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A novel platform for creating digital PCR assays to detect genetic translocations and its application to the initial diagnosis of cancer. Lund, Helen Louise

Abstract

Chromosomal translocations can cause cancer, often through the formation of fusion genes that code for an unnatural tyrosine kinase that promotes constitutive activation of a signaling pathway controlling cell proliferation and differentiation. For example, the diagnostic hallmark of chronic myelogenous leukemia (CML) is an oncogene fusion formed from a reciprocal translocation (t(9;22)(q34.1;q11.2)) between chromosomes 9 and 22 that results in an altered chromosome 22q known as the Philadelphia chromosome. Approximately 95% of all CML patients harbor the gene fusion, BCR-ABL, which is formed via a double stranded break (DSB) within both the Abelson oncogene 1 (ABL) on chromosome 9q, which codes for a non-receptor tyrosine kinase (ABL), and the breakpoint cluster region gene (BCR) on chromosome 22q. BCR-ABL encodes a constitutively active tyrosine kinase BCR-ABL responsible for the uncontrolled proliferation associated with chronic myelogenous leukemia. The identification of these translocation events and/or associated fusion genes in clinical samples is critical to ensure the appropriate treatment for patients where the drug and related course of therapy target an activated fusion kinase. Clinical detection of complex chromosomal rearrangements is often conducted using fluorescence in situ hybridization (FISH). The FISH analysis, though effective, offers relatively poor sensitivity while being expensive, time-consuming and technically challenging to perform. Here we have developed and validated a new general platform for creating assays against complex chromosomal rearrangements, including both reciprocal and non-reciprocal translocations. It utilizes droplet digital PCR (ddPCR) technology in lieu of FISH to quantify the rearrangement of proto-oncogenes that undergo rearrangement as part of the translocation event. The platform is applied to the creation of two new assays of potential clinical use in cancer diagnostics or theranostics. The first provides a reliable and sensitive measure of DSBs within the major breakpoint region of BCR (M-BCR), permitting initial diagnosis of CML through unequivocal detection of the BCR-ABL fusion gene to a frequency of 0.25%. The second provides for the highly sensitive detection of DSBs in the anaplastic lymphoma kinase (ALK) gene that result in a non-reciprocal (inversion) translocation (inv(2)(p21;p23)) associated with an ALK-positive non-small cell lung cancer (NSCLC).

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