Multiplex sequencing for EZH2, CD79B, and MYD88 mutations using archival cytospin preparations from B-cell non-Hodgkin lymphoma aspirates previously tested for MYC rearrangement and IGH/BCL2 translocation

作者：Santos Gilda da Cunha^*; Saieg Mauro Ajaj; Ko Hyang Mi; Geddie William R; Boerner Scott L; Craddock Kenneth J; Crump Michael; Bailey Denis

来源：Cancer Cytopathology, 2015, 123(7): 413-420.

DOI：10.1002/cncy.21535

• cytospin preparation
• B-cell non-Hodgkin lymphoma
• fine needle biopsy
• DNA mutational analysis
• EZH2
• CD79B
• MYD88
• MYC
• IGH
• BCL2
• MassARRAY spectrometry

BACKGROUNDGene rearrangements and specific translocations define some B-cell non-Hodgkin lymphoma (NHL) subtypes. Genome-wide mutational studies have revealed recurrent point mutations with prognostic implications. The goals of this study were to evaluate the feasibility of applying a multiplex mutation assay to archival cytospin preparations (CPs) and to investigate the rate of EZH2, CD79B, and MYD88 mutations in B-cell NHL samples previously tested for MYC rearrangement and/or IGH/BCL-2 translocation.
METHODSDNA was extracted from archival CPs of B-cell NHL cases with previous fluorescence in situ hybridization (FISH) assays for MYC rearrangement and/or IGH/BCL-2 translocation. Multiplex sequencing was performed for the detection of EZH2 (Y641), CD79B (Y196), and MYD88 (L265) mutations. Sanger sequencing was applied to samples with positive results and failed assays.
RESULTSEighty-eight archival CPs were available from 40 patients. Alterations detected by FISH were: MYC rearrangement (10 cases), IGH/BCL-2 translocations (21 cases), dual translocations (6 cases), and other abnormalities for IGH/BCL-2 (23 cases) and for MYC (16 cases). DNA concentration ranged from 1.88 to 62.85 ng/mu L (mean, 9.46 ng/mu L). Successful results were obtained in 88.0% of the specimens submitted to multiplex sequencing. With Sanger sequencing, 2 additional mutated cases were found, and all cases with mutations were confirmed. Eight specimens showed mutations: 6 for EZH2, 1 for CD79B, and 1 for MYD88. Among them, 5 cases showed concurrent MYC and/or IGH/BCL-2 translocations and 2 revealed abnormal signals of IGH/BCL-2 and MYC.
CONCLUSIONSCPs archived for up to 6 years are a reliable source of high-quality genomic material for multiplex sequencing. Almost all B-cell NHL with point mutations showed concurrent chromosomal abnormalities. Cancer (Cancer Cytopathol) 2015;123:413-20. (c) 2015 American Cancer Society. Cytospin preparations (CP) stored at -20 degrees C for up to 6 years are a reliable source of high-quality genomic material for multiplex sequencing. Successful detection of EZH2 (Y641), CD79B (Y196), and MYD88 (L265) mutations can be achieved using material scraped from archival CP obtained from B-cell non-Hodgkin lymphoma aspirates of cases previously tested for MYC rearrangement and IGH/BCL2 translocation.

• 出版日期2015-7