Low Copy Number DNA Template Can Render Polymerase Chain Reaction Error Prone in a Sequence-Dependent Manner

Akbari, Mansour; Hansen, Marianne Doré; Halgunset, Jostein; Skorpen, Frank; Krokan, Hans E.

doi:10.1016/s1525-1578(10)60006-2

Cited by 97 publications

(71 citation statements)

References 16 publications

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“…Finally, statistical analysis showed that the majority of these active TP53 mutants were aggregated in a small number of publications associated with the use of DNA extracted from FFPE tissue and nested PCR (13). It has now been clearly established that this type of DNA material can lead to amplification and sequencing errors if control experiments are not carefully performed (24,25).…”

Section: Discussionmentioning

confidence: 99%

Data-driven unbiased curation of the TP53 tumor suppressor gene mutation database and validation by ultradeep sequencing of human tumors

Edlund

Larsson

Ameur

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Cancer mutation databases are expected to play central roles in personalized medicine by providing targets for drug development and biomarkers to tailor treatments to each patient. The accuracy of reported mutations is a critical issue that is commonly overlooked, which leads to mutation databases that include a sizable number of spurious mutations, either sequencing errors or passenger mutations. Here we report an analysis of the latest version of the TP53 mutation database, including 34,453 mutations. By using several data-driven methods on multiple independent quality criteria, we obtained a quality score for each report contributing to the database. This score can now be used to filter for high-confidence mutations and reports within the database. Sequencing the entire TP53 gene from various types of cancer using next-generation sequencing with ultradeep coverage validated our approach for curation. In summary, 9.7% of all collected studies, mostly comprising numerous tumors with multiple infrequent TP53 mutations, should be excluded when analyzing TP53 mutations. Thus, by combining statistical and experimental analyses, we provide a curated mutation database for TP53 mutations and a framework for mutation database analysis.cancer genetics | genomic | locus-specific database C onventional sequencing using Sanger's methodology has allowed for the discovery of genetic alterations in cancer genes (1). Next-generation sequencing (NGS) techniques have expanded this knowledge by providing a more complete description of each type of alteration, including copy-number variations, translocations, and missense mutations (2, 3). The majority of these mutations are passenger mutations (or hitchhiking mutations) that have no active role in cancer progression and are only coselected with the driver mutations (4).Since the first publication on TP53 mutations in 1989, more than 2,700 articles have been published describing more than 35,000 TP53 mutations in various tumor types and cell lines (5, 6). TP53 mutation studies have applied a variety of analyses, including molecular epidemiology, clinical surveys, and structural analyses (7,8). Such studies require highly curated TP53 mutation data from the Locus Specific Database (LSDB) established and maintained since 1989 (9, 10).The unique feature of TP53 compared with other tumor-suppressor genes is its mode of inactivation. Although most tumorsuppressor genes are inactivated by mutations, leading to absence of the protein (or synthesis of a truncated product), more than 80% of TP53 alterations are missense mutations encoding a stable full-length protein (11). Moreover, each tumor generally harbors a single mutation in the TP53 gene that reduces the transactivation activity of the TP53 protein, leading to loss of its antiproliferative and proapoptotic properties.Previous studies have raised concerns about the accuracy of the various TP53 databases, because they include all mutations published in peer-reviewed journals (12)(13)(14). Statistical analysis showed that the use of n...

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Section: Discussionmentioning

confidence: 99%

Data-driven unbiased curation of the TP53 tumor suppressor gene mutation database and validation by ultradeep sequencing of human tumors

Edlund

Larsson

Ameur

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Unreliable genotyping can be generated when the starting amount of DNA is low. 37 Artifactual mutations can be observed if nucleotide misincorporation occurs during the first PCR cycle, 38 or if chemical modifications have been induced by formalin fixation. [39][40][41] To reduce such artifacts, it is recommended to use a minimum of 1 mg of formalin-fixed paraffin-embedded-recovered DNA and to confirm each identified mutation by an independent analysis.…”

Section: Discussionmentioning

confidence: 99%

“…[39][40][41] To reduce such artifacts, it is recommended to use a minimum of 1 mg of formalin-fixed paraffin-embedded-recovered DNA and to confirm each identified mutation by an independent analysis. 38,42 In our practice, manual dissection of pre-treatment biopsies allowed the recovery of an amount of DNA that was enough for our study protocol. Unlike Lamy et al, 42 we did not observe artifactual mutations, even when using small amounts of template DNA.…”

Section: Discussionmentioning

confidence: 99%

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

Boissière‐Michot¹,

Lopez-Crapez²,

Frugier³

et al. 2012

Modern Pathology

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KRAS status assessment is mandatory in patients with metastatic colorectal cancer before therapy with antiepidermal growth factor receptor monoclonal antibodies, as KRAS mutations are associated with resistance to this treatment. However, KRAS genotyping may be very challenging in case of poor tumor cellularity, particularly when major tumor regression is achieved in locally advanced rectal adenocarcinomas after radiochemotherapy. We aimed at identifying the most reliable strategy to detect KRAS mutations in such samples. DNA was extracted from 31 surgical specimens with major tumor regression, following manual dissection, and from paired pre-treatment biopsies and analyzed by high-resolution melting. DNA samples displaying altered melting curve shapes were then sequenced. Samples with unmodified melting curves or wild-type sequence were further investigated by using an allele-specific PCR assay (TheraScreen) and laser microdissection (followed by high-resolution melting and sequencing analyses). In the 31 post-radiochemotherapy surgical specimens, seven KRAS mutations were identified by high-resolution melting analysis/sequencing. One additional mutation was detected by the TheraScreen assay and two mutations, including the one identified by the TheraScreen assay, were detected following laser microdissection. Altogether, 9/31 surgical specimens (29%) presented KRAS mutations. In the manually dissected pre-treatment biopsies, 12 mutations (39%) were identified by high-resolution melting analysis and sequencing. No additional mutations were found by using the TheraScreen assay or laser microdissection. These results indicate that, in the case of post-radiochemotherapy surgical specimens of colorectal cancer with low tumor cellularity, pre-treatment biopsies might represent the most cost-effective option for reliable KRAS genotyping. The use of more sensitive assays, such as allelespecific PCR or laser microdissection, can be envisaged but with higher costs and longer delays.

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“…20,21 As a consequence, several whole tissue sections would be required for a micro-dissection to yield sufficient amounts of DNA to reliably perform a PCR. To decrease the risk of PCR artifacts and to avoid wasting of valuable multilocular cystic renal cell carcinoma tissue, we punched directly in the cyst walls using a hollow needle with a diameter of 0.6 mm.…”

Section: Discussionmentioning

confidence: 99%

VHL mutations and dysregulation of pVHL- and PTEN-controlled pathways in multilocular cystic renal cell carcinoma

et al. 2011

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Multilocular cystic renal cell carcinoma is a rare renal cell carcinoma with an excellent prognosis. To clarify the relationship with typical clear cell renal cell carcinoma, we evaluated 15 cases of multilocular cystic renal cell carcinomas diagnosed according to the 2004 WHO classification. Von Hippel Lindau (VHL) gene mutations were determined by whole genome amplification and direct sequencing. Carbonic anhydrase 9 (CAIX), a hypoxiainducible factor (HIF) target, paired box gene 2 (PAX2), cyclin-dependent kinase inhibitor p27 and glycogen synthase kinase 3-b (GSK3b) were immunohistochemically evaluated as members of the VHL protein (pVHL)-and phosphatase and tensin homolog (PTEN)-controlled pathways. VHL mutations were identified in 3 of 12 (25%) tumors. Inactivated GSK3b, decreased PTEN expression and PAX2 positivity were observed in the vast majority of the multilocular cystic renal cell carcinomas. Strong nuclear staining of p27 was seen in 14 of 15 cases. Compared with multilocular cystic renal cell carcinomas, expression frequencies of PAX2, p-GSK3b, PTEN and CAIX were similar in a set of low-grade, early-stage clear cell renal cell carcinomas, whereas only 30% had strong p27 positivity. These results are consistent with the hypothesis that multilocular cystic renal cell carcinomas are related at the molecular level with clear cell renal cell carcinomas. Maintenance of a strong subcellular p27 expression in all multilocular cystic renal cell carcinomas analyzed may in part explain the excellent prognosis of these tumor patients.

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Low Copy Number DNA Template Can Render Polymerase Chain Reaction Error Prone in a Sequence-Dependent Manner

Cited by 97 publications

References 16 publications

Data-driven unbiased curation of the TP53 tumor suppressor gene mutation database and validation by ultradeep sequencing of human tumors

Data-driven unbiased curation of the TP53 tumor suppressor gene mutation database and validation by ultradeep sequencing of human tumors

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment

VHL mutations and dysregulation of pVHL- and PTEN-controlled pathways in multilocular cystic renal cell carcinoma

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