REARRANGEMENT OF THE BCL-2 GENE IN FOLLICULAR LYMPHOMA - DETECTION BYPCR IN BOTH FRESH AND FIXED TISSUE SAMPLES

Rearrangement of the BCL-2 gene is the molecular consequence of the t( 14;18) chromosomal translocation, which is found in approximately 60-9 0% of follicular lymphomas. To investigate the ability of the polymera se chain reaction (PCR) to detect this rearrangement in fixed-tissue s amples, we studied 48 cases of follicular lymphoma using DNA extracted from paired samples of fresh-frozen tissue and formalin-fixed, paraff in-embedded tissue. A standard phenol-chloroform DNA extraction method was used for both types of tissue. Rearrangements of the major breakp oint region (MBR) and minor cluster sequence (MCS) were examined. Thre e segments of the human beta-globin gene were also amplified to estima te the degree of DNA degradation in the fixed-tissue samples. PCR of f resh-tissue (intact) DNA revealed amplifiable products in 29 of the 48 follicular lymphomas (60%), whereas the fixed-tissue (degraded) DNA s tudies were positive in 24 (50%). MBR products were detected in 24 fre sh-tissue samples, and varied from 80 bp to > 1.5 kb. Twenty of these cases yielded MBR products in the corresponding fixed-tissue DNA, rang ing from 80 to 276 bp. Five fresh-tissue and four fixed-tissue samples produced MCS segments that ranged from 340 bp to 1.2 kb. Four of the five samples with no detectable MBR or MCS translocations using degrad ed DNA had products greater than 1.0 kb in the fresh-tissue studies. A 175-bp segment of the beta-globin gene was amplified in all 29 fixed- tissue samples; a 324 bp fragment was produced in 20 samples (69%), an d a 676 bp segment was detected in 13 (45%). These studies show that P CR performed with DNA extracted from formalin-fixed tissue can detect and accurately size approximately 80% of the BCL-2 gene rearrangements evident with intact high-molecular-weight DNA, and that the majority of falsely negative fixed-tissue samples have translocation products a bove 1.0 kb. Furthermore, by amplifying three successively larger segm ents of the human beta-globin gene, we have shown that the degradative effect of formalin fixation on DNA extracted from clinical samples is gradual and progressive, at least up to a size of 676 nucleotides.