Acute megakaryoblastic leukemia and severe pulmonary fibrosis in a child with down syndrome: Successful treatment with ultra low‐dose cytarabine using <i>GATA1</i> mutation to monitor minimal residual disease
Authors: Hiroshi Moritake, Ai Yamada, Yasuhiro Kimoto, Daisuke Sawa, Hidemi Shimonodan, Hiroyuki Nunoi
Journal: American Journal of Hematology (2012)

Children with Down syndrome (DS) have a high risk of acute megakaryoblastic leukemia (AMKL). Unlike AMKL in children without DS, AMKL-DS shows a uniquely favorable response to chemotherapy, especially to cytarabine. Since increase in treatment intensity in AMKL-DS enhances treatment-related mortality and decreases survival rate, lowering intensity of chemotherapy has been attempted. However, the risk-adapted appropriate intensity is not known. The authors describe an AMKL-DS case who suffered from severe pulmonary fibrosis and required tracheostomy. The authors identified the unique GATA1 mutation site in the leukemic cells of the patient and succeeded in performing real-time quantitative polymerase chain reaction (RQ-PCR) for measurement of minimal residual disease (MRD). To prevent fatal respiratory infection during myelosuppression with conventional aggressive chemotherapy, the patient basically received 12 courses consisting of 14 days of ultra low-dose cytarabine (10 mg/m2/dose, twice per day) for 12 months using the GATA1 mutation to monitor MRD and has been in complete remission for more than 23 months after achieving her first complete remission. Ultra low-dose cytarabine treatment using GATA1 mutation by RQ-PCR to monitor MRD is an attractive candidate treatment in AMKL-DS. Acute megakaryoblastic leukemia (AMKL) occurs with an estimated 500-fold increase in children with Down syndrome (DS) when compared with non-DS children [1]. Transient myeloproliferative disorder (TMD), a clonal disorder affecting the megakaryocytic lineage, is observed in 10% of infants with DS but may actually be higher because some affected fetuses die in utero [2]. TMD usually resolves spontaneously within 2–3 months, but 20–30% of patients develop overt AMKL within 4 years post-TMD [2]. The megakaryoblasts in TMD and AMKL are morphologically and immunohistochemically similar, and share common karyotypic abnormalities, suggesting that AMKL derives from TMD blasts [3, 4]. The GATA1 gene encodes a zinc-finger transcription factor that is essential for normal erythroid and megakaryocytic differentiation [5, 6]. Somatic mutations of GATA1 are found in almost all cases of DS associated TMD and AMKL [7]. Both trisomy 21 and GATA1 mutations can be somatically acquired in utero, and they are necessary for the development of TMD and an additional genetic hit may be acquired for the development of AMKL. Measurements of minimal residual disease (MRD) have become a crucial component of contemporary treatment protocols in leukemia. Since GATA1 mutations in AMKL-DS are clone-specific, they are valuable targets for MRD [8]. Before the 1990s, most patients with AMKL-DS were treated outside clinical trials and received suboptimal therapy, resulting in dismal outcomes. The first report that there was a significantly better outcome for children with AMKL-DS came from the Pediatric Oncology Group in 1992 [9]. AMKL-DS shows a uniquely favorable response to chemotherapy in contrast to the general population [9-12]. Increased sensitivity of myeloid leukemic blasts in DS to cytarabine was demonstrated in vitro [13, 14]. Reducing intensity or prolonging intervals between chemotherapy courses have proved to be successful for AMKL-DS [11, 12]. The authors present a case with AMKL and severe pulmonary fibrosis in a child with DS who received ultra low-dose cytarabine using the GATA1 mutation to monitor MRD. A 2.4-kg female was born at 36-weeks gestation. Physical examination showed hepatosplenomegaly in addition to phenotypic features of DS. She presented with mild respiratory distress and required oxygen, but not artificial surfactant supplement. The blood count was as follows: hemoglobin 15.9 g/dL, platelets 788 × 109/L, and white blood cell count (WBC) 100.5 × 109/L with 72% blasts showing distinct blebs in cytoplasms. Flow cytometric analysis revealed these cells were positive for CD7, CD34, CD41a, and CD61. The patient was diagnosed with TMD. Serological findings showed increased levels of asparate aminotransferase 95 IU/L, alanine aminotransferase 149 IU/L, and lactate dehydrogenase 3,681 IU/L. Peripheral blood blasts disappeared within 2 months and hepatic function normalized within 1 month without chemotherapy. However, her respiratory status gradually deteriorated to progressive pulmonary fibrosis and continuous oxygen flow was increased. Her serum Krebs von den Lungen-6 (KL-6) level, which is a glycoprotein secreted by type II alveolar pneumocytes and bronchiolar epithelial cells and has been described as a promising biomarker for interstitial lung disease [15], was extremely high 14,500 U/ml (<500 U/mL). At 7 months of age, the patient received prednisolone 2 mg/kg/day, although the etiology of pulmonary fibrosis had not been elucidated. The efficacy was modest and dyspnea progressed increasing the need for oxygen. At 13 months of age, the patient developed pallor and intubation was performed. CBC showed anemia (4.5 g/dL), thrombocytopenia (38 × 109/L), and elevated WBC 26.2 × 109/L with 8% blasts. Bone marrow analysis revealed increased megakaryoblasts (21%). The blast population expressed megakaryocyte-associated antigens similar to those seen in the TMD. Cytogenetic analysis revealed karyotype 54, XX, +8, +14, +15, +19, +21, +21, +21, +22. There was a 25-bp deletion in the splice acceptor site at 3′ intron/exon boundary in GATA1 intron 1 (Fig. 1A). Given these clinical and laboratory findings, the patient was diagnosed with AMKL. Chest computed tomography revealed diffuse crazy-paving pattern (Fig. 2). Considering her respiratory state, conventional aggressive chemotherapy could prove fatal, if myelosuppression is complicated with respiratory infection. Therefore, the authors successfully designed patient clone-specific TaqMan probe encompassing the unique GATA1 mutation region and primers for the surrounding region. Sensitivity of MRD could be established at 10−3 to 10−4 (Fig. 1B). Intravenous injections of ultra low-dose cytarabine 10 mg/m2 once per day for 12 days as induction therapy led to complete remission (CR). The level of MRD declined under the detection limit (between 10−3 and 10−4) after this induction therapy. After achieving CR, the authors tried to double the dose of intravenous cytarabine to 10 mg/m2 twice per day for 14 days while using GATA1 to monitor MRD (Table I). At 15 months of age, after two courses of chemotherapy, tracheostomy was performed for long-term respiratory support (Table I). Transient anemia requiring one red blood cell transfusion per course and mild thrombocytopenia (platelet count above 75 × 109/L) were the only recognized adverse reactions of chemotherapy throughout the entire course. All 12 courses of chemotherapy was complicated by pulmonary bacterial infections requiring intravenous antibiotics. Pseudomonas aeruginosa was isolated in sputum cultures in each course. However, these were not fatal in most courses because neutrophil counts were always kept above 1.0 × 109/L except for the induction phase. The duration of chemotherapy was shortened in the 6th and the 12th courses, because of complications with severe pulmonary Pseudomonas aeruginosa infection and respiratory synctial virus infection, respectively (Table I). MRD levels were examined before initiation of the next chemotherapy course and remained less than 0.1% for 1 year after achieving undetectable levels before the second course (Table I). The patient has been in CR for more than 23 months after achieving her first CR. (A) GATA1 gene, mRNA, and truncated protein GATA1s. Genomic DNA encoding GATA1 is shown. The exons are depicted as boxes and numbered. The open parts of the exons are 5′ and 3′ noncoding regions. The shaded areas are coding regions. Positions of the two translational start sites (ATG) are marked by arrowheads. The sequence in splice acceptor site at the 3′ intron/exon boundary between intron1 and exon 2 is described. The deletion of 25 bp in intron 1 and exon 2 is shown in large letters. The effect of the deletion is the production of truncated protein GATA1s. NF and CF represent N-terminal zinc finger and C-terminal zinc finger, respectively. (B) Real-time polymerase chain reaction plot of cycle number vs. fluorescence shows mutation-specific GATA1 amplification. Curves represent dilutions of the patient bone marrow DNA at diagnosis from 1 × 10−1 to 5 × 10−4. All bone marrow samples of the patient after induction therapy were not amplified as well as water and peripheral blood of a healthy volunteer as negative control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Chest radiograph and high-resolution computed tomography (HRCT) scan. Chest X-ray shows diffuse bilateral interstitial infiltrates. HRCT scan shows bilateral diffuse ground-glass attenuation and inter- and intralobular septal thickening (crazy-paving pattern) and emphysema in the peripheral lung fields. Before the 1990s, most patients with AML-DS were treated outside clinical trials, resulting in dismal outcomes. After the clinical trials in the 1990s, it became apparent that induction failure and relapse are rare, but treatment-related deaths are frequent [9]. Recently, the 5-year event-free survival has exceeded 80% with reduction of treatment-related deaths by reducing treatment intensity [11, 12]. Our patient had severe pulmonary complication. Surfactant protein B (SP-B) and surfactant protein C (SP-C) are hydrophobic proteins that interact with surfactant lipids to facilitate adsorption to the air–liquid interface. ATP-binding cassette sub-family A member 3 (ABCA3) are highly expressed in alveolar epithelial cells at the limiting membrane of lamellar bodies and may play a role in lipid homeostasis of lamellar bodies. Mutations in these genes involved in surfactant metabolism have been reported in pediatric pulmonary fibrosis [16]. Pulmonary alveolar proteinosis is a rare syndrome characterized by intra-alveolar accumulation of surfactant components and cellular debris with interstitial inflammation or fibrosis. Granulocyte-macrophage colony-stimulating factor (GM-CSF) pathway has been shown to be involved in the disease pathogenesis [17]. The authors excluded genetic disorders that elicit progressive pulmonary fibrosis and alveolar proteinosis, such as SP-B, SP-C, ABCA3, and GM-CSF receptor α and β chain by mutation analysis using genomic DNA and total RNA from peripheral blood mononuclear cells (Supporting Information Table S1). The authors also examined her serum anti-GM-CSF antibody and excluded idiopathic pulmonary proteinosis [18]. Furthermore, the authors analyzed 28 cytokines and chemokines in the patient's serum at diagnosis of AMKL. Among them, only two chemokines [interferon γ-inducible protein (IP10) and monocyte chemoattractant protein-1 (MCP1)] were significantly elevated (data not shown). IP10 is a chemokine, which functions as an inhibitor of pulmonary fibrosis [19]. Inversely, MCP1 is a chemokine working as an inducer of pulmonary fibrosis [20]. On the other hand, TGFβ1 and PDGFββ, which are produced by megakaryoblasts and cause organ fibrosis in TMD, were within normal limits [21-23]. The liver is widely known as a representative fibrogenic organ in TMD. There are several reports that other organs, such as the kidney, pancreas, and the lung were involved with severe fibrosis [23-25]. The possibility of TMD-related pulmonary fibrosis in our patient could not be completely excluded, however, the probability is low because TMD-related organ fibrosis usually occurs with fatal hepatic fibrosis [23-25]. No severe hepatic fibrosis was found at the time of TMD and AMKL, and hepatic function has been within normal limits from age 1 month after resolution. The etiology of her progressive pulmonary disorder still has not been elucidated. Lung biopsy in addition to liver biopsy is necessary to elucidate the cause of her pulmonary fibrosis, however, these procedures themselves, especially lung biopsy, may prove fatal in our patient. In general, children with DS have a variety of complications, some of them hindering aggressive conventional chemotherapy, as with our patient. Ultra low-dose cytarabine therapy was definitely effective for our patient, because the level of MRD declined under the detection limit (between 10−3 and 10−4) after a single course of 10 mg/m2 of cytarabine once per day for 12 days as induction therapy. Al-Ahmari et al. [26] has already demonstrated that an ultra low-dose cytarabine-based regimen including vincristine and retinylpalmitate for 2 years was a curable strategy for more than half of AMKL-DS patients. Tchernia et al. [27] also reported that single ultra low-dose cytarabine treatment for 2 years cured three patients among seven patients. Our case is the first reported AMKL-DS treated by single ultra low-dose cytarabine for 1 year only using the GATA1 mutation to monitor MRD. Tandonnet et al. [28] suggests that myeloid leukemia-DS patients had to be offered standard chemotherapy including cytarabine and anthracyclines with significant dose reduction rather than a low-dose cytarabine-based protocol. The intensity of chemotherapy needed to cure patients differs among individuals. GATA1 MRD is useful for treating safely yet keeping efficacy of the treatment, whatever treatment strategy is selected. Our report suggests that ultra low-dose cytarabine therapy while monitoring GATA1 MRD is a useful treatment option for AMKL-DS patients at high risk of infectious complications and may be used in a larger concept of a risk-stratified treatment approach in AMKL-DS. Karyotype analysis was performed using a bone marrow sample. For a cytogenetic study, chromosomes were prepared using standard techniques and analyzed by Trypsin–Giemsa banding as described earlier [29]. Karyotypes were determined according to the International system for Cytogenetic Nomenclature. Genomic DNA and total RNA were prepared from bone marrow and peripheral blood obtained from the patient using Puregene DNA isolation kit (Gentra Systems Inc, Minneapolis, MN) and TRIzol (Invitrogen, Carlsbad, CA). To analyze the GATA1 gene, the coding exon 2 and the adjacent intronic regions were amplified by PCR. The primers used were as follows: gata4642-F 5′TGGAGGAGAAAAAGGAAGCA3′ and gata5924-R 5′CTCCATACAGTTGAGCAATGGG3′. Other primer sequences are listed in Supporting Information Table S1. PCR amplification was performed in a 50-μL reaction volume using PfuUltra II fusion HS DNA polymerase according to the manufacture's protocol (Stratagene, La Jolla, CA). Thermal cycling was performed with an initial denaturation at 95°C for 2 min, 30 cycles of denaturation at 95°C for 20 s, annealing at 55°C for 20 s, and extension at 72°C for 30 s, and then a final step at 72°C for 3 min. PCR products after gel purification were directly sequenced with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). Sequencing was performed with an ABI model 3130XL (Applied Biosystems). Serum GM-CSF autoantibody concentration was measured by ELISA as described earlier [30]. Serum cytokine and chemokine analyses were performed on blood collected from the patient at the time of diagnosis of AMKL. Blood was collected in a serum-separator collection tube, centrifuged for 10 min at 1,320 rpm, and the serum was removed and stored at −70°C until assayed. Twenty-eight cytokines and chemokines (IL1β, IL1ra, IL2, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IL13, IL15, IL17, Eotaxin, FGF basic, G-CSF, GM-CSF, TNFα, TGFβ1, IFNγ, IP10, MCP1, MIP1α, MIP1β, PDGFββ, VEGF, and RANTES) were assayed using the Bio-Plex suspension array system (Bio-Rad Laboratories, Hercules, CA). The assay was performed according to the manufacture's instructions. Sera obtained from volunteer healthy donors were used as controls. Patient samples taken after diagnosis of AMKL were examined for evidence of GATA1 mutation persistence using real-time PCR using the Lightcycler. GATA1 mutation-specific primers and probe were as follows: forward primer 5′ GGAGAAATATGGAGACTGAGGTGATG 3′, reverse primer 5′ CAGGCCAGGGAACTCCATG 3′, and TaqMan MGB probe 5′ TAACCTGCAGAAATC 3′. For real-time PCR, samples were tested in duplicate. Standard curves derived from plasmid, which had patient-specific GATA1 mutation sequence. Sensitivity was determined as the lowest dilution in the sample at the diagnosis diluted in normal DNA. The authors are grateful to Ms. Megumi Obara for her technical assistance and Dr. Kaede Yanagita for her useful comments on this manuscript. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Hiroshi Moritake*, Ai Yamada*, Yasuhiro Kimoto*, Daisuke Sawa*, Hidemi Shimonodan*, Hiroyuki Nunoi*, * Division of Pediatrics, Department of Reproductive and Developmental Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.