Mutations of NPM1 have recently been described as one of the most frequent genetic lesions in AML, occurring in 50–60% of adult AML with normal karyotype 6 7 . Additional evaluation has shown that mutations in NPM1 are rare in other risk groups of AML and in one study, no NPM1 mutations were shown in patients with favorable cytogenetics 7 . The NPM1 gene encodes a nucleo-cytoplasmic shuttling protein that regulates the ARF-p53 tumor-supressor pathway 8 9 . Mutations in this gene result in an abnormal accumulation of the NPM1 protein in cytoplasm. Two types of mutations have been described to date. The first and most frequent mutations consists of a 4-nucleotide (nt) insertion (YWTG; YUPAC code) downstream from nucleotide 959; the second is deletion of a GGAGG sequence at positions 965 through 969 and substitution with 9 extra nt (GenBank accession no. NM_002520). Both mutations lead to aberrant cytoplasmic localization of NPM1 as shown after immunostaining with anti-NPM1 monoclonal antibodies. This is caused by open reading frameshift mutations that lead to either the disruption of the NPM1 nucleolar-localization signal or the generation of a leucine-rich nuclear export motif. In all mutated cases, the resulting frameshift led to a product five amino acids longer with the new C-terminal tail CFSQVSLRK, peculiar to the NPM1-mutated product 7 .

Recent studies in cell lines and knockout mice have shown that NPM1 is involved in the control of genomic stability and contributes to growth-suppressing pathways through its interaction with ARF. Therefore, the loss of NPM1 expression can contribute to tumorgenesis 10 . Several methods are suitable for detecting NPM1 gene mutation, including molecular and immunohistochemical studies 7 11 12 13 .

NPM1 gene mutations appear to occur more frequently in adult female AML patients 14 15 16 and also tend to be associated with: a) higher white blood cell count, b) monocytic differentiation (in particular FAB M5b AML subtype) 17 , c) wide morphologic spectrum, d) multilineage involvement 9 , e) lack of CD34/CD34-negativity 7 9 11 , f) normal cytogenetics 18 , g) a decreased prevalence of CEBPα mutations 17 , h) high frequency of FLT3-ITD gene mutation 18 and i) a trend toward favorable clinical outcome, especially in patients without a FLT3 gene mutation 7 15 . Patients with only an NPM1 mutation exhibit higher complete remission (CR) and significantly better OS 14 15 16 , event free survival (EFS) 15 , and disease free survival (DFS) as well as a lower cumulative incidence of relapse 6 16 . The various study outcomes of risk associated with NPM1 status are summarized in Table 2.

Detection of NPM1 gene mutations may be useful in the dissection of the heterogeneous group of AML patients with normal karyotype into prognostically different subgroups 7 . Further, due to their frequency and stability, NPM1 mutations may become a new tool for monitoring minimal residual disease in AML-patients with a normal karyotype 9 .

FLT3 is a tyrosine kinase that is primarily expressed on hematopoietic progenitor cells and functions in the proliferation and differentiation of these cells. FLT3 is the most commonly mutated gene in AML with the mutation occurring in approximately 30–40% of AML patients 19 . The most common mutation consists of an internal tandem duplication (FLT3-ITD) in the juxtamembrane domain of the FLT3 gene. FLT3-ITD results in a constitutively active FLT3 protein that promotes Stat 5 phosphorylation. The net consequence of FLT3/Stat5 constitutive activation is uncontrolled hematopoietic cell proliferation 20 . AML patients who carry the FLT3-ITD mutation appear to have poorer clinical outcomes. Adult patients usually have a higher prevalence of FLT3-ITD than pediatric AML patients. This observation may partially explain why adult AML has a poorer clinical outcome than pediatric AML. Clinically, AML patients with FLT3-ITD tend to have higher WBC counts and an increase percentage of leukemic blasts 21 . A missense mutation in the activation loop (FLT3-ALM) of the second tyrosine kinase domain of FLT3 at Asp835 leads to another common FLT3 mutant (FLT3-TKD) that is found in approximately 5–10% of AML patients 22 . Although the clinical significance of this FLT3 mutation especially in NC-AML is not yet clear, several studies indicate that it is also an adverse prognostic indicator 19 21 . The associated risk of FLT3 status determined by these studies are summarized in Table 3.

Particularly in AML patients with normal cytogenetics, FLT3-ITD status is important in assessing the prognosis of patients. Several studies have demonstrated that FLT3-ITD in NC-AML patients correlates with an adverse prognosis for both DFS and OS 23 24 25 . Not only does the presence of FLT3-ITD impart a poor prognosis, but the size of the internal tandem duplication is significant. The duplication can range in size from three to hundreds of nucleotides and longer duplications correlate with a worse OS 26 . In addition to the mutant allele, the status of the wild-type allele in patients with FLT3-ITD has been demonstrated to have prognostic significance. Patients who lack the wild-type allele have a worse prognosis 27 . In those who express the wild-type allele, the ratio of the mutant to wild-type level of FLT3-ITD has a strong correlation to survival. In one study, patients with a high mutant to wild-type ratio (defined as greater than 0.78) had a significantly shorter OS and DFS than those with a lower ratio. The DFS and OS for patients with a lower ratio were no different than the group of patients without FLT3 abnormalities 28 .

In addition to mutation of FLT3 and the decreased expression of the wild-type allele, over-expression of FLT3 in the absence of mutation has also been observed in AML patients. Over-expression of FLT3 even in the absence of FLT3-ITD is also an unfavorable prognostic factor for OS 29 . As FLT3-ITD is an adverse prognostic factor, it has been speculated that patients with this genetic abnormality should be considered for more intensive therapy. However, a large study of 1135 adult patients with AML including 25% with FLT3-ITD, there was no improvement in outcome based upon whether or not a patient with FLT3-ITD received a transplant in first complete remission 30 .

The association of FLT3 mutations have also been evaluated in patients with favorable cytogenetics (t(8;21), inv16, t(15;17)). FLT3 mutations were noted in 15 of 17 patients studied. Of these patients, 41% of those with t(15;17) were associated with FLT3 mutations and only 9% of cases with inv16. Those with PML-RARα had decreased to no CD11c or HLA-DR expression. However, this study did not illustrate significant correlations with outcomes and FLT3 mutational status 31 .

As FLT3 mutations lead to constitutively activated signaling, much work has been performed to develop small molecule FLT3 inhibitors. Unfortunately FLT3 inhibitors have thus far shown disappointing results as remission induction has been short lived. Nevertheless, there is optimism that FLT3 inhibitors may be more efficacious when used in combination therapies 32 . Besides being a useful as a prognostic marker and a therapeutic target, FLT3-ITD has also been used for minimal residual disease monitoring 33 .

MLL is frequently rearranged in AML and ALL and has been found in combination with greater than 30 different genes. The most frequent rearrangements in the current published series were unbalanced translocations leading to loss of chromosomal material. Overall, loss of 5q and/or 7q chromosomal material seemed the more common event, and losses of 5q, 7q, and 17p in combination were observed in many cases. Overrepresented chromosomal material from 8q, 11q23, 21q, and 22q was found recurrently and in several cases this was due to the amplification of the MLL (located at 11q23) and AML1/RUNX1 (located at 22q22) genes 34 . MLL encodes a histone methyltransferase that plays a role in hematopoiesis by regulating homeobox genes. In mice heterozygous for MLL, both hematopoietic abnormalities are found as well as decreased Hox gene expression 35 .

In addition to rearrangements, the MLL gene can also undergo partial tandem duplications of exons 5–11 or exons 5–12 and produce an elongated protein. This abnormal protein, which contains a DNA-binding and transcriptional repression domain, can suppress the expression of the wild-type allele by an unknown mechanism. Interestingly, the silencing of wild-type MLL in blasts positive for MLL-PTD was reversed by DNA methyltransferase and histone deacetylase inhibitors 36 . These findings indicate the potential therapeutic role of the DNA methyltransferase and histone deacetylase inhibitors such as decitabine and valpoic acid in these AML patients.

In one large study of 247 young adult patients with AML, MLL-PTD was found in 7.7% of patients. In this study, MLL-PTD was an adverse prognostic indicator as the median remission duration was 19 months in the absence of MLL-PTD and 7.75 months in its presence 37 . In general, the majority of studies indicate that MLL-PTD is poor prognostic indicator in NC-AML including median survival and relapse-free interval 38 . Additionally, the use of MLL-PTD for minimal residual disease monitoring has been shown to be effective in detecting relapse prior to clinical manifestations 39 .

CEBPα is an essential transcription factor for granulocytic differentiation as demonstrated by CEBPα-null mice that lack mature granulocytes 40 41 . Studies have reported N- and C-terminal CEBPα mutations in approximately 15% to 20% of AML 42 . The mutant proteins act in a dominant-negative manner to block DNA binding and transactivation of granulocyte target genes resulting in the failure of granulocytic differentiation 40 . Patients with a CEBPα mutation have higher hemoglobin levels, lower platelet counts, higher blast counts, and are less likely to present with lymphadenopathy or extramedullary leukemia compared to patients without a CEBPα mutation.

Suprisingly, as CEBPα is required for differentiation, mutation of CEBPα is correlated with beneficial effects on remission, CR duration 42 , event-free survival 17 , DFS 25 , and OS 41 . While there is no significant difference in CR rates between patients with and without CEBPα mutations 25 42 , mutations are associated with a significantly reduced hazard ratio for death and event free survival 41 . CEBPα mutations appear to be an independent prognostic factor even in the presence of FLT3 and MLL mutations. Studies have shown that there is no significant overlap between the patients with CEBPα mutation and patients with FLT3-ITD or MLL-PTD mutations, suggesting that CEBPα mutations define a distinct biologic subclass of NC-AML 42 . CEBPα mutation is an independent prognostic marker for OS irrespective of age, MLL-PTD, and FLT3-ITD status 43 and is another marker that permits the division of NC-AML into distinct clinical groups 25 .

While NPM1, FLT3, MLL-PTD, CEPBα are all mutations noted in NC-AML, additional genetic abnormalities are noted in the form of over-expression. More detailed discussion of BAALC, MN1, ERG-1, and AF1q give insight to over-expression of genes in NC-AML.

BAALC has also been found to be an important adverse prognostic factor in NC-AML. Though little is known about the biological function of BAALC, it is highly expressed in hematopoietic precursor cells as well as leukemic blasts and is down-regulated during differentiation. BAALC has been postulated to function in the cytoskeleton network due to its cellular location 44 45 . Several studies have demonstrated that high BAALC expression is a poor prognostic indicator in NC-AML for such factors as OS, DFS, and resistant disease 46 47 . In one study of 86 AML patients with NC-AML, high expression of BAALC was found to be an independent risk factor for both inferior OS (1.7 vs. 5.8 years) and DFS (1.4 vs 7.3 years). Different post-remission strategies among patients with different level of BAALC expression (consolidation, autologous and allogeneic stem cell transplantation) have no influence on OS. However, high BAALC expressive patients who underwent allogeneic stem cell transplantation have lower cumulative relapse rate compared to those who underwent autologous stem cell transplantation 47 .

MN1 is an oncoprotein that has been found to function as a transcription coactivator. In AML it has been found as part of the translocation t(12;22)(p13;q11) which leads to the MN1-TEL fusion gene 48 . In animal models, the MN1-TEL fusion gene collaborates with HOXA9 to induce AML 49 . Recently, high levels of expression of MN1 have been found to be a prognostic marker in NC-AML. Though the exact function of MN1 in hematopoietic cells is unclear, it is another protein that is highly expressed in hematopoietic cells and is down-regulated during differentiation. In a study of 142 adult patients with NC-AML, high MN1 expression was significantly related to unmutated NPM1, poor response to initial induction chemotherapy, high relapse rate, risk free survival, and OS. In multivariate analysis, high MN1 expression was an independent prognostic marker 50 .

ERG is a member of the ETS family of transcription factors. High ERG expression is associated with the upregulation of many genes which are involved in cell proliferation, differentiation, and apoptosis 51 . The ERG gene is a recently identified molecular marker predicting adverse outcome of NC-AML patients. Over-expression of the ERG gene was first discovered in patients with complex karyotypes and abnormal chromosome 21 34 52 . Marcucci, et al showed that in patients less than 60 years old with de novo NC-AML, those patients expressing the highest levels of ERG (the top 25%) have a worse cumulative incidence of relapse (CIR) and OS. In this analysis, ERG over-expression predicted a shorter survival only in patients with low BAALC expression. Though more study is needed to confirm these results, ERG over-expression in NC-AML not only predicts an adverse clinical outcome, but also appears to be associated with a specific molecular signature 51 .

As the ERG gene is located on chromosome 21, it has been speculated that ERG expression may play a role in the pathogenesis of acute leukemia in patients with Down's syndrome. Patients with Down's syndrome are known to have a higher incidence of acute leukemia 53 . The high ERG expression may also be related to acute megakaryoblastic leukemia (FAB-M7) which is associated with trisomy 21 54 .

Two small studies conducted by Tse et al suggest that elevated AF1q expression is associated with poor outcomes both in pediatric AML and adult myelodysplastic syndrome (MDS) 55 56 . In the pediatric study, AF1q expression in AML patients varied from 0 to 154-fold compared with normal marrow and increasing AF1q expression level was associated with worsening survival with a hazard ratio of 1.02 per fold in AF1q expression (p = 0.032). High AF1q expression was related to poor survival in univariate and multivariate models without association with any specific adverse cytogenetics 55 . The AF1q expression levels in the MDS study (total of 47 patients) suggested a statistically significant correlation with IPSS and AF1q expression level in high risk MDS 56 . Consistent with the findings in the pediatric AML study, MDS patients with high AF1q expression have an increased hazard ratio of death from MDS and relapse after allogeneic stem cell transplantation with correlation of specific poor cytogenetics. These observations led to a hypothesis that elevated AF1q expression might serve an adverse molecular marker for poor prognosis in AML patients with normal cytogenetics.

Recently, Strunk et al examined AF1q expression in 290 adult NC-AML patients (aged < 60). They found NC-AML patients with low AF1q expression (AF1q^low) had better OS (p = 0.026) and CR rate with initial induction chemotherapy (p = 0.06) compared to high AF1q expressing patients (AF1q^high). The AF1q^high patients had a significantly greater incidence of concurrent FLT3-ITD. A subgroup of the AF1q^high patients who received allogeneic stem cell transplantation (SCT) had a significant better relapse-free survival compared to patients who received chemotherapy/autologous SCT (p = 0.04). This suggests that high AF1q expression is a poor prognostic marker for adult NC-AML patients and may help direct post-induction treatment strategies.

Normal cytogenetics are detected pretreatment in approximately 45% of patients with de novo acute myeloid leukemia; thus, this constitutes the single largest cytogenetic group of AML. Recently, molecular genetic alterations with prognostic significance have been reported in these patients. They include internal tandem duplication of the FLT3 gene, partial tandem duplication of the MLL gene, mutations of the CEBPα and NPM1 genes and aberrant expression of the BAALC, ERG and MN1 genes. Additionally, gene-expression profiling has been applied to identify prognostically relevant subgroups 57 .

Gene expression studies have identified that the majority of NC-AML patients fall into specific clusters that exhibit similar gene expression profiles. The identification of these clusters may not only be useful for diagnostic purposes, but also may help guide prognosis and therapeutic approaches. For example, NC-AML patients were found to up-regulate class I homeobox A and B gene families 58 . In another study, DNA microarray experiments identified two distinct subgroups of NC-AML including one that was closely related to the gene signatures observed in AML with translocations. In this study, NC-AML patients in the "translocation-like" group had a superior prognosis to the other group 57 . Similarly, a separate study also found two distinct gene expression clusters in NC-AML patients with significantly different survival. Using a panel of 133 genes, it was possible to predict the clinical outcome of NC-AML patients 59 . NC-AML patients in the cluster with worse survival were more likely to harbor FLT3 mutations and were more commonly diagnosed with specific AML subtypes (FAB M1 and M2). Bullinger's observation was recently validated by a Cancer and Leukemia Group B (CALGB) study that used a different microarray platform and had a longer follow up 60 . Gene-expression profiling allows a comprehensive classification of AML that includes previously identified genetically defined subgroups and a novel cluster with an adverse prognosis 59 . In the future, gene expression profiling studies will likely play a role clinically in molecularly risk stratifying NC-AML patients as well as further elucidating the biology of NC-AML.

Minimal residual disease (MRD) can provide an early indication of potential relapse in AML post-treatment. There has been some initial evaluation of the utility of FLT3-ITD, CEBPα, ERG, NPM1, and MLL in MRD whereas AF1q, MN-1, and BAALC have not. The impact of FLT3 was analyzed in 11 patients. All of the six patients with positive quantitative real-time polymerase chain reaction (RQ-PCR) post-treatment eventually relapsed 33 . Real-time quantitative PCR has also been used to evaluate MRD in patients carrying NPM1 mutations at time of diagnosis. Decreasing NPM1 copy number correlated with response to therapy and in four cases followed post-therapy, rising copy number preceded hematological relapse 13 . Additional evaluation in the post-transplant setting showed that all patients who remained NPM1 mutant positive after transplant relapsed and all those who had increases in mutation copies post-transplant relapsed as well 61 . Comparison of CEBPα mutational status between diagnosis and relapse in AML was first investigated by Tiesmeier et al 62 . Two of 26 patients that relapsed had mutated CEBPα which persistent post-treatment suggesting a concordance between presentation and relapse. As approximately 60% of CEBPα mutations are insertion or deletions, they are an amenable to MRD monitoring by RQ-PCR 63 . One case study have also reported the utility of RT-PCR for detecting ERG MRD. The patient had a negative ERG fusion gene after transplant which then recurred at time of relapse 64 . A larger analysis of 19 patients conducted by Kong et al. noted four types of TLS/FUS-ERG chimeric transcripts via RT-PCR. The transcripts were detectable at diagnosis as well as during remission and relapse suggesting resistance to conventional chemotherapy 65 . Finally, RT-PCR has also been used to evaluate expression levels of partial tandem duplications in the MLL gene. Expression levels in 16 patients were analyzed at time of diagnosis and at relapse and found to be equivalent. Additionally, molecular relapse was detected 35 days before clinical relapse in two patients 39 . Thus, MLL-PTD was suggested as a target for MRD detection. Currently, MRD monitoring via these molecular markers is not commercially available to the community physician, but these studies provide insight to their future potential in MRD monitoring.