A High Frequency of Sequence Alterations Is Due to Formalin Fixation of Archival Specimens

Williams, Cecilia; Pontén, Fredrik; Moberg, Catherine; Söderkvist, Peter; Uhlén, Mathias; Pontén, Jan; Sitbon, Gisela; Lundeberg, Joakim

doi:10.1016/s0002-9440(10)65461-2

Cited by 455 publications

(374 citation statements)

References 11 publications

(12 reference statements)

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“…There have been arguments against using extremely small amounts of DNA for PCR-based mutational analysis as artifactual mutations have been described especially in DNA extracted from paraffinembedded tissue. [34][35][36][37] However, improved methods using DNA extraction kits specially adapted for laser capture microdissected specimens from paraffinembedded tissues, and more sensitive and reliable detection methods such as pyrosequencing and using small PCR amplicons (150-200 bp) have improved downstream analysis for samples with small amounts of DNA. To ensure we did not interpret false-positive results as true mutations, all our assays were performed in duplicate.…”

Section: Discussionmentioning

confidence: 99%

EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection

Roy‐Chowdhuri

Pham

et al. 2012

Modern Pathology

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The discovery of activating mutations in EGFR and KRAS in a subset of lung adenocarcinomas was a major advance in our understanding of lung adenocarcinoma biology, and has led to groundbreaking studies that have demonstrated the efficacy of tyrosine kinase inhibitor therapy. Fine-needle aspirates and other cytologic procedures have become increasingly popular for obtaining diagnostic material in lung carcinomas. However, frequently the small amount of material or sparseness of tumor cells obtained from cytologic preparations limit the number of specialized studies, such as mutation analysis, that can be performed. In this study we used laser capture microdissection to isolate small numbers of tumor cells to assess for EGFR and KRAS mutations from cell block sections of 19 cytology samples from patients with known lung adenocarcinomas. We compared our results with previous molecular assays that had been performed on either surgical or cytology specimens as part of the patient's initial clinical work-up. Not only were we able to detect the identical EGFR or KRAS mutation that was present in the patient's prior molecular assay in every case, but we were also able to consistently detect the mutation from as few as 50 microdissected tumor cells. Furthermore, isolating a more pure population of tumor cells resulted in increased sensitivity of mutation detection as we were able to detect mutations from laser capture microdissection-enriched cases where the tumor load was low and traditional methods of whole slide scraping failed. Therefore, this method can not only significantly increase the number of lung adenocarcinoma patients that can be screened for EGFR and KRAS mutations, but can also facilitate the use of cytologic samples in the newly emerging field of molecular-based personalized therapies.

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Section: Discussionmentioning

confidence: 99%

EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection

Roy‐Chowdhuri

Pham

et al. 2012

Modern Pathology

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“…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. 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.…”

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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“…The denaturation of DNA at AT-rich regions as a result of formaldehyde fixation, with the subsequent generation of free pyrimidine and purine residues, is well documented in the literature. [60][61][62][63] As a result of this phenomenon, Taq polymerase tends to insert adenosines when no template base is present, thus producing artificial mutations that are subsequently amplified throughout the PCR process. The number of errors introduced by Taq polymerase in fresh tissue samples is in the order of 1 in every 10 5 base pairs, whereas in FFPE, it has been estimated at 1 in 500.…”

Section: Discussionmentioning

confidence: 99%

KLF6 and TP53 mutations are a rare event in prostate cancer: distinguishing between Taq polymerase artifacts and true mutations

et al. 2008

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Krü ppel-like factor 6 (KLF6) has been reported to act as a tumor suppressor gene involved in the regulation of the cell cycle by activating p21 in a p53-independent manner. Many studies suggest that KLF6 is inactivated by allelic loss and somatic mutation. However, there is a high variability in the reported frequency of mutations (from 1 to 55%). TP53 also regulates the cell cycle through the activation of p21. In prostate cancer, the reported frequency of TP53 mutations ranges from 3 to 42%. In all these reports, there is a considerable degree of methodological heterogeneity. Our aim was to determine the frequency of KLF6 and TP53 mutations in a welldefined group of prostate tumors with different stages and Gleason grades. The four exons of KLF6 and exons 4-9 of TP53 were studied in 103 cases, including 90 formalin-fixed, paraffin-embedded (FFPE) and 13 frozen samples. All tumors were analyzed through PCR and direct sequencing. All changes found were confirmed by a second independent PCR and sequencing reaction. For KLF6, mutation (E227G) was only detected in one tumor (1%) and for TP53, three different mutations (L130H, H214R, and Y234C) were detected in five tumors (5%). This low mutation index is in keeping with recent papers on the subject. Our study strongly supports the notion that KLF6 and TP53 mutations are not frequent events in prostate cancer. When using FFPE tissues, it is mandatory to perform at least two independent rounds of PCR and sequencing to confirm mutations and exclude Taq polymerase-induced artifacts. Two of these genes are Krü ppel-like factor 6 (KLF6) and TP53.KLF6 is a transcription factor that interacts with DNA through three zinc-fingers in its COOHterminal domain. KLF6 belongs to the KLF family, a family that is broadly involved in cell differentiation, development, growth-related signal transduction, cell proliferation, apoptosis, and angiogenesis. 5 It has been suggested that KLF6 could be involved in the regulation of the cell cycle by activating p21 in a p53-independent manner. 6 This has been proven in several in vitro 5 and in vivo assays. 7 KLF6 is believed to regulate cancer development and progression through the downregulation of the c-Jun oncoprotein 8 and the activation of E-cadherin. 9 The reported frequency of KLF6 mutations in different types of human cancer varies in the different published studies. [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] In prostate cancer, four studies have analyzed the frequency of KLF6 mutations. 6,[30][31][32] In the first published study, Narla et al 6 showed a very high frequency, 55%, whereas in the following study, Chen et al 30 obtained a lower frequency, around 15%. The results of two recent reports provided frequencies of 0 and 1%. 31,32 It should be noted that there were considerable

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A High Frequency of Sequence Alterations Is Due to Formalin Fixation of Archival Specimens

Cited by 455 publications

References 11 publications

EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection

EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection

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

KLF6 and TP53 mutations are a rare event in prostate cancer: distinguishing between Taq polymerase artifacts and true mutations

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