Background

Myelodysplastic syndrome (MDS) comprises a heterogeneous group of clonal neoplastic blood diseases characterized by ineffective hematopoiesis, peripheral cytopenias, bone marrow dysplasias and an increased risk of acute myeloid leukemia (AML) [1]. Pediatric MDS is an uncommon disorder accounting for 4–9% of hematologic malignancies [2, 3] and it may be associated with inherited bone marrow failure (BMF) disorders such as Fanconi Anemia (FA) [4].

First described in 1920 by the pediatrician Guido Fanconi [5], nowadays, it is known that FA is a cancer-prone chromosomal instability disorder with diverse clinical symptoms. The congenital anomalies may include skeletal defects, classically abnormal thumb or radius, short stature, café-au-lait spots and endocrinopathies. FA is a rare autosomal and X-linked genetic disease, with a wide variety of symptoms, characterized by congenital abnormalities, progressive BMF and increased cancer risk, which can be difficult to diagnose [5,6,7].

At the molecular level, 21 FA-related genes that constitute FA-BRCA pathway were identified [6]. Proteins encoded by FA-related genes play important roles in various cellular functions, including DNA repair, detoxification of reactive oxygen species and aldehydes, energy metabolism and both pro-inflammatory and myelosuppressive cytokine homeostasis [8].

Since the first clinical report of FA, important laboratory and clinical advances were incorporated in the diagnosis and treatment, such as the DEB test and the application of HSCT, with a low dosage chemotherapy regimen for children with FA [9,10,11].

Supportive care using hematopoietic growth factors such as EPO, G-CSF or androgens such as oxymetholone to boost blood cell production, provides transient benefit in some patients. Although, the allogeneic HSCT remains the main treatment approach to advanced marrow failure in FA patients [11, 12]. In this context, some preclinical studies using gene therapy in an attempt to improve the life quality and survival of patients with FA have been performed [12].

Cytogenetic studies in the bone marrow have an important role in the therapeutic decision, mainly in the indication for HSCT. A better understanding of the clinical relevance and biological implications of clonal chromosomal alterations in FA patients was achieved over the last decade by the incorporation of molecular cytogenetic technologies in addition to classical karyotyping [11, 13]. This has led to the identification of some specific chromosomal alterations in FA patients, their prognostic value and association with the risk of evolution to MDS and/or AML. The most frequent chromosomal alterations in bone marrow of patients with FA are gains of the chromosomal regions 1q and 3q, and partial or complete loss of chromosome 7. Besides, monosomy 7 and del(7q) have been associated with poor prognosis and progression to leukemia. A few reports also suggested that gains of 3q are associated with progression to MDS/AML and overall presenting poor prognosis [13, 14].

Alterations involving del(11q) are uncommon in FA and MDS, so the prognostic value of this alteration remains uncertain [13, 15]. In this region, there are genes such as Ataxia Telangiectasia Mutated gene [ATM (11q22)] and lysine methyltransferase 2A [KMT2A (11q23)] that have been described as having an important role in the pathogenesis of MDS. The ATM gene acts on the regulation of the cell cycle after a DNA damage is recognized [15, 16]. On the other hand, the KMT2A gene encodes a protein that is involved in chromatin remodeling and positively regulates multiple homeobox transcription factors, also it is highly associated with the development of AML [17].

Given the high incidence of hematological complications of FA patients, BM surveillance for morphological and cytogenetic changes provides an important contribution to the clinical decision [11]. However, there are only a few studies in patients with FA showing the bone marrow chromosomal alterations analyzed by classical and molecular cytogenetics associated with evolution to MDS and AML [13, 14, 17,18,19]. Here, we describe an uncommon yet unreported t(9;11)(p24;q22) with monoallelic loss of ATM and KMT2A genes, defined by classical cytogenetic and FISH analysis, in a child with MDS/AML who evolved from FA associated with poor clinical outcome.

Case presentation

A five-year-old male patient with recurrent infections and persistent anemia was admitted at the National Cancer Institute, Rio de Janeiro, Brazil. Physical examination showed a small stature (<P2); hyperpigmentation around the eyes; enophthalmia; multiple cafe-au-lait spots; hypoplasia of the thenar eminence accompanies left thumb hypoplasia. Laboratory findings: Hb 9.1 g/dl (age-adjusted 13.5–18.0 g/dl), platelet count 40 × 109/l (150-400 × 109/l) and white blood cell count 7.6 × 109/l (age-adjusted range 4-10 × 109/l). BM findings: hypocellularity and normal karyotype by G-banding, according to the International System form Human Cytogenomic Nomenclature (ISCN 2016) [20]. Clinical genetic exams were done at Medical Genetic Department, Fernandes Figueira National Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil. Chromosome breakage test cytogenetic analysis was performed in peripheral lymphocytes during 72 h of cultures exposed to DEB (0.1 μg/ml), according to Auerbach [9]. This analysis demonstrated spontaneous chromosome breakage - 0.16 breaks per cell (reference 00.00–0.08) and DEB-induced chromosome breakage - 2,32 breaks per cell (reference 0.00–0.08), confirming the FA diagnosis. He was hospitalized due to the persistent anemia and progressive neutropenia. Oxymetholone (50 mg/day) was the initial treatment. A partial hematological response was achieved, even with oxymetholone dose reductions due to liver toxicity. However, the hematological parameters worsened and erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF) were associated, as well as danazol 200 mg/day was introduced. He also received multiple blood transfusions during the treatment but no satisfactory response was achieved. At this time, BM evaluation revealed dysplastic megakaryocytes, intense dyserythropoiesis and 11% of myeloblasts. Immunophenotypic analysis of BM cells also revealed 11% of myeloblasts expressing CD34/CD13/CD11b (54.25%), HLA-DR/CD33/CD7 (31,54%), dysplastic erythropoiesis (CD36/CD71/CD235a), dysgranulopoiesis (CD13/CD16/CD11b/Cd33/CD64/CD15,CD45) and monocytic lineage expressing CD14/CD64/CD36/HLA-DR,CD45. Cytogenetic analysis with G-band technique in bone marrow cells showed an abnormal karyotype: 46,XY,der(9)t(9;11)(p24;q?22)[9]/46,XY[12] (Fig. 1a). The diagnosis was MDS, refractory anemia with excess of blasts (RAEB). Fluorescence in situ hybridization (FISH) was performed to analyze some genes that may be altered during chromosomal rearrangement. So, we investigated the CDKN2A gene (located in 9p) and the ATM and KMT2A genes (located in 11q22 and 11q23, respectively), due to the important role they play during leukemogenesis. The FISH analysis for the CDKN2A gene showed two normal signals (Fig. 1b). It was observed a monoallelic loss of KMT2A and ATM genes (Fig. 1c and d, respectively). The final karyotype with G-banded and FISH analysis, according to the ISCN 2016 [20], was: 46,XY,der(9)t(9;11)(p24;q?22),der(11)t(9;11)(p24;q?22)[9].ish del(11)(q22.3q23)(ATM-)(KMT2A-)[7]. Treatment with decitabine was initiated and well tolerated, although progression to AML occurred 3 months later. He had no remission with systemic chemotherapy. The patient died 8 months after the diagnosis of RAEB-t, due to disease progression and infectious complications. Allogeneic HSCT was indicated early in the diagnosis, but non-consanguineous parents were available and no donor was found.

Fig. 1
figure 1

a G-banded showing the der(9)t(9,11)(p24;q?22). The black arrows point the gain of chromosome material on 9p and the loss on 11q; b FISH analysis using LSI p16 probe [LSI p16 (9p21), red signal/CEP 9 (9p11-q11), green signal, Dual Color Probe, Vysis] and inverted DAPI, showing that CDKN2A gene was normal in a metaphase. The black arrows shows both CDKN2A normal signals; c FISH analysis using LSI KMT2A Dual Color break apart rearrangement probe Vysis, showing the monoallellic loss of the KMT2A gene with the loss of one signal in interphase nuclei (white arrows) and in metaphase using inverted DAPI (black arrow). d FISH using LSI ATM (11q22) spectrum orange probe, Vysis showing the monoallellic loss of ATM with the loss of one signal in interphase nuclei (white arrows) and in metaphase using inverted DAPI (black arrow)

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Discussion and conclusions

Bone marrow failure occurs with a median age of 8 years and remains the primary cause of morbidity and mortality in FA [12]. The FA cells have a reduced fidelity in processing DNA double-strand breaks. This specific intrinsic susceptibility might, together with extrinsic factors, influence the course of the disease, resulting in the outgrowth of clones with chromosomal aberrations in the bone marrow cells [6]. Nevertheless, there is a cytogenetic clonal fluctuation in patients with FA, since clones often can disappear, reappear, evolve or be replaced by entirely new clones [10]. Therefore, the significance and the predictive value of such clonal alterations with respect to malignant progress are not fully understood [6].

Rochowski et al., (2012) hypothesized that unique bone marrow cytogenetic clones may distinguish patients with FA and AML from patients with de novo AML. Some chromosomal abnormalities as the gain of 1q, 3q, 13q and partial loss of 7q, 20q, 11q or complete loss of chromosome 7 are more frequent in patients with FA who showed progression to AML. While others like trisomy 8, t(8;21), t(9;11), t(6;9) and inv.(16) have been described exclusively in patients with de novo AML [21]. In this study, the t(9;11) was described in MDS/AML secondary from FA. Nevertheless, it is important to note that the breakpoints involved in this chromosomal translocation were not yet reported [22, 23]. In literature, the recurrent breakpoints involved in the t(9;11) in de novo AML patients were: t(9;11)(q34;q23) with FNBP1/KMT2A gene rearrangement [24]; t(9;11)(q34;p15) with NUP98/PRRX2 [25]; t(9;11)(q34;q23) with AF9q34/MLL [26] t(9;11)(p22;p15) with NUP98/PSIP1 [27]; and t(9;11)(p21;q23) with MLLT3/KMT2A [28]; t(9;11)(q34;q23) with DAB2IP/KMT2A [29].

In our study, the patient with FA showed the acquisition of a der(9)t(9;11)(p24;q?22), der(11)t(9;11)(p24;q?22) associated with evolution from MDS to AML. In FISH analysis, it was detected the monoallelic loss of ATM gene. The ATM gene is located at 11q22 region. This gene encodes a serine/threonine protein kinase, a critical enzyme in the regulation of the stress response to DNA damage, especially double-strand DNA breaks and it is also involved in cell cycle control. The ATM gene appears to act as a “caretaker” of the genome [16]. The loss of ATM has been described as having contributed to the increased need for transfusion in patients with MDS associated with del(11q) [15]. Currently, it has been demonstrated the joint and reciprocal action of ATM and FA proteins in the DNA repair pathway during replication [30, 31].

In our study, the FISH analysis also showed the monoallelic loss of the KMT2A gene. The KMT2A locus is involved in more than 60 different chromosomal translocations in pediatric acute leukemias [17]. In AML, the t(9;11) usually results from KMT2A gene translocation, being the (p22;q23) the breakpoint frequently described [11]. Interestingly, it was detected the monoallellic deletion of KMT2A gene in our study. Wang et al. showed that deletions of chromosome 11q lack cryptic KMT2A rearrangements in patients with MDS and suggested that loss of tumor suppressor genes located in 11q through deletions and secondary allelic loss of heterozygosity is likely more important in pathogenesis and disease progression [15]. The detection of all possible types of KMT2A cyto-molecular abnormalities is of key importance for the identification of biological subgroups, which may differ in clinical outcome [30].

In the literature, data on FISH studies in FA are quite limited [14]. In our study, the analysis by the FISH technique was essential allowing the identification at the molecular level of the monoallelic deletion involving the KMT2A and ATM genes. With the FISH results it was possible to note that this chromosomal abnormality was not a simple reciprocal translocation involving the regions of chromosomes 9p24 and 11q22, but an unbalanced chromosomal alteration due to the monoallelic loss of ATM and KMT2A genes. FISH continues to be considered an important technique for molecular investigation of small or hidden chromosomal abnormalities [32].

Taken together, the monoallelic deletions of the genes KMT2A and ATM, probably, contributed to the defects in the genomic instability of the chromatin remodeling, conferring a poor prognosis, being associated to a rapid leukemic transformation and a poor response to treatment. Furthermore, with our results we contribute to the literature showing an yet unreported der(9)t(9;11)(p24;q?22),der(11)t(9;11)(p24;q?22) with monoallelic loss of ATM and KMT2A genes.