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Detection of cell-free, tumor-derived DNA mutations prior to clinical diagnosis of breast cancer

English title Detection of cell-free, tumor-derived DNA mutations prior to clinical diagnosis of breast cancer
Applicant Toniolo Paolo
Number 124958
Funding scheme Project funding (Div. I-III)
Research institution Institut Universitaire de Médecine Sociale et Préventive - IUMSP CHUV et Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Cancer
Start/End 01.04.2009 - 31.12.2012
Approved amount 375'000.00
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All Disciplines (2)

Molecular Biology

Keywords (11)

breast cancer; female; somatic mutations; cell-free DNA; MALDI-TOF MS; early detection; blood based test; mass spectrometry;; biological banks; prospective cohort

Lay Summary (English)

Lay summary
Early diagnosis of breast cancer rests on mammography, a technique that has undoubtedly contributed to reducing mortality, but suffers from certain limitations: provides no real benefits to women before age 50, exposes them to ionizing radiation and has elevated monetary and emotional costs. A desirable tool would be based on a simple-to-administer blood test. Considering that even a small tumor undetectable at mammography continuously releases millions of neoplastic cells into the bloodstream, a possible approach would be to detect in circulation and quantify such cells or, preferably, DNA fragments from broken cells carrying the marks (mutations) of its presence. A laboratory technique called mass spectrometry makes now feasible to identify given mutations in circulation, in particular those in the 140 genes that appear to be breast cancer-specific (CAN-genes). This research proposes to explore if CAN-genes mutations in blood signal the presence of an undetected breast cancer. To this end, blood samples will be obtained from the North Sweden Health and Disease Study preserved within the Biological Bank of the University of Umeå, Sweden. In this prospective cohort, a large number of women were recruited when free of any cancer, and blood specimens stored frozen. Women were then followed-up for many years to identify those who developed breast cancer. In this initial phase, 90 plasma specimens will be utilized from those diagnosed with breast cancer within 6 months after the initial blood draw, a time interval in which the breast cancer subsequently diagnosed was certainly already present. An equal number of specimens from women who will have remained free of any cancer for 10 or more years following the initial blood draw (thus, truly free of cancer) will serve as controls. Stored blood samples are analyzed to identify all CAN-genes mutations and determine if they occur in different patterns in breast cancer patients as compared to controls.If successful, the study will help establish the principle that the detection of specific mutations has the potential to be developed into a powerful, practical and low-cost new tool in the early detection of breast cancer. In a subsequent phase, the initial observations will be validated in different plasma samples in the same population in order to confirm the goodness of initial observations and will be extended to blood samples drawn within increasing time ahead of clinical diagnosis, thus determining the potential of the proposed approach as a new early detection tool.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants



Assessing the value of CAN-gene mutations using MALDI-TOF MS
Kohler C, Tavelin B, Fan AXC, Radpour R, Barekati Z, Levi F, Zhong XY, Lenner P, Toniolo P (2011), Assessing the value of CAN-gene mutations using MALDI-TOF MS, in JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY, 137(8), 1239-1244.
Methylation profile of TP53 regulatory pathway and mtDNA alterations in breast cancer patients lacking TP53 mutations
Barekati Z, Radpour R, Kohler C, Zhang B, Toniolo P, Lenner P, Lv Q, Zheng H, Zhong XY (2010), Methylation profile of TP53 regulatory pathway and mtDNA alterations in breast cancer patients lacking TP53 mutations, in HUMAN MOLECULAR GENETICS, 19(15), 2936-2946.


We are proposing a uniquely innovative project seeking to develop a potentially revolutionary approach to the early detection of breast cancer, the most common malignancy of women in Switzerland and worldwide. The project is based on an established collaboration between investigators at the CHUV in Lausanne and at the University Women’s Hospital of the University of Basel with the essential contribution of investigators at the University of Umeå, Sweden.Background: The early detection of breast cancer rests by and large on mammography, a technique the effectiveness of which is undermined by sub-optimal predictive value, patients’ exposure to radiation and high costs, both direct and indirect. Blood-based bioassays built on measurements of biological/molecular components may lead to earlier detection of lesions through enhanced sensitivity and specificity, along with contained costs, improved efficiency and minimal, if any, discomfort.Theoretically, rare breast cancer cells in circulation should be identifiable well before the tumor reaches clinically detectable size, but the technical complexities of growing rare tumor cells in circulation greatly limit the sensitivity and specificity of such an approach. In recent times. the ability to detect and quantify circulating cell-free DNA (cfDNA) has greatly improved. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a robust technology that is perfectly adaptable to the detection of single-nucleotide variations on nuclear DNA. Recently, the first genome-wide screen of clonal mutations in breast cancer has led to the identification of 140 so-called CAN-genes mutations distinguishable from non-tumor derived cell-free DNA by allele-specific PCR. Although the CAN-genes approach was conceived in order to better understand the biology of tumor growth with an eye to the development of individualized treatments, we believe that progress in cfDNA detection along with the availability of tumor-specific CAN-genes detectable through a novel mass spectrometry approach opens up incredible new possibilities in the detection of rare signature mutations of yet undiagnosed malignancies in circulation and therefore a novel, potentially powerful tool in early detection.Objectives: The purpose of this collaborative study is to determine the potential of the proposed breast cancer CAN-gene mutations to be developed as a blood-based bioassay to signal the presence of an as yet undetected invasive breast cancer in pre-clinical stage. The proposal is both highly innovative and high-impact in that it introduces a totally new paradigm in utilizing newly developed, powerful technologies that may have a dramatic impact in the early detection of breast cancer and in reducing its major complications and mortality. The study will be carried out taking advantage of the unique resources of a long-term prospective cohort study at the University of Umeå, Sweden, with access to well characterized criopreserved plasma samples drawn in advance of the clinical diagnosis of breast cancer, as well as tumor tissue samples obtained at subsequent diagnosis.The main research hypothesis underlying the project is that specific patterns of the proposed CAN-genes when detected in circulation, are indicative of the presence of an invasive breast cancer at pre-clinical stage and, in addition, carry information concerning the specific pathway(s) affected. We hypothesize also that our approach will be particularly effective in the early identification of subjects subsequently diagnosed with breast cancer at stages (II and III) more advanced, and with worse prognosis than Stage I, a circumstance which would have an especially large impact in reducing the burden of breast cancer. Finally, the inclusion of these more advanced cases allows us to hypothesize that patterns of CAN-genes mutations and/or specific pathways could be identified that are associated with tumor aggressiveness. The research’s specific aims are the following:1)To develop a prediction model of breast cancer based on the intensity of all the 332 observed mutations in the 140 CAN-genes of interest: 90 cases with blood samples drawn in advance of clinical diagnosis + 90 non-cancer controls.2)To compare CAN-genes analyses between plasma and tissue samples in the same individual: 25 study subjectsExperimental design: We will develop a prediction model based on the entire set of 332 mutations in the 140 breast cancer CAN-genes in breast cancer cases and in suitable controls. We will than compare the presence of the most informative mutations between blood and cancer tissue specimens from a subset the same study subjects.All information and biological samples are already available. Two groups of study subjects will be considered: a) Cases: 90 women diagnosed with invasive breast cancer at any stage (except stage IV, metastatic); and b) Controls: 90 women never diagnosed with any cancer until ten years following baseline blood draw. Individually matched cases and controls will be selected from among cohort members who had blood drawn within 180 days (6 months) preceding the date of diagnosis of a case. Fifty percent of cases will be Stage I and 50% Stage II or III. Plasma samples (1.5 ml) stored at the Biobank in Umeå, will be retrieved from storage, prepared and labeled appropriately. Fixed tumor tissue from the same subjects will be prepared by a pathologist and all specimens (plasma samples frozen) will be shipped to the laboratory in Basel for analyses. Matched cases and controls will be allocated to the same laboratory run in order to be analyzed the same day by the same technicians. De-identified databases containing all information, including all laboratory results will be created at the CHUV in Lausanne where statistical analyses will be performed.Laboratory Methods: MALDI-TOF MS (matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry) provides accurate and direct measurements of molecular weights of nucleic acids at about 100-fold higher resolution than capillary sequencing. MALDI-TOF MS is being used extremely successfully within the Basel group to genotype single nucleotide polymorphisms in fetal DNA in maternal circulation for the non-invasive prenatal diagnosis of genetic conditions. The technique can differentiate between two alleles differing by as little as one base robustly and with high automation. The iPLEX Gold Assay enables multiplex PCR and up to 36-40 primer extension reactions per assay, so that 36-40 mutations can be detected simultaneously in a single reaction. DNA sequences are first amplified by PCR in order to distinguish between the normal and mutant alleles. Single base extension reaction is performed with unlabeled ddNTPs resulting in two extension products of different molecular weights depending on the allele. Expected Value of the Proposed ProjectNew blood based bioassays based on measurements of biological components are badly needed in order to overcome the limitations and costs of traditional imaging in the early detection of breast cancer. The proposed project relies on the considerable expertise in the detection and quantification of cfDNA fragments, as well as their novel MALDI-TOF MS-based approach to the measurement of the molecular weight of nucleid acids of Dr. Zhong’s group. It relies also heavily on the availability of a large number of well-characterized clinical and pre-clinical biological samples from women with breast cancer in the prospective cohort at the University of Umeå. Observations from the proposed research could lead to a completely novel approach to the early detection of breast cancer based on minimally invasive biological measurements with potentially elevated predictive value, rapidity of execution and very low costs.