Comparison of MLL Fusion Genes Expression among the Cytogenetics Abnormalities of Acute Myeloid Leukemia and Their Clinical Effects

Mixed-lineage leukemia (MLL) is a subtype of acute myeloid leukemia with more aggressive prognosis than other subtypes. Translocations of MLL gene with other partner genes, forming the MLL-fusion proteins (MLL-FPs), are the main characteristics of MLL leukemia. Many studies have demonstrated that MLL-FPs such as: MLL-AF4, MLL-AF6, MLL-AF9, MLL-AF10, MLL-ENL, MLL-ELL, MLL-EPS15, as well as partial tandem duplication are the most common abnormalities that play significant role in MLL-rearranged leukemia. Gene expression profiles from 197 patients and 180 clinical data were downloaded from TCGA database. R statistical program has classified clinical and genomic data simultaneously according to cytogenetic abnormalities. As a result of this analysis, the most frequent 47 MLLFPs genes expression have been detected and compared with other cytogenetic abnormalities such as t(4;11), t(9;11), t(8;21), t(15;17), complex, inversion 16, trisomy 8 and cytogenetically normal AML. 35 out of 46 MLL-FPs genes presented with abnormal gene expression profile. This study showed that MLL-FPs are not just active and related with MLL, but also with other subtypes of AML.


Introduction
Acute myeloid leukemia (AML) is a clonal hematopoietic disorder that may be derived from either a hematopoietic stem cell or a lineagespecific progenitor cell [1]. Hematopoietic tissues have a potential to produce various types of malignancies, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL) [2]. Acute leukemia is one of the most common types of leukemia among adults and constitutes 97% of all childhood malignancies, which show clonal expansion and changing specific stages of normal myeloid and lymphoid hematopoiesis [3]. Although cytogenetic analysis has been used to identify the pathogenesis of acute myeloid leukemia (AML) for more than decades, clinically defined subtypes are very heterogeneous diseases and difficult to characterize in a same group [4,5]. The cytogenetic of the subtypes and chromosome translocation are the main key properties to distinguish the disease and prognostic factors of AML [1,6,7]. The AML and ALL cytogenetic reports have revealed that many non-random chromosome abnormalities included specific genes that implicated in the process of leukemogenesis [8].
Lastly, the new term mixed-lineage leukemia was added to the literature [9]. MLL gene plays a positive regulator of global gene expression in early embryonic development and hematopoiesis [10]. The gene encodes a very important epigenetic transcription factors, such as HOX genes [11]. In addition to that, MLL fusion proteins, main properties of MLL, are produced by chromosomal translocations, which affect the MLL gene at 11q23 [12]. If AML includes MLL chromosomal rearrangements and produces fusion proteins, it is assumed to be followed by poor prognosis and aggressive for infants [13][14][15]. The fusion proteins are observed in both hematological and solid tumors as breaking within genes on each chromosome [7]. The translocation genes involved in AML might be transcription regulators, which determine the cellular development and cell fate [16]. There are more than 80 different partner genes, but the most commonly observed MLL-FPs are MLL-AF4 in ALL, MLL-AF9 and MLL-AF10 in AML and MLL-ELL in MLL [17].
Although it is well known that the fusion proteins activity are specific reasons for The previous studies show that statistical analysis of genomic data and clinical research should have been done together to understand better of new type of leukemia [18][19][20][21]. Development of MLL, the research has been done to show the potential correlation between the proteins and other subtypes. Finding the expression activity of the fusion genes is the main target of the research. The main idea of the research is to see the genes expression profile of MLL-FPs in other subtypes of AML and potential relation between them.

Materials and Methods
The cancer genome atlas, R, HCE 3.

5, Genevestigator
Gene expression and clinical data of acute myeloid leukemia are the main material for the research, which are downloaded from TCGA database, https://tcga-data.nci.nih.gov/tcga/. The data have been mined by different computational programs and web tools ( Figure 1). The statistical analyses and data comparison have been done by R statistical program (https://www.r-project.org), which is created for this particular research to analyze the data. The program categorizes genes which represent different expression profiles in the subtype of AML. HCE 3.5 (http://www.cs.umd.edu/hcil/hce/) has been used to cluster gene expressions. According to the clinical data, the different subtypes have been found and abnormally expressed MLL fusion genes were detected. Synchronization of the clinical and expression data is the key part of the method.

Clinical data
AML clinical data have been used to find subtypes which are made of distinct cytogenetic abnormalities. Patients have different chromosomal abnormalities which are detected, identified and separated into different types: t(4;11), t(9;11), t(8;21), t(15;17), complex, inversion 16, trisomy 8 and AML. TCGA ID numbers of each patient is very useful for matching with their gene expression value. Therefore, same IDs have been used to find the correct gene expression value in the subtype of AML. The clinical data were prepared to find their gene expression values.

Gene expression data according to AML subtypes
197 AML patients' expression values and 19798 genes were compared within each subtype. According to cytogenetic abnormalities which were derived from the clinical data, patients have been separated and categorized into subgroups of AML ( Figure 1).
The IDs of each subgroup member is used to find their gene expression value. Then the abnormally high and low expressed genes of the subtypes have been compared among each other and average value (  Although there are more than 80 MLL fusion encoded genes [15], the 46 most frequently found have been analyzed. The R program categorized and selected the genes depending on their expression abnormalities of AML cytogenetic disorders.

Clinical outcome of the subtypes
The patients of the subtypes show different survival rate (Figure 3). While t (15,17) and inv16 have the highest survival rate, t(9:11) and t(4:11) have the lowest survival rate. It is obvious that patients with the subtypes involving MLL translocation on 11q23, have the worst prognosis.

Gene expression result among subtypes
The comparison of 46 mostly common MLL-FPs gene expression shows different profile and abnormal deviations (  Table 2: MLL fusion genes expression in different subtypes of AML.
The black and gray cells represent the high expressed and low expressed values of the genes, respectively. The comparison is done between average of the gene expression and the subtypes.

MLL-FP genes and clinical outcome
According to the AML subtypes, clinical outcome shows high heterogeneity. While t (15,17) and inv16 have the highest survival rate and relatively good prognosis, t(9:11) is lethal and leads to very bad prognosis ( Figure 3).

Discussion
However, it is well known that the genes responsible for MLL-FPs are important key factor for MLL leukemogenesis, the research has been done to investigate the expression profile in other subtypes of AML. 46 most commonly found MLL-FPs genes have been compared among the subtypes that the gene expression profile is presented as high and low ( Table 2). Analysis of variations in gene expression in different cytogenetic abnormalities showed that significant number of genes is down-regulated in t (15;17). Furthermore, there are some genes which have opposite direction expressions. Therefore, MLL-FPs show diverse expression profiles in different subtypes of AML.
Despite of the fact that MLL is a new subtype of AML leukemia, there is no significant and efficient individual therapy so far. MLL is a result of crucial change in genetic conformation by the translocation and MLL protein complex that can lead to leukemogenesis. The analysis of MLL-FPs might be prospective target to find common properties and patterns between MLL type and other subtypes of AML. Moreover, we can find the correlation between abnormal expression of the genes and clinical outcome among all the subtypes.

Conclusion
MLL is very aggressive type and there is no strong therapy. The bad prognosis and clinical outcome may be correlated with the fusion proteins activity. That`s why the gene expression profile might be used to find therapeutic target genes and help us to see the relation between the subtypes of AML and MLL-FPs. The major contribution of the research is to find the reason of different clinical outcome and survival rate among the subtypes of AML and MLL-FP activity.