Preferential Amplification of Apoptotic DNA from Plasma: Potential for Enhancing Detection of Minor DNA Alterations in Circulating DNA

Mamon, Harvey J.; Hader, Carlos; Li, Jin; Wang, Lilin; Kulke, Matthew H.; Amicarelli, Giulia; Shehi, Erlet; Adlerstein, Daniel; Röper, Kristin; Killion, Leah; Hooshmand, Susanne M.; Makrigiorgos, G. Mike

doi:10.1373/clinchem.2008.104612

Cited by 12 publications

(15 citation statements)

References 4 publications

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“…For whole genome amplification plasma DNA was processed either by a blunt-end ligation method described previously (19) or by an alternative method that favors the amplification of small, tumor-derived DNA (20). Whole genome amplification was carried out using GenomiPhi V2 DNA Amplification Kit (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Noninvasive Detection of EGFR T790M in Gefitinib or Erlotinib Resistant Non–Small Cell Lung Cancer

Kuang

Rogers

Yeap

et al. 2009

Clinical Cancer Research

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216

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Purpose: Tumors from 50% of epidermal growth factor receptor (EGFR) mutant non^small cell lung cancer patients that develop resistance to gefitinib or erlotinib will contain a secondary EGFR T790M mutation. As most patients do not undergo repeated tumor biopsies we evaluated whether EGFR T790M could be detected using plasma DNA. Experimental Design: DNA from plasma of 54 patients with known clinical response to gefitinib or erlotinib was extracted and used to detect both EGFR-activating and EGFR T790M mutations. Forty-three (80%) of patients had tumor EGFR sequencing (EGFR mutant/wild type: 30/13) and seven patients also had EGFR T790M gefitinib/erlotinib-resistant tumors. EGFR mutations were detected using two methods, the Scorpion Amplification Refractory Mutation System and the WAVE/Surveyor, combined with whole genome amplification. Results: Both EGFR-activating and EGFR T790M were identified in 70% of patients with known tumor EGFR-activating (21 of 30) or T790M (5 of 7) mutations. EGFR T790M was identified from plasma DNA in 54% (15 of 28) of patients with prior clinical response to gefitinib/erlotinib, 29% (4 of 14) with prior stable disease, and in 0% (0 of 12) that had primary progressive disease or were untreated with gefitinib/erlotinib. Conclusions: EGFR T790M can be detected using plasma DNA from gefitinib-or erlotinibresistant patients. This noninvasive method may aid in monitoring drug resistance and in directing the course of subsequent therapy.

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Section: Methodsmentioning

confidence: 99%

Noninvasive Detection of EGFR T790M in Gefitinib or Erlotinib Resistant Non–Small Cell Lung Cancer

Kuang

Rogers

Yeap

et al. 2009

Clinical Cancer Research

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216

173

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“…Plasma-circulating DNA isolated from a normal volunteer was tested in parallel. The pair of plasma-circulating DNA was pre-amplified via LM-PCR using the method we described (27). To exclude the possibility of a polymerase-introduced error during the LM-PCR pre-amplification step, the mutation was also validated using fast -COLD-PCR directly from unamplified DNA isolated from plasma (not shown).…”

Section: Resultsmentioning

confidence: 99%

“…Additionally, we also tested plasma-circulating DNA (obtained from a radiation therapy patient following informed consent and IRB approval) that demonstrated a TP53 mutation. The plasma-circulating DNA was pre-amplified by ligation-mediated PCR as previously described (27), along with plasma-circulating DNA from a healthy donor, Supplementary Table 2.…”

Section: Methodsmentioning

confidence: 99%

Temperature-Tolerant COLD-PCR Reduces Temperature Stringency and Enables Robust Mutation Enrichment

et al. 2012

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BACKGROUND Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3°C. This precludes simultaneous mutation enrichment in sequences of substantially different melting-temperature (Tm) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT-COLD-PCR) approach that reduces this obstacle. METHODS Thermo-cycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR are described. This approach enables enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation is provided for KRAS and TP53 exons 6–9 using dilutions of mutated DNA, clinical cancer samples and plasma-circulating DNA. RESULTS A single thermocycling program with a denaturation-temperature window of 2.5–3.0°C enriches mutations in all DNA amplicons simultaneously, despite their different Tms. Mutation enrichments of 6–9-fold were obtained using TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other two forms of COLD-PCR, fast-COLD-PCR and ice-COLD-PCR. CONCLUSIONS Low-level mutations in diverse amplicons with different Tm can be mutation-enriched via TT-COLD-PCR provided that their Tms fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons, and increases significantly the versatility of COLD-PCR.

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“…In-house-prepared ssDNA adaptors 12-mers and 24-mers were added to the phosphorylated amplicons, and ligation was applied using the dsDNA adaptors as described (30). Fast-TT-COLD-PCR was then applied using the protocols described in Supplemental Table 2 and Supplemental Table 4.…”

Section: Methodsmentioning

confidence: 99%

“…cfDNA recovered from plasma was amplified by ligation mediated-PCR as described (30). A TP53 exon 8 gene region from DNA containing a 1% mutation (c.818G>A) was separately pre-amplified using primer set 115-P20 (Supplemental Table 3).…”

Section: Methodsmentioning

confidence: 99%

Single-Tube, Highly Parallel Mutation Enrichment in Cancer Gene Panels by Use of Temperature-Tolerant COLD-PCR

Castellanos-Rizaldos¹,

Richardson

Lin

et al. 2015

Clinical Chemistry

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BACKGROUND Multiplexed detection of low-level mutations presents a technical challenge for many technologies, including cancer gene panels used for targeted-re-sequencing. Analysis of mutations below ~2–5% abundance in tumors with heterogeneity, samples with stromal contamination, or biofluids, is problematic due to increased ‘noise’ from sequencing errors. Technologies that reduce noise via deep-sequencing unavoidably reduce throughput and increase cost. Here we provide proof-of-principle that COLD-PCR technology enables multiplex low-level mutation detection in cancer gene panels while retaining throughput. METHODS We have developed a multiplex temperature-tolerant-COLD-PCR (fast-TT-COLD-PCR) approach that uses cancer gene panels developed for massively parallel sequencing. Following a multiplex pre-amplification from genomic DNA we attach tails to all amplicons and perform fast-TT-COLD-PCR. This approach gradually increases denaturation temperatures in a step-wise fashion, such that all possible denaturation temperatures are encompassed. By introducing modified nucleotides, fast-COLD-PCR is adapted to enrich for Tm-increasing as well as Tm-decreasing mutations over all amplicons, in a single tube. RESULTS Using custom-made and commercial gene panels containing 8, 50, 190 or 16,000 amplicons we demonstrate that fast-TT-COLD-PCR enriches mutations on all examined targets simultaneously. Incorporation of dITP/dDTP in place of dGTP/dATP enables enrichment of Tm-increasing mutations. Serial dilution experiments demonstrate a limit-of-detection of ~ 0.01–0.1% mutation abundance using Ion-Torrent and 0.1–0.3% using Sanger sequencing. CONCLUSIONS Fast-TT-COLD-PCR improves the limit of detection of cancer gene panels by enabling mutation enrichment in multiplex, single tube reactions. This novel adaptation of COLD-PCR converts subclonal mutations to clonal, thereby facilitating detection and subsequent mutation sequencing.

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Preferential Amplification of Apoptotic DNA from Plasma: Potential for Enhancing Detection of Minor DNA Alterations in Circulating DNA

Cited by 12 publications

References 4 publications

Noninvasive Detection of EGFR T790M in Gefitinib or Erlotinib Resistant Non–Small Cell Lung Cancer

Noninvasive Detection of EGFR T790M in Gefitinib or Erlotinib Resistant Non–Small Cell Lung Cancer

Temperature-Tolerant COLD-PCR Reduces Temperature Stringency and Enables Robust Mutation Enrichment

Single-Tube, Highly Parallel Mutation Enrichment in Cancer Gene Panels by Use of Temperature-Tolerant COLD-PCR

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