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Analysis of cancer mutation signatures in blood by a novel ultra-sensitive assay: monitoring of therapy or recurrence in non-metastatic breast cancer.

Chen Z, Feng J, Buzin CH, Liu Q, Weiss L, Kernstine K, Somlo G, Sommer SS - PLoS ONE (2009)

Bottom Line: The utility of this method is illustrated in two ways. 1) We demonstrate that two EGFR deletions commonly found in lung cancers are not present in tissue from four normal human lungs (10(7) copies of gDNA each) or in blood samples from 10 healthy individuals (10(7) copies of gDNA each).MAP has an analytical selectivity of one part per billion for detection of MIDIs and an analytical sensitivity of one molecule.MAP provides a general tool for monitoring ultra-rare mutations in tissues and blood.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, City of Hope National Medical Center, Duarte, California, United States of America.

ABSTRACT

Background: Tumor DNA has been shown to be present both in circulating tumor cells in blood and as fragments in the plasma of metastatic cancer patients. The identification of ultra-rare tumor-specific mutations in blood would be the ultimate marker to measure efficacy of cancer therapy and/or early recurrence. Herein we present a method for detecting microinsertions/deletions/indels (MIDIs) at ultra-high analytical selectivity. MIDIs comprise about 15% of mutations.

Methods and findings: We describe MIDI-Activated Pyrophosphorolysis (MAP), a method of ultra-high analytical selectivity for detecting MIDIs. The high analytical selectivity of MAP is putatively due to serial coupling of two rare events: heteroduplex slippage and mis-pyrophosphorolysis. MAP generally has an analytical selectivity of one mutant molecule per >1 billion wild type molecules and an analytical sensitivity of one mutant molecule per reaction. The analytical selectivity of MAP is about 100,000-fold better than that of our previously described method of Pyrophosphorolysis Activated Polymerization-Allele specific amplification (PAP-A) for detecting MIDIs. The utility of this method is illustrated in two ways. 1) We demonstrate that two EGFR deletions commonly found in lung cancers are not present in tissue from four normal human lungs (10(7) copies of gDNA each) or in blood samples from 10 healthy individuals (10(7) copies of gDNA each). This is inconsistent, at least at an analytical sensitivity of 10(-7), with the hypotheses of (a) hypermutation or (b) strong selection of these growth factor-mutated cells during normal lung development leads to accumulation of pre-neoplastic cells with these EGFR mutations, which sometimes can lead to lung cancer in late adulthood. Moreover, MAP was used for large scale, high throughput "gene pool" analysis. No germline or early embryonic somatic mosaic mutation was detected (at a frequency of >0.3%) for the 15/18 bp EGFR deletion mutations in 6,400 individuals, suggesting that early embryonic EGFR somatic mutation is very rare, inconsistent with hypermutation or strong selection of these deletions in the embryo. 2) The second illustration of MAP utility is in personalized monitoring of therapy and early recurrence in cancer. Tumor-specific p53 mutations identified at diagnosis in the plasma of six patients with stage II and III breast cancer were undetectable after therapy in four women, consistent with clinical remission, and continued to be detected after treatment in two others, reflecting tumor progression.

Conclusions: MAP has an analytical selectivity of one part per billion for detection of MIDIs and an analytical sensitivity of one molecule. MAP provides a general tool for monitoring ultra-rare mutations in tissues and blood. As an example, we show that the personalized cancer signature in six out of six patients with non-metastatic breast cancer can be detected and that levels over time are correlated with the clinical course of disease.

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MAP analytical selectivity is related to the number of mismatched nucleotides using the EGFR 15 bp deletion as a model.The analytical selectivity of MAP is higher than 1×109 when the number of mismatched nucleotides is 2–5, but sharply lower when the number of mismatched nucleotides is 7 or more. Analytical Sensitivity: Mutant DNA is serially diluted to 100, 10, 4, 2, 1, 1/2, 1/4 copies of template. The analytical sensitivity of the reaction is the minimum copy number of a mutant DNA that generates a detectable product when the primer matches the mutant template. The absence of a signal at one copy and the presence of a signal at ½ or ¼ copy are consistent with the Poisson distribution of expected signal resulting from dilution of DNA. Analytical Specificity: Wild type DNA is serially diluted from 1010 to 103 copies. The analytical specificity of the reaction is the maximum copy number of the mismatched (wt) template that does not result in a detectable product when the primer mismatches the wild-type template. Analytical selectivity is the ratio of analytical specificity to analytical sensitivity. Negative controls do not contain targeted DNA. M: ФX174 DNA/HaeIII Marker.
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pone-0007220-g002: MAP analytical selectivity is related to the number of mismatched nucleotides using the EGFR 15 bp deletion as a model.The analytical selectivity of MAP is higher than 1×109 when the number of mismatched nucleotides is 2–5, but sharply lower when the number of mismatched nucleotides is 7 or more. Analytical Sensitivity: Mutant DNA is serially diluted to 100, 10, 4, 2, 1, 1/2, 1/4 copies of template. The analytical sensitivity of the reaction is the minimum copy number of a mutant DNA that generates a detectable product when the primer matches the mutant template. The absence of a signal at one copy and the presence of a signal at ½ or ¼ copy are consistent with the Poisson distribution of expected signal resulting from dilution of DNA. Analytical Specificity: Wild type DNA is serially diluted from 1010 to 103 copies. The analytical specificity of the reaction is the maximum copy number of the mismatched (wt) template that does not result in a detectable product when the primer mismatches the wild-type template. Analytical selectivity is the ratio of analytical specificity to analytical sensitivity. Negative controls do not contain targeted DNA. M: ФX174 DNA/HaeIII Marker.

Mentions: The common 15 bp or 18 bp deletions in the epidermal growth factor receptor (EGFR) gene, commonly found in 5–20% of patients with non-small cell lung cancers [10], were chosen as models to explore the analytical sensitivity and analytical selectivity of MAP. In MAP, both downstream and upstream primers are blocked with a dideoxynucleotide (P*) and separated by 50 to 300 bp. The mutation-specific primers match the mutant and overlap the deletion junction so that two or more nucleotides mismatch the wild type sequence (Fig. 1A). When mutant-specific primers for the 15 bp deletion contain multiple mismatches (2–5 bases) with the wild-type template, the analytical selectivity of the assay is >109 (Fig. 2B–D, Table S2) and 100,000-fold greater than that observed in PAP-A, which contains only one base mismatch at the 3′ end (analytical selectivity ≤104) (Fig. 2A).


Analysis of cancer mutation signatures in blood by a novel ultra-sensitive assay: monitoring of therapy or recurrence in non-metastatic breast cancer.

Chen Z, Feng J, Buzin CH, Liu Q, Weiss L, Kernstine K, Somlo G, Sommer SS - PLoS ONE (2009)

MAP analytical selectivity is related to the number of mismatched nucleotides using the EGFR 15 bp deletion as a model.The analytical selectivity of MAP is higher than 1×109 when the number of mismatched nucleotides is 2–5, but sharply lower when the number of mismatched nucleotides is 7 or more. Analytical Sensitivity: Mutant DNA is serially diluted to 100, 10, 4, 2, 1, 1/2, 1/4 copies of template. The analytical sensitivity of the reaction is the minimum copy number of a mutant DNA that generates a detectable product when the primer matches the mutant template. The absence of a signal at one copy and the presence of a signal at ½ or ¼ copy are consistent with the Poisson distribution of expected signal resulting from dilution of DNA. Analytical Specificity: Wild type DNA is serially diluted from 1010 to 103 copies. The analytical specificity of the reaction is the maximum copy number of the mismatched (wt) template that does not result in a detectable product when the primer mismatches the wild-type template. Analytical selectivity is the ratio of analytical specificity to analytical sensitivity. Negative controls do not contain targeted DNA. M: ФX174 DNA/HaeIII Marker.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2749210&req=5

pone-0007220-g002: MAP analytical selectivity is related to the number of mismatched nucleotides using the EGFR 15 bp deletion as a model.The analytical selectivity of MAP is higher than 1×109 when the number of mismatched nucleotides is 2–5, but sharply lower when the number of mismatched nucleotides is 7 or more. Analytical Sensitivity: Mutant DNA is serially diluted to 100, 10, 4, 2, 1, 1/2, 1/4 copies of template. The analytical sensitivity of the reaction is the minimum copy number of a mutant DNA that generates a detectable product when the primer matches the mutant template. The absence of a signal at one copy and the presence of a signal at ½ or ¼ copy are consistent with the Poisson distribution of expected signal resulting from dilution of DNA. Analytical Specificity: Wild type DNA is serially diluted from 1010 to 103 copies. The analytical specificity of the reaction is the maximum copy number of the mismatched (wt) template that does not result in a detectable product when the primer mismatches the wild-type template. Analytical selectivity is the ratio of analytical specificity to analytical sensitivity. Negative controls do not contain targeted DNA. M: ФX174 DNA/HaeIII Marker.
Mentions: The common 15 bp or 18 bp deletions in the epidermal growth factor receptor (EGFR) gene, commonly found in 5–20% of patients with non-small cell lung cancers [10], were chosen as models to explore the analytical sensitivity and analytical selectivity of MAP. In MAP, both downstream and upstream primers are blocked with a dideoxynucleotide (P*) and separated by 50 to 300 bp. The mutation-specific primers match the mutant and overlap the deletion junction so that two or more nucleotides mismatch the wild type sequence (Fig. 1A). When mutant-specific primers for the 15 bp deletion contain multiple mismatches (2–5 bases) with the wild-type template, the analytical selectivity of the assay is >109 (Fig. 2B–D, Table S2) and 100,000-fold greater than that observed in PAP-A, which contains only one base mismatch at the 3′ end (analytical selectivity ≤104) (Fig. 2A).

Bottom Line: The utility of this method is illustrated in two ways. 1) We demonstrate that two EGFR deletions commonly found in lung cancers are not present in tissue from four normal human lungs (10(7) copies of gDNA each) or in blood samples from 10 healthy individuals (10(7) copies of gDNA each).MAP has an analytical selectivity of one part per billion for detection of MIDIs and an analytical sensitivity of one molecule.MAP provides a general tool for monitoring ultra-rare mutations in tissues and blood.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, City of Hope National Medical Center, Duarte, California, United States of America.

ABSTRACT

Background: Tumor DNA has been shown to be present both in circulating tumor cells in blood and as fragments in the plasma of metastatic cancer patients. The identification of ultra-rare tumor-specific mutations in blood would be the ultimate marker to measure efficacy of cancer therapy and/or early recurrence. Herein we present a method for detecting microinsertions/deletions/indels (MIDIs) at ultra-high analytical selectivity. MIDIs comprise about 15% of mutations.

Methods and findings: We describe MIDI-Activated Pyrophosphorolysis (MAP), a method of ultra-high analytical selectivity for detecting MIDIs. The high analytical selectivity of MAP is putatively due to serial coupling of two rare events: heteroduplex slippage and mis-pyrophosphorolysis. MAP generally has an analytical selectivity of one mutant molecule per >1 billion wild type molecules and an analytical sensitivity of one mutant molecule per reaction. The analytical selectivity of MAP is about 100,000-fold better than that of our previously described method of Pyrophosphorolysis Activated Polymerization-Allele specific amplification (PAP-A) for detecting MIDIs. The utility of this method is illustrated in two ways. 1) We demonstrate that two EGFR deletions commonly found in lung cancers are not present in tissue from four normal human lungs (10(7) copies of gDNA each) or in blood samples from 10 healthy individuals (10(7) copies of gDNA each). This is inconsistent, at least at an analytical sensitivity of 10(-7), with the hypotheses of (a) hypermutation or (b) strong selection of these growth factor-mutated cells during normal lung development leads to accumulation of pre-neoplastic cells with these EGFR mutations, which sometimes can lead to lung cancer in late adulthood. Moreover, MAP was used for large scale, high throughput "gene pool" analysis. No germline or early embryonic somatic mosaic mutation was detected (at a frequency of >0.3%) for the 15/18 bp EGFR deletion mutations in 6,400 individuals, suggesting that early embryonic EGFR somatic mutation is very rare, inconsistent with hypermutation or strong selection of these deletions in the embryo. 2) The second illustration of MAP utility is in personalized monitoring of therapy and early recurrence in cancer. Tumor-specific p53 mutations identified at diagnosis in the plasma of six patients with stage II and III breast cancer were undetectable after therapy in four women, consistent with clinical remission, and continued to be detected after treatment in two others, reflecting tumor progression.

Conclusions: MAP has an analytical selectivity of one part per billion for detection of MIDIs and an analytical sensitivity of one molecule. MAP provides a general tool for monitoring ultra-rare mutations in tissues and blood. As an example, we show that the personalized cancer signature in six out of six patients with non-metastatic breast cancer can be detected and that levels over time are correlated with the clinical course of disease.

Show MeSH
Related in: MedlinePlus