H-RAS 1 minisatellite rare alleles: a genetic susceptibility and prognostic factor for non-Hodgkin's lymphoma.

Abstract

The dramatic rise in non-Hodgkin’s lymphoma (NHL) incidence and mortality throughout the world (1,2) has prompted investigators to search for biomarkers that might indicate an increased predisposition for NHL development. Moreover, although combination chemotherapy has improved the outcome of patients with intermediateand highgrade NHL (3,4), a substantial fraction of these patients still achieve only a partial remission and/or will ultimately relapse. If such patients could be identified at diagnosis by means of new prognostic markers, they might benefit from strategies other than conventional chemotherapy. The H-RAS 1 proto-oncogene has an associated hypervariable minisatellite that is believed to be capable of influencing H-RAS 1 gene transcription (5,6). Rare alleles of this minisatellite, H-RAS 1 VNTR (variable number of tandem repeats), have been associated with a predisposition to acute leukemia (7) and carcinomas of the lung (8–10), testis (11), breast (7,12), colorectum (7), and urinary bladder (7); however, associations with clinical data are not available in any of these studies. This hypervariable minisatellite, which maps 1 kilobase downstream from the polyadenylation signal of the human H-RAS 1 proto-oncogene, originated through the tandem replication of a 28-base-pair (bp) consensus motif (13). Population genetic analysis has defined this as a complex locus, in which several dozen rare H-RAS 1 VNTR alleles have arisen from mutation of four common alleles (a1, a2, a3, and a4). The rare alleles seem to differ from the common ones not only in the number of 28bp repeat motifs that comprise them (14,15) but also in the interspersion pattern of the five different types of 28-bp repeat motifs described thus far in the VNTR sequence (16). The distribution of allelic frequencies in matched populations of cancer patients and cancerfree control subjects has indicated that this process of allele mutation may not be merely fortuitous. Rare H-RAS 1 alleles appear, in aggregate, twice as often in the genomes of cancer patients (17– 19), an observation that now rests on a sample size of approximately 8500 alleles from 23 independent studies (7). This association has suggested the possibility that H-RAS 1 VNTR may interact with cellular elements that control gene expression. In fact, it has already been demonstrated that at least four members of the rel/NF-kB family of transcription factors bind this H-RAS 1 minisatellite and that, like other NF-kB binding sites, H-RAS 1 VNTR displays promoterand cell-type-specific transcriptional regulatory activity (5). It is hypothesized that, by binding rel/NF-kB factors (6), the H-RAS 1 minisatellite is capable of activating and repressing HRAS 1 gene transcription. Moreover, the fact that variations in the interspersion pattern of the 28-bp repeat motifs in rare alleles were observed to be different from those in common alleles (16) prompted us to hypothesize that this might be the biologic basis for a different behavior of the lymphomas in patients bearing rare alleles. To our knowledge, neither a comprehensive study on genetic predisposition to NHL in relation to H-RAS 1 VNTR genotypes nor clinical data on the relationship between rare H-RAS 1 VNTR alleles and the prognosis of patients has ever been presented. We were interested, therefore, to determine whether, in a cohort of 125 well-defined cases of NHL with adequate clinical follow-up, we could establish rare H-RAS 1 VNTR alleles as both a new inherited genetic marker of NHL susceptibility and a new independent prognostic factor for this disease. As inherited genetic factors, H-RAS 1 VNTR alleles can easily be analyzed in DNA from peripheral blood lymphocytes by means of a polymerase chain reaction (PCR)-based assay, thus eliminating the need for tumor tissue samples. Although most previous studies (7,8,10,11) of this locus were based on Southern blot analysis, we employed the PCR assay and long-gel electrophoretic procedures, which are easier to perform and offer much higher resolution. Rare alleles were differentiated from common ones (a1, a2, a3, and a4) by shifts in electrophoretic mobility (Fig. 1). We obtained peripheral blood samples from 125 patients with NHL and 446 cancer-free control subjects at the Hematology Service of the Hospital Universitari Germans Trias i Pujol of Badalona (Barcelona, Spain). Sequential, unrelated blood donors, as well as other healthy volunteers, were recruited as the controls. Although this was an unmatched case–control study, there were no statistically significant differences between the control subjects and the patients with respect to age, sex, or ethnic background. All study subjects were Spanish Caucasian residents of the surrounding area of Badalona and had no prior history of cancer. Human immunodeficiency virus (HIV)-positive patients were excluded from the study. However, in our sample of 125 patients, little is known either about the role of potential oncogenic viruses other than HIV or about exposure to carcinogens such as 1,3-butadiene, which is used in synthetic rubbers and which has been detected in industrial exhaust and cigarette smoke.