De novo adult acute myeloid leukemia with two new mutations in juxtatransmembrane domain of the FLT3 gene: a case report

Background Approximately 30% of adult acute myeloid leukemia (AML) acquire within fms-like tyrosine kinase 3 gene (FLT3) internal tandem duplications (FLT3/ITDs) in their juxtamembrane domain (JMD). FLT3/ITDs range in size from three to hundreds of nucleotides, and confer an adverse prognosis. Studies on a possible relationship between of FLT3/ITDs length and clinical outcomes in those AML patients were inconclusive, yet. Case presentation Here we report a 54-year-old Arab male diagnosed with AML who had two FLT3-ITD mutations in addition to NPM1 mutation. Cytogenetic approaches (banding cytogenetics) and fluorescence in situ hybridization (FISH) using specific probes to detect translocations t(8;21), t(15;17), t(16;16), t(12;21), and deletion del(13q)) were applied to exclude chromosomal abnormalities. Molecular genetic approaches (polymerase chain reaction (PCR) and the Sanger sequencing) identified a yet unreported combination of two new mutations in FLT3-ITDs. The first mutation induced a frameshift in JMD, and the second led to a homozygous substitution of c.1836T>A (p.F612L) also in JMD. Additionally a NPM1 type A mutation was detected. The first chemotherapeutic treatment was successful, but 1 month after the initial diagnosis, the patient experienced a relapse and unfortunately died. Conclusions To the best of our knowledge, a combination of two FLT3-ITD mutations in JMD together with an NPM1 type A mutation were not previously reported in adult AML. Further studies are necessary to prove or rule out whether the size of these FLT3-ITDs mutations and potential other double mutations in FLT3-ITD are correlated with the observed adverse outcome.


Background
In patients with acute myeloid leukemia (AML) genetic diagnostics were performed in the past mainly by cytogenetics and molecular cytogenetics. In recent years also tumor markers were added, which rely on molecular genetic methods [1].
The fms-like tyrosine kinase 3 (FLT3) gene encodes a class III tyrosine kinase receptor for the FLT3 ligand, which is normally expressed in CD34 + ‫‬ hematopoietic stem/progenitor cells, and plays a fundamental role in both normal and leukemic hematopoiesis [2]. Internal tandem duplications (ITDs) of the FLT3 gene (FLT3/ ITDs) represent one of the most common molecular abnormalities in patients with AML. They are detectable in around 25-30% of all patients [3,4]. ITDs consist of in-frame insertions of duplicated sequences localized in the juxtamembrane domain (JMD) of the FLT3 molecule. Their presence results in a constitutive, ligand independent activation of the tyrosine kinase activity of the FLT3 receptor; this is responsible for abnormal proliferation and differentiation of leukemic stem cells [2]. In AML constitutive activation of kinase domain happens due to disruption of auto-inhibitory interaction between JMD and the activation loop, which normally stabilizes the inactive kinase, and at the same time protects ATP binding pocket [5,6]. FLT3/ITDs also protect leukemic cells from the damaging chemotherapeutic agents [7].
It was suggested that an increasing length of additional sequences like ITDs may influence the grade of tyrosine kinase activity of the FLT3 receptor, and could (1) lead progressively to increasing activation levels and (2) to worsen the overall survival (OS) in affected patients. Still, the results of studies which investigated the impact of ITD length on the clinical outcome are contradictory. Some studies confirming the aforementioned assumption [8][9][10][11][12], whereas others contradicted [13,14].
Presence of FLT3/ITDs has been associated with an increased initial peripheral white blood cell (WBC) count and percentage of blast cells in bone marrow, a reduced disease-free survival (DFS) and OS, and a high relapse rate with an overall adverse prognosis. However, the rate of complete remission (CR) was not significantly affected [15][16][17]. Thus, a prognostic significance of FLT3/ ITDs has been suggested [8]. According to the National Comprehensive Cancer Network and the European Leu-kemiaNet (ELN) 2017, AML cases with cytogenetically normal karyotypes and FLT3/ITD mutation have a poor prognosis.
Besides in FLT3/ITD, mutations in nucleophosmin 1 (NPM1) gene represent the second most frequent molecular aberration in AML patients [18]. A combined status of mutated NPM1 and the wild type FLT3 gene (NPM1 + /FLT3 − ) is a well-established favorable risk factor in younger adult patients, with less probability of relapse and prolonged survival [19][20][21]; these patients are not obliged to receive allogeneic hematopoietic stem cell transplantation [22]. Otherwise, co-occurrence of FLT3/ITD and NPM1 mutations was suggested to partially improve response rates, DFS and OS outcomes compared to AML-patients having exclusively FLT3/ ITD mutations. However, cases with mutations in FLT3/ ITD and NPM1 have worse prognosis than those having (NPM1 + /FLT3 − ) [23].
Here, we present a unique case with two FLT3-ITDs mutations in JMD and an NPM1 type A gene mutation associated with adverse outcome.

Case presentation
In October 2019, a 54-year-old Arab male patient presented with 2 months history of fatigue, orthostatic hypotension followed by bruising on the lower right extremity, melena (present for one month only) and dyspnea II. Physical examination and computer tomographic scan showed hepatomegaly (4 cm). He had no familial history of malignancies and no social and environmental history of exposure to toxins or animals. Initial laboratory evaluation of peripheral blood (PB) revealed white blood cells count (WBC) of 26.3 × 10 9 /l (10% were blasts). Pathologic examination of bone marrow (BM) aspirate characterized hybercellularity with 60% of blasts. Flow cytometric (FCM) analysis classified this case as AML-M2 according to world health organization (WHO) classification. The abnormal cell population (60%) was positive for CD45 dim , CD34, HLADr, CD13, CD33 and expressed CD117 heterogeneously. Blasts cell population was negative for CD3, CD117, CD14, cCD3, cCD79a, CD14, CD11c, CD38, CD64, CD32, CD7, CD19, CD10, and CD5.
The patient was given standard treatment for AML including 3 + 7 induction chemotherapy (daunorubicin 60 mg/m 2 for 3 days and cytarabine 200 mg/m 2 for 7 days). One month later, under treatment with 3 + 7 protocol, the patient relapsed, i.e. his PB showed a WBC of 107 × 10 9 /l, anemia (hemoglobin level (Hgb) = 8.8 g/dl) and thrombocytopenia (Plt 93 × 10 9 /l). The patient was given re-induction with 3 + 7 chemotherapy protocol (for more details see Table 1). Less than one month after relapse, the patient acquired additional severe symptoms such as neutropenia, neutropenic enterocolitis, and diabetes insipidus, and the patient unfortunately passed away due to respiratory and cardiac arrest. No autopsy was performed. The patient's brother agreed with the scientific evaluation of this case and the study was approved by the ethical committee of Pharmacy faculty at Damascus University, Ministry of High Education, Syria review Board, No. 2/2019. Chromosome analysis using GTG-banding was performed on BM sample taken prior to chemotherapy according to standard protocols [24]. A normal male karyotype was diagnosed. Fluorescence in situ hybridization (FISH) using specific probes to detect translocations t(8;21), t(15;17), t(16;16), t(12;21), and deletion del(13q), were applied to excluded chromosomal abnormalities, too, as previously reported [24].
For molecular analyses, whole genomic DNA was extracted from PB cells (EDTA-blood) prior to chemotherapy treatment. Polymerase chain reaction (PCR) amplification of genomic DNA and Sanger sequencing were used to screen for the presence of mutations of the following genes: FLT3/ITD (exons 11 and 12), FLT3-KTD and NPM1; using specific primers for each mutation previously reported [25]. ITDs were confirmed by Sanger sequence analysis; the wild-type band of 330 bp length, and other differently sized PCR products were identified in our patient (Fig. 1) using the ABI Prism 310 genetic Table 1

G A ATA T G A T C T C A A AT G G G A G T T T C C A A G A G A A A A T T T A G A G T T A G G (p.D593-F594insREYEYDLKWEFPRENLEF).
mutation 2: homozygous substitution c.1836T>A (p.F612L).
A D835 mutation was not detected by FLT3-KTD test in our patient. However, he had also NPM1 type A mutation (data not shown).

Discussion and conclusions
Here we report the first case of an adult AML patient with normal karyotype, who had one NPM1 type A and two frameshift FLT3-ITD mutations. The first frameshift FLT3-ITD was never reported before (COSMIC database for somatic samples from hematopoietic and lymphoid tissue), whereas the second mutation has already been observed, but as heterozygous variant (COSV54057677).
The present case supports previous findings [8][9][10][11][12], which suggested that long ITDs are associated with adverse OS, a higher incidence for relapse and a negative impact on clinical outcomes in AML patients post chemotherapy.
Of special interest, is the suggestion that the complications of intense chemotherapy, as observed in our patient (see Table 1), could also have been promoted by the observed combination of mutation events. The observed neutropenia, is associated with the risk for developing serious and complicated infections or even sepsis [26][27][28]. Also neutropenic enterocolitis (NE), a necrotizing process usually localized to the ascending colon, cecum, and terminal ileum [29,30] can appear in 15% of AML patients treated with a combination of Idarubicin and Cytosine arabinoside [31], It also associated with increased mortality [32]. Finally, central diabetes insipidus (DI) is a rare complication in AML and myelodysplastic syndrome (MDS) cases with less than 100 cases reported in literature [33]. Central DI can precede the diagnosis of AML or MDS or it can manifest during treatment and was thought to confer a poor prognosis [33]. The pathogenesis of central DI in AML and MDS may be secondary to leukemic infiltration of the infundibulum, hemorrhage, thrombosis, infection, or autoimmunity [34].