With the gradual development of research on AML, the heterogeneity in the disease is becoming more common, and cytogenetics is increasingly recognized as an independent predictor of prognosis in AML patients. However, though many prognostic factors have been identified, there is more than 50% of patients do not have cytogenetic marker, which are cytogenetically normal AML patients.[9, 10] The good news is many gene abnormalities that escape cytogenetic detection have been discovered, like CEBPA, NPM1, KIT, having great significance in predicting prognosis.[14, 15] In addition, the role of gene methylation in prognosis has also been discovered, like high GADD45A methylation predicting low survival of AML patients.[21] Therefore, new gene abnormalities are still required for further discovery, in order to provide clues for predicting prognosis, studying the pathogenesis and exploring new therapeutic targets. In this study, we integrated survival of 75 TCGA samples with CN-AML and 240 GEO CN- AML samples, examined the effect of RASGEF1A expression on CN-AML patients’ prognosis, including patients received chemotherapy or Allo-HSCT, and the influence of RASGEF1A methylation levels on CN-AML samples’ prognosis.
In this study, we found that RASGEF1A can be used as a CN-AML patients’ prognostic factor. We compared the OS and EFS of 75 TCGA samples with CN-AML and 240 CN-AML patients from GEO database with or without high RASGEF1A expression, including CN-AML patients’ overall prognosis and respective prognosis of AML patients received chemotherapy or Allo-HSCT. The study showed that the prognosis of patients in RASGEF1A-high expression group of RASGEF1A was poorer than the RASGEF1A-low expression group (P < 0.0001, Fig. 1and Fig. 2), indicating that the RASGEF1A gene expression level is a strong adverse factor for CN-AML samples’ prognosis. We can examined the 75 CN-AML samples’ baseline characteristics in Table 1. Two group had same level in gender, age, race, FAB type of AML, induction program, status before transplant, and other gene mutations, showing that the results of this study group are not affected by other factors and have high credibility. Furthermore, The BeatAML CN-AML patients with RASGEF1A-high have better sensitivity to Crenolanib and in multivariate analysis, we can see that the effect of RASGEF1A expression on prognosis is strong and meaningful.
In addition, we studied the association between the methylation of RASGEF1A gene and patients survival rates. Just as the high methylation level of GADD45A can indicate adverse prognosis, we discovered the high methylation of RASGEF1A also suggests unfavorable prognosis in CN-AML patients.[21] As can be seen from Fig. 3A, the EFS and OS of patients are much better in RASGEF1A-high methylation group than RASGEF1A-low methylation group (P < 0.0001, Figure. 3A). Not only that, but we also found the same results in patients receiving chemotherapy or Allo-HSCT, high level of methylation suggests favorable prognosis of AML patients receiving chemotherapy (P < 0.0001, Figure. 3B) or Allo-HSCT (P < 0.0001, Figure. 3C). Therefore, we can say that the level of RASGEF1A methylation is a new prognostic factor of AML patients.
Furthermore, we found that the integrative analysis of gene expression and methylation can provide a more accurate method of prognostic classification. In Fig. 4, we can see that CN-AML samples can be separated into 4 groups by RNA level and methylation level, and the difference in EFS and OS among the 4 groups were statistically significant (P < 0.0001, Figure. 4). In the left side of Figure.4, it is easily to find that RASGEF1A-low expression RASGEF1A-high methylation group (G3) had the best EFS and RASGEF1A-high expression RASGEF1A-low methylation group (G2) had the worst EFS among 4 groups, consistent with previous conclusions. Due to the small overall sample size, there is only one patient in G1 group in the OS (Figure. 4, right side), we have to say individual factors may have a great impact on the outcome. Despite this, we can find that the G3 group had the best OS and G2 had the worst OS among other three groups similarly. It has been found that methylation can affect survival not only by regulating gene expression, but also through some other mechanisms.[34] This shows that the two may reflect different processes in the pathogenesis, while the integrative analysis may be more comprehensive. This method has been applied in other diseases and many new discoveries have been harvested. The new strategies to overcome tamoxifen resistance may be discovered by analyzing the DNA expression and methylation features of cancer stem cells.[35] By investigating the DNA imbalance and methylation profiles of myeloma cells, it was found that the genomic heterogeneity always present from diagnosis to relapse.[36] The integrative analysis in soft tissue sarcomas, helped to find new biomarkers associated with pathogenesis.[37] The integrative analysis of gene expression level and methylation level of RASGEF1A allows CN-AML patients to be more accurately graded, making the prognosis prediction more accurate and even providing a more accurate direction for treatment. The CN-AML patients with RASGEF1A-high expression level have better sensitivity to Crenolanib.
RASGEF1A is an important guanine nucleotide exchange factors of Ras. Previous studies have exposed that RASGEF1A high expression is related to the survival and migration of intrahepatic cholangiocarcinoma cells, and our study has shown a correlation with the prognosis of patients with AML.[29] This may suggests that this gene further contribute to the pathogenesis of AML and is associated with some malignant diseases. At the same time, this gene was detected in the bone marrow and other 26 detected tissues, especially in brain, lymph nodes, spleen and testis.[25–30] We can guess that there are also potential correlations with RASGEF1A gene and diseases in those tissues, which requires further studies.
What is regrettable is that this experiment does not involve molecular mechanisms. To date, no prospective studies have proved the clinical significance of RASGEF1A in the treatment of CN-AML.
In conclusion, the high RASGEF1A expression level and low methylation level suggest poor survival and adverse prognosis for CN-AML patients. The integrative analysis of RNA and methylation level can provide a more accurate classification for prognosis. The CN-AML patients with RASGEF1A high expression level have better sensitivity to Crenolanib. Low RASGEF1A expression is a favorable prognostic factor for AML patients receiving chemotherapy or Allo-HSCT.