Herein, we describe the establishment and characterization of the first mixed-phenotype acute leukemia cell line (JIH-5). The JIH-5 cell line was established from leukemia cells with B lymphoid/myeloid phenotype from a female mixed-phenotype acute leukemia patient. JIH-5 cells exhibit an immunophenotype comprised of myeloid and B lymphoid antigens. Whole-exome sequencing revealed somatic mutations in nine genes in JIH-5 cells. Transcriptional sequencing of JIH-5 cells identified EP300-ZNF384 fusion transcript, which is a recurrent alteration in B cell acute lymphoblastic leukemia. Our results suggest that the JIH-5 cell line may serve as a tool for the study of mixed-phenotype acute leukemia or EP300-ZNF384.
Hinweise
Nana Ping Huiying Qiu and Qian Wang contributed equally to this work.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SC was the principal investigator. NP, HQ, and QW performed most of the experiments. SE, CR, and RM performed the cytogenetic and gene expression array analysis. SC, HD, and RM wrote the manuscript. All authors read and approved the final manuscript.
Abkürzungen
B-ALL
B cell acute lymphoblastic leukemia
CNA
copy number alterations
EBV
Epstein-Barr virus
FCM
flow cytometry
FISH
fluorescence in situ hybridization
MOAP
mitoxantrone, cytosine arabinoside, vindesine, and dexamethasone
MPAL
mixed-phenotype acute leukemia
NGS
next-generation sequencing
NuRD
nucleosome remodeling and deacetylase
PCR
polymerase chain reaction
SKY
spectral karyotype
T-ALL
T cell acute lymphoblastic leukemia
Findings
In a minority of patients with acute leukemia, it is difficult to determine the lineage origin because of the expression of both lymphoid and myeloid lineage-specific antigens [1‐5]. The 2008 World Health Organization (WHO) classification introduced a new designation for this entity, mixed-phenotype acute leukemia (MPAL) [6]. Tumor cells are characterized by various biomarkers, such as cytogenetic, molecular genetic, or epigenetic aberrations [7‐11]. However, the pathogenesis and optimal therapy of patients with MPAL remain largely undefined. Although several leukemia cell lines were once reported as BAL cell lines [12‐17], none fulfill the WHO 2008 criteria for MPAL. So far, no cell line established from patients with MPAL has been reported. Recently, we established the first human MPAL cell line, JIH-5. Herein, we present the phenotypic, genetic, and functional properties of JIH-5 cells. We applied next-generation sequencing (NGS) technology to unravel the transcriptome of JIH-5 cells.
A 21-year-old female with MPAL was admitted to our hospital in December 2008. Bone marrow sample was obtained from the patient with informed consent in December 2009 during the second relapse. Mononuclear cells were cultured in Iscove’s Modified Dulbecco’s Medium with 20 % fetal calf serum. The leukemia cells exhibited gradual cell proliferation 2 months after primary culture was initiated. The cell line was designated JIH-5. JIH-5 cells were tolerant to freezing in defined medium, storage in liquid nitrogen, thawing, and subsequent expansion. JIH-5 cells grow as single cells in suspension culture.
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JIH-5 cells exhibit medium-sized spheroidal morphologies and large round nuclei with fine nuclear chromatin (Fig. 1a). The immunoprofiles of the JIH-5 cells are summarized in Table 1. JIH-5 cells express typical antigens of myeloid lineages (CD13, CD15, CD33, and cMPO), as well as antigens of B lymphoid lineages (CD10, CD19, CD22, CD23, and cCD79a) (Fig. 1b). The negative polymerase chain reaction (PCR) results with EBV and mycoplasma specific primers excluded EBV and mycoplasma contamination. The colony formation rate of JIH-5 cells was 1.41 % by semi-solid methylcellulose clonogenic assay. Tumor masses were found in one of six mice injected with JIH-5 cells after 83 days. The genetic identity of JIH-5 cells was compared to BM cell sample from the patient using short tandem repeat PCR. The results of authentication analysis indicated that the JIH-5 cells remained genetically identical to the founding tumor cells.
Table 1
Immunophenotypic characterization of the JIH-5 cells and the primary leukemia cells
Antigen (CD)
Primary leukemia cells (%)
JIH-5 cells (%)
At presentation
At the second relapse
T/NK cell markers
CD2
0.2
0.5
16.4
CD3
ND
ND
1.1
CD5
ND
ND
1.1
CD7
0.3
0.6
0
CD56
ND
ND
1.4
cCD3
ND
0
0.3
B cell markers
CD10
5.6
11.6
34.8
CD19
99.8
93.9
90.4
CD20
0.1
0.5
1.7
CD22
ND
ND
73.6
CD23
ND
ND
53.3
FMC-7
ND
ND
2.1
cCD79a
97.6
93.6
80.8
Myeloid markers
CD13
28.9
72.2
79.2
CD14
0.2
1.0
2.1
CD15
14.1
7.3
4.2
CD33
85.2
80.9
78.6
CD64
ND
ND
5.6
MPO
11.9
59.1
66.8
Progenitor markers
CD34
92.5
95.3
13.6
CD38
ND
ND
30
CD117
0.1
0.8
3.6
HLA-DR
65.8
11.2
52.2
Adhesion markers
CD11b
ND
ND
0.9
Erythroid markers
CD71
ND
ND
5.3
GPA
ND
ND
1.3
Megakaryocytic markers
CD41
ND
ND
47.2
CD61
ND
ND
1.1
Plasma cell markers
CD138
ND
ND
1.8
ND not done
×
Combined G-banding and spectral karyotype (SKY) yielded the following karyotype for JIH-5: 46,XX,del(2)(q33)t(2;2)(p22;q37),t(4;5)(q35;q35),t(5;8)(q32;q22),der(6)del(6)(p21p22)t(6;10)(p23;q23),t(7;21)(p15;q21),der(9)del(9)(p21)del(9)(q34.2),der(10)t(6;10),t(12;22)(p13;q13),der(17)t(17;17)(p13;q22),del(19)(q13) (Fig. 2a, b). A total of ten copy number alterations (CNA) were detected by a-CGH. Both fluorescence in situ hybridization (FISH) and a-CGH analysis showed a microdeletion affecting ETV6 gene (Fig. 2c, d). No mutations were detected in 15 acute leukemia-related genes by direct sequencing of PCR products in JIH-5 cells. The global expression profile of JIH-5 was compared to leukemic blast cells from the patient, and a range of cell lines representing B and T cell acute lymphoblastic leukemia (T-ALL). The results indicate that transcriptionally, JIH-5 cells more closely resemble cell lines of B rather than T-ALL origin.
×
We captured and sequenced exomes from the paired sample of JIH-5 cells and control specimen in remission. We detected somatic tumor-specific mutations in a total of nine genes (eight missense and one nonsense mutations), including ABCA8, BCHE, CALCA, CSTF2, FPR1, KCNJ8, MAFB, STMN1, and TAAR8; all were heterozygous in JIH-5 cells. Bioinformatic evaluation of the transcriptional sequencing data and RT-PCR verification revealed six novel fusions, comprising three acting as translocations: EP300 (at 22q13) with both the adjacent ZNF384 and CHD4 (12p13), MSH2 (2p21) with NLK (17q11), and three microdeletions, HACL1-COLQ (3p25), HDAC8-CITED1 (Xq13), and POLA2-CDC42EP2 (11q13). Interestingly, the EP300 gene was found to fuse simultaneously with two partner genes located in 12p13, CHD4, and ZNF384 (Fig. 3a). Further FISH analysis with BAC and fosmid clones flanking EP300, CHD4, and ZNF384 confirmed breakpoints within CHD4 and EP300 due to a complex, apparently insertional, rearrangement involving 12p13 and 22q13 (Fig. 3b). Mutations of EP300 have been detected in Rubinstein-Taybi syndrome and some solid tumors [18‐22]. The EP300 was found to be fused with MLL in an AML patient harboring t(11;22)(q23;q13) [23]. CHD4 encodes a catalytic subunit of the NuRD complex and plays an important role in transcriptional regulation, chromatin assembly, and DNA damage repair [24]. The ZNF384 gene has been observed recurrently fused with EWSR1, TAF15, or E2A in acute leukemia [25, 26]. Recently, the EP300-ZNF384 was identified as a recurrent aberration in B cell acute lymphoblastic leukemia (B-ALL) [27]. The genetic abnormalities found in JIH-5 cells are detailed in Table 2.
Table 2
Synopsis of data on the JIH-5 cell line
Parameter
JIH-5
Clinical data
Patient
21-year-old female
Diagnosis
MPAL
Treatment status
At the second relapse
Specimen
BM
Year of establishment
2009
Culture characterization
Culture medium
IMDM + 20 % FCS
Growth pattern
Single cells in suspension
Doubling time
97 h
Optimal cell density
1 × 106cells/ml
Optimal split
1:3 every 3–4 days
Cryopreservation
In 70 % medium, 20 % FCS, 10 % DMSO
Morphology
medium-sized spheroidal morphologies
Viral status
Negative for EBV
Contamination
Negative for mycoplasma
Authentication
Yes (by DNA finger printing, cytogenetic characteristics, immunoprofile)
In summary, we established a novel MPAL cell line, JIH-5, and characterized its biologic background comprehensively to show a novel oncogenomic gene fusion together with an associated cluster of mutations. Our findings suggested that the JIH-5 cell line may serve as a tool for the study of MPAL or EP300-ZNF384.
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Acknowledgments
This work was supported by grants from the National Key Scientific Projects of China (2011CB933501), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Natural Science Foundation of China (81070416, 81270617), the Jiangsu Provincial Special Program of Medical Science (BL2012005), the Jiangsu Province’s Key Medical Center (ZX201102), the National Public Health Grand Research Foundation (No.201202017), and the Jiangsu Province Natural Science Fund for Distinguished Young Scholars (BK2012006).
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SC was the principal investigator. NP, HQ, and QW performed most of the experiments. SE, CR, and RM performed the cytogenetic and gene expression array analysis. SC, HD, and RM wrote the manuscript. All authors read and approved the final manuscript.
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