A summary of the molecular testing recommended in acute myeloid leukemia

Advances in Next-Generation Sequencing technologies (NGS) are revealing germline and somatic mutations that, together with karyotype, determine the diagnosis and subtype of Acute Myeloid Leukemia (AML). Molecular testing is also essential for the genetic risk stratifi cation of patients with AML, in particular for those with normal karyotype AML (CN-AML), a large and highly heterogeneous group of patients. Patients determined to be at high risk could benefi t from a more aggressive fi rst-line therapy, or a more directed therapy, such as midostaurin (for FLT3-mutated AML) or ivosidenib (for IDH1-mutated AML). Here, we will summarize the molecular testing currently recommended in AML and introduce new mutations that may have prognostic value and clinical application in the near future. Review Article A summary of the molecular testing recommended in acute myeloid leukemia Ruth Stuckey1, Cristina Bilbao-Sieyro1,2 and María Teresa Gómez-Casares1,3* 1Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrín, 35019 Las Palmas de Gran Canaria, Spain. 2Morphology Department, Universidad de Las Palmas de Gran Canaria, 35016 Las Palmas de Gran Canaria, Spain 3Department of Medical Sciences, Universidad de Las Palmas de Gran Canaria, 35016 Las Palmas de Gran Canaria, Spain Received: 01 April, 2020 Accepted: 17 April, 2020 Published: 18 April, 2020 *Corresponding author: Dr. María Teresa GómezCasares, MD, PhD, Hematology Department, Hospital Universitario de Gran Canaria Dr. Negrín, 35019 Las Palmas de Gran Canaria, Spain, Tel: +34 928 479433; E-mail:


Introduction
Acute Myeloid Leukemia (AML) is a complex hematological neoplasm, characterized by the World Health Organization (WHO) into multiple subtypes [1,2]. Cytogenetic analysis is essential for AML diagnosis since certain subtypes are characterized by numerical chromosome abnormalities or gross translocations, including loss of chromosome 5 or the common structural aberration t(8;21) (q22;q22), leading to production of the AML-ETO fusion gene, respectively. In fact, according to the National Comprehensive Cancer Network (NCCN), "karyotype represents the single most important prognostic factor for predicting remission rates, relapse risks and overall survival outcomes" [3].
In recent years, signifi cant progress has been made in the biological understanding of AML [4]. In particular, as a result of advances in Next-Generation Sequencing technologies (NGS), genetic studies are revealing an ever-rising number of somatic mutations that help to determine both the phenotypic and prognostic heterogeneity of this pathology [5].
The increasingly widespread use of NGS technology with gene panels in clinics means that the simultaneous analysis of mutations in a high number of genes is both practical and economically feasible [5]. Moreover, the incorporation of molecular markers into current prognostic algorithms is permitting the more precise risk stratifi cation of patients.

To inform diagnosis
Advances in NGS technologies have driven the inclusion of several new AML subtypes according to their associated genetic alterations, highlighting the importance of molecular testing for a complete diagnosis. For instance, the WHO included the categories of "AML with mutated NPM1" and "AML with biallelic mutations of CEBPA" in 2008 [1], and "AML with mutated RUNX1" and "AML with BCR-ABL1" were provisionally recognized as new entities in the 2016 revision [2]. Although the latter entity is not frequent (less than 1% of all AML cases [7,8]), de novo AML patients identifi ed as having this subtype may benefi t from treatment with tyrosine kinase inhibitors [9,10].

Germline mutations
The presence of germline mutations in several genes have been shown to cause a predisposition to myeloid neoplasms, including AML. These observations led to the inclusion of a new category of myeloid neoplasms with germline predisposition ( selection of a suitable donor if the patient is a candidate for Hematopoietic Stem Cell Transplantation (HSCT). Germline mutations can also confer a higher risk of relapse in AML or a predisposition to the development of a secondary leukemia, including therapy-related leukemias [12]. For example, between 5% and 7% of patients with CEBPA-mutated AML harbor germline mutations [13] and although the majority reach complete remission following chemotherapy, recurrent disease is frequent [13].
If a positive result is detected, it is recommended that genetic counseling be offered to the patient's family members for the prevention and/or early detection of germline myeloid neoplasms [11].

The importance of studying TP53
In the case of TP53, the study of mutations in this gene is of particular importance given that their presence predicts a particularly poor outcome for the patient [4,14,15]. Mutations in the TP53 gene locus may be point mutations or deletions of 17p13 of a range of sizes, and either somatic or germline [16].
TP53 mutations usually coincide with a complex karyotype [14,15]. However, their presence in conjunction with a monosomal karyotype or aneuplodies such as-5/5q and -7/7q-themselves associated with high risk-is independent and additive in terms of risk stratifi cation, giving rise to an extremely adverse prognosis [4,15].
Recently, the presence of ASXL1 and RUNX1 mutations were also included in the high-risk category of AML due to their association with a worse prognosis and poor survival, respectively [2]. Germline mutations of the latter are associated with familial platelet disorder with associated myeloid malignancy, a disorder of abnormal hematopoiesis that confers affected individuals with a higher risk of AML [17]. Moreover, germline mutations in ASXL1 [18], have been reported as causing a predisposition to myelodysplastic syndrome and/ or AML. However, further studies are required to confi rm the impact of germline mutations in ASXL1, as well as other germline mutations such as SRP72 [19], and CBFA2 [20] before their possible inclusion in congenital AML genetic testing recommendations in the future.

Risk stratifi cation
The genetic risk stratifi cation of patients with AML is especially useful to determine the response to therapy of patients with normal karyotype AML (CN-AML), a particularly heterogeneous group of patients representing 40%-50% of all new cases. with CN-AML due to its prognostic value [2,3,6]. Specifi cally, numerous studies have reported higher relapse rates and worse Table 1: Classifi cation of myeloid neoplasms with germline predisposition. Adapted from [2].

With pre-existing platelet disorders and With other organ dysfunction.
AML with CEBPA germline mutation When there is a suspicion that a patient may harbor congenital AML-due to a family history of neoplasia, an exceptionally early age of AML presentation, or the presence of various tumors-the ELN recommends the genetic analysis of the genes RUNX1, CEBPA, GATA2, DDX41, ANKRD26, and ETV6 [6]. Mutations in these genes are just some that bestow a predisposition to the development of AML; others include mutations in genes associated with a general susceptibility to cancer, such as the Fanconi anemia genes, or TP53 and BRCA1/ BRCA2 [6,[11][12][13], often mutated in therapy-related leukemias [12].
The analysis of germline mutations is critical to inform the Citation: Stuckey  Inhibitor (TKI) gilteritinib has also been approved by the FDA for the treatment of relapsed or refractory FLT3-mutated AML [27].
Despite this, a 2017 survey carried out among AML experts in Europe and the United States reported that only 51.4% of specialists evaluate FLT3-ITD in all patients [28]; while at the national level, a recent survey among AML experts in Spanish clinics revealed that 7.5% "never" or "sometimes" carried out the analysis of FLT3 mutations [29]. These data demonstrate that there is still a lack of understanding of the prognostic and therapeutic indications of the presence of FLT3 mutations. It is important that mutational analysis is carried out promptly so that patients identifi ed as high risk can benefi t from a more aggressive fi rst-line therapy, or a more directed therapy in some cases. For example, patients with a positive result for FLT3-ITD (with a ratio > 0.5) may be candidates for the addition of the TKI midostaurin to their chemotherapy regime or for HSCT.

Relapsed and refractory AML
In addition to its role in the accurate diagnosis of patients with AML, mutational analysis is also very informative in cases Currently, there is a difference between the NCCN and ELN recommendations on molecular testing in AML. Specifi cally, in  [3,6]. the 2017 update the ELN did not include a recommendation for the analysis of mutations in IDH1 and IDH2 in the evaluation of AML at diagnosis due to lack of evidence [6]. However, shortly after its publication, the FDA approved two directed therapies, ivosidenib (AG-120, Tibsovo®) against IDH1-mutated AML andenasidenib (AG-221, IDHIFA®) against IDH2-mutated AML [32,33]. Thus, in 2018 the NCCN included the recommendation for the analysis of mutations in the IDH1 and IDH2 genes because of the availability of FDA-approved directed therapies [30].

Minimal residual disease MRD
Minimal Residual Disease (MRD) has been shown to be a very important prognostic factor. High levels after chemotherapy are associated with relapse and so patients with a positive MRD are considered to be at risk [34]. The analysis of mutations, such as insertions and deletions in NPM1, permit their monitoring as a molecular marker of MRD [35]. The ELN's current recommendations describe the applicability of MRD testing for the follow-up of AML patients because it allows different therapeutic options to be optimized and personalized for patients with high MRD, such as an indication for HSCT or not [6].

Updating of risk stratifi cation algorithms
The list of mutations in genes with prognostic value is continually increasing. For example, studies have demonstrated that certain MCM7 polymorphisms are associated with relapse and overall survival in AML patients [36], while the partial tandem duplication of MLL (MLL-PTD) confers worse prognosis to patients with CN-AML [37]. In the future it's probable that these or other genes could be incorporated into risk algorithms to help stratify patient subgroups, but only when suffi cient evidence exists to support their prognostic value [4,6,37].

Prognostic relevance of mutations according to age
Finally, another possible future modifi cation to the NCCN and ELN's recommendations could be the inclusion of risk stratifi cation algorithms not only optimized to the mutational profi le of the patient but also to the patient's age.
In general, older patients with AML tend to have poorer results with standard chemotherapy regimens [38,39]. This could be caused by adverse karyotypes that are more common and/or the higher number of mutations in older patients with AML than younger patients with AML [4], although the association between the number of mutations and prognosis continues to be a matter of debate.
A phenomenon called clonal hematopoiesis of indeterminate potential (CHIP), which is intrinsic to aging, is characterized by the accumulation of mutations in the DNMT3A, TET2 and ASXL1 genes (collectively known as DTA mutations). A positive correlation exists between age and the presence of DTA mutations, even in healthy individuals [40,41], although studies have found an association between the accumulation of DTA mutations and a higher risk for developing myeloid neoplasms, including AML, as well as cardiovascular pathologies [40][41][42].
Nevertheless, the detection of DTA mutations in AML patients in remission is not associated with relapse [43].
It still remains to be determined whether the presence of different mutations in patients aged over 65 years of age has the same prognostic value as in patients aged under 65 years, although several groups are actively investigating this matter. For instance, preliminary data from the Alliance group indicates that mutations in the splicing factor SF1 may refi ne the prognosis of NPM1-mutated AML patients aged over 60 years [44].