Detection of Loss of A20 Gene by FICTION in Hodgkin's Lymphoma Cases.

A20 (also called TNFAIP3) gene encodes an ubiquitin-modifying enzyme involved in termination of NF-kB responses, and that is negative regulator of NF-kB. We previously reported that A20 is a common genetic target in B-cell lymphomas by using a genome–wide analysis of genetic lesions in 238 B-cell lymphomas. [Kato et al., Nature 459; 712, 2009] In the study, we showed that three Hodgkin's lymphoma (HL)-derived cell lines have deletion of both alleles or loss of one allele and a mutated allele of A20 gene by high-density typing using CNAG/AsCNAR algorism, which enabled accurate determination of allele-specific copy numbers, and thus allowed for sensitive detection of loss of heterozygosity (LOH) without apparent copy-number reduction, even in the presence of up to 70–80% normal cell contamination. Twenty-four primary samples from HL were also analyzed for the mutations and microdissected CD30-positive tumor cells (Hodgkin-Reed-Sternberg (HRS) cells) revealed one intronic and four missense mutations. However, we might have missed the homozygous deletions, which are shown in other B-cell lymphoma subtypes, and underestimates the frequency of involvement of A20 gene in primary HL cases.

The objects were 12 of the 24 samples of our previous cohort. The samples included one case that showed mutation in the previous study. We tried to detect A20 gene deletion in HRS cells with the combination of anti-CD30 immunofluorescence with FISH (fluorescence immunophenotyping and interphase cytogenetics as a tool for the investigation of neoplasm: FICTION) method. BAC clones suitable for FISH analysis of A20 gene were screened. A BAC clone RP11-783B20 (TNFAIP3 locus, 6q23) labeled with spectrum orange by nick translation, was selected because of the best signal/noise ratio. CEP 6 Spectrum Green Probe was used to detect centromere of chromosome 6 (6p11.1-q11). Approximately 4 micrometer thick slides were immunostained with anti-CD30 antibody and FISH was performed. As the diameter of HRS cells is known to be several times more than the 4 micrometer thickness of the section slides suitable for FISH, ratio of A20/CEP 6 signals were calculated and the A20 gene status was evaluated in CD30 positive cells. A20 and CEP6 signals were also counted in CD30 negative cells and used as a control. We counted signals of 30(15–55) CD30 positive cells and 50 CD30 negative cells.

In 9 cases among 12 cases, signals were successfully counted. The average number of CEP6 signals on the CD30 negative cells was 1.53, which showed that a whole cell is located on the section slides. The ratio of A20/CEP6 signals on these CD30 negative cells were approximately 1.0 confirming the quality of FISH signals. As predicted, the average number of the CEP6 signals on the HRS cell was approximately 1.21, which suggested that one HRS cell is cut in the middle and divided into pieces during preparation of the section slides. In these CD30 positive cells, the A20/CEP6 ratio was less than 1.0; the cells contained less A20 gene signals than CEP6 signals. The relative numbers of signals determined by FICTION in 9 cases are shown in the table. We knew that the case #3 had A 20 gene mutation determined by direct sequence analysis of micro-dissected CD30 positive cells. As the sequence analysis showed no residual wild A20 genes, the relative ratio of 0.71 was considered as a consequence of loss of normal allele. Thus, the cut-off level of 0.71 was set to define deletion of at least one allele. Two cases showed that the A20/CEP6 signal ratio was more than this figure (0.90 in case #1, and 0.80 in case #4). Except for these 2 cases, 7 out of 9 cases were judged to have deletions of A20 gene. Notably case #6 had the ratio of 0.14, which suggested that virtually complete loss of A20 gene had occurred. The sequence analysis had missed the deletions probably due to the contaminated surrounding cells.

We found a frequent deletion of A20 gene in HL cases including a case with a complete loss. The involvement A20 gene deletion associated with the pathogenesis of HL might be more frequent than previously suggested by the sequence analysis.

No relevant conflicts of interest to declare.