Low frequency of FAS mutations in Reed-Sternberg cells of Hodgkin's lymphoma

Abstract

Reed-Sternberg (RS) cells, the neoplastic elements of Hodgkin's lymphoma (HL), usually lack B-cell receptor expression. Normal germinal center B cells, with lack of or low-affinity B-cell receptor expression, are eliminated via FAS-induced apoptosis. RS cells express FAS, but are rescued from apoptosis by a transforming event. It is known that HL-derived cell lines are resistant to FAS-mediated apoptosis. To investigate potential causes for this resistance, FAS mutations and c-FLIP expression were studied in four HL-derived cell lines and 20 cases of HL. L1236 was found to have a splice donor site mutation in intron 7 that resulted in an aberrantly spliced FAS transcript. Screening of microdissected RS cells revealed loss of heterozygosity for a known exon 7 polymorphism in two of six informative cases indicating loss of one FAS allele. In one of the two cases with loss of heterozygosity a hemizygous mutation was detected in exon 9. c-FLIP expression was observed in all HL cell lines and in RS cells of all HL cases. Our data show that FAS mutations are rare and suggest that overexpression of c-FLIP, which was present in all cases, is involved in the resistance to FAS-mediated apoptosis.

Figure 1.

DGGE analyses of the HL samples. A: Exon 7 analysis for cases 4, 23, and 34. For cases 4 and 23 LOH can be observed on comparison of reactive and RS cells microdissected from HL-involved tissue. The duplicate analysis of case 4 was analyzed in a separate DGGE analysis (result not shown). For case 34 an aberrant banding pattern was present in RS cells in one of a duplicate experiment. B: Exon 9 analysis for cases 4 and 18. For case 18 an aberrant banding pattern was present in RS cells in one of a triplicate experiment. For case 4 an aberrant homoduplex band was detected without presence of the wild-type homoduplex band. Shown is the inverted image of an ethidium bromide-stained DGGE gel.

Figure 2.

DGGE analyses of the HL-derived cell line L1236. A: DGGE analysis of exon 7. A heterozygous banding pattern representing the known exon 7 polymorphism was detected for L591 and DEV, whereas a homozygous pattern was observed for L428. An aberrant four-banding pattern was observed for L1236. B: Reamplification and digestion with PstI revealed presence of an undigested band (255 bp) representing the normal allele and two smaller fragments (140 and 115 bp) representing the allele carrying the mutation for L1236. C: DGGE analysis of the exon 6 to 8 RT-PCR product showing an aberrant four-banded pattern for L1236 and a normal band for L428. Shown is the inverted image of ethidium bromide-stained agarose or DGGE gels.

Figure 3.

c-FLIP analyses in HL-derived cell lines and cases. A: RT-PCR for c-FLIP long (30 cycles) and short (35 cycles) splice forms. GAPDH housekeeping gene (18 cycles) was used as a control for RNA loading and quality. B: c-FLIP immunohistochemical staining of L428. A cytoplasmic staining can be observed in all cells. C: c-FLIP immunohistochemistry of a MCHL case. A cytoplasmic staining is present in the RS cells. Original magnifications, ×630 (B and C).