Errors in DNA replication and environmental factors, such as ionizing radiation and exposure to chemicals, often lead to double-stranded DNA breaks in the human genome. The active enzymatic DNA repair system in cells often lead to the fusion of sections of unrelated chromosomes, or to the fusion of different regions of the same chromosome to each other. Depending on the location of the breakpoints, these rearrangements often result in disruption or misregulation of normal gene function. During the past few decades, clinical studies have found that specific chromosomal translocations are the primary cause of various cancers, including lymphomas, leukemias, solid tumors, and sarcomas. Identification of these recurrent gene fusions has enabled the development of drugs that specifically target the products of these gene fusions. It is therefore of great importance to detect the underlying gene fusion at early stages in the disease process, so that appropriate treatment can be started in time. Due to the stochastic nature of gene expression, different cells in the same tissue have different amounts of gene fusion products (mRNAs) at any given time. Therefore, there is a need for a method that can quantify the number of gene fusion transcripts that are present in individual cells. We will develop a novel method for the detection of these gene fusion products, which uses fluorescence in situ hybridization probes that bind specifically to different regions of fused mRNAs. Multiple probes are designed to bind in close proximity to the target region in the mRNA, and each probe in the set is labeled singly with the same type of fluorophore. This method enables each mRNA molecule to be seen as a bright, diffraction-limited spot in a fluorescence microscope. A modification of this method will be developed to enable detection of short fragments of mRNAs as well. Different mRNA species can be distinguished from each other, using probe sets labeled with differently colored fluorophores. Coupling this single-molecule imaging technique with automated image-analysis computer programs, the location of affected cells among the normal cells in a tissue sample can be readily determined, and the number of gene fusion transcripts in each individual affected cell can be counted. This single-molecule imaging technique has already been successfully employed to understand the localization patterns of mRNA species in diverse biological systems. Using this technique to detect and count gene fusion transcripts in cancers will open up a new avenue for the characterization of cancer cells. Most importantly, the successful implementation of this novel method will significantly improve our ability to study the efficacy of drugs, and will enable the monitoring of responses to treatment, particularly in patients with solid tumors and sarcomas.
The objective of this project is to develop a method for the detection of tumor-causing chromosomal translocations and rearrangements by imaging individual molecules of the resulting gene fusion transcripts. In this method, sets of approximately 50 different singly labeled probes are hybridized to selected segments of a target mRNA. With the deposition of multiple labels, individual mRNA molecules are rendered so intensely fluorescent that they can be detected as diffraction-limited spots in a fluorescence microscope. Different mRNA species can be imaged simultaneously using probe sets labeled with differently colored fluorophores. If a chromosomal rearrangement results in the synthesis of recombinant mRNAs that possess portions of two separate mRNAs that are imaged with differently colored probe sets, then the recombinant mRNAs are seen in the microscope as individual spots lit up in both colors. I will adopt this method for the detection of gene fusion transcripts that are found in cancers as a result of chromosomal rearrangements. I will also modify the probe design to image mRNA, which are shorter in length and cannot be imaged with the current method. These extremely precise in situ hybridization probes will light up tumor cells in healthy tissue to provide anatomical information of the tumor, and will be valuable for assessing disease progression and response to therapy.
