Restriction fragment length polymorphism of the L-myc gene is not a prognostic factor in bladder cancer patients

The L-myc restriction fragment length polymorphism has been suggested to be of prognostic significance in some types of primary tumours. We examined the prognostic and susceptibility significance of the L-myc genotype in a group of 98 bladder cancer patients. The L-myc genotype did not correlate with any pathologic parameter and does not offer any clinical utility in patients with bladder cancer. © 1999 Cancer Research Campaign


MATERIALS AND METHODS
Ninety-eight patients (88 males, 10 females, aged 38-91), diagnosed at Fundació Puigvert as having primary bladder cancer, were studied for L-myc restriction fragment length polymorphism (RFL); P 53 were superficial bladder tumours and 45 were invasive tumours. In addition, 110 normal DNA specimens from healthy blood donors (66 males, 44 females, aged 2-81) were analysed to investigate the genotype frequency of L-myc proto-oncogene.
High molecular weight DNA was isolated by standard phenol-chloroform methods. Restriction digests of DNA were performed with endonuclease Eco RI under standard conditions, electrophoresed in a 0.8% agarose gel, denaturated, transferred to Hybond® nylon membrane (Amersham) and hybridized with a 32 P-labelled probe by the method essentially described by Southern (1975).
All results obtained in these studies were analysed for statistical significance by the χ 2 test with Yates' correction for adjustment when necessary. Differences between the two populations were judged significant at P < 0.05. Kaplan-Meier curves were constructed to analyse patient survival between different genotypes (log-rank test).

RESULTS
Eco RI-digested DNA probed with the L-myc probe results in two fragments of 10 kb (L) and 6.6 kb (S), which are due to an Eco RI restriction site polymorphism ( Figure 1). The distribution of the three genotypes (LL, LS, SS) in the control and patient groups is shown in Table 1. Chi-squared analysis showed that there was no difference between the distribution of the three genotypes of patient group and controls, and all are in accordance with Hardy-Weinberg equilibrium.
When the patients in the current study were examined for a relationship between the presence of S-alleles (LS or SS individuals) and distant metastasis, no association was found (P = 0.51). Nor was there a significant association between homozygosity for the

Short communication
Restriction fragment length polymorphism of the L-myc gene is not a prognostic factor in bladder cancer patients S-allele and nodal metastasis (P = 0.46). Further analysis revealed no significant associations with grade of differentiation of tumours, TNM stages, number of previous and subsequent relapses, and urethral and prostate involvement (Table 2). No association was observed between L-myc RFLP and survival of bladder cancer patients ( Figure 2).

DISCUSSION
An Eco RI polymorphism of the L-myc proto-oncogene was reported with the initial description of the gene (Nau et al, 1985) and the site of the polymorphism subsequently located in the second intron (Kaye et al, 1988). Studies of this polymorphism have linked it to both cancer susceptibility and prognosis. The role of L-myc in cancer susceptibility has been implied by studies showing differences in genotype frequencies between cancer patients and controls Dolcetti et al, 1991;Crossen et al, 1994). The role of L-myc in prognosis has been suggested by studies showing that, among cancer patients, those that carry an Sallele (either LS or SS genotype) have earlier lymph node involvement or metastasis, or poorer survival than those who have genotype LL (Kakehi and Yoshida, 1989;Champeme et al, 1992;Kawashima et al, 1992). In contrast, Taylor et al (1993) reported a protective effect for the SS genotype in hepatocellular carcinoma. Hence, the association of the L-myc genotype with susceptibility and prognosis appears to vary with tumour type.  In this study, we examined the relationship between the L-myc RFLP pattern and the clinical features of bladder cancer in 98 cases. A preponderance of the LL, LS or SS fragments was not observed in the bladder cancer patients compared to normal healthy individuals; thus, presence of a particular allele did not indicate predisposition to bladder cancer. Similar L-myc allelic frequencies were also found in previously reported analyses.
Our results fail to support the hypothesis that the L-myc locus is involved in a genetic predisposition to bladder cancer. These results confirm those of Ikeda et al in colorectal cancer (1988), Tefre et al in lung cancer in Norway (1990), Ishizaki et al (1990) and Champeme et al (1992) in breast cancer, Saranath et al (1990) in oral cancer, Weston et al (1992) in lung cancer in the USA, Mironov et al (1994) in gastric cancer in Russia and Presti et al (1996) in renal cancer. On the other hand L-myc RFLP is correlated with the extent of metastasis of lung cancer in Japan (Kawashima et al, 1988(Kawashima et al, , 1992 and in renal cancer (Kakehi and Yoshida, 1989); Ishizaki et al (1990) also reported that the presence of the S-allele is associated with poor prognosis due to metastasic lesion in gastric cancer. Kato et al (1990) proposed that Japanese men with the S-allele may be prone to development of osteosarcoma and Saranath et al (1990) observed that patients with oral cancer with a genotype including an S fragment are more likely to develop a poorly to moderately differentiated tumour, or a larger tumour, than patients without an S fragment.
Our results show that the L-myc genotype does not correlate with tumour grade or stage in patients with bladder carcinoma. No significant correlation was observed between the L-myc genotype and the presence of nodal metastases or distant metastases at the time of surgery or with subsequent disease relapse.
This study did not demonstrate an association between the L-myc genotype and disease status in bladder cancer. It seems that L-myc polymorphism is not suitable as a prognostic and susceptibility marker in the bladder cancer patients studied.