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


RESULTS
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.
Department of Molecular Genetics, City of Hope National Medical Center, Duarte, California, United States of America.
Zhenbin
Z
Chen
Jinong
J
Feng
Carolyn H
CH
Buzin
Qiang
Q
Liu
Lawrence
L
Weiss
Kemp
K
Kernstine
George
G
Somlo
Steve S
SS
Sommer
D000818:Animals; D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000097:blood* / Q000175:diagnosis / Q000235:genetics* / Q000473:pathology* / Q000628:therapy; D004247:DNA / Q000097:blood*; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D066246:ErbB Receptors / Q000378:metabolism; D005260:Female; D006801:Humans; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000097:blood* / Q000235:genetics*; D009360:Neoplastic Cells, Circulating / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D012008:Recurrence; D012680:Sensitivity and Specificity
D014408:Biomarkers, Tumor; D017931:DNA Primers; D004247:DNA; D066246:ErbB Receptors