Abstract
Chromosomal microdeletions frequently cause loss of prognostically relevant tumor suppressor genes in hematologic malignancies; however, detection of minute deletions by conventional methods for chromosomal analysis, such as G-banding and fluorescence in situ hybridization (FISH), is difficult due to their low resolution. Here, we describe a new diagnostic modality that enables detection of chromosomal microdeletions, using CDKN2A gene deletion in B cell lymphomas (BCLs) as an example. In this method, which we refer to as amplified-FISH (AM-FISH), a 31-kb fluorescein isothiocyanate (FITC)-conjugated DNA probe encoding only CDKN2A was first hybridized with the chromosome, and then labeled with Alexa Fluor 488-conjugated anti-FITC secondary antibody to increase sensitivity. CDKN2A signals were equally identifiable by AM-FISH and conventional FISH in normal mononuclear blood cells. In contrast, when two BCL cell lines lacking CDKN2A were analyzed, CDKN2A signals were not detected by AM-FISH, whereas conventional FISH yielded false signals. Furthermore, AM-FISH detected CDKN2A deletions in two BCL patients with 9p21 microdeletions, which were not detected by conventional FISH. These results suggest that AM-FISH is a highly sensitive, specific, and simple method for diagnosis of chromosomal microdeletions.
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Acknowledgements
The authors thank K. Ueda, N. Inada, and T. Ikawa for their excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT KAKENHI 16K09856) (MT); by the National Cancer Center Research and Development Fund (29-A-3); by a grant (Practical Research for Innovative Cancer Control) from the Japan Agency for Medical Research and Development (AMED) (17ck0106348h0001) (J.K.), and a Grant-in-Aid for Young Scientists (B) (JSPS KAKENHI Grant Number JP16K21284 (Y.C.)).
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Mizuno, Y., Chinen, Y., Tsukamoto, T. et al. A novel method of amplified fluorescent in situ hybridization for detection of chromosomal microdeletions in B cell lymphoma. Int J Hematol 109, 593–602 (2019). https://doi.org/10.1007/s12185-019-02617-x
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DOI: https://doi.org/10.1007/s12185-019-02617-x