Acute Myeloid Leukemia with Complex … Myeloid Leukemia with Complex Hypodiploidy and ... (DEB), and melphalan [4], ... BM study revealed mark-Published in: Annals of Clinical and

Acute Myeloid Leukemia with Complex Hypodiploidy andLoss of Heterozygosity of 17p in a Boy with Fanconi Anemia

Hye In Woo,1 Hee-Jin Kim,1 Soo Hyun Lee,2 Keon Hee Yoo,2 Hong Hoe Koo,2 and Sun-Hee Kim1

Departments of 1Laboratory Medicine & Genetics and 2Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea

Abstract. Fanconi anemia (FA) is a congenital bone marrow failure syndrome in association with increased susceptibility to malignancy. We report the first in-depth description of a boy with FA who developed acute myeloid leukemia with complex hypodiploidy karyotype after successful stem cell transplantation. Of note, the leukemic cells consistently showed loss of heterozygosity (LOH) of the short arm of chromosome 17 (17p), which harbors the TP53 tumor suppressor gene. The complex hypodiploidy karyotype of the leukemic cells with LOH for 17p may represent a unique karyotypic profile that reflects genomic instability and thereby confers poor prognosis.

Address correspondence to Hee-Jin Kim, M.D., Ph.D., Department of Laboratory Medicine & Genetics, or Hong Hoe Koo, M.D., Ph.D., Department of Pediatrics, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul, Korea, 135-710; e-mail: [email protected], or [email protected].

Introduction

Fanconi anemia (FA) is a genetically and phenotypically heterogeneous congenital disorder characterized by a high incidence of physical abnor-malities, bone marrow failure (BMF), and predisposition to malig-nancy, especially acute myeloid leukemia (AML) [1-3]. Due to the hypersensitivity of FA cells to DNA cross-linking agents such as cis-platin, mitomycin C (MMC), diep-oxybutane (DEB), and melphalan [4], chemotherapy using such agents as cyclophosphamide is challenging in patients with FA [5]. The only curative treatment for BMF in FA is stem cell transplantation (SCT). However, it has been suggested that the risk to develop malignancies, particularly solid cancers, is increased after SCT [6-8]. In this report, the authors describe a 4-yr-old boy with FA who developed AML with a complex hypodiploidy karyotype (or complex karyotype [CK] with hypodiploidy) after SCT.

To our knowledge, this is the first in-depth description of the clinical course and cytogenetic changes of AML in FA.

Case report

A 4-yr-old boy was admitted because of bleeding tendency for 2 months. Initial laboratory evaluation showed Hb 7.9 g/dl, white blood cells 3.54 x 109/L (absolute neutrophil count 0.46 x 109/L), and platelets 40 x 109/L. Peripheral blood smear revealed neither blasts nor dysplastic features. BM study revealed mark-edly decreased cellularity (~10%) without apparent dysplastic features. Cytogenetic analysis using BM cells showed a normal karyotype, 46,XY[20]. He had no physical stig-mata indicative of congenital BMF syndrome such as FA. However, a chromosomal breakage study using DEB and MMC showed chromo-somal instability (3.80 breaks/cell) compared with a normal control (0.30 breaks/cell). Based on this

fiinding along with BMF, the diagnosis of FA was made. To identify the causative mutations of FA in the patient, we performed molecular genetic analyses for 3 FA genes that account for the majority of the disease mutations, FANCA, FANCC, and FANCG genes, by direct sequencing of the patient’s DNA. As a result, we observed no mutations. He underwent an unrelated sex-matched allogeneic bone marrow SCT after two courses of 250 cGy total body irradiation (TBI) in addition to cyclophos-phamide (CY) and anti-thymocyte globulin (ATG). Evaluation of chi-merism status using short tandem repeat (STR) markers showed complete engraftment of donor cells (Table 1). The follow-up course of the patient was uneventful until 1 year after SCT, when recipient DNA was detected at 7.7% on chimerism study (Table 1, BM #5). One month thereafter, a BM study revealed

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Fig. 1. Aspirate smear findings on serial bone marrow study (x1,000). (A) Blasts at the initial diagnosis of AML (BM study #6 in Table 1). (B) Increased blasts with micromegakaryocytes at 1st relapse (BM study #12 in Table 1).

Table 1. Clinical course of the patient represented by cytogenetic and molecular tests on serial bone marrow studies (*FISH and †STR analysis).

BM # Diagnosis Blasts Karyotype Monosomy Recipient (%) 7 (%)* DNA (%)†

1 Dx of AA/FA <5 46,XY[20] - -

2 Post-SCT 1 mo <5 46,XY[20] - 0

3 Post-SCT 3 mo <5 46,XY[20] - 0

4 Post-SCT 6 mo <5 46,XY[20] - 0

5 Post-SCT 12 mo <5 46,XY[20] - 7.7

6 Post-SCT 13 mo; 21 No mitotic cells - 16.3 Dx AML7 1 mo from Dx AML <5 46,XY[20] - 0

8 2 mo from Dx AML <5 46,XY[20] - 0

9 3 mo from Dx AML <5 46,XY[20] - 0

10 5 mo from Dx AML <5 46,XY[20] - 0

11 12 mo from Dx AML <5 42,X,-Y,+3,add(5)(q13),-6,-7,add(11)(p15),add(12)(p13),der(13)t(13;15) (p11.2;q15),-15,add(15)(p11.2),-17,-17,-21,+2mar[2]/46,XY[18] 0 1.4

12 15 mo from Dx AML; 50 42,X,-Y,add(5)(q13),-6,add(7)(q22),add(11)(p15)x2,+add(11)(q25),der(13)t(13;15) Relapse of AML (p11.2;q15),-15,-17,-17 [13]/41,sl,add(3)(q12),del(5)(q32),-add(11)(p15),+mar[7] 46.5 32.7

13 16 mo from Dx AML; <5 46,XY[20] 0 -

14 18 mo from Dx AML; 2nd relapse of AML 9 46,XY[40] 1 2.9

15 19 mo from Dx AML; 1 mo after 2nd SCT <5 46,XY[20] 0 0

16 20 mo from Dx AML; 2 mo after 2nd SCT <5 46,XY[2] 0 0

17 21 mo from Dx AML; 3 mo after 2nd SCT <5 46,XY[8] 1 0

18 24 mo from Dx AML; 6 mo after 2nd SCT <5 46,XY[20] 0 0

19 26 mo from Dx AML; 8 mo after 2nd SCT; 12 40,X,-Y,add(3)(q12),add(5)(q13),-6,add(7)(q22),add(11)(p15), 3rd relapse of AML add(11)(q25),der(13)t(13;15)(p11.2;q15),-15,-15,-16,-17,add(17)(p13), -18,add(19)(p13.3),-22,+2mar[5]/46,XY,del(3)(p21)[2]/46,XY[13] 5.5 3.3

BM, bone marrow; FISH, fluorescence in situ hybridization; STR, short tandem repeat; Dx, diagnosis; AA, aplastic anemia; FA, Fanconi anemia; SCT, stem cell transplantation; AML, acute myeloid leukemia; mar, marker chromosome.

AML with complex karyotype in Fanconi anemia 67

Fig. 2. Representative karyo-types of the patient showing complex karyotype with deletions of chromosome 5q, 7q and -17. (A) At 1st relapse of AML showing 42,X,-Y,add(5)(q13),-6,add(7)(q22),add(11)(p15)x2,+add(11)(q25),der(13)t(13;15)(p11.2;q15), -15,-17,-17[13]/41,sl,add(3)(q12),del(5)(q32),-add(11) (p15),+mar[7] (BM study #12, Table 1). (B) At 3rd relapse of AML showing 40,X,-Y,add(3)(q12),add(5)(q13),-6,add(7)(q22),add(11)(p15),add(11)(q25),der(13)t(13;15)(p11.2;q15),-15,-15,-16,-17,add(17)(p13),-18,add(19)(p13.3),-22,+2mar [5]/46,XY,del(3)(p21) [2]/46,XY[13] (BM study #19, Table 1). Arrows indicate the cytogenetic aberrations.

increased blasts up to 21% of total nucleated cells, with mixed chimer-ism (patient’s DNA 16.3%). Flow cytometric analyses for the immuno-phenotype of the blasts showed CD13+, CD33+, CD15+, CD34+, CD64+, CD65+, CD117+, HLA-DR+, and MPO+, and the diagnosis of AML was made. Due to the poor quality of the specimen, differential counts of BM cells and cytogenetic analysis were not successful and therefore, morphological or cyto-genetic sub-classification of AML was impossible.

The patient received reduced-dose induction chemotherapy with the IDA-FLAG regimen (idarubicin, fludarabine, cytarabine, G-CSF) and stem cell boost. He achieved complete remission. However, BM study 12 months after the diagnosis of AML revealed complex cyto-genetic abnormalities, despite no apparent increase of leukemic blasts (BM #11). Three months thereafter, BM blasts were increased up to 50% with dysplastic features in mega-karyocytes (Fig. 1). Cytogenetic analysis revealed CK with hypo-

diploidy including deletion of 5q, 7q, and 17 (Fig. 2). FISH analysis showed 46.5% nuclei with loss of chromosome 7. Despite chemo-therapy with cytarabine and idarub-icin, the patient experienced a 2nd relapse of AML 3 months after the 1st relapse. BM study revealed blasts up to 9% with dysplastic megakaryo-cytes. At that time, neither cyto-genetic analysis nor FISH study showed chromosomal abnormalities, which was considered to be due to peripheral blood dilution. In line with this, a chimerism study showed

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patient’s DNA only at 2.9%. He received conditioning chemotherapy with fludarabine and iv busulfan in addition to etoposide and a 2nd unrelated SCT from a different donor. Following complete chimeri-sm status for 8 months, he eventually experienced a 3rd relapse of AML. Cytogenetic analysis again revealed CK with hypodiploidy with further clonal evolution. Of note, deletions of 5q, 7q, and 17p were consistent aberrations in all three events (BM #11, #12, and #19). In particular, both copies of 17p were lost, either in the form of -17 or add(17)(p13), which suggested loss of both copies of the TP53 gene on 17p13.1. Despite additional care, the patient expired from septic shock 27 months after the diagnosis of AML.

Discussion

The diagnosis of FA underlying BMF in our patient was based on the result of a chromosomal break-age study. He had no physical abnor-malities suggestive of FA. It has been reported that 25-40% of patients with FA do not have physical stigmata. On the other hand, the cumulative incidence of BMF was reported to be up to 90% by age 40 to 48 years [8]. Thus, investigation for FA including chromosomal breakage study is indicated in pediatric and also adult patients with idiopathic BMF without physical stigmata. The diagnosis of FA is important in patients with cytopenia because of the underlying genomic instability that increases susceptibility to develop cancers and warrants dose adjustment in SCT or when treating the cancer. Genetically, FA is a heterogeneous group of disorders with up to 13 causative genes identified hitherto (http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi? book=gene&part=fa). Among them, FANCA, FANCC, and FANCG account for more than 80%. In our

patient, we observed no mutations in those three genes. We speculate that the underlying genetic defect in the patient would be mutations other than point mutations in those genes or mutations in other genes. SCT is the only curative treat-ment for BMF in FA, but the dose of conditioning regimen needs to be reduced due to the intrinsic hyper-sensitivity of cells of FA patients to genotoxic agents [9]. Tan et al [10] found that the 5-year survival rate in patients using a conditioning regimen of low dose CY and limited field irradiation is greater than 75%, and they reported successful results of conditioning using CY, ATG, and fludarabine. Our patient received SCT from an unrelated donor with reduced doses of chemo-therapy, and successfully achieved complete chimerism. However, recipient cells emerged 1 year after SCT, and shortly thereafter the patient developed AML. We speculate that the leukemic cells were of recipient origin rather than of donor origin based on the observation of the trend of changes of BM blasts in accordance with that of % recipient DNA on STR analysis (Table 1). Interestingly, while AML is the most common malignancy in patients with FA, studies on malig-nancies following SCT report mostly solid cancers and lymphoid malignancies [11]. Data from the International Fanconi Anemia Registry (IFAR) documented AML in 8% of FA patients, but unfortun-ately, without information whether the disease developed before or after SCT [12]. Recent reports described two patients with FA with myelo-dysplastic syndrome who received allogeneic SCT and achieved complete engraftment, but they experienced relapse of myelodys-plastic syndrome and evolution to AML [10,13]. To our knowledge, the present case is the first report of

AML developing after SCT in FA without obvious precedent myelo-dysplastic syndrome and with in-depth description of serial cyto-genetic changes. Although a few reports described improved outcomes of AML in FA patients, the prognosis of AML in FA patients has remained poor, which may be due to the underlying genomic instability [10,14]. The karyotype of AML in our patient was very complex (more than five aberrations) with hypo-diploidy, representing genomic instability. It was hard to determine whether the CK was from the leukemic blasts or not. However, the appearance and proportion of CK in accordance with the leukemic blasts in BM (Table 1) suggests that the CK represented genomic instability of the leukemic blasts in FA rather than non-leukemic recipient cells, possibly triggered by the condition-ing treatment for SCT. CK is encountered in 10-12% of AML, and most studies on AML with CK have involved adult patients, reporting the poorest prognosis of disease [15-18]. Of note, the modal number in up to 55-75% of cases of AML with CK was hypodiploidy, with consistent loss of chromosome segments 5q, 7q, and 17p. In particular, the loss of heterozygosity (LOH) of the TP53 gene on 17p13.1 (MIM# 191170), either in the form of gene deletion or mutation, is considered to play a critical role in the marked genome instability in AML with CK [18]. The leukemic cells of our patient also had CK with hypodiploidy, with loss of 5q, 7q, and both copies of 17p (LOH). This suggests that the molecular patho-physiology of AML in FA is in close relation with AML with CK in elderly patients. As in their elderly counterparts, pediatric patients with AML with CK also had poor prognosis with a high relapse rate [19,20]. Deletions of 5q/7q are also

AML with complex karyotype in Fanconi anemia 69

recurrent cytogenetic aberrations in myelodysplastic syndrome. Our patient had no overt MDS prior to the diagnosis of AML, and the deletions were observed in the context of CK. In summary, we describe a patient with FA who developed AML after SCT without the antecedence of obvious myelodys-plastic syndrome despite frequent BM studies. Therefore, careful follow-up is needed in FA without dysplastic features of BM. The CK with hypodiploidy including LOH of 17p of leukemic cells, as observed in our patient, may represent a unique karyotypic profile that reflects genomic instability and confers a poor prognosis. Compre-hensive data from more cases of AML in FA are needed to elucidate and manage this challenging situation in pediatric hemato-oncology.

Acknowledgement

This study was supported by the Samsung Medical Center Clinical Research Development Program Grant #CRS-108-02-1.

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