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Retrospective analysis of 36 fusion genes in 2479 Chinese patients of de novo acute lymphoblastic leukemia
Leukemia Research 72 (2018) 99–104
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Research paper
Retrospective analysis of 36 fusion genes in 2479 Chinese patients of de novo acute lymphoblastic leukemia
T
Xue Chena, Fang Wanga, Yang Zhanga, Mangju Wangb, Wenjun Tianc, Wen Tenga, Xiaoli Maa, ⁎ Lei Guoa, Jiancheng Fanga, Ying Zhangb, Ping Zhub, Hongxing Liua,d, a
Pathology & Laboratory Medicine Division, Hebei Yanda Lu Daopei Hospital, Langfang, 065201, China Department of Hematology, Peking University First Hospital, Beijing, 100034, China c Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 250021, China d Beijing Lu Daopei Institute of Hematology, Beijing, 100176, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Acute lymphoblastic leukemia Fusion genes Pediatric Adult
Fusion genes are major molecular biological abnormalities in hematological malignancies. To depict the common recurrent gene-fusion landscape in acute lymphoblastic leukemia (ALL), 36 recurrent fusion genes in hematologic malignancies were assessed using multiplex-nested RT-PCR in 2479 patients with de novo ALL. 17 kinds of distinct fusion genes were detected in 712 (28.72%) cases. Co-occurrence of different fusion genes was observed in 6 (0.24%) patients. Incidence of fusion genes in B-ALL was significantly higher than in T-ALL (31.40% vs. 14.50%, P < 0.001). Pediatric ALL had higher prevalence of ETV6-RUNX1, TCF3-PBX1, and STILTAL1, while BCR-ABL1 and SET-NUP214 were more common in adult ALL. BCR-ABL1, TCF3-PBX1, KMT2A-AFF1 and ETV6-RUNX1 were more frequent in B-ALL. On the contrary, KMT2A-MLLT4, SET-NUP214 and STIL-TAL1 were of higher incidence in T-ALL. In comparison with Western cohorts, the incidence of BCR-ABL1 (5.94%) was much higher in our series, while the occurrence of ETV6-RUNX1 (13.19%) was significantly lower in pediatric BALL patients in our study than in Western reports. This study provides a genetic landscape of common fusion genes in ALL patients and may serve as a foundation for further improvement of a fusion gene screening panel for clinical applications.
1. Introduction Fusion genes are the main molecular biological abnormalities of hematological malignancies. They exist stably with tumor cells and are well established as diagnostic and prognostic markers for leukemia. They can also be used as molecular markers for monitoring minimal residual disease (MRD) with high sensitivity [1–4]. Based on their essential role in the mechanisms of tumorigenesis, the WHO classification of neoplastic diseases of the hematopoietic and lymphoid tissues has incorporated some fusion genes into the diagnostic criteria for leukemia since 2000 [5]. Over the past several decades, a growing number of fusion genes have been identified, dozens of which are common in hematological malignancies. Of note, most patients are positive of only one kind of fusion gene. The laboratory of hematology of Peking University First Hospital began to carry out fusion genes screening using multiplexnested reverse transcription-PCR (RT-PCR) method according to Pallisgaard et al. [6] with modification in clinical practice since
⁎
September 2002 and this screening method has been applied in many hospitals nationwide ever since then. Nowadays, screening multiple fusion genes simultaneously and then quantitatively monitoring the positive ones has become a routine clinical application. Hebei Yanda Lu Daopei Hospital carried out fusion genes screening service in leukemia since its establishment. Both the two hospitals accumulated a large number of cases and data on fusion genes. Hence, we performed this study to analyze the panoramic incidence of 36 common fusion genes in 2479 patients of de novo acute lymphoblastic leukemia during a 15-year period. The differences in spectrum of these fusion genes between different age groups and different immunophenotypes were systematically compared. Differences in both incidence and spectrum of these fusion genes between Chinese and Western cohorts were also compared.
Corresponding author at: Pathology & Laboratory Medicine Division, Hebei Yanda Lu Daopei Hospital, 6 Sipulan Road, Langfang, 065201, China. E-mail address: [email protected] (H. Liu).
https://doi.org/10.1016/j.leukres.2018.08.009 Received 13 June 2018; Received in revised form 7 August 2018; Accepted 12 August 2018 Available online 15 August 2018 0145-2126/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Fusion genes detected in the cohort of 2479 patients. (A) 17 distinct fusion genes detected in 2479 patients. The plot was created using Circos software. Ribbon widths are proportional to the frequency of fusion genes. Chromosomes were individually colored and are arranged clockwise from chromosome 1 to 22, starting with chromosome 1 at 12 o’clock. (B) Positive rates of each fusion gene detected in 2479 patients.
2. Materials and methods
the interpretation table provided in Table S4.
2.1. Case selection
2.4. Statistical analysis
From September 2002 to April 2017, a total of 2479 patients diagnosed with de novo ALL in Peking University First Hospital and Hebei Yanda Lu Daopei Hospital were included in this study. Among them, 1636 (65.99%) were children (< 19 years, 1003 males and 633 females, median age 6 years, range 2 months-18 years) and 843 (34.01%) were adults (≥19 years, 514 males and 329 females, median age 49 years, range 19–91 years). 2086 (84.15%) were B-ALL (1380 children and 706 adults) and 393 (15.85%) were T-ALL (256 children and 137 adults). The diagnosis of ALL was according to the WHO 2016 classification of tumors of hematopoietic and lymphoid tissues [7]. The studies were approved by the ethics committee at the two hospitals. Written informed consents for medical record review were obtained from all patients or their guardians in accordance with the Declaration of Helsinki.
The χ2 and Fisher’s exact tests were used to compare differences of frequencies of fusion genes between different age groups, different ALL subtypes, and different populations using SPSS statistics (version 20, IBM Corp., Armonk, NY). Two-sides P < 0.05 were considered to be statistically significant. 3. Results 3.1. Spectrum and incidence of fusion genes in ALL patients Here, 17 kinds of distinct fusion genes were detected in 712 (28.72%) of the 2479 patients. Patients who were positive of BCRABL1, TCF3-PBX1, KMT2A-AFF1, FUS-ERG, KMT2A-MLLT1, KMT2AMLLT4, KMT2A-MLLT3, KMT2A-EPS15, KMT2A-MLLT11, TCF3-HLF, and ETV6-PDGFRB all showed corresponding chromosomal abnormalities. On the contrary, patients who were positive of ETV6-RUNX1, STIL-TAL1, KMT2A-PTD, SET-NUP214, and ETV6-ABL1 could not be diagnosed by routine cytogenetics analysis. Two of the three patients who were positive of KMT2A-MLLT10 had t(10;11)(p12;q23), while no karyotype abnormality was found in the other patient. Five fusion gens with relative high incidences were: BCR-ABL1 (10.25%), ETV6-RUNX1 (7.54%), TCF3-PBX1 (4.72%), STIL-TAL1 (1.90%) and KMT2A-AFF1 (1.41%). Positive rates of the remaining fusion genes were all below 1.00% (Fig. 1). Six cases with co-existence of two different fusion genes were identified (three adults and three children), accounting for 0.24% of all cases enrolled in this study and 0.84% of all positive cases. Four of them were B-ALL and were positive for BCR-ABL1 (e1a2)+KMT2A-PTD, BCR-ABL1 (e1a2)+FUS-ERG, FUS-ERG+ETV6-RUNX1, and FUSERG+SET-NUP214, respectively. The other two cases were T-ALL and were positive for STIL-TAL1+BCR-ABL1 (e1a2) and STIL-TAL1+ETV6PDGFRB, separately. The experimental procedures performed on these patients were carefully examined and repeated in different laboratories to eliminate false positive due to PCR product contaminations.
2.2. RNA extraction and cDNA synthesis Total RNA was isolated from peripheral blood or bone marrow of patients by the guanidinium thiocyanate-phenol chloroform method using a TRIZOL reagent according to the manufacturer’s recommendations (Invitrogen Corporation, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using M-MLV Reverse Transcriptase (Promega Corporation, Madison, WI, USA) or a Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. 2.3. Multiplex-nested RT-PCR A multiplex-nested RT-PCR strategy was designed to detect 36 fusion genes which were commonly found in leukemia patients according to Pallisgaard et al. [6] with modification of some of the primers. About 4–8 bases which were complementary to the 3’ end of the corresponding primer were added to the 5’ end of each primer so as to reduce nonspecific amplification before the PCR cycles and improve sensitivity and specificity of the reactions. Details of the fusion transcripts and the number of splice variants of them were listed in Table S1. All primers used were listed in Tables S2–S3. The specific fusion gene and splice variant was identified by comparing the size of the PCR segment with
3.2. Difference of gene-fusion spectrum between pediatric and adult cases In the group of 843 adult ALL, 11 kinds of distinct fusion genes were 100
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Fig. 2. Fusion genes detected in ALL patients of different age groups. (A) Frequencies of positive fusion genes in adult ALL. (B) Frequencies of positive fusion genes in pediatric ALL. Table 1 Distribution of positive fusion genes in 2479 Chinese ALL patients. Total, n (%)
Total BCR-ABL1 ETV6-RUNX1 TCF3-PBX1 STIL-TAL1 KMT2A-AFF1 KMT2A-PTD FUS-ERG KMT2A-MLLT1 SET-NUP214 TCF3-HLF KMT2A-MLLT10 ETV6-PDGFRB KMT2A-MLLT4 KMT2A-MLLT3 KMT2A-EPS15 KMT2A-MLLT11 ETV6-ABL1 KMT2A-PTD/ KMT2A-FG
2479 (100) 254 (10.25) 187 (7.54) 117 (4.72) 47 (1.90) 35 (1.41) 20 (0.81) 17 (0.69) 11 (0.44) 11 (0.44) 6 (0.24) 3 (0.12) 3 (0.12) 2 (0.08) 2 (0.08) 1 (0.04) 1 (0.04) 1 (0.04) 75 (3.03)
Sex, n (%)
Age, years, n (%)
Immunophenotype, n (%)
Male
Female
< 19
≥19
P-value
B-ALL
T-ALL
P-value
1517 (61.19) 143 106 64 37 14 13 9 7 7 1 2 2 2 1 0 1 1 40
962 (38.81) 111 81 53 10 21 7 8 4 4 5 1 1 0 1 1 0 0 35
1636 (65.99) 83 (5.07) 182 (11.12) 88 (5.38) 38 (2.32) 22 (1.34) 11 (0.67) 13 (0.79) 8 (0.49) 2 (0.12) 6 (0.37) 3 (0.18) 1 (0.06) 1 (0.06) 2 (0.12) 1 (0.06) 1 (0.06) 1 (0.06) 49 (3.00)
843 (34.01) 171 (20.28) 5 (0.59) 29 (3.44) 9 (1.07) 13 (1.54) 9 (1.07) 4 (0.47) 3 (0.36) 9 (1.07) 0 0 2 (0.24) 1 (0.12) 0 0 0 0 26 (3.08)
< 0.001 < 0.001 0.035 0.030 0.721 0.344 0.448 0.759 0.002 0.102 0.555 0.268 1.000 0.551 1.000 1.000 1.000 0.902
2086 (84.15) 253 (12.13) 187 (8.96) 117 (5.61) 0 35 (1.68) 19 (0.91) 17 (0.81) 9 (0.43) 6 (0.29) 6 (0.29) 3 (0.14) 2 (0.10) 0 2 (0.10) 1 (0.05) 1 (0.05) 1 (0.05) 70 (3.36)
393 (15.85) 1 (0.25) 0 0 47 (11.96) 0 1 (0.25) 0 2 (0.51) 5 (1.27) 0 0 1 (0.25) 2 (0.51) 0 0 0 0 5 (1.27)
< 0.001 < 0.001 < 0.001 < 0.001 0.004 0.350 0.093 0.689 0.020 0.598 1.000 0.404 0.025 1.000 1.000 1.000 1.000 0.024
3.4. BCR-ABL1 fusion gene
observed in 252 (29.89%) patients. In the group of 1636 pediatric ALL, 17 kinds of distinct fusion transcripts were detected in 460 (28.12%) cases (Fig. 2). There was no significant difference in the frequency of fusion genes between adult and pediatric ALL (P = 0.373). Pediatric ALL had higher prevalence of ETV6-RUNX1, TCF3-PBX1, and STILTAL1, while BCR-ABL1 and SET-NUP214 were more common in adult ALL (Table 1).
Different breakpoints in the BCR or ABL1 gene generate different isoforms of BCR-ABL1 fusion transcript. The most common three types of them (e13a2, e14a2 and e1a2) were included in our screening panel. BCR-ABL1 was the most common fusion gene in this study, which was detected in 254 (10.25%) cases. 251of them carried BCR-ABL1 only, the remaining 3 cases concurrently harbored other fusion genes. Except for one T-ALL case, who was positive of both STIL-TAL1 and BCR-ABL1 (e1a2), the other 253 cases were all B-ALL. In all patients positive of BCR-ABL1, 77.56% had e1a2, 12.60% had e14a2, 9.84% had e13a2. All these three types of BCR-ABL1 genotypes were more common in adult ALL than in pediatric cases (P < 0.001).
3.3. Difference of gene-fusion spectrum between B-ALL and T-ALL In the group of 2086 B-ALL, 15 kinds of distinct fusion transcripts were found in 655 cases (31.40%). In the group of 393 T-ALL, 7 kinds of distinct fusion genes were detected in 57 patients (14.50%) (Fig. 3). Incidence of fusion genes in B-ALL was significantly higher than in TALL (P < 0.001). BCR-ABL1, TCF3-PBX1, KMT2A-AFF1 and ETV6RUNX1 were more frequent in B-ALL. On the contrary, KMT2A-MLLT4, SET-NUP214 and STIL-TAL1 were of higher incidence in T-ALL (Table 1).
3.5. Distribution of fusion genes in different age groups We investigated the frequency of fusion genes in different age groups according to the age categories under 1 years, 1–9 years, 10–14 years, 15–19 years, 20–29 years, 30–39 years, 40–49 years, 50–59 101
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Fig. 3. Fusion genes detected in ALL patients of different subtypes. (A) Frequencies of positive fusion genes in B-ALL. (B) Frequencies of positive fusion genes in TALL.
Fig. 4. Age-related distribution of the major fusion genes. X-axis: age group in years. Y-axis: percentage of patients positive of fusion genes. The different colors of the columns represent diverse fusion genes.
compared between our series and Western cohorts in pediatric and adult cases, respectively. In pediatric B-ALL patients, the incidence of BCR-ABL1 was much higher in this study than in Western reports (5.94% vs. 1.5%–3%, all P < 0.05) [8–11].On the contrary, the occurrence of ETV6-RUNX1 in Chinese pediatric B-ALL was significantly lower than that in Western cohorts (13.19% vs. 18.8–25%, all P < 0.05) [8–11]. No significant differences were observed in adult ALL patients.
years, and 60 years and older. The number of cases and distribution of different fusion genes in each age group were displayed in Fig. 4. The primary fusion gene in infants younger than 1 year was KMT2A-AFF1, with a positive rate of 46.67% which was significantly higher than in other age groups. With the exception of 15 infant ALL, patients of 30–59 years had the highest frequency of fusion genes. The occurrence of BCR-ABL1 increased with the growth of age and peaked in patients between 50–59 years. On the contrary, the incidence of ETV6-RUNX1 peaked at 1–9 years and declined with age. STIL-TAL1 was mainly observed in patients no older than 29 years.
4. Discussion Recurrent fusion genes in hematological tumors are major genetic variants that contribute to tumorigenesis. These exist stably within tumor cells and have been used as molecular markers for the diagnosis, classification, risk assessment and targeted therapy of leukemia. Although fusion genes have definite clinical significance, their
3.6. Comparison of common fusion genes between our results and Western reports Incidences of the five most common fusion genes, including BCRABL1, ETV6-RUNX1, TCF3-PBX1, STIL-TAL1, and KMT2A-AFF1, were 102
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lymphoid tissues as a provisional tissues [7]. BCR-ABL1-like ALL is characterized by a wide range of genetic alterations that dysregulate several cytokine receptor and kinase signaling pathways (predominantly ABL-class and Janus kinases), including gene rearrangements involving genes including ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, and PDGFRB [18,19]. It is a common subtype of B-ALL, representing 7%–25% of new diagnoses, but the incidence of a specific fusion gene is rather rare [20–24]. We detected ETV6-ABL1 and ETV6PDGFRB in our screening panel and only three patients (0.12%) were found to be positive of ETV6-PDGFRB and one patient (0.04%) was found to be positive of ETV6-ABL1. We use “fusion gene family (FGfamily)” to describe this kind of fusions which involved one constant gene and multiple fusion partners. Therefore, it is important to design effective ways to detect all members of one FG-family so as to help molecular diagnosis, risk stratification and MRD monitoring of leukemia patients. Besides fluorescence in situ hybridization (FISH) utilizing breakapart probes, hybrid capture-based targeted next-generation sequencing (NGS), anchored multiplex PCR-based targeted NGS, and whole transcriptome sequencing [19], we are now exploring much more economical and practical methods to detect members of the FGfamilies reported in leukemia (data not shown in this paper).
relationships with disease phenotypes are often quite complicated, and the positive rate of a specific fusion gene is rather limited. Panel screening of several common fusion genes has effectively solved the problem of low positive rate of single fusion gene detection. Multiplexnested RT-PCR offers rapid turnaround time and enables testing of a wide range of possible gene fusions. Nevertheless, novel gene partners remain undetectable by standard RT-PCR methods. Moreover, fusions that are rarely reported are not included in our screening panel. In this study, we simultaneously detected 36 common fusion genes in hematological malignancies and retrospectively analyzed the results of both children and adults in a large cohort of 2479 Chinese de novo ALL patients during a 15-year period. Fusion genes were detected in 28.72% of all patients and a significantly higher fraction of positive patients was observed in B-ALL than in T-ALL (31.40% vs. 14.50%, P < 0.001). Several fusion genes, including BCR-ABL1, TCF3-PBX1, KMT2A-AFF1, and ETV6-RUNX1, were more common in B-ALL. On the contrary, KMT2A-MLLT4, SETNUP214, and STIL-TAL1 were more frequently in T-ALL. The incidences and frequencies of fusion genes were also associated with age. With the exception of 15 infant ALL, patients of 30–59 years had the highest frequency of fusion genes. Pediatric ALL had higher prevalence of ETV6-RUNX1, TCF3-PBX1 and STIL-TAL1, while BCR-ABL1 and SETNUP214 were more common in adult ALL. There were also some differences in the incidences of fusion genes between our series and Western cohorts. BCR-ABL1, which was the most common fusion gene in this study, had a higher incidence in our cohort than in European and American populations in pediatric patients. Moreover, the incidence of ETV6-RUNX1 was much lower in pediatric patients in our series than that in Western reports. These results indicate that regional and racial differences in the pathogenesis of ALL exist. Data in this study showed that BCR-ABL1 was the most common fusion gene in ALL. The majority of BCR-ABL1+ ALL were B-ALL, except one T-ALL who was also positive of STIL-TAL1. The incidence of BCR-ABL1 in adult B-ALL was 24.22%, which was similar to the reports from Europe and the United States (15–37.97%) [12–14]. Frequencies of diverse BCR-ABL1 transcripts in 2498 adult B-ALL had been investigated by various German Multicenter ALL study group. In all patients positive of BCR-ABL1, 66.26% had e1a2, 31.19% had e13a2 or e14a2, 1.66% had e1a2 in combination with e14a2 or e13a2 [15]. A single center study from China had reported the results of BCR-ABL1 isotypes in 100 adult B-ALL. 64.00% had e1a2, 22.00% had e13a2 or e14a2, another 14.00% had e1a2 in combination with e14a2 or e13a2 [16]. In this survey, we identified that 70.76% of adult BCR-ABL1+ BALL expressed e1a2, the remaining 29.24% expressed e13a2 or e14a2. No patients expressed e1a2 in combination with e14a2 or e13a2 were detected. The reason for the rare co-existence of different BCR-ABL1 transcripts in our study may be that we strictly verified the experimental process and repeated in different laboratories when encountered rare situations to maximally eliminate false positive due to PCR product contaminations. In this study, we find that in certain group of fusion genes, the positive rate of one specific fusion is fairly low, but the occurrence of the total group is not rare. As in the case of fusion genes involving the KMT2A gene, which includes 135 different KMT2A rearrangements and 94 fusion partners are now characterized at the molecular level [17]. Our screening included partial tandem duplications of specific KMT2A gene portions (KMT2A-PTD) and ten gene fusions (KMT2A-FG) which are reported to be the most common aberrations involving KMT2A [17]. Except for KMT2A-AFF1, which was detected in 1.41% of patients, the incidence of KMT2A-PTD and the other seven types of KMT2A-FG were all below 1%, and no KMT2A-ELL, KMT2A-MLLT6, or KMT2AMLLT7 was detected. Our results further confirm the fact that although KMT2A-FG/PTD is quite common in acute leukemia, the positive rate of a specific fusion gene involving KMT2A is rather rare. Another example is BCR-ABL1-like ALL, which is now incorporated into the 2016 revision of WHO classification of neoplastic diseases of the hematopoietic and
5. Conclusions Our findings delineate the frequencies and distributions of common fusion genes in ALL cases and confirmed different incidences between age groups and races, and it will help to provide a foundation in data for further improvement of a fusion gene screening panel in clinical applications. Conflicts of interest The authors have no conflicts of interest to disclose. Contributions X.C. designed the study, analyzed and interpreted the data and drafted the manuscript. F.W., Y.Z. and W.T. performed patient management and collected clinical information of eligible patients from the Hebei Yanda Lu Daopei Hospital. M.W. and P.Z. performed patient management and collected clinical information of eligible patients from Peking University First Hospital. X.M., L.G., and Y.Z. carried out experiments and analyzed experimental data. J.F. made some of the figures. H.L. designed the study and revised the manuscript critically. All authors read and approved the final manuscript. Acknowledgments The authors thank Qing Yin, Hao Wang and Wei Zhang for their work in clinical data collection and experimental operation. This work was supported in part by grants from the National Natural Science Foundation of China (81370612) (P.Z.) and the Youth Science Fund Project of the National Natural Science Foundation of China (81102211) (M.W.). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.leukres.2018.08.009. References [1] T. Szczepanski, C.J. Harrison, J.J. van Dongen, Genetic aberrations in paediatric acute leukaemias and implications for management of patients, Lancet Oncol. 11 (9) (2010) 880–889, https://doi.org/10.1016/S1470-2045(09)70369-9. [2] H. Inaba, M. Greaves, C.G. Mullighan, Acute lymphoblastic leukaemia, Lancet 381
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Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Research paper
Retrospective analysis of 36 fusion genes in 2479 Chinese patients of de novo acute lymphoblastic leukemia
T
Xue Chena, Fang Wanga, Yang Zhanga, Mangju Wangb, Wenjun Tianc, Wen Tenga, Xiaoli Maa, ⁎ Lei Guoa, Jiancheng Fanga, Ying Zhangb, Ping Zhub, Hongxing Liua,d, a
Pathology & Laboratory Medicine Division, Hebei Yanda Lu Daopei Hospital, Langfang, 065201, China Department of Hematology, Peking University First Hospital, Beijing, 100034, China c Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, 250021, China d Beijing Lu Daopei Institute of Hematology, Beijing, 100176, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Acute lymphoblastic leukemia Fusion genes Pediatric Adult
Fusion genes are major molecular biological abnormalities in hematological malignancies. To depict the common recurrent gene-fusion landscape in acute lymphoblastic leukemia (ALL), 36 recurrent fusion genes in hematologic malignancies were assessed using multiplex-nested RT-PCR in 2479 patients with de novo ALL. 17 kinds of distinct fusion genes were detected in 712 (28.72%) cases. Co-occurrence of different fusion genes was observed in 6 (0.24%) patients. Incidence of fusion genes in B-ALL was significantly higher than in T-ALL (31.40% vs. 14.50%, P < 0.001). Pediatric ALL had higher prevalence of ETV6-RUNX1, TCF3-PBX1, and STILTAL1, while BCR-ABL1 and SET-NUP214 were more common in adult ALL. BCR-ABL1, TCF3-PBX1, KMT2A-AFF1 and ETV6-RUNX1 were more frequent in B-ALL. On the contrary, KMT2A-MLLT4, SET-NUP214 and STIL-TAL1 were of higher incidence in T-ALL. In comparison with Western cohorts, the incidence of BCR-ABL1 (5.94%) was much higher in our series, while the occurrence of ETV6-RUNX1 (13.19%) was significantly lower in pediatric BALL patients in our study than in Western reports. This study provides a genetic landscape of common fusion genes in ALL patients and may serve as a foundation for further improvement of a fusion gene screening panel for clinical applications.
1. Introduction Fusion genes are the main molecular biological abnormalities of hematological malignancies. They exist stably with tumor cells and are well established as diagnostic and prognostic markers for leukemia. They can also be used as molecular markers for monitoring minimal residual disease (MRD) with high sensitivity [1–4]. Based on their essential role in the mechanisms of tumorigenesis, the WHO classification of neoplastic diseases of the hematopoietic and lymphoid tissues has incorporated some fusion genes into the diagnostic criteria for leukemia since 2000 [5]. Over the past several decades, a growing number of fusion genes have been identified, dozens of which are common in hematological malignancies. Of note, most patients are positive of only one kind of fusion gene. The laboratory of hematology of Peking University First Hospital began to carry out fusion genes screening using multiplexnested reverse transcription-PCR (RT-PCR) method according to Pallisgaard et al. [6] with modification in clinical practice since
⁎
September 2002 and this screening method has been applied in many hospitals nationwide ever since then. Nowadays, screening multiple fusion genes simultaneously and then quantitatively monitoring the positive ones has become a routine clinical application. Hebei Yanda Lu Daopei Hospital carried out fusion genes screening service in leukemia since its establishment. Both the two hospitals accumulated a large number of cases and data on fusion genes. Hence, we performed this study to analyze the panoramic incidence of 36 common fusion genes in 2479 patients of de novo acute lymphoblastic leukemia during a 15-year period. The differences in spectrum of these fusion genes between different age groups and different immunophenotypes were systematically compared. Differences in both incidence and spectrum of these fusion genes between Chinese and Western cohorts were also compared.
Corresponding author at: Pathology & Laboratory Medicine Division, Hebei Yanda Lu Daopei Hospital, 6 Sipulan Road, Langfang, 065201, China. E-mail address: [email protected] (H. Liu).
https://doi.org/10.1016/j.leukres.2018.08.009 Received 13 June 2018; Received in revised form 7 August 2018; Accepted 12 August 2018 Available online 15 August 2018 0145-2126/ © 2018 Elsevier Ltd. All rights reserved.
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Fig. 1. Fusion genes detected in the cohort of 2479 patients. (A) 17 distinct fusion genes detected in 2479 patients. The plot was created using Circos software. Ribbon widths are proportional to the frequency of fusion genes. Chromosomes were individually colored and are arranged clockwise from chromosome 1 to 22, starting with chromosome 1 at 12 o’clock. (B) Positive rates of each fusion gene detected in 2479 patients.
2. Materials and methods
the interpretation table provided in Table S4.
2.1. Case selection
2.4. Statistical analysis
From September 2002 to April 2017, a total of 2479 patients diagnosed with de novo ALL in Peking University First Hospital and Hebei Yanda Lu Daopei Hospital were included in this study. Among them, 1636 (65.99%) were children (< 19 years, 1003 males and 633 females, median age 6 years, range 2 months-18 years) and 843 (34.01%) were adults (≥19 years, 514 males and 329 females, median age 49 years, range 19–91 years). 2086 (84.15%) were B-ALL (1380 children and 706 adults) and 393 (15.85%) were T-ALL (256 children and 137 adults). The diagnosis of ALL was according to the WHO 2016 classification of tumors of hematopoietic and lymphoid tissues [7]. The studies were approved by the ethics committee at the two hospitals. Written informed consents for medical record review were obtained from all patients or their guardians in accordance with the Declaration of Helsinki.
The χ2 and Fisher’s exact tests were used to compare differences of frequencies of fusion genes between different age groups, different ALL subtypes, and different populations using SPSS statistics (version 20, IBM Corp., Armonk, NY). Two-sides P < 0.05 were considered to be statistically significant. 3. Results 3.1. Spectrum and incidence of fusion genes in ALL patients Here, 17 kinds of distinct fusion genes were detected in 712 (28.72%) of the 2479 patients. Patients who were positive of BCRABL1, TCF3-PBX1, KMT2A-AFF1, FUS-ERG, KMT2A-MLLT1, KMT2AMLLT4, KMT2A-MLLT3, KMT2A-EPS15, KMT2A-MLLT11, TCF3-HLF, and ETV6-PDGFRB all showed corresponding chromosomal abnormalities. On the contrary, patients who were positive of ETV6-RUNX1, STIL-TAL1, KMT2A-PTD, SET-NUP214, and ETV6-ABL1 could not be diagnosed by routine cytogenetics analysis. Two of the three patients who were positive of KMT2A-MLLT10 had t(10;11)(p12;q23), while no karyotype abnormality was found in the other patient. Five fusion gens with relative high incidences were: BCR-ABL1 (10.25%), ETV6-RUNX1 (7.54%), TCF3-PBX1 (4.72%), STIL-TAL1 (1.90%) and KMT2A-AFF1 (1.41%). Positive rates of the remaining fusion genes were all below 1.00% (Fig. 1). Six cases with co-existence of two different fusion genes were identified (three adults and three children), accounting for 0.24% of all cases enrolled in this study and 0.84% of all positive cases. Four of them were B-ALL and were positive for BCR-ABL1 (e1a2)+KMT2A-PTD, BCR-ABL1 (e1a2)+FUS-ERG, FUS-ERG+ETV6-RUNX1, and FUSERG+SET-NUP214, respectively. The other two cases were T-ALL and were positive for STIL-TAL1+BCR-ABL1 (e1a2) and STIL-TAL1+ETV6PDGFRB, separately. The experimental procedures performed on these patients were carefully examined and repeated in different laboratories to eliminate false positive due to PCR product contaminations.
2.2. RNA extraction and cDNA synthesis Total RNA was isolated from peripheral blood or bone marrow of patients by the guanidinium thiocyanate-phenol chloroform method using a TRIZOL reagent according to the manufacturer’s recommendations (Invitrogen Corporation, Carlsbad, CA, USA). Complementary DNA (cDNA) was synthesized using M-MLV Reverse Transcriptase (Promega Corporation, Madison, WI, USA) or a Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. 2.3. Multiplex-nested RT-PCR A multiplex-nested RT-PCR strategy was designed to detect 36 fusion genes which were commonly found in leukemia patients according to Pallisgaard et al. [6] with modification of some of the primers. About 4–8 bases which were complementary to the 3’ end of the corresponding primer were added to the 5’ end of each primer so as to reduce nonspecific amplification before the PCR cycles and improve sensitivity and specificity of the reactions. Details of the fusion transcripts and the number of splice variants of them were listed in Table S1. All primers used were listed in Tables S2–S3. The specific fusion gene and splice variant was identified by comparing the size of the PCR segment with
3.2. Difference of gene-fusion spectrum between pediatric and adult cases In the group of 843 adult ALL, 11 kinds of distinct fusion genes were 100
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Fig. 2. Fusion genes detected in ALL patients of different age groups. (A) Frequencies of positive fusion genes in adult ALL. (B) Frequencies of positive fusion genes in pediatric ALL. Table 1 Distribution of positive fusion genes in 2479 Chinese ALL patients. Total, n (%)
Total BCR-ABL1 ETV6-RUNX1 TCF3-PBX1 STIL-TAL1 KMT2A-AFF1 KMT2A-PTD FUS-ERG KMT2A-MLLT1 SET-NUP214 TCF3-HLF KMT2A-MLLT10 ETV6-PDGFRB KMT2A-MLLT4 KMT2A-MLLT3 KMT2A-EPS15 KMT2A-MLLT11 ETV6-ABL1 KMT2A-PTD/ KMT2A-FG
2479 (100) 254 (10.25) 187 (7.54) 117 (4.72) 47 (1.90) 35 (1.41) 20 (0.81) 17 (0.69) 11 (0.44) 11 (0.44) 6 (0.24) 3 (0.12) 3 (0.12) 2 (0.08) 2 (0.08) 1 (0.04) 1 (0.04) 1 (0.04) 75 (3.03)
Sex, n (%)
Age, years, n (%)
Immunophenotype, n (%)
Male
Female
< 19
≥19
P-value
B-ALL
T-ALL
P-value
1517 (61.19) 143 106 64 37 14 13 9 7 7 1 2 2 2 1 0 1 1 40
962 (38.81) 111 81 53 10 21 7 8 4 4 5 1 1 0 1 1 0 0 35
1636 (65.99) 83 (5.07) 182 (11.12) 88 (5.38) 38 (2.32) 22 (1.34) 11 (0.67) 13 (0.79) 8 (0.49) 2 (0.12) 6 (0.37) 3 (0.18) 1 (0.06) 1 (0.06) 2 (0.12) 1 (0.06) 1 (0.06) 1 (0.06) 49 (3.00)
843 (34.01) 171 (20.28) 5 (0.59) 29 (3.44) 9 (1.07) 13 (1.54) 9 (1.07) 4 (0.47) 3 (0.36) 9 (1.07) 0 0 2 (0.24) 1 (0.12) 0 0 0 0 26 (3.08)
< 0.001 < 0.001 0.035 0.030 0.721 0.344 0.448 0.759 0.002 0.102 0.555 0.268 1.000 0.551 1.000 1.000 1.000 0.902
2086 (84.15) 253 (12.13) 187 (8.96) 117 (5.61) 0 35 (1.68) 19 (0.91) 17 (0.81) 9 (0.43) 6 (0.29) 6 (0.29) 3 (0.14) 2 (0.10) 0 2 (0.10) 1 (0.05) 1 (0.05) 1 (0.05) 70 (3.36)
393 (15.85) 1 (0.25) 0 0 47 (11.96) 0 1 (0.25) 0 2 (0.51) 5 (1.27) 0 0 1 (0.25) 2 (0.51) 0 0 0 0 5 (1.27)
< 0.001 < 0.001 < 0.001 < 0.001 0.004 0.350 0.093 0.689 0.020 0.598 1.000 0.404 0.025 1.000 1.000 1.000 1.000 0.024
3.4. BCR-ABL1 fusion gene
observed in 252 (29.89%) patients. In the group of 1636 pediatric ALL, 17 kinds of distinct fusion transcripts were detected in 460 (28.12%) cases (Fig. 2). There was no significant difference in the frequency of fusion genes between adult and pediatric ALL (P = 0.373). Pediatric ALL had higher prevalence of ETV6-RUNX1, TCF3-PBX1, and STILTAL1, while BCR-ABL1 and SET-NUP214 were more common in adult ALL (Table 1).
Different breakpoints in the BCR or ABL1 gene generate different isoforms of BCR-ABL1 fusion transcript. The most common three types of them (e13a2, e14a2 and e1a2) were included in our screening panel. BCR-ABL1 was the most common fusion gene in this study, which was detected in 254 (10.25%) cases. 251of them carried BCR-ABL1 only, the remaining 3 cases concurrently harbored other fusion genes. Except for one T-ALL case, who was positive of both STIL-TAL1 and BCR-ABL1 (e1a2), the other 253 cases were all B-ALL. In all patients positive of BCR-ABL1, 77.56% had e1a2, 12.60% had e14a2, 9.84% had e13a2. All these three types of BCR-ABL1 genotypes were more common in adult ALL than in pediatric cases (P < 0.001).
3.3. Difference of gene-fusion spectrum between B-ALL and T-ALL In the group of 2086 B-ALL, 15 kinds of distinct fusion transcripts were found in 655 cases (31.40%). In the group of 393 T-ALL, 7 kinds of distinct fusion genes were detected in 57 patients (14.50%) (Fig. 3). Incidence of fusion genes in B-ALL was significantly higher than in TALL (P < 0.001). BCR-ABL1, TCF3-PBX1, KMT2A-AFF1 and ETV6RUNX1 were more frequent in B-ALL. On the contrary, KMT2A-MLLT4, SET-NUP214 and STIL-TAL1 were of higher incidence in T-ALL (Table 1).
3.5. Distribution of fusion genes in different age groups We investigated the frequency of fusion genes in different age groups according to the age categories under 1 years, 1–9 years, 10–14 years, 15–19 years, 20–29 years, 30–39 years, 40–49 years, 50–59 101
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Fig. 3. Fusion genes detected in ALL patients of different subtypes. (A) Frequencies of positive fusion genes in B-ALL. (B) Frequencies of positive fusion genes in TALL.
Fig. 4. Age-related distribution of the major fusion genes. X-axis: age group in years. Y-axis: percentage of patients positive of fusion genes. The different colors of the columns represent diverse fusion genes.
compared between our series and Western cohorts in pediatric and adult cases, respectively. In pediatric B-ALL patients, the incidence of BCR-ABL1 was much higher in this study than in Western reports (5.94% vs. 1.5%–3%, all P < 0.05) [8–11].On the contrary, the occurrence of ETV6-RUNX1 in Chinese pediatric B-ALL was significantly lower than that in Western cohorts (13.19% vs. 18.8–25%, all P < 0.05) [8–11]. No significant differences were observed in adult ALL patients.
years, and 60 years and older. The number of cases and distribution of different fusion genes in each age group were displayed in Fig. 4. The primary fusion gene in infants younger than 1 year was KMT2A-AFF1, with a positive rate of 46.67% which was significantly higher than in other age groups. With the exception of 15 infant ALL, patients of 30–59 years had the highest frequency of fusion genes. The occurrence of BCR-ABL1 increased with the growth of age and peaked in patients between 50–59 years. On the contrary, the incidence of ETV6-RUNX1 peaked at 1–9 years and declined with age. STIL-TAL1 was mainly observed in patients no older than 29 years.
4. Discussion Recurrent fusion genes in hematological tumors are major genetic variants that contribute to tumorigenesis. These exist stably within tumor cells and have been used as molecular markers for the diagnosis, classification, risk assessment and targeted therapy of leukemia. Although fusion genes have definite clinical significance, their
3.6. Comparison of common fusion genes between our results and Western reports Incidences of the five most common fusion genes, including BCRABL1, ETV6-RUNX1, TCF3-PBX1, STIL-TAL1, and KMT2A-AFF1, were 102
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lymphoid tissues as a provisional tissues [7]. BCR-ABL1-like ALL is characterized by a wide range of genetic alterations that dysregulate several cytokine receptor and kinase signaling pathways (predominantly ABL-class and Janus kinases), including gene rearrangements involving genes including ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, and PDGFRB [18,19]. It is a common subtype of B-ALL, representing 7%–25% of new diagnoses, but the incidence of a specific fusion gene is rather rare [20–24]. We detected ETV6-ABL1 and ETV6PDGFRB in our screening panel and only three patients (0.12%) were found to be positive of ETV6-PDGFRB and one patient (0.04%) was found to be positive of ETV6-ABL1. We use “fusion gene family (FGfamily)” to describe this kind of fusions which involved one constant gene and multiple fusion partners. Therefore, it is important to design effective ways to detect all members of one FG-family so as to help molecular diagnosis, risk stratification and MRD monitoring of leukemia patients. Besides fluorescence in situ hybridization (FISH) utilizing breakapart probes, hybrid capture-based targeted next-generation sequencing (NGS), anchored multiplex PCR-based targeted NGS, and whole transcriptome sequencing [19], we are now exploring much more economical and practical methods to detect members of the FGfamilies reported in leukemia (data not shown in this paper).
relationships with disease phenotypes are often quite complicated, and the positive rate of a specific fusion gene is rather limited. Panel screening of several common fusion genes has effectively solved the problem of low positive rate of single fusion gene detection. Multiplexnested RT-PCR offers rapid turnaround time and enables testing of a wide range of possible gene fusions. Nevertheless, novel gene partners remain undetectable by standard RT-PCR methods. Moreover, fusions that are rarely reported are not included in our screening panel. In this study, we simultaneously detected 36 common fusion genes in hematological malignancies and retrospectively analyzed the results of both children and adults in a large cohort of 2479 Chinese de novo ALL patients during a 15-year period. Fusion genes were detected in 28.72% of all patients and a significantly higher fraction of positive patients was observed in B-ALL than in T-ALL (31.40% vs. 14.50%, P < 0.001). Several fusion genes, including BCR-ABL1, TCF3-PBX1, KMT2A-AFF1, and ETV6-RUNX1, were more common in B-ALL. On the contrary, KMT2A-MLLT4, SETNUP214, and STIL-TAL1 were more frequently in T-ALL. The incidences and frequencies of fusion genes were also associated with age. With the exception of 15 infant ALL, patients of 30–59 years had the highest frequency of fusion genes. Pediatric ALL had higher prevalence of ETV6-RUNX1, TCF3-PBX1 and STIL-TAL1, while BCR-ABL1 and SETNUP214 were more common in adult ALL. There were also some differences in the incidences of fusion genes between our series and Western cohorts. BCR-ABL1, which was the most common fusion gene in this study, had a higher incidence in our cohort than in European and American populations in pediatric patients. Moreover, the incidence of ETV6-RUNX1 was much lower in pediatric patients in our series than that in Western reports. These results indicate that regional and racial differences in the pathogenesis of ALL exist. Data in this study showed that BCR-ABL1 was the most common fusion gene in ALL. The majority of BCR-ABL1+ ALL were B-ALL, except one T-ALL who was also positive of STIL-TAL1. The incidence of BCR-ABL1 in adult B-ALL was 24.22%, which was similar to the reports from Europe and the United States (15–37.97%) [12–14]. Frequencies of diverse BCR-ABL1 transcripts in 2498 adult B-ALL had been investigated by various German Multicenter ALL study group. In all patients positive of BCR-ABL1, 66.26% had e1a2, 31.19% had e13a2 or e14a2, 1.66% had e1a2 in combination with e14a2 or e13a2 [15]. A single center study from China had reported the results of BCR-ABL1 isotypes in 100 adult B-ALL. 64.00% had e1a2, 22.00% had e13a2 or e14a2, another 14.00% had e1a2 in combination with e14a2 or e13a2 [16]. In this survey, we identified that 70.76% of adult BCR-ABL1+ BALL expressed e1a2, the remaining 29.24% expressed e13a2 or e14a2. No patients expressed e1a2 in combination with e14a2 or e13a2 were detected. The reason for the rare co-existence of different BCR-ABL1 transcripts in our study may be that we strictly verified the experimental process and repeated in different laboratories when encountered rare situations to maximally eliminate false positive due to PCR product contaminations. In this study, we find that in certain group of fusion genes, the positive rate of one specific fusion is fairly low, but the occurrence of the total group is not rare. As in the case of fusion genes involving the KMT2A gene, which includes 135 different KMT2A rearrangements and 94 fusion partners are now characterized at the molecular level [17]. Our screening included partial tandem duplications of specific KMT2A gene portions (KMT2A-PTD) and ten gene fusions (KMT2A-FG) which are reported to be the most common aberrations involving KMT2A [17]. Except for KMT2A-AFF1, which was detected in 1.41% of patients, the incidence of KMT2A-PTD and the other seven types of KMT2A-FG were all below 1%, and no KMT2A-ELL, KMT2A-MLLT6, or KMT2AMLLT7 was detected. Our results further confirm the fact that although KMT2A-FG/PTD is quite common in acute leukemia, the positive rate of a specific fusion gene involving KMT2A is rather rare. Another example is BCR-ABL1-like ALL, which is now incorporated into the 2016 revision of WHO classification of neoplastic diseases of the hematopoietic and
5. Conclusions Our findings delineate the frequencies and distributions of common fusion genes in ALL cases and confirmed different incidences between age groups and races, and it will help to provide a foundation in data for further improvement of a fusion gene screening panel in clinical applications. Conflicts of interest The authors have no conflicts of interest to disclose. Contributions X.C. designed the study, analyzed and interpreted the data and drafted the manuscript. F.W., Y.Z. and W.T. performed patient management and collected clinical information of eligible patients from the Hebei Yanda Lu Daopei Hospital. M.W. and P.Z. performed patient management and collected clinical information of eligible patients from Peking University First Hospital. X.M., L.G., and Y.Z. carried out experiments and analyzed experimental data. J.F. made some of the figures. H.L. designed the study and revised the manuscript critically. All authors read and approved the final manuscript. Acknowledgments The authors thank Qing Yin, Hao Wang and Wei Zhang for their work in clinical data collection and experimental operation. This work was supported in part by grants from the National Natural Science Foundation of China (81370612) (P.Z.) and the Youth Science Fund Project of the National Natural Science Foundation of China (81102211) (M.W.). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.leukres.2018.08.009. References [1] T. Szczepanski, C.J. Harrison, J.J. van Dongen, Genetic aberrations in paediatric acute leukaemias and implications for management of patients, Lancet Oncol. 11 (9) (2010) 880–889, https://doi.org/10.1016/S1470-2045(09)70369-9. [2] H. Inaba, M. Greaves, C.G. Mullighan, Acute lymphoblastic leukaemia, Lancet 381
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