Int J Hematol (2015) 101:423–425 DOI 10.1007/s12185-015-1757-5

IMAGES IN HEMATOLOGY

Disappearance of double minute chromosomes with MYC amplification in relapsed acute myeloid leukemia after stem cell transplantation Katsuya Yamamoto · Kimikazu Yakushijin · Keiji Kurata · Yukinari Sanada · Shinichiro Kawamoto · Hiroshi Matsuoka · Hironobu Minami 

Received: 14 December 2014 / Revised: 2 February 2015 / Accepted: 2 February 2015 / Published online: 14 February 2015 © The Japanese Society of Hematology 2015

Double minute chromosomes (dmin) are small, acentric, extrachromosomal fragments that frequently mediate oncogene amplification and reflect rapid disease progression in human tumors. It has been shown that elimination of dmin can be induced by treating cancer cells with low concentrations of hydroxyurea or gemcitabine in vitro, resulting in reduced tumorigenicity and reversion of phenotype [1, 2]. Morphologically, the loss of dmin is facilitated by incorporation of dmin into micronuclei. However, this phenomenon has been reported only infrequently in primary tumor cells from patients [3]. A 58-year-old man was initially diagnosed with myelodysplastic syndrome, refractory cytopenia with multilineage dysplasia (MDS-RCMD). G-banding showed 44~45,XY,der(2;17)(q10;q10),inv(3)(p25q21),-5,-7,add(9) (p13),-11,-20,+mar1,+mar2,+mar3[cp8]/45,sl,+8[3]/46 ,XY[9]. After 11 months, the disease progressed to acute myeloid leukemia (AML). Peripheral blood values were as follows: hemoglobin 105 g/L, platelets 80 × 109/L and leukocytes 3.9 × 109/L with 22 % myeloblasts. Bone marrow was hypercellular with 46.6 % myeloblasts, which had many granules with vacuoles but no micronuclei (Fig. 1a). G-banding revealed the appearance of dmin (Fig. 1b): 42~45,XY,der(2;17)(q10;q10),inv(3)(p25q21),-5,-7,add(9) (p13),-11,-20,+mar1,+mar2,+mar3,0~85dmin[cp13]/42,sl ,add(1)(q21),add(1)(q32),+der(2)add(2)(p21)add(2)(q21),der(2;17),-inv(3),add(7)(p22),-12,-16,+mar4[2]/46,XY[5]. Spectral karyotyping (SKY) clarified the origin of marker

K. Yamamoto (*) · K. Yakushijin · K. Kurata · Y. Sanada · S. Kawamoto · H. Matsuoka · H. Minami  Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Graduate School of Medicine, 7‑5‑1 Kusunoki‑cho, Chuo‑ku, Kobe 650‑0017, Japan e-mail: [email protected]‑u.ac.jp

chromosomes, and revised the karyotype as follows (Fig.  2ab): 44,XY,-2,inv(3)(p25q21),der(5)del(5)(p?) del(5)(q?),der(7)(11?::7p13→7q22::7?::1?),add(9) (p13),-11,der(17)t(2;17)(q11.2;p11.2),der(20)t(2;20) (?;q11.2),2dmin[2]/42,sl,del(1)(q?),r(6),der(7) (7?::p13→ qter),-12,-16,-der(17)t(2;17),der(17) (2?::?::2?::17p11.2→17q23::12?),-2dmin[1]. Furthermore, dmin were shown to be derived from chromosomes 8. We next performed fluorescence in situ hybridization (FISH) with a probe for MYC located at 8q24 (Vysis LSI IGH/ MYC/CEP 8 Tri-Color Dual Fusion Probes), and detected multiple MYC signals on dmin (Fig. 1c). The patient underwent non-myeloablative cord blood transplantation (CBT) after conditioning with total body irradiation (TBI), fludarabine, and melphalan. However, the disease relapsed due to graft failure. G-banding at relapse after CBT showed 41~42,XY,2,inv(3)(p25q21),-4,der(5)del(5)(p?)del(5)(q?),der(7) (11?::7p13→7q22::7?::1?),add(9)(p13),-11,-12,-16,der(17) (2?::?::2?::17p11.2→17q23::12?),der(20)t(2;20)(?;q11.2) [cp5]/46,XY[15]. Thus, dmin disappeared, although other structural abnormalities remained. The patient next received an unrelated bone marrow transplant (u-BMT) after conditioning with TBI, fludarabine, and busulfan, but the disease progressed again. Unexpectedly, some of the myeloblasts had micronuclei (Fig. 1d). The similar karyotype did not include dmin (Fig. 1e): 40~43,XY,2,inv(3)(p25q21),-4,der(5)del(5)(p?)del(5)(q?),der(7) (11?::7p13→7q22::7?::1?),add(9)(p13),-11,-12,-16,der(17) (2?::?::2?::17p11.2→17q23::12?),der(20)t(2;20)(?;q11.2) [cp14]/41,sl,add(2)(q11.2),add(8)(q24),-add(9),+add(9) (p13)[5]/46,XY[1]. FISH also showed only three or four MYC signals (Fig. 1f). The patient succumbed to pneumonia 19 months after initial diagnosis.

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Fig.  1  a Bone marrow smear showing myeloblasts at the time of diagnosis of AML. Many orange granules and vacuoles are found in the cytoplasm (May-Grünwald-Giemsa staining, ×1000). b G-banded metaphase spreads from bone marrow cells at the time of diagnosis of AML. Multiple copies of dmin are present. c FISH with Vysis LSI IGH/MYC/CEP 8 Tri-Color Dual Fusion Probes (Abbott Molecular, Abbott Park, IL, USA) on metaphase spreads and interphase nuclei (inset) at the diagnosis of AML. Multiple (>20) MYC signals (red) are detected on the dmin. Two IGH (green) and two CEP 8 (blue) signals are also found on chromosomes 14 and 8, respectively. Interphase nuclei are also heavily labeled with MYC signals. Multiple (>20) MYC signals are found in 47 of 100 interphase cells. d Bone mar-

row smears showing myeloblasts with micronuclei at relapse after u-BMT. Black arrows indicate micronuclei (May-Grünwald-Giemsa staining, ×1000). e G-banded metaphase spreads from bone marrow cells at relapse after u-BMT. No dmin are observed. f FISH with Vysis LSI IGH/MYC/CEP 8 Tri-Color Dual Fusion Probes on metaphase spreads and interphase nuclei (inset) at relapse after u-BMT. Only three or four MYC signals (red) are detected. Similarly, three red, two green and two blue signals are observed in the interphase nucleus. Three or four MYC signals are observed in 52 of 100 interphase cells. MYC-labeled micronuclei could not be found in all 400 interphase cells examined

We have presented the disappearance of dmin with MYC amplification in AML at relapse after CBT/u-BMT. Initially, dmin emerged during the transformation from MDS to AML. However, dmin were not detected at relapse, and their loss was maintained thereafter. The decrease of dmin caused by a non-cytotoxic dose of hydroxyurea has been reported only in patients with advanced ovarian carcinoma [3]. To the best of our knowledge, this is the first case showing loss of dmin in a hematological malignancy. Similar to hydroxyurea and gemcitabine, fludarabine also belongs to the class of ribonucleotide reductase inhibitors. Furthermore, radiation has been shown to deplete amplified oncogenes via micronuclear capture of dmin [4]. Thus, the possibility remains that conditioning with TBI and fludarabine is associated with the loss of dmin induced by blast DNA damage [2]. Alternatively, it may be that the minor clone without dmin (Fig. 2b) predominated after CBT/u-BMT. In any case, independent of dmin, leukemic cells consistently retained complex common cytogenetic

abnormalities including inv(3)(p25q21), der(5)del(5)(p?) del(5)(q?), der(7)(11?::7p13→7q22::7?::1?), add(9)(p13), and der(20)t(2;20)(?;q11.2), which were ultimately responsible for leukemic progression. We have previously reported an AML case with micronuclei-associated MYC amplification, and suggested that micronuclei in interphase cells are closely linked to dmin in metaphase cells through MYC amplification [5]. Recently, Chinen et al. [6] also reported an additional AML case with dmin and micronuclei simultaneously. They clarified that the 8q24 amplicon included not only MYC but also NSMCE2 and 5′-PVT1, both of which were shown to be located in micronuclei. On the other hand, in the present case, micronuclei were found only after the disappearance of dmin. A similar inverse correlation between the numbers of micronuclei and dmin was observed in a patient with ovarian carcinoma [3]. Thus, careful morphological examination may be required to detect micronuclei even if dmin have disappeared.

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Fig. 2  Spectral karyotyping (SKY) of the metaphase spread after spectrum-based classification at the diagnosis of AML. Chromosomes were assigned a pseudocolor according to the measured spectrum. The grayscale images are reverse DAPI; the colored images are SKY. The two types of clones (a, b) are shown. Arrows indicate rearranged chromosomes. a 44,XY,-2,inv(3)(p25q21),der(5)del(5)(p?)

del(5)(q?),der(7)(11?::7p13→7q22::7?::1?),add(9)(p13),-11,der(17) t(2;17)(q11.2;p11.2),der(20)t(2;20)(?;q11.2),2dmin. b 42,XY,del(1) (q?),-2,inv(3)(p25q21),der(5)del(5)(p?)del(5)(q?),r(6),der(7) (7?::p13→qter),der(7)(11?::7p13→7q22::7?::1?),add(9)(p13),11,-12,-16,der(17)(2?::?::2?::17p11.2→17q23::12?),der(20)t(2;20) (?;q11.2)

Conflict of interest  The authors have no conflicts of interest.

patients with advanced ovarian carcinomas. Clin Cancer Res. 2001;7:1171–80. 4. Schoenlein PV, Barrett JT, Kulharya A, Dohn MR, Sanchez A, Hou DY, et al. Radiation therapy depletes extrachromosomally amplified drug resistance genes and oncogenes from tumor cells via micronuclear capture of episomes and double minute chromosomes. Int J Radiat Oncol Biol Phys. 2003;55:1051–65. 5. Yamamoto K, Okamura A, Sanada Y, Yakushijin K, Matsuoka H, Minami H. Micronuclei-associated MYC amplification in the form of double minute chromosomes in acute myeloid leukemia. Am J Hematol. 2013;88:717–8. 6. Chinen Y, Sakamoto N, Nagoshi H, Taki T, Maegawa S, Tatekawa S, et al. 8q24 amplified segments involve novel fusion genes between NSMCE2 and long noncoding RNAs in acute myelogenous leukemia. J Hematol Oncol. 2014;7:68.

References 1. Von Hoff DD, McGill JR, Forseth BJ, Davidson KK, Bradley TP, Van Devanter DR, et al. Elimination of extrachromosomally amplified MYC genes from human tumor cells reduces their tumorigenicity. Proc Natl Acad Sci USA. 1992;89:8165–9. 2. Yu L, Zhao Y, Quan C, Ji W, Zhu J, Huang Y, et al. Gemcitabine eliminates double minute chromosomes from human ovarian cancer cells. PLoS One. 2013;8:e71988. 3. Raymond E, Faivre S, Weiss G, McGill J, Davidson K, Izbicka E, et al. Effects of hydroxyurea on extrachromosomal DNA in

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