A novel method of amplified fluorescent in situ hybridization for detection of chromosomal microdeletions in B cell lymphoma

• Published in: International journal of hematology Vol. 109; no. 5; pp. 593 - 602
• Main Authors: Mizuno, Yoshimi, Chinen, Yoshiaki, Tsukamoto, Taku, Takimoto-Shimomura, Tomoko, Matsumura-Kimoto, Yayoi, Fujibayashi, Yuto, Kuwahara-Ota, Saeko, Fujino, Takahiro, Nishiyama, Daichi, Shimura, Yuji, Kobayashi, Tsutomu, Horiike, Shigeo, Taniwaki, Masafumi, Kuroda, Junya
• Format: Journal Article
• Language: English
• Published: Tokyo Springer Japan 01-05-2019; Springer Nature B.V
• Subjects: Amplification; B-cell lymphoma; Banding; Blood cancer; Blood cells; CDKN2A gene; Chromosome banding; Chromosome deletion; Clonal deletion; Deoxyribonucleic acid; Diagnostic systems; DNA; Fluorescein; Fluorescein isothiocyanate; Fluorescence; Fluorescence in situ hybridization; Gene deletion; Hematology; Lymphocytes B; Lymphoma; Medicine; Medicine & Public Health; Oncology; Original Article; Tumor suppressor genes; Fluorescence in situ hybridization (FISH); Microdeletion; Amplified-FISH; Hematologic malignancy; CDKN2A
• ISSN: 0925-5710; 1865-3774
• DOI: 10.1007/s12185-019-02617-x

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.