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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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A: MAP: Introducing multiple oligonucleotide mismatch into the 3′ end of a mutation-specific blocked primer.An example of a deletion mutant sequence (the common EGFR 15 bp deletion) is shown below the wild-type sequence (deleted sequence in brackets, red letters). The last three bases (3′) of a mutation-specific 3′ blocked primer (upstream) are complementary to the three bases (caa) just before the 5′ end of the deletion; the primer mismatches the wild-type sequence at the three bases (agc) at the 3′ end of the deleted region. Asterisks indicate the 3′ dideoxynucleotide of the blocked primers. The “X” represents mismatch between the mutant-specific primer and wild-type sequence. B: Serial coupling of two errors underlies the ultra-high analytical selectivity of PAP and MAP. PAP-A or Bi-PAP-A and MAP derive their high analytical selectivity from serial coupling of two events, but the events differ. The practical analytical specificity for PAP-A and Bi-PAP-A is limited by side reactions such as misincorporation from the extended generic PAP primer or the presence of DNA damage products such as deaminated cytosine or 8-oxo guanidine. In contrast, false positives in MAP require the serial coupling of DNA slippage and mis-pyrophosphorolysis within this distorted DNA structure.
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pone-0007220-g001: A: MAP: Introducing multiple oligonucleotide mismatch into the 3′ end of a mutation-specific blocked primer.An example of a deletion mutant sequence (the common EGFR 15 bp deletion) is shown below the wild-type sequence (deleted sequence in brackets, red letters). The last three bases (3′) of a mutation-specific 3′ blocked primer (upstream) are complementary to the three bases (caa) just before the 5′ end of the deletion; the primer mismatches the wild-type sequence at the three bases (agc) at the 3′ end of the deleted region. Asterisks indicate the 3′ dideoxynucleotide of the blocked primers. The “X” represents mismatch between the mutant-specific primer and wild-type sequence. B: Serial coupling of two errors underlies the ultra-high analytical selectivity of PAP and MAP. PAP-A or Bi-PAP-A and MAP derive their high analytical selectivity from serial coupling of two events, but the events differ. The practical analytical specificity for PAP-A and Bi-PAP-A is limited by side reactions such as misincorporation from the extended generic PAP primer or the presence of DNA damage products such as deaminated cytosine or 8-oxo guanidine. In contrast, false positives in MAP require the serial coupling of DNA slippage and mis-pyrophosphorolysis within this distorted DNA structure.

Mentions: A pair of primers with similar Tm values, each about 30 bases in length and separated by a 50∼300 bp sequence segment, was designed for each MAP assay to detect rare deletions. Each P* primer was modified by adding a dideoxynucleotide at the 3′ terminus as described previously [15]. The mutation-specific primer mismatched the wild type sequence at two to six bases, but matched the mutant sequence at these positions (Fig. 1A). For detecting a mutation in plasma, the size of the amplicon should be <100 bp because plasma DNA is highly degraded.


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)

A: MAP: Introducing multiple oligonucleotide mismatch into the 3′ end of a mutation-specific blocked primer.An example of a deletion mutant sequence (the common EGFR 15 bp deletion) is shown below the wild-type sequence (deleted sequence in brackets, red letters). The last three bases (3′) of a mutation-specific 3′ blocked primer (upstream) are complementary to the three bases (caa) just before the 5′ end of the deletion; the primer mismatches the wild-type sequence at the three bases (agc) at the 3′ end of the deleted region. Asterisks indicate the 3′ dideoxynucleotide of the blocked primers. The “X” represents mismatch between the mutant-specific primer and wild-type sequence. B: Serial coupling of two errors underlies the ultra-high analytical selectivity of PAP and MAP. PAP-A or Bi-PAP-A and MAP derive their high analytical selectivity from serial coupling of two events, but the events differ. The practical analytical specificity for PAP-A and Bi-PAP-A is limited by side reactions such as misincorporation from the extended generic PAP primer or the presence of DNA damage products such as deaminated cytosine or 8-oxo guanidine. In contrast, false positives in MAP require the serial coupling of DNA slippage and mis-pyrophosphorolysis within this distorted DNA structure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749210&req=5

pone-0007220-g001: A: MAP: Introducing multiple oligonucleotide mismatch into the 3′ end of a mutation-specific blocked primer.An example of a deletion mutant sequence (the common EGFR 15 bp deletion) is shown below the wild-type sequence (deleted sequence in brackets, red letters). The last three bases (3′) of a mutation-specific 3′ blocked primer (upstream) are complementary to the three bases (caa) just before the 5′ end of the deletion; the primer mismatches the wild-type sequence at the three bases (agc) at the 3′ end of the deleted region. Asterisks indicate the 3′ dideoxynucleotide of the blocked primers. The “X” represents mismatch between the mutant-specific primer and wild-type sequence. B: Serial coupling of two errors underlies the ultra-high analytical selectivity of PAP and MAP. PAP-A or Bi-PAP-A and MAP derive their high analytical selectivity from serial coupling of two events, but the events differ. The practical analytical specificity for PAP-A and Bi-PAP-A is limited by side reactions such as misincorporation from the extended generic PAP primer or the presence of DNA damage products such as deaminated cytosine or 8-oxo guanidine. In contrast, false positives in MAP require the serial coupling of DNA slippage and mis-pyrophosphorolysis within this distorted DNA structure.
Mentions: A pair of primers with similar Tm values, each about 30 bases in length and separated by a 50∼300 bp sequence segment, was designed for each MAP assay to detect rare deletions. Each P* primer was modified by adding a dideoxynucleotide at the 3′ terminus as described previously [15]. The mutation-specific primer mismatched the wild type sequence at two to six bases, but matched the mutant sequence at these positions (Fig. 1A). For detecting a mutation in plasma, the size of the amplicon should be <100 bp because plasma DNA is highly degraded.

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