Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

Keynote webinar | Spotlight on medication adherence

LIVE: Thursday 27th June 2024, 18:00-19:30 (CEST)
Join our expert panel to discover why you need to understand the drivers of non-adherence in your patients, and how you can optimize medication adherence in your clinics to drastically improve patient outcomes.
ADVANCED SEARCH
Log in
Top
BMC Cancer
Published in: BMC Cancer 1/2007

Open Access 01-12-2007 | Research article

High resolution melting for mutation scanning of TP53exons 5–8

Authors: Michael Krypuy, Ahmed Ashour Ahmed, Dariush Etemadmoghadam, Sarah J Hyland, Anna deFazio, Stephen B Fox, James D Brenton, David D Bowtell, Alexander Dobrovic, Australian Ovarian Cancer Study Group

Published in: BMC Cancer | Issue 1/2007

Login to get access

Abstract

Background

p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples.

Methods

We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status.

Results

One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons.

Conclusion

HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.
Appendix
Available only for authorised users
Literature
1.
Diller L, Kassel J, Nelson CE, Gryka MA, Litwak G, Gebhardt M, Bressac B, Ozturk M, Baker SJ, Vogelstein B, et al: p53 functions as a cell cycle control protein in osteosarcomas. Mol Cell Biol. 1990, 10 (11): 5772-5781.CrossRefPubMedPubMedCentral
2.
Deppert W, Buschhausen-Denker G, Patschinsky T, Steinmeyer K: Cell cycle control of p53 in normal (3T3) and chemically transformed (Meth A) mouse cells. II. Requirement for cell cycle progression. Oncogene. 1990, 5 (11): 1701-1706.PubMed
3.
Lin D, Shields MT, Ullrich SJ, Appella E, Mercer WE: Growth arrest induced by wild-type p53 protein blocks cells prior to or near the restriction point in late G1 phase. Proc Natl Acad Sci U S A. 1992, 89 (19): 9210-9214. 10.1073/pnas.89.19.9210.CrossRefPubMedPubMedCentral
4.
Yonish-Rouach E, Grunwald D, Wilder S, Kimchi A, May E, Lawrence JJ, May P, Oren M: p53-mediated cell death: relationship to cell cycle control. Mol Cell Biol. 1993, 13 (3): 1415-1423.CrossRefPubMedPubMedCentral
5.
Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB: Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A. 1992, 89 (16): 7491-7495. 10.1073/pnas.89.16.7491.CrossRefPubMedPubMedCentral
6.
Lowe SW, Ruley HE, Jacks T, Housman DE: p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell. 1993, 74 (6): 957-967. 10.1016/0092-8674(93)90719-7.CrossRefPubMed
7.
Goi K, Takagi M, Iwata S, Delia D, Asada M, Donghi R, Tsunematsu Y, Nakazawa S, Yamamoto H, Yokota J, Tamura K, Saeki Y, Utsunomiya J, Takahashi T, Ueda R, Ishioka C, Eguchi M, Kamata N, Mizutani S: DNA damage-associated dysregulation of the cell cycle and apoptosis control in cells with germ-line p53 mutation. Cancer Res. 1997, 57 (10): 1895-1902.PubMed
8.
Bunz F, Hwang PM, Torrance C, Waldman T, Zhang Y, Dillehay L, Williams J, Lengauer C, Kinzler KW, Vogelstein B: Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest. 1999, 104 (3): 263-269.CrossRefPubMedPubMedCentral
9.
Vogelstein B, Lane D, Levine AJ: Surfing the p53 network. Nature. 2000, 408 (6810): 307-310. 10.1038/35042675.CrossRefPubMed
10.
Petitjean A, Mathe E, Kato S, Ishioka C, Tavtigian SV, Hainaut P, Olivier M: Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat. 2007, 28 (6): 622-629. 10.1002/humu.20495.CrossRefPubMed
11.
Fujita M, Enomoto T, Inoue M, Tanizawa O, Ozaki M, Rice JM, Nomura T: Alteration of the p53 tumor suppressor gene occurs independently of K-ras activation and more frequently in serous adenocarcinomas than in other common epithelial tumors of the human ovary. Jpn J Cancer Res. 1994, 85 (12): 1247-1256.CrossRefPubMed
12.
Singer G, Stohr R, Cope L, Dehari R, Hartmann A, Cao DF, Wang TL, Kurman RJ, Shih Ie M: Patterns of p53 mutations separate ovarian serous borderline tumors and low- and high-grade carcinomas and provide support for a new model of ovarian carcinogenesis: a mutational analysis with immunohistochemical correlation. Am J Surg Pathol. 2005, 29 (2): 218-224. 10.1097/01.pas.0000146025.91953.8d.CrossRefPubMed
13.
Greenblatt MS, Chappuis PO, Bond JP, Hamel N, Foulkes WD: TP53 mutations in breast cancer associated with BRCA1 or BRCA2 germ-line mutations: distinctive spectrum and structural distribution. Cancer Res. 2001, 61 (10): 4092-4097.PubMed
14.
Amikura T, Sekine M, Hirai Y, Fujimoto S, Hatae M, Kobayashi I, Fujii T, Nagata I, Ushijima K, Obata K, Suzuki M, Yoshinaga M, Umesaki N, Satoh S, Enomoto T, Motoyama S, Nishino K, Haino K, Tanaka K, Japanese Familial Ovarian Cancer Study Group: Mutational analysis of TP53 and p21 in familial and sporadic ovarian cancer in Japan. Gynecol Oncol. 2006, 100 (2): 365-371. 10.1016/j.ygyno.2005.09.010.CrossRefPubMed
15.
Royds JA, Iacopetta B: p53 and disease: when the guardian angel fails. Cell Death Differ. 2006, 13 (6): 1017-1026. 10.1038/sj.cdd.4401913.CrossRefPubMed
16.
Alsner J, Yilmaz M, Guldberg P, Hansen LL, Overgaard J: Heterogeneity in the clinical phenotype of TP53 mutations in breast cancer patients. Clin Cancer Res. 2000, 6 (10): 3923-3931.PubMed
17.
Van Dyke T: Cancer biology: sense out of missense. Nature. 2005, 434 (7031): 287-288. 10.1038/434287a.CrossRefPubMed
18.
Soussi T, Lozano G: p53 mutation heterogeneity in cancer. Biochem Biophys Res Commun. 2005, 331 (3): 834-842. 10.1016/j.bbrc.2005.03.190.CrossRefPubMed
19.
Olive KP, Tuveson DA, Ruhe ZC, Yin B, Willis NA, Bronson RT, Crowley D, Jacks T: Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell. 2004, 119 (6): 847-860. 10.1016/j.cell.2004.11.004.CrossRefPubMed
20.
Lang GA, Iwakuma T, Suh YA, Liu G, Rao VA, Parant JM, Valentin-Vega YA, Terzian T, Caldwell LC, Strong LC, El-Naggar AK, Lozano G: Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell. 2004, 119 (6): 861-872. 10.1016/j.cell.2004.11.006.CrossRefPubMed
21.
22.
Laframboise S, Chapman W, McLaughlin J, Andrulis IL: p53 mutations in epithelial ovarian cancers: possible role in predicting chemoresistance. Cancer J. 2000, 6 (5): 302-308.PubMed
23.
Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ: High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem. 2003, 49 (6 Pt 1): 853-860. 10.1373/49.6.853.CrossRefPubMed
24.
Willmore-Payne C, Holden JA, Tripp S, Layfield LJ: Human malignant melanoma: detection of BRAF- and c-kit-activating mutations by high-resolution amplicon melting analysis. Hum Pathol. 2005, 36 (5): 486-493. 10.1016/j.humpath.2005.03.015.CrossRefPubMed
25.
Willmore-Payne C, Holden JA, Layfield LJ: Detection of epidermal growth factor receptor and human epidermal growth factor receptor 2 activating mutations in lung adenocarcinoma by high-resolution melting amplicon analysis: correlation with gene copy number, protein expression, and hormone receptor expression. Hum Pathol. 2006, 37 (6): 755-763. 10.1016/j.humpath.2006.02.004.CrossRefPubMed
26.
Nomoto K, Tsuta K, Takano T, Fukui T, Fukui T, Yokozawa K, Sakamoto H, Yoshida T, Maeshima AM, Shibata T, Furuta K, Ohe Y, Matsuno Y: Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis. Am J Clin Pathol. 2006, 126 (4): 608-615.CrossRefPubMed
27.
Krypuy M, Newnham GM, Thomas DM, Conron M, Dobrovic A: High resolution melting analysis for the rapid and sensitive detection of mutations in clinical samples: KRAS codon 12 and 13 mutations in non-small cell lung cancer. BMC Cancer. 2006, 6: 295-10.1186/1471-2407-6-295.CrossRefPubMedPubMedCentral
28.
29.
Miller SA, Dykes DD, Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988, 16 (3): 1215-10.1093/nar/16.3.1215.CrossRefPubMedPubMedCentral
30.
31.
Hensel CH, Xiang RH, Sakaguchi AY, Naylor SL: Use of the single strand conformation polymorphism technique and PCR to detect p53 gene mutations in small cell lung cancer. Oncogene. 1991, 6 (6): 1067-1071.PubMed
32.
Borresen AL, Hovig E, Smith-Sorensen B, Malkin D, Lystad S, Andersen TI, Nesland JM, Isselbacher KJ, Friend SH: Constant denaturant gel electrophoresis as a rapid screening technique for p53 mutations. Proc Natl Acad Sci U S A. 1991, 88 (19): 8405-8409. 10.1073/pnas.88.19.8405.CrossRefPubMedPubMedCentral
33.
Pignon JM, Vinatier I, Fanen P, Jonveaux P, Tournilhac O, Imbert M, Rochant H, Goossens M: Exhaustive analysis of the P53 gene coding sequence by denaturing gradient gel electrophoresis: application to the detection of point mutations in acute leukemias. Hum Mutat. 1994, 3 (2): 126-132. 10.1002/humu.1380030207.CrossRefPubMed
34.
Guldberg P, Nedergaard T, Nielsen HJ, Olsen AC, Ahrenkiel V, Zeuthen J: Single-step DGGE-based mutation scanning of the p53 gene: application to genetic diagnosis of colorectal cancer. Hum Mutat. 1997, 9 (4): 348-355. 10.1002/(SICI)1098-1004(1997)9:4<348::AID-HUMU8>3.0.CO;2-1.CrossRefPubMed
35.
Keller G, Hartmann A, Mueller J, Hofler H: Denaturing high pressure liquid chromatography (DHPLC) for the analysis of somatic p53 mutations. Lab Invest. 2001, 81 (12): 1735-1737.CrossRefPubMed
36.
Gross E, Kiechle M, Arnold N: Mutation analysis of p53 in ovarian tumors by DHPLC. J Biochem Biophys Methods. 2001, 47 (1-2): 73-81. 10.1016/S0165-022X(00)00153-6.CrossRefPubMed
Metadata
Title
High resolution melting for mutation scanning of TP53exons 5–8
Authors
Michael Krypuy
Ahmed Ashour Ahmed
Dariush Etemadmoghadam
Sarah J Hyland
Anna deFazio
Stephen B Fox
James D Brenton
David D Bowtell
Alexander Dobrovic
Australian Ovarian Cancer Study Group
Publication date
01-12-2007
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2007
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/1471-2407-7-168

Other articles of this Issue 1/2007

BMC Cancer 1/2007 Go to the issue

Research article

Curative and organ-preserving treatment with intra-arterial carboplatin induction followed by surgery and/or radiotherapy for advanced head and neck cancer: single-center five-year results

Research article

Cross-study analysis of gene expression data for intermediate neuroblastoma identifies two biological subtypes

Research article

Ductal carcinoma in situ of the breast (DCIS) with heterogeneity of nuclear grade: prognostic effects of quantitative nuclear assessment

Research article

Paradoxical antiproliferative effect by a murine mammary tumor-derived epithelial cell line

Research article

The progressive elevation of alpha fetoprotein for the diagnosis of hepatocellular carcinoma in patients with liver cirrhosis

Research article

HNPCC versus sporadic microsatellite-unstable colon cancers follow different routes toward loss of HLA class I expression
Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras
Prof. Fabrice Barlesi
Developed by: Springer Medicine
Image Credits