Blood Cells, Molecules and Diseases 52 (2014) 208–213
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Fluorescence in situ hybridization panel for monitoring of minimal residual disease in patients with double minute chromosomes Yongbum Jeon a,c, Seon Young Kim a,c, Miyoung Kim a,c, Hyun-Kyung Park b, Sang Ho Lee c, Cha Ja See c, Jiseok Kwon d, Dong Soon Lee a,c,d,⁎ a
Department of Laboratory Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea Department of Laboratory Medicine, Seoul Medical Science Institute, 7-14 Dongbinggo-dong Yongsan-gu, Seoul 140-809, Republic of Korea Department of Laboratory Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea d Cancer Research Institute, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea b c
a r t i c l e
i n f o
Article history: Submitted 11 August 2012 Revised 16 November 2012 Available online 7 November 2013 (Communicated by J. Rowley, M.D., 21 October 2013) Keywords: Double minute chromosome Oncogene NMYC Fluorescence in situ hybridization
a b s t r a c t A double minute chromosome (dmin) is a small fragment of extrachromosomal DNA bearing amplified genes observed in malignancies. We investigated the incidence and characteristics of dmins in hematologic malignancies, and the quantitative changes during the treatment follow-up. Once a dmin was observed in conventional Gbanding, it was characterized using fluorescence in situ hybridization (FISH) with the panel of MYC, NMYC, and MLL probes. Quantitative changes of malignant cells were measured using G-banding and FISH during the follow up. Dmins were observed in 1.23% of patients (6/489) at the initial diagnosis including 4 with MYC amplification, 1 with MLL and 1 with NMYC. All 6 had complex karyotypes and showed short overall survival (7.7 months). In follow-up specimens, FISH detected dmins in 11 cases out of which G-banding detected dmins in 9 cases. The number of dmins detected by FISH and G-banding did not correlate well. Amplification of NMYC in dmins is reported for the first time. A FISH panel composed of frequently amplified oncogenes (MYC, NMYC, and MLL) in dmins is useful for characterization of dmins. FISH is a sensitive method in detecting dmins and will be useful in monitoring of the minimal residual disease. © 2013 Elsevier Inc. All rights reserved.
Introduction
Materials and methods
A double minute chromosome (dmin) is a small chromatin body that consists of amplified genes in an extrachromosomal location. Genes amplified in dmins include MYC oncogene [1,2], MLL transcription factor [1,2], and other genes located at 11q23 [3]. MYC is the most frequently amplified gene, and MLL is also frequently amplified [1]. Though the precise incidence of dmins in human leukemia is uncertain, they range from less than 1% [4,5] to 10.3% [6], mostly being limited to acute myeloid leukemia (AML) [7]. In the present study, we investigated the incidence of dmins by G-banding in various hematologic malignancies and identified the amplified genes by FISH panel (MYC, NMYC, and MLL) performed on consecutive bone marrow specimens. To assess the utility of this FISH test for monitoring and assessment of minimal residual disease in patients with dmins, we compared the proportion of malignant cells measured by G-banding and those by FISH.
Patients
⁎ Corresponding author at: Department of Laboratory Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea Fax: + 82 2 747 0359. E-mail address: [email protected] (D.S. Lee). 1079-9796/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcmd.2013.10.008
The study included bone marrow aspirates of 489 patients [including 157 acute myeloid leukemia (AML), 65 myelodysplastic syndrome (MDS), 168 myeloproliferative neoplasm (MPN), and 99 acute lymphoblastic leukemia (ALL) patients] diagnosed at the Seoul National University Hospital from June 2006 to May 2010. G-banding and FISH Bone marrow aspirates were processed by conventional cytogenetic procedures using GTG (G-bands by trypsin using Giemsa) banding [8]. At least 20 metaphase cells were analyzed and karyotypes are given according to the International System for Human Cytogenetic Nomenclature 2009 (ISCN 2009) [9]. FISH studies were carried out according to the previously described procedure [10]. Three kinds of probes, LSI MYC Dual Color and Break Apart Rearrangement Probe (Vysis, Abbott Molecular Inc., Downers Grove, IL), LSI N-MYC Spectrum Orange Probe (Vysis), and LSI MLL Dual Color and Break Apart Rearrangement Probe (Vysis) were used. The FISH panel composed of 3 probes (MYC, NMYC, and MLL) was used to analyze all available initial and follow-up samples from 6 patients with dmins. For scoring of FISH, 300 cells were scored in
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Table 1 Serial follow-up of karyotypes and results of FISH panel in 6 patients with double minute chromosomes. Patient Age/Sex
Date
Diagnosis
Karyotype
BM blasts (%) Proportion of cells (%) with amplified oncogene by FISH & ISCN nomenclature
Proportion of cells (%) showing accompanying chromosomal abnormality
1
2006-03-30
AML (M2)
45,XY,del(5)(q15q33),der(9)t(9;17)(q13;q21)[9]/ 45,sl,der(7)t(7;9)(q22q13)[4]/90,sl × 2[3]/46,XY [2]
38.5%
59.0% with 5q deletion
2006-06-05
Remission
46,XY[1]
2.6%
2006-07-24
Relapse
68.9%
2006-08-22
Persistence
45,XY,del(5)(q15q33),der(9)t(9;17)(q22;q13), −17[5]/42–45,sl,dup(2)(q21q37),add(4)(p16), der(5;10)(q10;p10),dup(6)(q14q27),der(10) t(10;21)(p15;q11.2),add(19)(q13),15–100dmin [cp4]/46,XY[5] 46,XY,del(5)(q15q33)[1]/46,XY[18]
2006-11-13
Persistence
68.9%
2006-03-23
AML (M4)
69,XXY,del(5)(q15q33),der(7)t(7;9)(q22;q13), del(9)(q22),der(9;16)(q10;q10),der(9) t(9;17)(q13;q21),ins(9;?)(q12;?), dup(13)(q12q14)[12] 46,XY[11]
2006-06-26 2006-08-01
Remission Relapse
46,XY[16] 47,XY,inv(3)(p25q21),+13,2–50dmin[18]/46,XY [2]
0.0% 55.0%
2006-08-29
Persistence
46–47,XY,inv(3)(p25q21),+13,3–27dmin[14]/46, XY[6]
5.2%
2006-10-10
Persistence
47,XY,inv(3)(p25q21),+13,3–50dmin[18]/46,XY [3]
36.8%
2006-11-08
Persistence
46,XY,inv(3)(p25q21),2–40dmin[3]/47,XY, idem,+13[16]/46,XY[1]
15.3%
2010-04-12
AML with t(15;17)
46,XX,14pstk+,15pstk+,t(15;17)(q22;q21), 0–55dmin[16]/46,XX,14pstk+,15pstk[4]
60.0%
2010-05-14
Persistence
46,XX,14pstk+,15pstk + [30]
8.9%
2010-06-30
46,XX,14pstk+,15pstk + [20]
Not available
46,XX,del(5)(q13q33),inv(9)(p11q13),−13, der(18)t(13;18)(q14;q23),+add(?22)(q?13), 0–11dmin[5]/46,sl,add(16)(q?24)[4]/46,XX, inv(9)(p11q13)[4]
24.7%
2
3
59/M
73/M
69/F
5.2%
68.0%
4
68/F
2008-12-18
Inadequate specimen AML with myelodysplasiarelated change
5
29/F
2008-08-21
MDS, RAEB-1
46,XX,2–63dmin[12]/46,XX[8]
6.6%
2009-01-16
Persistence
46,XX,5–100dmin[16]/46,idem,del(9)(q22)[7]
16.1%
2009-03-19
Persistence
46,XX[21]
8.2%
2009-05-12
AML transformed from RAEB-1 Remission
46,XX,i(17)(q10),6–73dmin[20]
35.0%
46,XX[20]
0.4%
2008-05-13
Precursor T-cell ALL
46,XY,t(10;11)(p13;q21),i(17)(q10)[6]/47,sl,+4 [5]/46,XY[9]
91.4%
2008-06-25
Remission
46,XY[20]
1.3%
2009-06-24
6
8/M
MYC 46% nuc ish amp(myc)[31/ 100]/(myc × 5–50)[15/ 100] MYC 0% nuc ish(myc × 2)[300] MYC 27% nuc ish amp(myc)[19/ 100]/(myc × 4–15)[8/ 100] MYC 0% nuc ish(myc × 2)[300] MYC 88% nuc ish amp(myc)[12/ 100]/(myc × 10–50)[76/ 100] MYC 90% nuc ish amp(myc)[83/ 100]/(myc × 10–50)[7/ 100] Specimen not available MYC 82% nuc ish amp(myc)[52/ 100]/(myc × 3–50)[30/ 100] MYC 32% nuc ish amp(myc)[20/ 100]/(myc × 3–50)[12/ 100] MYC 66% nuc ish amp(myc)[43/ 100]/(myc × 3–50)[21/ 100] MYC 71% nuc ish(myc × 11–50) [57/100]/(myc × 3–10) [14/100] MYC 5% nuc ish amp(myc)[5/ 100] MYC 0% nuc ish(myc × 2)[300] MYC 0% nuc ish(myc × 2)[300] MLL 22% nuc ish(MLL × 3)[58/ 200]/(MLL × 20–50)[16/ 200]/(MLL × 10–20)[16/ 200]/(MLL × 4–10)[12/ 200] MYC 75% nuc ish(myc × 3–50) [75/100] MYC 85% nuc ish amp(myc)[85/ 100] MYC 0% nuc ish(myc × 2)[300] MYC 72.5% nuc ish(myc × 4–50) [145/200] MYC 1% nuc ish(myc × 10–25) [3/300] NMYC 0% nuc ish(MYCN × 2)[200]
Survival (months)
8
7.0% of 5q deletion 4.5% of 5q deletion
3.0% of 5q deletion 94.0% of 5q deletion
Not tested
60.5% of PML/RARA rearrangement 22.0% of PML/RARA rearrangement 0.0% of PML/RARA rearrangement 78.5% of 5q deletion and 35.5% of trisomy 1
10
4
2
0.0% of 9q deletion 13
16.0% of 9q deletion 0.3% of 9q deletion 0.0% of 9q deletion
0.0% of 9q deletion
80.5% of TEL gene deletion and 0.0% of iso 17q NMYC 0% 15.3% of TEL gene nuc ish(MYCN × 2)[200] deletion and 6.7%
9
(continued on next page)
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Table 1 (continued) Patient Age/Sex
Date
Diagnosis
Karyotype
BM blasts (%) Proportion of cells (%) with amplified oncogene by FISH & ISCN nomenclature
Proportion of cells (%) showing accompanying chromosomal abnormality
Survival (months)
of iso 17q 2008-09-02
Remission
46,XY[23]
1.3%
2008-11-03
Relapse
44,XY,del(5)(q31),+7,der(8)t(8;15)(p12;q12), −9,t(10;11;11)(p13;p15;q21),der(12;17)(q10; q10),−15,0–60dmin[18]/44,idem,i(17)(q10)[2]
63.9%
2009-02-18
Persistence
43,XY,del(5)(q31),+7,−9,t(10;11;11)(p13;p15; q21),der(12;17)(q10:q10),−15,−21,0–30dmin [4]/44,sl,der(8)t(8;15)(p12;q12),+21[15]/46,XY [2]
69.6%
each sample, and when sufficient numbers of interphase cells were not available, 50–100 cells were scored.
Results Six patients (1.23%) among 489 patients with hematologic malignancies were found to have dmin(s) in pretreatment by G-banding analysis. The frequencies among subtypes were 1.5% in MDS (1/65 patients), 2.6% in AML (4/157 patients), 0% in MPN (0/168 patients), and 1.0% in ALL (1/99 patients). Summaries of laboratory finding in 6 patients are described below (Table 1).
Case 1: acute myeloid leukemia (M2) A 59-year-old man was diagnosed with AML. Bone marrow study revealed 38.5% blasts with Auer rods (myeloperoxidase (MPO)+, CD13 +, CD33 +, CD34 + by flow cytometry). The karyotype was 45,XY,del(5)(q15q33),der(9)t(9;17)(q13;q21)[9]/44– 90,idem,der(7)t(7;9)(q22q13)[cp7]/46,XY[2]. Interphase FISH revealed amplified MYC signals in 46% of bone marrow nucleated cells and 5q deletion in 59% [nuc ish amp(MYC)[31/100]/(MYC × 5–50)[15/ 100]]. Four months after achieving morphologic remission, he relapsed. MYC amplification was observed in 27%, whereas 5q deletion was observed in 4.5%, suggesting differential clonal relapse. Despite continuing chemotherapy, the leukemia persisted without a reduction in the leukemic cell burden. A bone marrow specimen taken during the time of persistence revealed a persistence of leukemic cells with MYC amplification (88%) and a marked increase of cells with a 5q deletion (94%). He died 8 months after the initial diagnosis.
NMYC 0% 0.0% of TEL gene nuc ish(MYCN × 2)[200] deletion and 0.0% of iso 17q 73.0% of TEL gene NMYC 80% nuc ish amp(MYCN)[19/ deletion and 76.0% of iso 17q 50]/(MYCN × 10–30) [21/50] NMYC 77.5% 77.5% of TEL gene nuc ish amp(MYCN) deletion and 3.5% [155/200],(myc × 2) of iso 17q [200/200]
Case 3: acute myeloid leukemia (M3) A 69-year-old woman was diagnosed with AML (M3) and the bone marrow revealed 60% blasts and abnormal promyelocytes (MPO+, CD13+, CD33+). PML/RARA rearrangements were observed in 60.5% of bone marrow cells by FISH and the karyotype was 46,XX,14pstk+, 15pstk+,t(15;17)(q22;q21), 0–55dmin[16]/46,XX,14pstk+,15pstk[4]. Interestingly, G-banding showed the presence of cells with double minute chromosomes in 80% of the cells (Fig. 1), whereas interphase FISH revealed MYC amplification only in 5% of the bone marrow-nucleated cells (Fig. 2A). Metaphase FISH for MYC revealed numerous amplifications of MYC in dmins (Fig. 3B). After treatment with ATRA and idarubicin, a follow-up bone marrow study revealed a morphologic persistence of abnormal promyelocytes (8.9%), however, these blasts did not show MYC amplification by interphase FISH analysis. When metaphase FISH on post-treatment sample in persistent disease was also performed, no amplification of MYC was observed (Fig. 3C). We suspected the coexistence of amplification of an oncogene other than MYC, but MLL and NMYC FISH revealed no amplifications. The patient died of sepsis 4 months after the initial diagnosis. Case 4: acute myeloid leukemia with myelodysplasia-related changes A 68-year-old woman was diagnosed with AML with myelodysplasiarelated changes. Bone marrow aspirates revealed 24.7% blasts (MPO+, CD34+, CD117+), and the karyotype was 46,XX,del(5)(q13q33), inv(9)(p12q13),−13,der(18)t(13;18)(q14;q23),+add(?22)(q?13), 0– 11dmin[5]/45–55,sl,+ X,+ 1,+ 8,+ 11[cp7]/46,sl,add(16)(q?24)[4]/ 46,XX,inv(9)(p12q13)[4]. Interphase FISH revealed MLL amplification in 22% of total nucleated cells (Fig. 2B). The patient underwent demethylating agent therapy and died of the pulmonary hemorrhage in the ICU 2 months after the initial diagnosis.
Case 2: acute myeloid leukemia (M4) Case 5: myelodysplastic syndrome, refractory anemia with excess blasts A 73-year-old man was diagnosed with AML (M4) and the bone marrow revealed 68% blasts with frequent hyposegmented and hypogranulated neutrophils (MPO+, CD13+, CD33+, HLA-DR+). The karyotype was 46,XY[11] and interphase FISH revealed MYC amplification in 90% of the bone marrow-nucleated cells. The results of metaphase FISH showed amplification of MYC in dmins (Fig. 3A). Four months after achieving remission, relapse occurred. At relapse, G-banding revealed an additional chromosomal aberration of chromosome 3 and the karyotype was 47,XY,inv(3)(p25q21),+13,2–50dmin [18]/46,XY[2]. The bone marrow blasts were 55%, however, MYC amplification by FISH was observed in 82% of nucleated cells. Despite chemotherapy, remission was not achieved, and the patient died 10 months after the initial diagnosis.
A 29-year-old woman was diagnosed with RAEB. Bone marrow aspirate revealed 6.6% blasts and prominent dysgranulopoietic features, and bone marrow section revealed diffuse myelofibrosis (grade 2, according to 0–3 scale). The karyotype was 46,XX,2–63dmin[12]/46,XX[8], and interphase FISH revealed MYC amplification in 75% of the bone marrownucleated cells. After chemotherapy, follow-up bone marrow analysis revealed morphologic persistence of leukemic cells (16%), and the karyotype was 46,XX,5–100dmin[16]/46, idem,del(9)(q22)[7]. FISH with MYC probe and ABL1 (9q) probe revealed MYC amplification in 85% and 9q deletion in 16%. After 8 months, the patient had progressed to AML, and the karyotype was 46,XX,i(17)(q10),6–73dmin[20]. Of note, cells with MYC amplification by FISH were increased up to 72.5%, but
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Fig. 1. (A) G-banding of metaphase with numerous dmins (arrow) in bone marrow of a 69-year-old woman with acute promyelocytic leukemia at initial diagnosis (case 3). (B) Karyotype: 46,XX,14pstk+,15pstk+,t(15;17)(q22;q21),1–55dmin[16]/46,XX,14pstk+,15pstk [4].
clonal cells with a 9q deletion disappeared at the time of progression to AML. We inferred that clonal cells with MYC amplification transformed into AML. The patient died of pneumonia 13 months after the initial diagnosis. Case 6: precursor T lymphoblastic leukemia An 8-year-old male was diagnosed with precursor T lymphoblastic leukemia. Bone marrow aspirate revealed 91.4% blasts (TdT+,
CD34+, cCD3+, CD5+, CD4+, CD7+, CD33+, MPO−, CD117+). The karyotype was 46,XY,t(10;11)(p13;q21),i(17)(q10)[6]/47,sl,+4[5]/46, XY[9], and interphase FISH with TEL/AML1 probe revealed a cryptic TEL deletion in 80.5% of the bone marrow-nucleated cells. At the initial diagnosis, dmins were not observed either by FISH or G-banding. Five months after achieving remission, relapse occurred with an appearance of dmin by G-banding, and the karyotype was 44,XY, del(5)(q31),+ 7,der(8)t(8;15)(p12;q12),− 9,t(10;11;11)(p13;p15; q21),der(12;17)(q10;q10),− 15,0–60dmin[18]/44,idem,i(17)(q10)
Fig. 2. Fluorescent in situ hybridization for dmin using panel of MYC, MLL and NMYC. (A) Interphase FISH showing amplification of MYC signals (pink signal) and two CEP8 signals (green signal) in bone marrow of a 69-year-old woman with acute promyelocytic leukemia (case 3). (B) Interphase FISH with LSI MLL Dual Color probe showing partial amplification of 5′ portion of MLL gene (orange signal) in bone marrow of a 68-year-old woman with AML with myelodysplasia-related change (case 4). The yellow signal is from 3′ MLL. (C) Interphase FISH with NMYC amplification (pink color) in bone marrow of an 8-year-old male with T-ALL (case 5).
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Fig. 3. Fluorescent in situ hybridization for dmins using MYC in metaphase cells. (A) Metaphase FISH showing amplification of MYC signals (orange) and two CEP8 signals (green) in initial bone marrow of a 73-year-old man with acute myeloid leukemia (M4) (case 2). The karyotype was 46,XY[11].ish 8q24.1(myc × 10–50). (B) Metaphase FISH showing amplification of MYC signals (orange) and two CEP8 signals (green) in initial bone marrow of a 69-year-old woman with acute promyelocytic leukemia (case 3). The karyotype was 46, XX,14pstk+,15pstk+,t(15;17)(q22;q21),0–55dmin[16]/46,XX,14pstk+,15pstk[4].ish 8q24.1 (myc × 10–50). (C) Metaphase FISH using MYC with 2 normal MYC signals (pink) and 2 normal CEP8 signals (green) and no amplification of MYC in post-treatment bone marrow of case 3 in persistent disease. The karyotype was 46,XX,14pstk+,15pstk + [30].ish 8q24.1(myc × 2).
[2]. Interphase FISH revealed NMYC amplification in 85% of bone marrow-nucleated cells (Fig. 2C). TEL deletion was observed by FISH in 73% of BM cells and the amplification of NMYC amplification was observed in 80% of BM cells, although dmins were not observed by G-banding. We performed NMYC FISH retrospectively on a stored bone marrow specimen at initial diagnosis and NMYC amplification was not observed at that time. The patient died 4 months after the relapse. Discussion The frequencies of dmins in hematologic malignancies was 1.23% (6/489 patients). Among the 6 cases with dmins, 5 were AML or MDS, suggesting that dmins are more frequently associated with myeloid neoplasms. From the standpoint of detectability, FISH was superior to G-banding. Among the 6 cases, 2 (case 1 and 2) oncogene amplification was uncovered only by FISH, and dmins were not detected by Gbanding at the initial study. During follow-up after treatment, dmins were observed despite their absence at initial diagnosis. Retrospective FISH analysis with MYC, NMYC, and MLL in the initial diagnosis samples revealed the occult presence of oncogene amplification, which presented as dmins at relapse but were not detectable by G-banding. The proportion of marrow-nucleated cells with amplified MYC by FISH was 46% in case 1 and 90% in case 2. In contrast, case 3 showed a higher proportion of dmins by G-banding (80%) in comparison to that by FISH (5%). G-banding reveals the karyotype of proliferating cells, whereas interphase FISH reveals the karyotype of proliferating and quiescent cells. We infer that the high proliferative activity of the cells harboring dmins resulted in a high proportion of dmins+ cells in case 3. Another explanation is the possible presence of an oncogene other than MYC, NMYC, and MLL. Case 6 did not show oncogene amplification either by FISH or G-banding, and oncogene amplification was detected upon relapse, which suggests that the dmins were an evolutionary change during disease progression. Of note, all patients with dmins had accompanying complex cytogenetic abnormalities: 5q deletion [del(5)(q15q33), del(5)(q13q33), del(5)(q31)], 9q deletion [del(9)(q22)], t(10;11)(p13;q21), t(10;11;11) (p13;p15;q21), inv(3)(p25q21), inv(9)(p11q13), i(17)(q10), and other derivative chromosomes, including duplications, additions, and polysomies. These complex cytogenetic abnormalities are thought to be associated with an adverse prognosis, and the overall survival of patients with dmins was short (7.7 months). The proportion of cells with an amplified oncogene by FISH did not correlate with the proportion of cells with accompanying cytogenetic abnormalities. When we
compared the quantitative results of G-banding and FISH in serial specimens of the six patients with dmins, case 1 showed MYC amplification in 46% and 5q deletion in 59% of marrow-nucleated cells at the initial diagnosis. However, at relapse, 27% of marrow-nucleated cells showed MYC amplification, whereas 4.5% showed 5q deletion. Possible explanations for these findings include the existence of multiple clones (one with MYC amplification and another with 5q deletion) or that clonal cells harboring both MYC amplification and 5q deletion responded differentially to treatment. When we compared the blast proportion in marrow and the proportion of cells with oncogene amplification, they did not correlate with one another especially in the follow-up specimen. For example, case 5 had 6.6% marrow blasts and MYC amplification in 75% of marrownucleated cells at the initial diagnosis and 16.1% marrow blasts with MYC amplification in 85% of marrow-nucleated cells during follow-up, which is a marked discrepancy. NMYC amplification is a well-known adverse prognostic factor in neuroblastoma, irrespective of age and clinical stage [11]. Cells with NMYC amplification present either as double minutes or homogeneously staining regions (HSR) by G-banding [12]. The present study has identified the amplification of NMYC on dmins in a patient with precursor T ALL, which is the first report of NMYC amplification in this hematologic malignancy as far as we know. We recommend the use of FISH panel for MYC, NMYC, and MLL in patients with dmins at initial diagnosis and during follow-up. Once amplification of oncogenes on dmins is identified by FISH, monitoring with FISH using an appropriate probe can be useful for detecting minimal residual disease during follow-up. Conflict of interest disclosure The authors declare no competing financial interests. Acknowledgments This work was supported in part by (1) Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-0002257), (2) a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A120216). (3) a grant (10172KFDA993) from Korea Food & Drug Administration in 2012.
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References [1] L. Thomas, J. Stamberg, I. Gojo, Y. Ning, A.P. Rapoport, Double minute chromosomes in monoblastic (M5) and myeloblastic (M2) acute myeloid leukemia: two case reports and a review of literature, Am. J. Hematol. 77 (2004) 55–61. [2] P.E. Crossen, L.M. Savage, D.C. Heaton, M.J. Morrison, Characterization of the C-MYC amplicon in a case of acute myeloid leukemia with double minute chromosomes, Cancer Genet. Cytogenet. 112 (1999) 144–148. [3] P.E. Crossen, M.J. Morrison, P. Rodley, J. Cochrane, C.M. Morris, Identification of amplified genes in a patient with acute myeloid leukemia and double minute chromosomes, Cancer Genet. Cytogenet. 113 (1999) 126–133. [4] The Fourth International Workshop on Chromosomes in Leukemia: a prospective study of acute nonlymphocytic leukemia. Chicago, Illinois, U.S.A., September 2–7, 1982, Cancer Genet. Cytogenet. 11 (1984) 249–360. [5] S.N. Sait, M.U. Qadir, J.M. Conroy, S. Matsui, N.J. Nowak, M.R. Baer, Double minute chromosomes in acute myeloid leukemia and myelodysplastic syndrome: identification of new amplification regions by fluorescence in situ hybridization and spectral karyotyping, Genes Chromosomes Cancer 34 (2002) 42–47. [6] M.J. Marinello, M.L. Bloom, T.D. Doeblin, A.A. Sandberg, Double minute chromosomes in human leukemia, N. Engl. J. Med. 303 (1980) 704.
213
[7] Y.S. Li, Double minutes in acute myeloid leukemia, Int. J. Cancer 32 (1983) 455–459. [8] J.J. Yunis, Comparative analysis of high-resolution chromosome techniques for leukemic bone marrows, Cancer Genet. Cytogenet. 7 (1982) 43–50. [9] L.G. Shaffer, M.L. Slovak, L.J. Campbell, ISCN 2009: An International System for Human Cytogenetic Nomenclature (2009): Recommendations of the International Standing Committee on Human Cytogenetic Nomenclature, S. Karger, Basel, 2009. [10] D.S. Lee, Y.S. Lee, Y.S. Yun, Y.R. Kim, S.S. Jeong, Y.K. Lee, C.J. She, S.S. Yoon, H.R. Shin, Y. Kim, H.I. Cho, A study on the incidence of ABL gene deletion on derivative chromosome 9 in chronic myelogenous leukemia by interphase fluorescence in situ hybridization and its association with disease progression, Genes Chromosomes Cancer 37 (2003) 291–299. [11] A.T. Look, F.A. Hayes, J.J. Shuster, E.C. Douglass, R.P. Castleberry, L.C. Bowman, E.I. Smith, G.M. Brodeur, Clinical relevance of tumor cell ploidy and N-myc gene amplification in childhood neuroblastoma: a Pediatric Oncology Group study, J. Clin. Oncol. 9 (1991) 581–591. [12] M. Yoshimoto, S.R. Caminada De Toledo, E.M. Monteiro Caran, M.T. de Seixas, M.L. de Martino Lee, S. de Campos Vieira Abib, S.M. Vianna, S.T. Schettini, J. Anderson Duffles Andrade, MYCN gene amplification. Identification of cell populations containing double minutes and homogeneously staining regions in neuroblastoma tumors, Am. J. Pathol. 155 (1999) 1439–1443.
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Fluorescence in situ hybridization panel for monitoring of minimal residual disease in patients with double minute chromosomes Yongbum Jeon a,c, Seon Young Kim a,c, Miyoung Kim a,c, Hyun-Kyung Park b, Sang Ho Lee c, Cha Ja See c, Jiseok Kwon d, Dong Soon Lee a,c,d,⁎ a
Department of Laboratory Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea Department of Laboratory Medicine, Seoul Medical Science Institute, 7-14 Dongbinggo-dong Yongsan-gu, Seoul 140-809, Republic of Korea Department of Laboratory Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea d Cancer Research Institute, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea b c
a r t i c l e
i n f o
Article history: Submitted 11 August 2012 Revised 16 November 2012 Available online 7 November 2013 (Communicated by J. Rowley, M.D., 21 October 2013) Keywords: Double minute chromosome Oncogene NMYC Fluorescence in situ hybridization
a b s t r a c t A double minute chromosome (dmin) is a small fragment of extrachromosomal DNA bearing amplified genes observed in malignancies. We investigated the incidence and characteristics of dmins in hematologic malignancies, and the quantitative changes during the treatment follow-up. Once a dmin was observed in conventional Gbanding, it was characterized using fluorescence in situ hybridization (FISH) with the panel of MYC, NMYC, and MLL probes. Quantitative changes of malignant cells were measured using G-banding and FISH during the follow up. Dmins were observed in 1.23% of patients (6/489) at the initial diagnosis including 4 with MYC amplification, 1 with MLL and 1 with NMYC. All 6 had complex karyotypes and showed short overall survival (7.7 months). In follow-up specimens, FISH detected dmins in 11 cases out of which G-banding detected dmins in 9 cases. The number of dmins detected by FISH and G-banding did not correlate well. Amplification of NMYC in dmins is reported for the first time. A FISH panel composed of frequently amplified oncogenes (MYC, NMYC, and MLL) in dmins is useful for characterization of dmins. FISH is a sensitive method in detecting dmins and will be useful in monitoring of the minimal residual disease. © 2013 Elsevier Inc. All rights reserved.
Introduction
Materials and methods
A double minute chromosome (dmin) is a small chromatin body that consists of amplified genes in an extrachromosomal location. Genes amplified in dmins include MYC oncogene [1,2], MLL transcription factor [1,2], and other genes located at 11q23 [3]. MYC is the most frequently amplified gene, and MLL is also frequently amplified [1]. Though the precise incidence of dmins in human leukemia is uncertain, they range from less than 1% [4,5] to 10.3% [6], mostly being limited to acute myeloid leukemia (AML) [7]. In the present study, we investigated the incidence of dmins by G-banding in various hematologic malignancies and identified the amplified genes by FISH panel (MYC, NMYC, and MLL) performed on consecutive bone marrow specimens. To assess the utility of this FISH test for monitoring and assessment of minimal residual disease in patients with dmins, we compared the proportion of malignant cells measured by G-banding and those by FISH.
Patients
⁎ Corresponding author at: Department of Laboratory Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea Fax: + 82 2 747 0359. E-mail address: [email protected] (D.S. Lee). 1079-9796/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcmd.2013.10.008
The study included bone marrow aspirates of 489 patients [including 157 acute myeloid leukemia (AML), 65 myelodysplastic syndrome (MDS), 168 myeloproliferative neoplasm (MPN), and 99 acute lymphoblastic leukemia (ALL) patients] diagnosed at the Seoul National University Hospital from June 2006 to May 2010. G-banding and FISH Bone marrow aspirates were processed by conventional cytogenetic procedures using GTG (G-bands by trypsin using Giemsa) banding [8]. At least 20 metaphase cells were analyzed and karyotypes are given according to the International System for Human Cytogenetic Nomenclature 2009 (ISCN 2009) [9]. FISH studies were carried out according to the previously described procedure [10]. Three kinds of probes, LSI MYC Dual Color and Break Apart Rearrangement Probe (Vysis, Abbott Molecular Inc., Downers Grove, IL), LSI N-MYC Spectrum Orange Probe (Vysis), and LSI MLL Dual Color and Break Apart Rearrangement Probe (Vysis) were used. The FISH panel composed of 3 probes (MYC, NMYC, and MLL) was used to analyze all available initial and follow-up samples from 6 patients with dmins. For scoring of FISH, 300 cells were scored in
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Table 1 Serial follow-up of karyotypes and results of FISH panel in 6 patients with double minute chromosomes. Patient Age/Sex
Date
Diagnosis
Karyotype
BM blasts (%) Proportion of cells (%) with amplified oncogene by FISH & ISCN nomenclature
Proportion of cells (%) showing accompanying chromosomal abnormality
1
2006-03-30
AML (M2)
45,XY,del(5)(q15q33),der(9)t(9;17)(q13;q21)[9]/ 45,sl,der(7)t(7;9)(q22q13)[4]/90,sl × 2[3]/46,XY [2]
38.5%
59.0% with 5q deletion
2006-06-05
Remission
46,XY[1]
2.6%
2006-07-24
Relapse
68.9%
2006-08-22
Persistence
45,XY,del(5)(q15q33),der(9)t(9;17)(q22;q13), −17[5]/42–45,sl,dup(2)(q21q37),add(4)(p16), der(5;10)(q10;p10),dup(6)(q14q27),der(10) t(10;21)(p15;q11.2),add(19)(q13),15–100dmin [cp4]/46,XY[5] 46,XY,del(5)(q15q33)[1]/46,XY[18]
2006-11-13
Persistence
68.9%
2006-03-23
AML (M4)
69,XXY,del(5)(q15q33),der(7)t(7;9)(q22;q13), del(9)(q22),der(9;16)(q10;q10),der(9) t(9;17)(q13;q21),ins(9;?)(q12;?), dup(13)(q12q14)[12] 46,XY[11]
2006-06-26 2006-08-01
Remission Relapse
46,XY[16] 47,XY,inv(3)(p25q21),+13,2–50dmin[18]/46,XY [2]
0.0% 55.0%
2006-08-29
Persistence
46–47,XY,inv(3)(p25q21),+13,3–27dmin[14]/46, XY[6]
5.2%
2006-10-10
Persistence
47,XY,inv(3)(p25q21),+13,3–50dmin[18]/46,XY [3]
36.8%
2006-11-08
Persistence
46,XY,inv(3)(p25q21),2–40dmin[3]/47,XY, idem,+13[16]/46,XY[1]
15.3%
2010-04-12
AML with t(15;17)
46,XX,14pstk+,15pstk+,t(15;17)(q22;q21), 0–55dmin[16]/46,XX,14pstk+,15pstk[4]
60.0%
2010-05-14
Persistence
46,XX,14pstk+,15pstk + [30]
8.9%
2010-06-30
46,XX,14pstk+,15pstk + [20]
Not available
46,XX,del(5)(q13q33),inv(9)(p11q13),−13, der(18)t(13;18)(q14;q23),+add(?22)(q?13), 0–11dmin[5]/46,sl,add(16)(q?24)[4]/46,XX, inv(9)(p11q13)[4]
24.7%
2
3
59/M
73/M
69/F
5.2%
68.0%
4
68/F
2008-12-18
Inadequate specimen AML with myelodysplasiarelated change
5
29/F
2008-08-21
MDS, RAEB-1
46,XX,2–63dmin[12]/46,XX[8]
6.6%
2009-01-16
Persistence
46,XX,5–100dmin[16]/46,idem,del(9)(q22)[7]
16.1%
2009-03-19
Persistence
46,XX[21]
8.2%
2009-05-12
AML transformed from RAEB-1 Remission
46,XX,i(17)(q10),6–73dmin[20]
35.0%
46,XX[20]
0.4%
2008-05-13
Precursor T-cell ALL
46,XY,t(10;11)(p13;q21),i(17)(q10)[6]/47,sl,+4 [5]/46,XY[9]
91.4%
2008-06-25
Remission
46,XY[20]
1.3%
2009-06-24
6
8/M
MYC 46% nuc ish amp(myc)[31/ 100]/(myc × 5–50)[15/ 100] MYC 0% nuc ish(myc × 2)[300] MYC 27% nuc ish amp(myc)[19/ 100]/(myc × 4–15)[8/ 100] MYC 0% nuc ish(myc × 2)[300] MYC 88% nuc ish amp(myc)[12/ 100]/(myc × 10–50)[76/ 100] MYC 90% nuc ish amp(myc)[83/ 100]/(myc × 10–50)[7/ 100] Specimen not available MYC 82% nuc ish amp(myc)[52/ 100]/(myc × 3–50)[30/ 100] MYC 32% nuc ish amp(myc)[20/ 100]/(myc × 3–50)[12/ 100] MYC 66% nuc ish amp(myc)[43/ 100]/(myc × 3–50)[21/ 100] MYC 71% nuc ish(myc × 11–50) [57/100]/(myc × 3–10) [14/100] MYC 5% nuc ish amp(myc)[5/ 100] MYC 0% nuc ish(myc × 2)[300] MYC 0% nuc ish(myc × 2)[300] MLL 22% nuc ish(MLL × 3)[58/ 200]/(MLL × 20–50)[16/ 200]/(MLL × 10–20)[16/ 200]/(MLL × 4–10)[12/ 200] MYC 75% nuc ish(myc × 3–50) [75/100] MYC 85% nuc ish amp(myc)[85/ 100] MYC 0% nuc ish(myc × 2)[300] MYC 72.5% nuc ish(myc × 4–50) [145/200] MYC 1% nuc ish(myc × 10–25) [3/300] NMYC 0% nuc ish(MYCN × 2)[200]
Survival (months)
8
7.0% of 5q deletion 4.5% of 5q deletion
3.0% of 5q deletion 94.0% of 5q deletion
Not tested
60.5% of PML/RARA rearrangement 22.0% of PML/RARA rearrangement 0.0% of PML/RARA rearrangement 78.5% of 5q deletion and 35.5% of trisomy 1
10
4
2
0.0% of 9q deletion 13
16.0% of 9q deletion 0.3% of 9q deletion 0.0% of 9q deletion
0.0% of 9q deletion
80.5% of TEL gene deletion and 0.0% of iso 17q NMYC 0% 15.3% of TEL gene nuc ish(MYCN × 2)[200] deletion and 6.7%
9
(continued on next page)
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Table 1 (continued) Patient Age/Sex
Date
Diagnosis
Karyotype
BM blasts (%) Proportion of cells (%) with amplified oncogene by FISH & ISCN nomenclature
Proportion of cells (%) showing accompanying chromosomal abnormality
Survival (months)
of iso 17q 2008-09-02
Remission
46,XY[23]
1.3%
2008-11-03
Relapse
44,XY,del(5)(q31),+7,der(8)t(8;15)(p12;q12), −9,t(10;11;11)(p13;p15;q21),der(12;17)(q10; q10),−15,0–60dmin[18]/44,idem,i(17)(q10)[2]
63.9%
2009-02-18
Persistence
43,XY,del(5)(q31),+7,−9,t(10;11;11)(p13;p15; q21),der(12;17)(q10:q10),−15,−21,0–30dmin [4]/44,sl,der(8)t(8;15)(p12;q12),+21[15]/46,XY [2]
69.6%
each sample, and when sufficient numbers of interphase cells were not available, 50–100 cells were scored.
Results Six patients (1.23%) among 489 patients with hematologic malignancies were found to have dmin(s) in pretreatment by G-banding analysis. The frequencies among subtypes were 1.5% in MDS (1/65 patients), 2.6% in AML (4/157 patients), 0% in MPN (0/168 patients), and 1.0% in ALL (1/99 patients). Summaries of laboratory finding in 6 patients are described below (Table 1).
Case 1: acute myeloid leukemia (M2) A 59-year-old man was diagnosed with AML. Bone marrow study revealed 38.5% blasts with Auer rods (myeloperoxidase (MPO)+, CD13 +, CD33 +, CD34 + by flow cytometry). The karyotype was 45,XY,del(5)(q15q33),der(9)t(9;17)(q13;q21)[9]/44– 90,idem,der(7)t(7;9)(q22q13)[cp7]/46,XY[2]. Interphase FISH revealed amplified MYC signals in 46% of bone marrow nucleated cells and 5q deletion in 59% [nuc ish amp(MYC)[31/100]/(MYC × 5–50)[15/ 100]]. Four months after achieving morphologic remission, he relapsed. MYC amplification was observed in 27%, whereas 5q deletion was observed in 4.5%, suggesting differential clonal relapse. Despite continuing chemotherapy, the leukemia persisted without a reduction in the leukemic cell burden. A bone marrow specimen taken during the time of persistence revealed a persistence of leukemic cells with MYC amplification (88%) and a marked increase of cells with a 5q deletion (94%). He died 8 months after the initial diagnosis.
NMYC 0% 0.0% of TEL gene nuc ish(MYCN × 2)[200] deletion and 0.0% of iso 17q 73.0% of TEL gene NMYC 80% nuc ish amp(MYCN)[19/ deletion and 76.0% of iso 17q 50]/(MYCN × 10–30) [21/50] NMYC 77.5% 77.5% of TEL gene nuc ish amp(MYCN) deletion and 3.5% [155/200],(myc × 2) of iso 17q [200/200]
Case 3: acute myeloid leukemia (M3) A 69-year-old woman was diagnosed with AML (M3) and the bone marrow revealed 60% blasts and abnormal promyelocytes (MPO+, CD13+, CD33+). PML/RARA rearrangements were observed in 60.5% of bone marrow cells by FISH and the karyotype was 46,XX,14pstk+, 15pstk+,t(15;17)(q22;q21), 0–55dmin[16]/46,XX,14pstk+,15pstk[4]. Interestingly, G-banding showed the presence of cells with double minute chromosomes in 80% of the cells (Fig. 1), whereas interphase FISH revealed MYC amplification only in 5% of the bone marrow-nucleated cells (Fig. 2A). Metaphase FISH for MYC revealed numerous amplifications of MYC in dmins (Fig. 3B). After treatment with ATRA and idarubicin, a follow-up bone marrow study revealed a morphologic persistence of abnormal promyelocytes (8.9%), however, these blasts did not show MYC amplification by interphase FISH analysis. When metaphase FISH on post-treatment sample in persistent disease was also performed, no amplification of MYC was observed (Fig. 3C). We suspected the coexistence of amplification of an oncogene other than MYC, but MLL and NMYC FISH revealed no amplifications. The patient died of sepsis 4 months after the initial diagnosis. Case 4: acute myeloid leukemia with myelodysplasia-related changes A 68-year-old woman was diagnosed with AML with myelodysplasiarelated changes. Bone marrow aspirates revealed 24.7% blasts (MPO+, CD34+, CD117+), and the karyotype was 46,XX,del(5)(q13q33), inv(9)(p12q13),−13,der(18)t(13;18)(q14;q23),+add(?22)(q?13), 0– 11dmin[5]/45–55,sl,+ X,+ 1,+ 8,+ 11[cp7]/46,sl,add(16)(q?24)[4]/ 46,XX,inv(9)(p12q13)[4]. Interphase FISH revealed MLL amplification in 22% of total nucleated cells (Fig. 2B). The patient underwent demethylating agent therapy and died of the pulmonary hemorrhage in the ICU 2 months after the initial diagnosis.
Case 2: acute myeloid leukemia (M4) Case 5: myelodysplastic syndrome, refractory anemia with excess blasts A 73-year-old man was diagnosed with AML (M4) and the bone marrow revealed 68% blasts with frequent hyposegmented and hypogranulated neutrophils (MPO+, CD13+, CD33+, HLA-DR+). The karyotype was 46,XY[11] and interphase FISH revealed MYC amplification in 90% of the bone marrow-nucleated cells. The results of metaphase FISH showed amplification of MYC in dmins (Fig. 3A). Four months after achieving remission, relapse occurred. At relapse, G-banding revealed an additional chromosomal aberration of chromosome 3 and the karyotype was 47,XY,inv(3)(p25q21),+13,2–50dmin [18]/46,XY[2]. The bone marrow blasts were 55%, however, MYC amplification by FISH was observed in 82% of nucleated cells. Despite chemotherapy, remission was not achieved, and the patient died 10 months after the initial diagnosis.
A 29-year-old woman was diagnosed with RAEB. Bone marrow aspirate revealed 6.6% blasts and prominent dysgranulopoietic features, and bone marrow section revealed diffuse myelofibrosis (grade 2, according to 0–3 scale). The karyotype was 46,XX,2–63dmin[12]/46,XX[8], and interphase FISH revealed MYC amplification in 75% of the bone marrownucleated cells. After chemotherapy, follow-up bone marrow analysis revealed morphologic persistence of leukemic cells (16%), and the karyotype was 46,XX,5–100dmin[16]/46, idem,del(9)(q22)[7]. FISH with MYC probe and ABL1 (9q) probe revealed MYC amplification in 85% and 9q deletion in 16%. After 8 months, the patient had progressed to AML, and the karyotype was 46,XX,i(17)(q10),6–73dmin[20]. Of note, cells with MYC amplification by FISH were increased up to 72.5%, but
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Fig. 1. (A) G-banding of metaphase with numerous dmins (arrow) in bone marrow of a 69-year-old woman with acute promyelocytic leukemia at initial diagnosis (case 3). (B) Karyotype: 46,XX,14pstk+,15pstk+,t(15;17)(q22;q21),1–55dmin[16]/46,XX,14pstk+,15pstk [4].
clonal cells with a 9q deletion disappeared at the time of progression to AML. We inferred that clonal cells with MYC amplification transformed into AML. The patient died of pneumonia 13 months after the initial diagnosis. Case 6: precursor T lymphoblastic leukemia An 8-year-old male was diagnosed with precursor T lymphoblastic leukemia. Bone marrow aspirate revealed 91.4% blasts (TdT+,
CD34+, cCD3+, CD5+, CD4+, CD7+, CD33+, MPO−, CD117+). The karyotype was 46,XY,t(10;11)(p13;q21),i(17)(q10)[6]/47,sl,+4[5]/46, XY[9], and interphase FISH with TEL/AML1 probe revealed a cryptic TEL deletion in 80.5% of the bone marrow-nucleated cells. At the initial diagnosis, dmins were not observed either by FISH or G-banding. Five months after achieving remission, relapse occurred with an appearance of dmin by G-banding, and the karyotype was 44,XY, del(5)(q31),+ 7,der(8)t(8;15)(p12;q12),− 9,t(10;11;11)(p13;p15; q21),der(12;17)(q10;q10),− 15,0–60dmin[18]/44,idem,i(17)(q10)
Fig. 2. Fluorescent in situ hybridization for dmin using panel of MYC, MLL and NMYC. (A) Interphase FISH showing amplification of MYC signals (pink signal) and two CEP8 signals (green signal) in bone marrow of a 69-year-old woman with acute promyelocytic leukemia (case 3). (B) Interphase FISH with LSI MLL Dual Color probe showing partial amplification of 5′ portion of MLL gene (orange signal) in bone marrow of a 68-year-old woman with AML with myelodysplasia-related change (case 4). The yellow signal is from 3′ MLL. (C) Interphase FISH with NMYC amplification (pink color) in bone marrow of an 8-year-old male with T-ALL (case 5).
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Fig. 3. Fluorescent in situ hybridization for dmins using MYC in metaphase cells. (A) Metaphase FISH showing amplification of MYC signals (orange) and two CEP8 signals (green) in initial bone marrow of a 73-year-old man with acute myeloid leukemia (M4) (case 2). The karyotype was 46,XY[11].ish 8q24.1(myc × 10–50). (B) Metaphase FISH showing amplification of MYC signals (orange) and two CEP8 signals (green) in initial bone marrow of a 69-year-old woman with acute promyelocytic leukemia (case 3). The karyotype was 46, XX,14pstk+,15pstk+,t(15;17)(q22;q21),0–55dmin[16]/46,XX,14pstk+,15pstk[4].ish 8q24.1 (myc × 10–50). (C) Metaphase FISH using MYC with 2 normal MYC signals (pink) and 2 normal CEP8 signals (green) and no amplification of MYC in post-treatment bone marrow of case 3 in persistent disease. The karyotype was 46,XX,14pstk+,15pstk + [30].ish 8q24.1(myc × 2).
[2]. Interphase FISH revealed NMYC amplification in 85% of bone marrow-nucleated cells (Fig. 2C). TEL deletion was observed by FISH in 73% of BM cells and the amplification of NMYC amplification was observed in 80% of BM cells, although dmins were not observed by G-banding. We performed NMYC FISH retrospectively on a stored bone marrow specimen at initial diagnosis and NMYC amplification was not observed at that time. The patient died 4 months after the relapse. Discussion The frequencies of dmins in hematologic malignancies was 1.23% (6/489 patients). Among the 6 cases with dmins, 5 were AML or MDS, suggesting that dmins are more frequently associated with myeloid neoplasms. From the standpoint of detectability, FISH was superior to G-banding. Among the 6 cases, 2 (case 1 and 2) oncogene amplification was uncovered only by FISH, and dmins were not detected by Gbanding at the initial study. During follow-up after treatment, dmins were observed despite their absence at initial diagnosis. Retrospective FISH analysis with MYC, NMYC, and MLL in the initial diagnosis samples revealed the occult presence of oncogene amplification, which presented as dmins at relapse but were not detectable by G-banding. The proportion of marrow-nucleated cells with amplified MYC by FISH was 46% in case 1 and 90% in case 2. In contrast, case 3 showed a higher proportion of dmins by G-banding (80%) in comparison to that by FISH (5%). G-banding reveals the karyotype of proliferating cells, whereas interphase FISH reveals the karyotype of proliferating and quiescent cells. We infer that the high proliferative activity of the cells harboring dmins resulted in a high proportion of dmins+ cells in case 3. Another explanation is the possible presence of an oncogene other than MYC, NMYC, and MLL. Case 6 did not show oncogene amplification either by FISH or G-banding, and oncogene amplification was detected upon relapse, which suggests that the dmins were an evolutionary change during disease progression. Of note, all patients with dmins had accompanying complex cytogenetic abnormalities: 5q deletion [del(5)(q15q33), del(5)(q13q33), del(5)(q31)], 9q deletion [del(9)(q22)], t(10;11)(p13;q21), t(10;11;11) (p13;p15;q21), inv(3)(p25q21), inv(9)(p11q13), i(17)(q10), and other derivative chromosomes, including duplications, additions, and polysomies. These complex cytogenetic abnormalities are thought to be associated with an adverse prognosis, and the overall survival of patients with dmins was short (7.7 months). The proportion of cells with an amplified oncogene by FISH did not correlate with the proportion of cells with accompanying cytogenetic abnormalities. When we
compared the quantitative results of G-banding and FISH in serial specimens of the six patients with dmins, case 1 showed MYC amplification in 46% and 5q deletion in 59% of marrow-nucleated cells at the initial diagnosis. However, at relapse, 27% of marrow-nucleated cells showed MYC amplification, whereas 4.5% showed 5q deletion. Possible explanations for these findings include the existence of multiple clones (one with MYC amplification and another with 5q deletion) or that clonal cells harboring both MYC amplification and 5q deletion responded differentially to treatment. When we compared the blast proportion in marrow and the proportion of cells with oncogene amplification, they did not correlate with one another especially in the follow-up specimen. For example, case 5 had 6.6% marrow blasts and MYC amplification in 75% of marrownucleated cells at the initial diagnosis and 16.1% marrow blasts with MYC amplification in 85% of marrow-nucleated cells during follow-up, which is a marked discrepancy. NMYC amplification is a well-known adverse prognostic factor in neuroblastoma, irrespective of age and clinical stage [11]. Cells with NMYC amplification present either as double minutes or homogeneously staining regions (HSR) by G-banding [12]. The present study has identified the amplification of NMYC on dmins in a patient with precursor T ALL, which is the first report of NMYC amplification in this hematologic malignancy as far as we know. We recommend the use of FISH panel for MYC, NMYC, and MLL in patients with dmins at initial diagnosis and during follow-up. Once amplification of oncogenes on dmins is identified by FISH, monitoring with FISH using an appropriate probe can be useful for detecting minimal residual disease during follow-up. Conflict of interest disclosure The authors declare no competing financial interests. Acknowledgments This work was supported in part by (1) Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012-0002257), (2) a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A120216). (3) a grant (10172KFDA993) from Korea Food & Drug Administration in 2012.
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References [1] L. Thomas, J. Stamberg, I. Gojo, Y. Ning, A.P. Rapoport, Double minute chromosomes in monoblastic (M5) and myeloblastic (M2) acute myeloid leukemia: two case reports and a review of literature, Am. J. Hematol. 77 (2004) 55–61. [2] P.E. Crossen, L.M. Savage, D.C. Heaton, M.J. Morrison, Characterization of the C-MYC amplicon in a case of acute myeloid leukemia with double minute chromosomes, Cancer Genet. Cytogenet. 112 (1999) 144–148. [3] P.E. Crossen, M.J. Morrison, P. Rodley, J. Cochrane, C.M. Morris, Identification of amplified genes in a patient with acute myeloid leukemia and double minute chromosomes, Cancer Genet. Cytogenet. 113 (1999) 126–133. [4] The Fourth International Workshop on Chromosomes in Leukemia: a prospective study of acute nonlymphocytic leukemia. Chicago, Illinois, U.S.A., September 2–7, 1982, Cancer Genet. Cytogenet. 11 (1984) 249–360. [5] S.N. Sait, M.U. Qadir, J.M. Conroy, S. Matsui, N.J. Nowak, M.R. Baer, Double minute chromosomes in acute myeloid leukemia and myelodysplastic syndrome: identification of new amplification regions by fluorescence in situ hybridization and spectral karyotyping, Genes Chromosomes Cancer 34 (2002) 42–47. [6] M.J. Marinello, M.L. Bloom, T.D. Doeblin, A.A. Sandberg, Double minute chromosomes in human leukemia, N. Engl. J. Med. 303 (1980) 704.
213
[7] Y.S. Li, Double minutes in acute myeloid leukemia, Int. J. Cancer 32 (1983) 455–459. [8] J.J. Yunis, Comparative analysis of high-resolution chromosome techniques for leukemic bone marrows, Cancer Genet. Cytogenet. 7 (1982) 43–50. [9] L.G. Shaffer, M.L. Slovak, L.J. Campbell, ISCN 2009: An International System for Human Cytogenetic Nomenclature (2009): Recommendations of the International Standing Committee on Human Cytogenetic Nomenclature, S. Karger, Basel, 2009. [10] D.S. Lee, Y.S. Lee, Y.S. Yun, Y.R. Kim, S.S. Jeong, Y.K. Lee, C.J. She, S.S. Yoon, H.R. Shin, Y. Kim, H.I. Cho, A study on the incidence of ABL gene deletion on derivative chromosome 9 in chronic myelogenous leukemia by interphase fluorescence in situ hybridization and its association with disease progression, Genes Chromosomes Cancer 37 (2003) 291–299. [11] A.T. Look, F.A. Hayes, J.J. Shuster, E.C. Douglass, R.P. Castleberry, L.C. Bowman, E.I. Smith, G.M. Brodeur, Clinical relevance of tumor cell ploidy and N-myc gene amplification in childhood neuroblastoma: a Pediatric Oncology Group study, J. Clin. Oncol. 9 (1991) 581–591. [12] M. Yoshimoto, S.R. Caminada De Toledo, E.M. Monteiro Caran, M.T. de Seixas, M.L. de Martino Lee, S. de Campos Vieira Abib, S.M. Vianna, S.T. Schettini, J. Anderson Duffles Andrade, MYCN gene amplification. Identification of cell populations containing double minutes and homogeneously staining regions in neuroblastoma tumors, Am. J. Pathol. 155 (1999) 1439–1443.
