The+Role+of+DNMT3A+in+Acute+Myeloid+Leukemia 

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__**Research Objective**__

In our cancer project, we will be focusing on the role of //DNMT3A// gene mutation in the progression of Acute Myeloid Leukemia. As these studies have been published recently in the past few months, we will be analyzing how this new data fits in with previous studies on the prevalence of cancer stem cells leading to development of AML cancer. Further research into the differentiation of pre-leukemic stem cells would improve treatment methods by targeting the root causes of AML.

=__Introduction __=

Adult Acute Myeloid Leukemia Adult Acute Myeloid Leukemia (AML) is a cancer of the blood and bone marrow and it is the most common type of acute leukemia in adults. Normally, the bone marrow makes immature, blood stem cells that become mature blood stem cells later over time. A blood stem cell, that is essentially a multipotent stem cell derived from a hemotopoietic stem cell (see discussion below), can become a myeloid stem cell or a lymphoid stem cell. The myeloid stem cell can give rise to one of three types of mature blood cells: red blood cells, white blood cells that fight infection and disease, and platelets that form blood clots to stop bleeding. The lymphoid stem cell becomes a white blood cell. In AML, myeloid stem cells usually become immature blood cells called myeloblasts (myeloid blasts). These myeloid blasts are abnormal and do not become healthy, normal white blood cells. If too many of these stem cells become abnormal red and white blood cells or platelets, then they are called leukemia cells or blasts. These leukemia cells can then spread to other parts of the body, including the central nervous system, skin and gums (NCI, 2013). Hematopoeitic Stem Cells Hematopoietic Stem Cells (HSCs) are stem cells that reside primarily in the bone marrow of adult mammals but that can also be found in the  spleen, in peripheral blood circulation, and other tissues (NIH, 2011).  They are important in the constant renewal of the blood, capable of giving rise to billions of new blood cells each day that include macrophages, erythrocytes (red blood cells), platelets, leukocytes (white blood cells), B and T cells, and Natural Killer (NK) cells. Scientists have defined the four actions of an HSC: (1) it can renew itself; (2) proliferate and differentiate to replenish all functional types of blood cells (3) it can mobilize out of the bone marrow into circulation (or the reverse) (4) it can undergo programmed cell death, or apoptosis

Studies have also revealed there to be two kinds of HSCs: (1) long-term stem cells that are capable of self-renewal, and (2) short-term progenitor or precursor cells that can proliferate but that cannot differentiate into more than one cell type (NIH, 2011). For example, a blood progenitor cell may only be able to make another red blood cell. DNMT3a DNMT3a is a DNA methyltransferase that catalyzes the addition of methyl groups at a cytosine base in a 5'-CG-3' dinucleotide pair (CpG) around promoter regions of our DNA. AML cancer cells exhibit areas of hypomethylation and hypermethylation of CpG islands near these promoters, that inactivate transcription and silence tumor-supressor genes. DNMT3a is just one of an entire family of DNMTs, including DNMT1, DNMT3B, which also establish and maintain DNA methlyation (Ley, et al. 2010).  **Overview** <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;"><span style="color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">Over the past few decades, scientists could not identify the single, unifying origin of stem cells that gave rise to the human AML disease, due to the immense heterogeneity of these cells. In other words, their capability to proliferate and differentiate into numerous specialized cells in tissues of the body was unexplained. They were aware that the two potential sources of cells from which AML could arise were either the primitive, multipotent HSCs or the mature committed myeloid precursor cells downstream of the HSCs. From studies done on mice and humans, they even knew that it was these primitive, small populations of self-renewing leukemic stem cells that gave rise to the large population of mature leukemic blasts which lacked self-renewal capacity. Until recently this year, however, researchers did not know that these ancestral leukemic stem cells could aggressively out compete non-leukemic HSCs, leading to clonal expansion, and that these leukemic stem cells were found in high proportion of AML patients that carry mutations in //DNMT3A,// among other genes that, unlike AML blasts, survive chemotherapy and persist in the bone marrow at remission, providing a reservoir of cells that can result in AML recurrence (Potter, Greaves, 2014).

<span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">__<span style="font-family: 'Times New Roman',Times,serif;">Research __

<span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">Pre-leukemic Ancestor Cells <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;"> According to a report published in Nature in February, John E. Dick, PhD, a researcher at the Princess Margaret Cancer Center in Toronto and the University of Toronto in Canada, and his colleagues sequenced 103 commonly mutated leukemia genes in peripheral blood samples from 83 patients at diagnosis (Slush, et al. 2014). They identified mutations in the DMNT3A gene in AML cells in roughly 25% of the samples (mutant allele frequency ~50%). The researchers also found another mutation in the NPM1cgene, which occurred in 88% of the samples that contained mutations in DMNT3A. These findings were consistent with previous published data, but what the researchers didn't expect to find was that in the T cells of 15 patients, mutations in DMNT3A were found (low mutant allele frequency 1-20%) but with no evidence of mutations in NPM1c. It should be noted that two types of cells were involved in this study: normal T cells obtained from non-leukemic tissue that are of the lymphoid lineage, and AML cells of the myeloid lineage. The fact that mutated DNMT3A was found in both the T cells and the AML cells, whereas mutated NPM1c was found only in the latter (refer to Table 1), led the researchers to conclude the order in which mutations arise in the disease, with mutated DNMT3Aarising the earliest followed by mutations in NPM1c as well as in FLT3-ITD, another type of mutated gene found only in AML blasts but not in T cells. The important finding to take away from this study is that mutated DMNT3A is present in the pre-leukemic ancestral cells which give rise to both the T cells and the AML cells that are observed during diagnosis. <span style="font-family: 'Times New Roman',Times,serif;">Researchers isolated non-leukemic hematopoietic stem and progenitor cell populations from 11 mutated DNMT3A/NPM1c AML patients. Note that progenitor cells are relatively immature cells that are precursors to a fully differentiated cell of the same tissue type. The cells isolated from these patients were the hemotopoietic stem cells/multipotent progenitors (HSCs/MPPs), multilymphoid progenitors (MLPs), common myeloid progenitors (CMPs), granulocyte monocyte progenitors (GMPs), megakaryocyte erythroid progenitors (MEPs), as well as mature B, T and natural killer (NK) cells within a CD33- cell fraction and CD33+ AML blasts. These were highly purified, phenotypically normal cell populations that were assessed by ddPCR for allele frequencies of DNMT3Aand NPM1c, both of which were found together in CD33+ AML blasts but DNMT3A was also found alone at variable allele frequency (mean is 24.6%) across the spectrum of mature and progenitor cells.
 * = [[image:http://4.bp.blogspot.com/-oD3RgGD6xXE/U0YhXApHEOI/AAAAAAAAAY4/SXUWD6DCXT0/s1600/figure1b.png width="175" height="400" link="http://4.bp.blogspot.com/-oD3RgGD6xXE/U0YhXApHEOI/AAAAAAAAAY4/SXUWD6DCXT0/s1600/figure1b.png"]] ||
 * = <span style="display: block; font-family: Times,'Times New Roman',serif; font-size: small; text-align: justify;">Table 1. Frequency (%) of both mutated //DNMT3A// <span style="display: inline !important; font-size: small; text-align: justify;">and //NPM1c// <span style="display: inline !important; font-size: small; text-align: justify;">in isolated CD33+ AML blasts and T cells. A procedure called ddPCR (droplet digital PCR) was used to determine the mutant allele frequencies corresponding to the length of the bars in 17 patients with normal karotype AML. (Slush, Liran, Sasan Zandi, et al. 2014) ||

<span style="font-family: 'Times New Roman',Times,serif;"> The data below is for a single, representative patient no. 11 whose cells were examined for DNMT3A and NPM1c at three particular times: diagnosis, remission (3 months) and relapse to evaluate the role that mutated DNMT3A plays in the progression of the disease.

<span style="font-family: 'Times New Roman',Times,serif;">In this sample, the mutated DNMT3A in HSCs/MPPs was found at an allele frequency of 12-30% without mutated NPM1c, which according to the authors, is a high enough mutated DNMT3A frequency to have a significant clonal contribution as compared to non-mutated HSCs. It was also found that compared to diagnosis, the allele frequency of mutated DNMT3A alone was similar to or higher at remission and relapse. What’s interesting is that CD33+ leukemic blasts always contained both mutated DNMT3A andNPM1c at diagnosis, but CD33+ myeloid cells at remission contained only DNMT3A, indicating that they were not AML blasts but progeny of the mutated DNMT3A bearing progenitor cells with preserved ability to differentiate into myeloid lineage. At relapse, both mutations are again present in majority of the cells of patient no. 11 with the exception of HSCs/MPPs, in which a proportion contained only mutated DNMT3A alone.
 * = [[image:http://3.bp.blogspot.com/-vxOshR64f-c/U2b8_ItEYoI/AAAAAAAAAao/-8aR5eAzY5g/s1600/figure2b.png width="400" height="290" align="center" link="http://3.bp.blogspot.com/-vxOshR64f-c/U2b8_ItEYoI/AAAAAAAAAao/-8aR5eAzY5g/s1600/figure2b.png"]] ||
 * = <span style="font-family: 'Times New Roman',Times,serif;">Figure 1. Allele frequency of DNMT3A and NPM1c mutations in stem/progenitor, mature lymphoid and blast cell populations, as indicated, isolated from diagnosis (blue), remission (white) and relapse (red) samples of patient no. 11 as determined by droplet digital PCR (Shlush, Liran, Sasan Zandi, et al. 2014) ||

<span style="font-family: 'Times New Roman',Times,serif;"> Another patient no. 57 was a long-surviving patient so cells from both early and late (36 months) remission could also be examined.

<span style="font-family: 'Times New Roman',Times,serif;">There is a significant increase in mutated DNMT3A allele frequency in most cell populations over time (Figure 2). In addition, though not evident in the figure, a small proportion of CD33+ myeloid cells at late remission also contained both mutated DNMT3A and NPM1c found in diagnosis. This meant that somehow there had been a regrowth in the diagnostic leukemic clone or emergence of a new clone following an independent NPM1c mutation within the pool of pre-leukemic ancestral cells. This suggests that the mutated DNMT3A found in HSCs/MPPs at diagnosis was capable of multilineage differentiation that somehow evaded chemotherapy and gave rise to clones that presented themselves at remission, and could potentially serve as a “reservoir” for further evolution of these clonal cells that leads to relapse of the disease. <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">**DNMT3a Mutation Frequency** <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;"><span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">Approximately [|26%] of AML patients have a mutation in DNMT3a. Ley, et. al sequenced the 24 exons of DNMT3a from 188 AML bone marrow samples, with the frequency of mutations in the figure below. <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;"><span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">The most common mutation within mutations of DNMT3a is a missense at the R882 residue of the protein, either R882H or R882C. There are also less common nonsense, frameshift, and splice site mutations throughout the coding sequence. Finding a common mutation source suggests that there may be a common methylation pattern or similar cause of the mutated methyltransferase gene. However, studies so far have not found evidence that the effects of this one amino acid substitution are significant, as shown in the Kaplan-Meier curves in the next section. <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">**DNMT3a Mutation Survival** <span style="font-family: 'Times New Roman',Times,serif;">Once a DNMT3a mutation is found in a patient, it signifies that the leukemia has progressed enough that the cancer will have a poor outcome. <span style="font-family: 'Times New Roman',Times,serif; line-height: 1.5;">Acute myeloid leukemia is already an aggressive cancer, but with a DNMT3a mutation, the cancer progression occurs at a much faster rate. These Kaplan-Meier curves show that in overall AML cases, as well as within various groups of patients, the mutation of DNMT3a is a predictor of lower survival rates. DNMT3a mutations were found in 0 of the 215 AML cases categorized with favorable outcome (Ley, et al. 2010).
 * [[image:http://1.bp.blogspot.com/-ca52nUlW7Sg/U2b9qul7NLI/AAAAAAAAAaw/I7juW6GoqdI/s1600/fig2d.png width="268" height="320" align="center" link="http://1.bp.blogspot.com/-ca52nUlW7Sg/U2b9qul7NLI/AAAAAAAAAaw/I7juW6GoqdI/s1600/fig2d.png"]] ||
 * = <span style="font-family: 'Times New Roman',Times,serif;">Figure 2. DNMT3A mutant allele frequency in sorted cell populations isolated from diagnosis (0 months), early (3) and late (36) remission samples of patient no. 57 (Shlush, Liran, Sasan Zandi, et al. 2014) ||
 * = [[image:Screen Shot 2014-05-18 at 10.38.20 AM.png width="459" height="240"]] ||
 * = <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: center;">Figure 4. DNMT3a Mutations in 188 Patients with Acute Myeloid Leukemia. (Ley, Timothy J., et al, 2010) ||
 * = [[image:Screen Shot 2014-05-11 at 10.54.22 PM.png width="597" height="593" align="center"]] ||
 * = <span style="font-family: 'Times New Roman',Times,serif;">Figure 5. Overall Survival in AML patients with DNMT3a mutations. (Ley, Timothy J., et al, 2010) ||

<span style="font-family: 'Times New Roman',Times,serif;">DNMT3a mutation presence is a more significant predictor of cancer fatality than any other mutation, and persists despite age, which is the largest factor in survival. It is also possible that the reasons for dramatically low survival in DNMT3a mutations may be partly due to those patients not yet having an effective targeted treatment for their type of leukemia. <span style="font-family: 'Times New Roman',Times,serif;"><span style="font-family: 'Times New Roman',Times,serif;">The R882H mutation would be expected to disrupt normal functioning of DNMT3a in affecting transcriptional regulation of genes. Each of the Kaplan-Meier estimates show a separate curve for patients specifically with a R882 DNMT3a mutation, since it is the most frequent. Based on this data, the R882 mutation only has a very slight, if any, difference in survival when compared to other DNMT3a mutations. Other studies on the R882 mutations disagree on whether the mutation type causes gain-of-function or loss-of-function for the methyltransferase, but it is likely that both occur at different densities of CpG sites (Schoofs, 2014). Holz-Schietinger provided evidence behind this idea by finding that there is a loss of methylation at clustered CpG islands, and hypermethylation at isolated CpG locations ( Holz-Schietinger, 2014). <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;"> <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">**Treatment Methods** <span style="font-family: 'Times New Roman',Times,serif;">The current goal of AML chemotherapy is complete remission (CR) with return to normal hematopoiesis, and this goal has been successful to some extent over the past four decades, with the combined use of anthracycline (that is, daunorubicin or idarubicin) with cytarabine. The combination has a 60-70% success rate in newly diagnosed AML patients (Smith, et al. 2014). However, as with many systemic anti-cancer drugs, the disease manages to relapse in these patients, and so new and novel therapies need to be created.

<span style="font-family: 'Times New Roman',Times,serif;"><span style="font-family: 'Times New Roman',Times,serif;"> Age seems to play a significant role in whether the disease can be cured or go into remission. For patients under the age of 60, the combination of cytarabine and allogeneic hematopoietic cell transplantation (HCT) improves cure rates to 50% (Smith, et al. 2014). However, for individuals over the age of 60, the cure rate remains quite low. These elderly patients generally do not receive intensive chemotherapy in the first place, since the side effects outweigh their therapy benefits. The standard treatment of the anti-metabolite cytarabine, which inhibits DNA synthesis during the S phase of mitosis, also damages healthy cells with its high toxicity (Al-Ali, et al. 2014). . <span style="font-family: 'Times New Roman',Times,serif;">Hypomethylating agents, decitabine and azacitidine, are DNA methyltransferase inhibitors approved by the FDA for treatment of high-risk Myelodysplastic Syndrome. Since 1 out of 3 cases of MDS can progress into AML, and they share symptoms of having a high percentage of abnormal cells in the bone marrow, these drugs are currently undergoing clinical trials for use in AML (NCCN, 2014). Decitabine is the deoxy derivative of azacitidine, and both work by binding covalently to DNA methyltransferase in order to inhibit their ability to catalyze methylation (Al-Ali, et al. 2014). Their purpose is to allow the transcription of tumor-suppressor genes, to help regulate abnormal cell growth.

<span style="font-family: 'Times New Roman',Times,serif;">Despite similar side effects of anemia, thrombocytophenia, and neutrophenia, which are all conditions caused by the death of healthy blood cells, the NCCN panel considers azacitidine and decitabine to be less-intensive forms of chemotherapy, when compared to the standard drugs used (anthracycline, cytarabine) and recommends it for older AML patients, as well as patients with less severe conditions (NCCN, 2014).

<span style="font-family: 'Times New Roman',Times,serif;"> In a recent Experimental Hematology and Oncology 2014 paper by Smith, a retrospective comparative analysis study was conducted to study outcomes for AML patients treated with either decitabine (DEC) or azacitidine (AZA) between January 2006 and June 2012. 487 patients were eligible for this study and over 70% of patients in each cohort were at least 65 years old (mean age was AZA 70.3 ± 11.8 years, DEC 69.4 ± 11.6 years). According to the study, most patient characteristics were similar between the cohorts except that the decitabine cohort had significantly more hospitalizations than the azacitdine cohort (62% AZA, 71% DEC; p = 0.0323).

<span style="font-family: 'Times New Roman',Times,serif;"> Below is a Kaplan Meier graph, analyzing the overall survival (OS) of the two cohorts. <span style="font-family: 'Times New Roman',Times,serif;">Overall survival was significantly better in the AZA-treated cohort compared with patients in the DEC-treated cohort (10.1 months vs. 6.9 months respectively; p = 0.007, Figure 6) and treatment with azacitidine resulted in a significantly longer time to death when compared with decitabine treatment. The most crucial aspect of the entire study, however, was that patients were not randomly assigned to treatment and administration schedules (i.e. dosing and adherence) for each therapy were not controlled.
 * = [[image:http://2.bp.blogspot.com/-cknbjMcjcjQ/U3hXaD5WfiI/AAAAAAAAAbM/wIyKR0h8nNM/s1600/kaplancurve.png.jpg width="400" height="275" align="center" link="http://2.bp.blogspot.com/-cknbjMcjcjQ/U3hXaD5WfiI/AAAAAAAAAbM/wIyKR0h8nNM/s1600/kaplancurve.png.jpg"]] ||
 * = <span style="font-family: 'Times New Roman',Times,serif;">Figure 6. Overall Survival (OS) for AML patients treated with decitabine or azacitidine between January 2006 and June 2012 (B Douglas Smith 2014). ||

<span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">__<span style="font-family: 'Times New Roman',Times,serif;">Analysis __

<span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">**DNMT3a's Effect on HSC Differentiation** <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;">Since the hematopoietic stem cell precursor gives rise to both lineages of myeloid and non-myeloid cells, finding the same DNMT3a and NPM1 mutations in non-myeloid cells of AML suggests that the HSC founder cell's original driver mutation differentiates into the multiple lineages. DNMT3a has a critical role in allowing self-renewal and differentiation of HSCs, and its mutation leads to a significant decrease of the methyltransferase enzyme's catalytic activity when it cannot attach cytosine groups to methylate DNA as efficiently. The high degree of competitive self-renewal of the mutant cells replaces normally functioning HSCs. Cancer stem cells taking over the process of self-renewal for their own growth, driving leukemic progression forward, falls under the Tumor Microenvironment characteristic of the newer Hallmarks of Cancer (Hanahan, Weinberg, 2011). <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;"> <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">**DNMT3a's Impact on AML Treatment** <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;">Though still undergoing clinical trials, the use of hypomethylating agents in chemotherapeutic treatment shows promise for AML. Azacitidine and decitabine allow for a more targeted therapy, that aims to reverse the effects of hypermethylation on silenced tumor-suppressor genes. These drugs would also account for the pre-leukemic cells that are not targeted by standard chemotherapies, preventing remission from the pools of DNMT3a-mutated HSCs that otherwise could still hold the potential to progress back into AML after remission. The complication is that DNMT3a mutations do not have one defined aberrant methylation pattern, or result solely in a widespread hypermethylation of genes. While they may not be a cure, they are still a more effective alternative to the standard treatment of cytarabine. <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;"> <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,'Times New Roman',serif; text-align: justify;">**Conclusion** <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;">DNMT3a is a driver mutation that requires secondary mutations, such as NPM1, to progress from pre-leukemic cells into AML. The ability of these pre-leukemic cells to maintain reservoirs allows them to avoid detection and relapse into differentiation after AML remission. This mutation can be used as a marker in both early detection of pre-cancerous cells before they progress into AML, as well as for targeted chemotherapy. The complexity of the DNMT3a mutation's mechanisms still require further research to be understood, but its critical role in AML can already be used in developing more successful treatment. <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;">

=__<span style="font-family: 'Times New Roman',Times,serif;">Sources __=

<span style="font-family: 'Times New Roman',Times,serif;">Al-Ali, Haifa Kathrin, Nadja Jaekel, and Dietger Niederwieser. "The role of hypomethylating agents in the treatment of elderly patients with AML." Journal of Geriatric Oncology 5.1 (2014): 89-105. Journal of Geriatric Oncology. Web. 21 May 2014. <http://www.geriatriconcology.net/ article/S1879-4068(13)00086-6/fulltext>.

<span style="font-family: 'Times New Roman',Times,serif;">Ley, Timothy J., et al. "DNMT3A Mutations in Acute Myeloid Leukemia." The New England Journal of Medicine (2010): 2424-33. Web. 12 May 2014.

<span style="font-family: 'Times New Roman',Times,serif;">Hanahan, Douglas, and Robert Weinberg. "Hallmarks of Cancer: The Next Generation." Cell. 144.5 (2011): 646-74. Web. 13 Apr. 2014.

<span style="font-family: 'Times New Roman',Times,serif;"> Hematopoietic Stem Cells. In Stem Cell Information [World Wide Web site]. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services, 2011 [cited Sunday, April 13, 2014] Available at []

<span style="font-family: 'Times New Roman',Times,serif;">Holz-Shietinger, Celeste, Doug M. Matje, and Norbert O. Reich. "Mutations in DNA Methyltransferase (DNMT3A) Observed in Acute Myeloid Leukemia Patients Disrupt Processive Methylation." The Journal of Biological Chemistry 287.37 (2012): 30941-51. Web. 12 May 2014.

<span style="font-family: 'Times New Roman',Times,serif;">Im, A. P., A. R. Seghal, M. P. Carroll, B. D. Smith, D. E. Johnson, and M. Boyiadzis. "DNMT3A and IDH Mutations in Acute Myeloid Leukemia and Other Myeloid Malignancies: Associations with Prognosis and Potential Treatment Strategies." Leukemia 28.4 (2014): 727-980. Nature. Web. 12 May 2014. <http://www.nature.com/leu/journal/vaop/ncurrent/full/leu2014124a.html>.

<span style="font-family: 'Times New Roman',Times,serif;"> National Cancer Institute: PDQ® Adult Acute Myeloid Leukemia Treatment. Bethesda, MD: National Cancer Institute. Date last modified <12/26/2013>. Available at: []. Accessed <04/13/2014>.

<span style="font-family: 'Times New Roman',Times,serif;">National Comprehensive Cancer Network. "NCCN Guidelines Version 2.2014 Acute Myeloid Leukemia." Mar. 2014. PDF file.

<span style="font-family: 'Times New Roman',Times,serif;">Potter, Nicola E., and Mel Greaves. "Cancer: Persistence of leukaemic ancestors." Nature 12 Feb. 2014: 300-01. nature.com. Web. 13 Apr. 2014 <http://www.nature.com/nature/journal/v506/n7488/full/nature13056.html>.

<span style="font-family: 'Times New Roman',Times,serif;">Schoofs, T., W. E. Berdel, and C. Müller-Tidow. "Origins of aberrant DNA methylation in acute myeloid leukemia." Leukemia 28.1 (2014): 1-14. Nature. Web. 12 May 2014.

<span style="font-family: 'Times New Roman',Times,serif;"> Shlush, Liran, Sasan Zandi, et al. "Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia." Nature. 506.February (2014): 328-33. Web. 13 Apr. 2014.

<span style="font-family: 'Times New Roman',Times,serif;">Smith, Douglas B., Charles L Beach, Dalia Mahmoud, Laura Weber, Henry J Henk. [|Survival and hospitalization among patients with acute myeloid leukemia treated with azacitidine or decitabine in a large managed care population: a real-world, retrospective, claims-based, comparative analysis]. Exp Hematol Oncol. 2014; 3: 10. Published online 2014 March 25. doi: 10.1186/2162-3619-3-10PMCID: PMC3994315 <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;">

<span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;"> <span style="background-color: #ffffff; color: #333333; display: block; font-family: Times,"Times New Roman",serif; text-align: justify;">