Limits...
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.

Show MeSH

Related in: MedlinePlus

MAP detects deletions in human DNA with an analytical selectivity >108.Four assays were established to test the MAP analytical sensitivity and analytical specificity (labeled by copy number). The analytical sensitivities of each assay are one copy and analytical specificities are >1×108 copy. From top to bottom panels, primer mismatches with the wild type template were 4, 4, 4 and 6 nucleotides, respectively. The presence of a signal at a mean of one copy of the mutant template is predicted to be 63% based on the Poisson distribution due to random sampling, consistent with the absence of signal in some reactions with one copy of mutant template.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2749210&req=5

pone-0007220-g003: MAP detects deletions in human DNA with an analytical selectivity >108.Four assays were established to test the MAP analytical sensitivity and analytical specificity (labeled by copy number). The analytical sensitivities of each assay are one copy and analytical specificities are >1×108 copy. From top to bottom panels, primer mismatches with the wild type template were 4, 4, 4 and 6 nucleotides, respectively. The presence of a signal at a mean of one copy of the mutant template is predicted to be 63% based on the Poisson distribution due to random sampling, consistent with the absence of signal in some reactions with one copy of mutant template.

Mentions: The MAP analysis of other deletions in the EGFR, EGFR2 and p53 genes in human or rat demonstrated analytical selectivities >108–109 when P* mismatched the wild type sequences by 3–5 nucleotides (Fig. 3, Table S3). Four assays with 4–5 mismatches had analytical selectivities >109 and one assay with six mismatches had an analytical selectivity >108. MAP was also demonstrated to detect four additional types of mutations with multiple base mismatches with respect to the wild type sequence. The analytical selectivity of MAP was >1010 for an insertion, >109 for a tandem base mutation and for two indels, and >108 for a doublet (two single base substitutions separated by 5 bases) (Fig. 4, Table S3).


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 detects deletions in human DNA with an analytical selectivity >108.Four assays were established to test the MAP analytical sensitivity and analytical specificity (labeled by copy number). The analytical sensitivities of each assay are one copy and analytical specificities are >1×108 copy. From top to bottom panels, primer mismatches with the wild type template were 4, 4, 4 and 6 nucleotides, respectively. The presence of a signal at a mean of one copy of the mutant template is predicted to be 63% based on the Poisson distribution due to random sampling, consistent with the absence of signal in some reactions with one copy of mutant template.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007220-g003: MAP detects deletions in human DNA with an analytical selectivity >108.Four assays were established to test the MAP analytical sensitivity and analytical specificity (labeled by copy number). The analytical sensitivities of each assay are one copy and analytical specificities are >1×108 copy. From top to bottom panels, primer mismatches with the wild type template were 4, 4, 4 and 6 nucleotides, respectively. The presence of a signal at a mean of one copy of the mutant template is predicted to be 63% based on the Poisson distribution due to random sampling, consistent with the absence of signal in some reactions with one copy of mutant template.
Mentions: The MAP analysis of other deletions in the EGFR, EGFR2 and p53 genes in human or rat demonstrated analytical selectivities >108–109 when P* mismatched the wild type sequences by 3–5 nucleotides (Fig. 3, Table S3). Four assays with 4–5 mismatches had analytical selectivities >109 and one assay with six mismatches had an analytical selectivity >108. MAP was also demonstrated to detect four additional types of mutations with multiple base mismatches with respect to the wild type sequence. The analytical selectivity of MAP was >1010 for an insertion, >109 for a tandem base mutation and for two indels, and >108 for a doublet (two single base substitutions separated by 5 bases) (Fig. 4, Table S3).

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