M.Sc Thesis


M.Sc StudentBurck Priev Nitza
SubjectSingle-Molecule Detection of ctDNAs Mutations Directly from
Blood
DepartmentDepartment of Biomedical Engineering
Supervisor PROF. Amit Meller
Full Thesis textFull thesis text - English Version


Abstract

Cancer is a leading cause of death worldwide with >18M new cases diagnosed and >9.5M deaths annually. Despite the use of some early diagnostics screenings, over 50% of new cases are diagnosed at late stages (stage III or IV), significantly reducing life expectancy of cancer patients and creating immense load on the global healthcare system. 

Circulating tumor DNAs (ctDNAs) are fragments of DNA shed by tumors into the bloodstream. These fragments harbor tumor-specific aberrations, reflecting genetic alterations of the tumors such as point mutations, and can be recovered from plasma, serum and other body fluids. A tumor containing ~50•106 malignant cells releases enough DNA for detection by conventional methods, but importantly, this is well below the size required for clinical imaging modalities. Therefore, improved means for ctDNA analysis hold the potential to identify cancer emergence earlier.

Although use of ctDNAs as a biomarker is promising, its detection from liquid biopsy has been challenging. The required sensitivity is extremely high due to considerable background from cell free DNA derived into circulation from normal cells. The fraction of tumor-derived circulating free DNA (cfDNA) in early stage cancer can be as low as <0.1%, thus requires overcoming low signal to noise ratio (SNR) to enable the quantification of an extremely limited copy number of target DNA.

Herein, I will present a novel genotyping method developed in our lab for single molecule ctDNA analysis. Our approach takes advantage of a ligation based biochemical assay together with the high efficiency of solid-state Nanopore (ssNP) biosensors, that can probe extremely low DNA concentrations. The ligation assay provides specificity, creating a reporter molecule differentiation two DNA strands even if they differ only by a single nucleotide. To enable highly multiplexed mutation detection using ssNPs, we have developed an orthogonal optical sensing method in addition to electrical probing. In our technique, an electrical voltage is used to focus the reporter biomolecules to a nanoscale pore, and to control their translocation speed. On the other hand, optical information is obtained from the fluorescence signals emitted by the molecules during their passage through the pore. Thus, by encoding a unique optical signature for each mutation we can resolve them as they pass through the nanopore. Notably, the coincidently recording of the electrical and optical signals, can facilitate the signal processing and reduce the chances of false positive results. Finally, to validate our assay, an animal xenograft model was utilized. DNA holding or lacking specific mutations was engineered into an aggressive cancer cell line. The cells were injected into mice and left to grow and metastasize, giving rise to mice with identical tumors distinguished only by the target DNA sequence. DNA was extracted from mice plasma and analyzed using our ligation-nanopore assay.  This was accomplished in collaboration with the Schneider Lab of NYU medical school. In this work we show results of specific identification of two actionable mutations directly from blood. Thus, opening a new technological opportunity towards liquid biopsy for early diagnosis of cancer in the single molecule level.