GATA1 as a new target to detect minimal residual disease in both transient leukemia and megakaryoblastic leukemia of Down syndrome

Leukemia Research 29 (2005) 1353–1356

Brief communication

GATA1 as a new target to detect minimal residual disease in both transient leukemia and megakaryoblastic leukemia of Down syndrome Sharon R. Pine ∗ , Qianxu Guo, Changhong Yin, Somasundaram Jayabose, Oya Levendoglu-Tugal, M. Fevzi Ozkaynak, Claudio Sandoval Department of Pediatrics, New York Medical College, Basic Science Building Room 401, Valhalla, NY 10595, USA Received 9 December 2004; received in revised form 4 April 2005; accepted 5 April 2005 Available online 23 May 2005

Abstract Acquired mutations in exon 2 of the GATA1 gene are detected in most Down syndrome (DS) patients with transient leukemia (TL) and acute megakaryoblastic leukemia (AMKL). We sought to determine if GATA1 mutations can be utilized as markers for minimal residual disease (MRD). GATA1 mutations were screened by SSCP analysis and sequenced. Using GATA1 mutation-specific primers, follow-up bone marrow samples from four patients were assayed by quantitative PCR. We show that molecular monitoring of GATA1 mutations is possible in Down syndrome patients with TL and AMKL, and GATA1 could be a stable marker for MRD monitoring. © 2005 Elsevier Ltd. All rights reserved. Keywords: Transient leukemia; Acute megakaryoblastic leukemia; GATA1; Real-time PCR

1. Introduction Children with Down syndrome (DS) have a 500-fold increased risk of developing acute megakaryoblastic leukemia (AMKL) compared to non-DS children [1]. Transient leukemia (TL), a clonal disorder affecting the megakaryocytic lineage, is observed in 10% of infants with DS [1]. The megakaryoblasts in TL and AMKL are morphologically [2] and immunophenotypically [3] similar, and share common karyotypic abnormalities [4], suggesting that AMKL derives from TL blasts. Although most cases of TL spontaneously regress without clinical complications, some infants require low-dose chemotherapy. Up to 30% of patients with TL eventually develop AMKL, signifying that residual TL blasts can remain at sub-clinical levels after resolution. Currently, there are no clinical, hematological or cytogenetic differences to predict which patients with TL will develop AMKL or which patients with AMKL will relapse. Due to a lack of known suitable molecular markers in DS TL and AMKL, monitor∗

Corresponding author. Tel.: +1 914 594 3725; fax: +1 309 279 5833. E-mail address: sharon [email protected] (S.R. Pine).

0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.04.007

ing such patients for minimal residual disease (MRD) has not been feasible. A stable molecular target to monitor TL and AMKL in DS and to aid in the diagnosis of AMKL could be clinically valuable. Acquired somatic mutations within exon 2 of the GATA1 gene are believed to be present in the majority of DSassociated TL and AMKL and absent in non-DS AMKL and DS acute lymphoblastic leukaemia [5]. The GATA1 mutation may be a stable marker because the same molecular mutation is identified during the TL and AMKL phases [1,6,7], and because it disappears upon disease resolution [8]. We report for the first time that GATA1 mutations can be utilized as targets for monitoring TL disease resolution and patient response to AMKL-directed chemotherapy.

2. Methods 2.1. Patients and samples Five patients (four with DS; one with trisomy 21 mosaicism) were included in this study. Patient 1 had TL, patient

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Table 1 Analysis of GATA1 exon 2 and quantitative real-time PCR assessment of MRD Patient

Diagnosis

Sex

DNA source blasts in PB/BM (%)

GATA1 mutation (NM 002049)

Clone-specific primer sequence (5 to 3 )

1

TL

F

PB (85)

279del 3bp; ins 1bp

CAC AGC CAC CGC TGC TA

2

TL

F

PB (53) (24)a

3

AMKL

M

BM

4

AMKL

F

BM (67)

5

AMKL

M

BM

(ND)c

Time from diagnosis (months)

MRD level

Outcome, months

0.75 16

1 × 10−3 Negative

CR, 16

202del 2bp

CTC CAC ACC AGA ATC GGG TT

30

Negative

CR, 46

287ins 14bp

AGC TGC GTG GCT GCA GCT

−5 6b 9 13 18 20 21

20% 100% 1 × 10−4 Negative Negative Negative Negative

Second CR, 60

276del 57bp

AGC ACA GCC ACC GGT AAC

0.5 1 2.5 8 14

2 × 10−4 Negative Negative Negative Negative

CR, 48

ND

CR, 16

Wild-type

ND

1.5

TL, transient leukemia; AMKL, acute megakaryoblastic leukemia; PB, peripheral blood; BM, bone marrow; ND, not done; CR, complete remission. a This is the BM sample taken at the time of diagnosis of AMKL. b This patient experienced a BM relapse 6 months after diagnosis of AMKL. c BMA was fibrotic and accurate assessment of blast percentage could not be made.

2 had trisomy 21 mosaicism and TL, patient 3 had both TL and AMKL, and patients 4 and 5 had AMKL without prior known TL. Patients were treated at New York Medical College, Westchester Medical Center and diagnosis bone marrow (BM) or peripheral blood (PB) samples were obtained retrospectively. Fifteen serial samples were available for MRD testing. This study was approved by the Institutional Review Board of New York Medical College and informed consents were obtained.

PCR, samples were tested in duplicate. Standard curves derived from DNA samples from which the GATA1 mutation was identified. For limiting dilution PCR, samples were tested in replicates of 10. Sensitivity was determined as the lowest dilution in which both duplicates were positive (real-time PCR) or at least 4 of 10 replicates were positive (limiting dilution) as described [9,10]. Quantification of MRD was achieved after normalization to the ␤-globin gene [10].

2.2. Mutation analysis

3. Results

GATA1 exon 2 genomic DNA was amplified by PCR using primers previously published [1] and standard PCR conditions (35 cycles of 94 ◦ C for 10 s, 60 ◦ C for 10 s, and 68 ◦ C for 60 s). Amplification products were purified by SSCP analysis and stained with Gel-star (Cambrex, East Rutherford, NJ). Aberrant-sized bands were excised and directly sequenced [9].

We found GATA1 mutations in four of five patients each resulting in the introduction of a premature stop codon previously described in DS-TL and DS-AMKL patients [11], as shown in Fig. 1A and Table 1. We successfully designed patient clone-specific primers encompassing the unique mutation region (Table 1) and amplified DNA harboring the GATA1 mutation for all four patients by quantitative PCR (sensitivity, 1 × 10−4 for pts. 1, 2, and 4, and 1 × 10−5 for pt. 3). Clinical information and MRD results are summarized in Table 1.

2.3. Quantification of GATA1 in follow-up samples Patient samples taken after diagnosis of TL and/or AMKL were examined for evidence of GATA1 mutation persistence by real-time PCR using the Lightcycler [10] (pts. 1, 2, and 4) or by limiting dilution quantitative PCR [9] (pt. 3). GATA1 mutation-specific forward primers are listed in Table 1. Reverse primer sequences were the following: 5 -GACCTAGCCAAGGATCTCCA-3 (pts. 1 and 4); 5 -TGGAGGAAGCTGCTGCAT C-3 (pt. 2); 5 GACCTAGCCAAGGATCTCCATG-3 (pt. 3). For real-time

3.1. Patient 1 A 1-day-old girl was transferred because of leukocytosis and hepatomegaly. The BMA was consistent with TL. Cytarabine 1.5 mg/kg/dose was administered subcutaneously every 12 h for eight doses. Her clone-specific GATA1 mutation persisted in her BM 21 days after diagnosis at a level of 1 × 10−3 (Fig. 1B) and was absent in her PB 16 months after diagno-

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3.3. Patient 3 A 15-month-old male, who had TL as an infant, presented with pancytopenia and myelodysplasia. The BMA examination was difficult to interpret and showed poor myeloid maturation without blasts. However, when analyzed retrospectively by PCR, the BM was positive for a GATA1 mutation at a level of 20%. At 20 months of age, AMKL was diagnosed based on BM blasts, flow cytometry, and abnormal cytogenetics. The disease remitted after one cycle of chemotherapy. At 6 months after diagnosis, the leukemia recurred with 100% blasts, and 100% (quantified by PCR) of the BM harbored the same GATA1 mutation-positive cells found at AMKL diagnosis and 5 months prior to diagnosis. A second remission was attained after chemotherapy. Remission BM drawn 9 months after initial AMKL diagnosis was positive at 1 × 10−4 for the GATA1 mutation, although no blasts were found, and BM samples tested by PCR at 13, 18, 20, and 21 months after initial AMKL diagnosis were negative. He remains in remission 60 months after the initial AMKL diagnosis. 3.4. Patient 4

Fig. 1. Mutation of GATA1 in TL and AMKL. (A) SSCP of GATA1 exon 2 revealed the presence of mutations in patients 1–4. Only wild-type (Wt) GATA1 was found in patient 5. Arrows show the PCR products from Wt GATA1. (B) Real-time PCR plot of cycle number vs. fluorescence shows mutation-specific GATA1 amplification. Curves represent dilutions of patient 1 TL diagnosis peripheral blood DNA from 5 × 10−2 to 1 × 10−4 (in a background of 20,000 genomic copies of non-leukemic DNA), and patient 1 remission BM slide DNA from 3 weeks after diagnosis. Samples were amplified in duplicates. Negative controls were water and pooled normal BM (NBM) from 20 patients. The level of GATA1 mutation positive cells in the remission sample was determined to be 1 × 10−3 , after normalization to the ␤-globin gene (not shown).

sis. The TL resolved 8 weeks after diagnosis. She remains in remission 16 months after diagnosis.

An 11-month-old girl presented with fever, irritability, and pallor. Physical examination showed hepatosplenomegaly. BMA was consistent with a diagnosis of AMKL. Chemotherapy was administered. Her clone-specific GATA1 mutation remained in the BM 2 weeks after AMKL diagnosis at a level of 2 × 10−4 , and subsequently was undetected in the BM 1, 2.5, 8, and 14 months after diagnosis. She remains in remission 48 months after diagnosis. 3.5. Patient 5 A 27-month-old boy was diagnosed with AMKL. No GATA1 mutations were identified by SSCP. Amplification products were directly sequenced to confirm the wild-type configuration, and the remainder of GATA1 exons were analyzed and found to be wild-type (primer sequences available upon request). It is possible the fibrotic biopsy harbored too few blasts to detect a GATA1 mutation. Complete remission was achieved after chemotherapy. He remains in clinical remission 16 months after diagnosis.

3.2. Patient 2 4. Discussion A 1-day-old girl was transferred because of anemia, hyperleukocytosis and pericardial effusion. Physical examination showed a phenotypically normal infant with hepatosplenomegaly and an erythematous, papular rash. A diagnosis of TL was confirmed when the rash and organomegaly spontaneously resolved and the blood counts normalized after 8 weeks of age. PB obtained 2.5 years after diagnosis was negative for her clone-specific GATA1 mutation. At 46 months after diagnosis, the physical examination and blood counts remain normal.

Our data show that quantitative PCR amplification of patient clone-specific GATA1 mutations may be an important tool for monitoring resolution of TL and response to antiAMKL treatment in DS patients harboring mutations. Moreover, GATA1 mutations may be relevant in the diagnosis of AMKL when BM findings in patients with previous TL are equivocal. This would be particularly useful when diagnosis of AMKL in DS is complicated by myelofibrosis and the presence of myelodysplastic syndrome. The data support the

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contention that GATA1 mutations are stable markers because the same mutation was present before and during AMKL and AMKL relapse in patient 3. All four patients tested in this study eventually became negative for the clone-specific GATA1 mutation, suggesting that clone-specific PCRdetectable levels of GATA1 do not typically remain after disease resolution. Therefore, detection of GATA1 by PCR could have clinical significance. The sensitivity of the mutationspecific PCR assay is at least 1 × 10−4 , which is comparable to other molecular leukemia targets [10] and is adequate for monitoring patients. GATA1 mutation-specific real-time PCR is fast and reliable. Larger and prospective studies are needed to assess the clinical relevance of this new tool.

Acknowledgements This work was supported by a grant from the Children’s Cancer Fund (Millwood, New York). S. Pine contributed to concept and design, analysis and interpretation of data, and drafting of the article. Q. Guo collected and assembled the data. C. Yin collected and assembled the data. S. Jayabose provided study materials of patients, and gave final approval of the article. O. Tugal provided study materials of patients, and provided critical revisions and scientific content. M. Ozkaynak provided critical revisions and scientific content. C. Sandoval contributed to concept and design, and critical review and scientific content. References [1] Hitzler JK, Cheung J, Li Y, Scherer SW, Zipursky A. GATA1 mutations in transient leukemia and acute megakaryoblastic leukemia of Down syndrome. Blood 2003;101(11):4301–4.

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