Significance of spatial organization of chromosomes in the progression of acute myeloid leukemia
© The Author(s) 2017
Received: 12 June 2016
Accepted: 26 December 2016
Published: 20 April 2017
Leukemia ranks as one of the ten most fatal cancers . The mortality and incidence of this disease are associated with multiple factors, including environmental factors, sex, and age. Distinct genetic and chromosomal aberrations differentially affect the phenotype and prognosis of individuals with leukemia [2, 3]. The t(8;21)(q22;q22) translocation, which is observed in patients with acute myeloid leukemia with maturation (AML-M2, according to the French–American–British classification system), is characterized by the fusion of AML1 (acute myeloid leukemia factor 1, also referred to as RUNX1 [runt-related transcription factor 1]) on chromosome 21 and ETO (eight-twenty-one, also referred to as RUNX1T1 [runt-related transcription factor 1, translocated to 1]) on chromosome 8. Although the t(8;21)(q22;q22) translocation is associated with a favorable prognosis, relapse remains the primary cause of treatment failure . Real-time fluorescent quantitative polymerase chain reaction (RQ-PCR) is a powerful tool for monitoring the presence of residual disease and planning treatment strategies [5, 6]; however, this technique is not 100% accurate . The three-dimensional organization of chromosomes might be a valuable prognostic marker of the risk of relapse in patients with t(8;21)(q22;q22)-positive AML . To further study the utility of this approach, we evaluated bone marrow (BM) samples from a patient with t(8;21)(q22;q22)-positive AML-M2 before and after hematopoietic stem cell transplantation (HSCT) using three-dimensional fluorescence in situ hybridization (3D-FISH) and confocal laser scanning microscopy to delineate and analyze the spatial organization of the target chromosomes. AML1-ETO fusion transcripts detected by RQ-PCR were also discussed in this article.
The 35-year-old male patient was diagnosed with t(8;21)(q22;q22)-positive AML-M2 in January 2013 in Peking University Third Hospital. He was treated with induction chemotherapy and achieved the first complete remission (CR1) in March 2013. However, he deteriorated since November 2014 and relapsed in January 2015. The patient received induction chemotherapy again and achieved the second complete remission (CR2) in March 2015. Then he underwent HSCT in June 2015 and relapsed again in November 2015. Using aspiration, BM specimens (4 mL each) were extracted from the patient when he achieved CR2 (CR2 sample), 2 months after HSCT (post-HSCT sample), 3 months after HSCT (follow-up 1 sample), and 2 months after the first follow-up (follow-up 2 sample). The fifth BM sample (relapse) was extracted 2 weeks after the second follow-up, when the patient relapsed.
Mononuclear cells were isolated from the BM specimens using density gradient centrifugation, and total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using the ProFlex PCR System (Applied Biosystems, Foster City, CA, USA), and RQ-PCR experiments were conducted to detect the AML1-ETO fusion transcripts using the 7500 Real-time PCR System (Applied Biosystems) and TaqMan technology. Abelson (ABL) was used as the reference gene.
Results of RQ-PCR for AML1-ETO fusion transcript detection and 3D-FISH for chromosomal organization detection in a 35-year-old male patient with t(8;21)(q22;q22)-positive AML-M2
AML1-ETO levela detected with RQ-PCR (%)
Percentage of cells detected with 3D-FISH
Labeling chromosomes 8 and 21
Labeling chromosomes 8 and 18
Normal cells (%)
Proximal cells (%)
Malignant cells (%)
Normal cells (%)
Proximal cells (%)
8.6% (8.6% for 3g2r)
14.1% (11.3% for 3g2r)
12.3% (10.8% for 3g2r)
7.6% (6.1% for 3g2r)
After an incubation of 150 min at 37 °C in an environment containing 5% CO2, mononuclear cells adhered to two microscope slides, which were used for the spatial chromosomal organization analysis with 3D-FISH. One slide was used to label and analyze chromosomes 8 and 21 (translocation-associated), whereas the other slide was used to label and analyze chromosomes 8 and 18 (translocation-irrelevant) to reconstruct them in situ and analyze their spatial organization. Whole chromosome 8 fluorescein isothiocyanate (FITC)-conjugated probes, whole chromosome 21 tetramethylrhodamine (TRITC)-conjugated probes, and whole chromosome 18 TRITC-conjugated probes (Kreatech Diagnostics, Amsterdam, the Netherlands) were used to label chromosomes 8, 21, and 18, respectively. The nuclei were counterstained with diamidinophenylindole. 3D-FISH was conducted using a ThermoBrite S500 system (StatSpin, Inc., Westwood, MA, USA). The samples were denatured for 5 min at 75 °C, and the probes were hybridized for 48 h at 37 °C. Optical sections were acquired at room temperature using a Nikon A1Rsi confocal microscope (Nikon Corporation, Shinagawa-ku, Tokyo, Japan) equipped with a plan apo 100 ×/1.4 NA oil immersion objective. Images with an 80 nm × 80 nm resolution and 500 nm axial step size were obtained. A minimum of 60 cells from each slide were evaluated.
In our previous study, we evaluated BM samples from t(8;21)(q22;q22)-negative AML-M2 patients and donors with a normal karyotype . The percentage of malignant cells in all participants was <20%. In the first patient described in this study, 3D-FISH demonstrated that 4.2% of cells in the CR2 sample were malignant, indicating that the patient was in complete remission. After HSCT, RQ-PCR showed complete remission with no AML-ETO fusion transcripts. However, the patient unexpectedly relapsed 5 months after HSCT. 3D-FISH conducted with labeled probes targeting chromosomes 8 and 21 (translocation-associated) demonstrated that the percentage of malignant cells in the BM samples steadily increased after HSCT, indicating that the disease was progressing prior to the time of relapse identification. Among cells labeled with probes targeting chromosomes 8 and 18 (translocation-irrelevant), the percentage of malignant cells detected by 3D-FISH was <20% in all samples, and most malignant cells exhibited chromosome 8 breaks (3g2r in the nuclei) but not chromosome 18 breaks. Furthermore, in cells labeled with probes targeting chromosomes 8 and 21, breaks of both chromosomes were detected. These data indicate that the percentage of malignant cells in the BM samples can contribute to determining the disease prognosis. As shown in Fig. 3, all three patients showed similar phenomenon regarding the significance of malignant cells detected with 3D-FISH as an early warning of potential relapse.
In summary, the spatial organization of chromosomes may play an important role in the progression of AML. As 3D-FISH offers the advantage of labeling and detecting chromosomes in situ and reconstructing them three-dimensionally, it is a useful technique for investigating cancer-associated mechanisms.
XT contributed to the proposal, 3D-FISH experiment, data analysis, and preparation of the manuscript. YW contributed to the RQ-PCR analysis and helped revise the manuscript. DC, XK, and WM contributed to the conception and design of the study and helped revise the manuscript. All authors read and approved the final manuscript.
This study was supported by the National Natural Science Foundation of China (No. 11374180). We thanked Yu Guo and Jing Wang for the RQ-PCR experiment guidance.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets analyzed during the current study are available from the corresponding author on reasonable request.
Consent to participate
This study was approved by the ethics committee of Peking University Third Hospital. All patients have signed informed consent to participate in the study.
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