Abstract 3930: Radiation-induced dicentrics: Tracking by parallel sequencing

• Published in: Cancer research (Chicago, Ill.) Vol. 70; no. 8_Supplement; p. 3930
• Main Authors: Vaughan, Andrew T.M, Shih, Shyh-Jen, Singh, Sheetal, Do, To Uyen
• Format: Journal Article
• Language: English
• Published: 15.04.2010
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM10-3930

Abstract Radiation and cytotoxic drugs exert much of their lethal effects by creating aberrations, such as dicentrics, that are lethal at a subsequent mitosis. Their accurate enumeration in experimental and clinical settings would provide valuable information on the efficacy of radiation and radiation/chemotherapy combinations. In this study a method was sought that could rapidly and efficiently quantitate this process using next generation sequencing. The use of modern molecular technology for aberration analysis has been restricted by the random nature of the breaks induced in individual chromosome that inhibit their detection. To overcome this problem, a common fragile site at 11q23 that is readily cleaved in response to a broad range of cytotoxic agents was used. Using irradiated human TK6 lymphoblastoid cells and blood samples taken from patients undergoing chemotherapy, partner chromosomes that are ligated to this location were detected using inverse PCR to capture the breakpoint. In order to enhance the efficiency of the process, amplified inverse PCR material was processed for Solexa type parallel sequencing. Here individual amplicons, representing the total content of a treated sample, are attached to a flow cell and their sequence recorded by sequential additions of fluorescent bases. Each aberration was then reconstructed with reference to a standard human genome database. In one specific sample of treated TK6 cells, two translocations involving Chr. 2 and 18 and two dicentrics involving Chr. 3 and 13 were found linked to the Chr 11q23 fragmentation site. In addition, aberrations were found in patients’ blood 6 months after initiation of therapy. Thus translocations that are rarely lethal could be readily discriminated from dicentric type rearrangements that are predominately lethal. The advantage of this analytical process is that all normal sequence is also recorded allowing the fractional content of all aberrations to be recorded in response to dose or time. In order to mitigate concerns over the arbitrary nature of aberrations induced at a common fragile site, a zinc finger nuclease was constructed targeting a region adjacent to the fragile site at 11q23. With this zinc finger system repetitive DNA breaks at the predicted site generated micro rearrangements (insertions/deletions) that were produced at far higher rates than translocations or dicentric type rearrangements measured by parallel processing. In summary, a novel analytical system for detecting a lethal class (dicentrics) of chromosome rearrangements has been described that is suitable for therapy monitoring both in-vitro and in-vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3930.