DNA Degradation Test Predicts Success in Whole-Genome Amplification from Diverse Clinical Samples

Wang, Fengfei; Wang, Lilin; Briggs, Christine; Sicińska, Ewa; Gaston, Sandra M.; Mamon, Harvey J.; Kulke, Matthew H.; Zamponi, Raffaella; Loda, Massimo; Maher, Elizabeth A.; Ogino, Shuji; Fuchs, Charles S.; Jin, Li; Hader, Carlos; Makrigiorgos, G. Mike

doi:10.2353/jmoldx.2007.070004

Cited by 55 publications

(36 citation statements)

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“…This study shows that the assessment of DNA fragmentation provides important information on the amplification capacity of the extracted FFPE-DNA and on the reliability of the obtained results, in line with previous reports on methods involving relatively long PCR products [30], [31], [45] but also short ones, as is the case with Q-PCR assays [45]. Based on the degree of DNA fragmentation, FFPE samples can be distinguished into those with relatively well preserved DNA (favorable samples, roughly ¾ of our diagnostic cases) and those with very fragmented DNA (unfavorable samples, ¼ of our diagnostic cases).…”

Section: Discussionsupporting

confidence: 90%

Targeted KRAS Mutation Assessment on Patient Tumor Histologic Material in Real Time Diagnostics

et al. 2009

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BackgroundTesting for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations.Methodology/Principal FindingsTumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect ≤1% mutant cells, provided that samples yielded cycle thresholds (Ct) <29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%).Conclusions/SignificanceDiagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.

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Section: Discussionsupporting

confidence: 90%

Targeted KRAS Mutation Assessment on Patient Tumor Histologic Material in Real Time Diagnostics

et al. 2009

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“…Given the enormous potential of serum banks for genetic testing, forensics, and genome-wide association studies, there is a critical need to further optimize wholegenome amplification methods to improve fidelity when amplifying partially degraded templates. [43][44][45][46] …”

Section: Discussionmentioning

confidence: 99%

Performance of Whole-Genome Amplified DNA Isolated from Serum and Plasma on High-Density Single Nucleotide Polymorphism Arrays

Croft

Jordan

Patney

et al. 2008

The Journal of Molecular Diagnostics

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Defining genetic variation associated with complex human diseases requires standards based on highquality DNA from well-characterized patients. With the development of robust technologies for wholegenome amplification , sample repositories such as serum banks now represent a potentially valuable source of DNA for both genomic studies and clinical diagnostics. We assessed the performance of wholegenome amplified DNA (wgaDNA) derived from stored serum/plasma on high-density single nucleotide polymorphism arrays. Neither storage time nor usage history affected either DNA extraction or whole-genome amplification yields; however , samples that were thawed and refrozen showed significantly lower call rates (73.9 ؎ 7.8%) than samples that were never thawed (92.0 ؎ 3.3%) (P < 0.001). Genotype call rates did not differ significantly (P ‫؍‬ 0.13) between wgaDNA from never-thawed serum/plasma (92.9 ؎ 2.6%) and genomic DNA (97.5 ؎ 0.3%) isolated from whole blood. Approximately 400 ,000 genotypes were consistent between wgaDNA and genomic DNA, but the overall discordance rate of 4.4 ؎ 3.8% reflected an average of 11 ,110 ؎ 9502 genotyping errors per sample. No distinct patterns of chromosomal clustering were observed for single nucleotide polymorphisms showing discordant genotypes or homozygote conversion. Because the effects of genotyping errors on whole-genome studies are not well defined , we recommend caution when applying wgaDNA from serum/plasma to high-density single nucleotide polymorphism arrays in addition to the use of stringent quality control requirements for the resulting genotype data. (J Mol

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“…For whole genome amplification, plasma DNA was processed by a blunt-end ligation method as described (12, 13). Whole genome amplification was carried out using GenomiPhi V2 DNA amplification kit (GE Healthcare).…”

Section: Materiel and Methodsmentioning

confidence: 99%

Detection of EGFR mutations in plasma DNA from lung cancer patients by mass spectrometry genotyping is predictive of tumor EGFR status and response to EGFR inhibitors

et al. 2011

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Aims EGFR mutations now guide the clinical use of EGFR-targeted therapy in lung cancer. However, standard EGFR mutation analysis requires a minimum amount of tumor tissue, which may not be available in certain situations. In this study, we combined a mass spectrometry genotyping assay (Sequenom) with a mutant-enriched PCR (ME-PCR) to detect EGFR mutations in free plasma DNA from patients with lung cancer. Method DNAs were extracted from 31 plasma samples from 31 patients and analyzed by both methods for EGFR exon 19 deletion and EGFR L858R mutation. Results in plasma DNA samples were compared with EGFR mutation status obtained in tumor DNA (18/31 EGFR mutant). The relationship of EGFR mutation status in tumor and/or plasma samples to overall survival was assessed. Results The EGFR mutation status in plasma DNA was identical to the primary tumor in 61% of patients (19/31). By mass spectrometry genotyping, the plasma samples contained mutant DNA corresponding to 5/14 EGFR exon 19 deletions and 3/4 EGFR L858R mutations previously diagnosed in the matched tumors. Two samples were positive in plasma DNA but negative in primary tumor tissue. Results were similar for ME-PCR. For patients treated with erlotinib, overall survival was correlated with the presence of EGFR mutation in plasma and/or tumor tissue (p=0.002), with the two patients positive only in plasma DNA showing responses and favorable outcomes. Conclusion The detection of EGFR mutations in plasma DNA samples by mass spectrometry genotyping and ME-PCR is feasible. A positive EGFR result in plasma DNA has a high predictive value for tumor EGFR status and for favorable clinical course on EGFR-targeted therapy and could therefore be useful in guiding clinical decisions in patients with insufficient or unavailable tumor specimens.

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DNA Degradation Test Predicts Success in Whole-Genome Amplification from Diverse Clinical Samples

Cited by 55 publications

References 36 publications

Targeted KRAS Mutation Assessment on Patient Tumor Histologic Material in Real Time Diagnostics

Targeted KRAS Mutation Assessment on Patient Tumor Histologic Material in Real Time Diagnostics

Performance of Whole-Genome Amplified DNA Isolated from Serum and Plasma on High-Density Single Nucleotide Polymorphism Arrays

Detection of EGFR mutations in plasma DNA from lung cancer patients by mass spectrometry genotyping is predictive of tumor EGFR status and response to EGFR inhibitors

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