Novel BCR-ABL1 fusion and leukemic mutations of SETBP1, PAX5, and TP53 detected by next generation sequencing in chronic myeloid leukemia

ABSTRACT

Patients with BCR-ABL1 fusion genes are potential candidates for targeted therapy with tyrosine kinase inhibitor (TKI) imatinib. However, novel BCR-ABL1 fusion variants can be undetected by qRT-PCR-based routine molecular screening, affecting immediate patient management and proper treatment selection. In this study, we describe a case of chronic myeloid leukemia (CML) harboring a novel BCR-ABL1 variant gene. Although Fluorescent In situ Hybridization (FISH) analysis suggested Philadelphia (Ph) translocation, qRT-PCR screening failed to detect the presence of a functional fusion transcript, which is critical for selecting targeted therapy against BCR-ABL1 fusion with aberrant kinase activity. Meanwhile, G-band cytogenetic analysis was performed twice without a solid conclusion. To overcome the uncertainty whether TKIs should be used to treat this patient effectively, we performed whole genome sequencing (WGS) in a next-generation sequencing (NGS) platform and discovered an unusual e13a2-like BCR-ABL1 fusion with 9 ABL1 intron 1 nucleotides incorporated into the broken BCR exon 13 to form a novel chimeric exon, which has never been described previously based on the best of our knowledge. Based on FISH and NGS results, the patient was treated with imatinib, showing significant improvement. Moreover, we also detected novel genetic mutations in the known leukemic genes SETBP1, PAX5, and TP53, while their role in the leukemogenesis remains to be determined. In summary, we have identified BCR-ABL1 fusion and other genetic mutations in a diagnostically difficult case of CML, demonstrating that NGS is a powerful diagnostic tool when routine procedures are challenged.

Abbreviations

NGS

next-generation sequencing

WGS

whole genome sequencing

WES

whole exome sequencing

CML

chronic myeloid leukemia

FISH

Fluorescent In-Situ Hybridization

TKI

tyrosine kinase inhibitor

Introduction

Chronic myeloid leukemia (CML) is characterized with oncogenic BCR-ABL fusion, which is also detected in a small population of patients with acute lymphoblastic leukemia (ALL).¹ In CML, the Philadelphia translocations (Ph) of the recurrent genomic rearrangements between chromosome 22q11 and 9q34 usually occur in the intron regions of BCR and ABL1, resulting in the generation of BCR-ABL1 fusion genes. Subsequently, these gene variants transcribe chimeric mRNAs with a juxtaposition of e13a2, e14a2, e19a2, or e1a2.¹ These transcripts are well-characterized and frequently detected by screening methods based on real-time quantitative RT-PCR (qRT-PCR), which is considered as the gold standard for the diagnostic and follow-up examination of Ph positive leukemia due to its superior sensitivity and cost-effectiveness.² However, new and rare forms of BCR-ABL1 fusions have also been discovered and these novel fusion gene variants including those with a masked or undetermined Ph chromosome karyotyping, often cannot be identified by routine RT-PCR methods,^3-15 presenting a big challenge for conventional diagnostic approaches.

During the past decade, next-generation sequencing (NGS) has become an efficient and relatively affordable method for detecting pathogenic mutations for both research and clinical purposes.¹⁶ Using the NGS platform, both whole genome sequencing (WGS) and whole exome sequencing (WES) are powerful tools for the discovery of new disease genes and for the diagnosis of many hematological conditions, such as leukemia, with complex phenotypic and genotypic variations.^17,18 While conventional diagnostic technologies, such as PCR-based and antibody-based detection methods, are still useful to detect known targets, NGS can be used for detecting both identified and novel mutations. Moreover, targeted NGS applications have been developed and have greatly facilitated the usage of this technology as a daily diagnostic tool.¹⁶

In this study, we described a case of CML patient carried a novel variant BCR-ABL fusion that has never been described. Although Fluorescent In Situ Hybridization (FISH) suggested the incidence of BCR-ABL1 rearrangement, a functional mRNA transcript was unable to be detected using qRT-PCR-based diagnostic methods, which are designed for detecting those well-known and previously identified BCR-ABL1 fusion transcripts. Such inconclusive results will against the selection of tyrosine kinase inhibitor (TKI) as a targeted medicine against oncogenic BCR-ABL1. To overcome this obstacle and validate the presence of transcribe BCR-ABL1 fusion gene, we performed whole exome sequencing on the Illumina HiseqX platform, followed by customized RT-PCR and Sanger sequencing of subsequent amplicon. With those, we have identified a novel BCR-ABL1 fusion with an uncommon breakpoint in the exon e13 (also known as b2) rather than in the usual intron regions in the BCR gene. Remarkably, ABL1 intron 1 nucleotides were incorporated into the broken BCR exon 13 to form a novel chimeric exon with a new splicing site. The resulting fusion transcript, which is similar to those often observed in CML patients with e13a2 or e14a2 junctions, was confirmed by customized RT-PCR and DNA direct sequencing. Furthermore, we also detected genomic mutations in several tumorigenic-related genes, including SETBP1, PAX5, and TP53, which require additional studies to determine their pathogenic roles in leukemia. This study provided strong evidence to support that the NGS can be employed as an effective method to detect unknown genetic mutations, which are out of the reach of conventional RT-PCR-based screening methods, in cancer diagnosis.

Case report

A 31-year-old female patient was admitted into the hospital in December 2014. This patient had ongoing gum bleeding for nearly a year without known causes. She displayed or complained about fatigue, headache, insomnia, night sweats, anorexia, malaise and significant unintentional weight loss (∼10 Kg) during the last 6 months. She denied any food and medicine allergy, any alcohol and tobacco usage, and any family history for genetic diseases. She had no history of coronary heart disease, hypertension, and diabetes. At the time of hospital admission, the general physical examination revealed a normal body temperature at 36.5°C, a normal breathing rate at 18/minute, a normal blood pressure at 120/70 mmHg, and a normal heart beat at 80/minute with a regular rhythm. However, the abdominal exam revealed splenomegaly, but was otherwise unremarkable. The blood samples were obtained periodically from the patient for diagnostic and treatment assessment. A bone marrow aspiration and biopsy were performed on the day of the hospitalization. The initial blood count and bone marrow assessment indicated symptoms often observed in CML patients. This study was approved by the Institutional Ethics Committee of Shengjing Hospital of China Medical University. The written informed consent was provided by the patient for all required medical procedures and for publication of this study.

This patient showed symptoms that were frequently observed in CML patients, including fatigue, night sweats, malaise, weight loss, and splenomegaly.²⁰ A complete blood count during the initial diagnosis showed severe leukocytosis and thrombocytosis: leukocytes 371.4 × 10⁹/L, platelets 533 × 10⁹/L, and hemoglobin level 87 g/dL. Assessment of the peripheral blood smear indicated leukoerythroblastic changes with increased neutrophil granulocytes and the presence of circulating myeloblasts (4%), promyelocytes (12.5%), nucleated red blood cells (NRBCs), and immature granulocytes at variant stages including myelocytes (22.5%) and metamyelocytes (13%), normoblasts, basophils, and eosinophils (Fig. 1A). Consistently, examination of the bone marrow revealed remarkable hypercellularity in the granulocytic series and the presence of granulocytes with dysplastia and unevenly distributed granules (Fig. 1B). A significant deficit in erythropoiesis and slightly decreased lymphopoiesis were also observed. Immunogenotyping analysis indicated that 85.19% of cells were neutrophils, which expressed CD33, CD15, CD13, CD16, CD11b, CD38 (abnormally), CD64 (partially), while basophils, eosinophils, monocytes, and erythrocytes accounted for 1.90%, 5.45%, 2.42%, and 0.64%, respectively. This patient also showed hypocalcemia (calcium 2.08 mmol/L) and hyperphosphatemia (phosphate 1.85 mmol/L).

Figure 1.

Wright-Giemsa stain of periphery blood smear and bone marrow aspirate smears. (A) Images showing the presence of myeloblast (M), basophil (B), eosinophil (E), and monocytes (N) in periphery blood film. (B) Image showing granulocytic hypercellularity and dysplastia in a bone marrow aspirate smear.

FISH analysis (GP Medical, Beijing, China) showed that 45.5% of examined cells possessed a nucleus with the BCR-ABL1 fusion demonstrated by yellowish signals occupied by overlapped green (BCR) and red (ABL1) immunosignals. However, quantitative RT-PCR using the Leukemia Related Fusion Gene Detection Kit for BCR-ABL p210, p190, or p230 (Fluorescence RT-PCR) (Shanghai Yuanqi Bio-Pharmaceutical Co., Ltd., Shanghai, China) showed the absence of both p210 and p230 mRNA and negligible level of p190 product (0.02% normalized to ABL). Cytogenetic analysis was performed twice without any conclusive result. Thus, standardized lab procedures using conventional molecular diagnostic methods were unable to confirm the existence of transcribe BCR-ABL1 fusion gene and its resulting transcript in this patient's bone marrow sample.

To validate the presence of the BCR-ABL1 fusion gene and transcript as a potential therapeutic target, we performed WGS on the HiseqX platform (Illumina, San Diego, CA, USA), followed by customized qRT-PCR and Sanger sequencing. Our WGS analysis indicated that a novel BCR-ABL1 fusion gene variant was identified in this patient. The sequence around the breakpoint in the fusion variant is shown in Fig. 2A. As demonstrated in Fig. 2B (top), the breakpoint was located in the exon e13 in the BCR gene and in the intron 1 in the ABL1 gene. We next performed RT-PCR using a 5′ primer targeting BCR exon e13 sequence before the breakpoint (blue arrow head) and a 3′ primer targeting ABL exon 2 sequence. The RT-PCR amplicon was then sequenced via Sanger direct DNA sequencing. Based on the sequencing result (Fig. 2C), 9 nucleotides from ABL1 intron 1 downstream to the breakpoint were inserted into the new BCR-ABL1 hybrid mRNA between the sequences transcribed from BCR e13 and ABL1 exon a2. Therefore, this fusion generated a new splice site, which is located downstream to the abovementioned 9 nucleotides as indicated by a black arrowhead (Fig. 2B middle). To summarize, this novel fusion produced a transcription variant containing sequences derived from a novel chimeric exon between the BCR exon e13 and the ABL1 intron 1, followed by the ABL1 exon a2-transcribed sequence (Fig. 2B bottom).

Figure 2.

Detection of a novel BCR-ABL1 fusion using next-generation sequencing. (A) The sequence around the breakpoint in the fusion gene variant identified by NGS platform. The breakpoint and new splicing site were indicated with arrows. (B) Diagram showing the fusion of BCR and ABL1 genes and resulting mRNA transcript. (C) RT-PCR analysis showing that the novel fusion transcript contains part of BCR exon e13, 9 ABL intronic donor nucleotides, and ABL1 exon a2.

In addition to BCR-ABL1 fusion, we also detected nonsynonymous single nucleotide variants (SNV), including SETBP1 (exon6:c.G4398T:p.E1466D), PAX5 (exon7:c.C791T:p.T264I), and TP53 (exon4:c.C215G:p.P72R), in this patient's bone marrow sample.

The patient was initially treated with hydroxyurea (hydroxycarbamide) and omacetaxine mepesuccinate (also known as homoharringtonine or HHT) to improve blood count and reduce the CML-associated symptoms.²¹ Additional medicines were used to treat persistent low-grade fever that was observed daily during night and morning time. After the detection of a novel BCR-ABL1 variant, this patient was accordingly treated with imatinib at 400 mg/day as a starting dosage. However, the patient was intolerant to such dosage by showing significant side effects. Thus, the amount of imatinib was reduced to 300 mg/day, and eventually to 200 mg/day. Due to the financial difficulties, the patient didn't receive treatment with dasatinib, a more advanced drug targeting BCR-ABL1. In spite of these disadvantages, a partial response was achieved after 3 month's treatment with imatinib (200 mg/day) as evidenced by qRT-PCR showing that BCR-ABL1/ABL1 was reduced considerably by 4.1-fold (Fig. 3A). FISH analysis also indicated a decreased percent of cells displaying BCR-ABL1 fusion signals from the initial 45.5% to 17.5% (Fig. 3B). The patient's condition was significantly improved showing nearly normal blood counts and bone marrow characteristics (Fig. 3C and D). Due to this improvement, the patient was released from the hospital with continued usage of imatinib at 200 mg/day.

Figure 3.

Clinicopathological characterization after targeted therapy. Chronic treatment with imatinib at a reduced dosage at least partially improved clinicopathological features, showing reduced expression level of fusion transcript by qRT-PCR (A), decreased percentage of BCR-ABL1 fusion positive cells by FISH (B), and normal bone marrow characteristics (C and D).

Discussion

The breakpoint in the ABL1 gene is usually detected in the intron region between exon 1a and exon 2 at the 5′ end of the gene.¹ However, the breakpoint can also be detected in the ABL1 introns downstream to the exon 2, resulting in a fusion gene skipping exon 2. In the BCR gene, the breakpoint in the majority of CML patients is usually found in the introns within a region known as the major breakpoint cluster region (M-bcr), which covers exons 12 to 16 (historically named as b1 to b5).¹ The protein products encoded by the resulting fusion genes are described as p210^BCR-ABL fusion proteins with a junction termed as e13a2 or e14a2. In rare cases of CML, the breakpoint in the BCR gene can be detected in the intron, known as the minor bcr (m-bcr), between the 2 alternative exons e2′ and e2.²² In these cases, the fusion BCR-ABL transcripts are translated into, by splicing out exons e1′ and e2′, smaller 190-k_D fusion proteins p190^BCR-ABL with an e1a2 junction. A third type of fusion protein, a large p230^BCR-ABL1 product, is encoded by fusion gene variant with a 3′ end BCR breakpoint located in the region designated as µ-bcr between exons 19 and 20.^23-25 In all these cases, the breakpoints in the BCR gene were detected in the intron regions.

Interestingly, the breakpoint was also detected in the BCR exons in rare cases. Under these circumstances, additional nucleotides are inevitably required to generate a new splicing site for the broken exon and for retaining the new hybrid gene in-frame. It was reported that an inverted intron 1b was inserted in a fusion BCR-ABL gene, resulting in a fusion mRNA transcript with an e8a2 junction in a CML patient.⁹ In the other 2 CML cases, the donor sequence was derived from a third gene: one from the SPECC1L (KIAA0376) gene located 1 Mb telomeric to BCR⁷ and the other from the CABIN1 gene located 0.8 Mb telomeric to BCR, respectively.¹⁴ It has been suggested that these types of rare rearrangements with a breakpoint in BCR exons occur more frequently in exon e8.⁷ In our case, the breakpoint was identified in exon e13, and no third gene was found to be involved in gene rearrangement. However, it has not been determined whether the incorporation of novel sequence from either a previously non-coding intronic region or a third-party gene may confer any pathologic effect during tumorigenesis.

In the current study, we have identified a novel BCR-ABL1 fusion variant and its transcript with a novel chimeric exon formed by part of the BCR exon e13 sequence and the 9 donor ABL1 intronic nucleotides. To our knowledge, this novel fusion gene variant and its mRNA transcript have never been described previously. This study is also informative as it provides an excellent example for those rare cases containing uncharacterized genetic alternations that cannot be identified by routine diagnostic methods. Cytogenetic analysis, FISH, and qRT-PCR are common screening methods that are often used together for the detection of BCR-ABL1 fusions in leukemia. In the case described here, however, while FISH analysis suggested the existence of the BCR-ABL1 fusion gene, its transcript variants p210 and p230 were not detected by qRT-PCR, a diagnostic method routinely used in our hospital to detect common types of BCR-ABL1 fusions in leukemia. Although p190 mRNA variant was detected, but it was at a very low level, which has been reported previously as a minor alternative splicing product in p210 positive cases.^26,27 In addition, conventional cytogenetic analysis has also failed to detect Philadelphia chromosome, which was also reported previously in BCR-ABL1 positive cases for the technical or other unknown reasons.²⁰ Facing these difficulties, WGS in a NGS platform followed by customized RT-PCR and Sanger sequencing represents an excellent approach to overcome the obstacle.

Despite being a novel breakpoint in the BCR exon e13 and a unique insertion of ABL1 intron 1 donor sequence, the oncogenic property of this novel fusion gene variant is less likely to be affected. Like those common fusion variants with e13a2 and e14a2 junctions, this fusion variant should encode a BCR-ABL1 protein with an oncogenic ABL1 tyrosine kinase domain. Therefore, this patient should respond well to those ABL1 tyrosine kinase inhibitor (TKI)-based drugs, including prototype imatinib.^28-30 Indeed, treating this patient with imatinib led to a better outcome compared to the traditional treatment using hydroxyurea and HHT. Thus, NGS can be used in rare cases with uncertain genetic alternations. Moreover, the NGS platform, when it becomes affordable, can be used directly as a standardized method in the daily diagnostic practice in cancer.

Interestingly, novel genomic mutations in known leukemic genes, including SETBP1, PAX5, and TP53, were also detected through NGS. A study has reported that recurrent SETBP1 mutations (encoding a p.Gly870Ser alteration) were detected in patients with atypical chronic myeloid leukemia (aCML) lacking the BCR-ABL1 fusion, exhibiting higher white blood cell counts and worse prognosis.³¹ Further in vitro studies demonstrated that this mutant promoted cell proliferation via enhancing the expression of SETBP1 and SET protein and reducing PP2A activity, representing a leukemogenic gene in aCML and closely related diseases.³¹ Our study indicated that SETBP1 mutation, although at different location (encoding a p.E1466D alternation), can also be detected in CML with BCR-ABL1 fusion. However, it is not clear whether this mutation share any biological or pathological functions with the previously reported mutation. The lymphoid transcription factor gene PAX5 was frequently mutated in ALL and considered as a hallmark of B cell precursor ALL (B-ALL).^32,33 In this study, we reported that a novel PAX5 mutation was detected in a CML case. TP53 is one of the most mutated gene in nearly all type of cancers. In leukemia, TP53 mutations have been detected in patients with acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and ALL, predicting poor prognosis.^34-36

In conclusion, we have identified a novel fusion BCR-ABL1 gene variant using NGS in a CML patient, while qRT-PCR-based routine laboratory procedures failed to do so. Subsequently, the patient received proper targeted treatment accordingly, leading to a better outcome compared to the conventional chemotherapy. Moreover, we also detected somatic mutations in the genes SETBP1, PAX5, and TP53, which should initiate further studies to determine their pathological roles in leukemogenesis. Thus, our study provides valuable insights that can be used to facilitate both clinical practice and laboratory research in leukemia with novel and uncharacterized genetic mutations.

Disclosure of potential conflicts of interest.

No potential conflicts of interest were disclosed.

Funding

This work was supported in part by National Youth Science Foundation of China No. 81300420 (to S.F.).