A Novel Hodgkin Lymphoma Cell Line (KHL) Established from An Untreated Patient.

The paucity of Hodgkin cell/Reed-Sternberg cell in classical Hodgkin Lymphoma (HL) represents a general problem for molecular and cytogenetic studies. The established HL cell lines could be used for such studies. However, only about 10 cell lines have been established and all of them were isolated from patients in late stage of illness when the tumor had recurred. Only one of these cell lines is known to be positive for Epstein- Barr virus antigen. In addition, the pattern of chromosomal aberration in these cell lines is highly complex. Therefore, it is necessary to have a cell line established from the early stage of disease, without prior treatment and with less chromosomal aberration for research. A sample of left axillary lymph node from a 27-year-old male with early stage of HL, and without prior chemotherapy, was cultured in RPMI 1640 media supplemented with fetal calf serum for routine cytogenetic study. The culture, passed weekly, now in its 60^st week, 57^th passes is growing autonomously without supplement of any growth factor. The original lymph node biopsy showed a classical Hodgkin lymphoma, mixed cellularity (WHO Classification) and the Hodgkin cells/Reed-Sternberg cells were positive for CD30, 4+(100%), CD20, 2+(33%) and negative for CD3, CD15, CD43, ALK-1 antigen and epithelial membrane antigen. EBV nuclear antigen 1 DNA and RNA are detected by PCR method. The cell line is positive for CD30, CD20, and Epstein-Barr virus (EBV) latent membrane protein, but negative for CD3, CD79a, and EBV early antigen. By florescent insitu hybridization the cell line is negative for p53 deletion, ALK gene rearrangement, MLL gene deletion, and t(12;21). A ploidy analysis by flow cytometry shows 80.22% diploid, and 19.78% hyperploids. The cells have doubling time of 30 to 36 hours. The initial karyotypes were: 45~46, XY, i(3)(q10), der(6)t(3;6)(p11;q22), t(6;13) (p21;q32), del(7)(q32), i(14) (q10)[cp3]/46, XY, del(3)(p10), der(6)t(3;6), der(6)t(6;13), del(7)(q32), add(10)(p13), der(13)add(13)(p11.1)t(6;13), i(14)(q10)[3]/46, XY[16]. Metaphase preparations from the cell line showed 46, XY, absence of the above mentioned chromosomal abnormalities, but 97% (1422/1466 cells) of cells were diploid; 2.5% (36/1466 cells) of cells were tetraploid/near tetraploid, and 0.5% (8/1466 cells) of cells showed endore-duplication. Chromosomal comparative genomic hybridization of the cell line showed microdeletion on chromosome 5q34 and 13q22~31 region, and gain on 12q12.1. Single nucleotide polymorphism array showed no abnormalities. This newly established cell line is unique in:

1. it is the second cell line known to be positive for EBV antigen,

2. it shows no complex chromosomal aberration by conventional karyotyping or molecular genotyping, and

3. since the cell line showed mostly in diploid, and only 3% of the cells are tetraploid/near tetraploid and endoreduplication by conventional cytogenetic method, the cell line is ideal for the study of formation of hyperploid cells (i.e. Reed-Sternberg cells) from diploid cells.