{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T11:04:54Z","timestamp":1760267094384,"version":"3.44.0"},"reference-count":28,"publisher":"BMJ","issue":"7","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J Clin Pathol"],"accepted":{"date-parts":[[2011,3,20]]},"published-print":{"date-parts":[[2011,7]]},"abstract":"<jats:sec>\n                  <jats:title>Aims<\/jats:title>\n                  <jats:p>The <jats:italic>BCR-ABL1<\/jats:italic> T315I mutation imparts resistance to tyrosine kinase inhibitors currently available for treatment of chronic myelogenous leukaemia. Thus, quantitative monitoring of the emergence and expansion of T315I-positive subclones may be clinically useful. The goals of this study were to retrospectively review the authors' experience with Sanger sequencing-based <jats:italic>BCR-ABL1<\/jats:italic> kinase domain mutation testing, paying particular attention to the T315I mutation, and to develop an alternative test for relative quantification of T315I using pyrosequencing.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>The performance of a new T315I pyrosequencing assay was evaluated. Total RNA was isolated from whole blood and reverse-transcribed. The resulting cDNA was subjected to an initial round of PCR across the <jats:italic>BCR-ABL1<\/jats:italic> breakpoint followed by a second round to amplify the sequence flanking <jats:italic>ABL1<\/jats:italic> codon 315. The final PCR product was pyrosequenced to detect and quantify the T315I point mutation. Additional experiments were carried out to determine the effects of background untranslocated <jats:italic>ABL1<\/jats:italic> on assay sensitivity in samples with low tumour burden.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>The results show that T315I was the most commonly detected kinase domain mutation and was persistent in follow-up testing. All 26 specimens that tested positive by Sanger sequencing for the T315I mutation were also positive using the pyrosequencing test. Relative quantification data derived from pyrosequencing matched the approximate wild-type\/mutant ratios found by Sanger sequencing. Serial dilution experiments show sensitivity to 5% mutant allele. The authors also quantitatively assessed the influence of untranslocated <jats:italic>ABL1<\/jats:italic> in the sample background on the assay and found that it occurred at levels not likely to influence performance.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusion<\/jats:title>\n                  <jats:p>The described test is useful for detection and relative quantification of the T315I point mutation in chronic myelogenous leukaemia in a sensitive, specific and reproducible manner.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1136\/jcp.2011.089193","type":"journal-article","created":{"date-parts":[[2011,4,12]],"date-time":"2011-04-12T20:40:31Z","timestamp":1302640831000},"page":"618-625","source":"Crossref","is-referenced-by-count":6,"title":["A pyrosequencing-based test for detection and relative quantification of the <i>BCR-ABL1<\/i> T315I point mutation"],"prefix":"10.1136","volume":"64","author":[{"given":"Jonathan A","family":"Schumacher","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Philippe","family":"Szankasi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David W","family":"Bahler","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Albert K","family":"Ho","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Todd W","family":"Kelley","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"239","published-online":{"date-parts":[[2011,4,12]]},"reference":[{"key":"2025092503525438000_64.7.618.1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa022457"},{"key":"2025092503525438000_64.7.618.2","first-page":"513","article-title":"Allogeneic hematopoietic stem cell transplantation for chronic myeloid leukemia in Europe 2006: transplant activity, long-term data and current results. 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