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

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; Kuroda, Junya

doi:10.1007/s12185-019-02617-x

Cited by 4 publications

(5 citation statements)

References 35 publications

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“…Detection of CDKN2A deletion is important in routine practice as it effectively discriminates MPM from reactive mesothelial proliferations, and is associated with poor prognosis in MPM (3)(4)(5)(6). There are several approaches used to circumvent the false negative results in detecting chromosomal microdeletion, including use of smaller probes (as used in the present study), enhancement of FISH signals (13), multiple ligation-dependent probe amplification (MLPA) assay (11) and array comparative genomic hybridization (CGH) (10). Practical use of S-probe can be somewhat challenging as the target red signal is small owing to the short probe length (9); thus, lower hybridization efficiency can result in false positive results for CDKN2A deletion (9).…”

Section: Discussionmentioning

confidence: 98%

“…The smallest deletion is only 25 kb on the CDKN2A gene (10). Cases with CDKN2A microdeletion fail to exhibit high proportions of homo-d by FISH with L-probe, as the L-probe, which is designed to cover three genes in the 9p21 locus (CDKN2A, CDKN2B and MTAP) (10), is large enough to hybridize the non-deleted area next to the microdeleted CDKN2A gene, resulting in 'pseudo' hetero-d signals (9,13). In the present study, the false red signals were smaller compared with normal CEP9 green signals.…”

Section: Discussionmentioning

confidence: 99%

“…Microdeletion, defined as a deletion <200 kb, can induce false negative results of signal deletion in FISH assays, owing to redundant probe reactivity (9)(10)(11)(12)(13). Some of the high hetero-d signals may result from microdeletions in MPM (8).…”

Section: Introductionmentioning

confidence: 99%

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Short 57 kb CDKN2A FISH probe effectively detects short homozygous deletion of the 9p21 locus in malignant pleural mesothelioma

et al. 2021

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Homozygous deletion (homo-d) of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene is frequently found in malignant pleural mesothelioma (MPM). Fluorescence in situ hybridization (FISH) is commonly used to detect chromosomal deletion, and sometimes reveals more frequent heterozygous deletion (hetero-d) compared with homo-d. In clinical practice, such CDKN2A FISH results belong to the 'borderline' homo-d rate, which makes it difficult to definitively diagnose MPM. Microdeletion, [<200 kilobase (kb)], can induce a 'pseudo' hetero-d signal in FISH assays with long probes owing to redundant probe reactivity. Thus, the present study hypothesized that shorter FISH probes can effectively detect the small deletion status of the CDKN2A gene and increase homo-d rate in MPM, which has high hetero-d and low homo-d status. The present study aimed to evaluate the effectiveness of a shorter CDKN2A FISH probe in diagnosing MPM. CDKN2A FISH with either a 222 kb long probe (L-probe) or a 57 kb short probe (S-probe) was performed in four MPM cases with high hetero-d and low homo-d patterns. Furthermore, immunohistochemistry for methylthioadenosine phosphorylase (MTAP) and quantitative (q)PCR analyses were performed to confirm the microdeletion of the 9p21 locus. The results demonstrated that all four MPM cases retained MTAP protein expression. CDKN2A FISH with L-probe revealed high hetero-d (cases 1-4; 73.3, 37.1, 59.2 and 64.8%, respectively) and low homo-d (cases 1-4; 12.1, 12.4, 25.4 and 22.2%, respectively). CDKN2A FISH with S-probe revealed high homo-d (cases 1-4; 96.8, 90.0, 87.5 and 82.6%, respectively), with low hetero-d (cases 1-4; 0.0, 1.2, 1.2 and 4.3%, respectively). qPCR analysis demonstrated no allele deletions of the MTAP gene and two-allele deletions of the CDKN2A gene in 3/4 cases. Taken together, these results suggest that the S-probe detects the short homo-d of the 9p21 locus more effectively than the L-probe in MPM. This can assist in solving diagnostic difficulties in cases involving high hetero-d with low homo-d.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

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Short 57 kb CDKN2A FISH probe effectively detects short homozygous deletion of the 9p21 locus in malignant pleural mesothelioma

et al. 2021

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“…Conventional standard DC-FISH for IGH / CCND1 , IGH / FGFR3 , and IGH / MAF were separately performed for each sample as described previously [ 18 , 20 ]. Probes utilized for standard DC-FISH were Vysis LSI IGH / FGFR3 Dual Color Dual Fusion Probes, Vysis LSI IGH / CCND1 Dual Color Dual Fusion Probe, and Vysis LSI IGH / MAF Dual Color Dual Fusion Probes (Abbott, Abbott Park, IL).…”

Section: Methodsmentioning

confidence: 99%

Imaging flow cytometry-based multiplex FISH for three IGH translocations in multiple myeloma

et al. 2023

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Three types of chromosomal translocations, t(4;14)(p16;q32), t(14;16)(q32;q23), and t(11;14)(q13;q32), are associated with prognosis and the decision making of therapeutic strategy for multiple myeloma (MM). In this study, we developed a new diagnostic modality of the multiplex FISH in immunophenotyped cells in suspension (Immunophenotyped-Suspension-Multiplex (ISM)-FISH). For the ISM-FISH, we first subject cells in suspension to the immunostaining by anti-CD138 antibody and, then, to the hybridization with four different FISH probes for genes of IGH, FGFR3, MAF, and CCND1 tagged by different fluorescence in suspension. Then, cells are analyzed by the imaging flow cytometry MI-1000 combined with the FISH spot counting tool. By this system of the ISM-FISH, we can simultaneously examine the three chromosomal translocations, i.e, t(4;14), t(14;16), and t(11;14), in CD138-positive tumor cells in more than 2.5 × 104 nucleated cells with the sensitivity at least up to 1%, possibly up to 0.1%. The experiments on bone marrow nucleated cells (BMNCs) from 70 patients with MM or monoclonal gammopathy of undetermined significance demonstrated the promising qualitative diagnostic ability in detecting t(11;14), t(4;14), and t(14;16) of our ISM-FISH, which was more sensitive compared with standard double-color (DC) FISH examining 200 interphase cells with its best sensitivity up to 1.0%. Moreover, the ISM-FISH showed a positive concordance of 96.6% and negative concordance of 98.8% with standard DC-FISH examining 1000 interphase cells. In conclusion, the ISM-FISH is a rapid and reliable diagnostic tool for the simultaneous examination of three critically important IGH translocations, which may promote risk-adapted individualized therapy in MM.

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“…Conventional G-banding for metaphase spreads and fluorescence in situ hybridization (FISH) studies were performed, as described elsewhere [54]. Interphase FISH was performed using a Vysis CEP 3 (D3Z1) SpectrumOrange Probe and a Vysis CEP 7 (D7Z1) SpectrumGreen Probe (Abbott, Abbott Park, IL, USA) for the detection of centromeres of chromosome 3 (chr3) and chr7, respectively.…”

Section: Chromosomal Analysismentioning

confidence: 99%

Aberrant BUB1 Overexpression Promotes Mitotic Segregation Errors and Chromosomal Instability in Multiple Myeloma

Fujibayashi

Isa

Nishiyama

et al. 2020

Cancers

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Chromosome instability (CIN), the hallmarks of cancer, reflects ongoing chromosomal changes caused by chromosome segregation errors and results in whole chromosomal or segmental aneuploidy. In multiple myeloma (MM), CIN contributes to the acquisition of tumor heterogeneity, and thereby, to disease progression, drug resistance, and eventual treatment failure; however, the underlying mechanism of CIN in MM remains unclear. Faithful chromosomal segregation is tightly regulated by a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1). In this study, we found that BUB1 was overexpressed in patient-derived myeloma cells, and BUB1 expression was significantly higher in patients in an advanced stage compared to those in an early stage. This suggested the involvement of aberrant BUB1 overexpression in disease progression. In human myeloma-derived cell lines (HMCLs), BUB1 knockdown reduced the frequency of chromosome segregation errors in mitotic cells. In line with this, partial knockdown of BUB1 showed reduced variations in chromosome number compared to parent cells in HMCLs. Finally, BUB1 overexpression was found to promote the clonogenic potency of HMCLs. Collectively, these results suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers and, thus, was involved in CIN in MM.

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

Cited by 4 publications

References 35 publications

Short 57 kb CDKN2A FISH probe effectively detects short homozygous deletion of the 9p21 locus in malignant pleural mesothelioma

Short 57 kb CDKN2A FISH probe effectively detects short homozygous deletion of the 9p21 locus in malignant pleural mesothelioma

Imaging flow cytometry-based multiplex FISH for three IGH translocations in multiple myeloma

Aberrant BUB1 Overexpression Promotes Mitotic Segregation Errors and Chromosomal Instability in Multiple Myeloma

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