 were negative. Neuroendocrine cells stained positively for synaptophysin and AE1/AE3 and negatively for chromogranin and mucicarmine. Hepatic metastases showed a similar immunohistochemical profile. Molecular analysis revealed a G13D KRAS mutation. BRAF mutational testing was negative and microsatellite instability was not detected. The patient had rapid disease progression on chemotherapy and died 60 d after presentation. Although the G13D KRAS mutation normally predicts an intermediate outcome, the aggressive tumor behavior suggests other modifying factors in rare types of colonic carcinomas.
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Matsukuma S, Yoshihara M, Suda T, Shiozawa M, Akaike M, Ishikawa T, Koizume S, Sakuma Y, Miyagi Y. Differential detection of KRAS mutations in codons 12 and 13 with a modified loop-hybrid (LH) mobility shift assay using an insert-type LH-generator. Clin Chim Acta 2011; 412:1874-8. [PMID: 21741959 DOI: 10.1016/j.cca.2011.06.030] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2011] [Accepted: 06/23/2011] [Indexed: 12/30/2022]
Abstract
BACKGROUND The loop-hybrid mobility shift assay (LH-MSA) was previously developed for the rapid detection of the EGFR mutation L858R for predicting clinical responses to gefitinib in lung cancer. Recently, clinical importance of determining KRAS mutations has been demonstrated in colorectal tumors as tumors harboring mutated KRAS genes were not responsive to therapy with EGFR-targeted antibodies such as cetuximab. METHODS We developed a new version of the LH-MSA using an insert-type LH generator that was capable of detecting all 12 KRAS mutations in codons 12 and 13. RESULTS Feasibility evaluation was performed with this new LH-MSA on 215 colorectal cancer specimens. KRAS codon 12 mutations were detected in 23% specimens and codon 13 mutations in 6.5% specimens by LH-MSA at a rate better than by direct sequencing. CONCLUSIONS Using the new method, the G13D mutation was readily distinguishable from other KRAS mutations in codon 12 and, therefore, would be advantageous for clinical applications.
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Affiliation(s)
- Shoichi Matsukuma
- Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Yokohama, Japan
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11
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Kristensen LS, Daugaard IL, Christensen M, Hamilton-Dutoit S, Hager H, Hansen LL. Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products. Hum Mutat 2010; 31:1366-73. [PMID: 20848649 DOI: 10.1002/humu.21358] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2010] [Accepted: 08/16/2010] [Indexed: 01/06/2023]
Abstract
Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low-level tumor specific KRAS mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation-containing templates based on the lower melting temperature of mutant homoduplexes versus wild-type homoduplexes. We have developed a fast COLD-PCR and high-resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD-PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen(®) KRAS mutation kit. The sensitivity was increased by 5- to 100-fold for melting temperature decreasing mutations when using COLD-PCR compared to standard PCR. Mutations, undetectable by the TheraScreen(®) kit in clinical samples, were detected by COLD-PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD-PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments.
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Affiliation(s)
- Lasse S Kristensen
- Institute of Human Genetics, University of Aarhus, The Bartholin Building, Aarhus C, Denmark.
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Yano Y, Yano T, Kinoshita A, Matoba A, Hasuma T, Wanibuchi H, Morimura K, Otani S, Fukushima S. Sensitive quantitative assay for point mutations in the rat H-ras gene based on single nucleotide primer extension. Exp Ther Med 2010; 1:657-661. [PMID: 22993590 DOI: 10.3892/etm_00000103] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2010] [Accepted: 05/31/2010] [Indexed: 01/04/2023] Open
Abstract
Point mutations in oncogenes and tumor suppressor genes occur at early stages in the carcinogenic process. Point mutations in ras family oncogenes are the most common mutational events in several types of human cancer, and are available as molecular markers for the detection of cancer cells in carcinogenicity bioassay systems as well as in clinical samples. Although several techniques are utilized to detect point mutations in carcinogenicity bioassay systems, the sensitivity is too low to determine a small number of mutations. In order to overcome the disadvantage and to sensitively determine gene mutation rates for in vivo carcinogenicity bioassays of presumptive carcinogens, we established a Thermosequenase Cycle End Labeling (TCEL) method, a sensitive approach based on single nucleotide primer extension. One of the characteristics of the method is a high sensitivity of 1:100,000, ten times the sensitivity of the mutant allele-specific amplification now commonly employed. Using TCEL, we here quantified H-ras mutations in the livers of rats treated with a genotoxic carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. Our findings suggest that this method may be applied for many genetic targets as a component in vivo.
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Mutational analysis of K-ras codon 12 in blood samples of patients with acute myeloid leukemia. Leuk Res 2010; 34:883-91. [DOI: 10.1016/j.leukres.2010.02.023] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2009] [Revised: 02/02/2010] [Accepted: 02/22/2010] [Indexed: 11/17/2022]
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Kinoshita E, Kinoshita-Kikuta E, Koike T. Phosphate-affinity polyacrylamide gel electrophoresis for SNP genotyping. Methods Mol Biol 2009; 578:183-92. [PMID: 19768594 DOI: 10.1007/978-1-60327-411-1_11] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/15/2023]
Abstract
We introduce a genotyping method which relies on the use of a 1:1 mixture of 5'-phosphate-labeled and nonlabeled allele-specific primers for polymerase chain reaction (PCR). The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (possessing the same number of base pairs) during phosphate-affinity polyacrylamide gel electrophoresis (PAGE). The phosphate-affinity site in the gel is represented by an immobilized phosphate-binding tag molecule [i.e., a polyacrylamide-bound dizinc(II) complex], which selectively captures the 5'-phosphate-labeled allele-specific product compared with the corresponding nonlabeled one. The DNA migration bands obtained can be visualized by ethidium bromide staining. We demonstrate the genotyping of a single-nucleotide polymorphism reported in a human cardiac sodium channel gene, SCN5A, using the phosphate-affinity PAGE.
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Affiliation(s)
- Eiji Kinoshita
- Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan
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15
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Godai TI, Suda T, Sugano N, Tsuchida K, Shiozawa M, Sekiguchi H, Sekiyama A, Yoshihara M, Matsukuma S, Sakuma Y, Tsuchiya E, Kameda Y, Akaike M, Miyagi Y. Identification of colorectal cancer patients with tumors carrying the TP53 mutation on the codon 72 proline allele that benefited most from 5-fluorouracil (5-FU) based postoperative chemotherapy. BMC Cancer 2009; 9:420. [PMID: 19954513 PMCID: PMC2796677 DOI: 10.1186/1471-2407-9-420] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2009] [Accepted: 12/02/2009] [Indexed: 01/18/2023] Open
Abstract
BACKGROUND Although postoperative chemotherapy is widely accepted as the standard modality for Dukes' stage C or earlier stage colorectal cancer (CRC) patients, biomarkers to predict those who may benefit from the therapy have not been identified. Previous in vitro and clinical investigations reported that CRC patients with wild-type p53 gene (TP53)-tumors benefit from 5-fluorouracil (5-FU) based chemotherapy, while those with mutated TP53-tumors do not. However, these studies evaluated the mutation-status of TP53 by immunohistochemistry with or without single-strand conformation polymorphism, and the mutation frequency was different from study to study. In addition, the polymorphic status at p53 codon 72, which results in arginine or proline residues (R72P) and is thought to influence the function of the protein significantly, was not examined. METHODS To evaluate the significance of the TP53 mutation as a molecular marker to predict the prognosis of CRC patients, especially those who received postoperative chemotherapy, we examined the mutation by direct sequencing from fresh CRC tumors and evaluated the R72P polymorphism of the mutated TP53 by a combined mutant allele- and polymorphic allele-specific polymerase chain reaction (PCR). RESULTS The TP53 mutation occurred in 147 (70%) of 211 Japanese CRC tumors. The mutation was observed in 93 (63%) tumors on the R72 allele and in 54 (37%) tumors on the P72 allele. Although the alterations to TP53 have no prognostic significance for CRC patients overall, we found that Dukes' stage C CRC patients who did not receive postoperative chemotherapy and carried the mutated TP53-R72 showed significantly longer survival times than those with the mutated TP53-P72 when evaluated by overall survival (p = 0.012). CONCLUSION Using a combined mutant allele- and polymorphic allele-specific PCR, we defined the codon 72 polymorphic status of the TP53 mutated allele in Japanese CRC patients. We raised a possibility that Dukes' stage C colorectal cancer patients with tumors carrying TP53 mutation, especially the P72 allele, benefited from 5-FU based postoperative chemotherapy.
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Affiliation(s)
- Ten-i Godai
- Department of Gastrointestinal Surgery, Kanagawa Cancer Center Hospital, 1-1-2 Nakao, Asahi-ku, Yokohama, Japan.
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Milbury CA, Li J, Makrigiorgos GM. PCR-based methods for the enrichment of minority alleles and mutations. Clin Chem 2009; 55:632-40. [PMID: 19201784 DOI: 10.1373/clinchem.2008.113035] [Citation(s) in RCA: 141] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND The ability to identify low-level somatic DNA mutations and minority alleles within an excess wild-type sample is becoming essential for characterizing early and posttreatment tumor status in cancer patients. Over the past 2 decades, much research has focused on improving the selectivity of PCR-based technologies for enhancing the detection of minority (mutant) alleles in clinical samples. Routine application in clinical and diagnostic settings requires that these techniques be accurate and cost-effective and require little effort to optimize, perform, and analyze. CONTENT Enrichment methods typically segregate by their ability to enrich for, and detect, either known or unknown mutations. Although there are several robust approaches for detecting known mutations within a high background of wild-type DNA, there are few techniques capable of enriching and detecting low-level unknown mutations. One promising development is COLD-PCR (coamplification at lower denaturation temperature), which enables enrichment of PCR amplicons containing unknown mutations at any position, such that they can be subsequently sequenced to identify the exact nucleotide change. SUMMARY This review summarizes technologies available for detecting minority DNA mutations, placing an emphasis on newer methods that facilitate the enrichment of unknown low-level DNA variants such that the mutation can subsequently be sequenced. The enrichment of minority alleles is imperative in clinical and diagnostic applications, especially in those related to cancer detection, and continued technology development is warranted.
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Affiliation(s)
- Coren A Milbury
- Department of Radiation Oncology, Division of Medical Physics and Biophysics, and Division of DNA Repair and Genome Stability, Dana Farber/Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA 02115, USA
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17
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Clinical significance of cathepsin E in pancreatic juice in the diagnosis of pancreatic ductal adenocarcinoma. J Gastroenterol Hepatol 2008. [DOI: 10.1046/j.1440-1746.2000.2351.x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 04/08/2023]
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Tatsumi K, Mitani Y, Watanabe J, Takakura H, Hoshi K, Kawai Y, Kikuchi T, Kogo Y, Oguchi-Katayama A, Tomaru Y, Kanamori H, Baba M, Ishidao T, Usui K, Itoh M, Cizdziel PE, Lezhava A, Ueda M, Ichikawa Y, Endo I, Togo S, Shimada H, Hayashizaki Y. Rapid screening assay for KRAS mutations by the modified smart amplification process. J Mol Diagn 2008; 10:520-6. [PMID: 18832461 DOI: 10.2353/jmoldx.2008.080024] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.
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Affiliation(s)
- Kenji Tatsumi
- Genome Exploration Research Group (Genome Network Project Core Group), RIKEN Genomic Sciences Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, Japan.
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Kimura K, Nagasaka T, Hoshizima N, Sasamoto H, Notohara K, Takeda M, Kominami K, Iishii T, Tanaka N, Matsubara N. No duplicate KRAS mutation is identified on the same allele in gastric or colorectal cancer cells with multiple KRAS mutations. J Int Med Res 2007; 35:450-7. [PMID: 17697521 DOI: 10.1177/147323000703500403] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Codon 12 and 13 mutations in 170 colorectal cancer (CRC) and 66 gastric cancer (GC) specimens were analysed by an 'enriched' polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All identified mutations were verified by direct sequencing of the second PCR products. Among the 170 CRC specimens, mutations were identified in 47 (28%) and 13 (7.6%) cases in codons 12 and 13, respectively. In the 66 GC specimens examined, however, mutations in codons 12 and 13 were only detected in two (3.0%) and one (1.5%) cases, respectively. Mutations in both codon 12 and 13 were found in 3/170 (1.8%) CRCs and 1/66 (1.5%) GCs. Duplicate mutations were never identified in the same allele, which was confirmed by direct sequencing of the second amplified products. The majority of colorectal and gastric cancer cells with KRAS mutations are homogeneous because they have the same KRAS mutation. A few colorectal or gastric cancers, however, showed heterogeneity, as verified by the fact that single mutations were identified in the same allele.
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Affiliation(s)
- K Kimura
- Department of Gastroenterological Surgery and Surgical Oncology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
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Katsumata K, Sumi T, Mori Y, Hisada M, Tsuchida A, Aoki T. Detection and evaluation of epithelial cells in the blood of colon cancer patients using RT-PCR. Int J Clin Oncol 2007; 11:385-9. [PMID: 17058136 DOI: 10.1007/s10147-006-0590-5] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2005] [Accepted: 05/25/2006] [Indexed: 11/24/2022]
Abstract
BACKGROUND As a mode of colorectal cancer recurrence, liver metastasis plays an important role. One of the factors reported to predict liver metastasis is the detection of trace amounts of tumor cells in the blood. For this purpose, cancer cell-induced cytokeratins (CKs) are generally identified, using the reverse transcriptase-polymerase chain reaction (RT-PCR). In the present study, we aimed to detect trace amounts of tumor cells, based on CK20, in the circulating venous blood, and we examined pathological factors, liver metastasis, and prognosis. METHODS The subjects were 57 colorectal cancer patients who had undergone operation. We examined the cancer-induced marker (CK20) in circulating venous blood by RT-PCR and investigated the relationships between this marker, pathological factors, and prognosis. RESULTS Detection ratio of CK20 mRNA was 42.1%, and CK20 was significantly correlated with the pathological factor of lymph node metastasis (P = 0.037). The 5-year survival rate for CK20-positive patients was 62.5% and that for the CK20-negative patients was 87.5%; there was a significant difference (P = 0.048) between the two groups. Recurrence was recognized in six patients; two were positive for CK20 and four were negative for CK20. CONCLUSIONS These findings indicate that CK20 is strongly related to lymph node metastasis and prognosis, suggesting its usefulness for the diagnosis of colorectal cancer recurrence. However, CK20 did not predict liver metastasis.
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Affiliation(s)
- Kenji Katsumata
- The Third Department of Surgery, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo, 160-0022, Japan.
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21
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Matsukuma S, Yoshihara M, Kasai F, Kato A, Yoshida A, Akaike M, Kobayashi O, Nakayama H, Sakuma Y, Yoshida T, Kameda Y, Tsuchiya E, Miyagi Y. Rapid and simple detection of hot spot point mutations of epidermal growth factor receptor, BRAF, and NRAS in cancers using the loop-hybrid mobility shift assay. J Mol Diagn 2006; 8:504-12. [PMID: 16931592 PMCID: PMC1867624 DOI: 10.2353/jmoldx.2006.060030] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed.
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Affiliation(s)
- Shoichi Matsukuma
- Division of Molecular Pathology and Genetics, Kanagawa Cancer Center Research Institute, Nakao 1-1-2, Asahi-ku, Yokohama 241-0815, Japan.
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22
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Sugio K, Uramoto H, Ono K, Oyama T, Hanagiri T, Sugaya M, Ichiki Y, So T, Nakata S, Morita M, Yasumoto K. Mutations within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking. Br J Cancer 2006; 94:896-903. [PMID: 16552419 PMCID: PMC3216424 DOI: 10.1038/sj.bjc.6603040] [Citation(s) in RCA: 105] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
Somatically acquired mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer are associated with significant clinical responses to gefitinib, a tyrosine kinase inhibitor that targets EGFR. We screened the EGFR in 469 resected tumours of patients with lung cancer, which included 322 adenocarcinomas, 102 squamous cell carcinomas, 27 large cell carcinomas, 13 small cell carcinomas, and five other cell types. PCR with a specific condition was performed to identify any deletion in exon 19, while mutant-allele-specific amplification was performed to identify a mutation in codon 858 of exon 21. EGFR mutations were found in 136 cases (42.2%) with adenocarcinoma, in one case with large cell carcinoma, and in one case with pleomorphic carcinoma. An in-frame deletion in exon 19 was found in 62 cases while an L858R mutation was found in 77 cases. In the 322 cases with adenocarcinoma, these mutations were more frequently found in women than in men (P=0.0004), in well differentiated tumours than in poorly differentiated tumours (P=0.0014), and in patients who were never smokers than in patients who were current/former smokers (P<0.0001). The mutation was more frequently observed in patients who smoked ⩽20 pack-year, and in patients who quit at least 20 years before the date of diagnosis for lung cancer. The K-ras mutations were more frequently found in smokers than in never smokers, and in high-dose smokers than in low-dose smokers. In conclusion, the mutations within the tyrosine kinase domain of EGFR were found to specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking.
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Affiliation(s)
- K Sugio
- Second Department of Surgery, University of Occupational and Environmental Health, 1-1 Iseigaoka, Kitakyushu, 807-8555 Japan.
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23
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Yamazaki Y, Chiba I, Hirai A, Satoh C, Sakakibara N, Notani KI, Iizuka T, Totsuka Y. Clinical value of genetically diagnosed lymph node micrometastasis for patients with oral squamous cell carcinoma. Head Neck 2006; 27:676-81. [PMID: 15957194 DOI: 10.1002/hed.20200] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
Abstract
BACKGROUND The purpose of this study was to investigate the incidence and clinical significance of genetically diagnosed lymph node micrometastasis for patients with oral squamous cell carcinoma (SCC). METHODS A total of 495 lymph nodes obtained from 21 patients with primary oral SCCs that had p53 mutations were examined for corresponding p53 mutations in lymph nodes using mutant allele-specific amplification (MASA). RESULTS Among 476 histologically negative nodes, 44 were scored as positive for metastasis by MASA. All 19 histologically positive lymph nodes were genetically positive. Four of the 10 pN0 cases and nine of the 11 pN-positive cases had genetically positive micrometastases. Four patients who had five or more genetically positive lymph nodes located in three or more levels, three with disease staged as pN0 or pN1, died of cancer. CONCLUSIONS These results indicate that a high rate of micrometastasis in cervical lymph nodes of oral SCCs and patients with multiple or lower neck spread of micrometastases have a poor prognosis; they should be treated with postoperative adjuvant therapy.
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Affiliation(s)
- Yutaka Yamazaki
- Oral Diagnosis and Medicine, Graduate School of Dental Medicine, Hokkaido University, Kita-13 Nishi-7, Sapporo, 060-8586, Japan.
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24
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Kappel S, Kandioler D, Steininger R, Längle F, Wrba F, Ploder M, Berlakovich G, Soliman T, Hetz H, Rockenschaub S, Roth E, Mühlbacher F. Genetic detection of lymph node micrometastases: a selection criterion for liver transplantation in patients with liver metastases after colorectal cancer. Transplantation 2006; 81:64-70. [PMID: 16421478 DOI: 10.1097/01.tp.0000189711.98971.9c] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
BACKGROUND Liver transplantation for nonresectable liver metastases from colorectal cancer was abandoned in 1994 on account of high recurrence rates. The aim of this study was to investigate whether the genetic detection of micrometastases in histologically negative lymph nodes of the primary colon cancer could be applied to select patients for liver transplantation. METHODS We analyzed 21 patients with colorectal cancer who had undergone liver transplantation between 1983 and 1994 for liver metastases. Eleven patients were histologically lymph node negative at the time of surgery; ten patients with lymph node metastases served as control group. DNA sequencing was used to screen tumor material for p53 and K-ras mutations. Mutant allele-specific amplification (MASA) was then used to search for micrometastases in DNA from regional lymph nodes of the primary colorectal cancer. RESULTS p53 and K-ras mutations were detected in 12 (57%) and 3 (14%) of 21 patients in the colorectal cancer, respectively. The mutations were confirmed in the corresponding liver metastases. Of 11 patients with histologically negative lymph nodes, nine were eligible for MASA due to presence of p53 or K-ras mutation. MASA revealed six of nine patients to be genetically positive for micrometastases. Three patients were both genetically and histologically negative. These three patients showed a significantly longer overall survival (P = 0.011) of 4, 5, and 20 years, respectively. CONCLUSIONS We conclude that the genetic detection of micrometastases by MASA could be a powerful prognostic indicator for selecting patients with colorectal liver metastases who could benefit from liver transplantation.
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Affiliation(s)
- Sonja Kappel
- Department of Surgical Research, Medical University of Vienna, Vienna, Austria
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25
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Däbritz J, Hänfler J, Preston R, Stieler J, Oettle H. Detection of Ki-ras mutations in tissue and plasma samples of patients with pancreatic cancer using PNA-mediated PCR clamping and hybridisation probes. Br J Cancer 2005; 92:405-12. [PMID: 15655549 PMCID: PMC2361834 DOI: 10.1038/sj.bjc.6602319] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
In the present study, we combined the PCR-clamping approach with melting curve analysis using mutant specific hybridisation probes and wild-type specific peptide nucleic acids (PNAs) to determine the genotypes of the most frequent point mutation in codon 12 of the proto-oncogene Ki-ras in tissue and plasma samples of patients with pancreatic cancer. The sensitivity of our assay was 1–5 × 10−5. The melting curve analysis of tissue samples of four patients revealed two valine mutations, one none-valine mutation and one wild-type sequence. Ki-ras alterations were found in 28% of DNAs (18 out of 64) of nonrelated plasma samples of 10 patients with ductal adenocarcinoma of the pancreas. The valine mutation was the predominantly detected gene alteration (83%). Out of ten patients investigated, four patients (40%) became positive during clinical observation with respect to Ki-ras mutation. All four patients exhibited progressive disease and high levels of tumour marker CA 19-9. In conclusion, the one-step procedure discribed may be a useful clinical tool for analysing Ki-ras point mutations in tissue and plasmas samples. In addition, this method can be adapted for simultanous detection of multiple mutations and quantitation.
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Affiliation(s)
- J Däbritz
- Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Medizinische Klinik und Poliklinik m.S. Hämatologie und Onkologie, Augustenburger Platz 1, 13353 Berlin, Germany
| | - J Hänfler
- Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Medizinische Klinik und Poliklinik m.S. Hämatologie und Onkologie, Augustenburger Platz 1, 13353 Berlin, Germany
- Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Medizinische Klinik und Poliklinik m.S. Hämatologie und Onkologie, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail:
| | - R Preston
- Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Medizinische Klinik und Poliklinik m.S. Hämatologie und Onkologie, Augustenburger Platz 1, 13353 Berlin, Germany
| | - J Stieler
- Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Medizinische Klinik und Poliklinik m.S. Hämatologie und Onkologie, Augustenburger Platz 1, 13353 Berlin, Germany
| | - H Oettle
- Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Medizinische Klinik und Poliklinik m.S. Hämatologie und Onkologie, Augustenburger Platz 1, 13353 Berlin, Germany
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Yamada S, Yashiro M, Maeda K, Nishiguchi Y, Hirakawa K. A novel high-specificity approach for colorectal neoplasia: Detection of K-ras2 oncogene mutation in normal mucosa. Int J Cancer 2005; 113:1015-21. [PMID: 15514939 DOI: 10.1002/ijc.20666] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
There is an important need for a high-specificity approach to colorectal cancer. Approximately 50% of colorectal tumors contain K-ras gene mutations, which occur as an early step in carcinogenesis. K-ras mutations were detectable not only in tumors but also in microscopically normal colorectal mucosa close to carcinomas in some patients with colorectal cancer. This is the first systematic analysis of K-ras mutations in normal colonic mucosa at multiple consistently-selected locations. A total of 480 normal colonic mucosal samples were obtained from 80 subjects, including 65 patients with sporadic colorectal cancer and 15 controls in whom a colorectal neoplasm was ruled out endoscopically. Normal mucosal samples were obtained at multiple consistently-selected locations using biopsy forceps during colonoscopy. Mutant allele-specific amplification (MASA)-PCR was performed; this could detect a K-ras mutation in normal colonic mucosa even though it was only sparsely present. The K-ras mutation was found in histologically normal mucosa from colorectal cancer patients (20 of 65 cases; 41 of 390 loci) by MASA-PCR, especially frequent (51%; 19 of 37 cases) when the tumor showed a K-ras mutation. In contrast, no mutation was found in normal mucosa from 15 controls (90 loci). K-ras mutation in normal mucosa showed a significant association with the presence of colorectal cancer (p = 0.008). The specificity of the MASA-PCR method for colorectal neoplasms was thus 100%. We conclude that detection of K-ras mutations in normal colonic mucosa might serve as a high-specificity approach to colorectal cancer.
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Affiliation(s)
- Shinobu Yamada
- Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Abenoku, Osaka, Japan
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27
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García-Olmo DC, Gutiérrez-González L, Ruiz-Piqueras R, Picazo MG, García-Olmo D. Detection of circulating tumor cells and of tumor DNA in plasma during tumor progression in rats. Cancer Lett 2005; 217:115-23. [PMID: 15596302 DOI: 10.1016/j.canlet.2004.06.043] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2004] [Revised: 06/15/2004] [Accepted: 06/19/2004] [Indexed: 10/26/2022]
Abstract
The role and clinical significance of circulating tumor cells and of tumor DNA in the plasma have not yet been clarified. In the present study, we compared rates of detection of tumor-derived DNA in the buffy coat to those in plasma from tumor-bearing rats, and we attempted to correlate these rates with the progression of tumors. We injected DHD/K12-PROb cancer cells subcutaneously into BD-IX rats and divided the animals into six groups according to the time between the injection of tumor cells and euthanasia. After euthanasia, macroscopic metastases were assessed and samples of blood and lung were collected. We used mutant allele-specific amplification by PCR to detect tumor-derived DNA. We detected tumor DNA in lung samples from the first week after inoculation, in plasma from the third week and in the buffy coat from the fifth week. All animals analyzed on the 11th week had macro- or micrometastases in their lungs. Regardless of group, the rate of PCR-positive plasma samples was significantly higher than that of circulating tumor cells (P=0.005). In animals with metastases, this difference was also statistically significant (P=0.008). However, neither the detection of tumor DNA in the plasma nor the presence of circulating tumor cells was strongly correlated with the presence of metastases. Thus, cell-free tumor DNA was detected sooner and more frequently than circulating tumor cells and the dissemination of tumor DNA in the plasma seems to be much more common than detectable hematogenic tumor cells during the spread of colorectal cancer.
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Affiliation(s)
- Dolores C García-Olmo
- Experimental Research Unit, Hospital General Universitario de Albacete, Unidad de Investigación, C/ Hermanos Falcó 37, 02006 Albacete, Spain.
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Shi C, Eshleman SH, Jones D, Fukushima N, Hua L, Parker AR, Yeo CJ, Hruban RH, Goggins MG, Eshleman JR. LigAmp for sensitive detection of single-nucleotide differences. Nat Methods 2004; 1:141-7. [PMID: 15782177 DOI: 10.1038/nmeth713] [Citation(s) in RCA: 101] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2004] [Accepted: 09/02/2004] [Indexed: 02/07/2023]
Abstract
We developed the LigAmp assay for sensitive detection and accurate quantification of viruses and cells with single-base mutations. In LigAmp, two oligonucleotides are hybridized adjacently to a DNA template. One oligonucleotide matches the target sequence and contains a probe sequence. If the target sequence is present, the oligonucleotides are ligated together and detected using real-time PCR. LigAmp detected KRAS2 mutant DNA at 0.01% in mixtures of different cell lines. KRAS2 mutations were also detected in pancreatic duct juice from patients with pancreatic cancer. LigAmp detected the K103N HIV-1 drug resistance mutation at 0.01% in plasmid mixtures and at approximately 0.1% in DNA amplified from plasma HIV-1. Detection in both systems is linear over a broad dynamic range. Preliminary evidence indicates that reactions can be multiplexed. This assay may find applications in the diagnosis of genetic disorders and the management of patients with cancer and infectious diseases.
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Affiliation(s)
- Chanjuan Shi
- Department of Pathology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, Maryland 21205, USA
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Nakamura M, Ando Y, Nagahara S, Sano A, Ochiya T, Maeda S, Kawaji T, Ogawa M, Hirata A, Terazaki H, Haraoka K, Tanihara H, Ueda M, Uchino M, Yamamura K. Targeted conversion of the transthyretin gene in vitro and in vivo. Gene Ther 2004; 11:838-46. [PMID: 14961068 DOI: 10.1038/sj.gt.3302228] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Familial amyloidotic polyneuropathy (FAP) is the common form of hereditary generalized amyloidosis and is characterized by the accumulation of amyloid fibrils in the peripheral nerves and other organs. Liver transplantation has been utilized as a therapy for FAP, because the variant transthyretin (TTR) is predominantly synthesized by the liver, but this therapy is associated with several problems. Thus, we need to develop a new treatment that prevents the production of the variant TTR in the liver. In this study, we used HepG2 cells to show in vitro conversion of the TTR gene by single-stranded oligonucleotides (SSOs), embedded in atelocollagen, designed to promote endogenous repair of genomic DNA. For the in vivo portion of the study, we used liver from transgenic mice whose intrinsic wild-type TTR gene was replaced by the murine TTR Val30Met gene. The level of gene conversion was determined by real-time RCR combined with mutant-allele-specific amplification. Our results indicated that the level of gene conversion was approximately 11 and 9% of the total TTR gene in HepG2 cells and liver from transgenic mice, respectively. Gene therapy via this method may therefore be a promising alternative to liver transplantation for treatment of FAP.
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Affiliation(s)
- M Nakamura
- Department of Laboratory Medicine, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860-0811, Japan
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30
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Wang Y, Yamaguchi Y, Watanabe H, Ohtsubo K, Motoo Y, Sawabu N. Detection of p53 gene mutations in the supernatant of pancreatic juice and plasma from patients with pancreatic carcinomas. Pancreas 2004; 28:13-9. [PMID: 14707724 DOI: 10.1097/00006676-200401000-00002] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
AIM The sensitivity of pure pancreatic juice (PPJ) cytology for the diagnosis of pancreatic carcinoma (PCa) is still low. The usefulness of genetic analyses of PPJ seems to be limited because of insufficient sensitivity or false positivity. To improve the molecular diagnosis of PCa, we analyzed mutations of p53 together with K-ras in DNA extracted not only from the sediment but also from the supernatant of PPJ samples. METHOD Polymerase chain reaction-single-strand conformation polymorphism and direct sequencing were used for analyses of p53 mutations in exons 5-8. K-ras mutations at codon 12 were examined by mutant allele-specific amplification. RESULTS In PPJ supernatant from patients with PCa, p53 and K-ras mutations were detected in 42.9% (9 of 21) and 81.0% (17 of 21) of cases, respectively. The incidence of p53 and K-ras mutations in the sediment was 28.6% and 71.4%, respectively. By a combination assay with supernatant and sediment, p53 mutations were detected in 52.4% (11 of 21) of PCa cases. Moreover, p53 mutations were detected in 7 of 15 (46.7%) cases of PCa in which the cytologic diagnosis was negative. Among 25 patients with chronic pancreatitis (CP), none harbored mutant p53, although K-ras mutations were detected at an incidence of 28% (7 of 25) in the supernatant and 20% (5 of 25) in the sediment. In addition, mutant bands of p53 in plasma were detected in 2 of 11 patients with PCa in whom p53 mutations were detectable in PPJ. CONCLUSION These results suggest that the sensitivity of detection for p53 mutations with high cancer specificity could be improved by using the Sup in PPJ samples of PCa. Genetic analysis of p53 could complement PPJ cytology. p53 mutations were detectable in PCa from plasma samples.
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Affiliation(s)
- Ying Wang
- Department of Internal Medicine and Medical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan
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31
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Hachisuga T, Miyakawa T, Tsujioka H, Horiuchi S, Emoto M, Kawarabayashi T. K-ras mutation in tamoxifen-related endometrial polyps. Cancer 2003; 98:1890-7. [PMID: 14584071 DOI: 10.1002/cncr.11728] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
BACKGROUND K-ras mutation is thought to occur at an early stage of neoplastic progression in the endometrium. The authors investigated mutations in codon 12 of K-ras in tamoxifen (TAM)-related endometrial polyps. METHODS DNA was extracted from 11 frozen endometrial polyps from TAM-treated patients with breast carcinoma. Mutations were detected using the mutant allele-specific amplification method. The results subsequently were analyzed for correlations with immunohistochemical data that were obtained using antibodies against estrogen receptors (ERs; alpha and beta forms), progesterone receptors (PRs; A and B forms), and Ki-67. RESULTS Mutations in codon 12 of K-ras were observed in 7 of 11 TAM-related endometrial polyps. Expression levels of ER-alpha and PR-B were high in the glandular epithelium and low in the stroma. PR-A expression was high in both the glandular epithelium and the stroma. In the glandular epithelium, expression of ER-beta appeared to be lower than expression of ER-alpha. The Ki-67 index in the glandular epithelium ranged from 2 to 38, whereas the index ranged from 0 to 4 in the stroma (P < 0.01). CONCLUSIONS The incidence of mutations in codon 12 of K-ras in TAM-related endometrial polyps (64%) was greater than the incidence of these same mutations in sporadic endometrial hyperplasias (4.5-23%). High expression levels of ER-alpha, PR-A, and PR-B in the glandular epithelium were observed in all polyps, regardless of K-ras codon 12 mutation status and Ki-67 index. The authors' findings may support the hypothesis that the polyp-carcinoma sequence partly indicates the development of endometrial carcinoma in postmenopausal women who have been treated with TAM.
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Affiliation(s)
- Toru Hachisuga
- Department of Obstetrics and Gynecology, School of Medicine, Fukuoka University, Fukuoka, Japan.
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Shono Y, Tanimura H, Iwahashi M, Tsunoda T, Tani M, Tanaka H, Matsuda K, Yamaue H. Specific T-cell immunity against Ki-ras peptides in patients with pancreatic and colorectal cancers. Br J Cancer 2003; 88:530-6. [PMID: 12592366 PMCID: PMC2377177 DOI: 10.1038/sj.bjc.6600697] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Mutations of codon 12 in the Ki-ras gene are frequently found in pancreatic and colorectal cancers. It has been demonstrated that human T-cells have the potential to recognise tumours expressing mutated ras-derived peptides. However, it remains unclear whether T-cells from a given individual can recognise the mutant peptides, which are expressed in that individual's tumour tissues. Mutations of the Ki-ras oncogene were analysed by the mutant-allele-specific amplification (MASA) method in pancreatic and colorectal tumour tissues, and T-cell responses against mutated Ki-ras-derived peptides were measured by [(3)H]thymidine incorporation and IFN-gamma production assays. Specific T-cell responses against Ki-ras-products were found in cancer patients, whereas no immune response was observed in normal individuals (P<0.01). Six of the eight pancreatic cancer patients (75%) and nine of 26 colorectal cancer patients (35%) had T-cell responses to mutated Ki-ras-derived-peptides. T-cell response in a given individual cannot recognise the same mutated ras peptide, which is expressed in that individual's tumour tissues. However, pancreatic and colorectal cancer patients have T-cell immunity against Ki-ras-peptides, and this provides potential target for cancer immunotherapy.
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Affiliation(s)
- Y Shono
- Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan
| | - H Tanimura
- Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan
| | - M Iwahashi
- Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan
| | - T Tsunoda
- Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan
| | - M Tani
- Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan
| | - H Tanaka
- Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan
| | - K Matsuda
- Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan
| | - H Yamaue
- Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan
- Second Department of Surgery, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama 641-8510, Japan. E-mail:
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Namiki Y, Endoh D, Kon Y. Genetic mutation associated with meiotic metaphase-specific apoptosis in MRL/MpJ mice. Mol Reprod Dev 2003; 64:179-88. [PMID: 12506350 DOI: 10.1002/mrd.10208] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022]
Abstract
It has been reported that the MRL/MpJ mouse strain shows several unique phenotypes, including rapid wound healing, inherent collagen disease, heat shock-resistant spermatocytes, and metaphase-specific apoptosis (Msa) in the testis. In the present study, we found the genetic mutation associated with Msa by chromosomal mapping with 555 backcross progeny. The Sertoli cell index of abnormal metaphasic spermatocytes was clearly divided into two groups in the first 200 male backcross progeny, which were created by mating female F1 (female C57BL/6 x male MRL/MpJ) with male MRL/MpJ mice, indicating that Msa was caused by only one gene. The result of chromosomal mapping throughout the 555 backcross progeny by using microsatellite markers and single nucleotide polymorphism (SNP) revealed that Msa was mapped on the telomeric region of chromosome 1 and was significantly linked with exonuclease 1 (Exo1) and choroideremia-like (rab escort protein 2) (Chml/Rep2) genes. It was found that the Chml/Rep2 gene was not a candidate for Msa by means of the nucleotide sequences of several inbred strains. On the Exo1 gene in strain MRL/MpJ, but not in other strains, it was surprisingly noted that the truncated forms (tr1-Exo1 and tr2-Exo1) were expressed in all tissues examined as well as normal Exo1 by reverse transcriptase-polymerase chain reaction (RT-PCR). Additionally, the truncated forms of the Exo1 gene were suggested to be transcribed by alternative splicing of the 9th exon, possibly resulting from nucleotide substitution of the branch site existing in the 8th intron. These results suggested that the testicular meiotic Msa in MRL/MpJ mice was a unique phenotype caused by incomplete alternative splicing of the Exo1 gene.
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Affiliation(s)
- Yuka Namiki
- Laboratory of Experimental Animal Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
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Sugiyama T, Osaka M, Koami K, Maeda S, Ueda N. 7,12-DMBA-induced rat leukemia: a review with insights into future research. Leuk Res 2002; 26:1053-68. [PMID: 12443876 DOI: 10.1016/s0145-2126(02)00045-0] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
7,12-Dimethylbenz[a]anthracene (DMBA) elicits leukemia in Long-Evans rats (LE). This leukemia is mostly erythroblastic and 30% of leukemias have total and partial trisomy of #2 chromosome and the rest have diploid karyotype. The common duplication site is in 2q26-q34 and N-ras gene is located in 2q34. 7,8,12-Trimethylbenz[a]anthracene (TMBA) also induces similar leukemias. These leukemias reveal a highly specific mutation of N-ras gene as in human leukemias. N-ras mutation is induced 48h after DMBA treatment. Wild type N-ras allele is frequently lost in diploid leukemias but not in trisomy type. Therefore, a gene dosage problem related to the mutant N-ras gene is involved in development of leukemia. Some secondary genetic rearrangements involving abl and H-ras are also observed in cultured leukemia cells. DMBA-induced chromosome aberrations as well as leukemia are enhanced by erythropoietin and blocked by Sudan III given prior to DMBA treatment. This leukemia will provide an important tool for chemical carcinogenesis and leukemia studies.
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Fernández-Vega C, García-Olmo DC, Ballesteros MA, García-Olmo D. Development of a simple and sensitive technique for detection of point mutations in the K-ras oncogene. Mol Biotechnol 2002; 22:115-21. [PMID: 12405259 DOI: 10.1385/mb:22:2:115] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
We sought to develop a simple and sensitive method based on mutant allele-specific amplification (MASA) for the detection of point mutations in the k-ras oncogene in blood samples. We used MASA and three nested MASA methods to detect a point mutation (GGT-->GAT) in rat DHD cells at codon 12 of exon 1 of the k-ras gene. MASA allowed us to detect one k-ras mutated cell on a background of 10(7) normal cells. The third nested-MASA (nested-MASA.c) method that we developed allowed us to detect one mutated cell among 10(10) normal cells. Our methods should allow the detection of small amounts of mutant k-ras DNA in tissue, serum, and plasma, combining speed with efficiency and specificity.
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36
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Lecomte T, Berger A, Zinzindohoué F, Micard S, Landi B, Blons H, Beaune P, Cugnenc PH, Laurent-Puig P. Detection of free-circulating tumor-associated DNA in plasma of colorectal cancer patients and its association with prognosis. Int J Cancer 2002; 100:542-8. [PMID: 12124803 DOI: 10.1002/ijc.10526] [Citation(s) in RCA: 217] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Tumor cells are characterized by specific genetic alterations. When such genetic alterations are identified in body fluid including plasma, regardless of the presence of detectable tumor cells, it shows the existence of free-circulating tumor-associated DNA. The objective of our study was to assess the prognostic value of free-circulating tumor-associated DNA in colorectal cancer patients' plasma. The first step of our work was to find common genetic alterations in tumors that would subsequently be used for plasma DNA screening. We focused on KRAS2 mutations in codons 12 and 13 by the mutant allele-specific amplification (MASA) method and p16 hypermethylation by the methylation-specific polymerase chain reaction (MSP) method. Patients with a tumor presenting either alteration were selected for plasma screening; 58 tumors were analyzed for KRAS2 mutations and tested for p16 gene promoter methylation. Survival and recurrence rates were assessed in patients with and without free-circulating tumor-associated DNA alterations in plasma. Of the 58 tumors analyzed, 39 (67%) demonstrated either one or both of the studied genetic alterations. Twenty-two (38%) were mutated at KRAS2, and an identical alteration was detected in 10 (45%) of the 22 corresponding plasma samples. Thirty-one (53%) had p16 gene promoter hypermethylation that could also be detected in the plasma in 21 cases (68%). Among the 39 patients who had one or the other alteration in tumor DNA, 37 had at least one reliable plasma test. In 26 (70%) of the 37 patients, free-circulating tumor-associated DNA was detected in plasma. The 2-year overall survival rate was 48% in the group where free-circulating tumor-associated DNA was detected in plasma and 100% in the one where free-circulating tumor-associated DNA was not detected in plasma (p < 0.03). Among these 37 patients, 25 patients had a stage I, II or III disease. In this subgroup of patients, the 2-year recurrence-free survival rate for the 17 patients with free-circulating tumor-associated DNA detected in plasma was 66%, compared to 100% for the 8 patients without free-circulating tumor-associated DNA detected in plasma (p = 0.044). The presence of free-circulating tumor-associated DNA in plasma seems to be a relevant prognostic marker for patients with colorectal cancer and may be used to identify patients with a high risk of recurrence.
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Affiliation(s)
- Thierry Lecomte
- Laboratoire de Toxicologie Moléculaire INSERM, Paris, France
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Ito-Harashima S, Hartzog PE, Sinha H, McCusker JH. The tRNA-Tyr gene family of Saccharomyces cerevisiae: agents of phenotypic variation and position effects on mutation frequency. Genetics 2002; 161:1395-410. [PMID: 12196388 PMCID: PMC1462226 DOI: 10.1093/genetics/161.4.1395] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Extensive phenotypic diversity or variation exists in clonal populations of microorganisms and is thought to play a role in adaptation to novel environments. This phenotypic variation or instability, which occurs by multiple mechanisms, may be a form of cellular differentiation and a stochastic means for modulating gene expression. This work dissects a case of phenotypic variation in a clinically derived Saccharomyces cerevisiae strain involving a cox15 ochre mutation, which acts as a reporter. The ochre mutation reverts to sense at a low frequency while tRNA-Tyr ochre suppressors (SUP-o) arise at a very high frequency to produce this phenotypic variation. The SUP-o mutations are highly pleiotropic. In addition, although all SUP-o mutations within the eight-member tRNA-Tyr gene family suppress the ochre mutation reporter, there are considerable phenotypic differences among the different SUP-o mutants. Finally, and of particular interest, there is a strong position effect on mutation frequency within the eight-member tRNA-Tyr gene family, with one locus, SUP6, mutating at a much higher than average frequency and two other loci, SUP2 and SUP8, mutating at much lower than average frequencies. Mechanisms for the position effect on mutation frequency are evaluated.
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Affiliation(s)
- Sayoko Ito-Harashima
- Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA
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Dang RKB, Anthony RS, Craig JIO, Leonard RCF, Parker AC. Limitations of the use of single base changes in the p53 gene to detect minimal residual disease of breast cancer. Mol Pathol 2002; 55:177-81. [PMID: 12032228 PMCID: PMC1187170 DOI: 10.1136/mp.55.3.177] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
BACKGROUND/AIMS Peripheral blood progenitor cell (PBPC) transplantation is frequently used in the treatment of malignant diseases, but contamination of the graft by tumour cells is a real concern and may lead to disease relapse. The feasibility of applying heterogeneous single base genetic changes as tumour specific markers to detect minimal residual disease in PBPC harvests was studied, using the p53 gene and breast cancer as models. METHODS Tumour tissues from 51 patients with cellular aliquots from PBPC harvests available were studied. Thirty eight patients had metastatic disease or were at high risk of metastasis, and 13 had high risk stage II/III disease with four or more involved axillary lymph nodes. Tumour DNA was screened for p53 mutations in exons 5 to 9, using denaturing gradient gel electrophoresis, followed by sequencing. Based on sequence information, allele specific primers were designed for each mutation and the non-radioisotopic, amplification refractory mutation system (ARMS) was used to screen DNA from PBPC harvests for minimal residual disease. Attempts were made to optimise each system, based on parameters determined using the T47D breast cancer cell line with a confirmed point mutation in codon 194. RESULTS Twelve different somatic mutations were found, two of which could not be sequenced. The remainder were point mutations. Only five of the 10 ARMS systems were successfully optimised, and minimal residual disease detection sensitivities ranged from one copy of tumour DNA in 10(2) to 10(3) copies of wild-type DNA. Using ARMS, three of five patients and eight of 12 of their PBPC harvests showed minimal residual disease. CONCLUSIONS These results suggest that the use of single base genetic changes in minimal residual disease detection is relatively insensitive and is limited to a small number of patients and to certain mutations. In addition, it is labourious and therefore unlikely to play an important role in clinical practice.
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Affiliation(s)
- R K B Dang
- Department of Haematology, Western General Hospital, Edinburgh EH4 2XU, UK.
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Hamajima N, Saito T, Matsuo K, Tajima K. Competitive amplification and unspecific amplification in polymerase chain reaction with confronting two-pair primers. J Mol Diagn 2002; 4:103-7. [PMID: 11986401 PMCID: PMC1906991 DOI: 10.1016/s1525-1578(10)60688-5] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is an inexpensive, time-saving genotyping method that is applicable for most single nucleotide polymorphisms. To date, we have applied PCR-CTPP successfully for the genotyping of more than 30 polymorphisms. This paper demonstrates the differences in DNA amplification among different annealing temperatures of PCR-CTPP with given melting temperatures for four primers. The NQO1 C609T (Pro187Ser) polymorphism was used as an example. Two sets of four primers were applied for PCR-CTPP; the first set with different melting temperatures (Tms), and the second with similar Tms. The comparisons with one-pair primer PCR (allele-specific PCR) revealed that PCR-CTPP amplified DNA more specifically than allele-specific PCR. The primers with different Tms caused competitive DNA amplification for heterozygous genotype. Four primers with similar Tms amplified both alleles unspecifically at a lower annealing temperature, while the same DNA samples were correctly genotyped under an optimal annealing temperature. These findings are unique for PCR-CTPP, and important characteristics when the primers and annealing temperatures in PCR-CTPP are designed. The knowledge of these characteristics will extend the applicability of PCR-CTPP for polymorphism genotyping.
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Affiliation(s)
- Nobuyuki Hamajima
- Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, 1-1 Kano-koden, Chikusa-ku, Nagoya 464-8681, Japan.
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Yamaguchi K, Chijiiwa K, Torata N, Kinoshita M, Tanaka M. Telomerase activity, P53 mutation and Ki-ras codon 12 point mutation of the peripheral blood in patients with hepato pancreato biliary diseases. HPB (Oxford) 2002; 4:75-82. [PMID: 18332928 PMCID: PMC2020534 DOI: 10.1080/136518202760378434] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND With progress in molecular biology, the presence of telomerase activity, P53 mutation and Ki-ras codon 12 point mutation has been reported in malignant tumours of the liver, pancreas and biliary tree. The purpose of this paper is to clarify the clinical implications of finding these three biomarkers in the peripheral blood of affected patients. METHODS Telomerase activity, P53 mutation, and Ki-ras codon 12 point mutation in the peripheral blood were examined among 86 patients with hepato pancreato biliary disease, both benign and malignant, and the results were compared with clinical findings. RESULTS Of 20 patients with benign conditions, only one patient with intraductal papillary adenoma showing severe dysplasia exhibited a biomarker (telomerase activity) in the peripheral blood. In total, there were 66 patients with various HPB carcinomas. Of 56 cancer patients studied pre-operatively, 16 were positive for more than one biomarker, 13 were positive for telomerase activity, 4 for P53 mutation (three at exon 7 and another at exon 8), and 2 for Kiras codon 12 point mutation (both in the second letter). Twelve of the 16 biomarker-positive patients had stage IV disease as opposed to 23 of 40 biomarker-negative patients. The resectability rate of the cancer was 38% in positive patients and 50% in negative patients. The one-year survival rate after resection was zero in positive patients and 15% in negative patients, but the difference was not significant (P=0.65). Of 32 patients with liver metastasis at the time of the molecular examination, eight were positive and 24 negative. Of 34 patients without liver metastasis, nine were positive and 25 negative. The development of subsequent liver metastases in those without them at the start was not significantly different in those with and without biomarkers (56 vs 36%: P=0.31). CONCLUSIONS The three novel biomarkers of the peripheral blood seemed to be of little value for screening of early malignant HPB neoplasms but may help to predict liver metastasis.
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Affiliation(s)
- Koji Yamaguchi
- Department of Surgery and Oncology, Graduate School of Medical SciencesFukuoka 812-8582Japan
| | - Kazuo Chijiiwa
- Department of Surgery and Oncology, Graduate School of Medical SciencesFukuoka 812-8582Japan
| | - Nobuhiro Torata
- Department of Surgery and Oncology, Graduate School of Medical SciencesFukuoka 812-8582Japan
| | - Moritoshi Kinoshita
- Gene-Diagnostic Center, Otsuka Assay Laboratories, Otsuka Pharmaceutical Co. LtdTokushima 771-0195Japan
| | - Masao Tanaka
- Department of Surgery and Oncology, Graduate School of Medical SciencesFukuoka 812-8582Japan
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Park TJ, Han SU, Cho YK, Paik WK, Kim YB, Lim IK. Methylation of O(6)-methylguanine-DNA methyltransferase gene is associated significantly with K-ras mutation, lymph node invasion, tumor staging, and disease free survival in patients with gastric carcinoma. Cancer 2001; 92:2760-8. [PMID: 11753949 DOI: 10.1002/1097-0142(20011201)92:11<2760::aid-cncr10123>3.0.co;2-8] [Citation(s) in RCA: 89] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
BACKGROUND O(6)-methylguanine-DNA methyltransferase (MGMT) can remove O(6)alkylG DNA adducts. If they are not removed, then the adducts mispair with T during DNA replication, resulting in G-to-A mutation. Interrelations between MGMT gene inactivation by promoter methylation, K-ras mutation, and clinicopathologic features in patients with gastric carcinoma were studied. METHODS Surgically removed tumor tissues from 79 patients were analyzed with MGMT methylation by genomic DNA modification and methylation specific polymerase chain reaction analysis, K-ras mutation by mutant allele specific amplification, TNM classification according to the International Union Against Cancer system, and MGMT protein expression by immunohistochemistry. RESULTS MGMT-promoter methylation was found in 18 of 79 tumors. Among those 18 tumors, K-ras mutations were found in 33% and 11% of tumors at codons 12 and 13, respectively, corresponding to 20 times and 7 times greater rates of mutation compared with unmethylated tumors. MGMT methylation was associated significantly with lymph node invasion (P < 0.01), tumor stage (P < 0.03) and 5-year disease free survival (P < 0.02). MGMT protein expression was detected in intestinal metaplasia and adenocarcinoma samples, whereas no expression was detected in normal foveolar cells. CONCLUSIONS MGMT-promoter methylation in patients with gastric carcinoma was associated significantly with point mutations of K-ras at codons 12 and 13, lymph node invasion, tumor stage, and disease free survival. These associations indicate a significant role of MGMT methylation during gastric carcinogenesis.
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Affiliation(s)
- T J Park
- Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, Korea
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Ha A, Watanabe H, Yamaguchi Y, Ohtsubo K, Wang Y, Motoo Y, Okai T, Wakabayahi T, Sawabu N. Usefulness of supernatant of pancreatic juice for genetic analysis of K-ras in diagnosis of pancreatic carcinoma. Pancreas 2001; 23:356-63. [PMID: 11668203 DOI: 10.1097/00006676-200111000-00004] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
AIMS To ascertain whether analysis of K-ras mutations at codon 12 (KRM) in the supernatant of pure pancreatic juice (PPJ) is more useful for the diagnosis of pancreatic carcinoma (PCa) than that in sediment, the authors analyzed KRM in DNA extract from not only the sediment but also the supernatant of PPJ and compared the results. METHODOLOGY PPJ was collected endoscopically from 19 patients with PCa and 25 patients with chronic pancreatitis (CP). DNA was extracted from the supernatant and the sediment of PPJ. Mutant allele-specific amplification (MASA) was performed for KRM analysis with the DNA extracts from these samples. RESULTS The incidence of KRM in the supernatant of PPJ was 89% (17 of 19) in patients with PCa and 28% (7 of 25) in patients with CP, whereas that in the sediment was 79% (15 of 19) in patients with PCa and 20% (5 of 25) in patients with CP. Although there was no significant difference in KRM incidence between supernatant and sediment, the positive rate of KRM was higher in the former. Additionally, with regard to the PCa cases, KRM were found in the supernatant alone in four cases and in the sediment alone in two cases. Consequently, by a combination assay, all of the patients with PCa showed KRM in either the supernatant or sediment of PPJ. Although there was no relation between the incidence of KRM in PPJ and the location and size of tumor, and clinical stage of carcinoma in the patients with PCa, two patients with clinical stage I disease showed KRM in the supernatant. CONCLUSION These results suggest that the positive rate of KRM in the supernatant is not lower than that in the sediment, and simultaneous analysis of KRM in the supernatant and sediment of PPJ enhances the genetic diagnosis of PCa.
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Affiliation(s)
- A Ha
- Department of Internal Medicine and Medical Oncology, Cancer Research Institute, Kanazawa University, 4-86, Yoneizumi, Kanazawa 921-8044, Japan
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Abstract
Most spinal muscular atrophy patients lack both copies of SMN1 exon 7 and most carriers have only one copy of SMN1 exon 7. We investigated the effect of SMN1/SMN2 heteroduplex formation on SMN gene dosage analysis, which is an assay to determine copy number of SMN1 exon 7 that utilizes multiplex quantitative polymerase chain reaction (PCR) with DraI digestion to differentiate SMN1 from SMN2. Heteroduplex formation in PCR is a well-described phenomenon. In addition to demonstrating the presence of heteroduplexes by sequence analysis of purified SMN1 bands, we compared the SMN1 signals in various genotype groups (total n = 260) to those in a group lacking SMN2 (n = 13), and we estimated the relative amounts of SMN1/SMN2 heteroduplexes. The SMN1 signal increased as SMN2 copy number increased despite a constant SMN1 copy number, although not all pairwise comparisons showed a statistically significant difference in the SMN1 signal. In conclusion, SMN1/SMN2 heteroduplexes form in SMN gene dosage analysis, falsely increasing the SMN1 signal. External controls for SMN gene dosage analysis should be chosen carefully with regard to SMN2 copy number. The effect of heteroduplex formation should be considered when performing quantitative multiplex PCR.
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Affiliation(s)
- S Ogino
- Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA
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Osanai M, Miyokawa N, Tamaki T, Yonekawa M, Kawamura A, Sawada N. Adenocarcinoma arising in gastric heterotopic pancreas: clinicopathological and immunohistochemical study with genetic analysis of a case. Pathol Int 2001; 51:549-54. [PMID: 11472568 DOI: 10.1046/j.1440-1827.2001.01240.x] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Heterotopic pancreas in the stomach is a relatively common congenital condition, but the risk of malignant transformation is extremely low. In this study, we describe a case of adenocarcinoma arising from a gastric heterotopic pancreas and we consider its morphological and immunohistochemical features and genetic analysis, in order to examine its histogenesis. This unusual sequela was seen in a 57-year-old woman. Image studies showed a protruding lesion with a central ulcer located in the lesser curvature from the angle to the body of the stomach. A biopsy specimen confirmed this lesion as adenocarcinoma before total gastrectomy. The tumor showed mixed patterns of solid neoplastic-cell proliferation and moderately differentiated glandular structures, and also showed transitional lesions to obvious malignancy, that is, dysplasia, or adenocarcinoma in situ. Neoplastic cells had positive immunoreactivity for carbohydrate antigen (CA) 19-9, mucin (MUC) 1, and insulin, and the mutant allele-specific amplification method revealed a point mutation at K-ras codon 12 (GGT [Gly]-->GAT [Asp]), which is the most common mutational change observed in patients with pancreatic carcinoma. The features of the present case provide clear evidence that this tumor originated from heterotopic pancreatic tissue rather than from gastric epithelium.
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Affiliation(s)
- M Osanai
- Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan.
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Etoh T, Ueo H, Inoue H, Sato K, Utsunomiya T, Barnard GF, Kitano S, Mori M. Clinical significance of K-Ras mutations in intraoperative tumor drainage blood from patients with colorectal carcinoma. Ann Surg Oncol 2001; 8:407-12. [PMID: 11407514 DOI: 10.1007/s10434-001-0407-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
BACKGROUND Recurrent and metastatic carcinoma of the colorectum remains a major problem. This may be ascribed to the presence of micrometastasis at diagnosis. The purpose of this study was to analyze prospectively the clinical value of detecting K-ras mutations in the perioperative circulating blood from patients with colorectal carcinoma. METHODS Twenty-four patients whose tumor carried mutations in codon 12 of the K-ras gene were studied for the presence of cancer cells in perioperative blood samples, in particular, tumor drainage samples. A detection assay using CD45 immunomagnetic separation plus nested mutant allele specific amplification (MASA) was performed. RESULTS K-ras mutations in CD45 negative cells in tumor drainage blood were detected in 7 (29.2%) of 24 patients. There was no significant relationship between the presence of a K-ras mutation and clinicopathological features. Four (57.1%) of the seven patients with a positive K-ras mutation in drainage blood had early recurrent disease. Of the 17 patients with no K-ras mutation, none developed metastatic disease. The recurrence rate of the K-ras mutation positive group was higher than that of the K-ras mutation negative group (P < .01). There was a significant difference, regarding prognosis, between K-ras mutation positive and negative groups (P < .01). CONCLUSIONS This preliminary study demonstrates that the detection of circulating cancer cells in the tumor drainage blood by our new assay system may provide a predictor of recurrence and metastasis of colorectal cancer.
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Affiliation(s)
- T Etoh
- Department of Surgery, Medical Institute of Bioregulation, Kyushu University, Beppu, Japan
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Böckmann B, Grill HJ, Giesing M. Molecular characterization of minimal residual cancer cells in patients with solid tumors. BIOMOLECULAR ENGINEERING 2001; 17:95-111. [PMID: 11222984 DOI: 10.1016/s1389-0344(00)00073-3] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The failure to reduce the mortality of patients with solid tumors is mainly a result of the early dissemination of cancer cells to secondary sites, which is usually missed by conventional diagnostic procedures used for tumor staging. PCR was shown to be superior to conventional techniques in detecting circulating tumor cells and micrometastases allowing the identification of one tumor cell in up to 10(7) normal cells in various sources such as blood, bone marrow, lymph nodes, urine or stool. The methods used are based on the detection of either genomic alterations in oncogenes and tumor suppressor genes or on the mRNA expression of tissue-specific and tumor-associated genes. The additional implementation of techniques for cancer cell purification had a significant impact on analytical sensitivity and specificity of MRCC detection. For patients with e.g. melanoma, breast, colorectal or prostate cancer it was demonstrated that the presence of disseminated cancer cells defines a subgroup of patients with reduced time to recurrence. The possibility to use easily accessible body fluids as a source for MRCC detection enables longitudinal observations of the disease. In this review we discuss the potential of molecular characterization of MRCC as a tool to improve prognostication, therapy selection and drug targeting as well as therapy monitoring.
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Affiliation(s)
- B Böckmann
- Institute for Molecular NanoTechnology, Berghäuser Strasse 295, 45659, Recklinghausen, Germany
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Ueyama H, Kumamoto T, Nagao S, Masuda T, Horinouchi H, Fujimoto S, Tsuda T. A new dysferlin gene mutation in two Japanese families with limb-girdle muscular dystrophy 2B and Miyoshi myopathy. Neuromuscul Disord 2001; 11:139-45. [PMID: 11257469 DOI: 10.1016/s0960-8966(00)00168-1] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
We found a new dysferlin gene mutation in two Japanese families, one with limb-girdle muscular dystrophy 2B and the other with Miyoshi myopathy. All patients in the limb-girdle muscular dystrophy 2B family showed apparent proximal dominant muscle atrophy and weakness, whereas a patient with Miyoshi myopathy in the second family showed distal muscle involvement at an early stage. The common clinical feature of all patients in both families was preferential involvement of calf muscles rather than the tibialis anterior muscle, which was confirmed by muscle computed tomography scan. All patients in both families shared the same homozygous alleles for chromosome 2p13 markers, and dysferlin gene analysis revealed a novel missense mutation, a G to A transition at nt 5882, which changed aspartic acid to asparagine at codon 1837. Allele-specific polymerase chain reaction analysis was used for confirmation of the mutation and for genotype analysis of the family members.
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Affiliation(s)
- H Ueyama
- Third Department of Internal Medicine, Oita Medical University, 1-1, Oita 879-5593, Hasama, Japan
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Kell MR, Winter DC, O'Sullivan GC, Shanahan F, Redmond HP. Biological behaviour and clinical implications of micrometastases. Br J Surg 2000; 87:1629-39. [PMID: 11122176 DOI: 10.1046/j.1365-2168.2000.01606.x] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND The most important prognostic determinant in cancer is the identification of disseminated tumour burden (metastases). Micrometastases are microscopic (smaller than 2 mm) deposits of malignant cells that are segregated spatially from the primary tumour and depend on neovascular formation (angiogenesis) to propagate. METHODS The electronic literature (1966 to present) on micrometastases and their implications in malignant melanoma and epithelial cancers was reviewed. RESULTS Immunohistochemical techniques combined with serial sectioning offer the best accuracy for detection of nodal micrometastases. Molecular techniques should be reserved for blood samples or bone marrow aspirates. Detection of micrometastases in regional lymph nodes and/or bone marrow confers a poor prognosis in epithelial cancers. The concept of sentinel node biopsy combined with serial sectioning and dedicated screening for micrometastases may improve staging procedures. Strategies against angiogenesis may provide novel therapies to induce and maintain micrometastatic dormancy. CONCLUSION The concept of micrometastases has resulted in a paradigm shift in the staging of epithelial tumours and our overall understanding of malignant processes.
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Affiliation(s)
- M R Kell
- Departments of Academic Surgery and Medicine, National University of Ireland, Cork University Hospital and Mercy Hospital, Cork, Ireland
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Morisaki H, Higuchi I, Abe M, Osame M, Morisaki T. First missense mutations (R388W and R425H) of AMPD1 accompanied with myopathy found in a Japanese patient. Hum Mutat 2000; 16:467-72. [PMID: 11102975 DOI: 10.1002/1098-1004(200012)16:6<467::aid-humu3>3.0.co;2-v] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Skeletal muscle AMP deaminase (AMPD: E.C. 3.5.4.6) deficiency is one of the most common inherited defects in the Caucasians, but not in Asians. Although a diagnosis of AMPD1 deficiency is indeed based on the reduced enzymatic activity, its clinical significance is still rather controversial since most subjects are asymptomatic. Alternative splicing of exon 2 in individuals who have inherited this defect is thought to provide a mechanism for phenotypic rescue that may explain the variability of clinical symptoms as we reported earlier. In this report we present the first case with a detectable defect of the AMPD1 gene in a Japanese patient with myopathy. Two missense mutations (R388W and R425H) in exon 9 and exon 10 of the AMPD1 gene were found. Prokaryotic expression showed a comparable amount of the AMPD1 peptides and undetectable AMPD activity in the constructs with these mutations. From this study, we have concluded that this patient is a compound heterozygote for AMPD1 mutant allele. This study also demonstrates the first reported instance of detectable dysfunction of the AMPD1 gene product, suggesting that AMPD1 indeed has a key role in muscle metabolism and function.
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Affiliation(s)
- H Morisaki
- Department of Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan.
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Mimori K, Mori M, Adachi Y, Antonyak MA, Kinoshita M, Kusaka H, Sugimachi K. Analysis of the genetic alterations in a case of juvenile multiple colon carcinoma with hypogammaglobulinemia. Ann Surg Oncol 2000; 7:692-5. [PMID: 11034248 DOI: 10.1007/s10434-000-0692-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
BACKGROUND We have previously reported the clinical characterization of a case of juvenile multiple colorectal carcinoma with hypogammaglobulinemia. Several recent studies have determined that agammaglobulinemia was caused by the loss of Bruton's tyrosine kinase (Btk) function. However, any genetic alterations associated with carcinoma formation in individuals with this immunodeficient disease have not been reported. METHODS DNA from eight carcinoma tissues and nine adenoma tissues from this reported case were examined for mutations in p53 by single strand conformation polymorphism analysis, K-ras by mutant allele specific analysis, and replication error or loss of heterozygosity of the TP53 locus on chromosome #17. RESULTS We found that p53 and K-ras were mutated in the carcinoma tissues. However, each tumor showed unequal and diverse results. CONCLUSIONS The progression of individual tumor was not due to a common genetic event caused directly under the influence of the primary disease at the genetic level.
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Affiliation(s)
- K Mimori
- Department of Surgery, Medical Institute of Bioregulation, and Surgery II, Kyushu University, Japan
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