| Objective:To study the possible relationship between the methylation status of Wnt antagonist genes, ID4 gene and clinical variables, and to determine their prognostic value in patients with myelodysplatic syndrome (MDS).Methods:The consecutive samples of 435 patients with de novo MDS in Sino-US Shanghai Leukemia Cooperative Group were collected by prospective methods between June 2003 and April 2007. The diagnosis was made according to the WHO (2001) criteria. We employed methylation-specific PCR (MSP) to examine the methylation status of Wnt antagonist genes and ID4 in 144 adult de novo MDS patients. Prognosis analysis also including:1) prognosis analysis on 285 patients with RCMD; 2) prognostic value of cytogenetic evolution in MDS patients; 3) prongnostic value of mean corpuscular volume (MCV) in MDS patients. All patients were followed until death or until the last follow-up data (April 30,3008). The median follow-up time was 25.1(5.5-53.2)months. Overall survival (OS) Leukemia-free survival (LFS) and time to progression (TTP) were predictive of prognosis. Distributions of OS or LFS or TTP curves were estimated according to the Kaplan-Meier method. Comparisons of OS or LFS or TTP between groups were evaluated by the log-rank test. The Cox regression model was used for multivariate survival analysis in order to identify the significant independent prognostic factors affecting OS or LFS. For all analyses.Results 1. Methylation of the gene promoters was observed in all six Wnt antagonist genes in 144 MDS patients. The methylation frequencies were as follows:41% for sFRP1,89.6% for sFRP2,43.1% for sFRP4,50.7% for sFRP5,44.4% for DKK-1, and 69.4% for DKK-3. A total of 104 (72.2%) patients had methylation of at least one of the six Wnt antagonists. For sFRP1, sFRP5 and DKK-1, aberrant methylation was more frequent in advanced stages of WHO subtypes (P<0.001, P<0.001, P= 0.007, respectively); In addition, the frequency of sFRP1 and sFRP5 methylation was significantly correlated with the IPSS risk score (P=0.038, P=0.011). Evaluation of the prognostic impact of epigenetic aberrations showed that sFRP1, sFRP4, and sFRP5 methylation had a significant impact on OS (P=0.001, P=0.009, P=0.015, respectively;). The median survival of patients with sFRP1, sFRP4, and sFRP5 methylation was 14.1 months,18.5 months, and 13.2 months, respectively, compared to 31.8 months,31.7 months, and 31.8 months for patients without methylation. Patients with sFRP5 methylation had a higher risk of leukemia evolution than those without sFRP5 methylation (P=0.018). Because the methylation status of sFRP1, sFRP4, and sFRP5 was predictive of prognosis, we further classified patients into four groups according to the number of these three genes that were involved in aberrant methylation:patients with none of these three genes methylaiton were included in the very low risk group, patients with only one of these three genes were included in the low risk group, patients with concurrent two of these of three genes were included in the intermidate risk group, and patients all these three genes methylaiton were included in the high risk group. Significantly different survival was observed among these four risk groups (P=0.002). Similarly, the classification based on the number of genes was also a useful predictor for leukemia evolution in patients with MDS (P =0.048).2. Of the 144 MDS patients evaluated,51 patients (35.4%) showed ID4 methylation. Patients with advanced stages of WHO subtypes (RAEB-1 and RAEB-2) exhibited a significantly higher frequency of ID4 methylation (38/83) compared with those with early stage subtypes (RA, RARS, RCMD, RCMD-RS,5q- syndrome, and MDS-U; 13/61, P=0.002). In addition, a significant correlation was also found between ID4 methylation and IPSS risk groups:patients with higher risk IPSS subgroups (Intermediated-2 and High risk) exhibited a significantly higher frequency of ID4 methylation than those with lower risk IPSS subgroups (Low and Intermediate-1 risk) (45.7% vs 26.0%, P=0.014). Also, there was a strong correlation between ID4 methylation and marrow blast levels (P=0.007). The median OS was 21.6 months for the all 144 patients:the difference in median survival time between the methylated ID4 group and unmethylated group was statistically significant (P=0.005) with median survival times of 12.2 months and 26.9 months, respectively. Furthermore, patients with ID4 methylation had higher frequency of leukemia evolution than those without ID4 methylation (P=0.008). Multivariate analysis showed that the status of ID4 methylation was an independent factor affecting LFS:MDS patients with ID4 methylation had higher risk of leukemic transformation (P=0.047, HR=2.11).3.1) Among the 285 patients with RCMD,252 patients had a marrow blast level of less than 3.5% and 33 patients had a level of 3.5% or more. A significant difference in survival times was observed between the two groups (P=0.001). The former group had longer survival, with the median survival time of 40.8 months compared to 23.7 months for the latter group. In addition, application of a marrow blast level cutoff of 3.5% in patients with RCMD could identify patients with a lower IPSS risk but with a potentially worse prognosis; 2) In 85 patients with complete cytogenetic data at initial diagnosis and from at least one time of later follow-up,18 patients presented with cytogenetic evolution. Choromosomes 8,5 and 1 were more commonly involved in cytogenetic evolution. Survival analysis showed that the median survival of patients in the group with cytogenetic evolution was 25.8 months, compared to 45.4 months for patients in the group without cytogenetic evolution (P=0.01). The same result was also found for TTP (time to progression):patients with cytogenetic evolution progressed more rapidly than those without cytogenetic evolution (P=0.007); 3) Multivariate analysis revealed that MCV of less than 100 fl (P=0.026, HR=1.75) were independent risk factors that affected the survival of patients with abnormal karyotypes.Conclusion:The methylation status of sFRP1,4,5 and ID4 was a useful prognostic marker and predicts poor prognosis in patients with MDS:methylation status of sFRP1, sFRP4, and sFRP5 was associated with poor OS in MDS (P=0.001, P= 0.009, and P=0.015, respectively); sFRP5 methylation also predicted a high risk of leukemia evolution (P=0.018). Using the number of these three genes present in a patient, we could classify patients into four risk groups with significantly different prognoses. In addition, several clinical factors were proved to be of prognostic significance in MDS patients:higher marrow blasts predicts prognosis in patients with RCMD; MCV was proved to be the independent prognostic factor for MDS patients with abnormal karyotypes; cytogenetic evolution correlates with poor poor prognosis in MDS patients.
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