TO THE EDITOR: Acute myeloid leukemia (AML) with t(8;21)(q22;q22.1), which results in RUNX1-RUNX1T1 gene fusion, is usually associated with a high rate of complete remission and long-term disease-free survival when treated with intensive consolidation therapy (e.g., high-dose cytarabine) [1, 2]. In contrast, AML with BCR-ABL1 accounts for <1% of all AML cases, and appears to be an aggressive disease with a poor response to traditional AML therapy or tyrosine kinase inhibitor therapy alone [3, 4]. However, concurrent RUNX-RUNX1T1 and BCR-ABL1 abnormalities are extremely rare in de novo AML. A treatment guideline has not been established for AML with concurrent RUNX1-RUNX1T1 and BCR-ABL1 fusions, and the treatment effect and prognosis are unknown. Here, we report a case of AML in which both genetic abnormalities were simultaneously detected using reverse transcription polymerase chain reaction (RT-PCR) and sequencing tests.

A 79-year-old male patient was admitted to the Department of Hematology and Oncology, Dong-A University Hospital, Busan, Korea, with cytopenias, which was confirmed by a blood test. He had a history of hypertension, chronic heart failure, unstable angina, diabetes, non-alcoholic liver disease, chronic kidney disease, and asthma. Three months prior to hospitalization for the cytopenias, he was admitted to the Department of Gastroenterology due to dyspnea on exertion and dizziness. Peripheral blood smear (PBS) revealed 4% blast cells along with hypogranulation of granulocytes. The patient and his family discussed further evaluations. Three months later, a follow-up PBS showed blast cells in 37% of the sample, leading to a recommendation for additional tests to rule in acute leukemia. The patient’s chief complaint was chest pain and dyspnea. Splenomegaly (125.41 mm) was observed on computed tomography. Blood tests showed a hemoglobin level of 8.3 g/dL, WBC count of 8,870/µL, and platelet count of 39,000/µL. Red blood cells were macrocytic and hyperchromic. The PBS confirmed 10% segment neutrophils, 22% lymphocytes, 4% monocytes, and 64% blast cells, without eosinophilia or basophilia. The blast cells occasionally showed singular Auer rod (Fig. 1A, B). Additionally, anisopoikilocytosis, mild polychromasia, and a few tear-drop cells were observed. During bone marrow aspiration, 43.8% blast cells were confirmed. The blast cells exhibited bread-like nucleus, salmon-colored granules, and occasional single long, sharp Auer rods (Fig. 1C, D). Hypogranulation was also observed. Special stains, including periodic acid schiff and nonspecific esterase butyrate, were negative, while myeloperoxidase, sudan black B, and specific esterase stains were strongly positive. Flow cytometry analysis revealed positive expression of CD13, CD34, CD56, CD71, CD117, MPO, and HLA-DR. Due to a suspicion of AML, additional tests were conducted. A G-banding chromosome analysis using bone marrow revealed a 46,XY,t(7;21;8)(p22;q22.1;q22)(21)/45,X,-Y,t(7;21;8) (p22;q22.1;q22) karyotype. Fluorescence in-situ hybridization (FISH) was performed for KMT2A, ERG1, del(7q)/CEP7, TP53, and RPN1-MECOM gene rearrangements; however, no positive findings were identified. An RT-PCR for 28 fusion transcripts showed the presence of RUNX-RUNX1T1 and BCR-ABL1(e1a2) minor fusion transcripts (Fig. 2A). A FISH was conducted for BCR-ABL1, as an additional confirmation, but there was no BCR-ABL1 rearrange-ment. Sequencing was performed for CEBPA, FLT3 ITD, FLT3 TKD, NPM1, and KIT genes, confirming single mutation in CEBPA and KIT mutations. Next generation sequencing confirmed a KIT mutation (p.ASP816Tyr by c.2466G>T). Direct sequencing confirmed breakpoints in the BCR exon 1 and ABL1 exon 2 (Fig. 2B). Based on the morphology of AML and presence of both RUNX1-RUNX1T1 and BCR-ABL1 fusion, a diagnosis of AML with RUNX1-RUNX1T1 and BCR-ABL1 fusion was established. A medical team started remission induction therapy using decitabine. After the first treatment, melena of unknown origin persisted. Eventually, the patient refused any further medical intervention, and chose to be discharged. The patient died two months later.

Figure 1. (A, B) PBS showing blast cells with occasional sharp and singular Auer rod (Wright-Giemsa stain, ×1,000). (C, D) Bone marrow aspiration smear showing blast cells with bread-like nucleus, salmon-colored granules, and occasional single, long, sharp Auer rods (Wright-Giemsa stain, ×1,000).

Figure 2. (A) Agarose gel electro-phoresis of the RT-PCR products showing RUNX1-RUNMX1T1 fusion transcript (size, 197 bp) in lane M4A, and BCR-ABL1 fusion tran-script (size, 320 bp) in M8F lane. (B) Sanger sequencing showing BCR-ABL1, b1a2 (size, 320 bp).

Recurrent genetic abnormalities in AML are associated with distinct clinicopathological features and have a prognostic significance. The most commonly identified abnormalities were balanced abnormalities. Most of these structural chromosomal rearrangements create a fusion gene encoding a chimeric protein that is required for leukemogenesis, but is usually insufficient [5]. Many of these diseases exhibit characteristic morphological and immunophenotypic features [6]. Experimental evidences suggest that in cases with rearrangements or mutations in genes that encode transcription factors implicated in myeloid differentiation, additional genetic abnormalities are necessary to promote proliferation or survival of neoplastic clones [7]. This additional abnormality is often caused by a mutation in a gene that encodes proteins that activate signal transduction pathways to promote proliferation and survival. In the revised Word Health Organization classification (2016) [8], it was agreed that structural chromosome rearrangement, such as t(8;21)(q22;q22);RUNX1-RUNX1T1 and inversion inv(16) (p13.1q22)/t(16;16)(p13;q22);CEFB-MYH11, represent “class II mutations” responsible for suppressed and/or altered differentiation as the main leukemic driver genetic event in core binding factor (CBF) leukemias. These mutations are assumed to cooperate with other “class I mutations” to increase the proliferation of mutated leukemic clones. It is extremely rare that BCR-ABL1, as a “class I mutation,” interacts with “class II mutations” in CBF leukemia [8-10]. However, RUNX1-RUNX1T1 belongs to the category of CBF AML and carries overall favorable prognosis. Conversely, AML with BCR-ABL1 accounts for <1% of de novo AML cases, and is known to be an aggressive disease with a poor response to standard AML therapy or tyrosine kinase inhibitor treatment alone. Four cases of AML with both mutations have been reported (Table 1) [10-13]. In 2022, both mutations (RUNX1-RUNX1T1 and BCR-ABL) were confirmed using karyotyping, FISH, and RT-PCR in a 34-year-old male patient. He underwent “3+7” imatinib induction chemotherapy followed by allogeneic stem cell transplantation (SCT), and his survival was confirmed up to 48 months after diagnosis [10]. However, the prognoses of the other three reported cases were poor. In 2009, a 34-year-old patient with osteosarcoma-related AML had both mutations confirmed using cytogenetic analysis and RT-PCR. The patient achieved partial remission with hydroxyurea and imatinib mesylate; however, the disease progressed four months later. Another partial remission was achieved with dacarbazine and themozolomide; however, the patient died after five months [11]. In 2017, a 39-year-old female patient initially harbored only one RUNX1-RUNX1T1 mutation, which was confirmed using PCR. However, BCR-ABL1 was detected 28 days after induction. The patient received cytarabine and imatinib treatment while planning allogeneic SCT, but he died of septic shock [12]. In a brief case reported in 2021, a 75-year-old male patient also received aggressive chemotherapy, as both mutations were confirmed using cytogenetic analysis, FISH, and RT-PCR. Unfortunately, the patient died one month later [13]. In the present case, BCR-ABL1 was not detected using chromosomal examination and FISH, but was only detected using PCR. It is speculated that these results appeared only in PCR because of small genetic abnormalities that were difficult to identify using FISH [14, 15]. Although clinical impact is unclear, limited studies suggest that BCR-ABL1 can cooperate with other mutation types as a “class I mutation” and may rarely co-occur in CBF-rearranged AML [13]. Additionally, although the patient refused further treatment, we confirmed a poor prognosis in this case. More case reports and studies are needed to establish treatment guidelines and prognoses for cases of AML co-existing with BCR-ABL1 and RUNX1-RUNX1T1.

Table 1 . Summary of published cases of AML co-existing with BCR-ABL1 and RUNX1-RUNX1T1 fusions and the present case..

No.	Year	Age/sex	Morphology	Karyotype	FISH/PCR	Outcome	Etc.	Ref.
1	2009	34/F	Large basophilic blasts with azurophilic granules	t(9;22)(q34;q11.2)	RUNX1-RUNX1T1(+)	Poor	Therapy (osteosarcoma) related AML	[11]
				t(8;21)(q22;q22)	BCR-ABL(+) (p190)
2	2017	39/F	Long slender Auer rods	t(8;21)(q22;q22) and loss of X	RUNX1-RUNX1T1(+)	Poor	Confirmed BCR-ABL1 in PCR after induction therapy	[12]
					BCR-ABL(+) (e1a3)
3	2021	75/M	Blast with marked vacuolation	t(8;14;21) (q22;111.2;122)	RUNX1-RUNX1T1(+)	Poor	-	[13]
					BCR-ABL(+)
4	2022	34/M	Myeloblast with Auer rods	RUNX1-RUNX1T1(+)	RUNX1-RUNX1T1(+)	CR	-	[10]
				BCR-ABL(+)	BCR-ABL(+) (p190)
5	2022	79/M	Myeloblast with long slender Auer rod	t(7;21;8)(p22;q22.1; q22)	RUNX1-RUNX1T1(+)	Poor		Present case
				t(7;21;8)(p22;q22.1; q22)	BCR-ABL(+) (e1a2)

Abbreviations: AML, acute myeloid leukemia; CR, complete remission; F, female; FISH, fluorescence in situ hybridization; M, male; PCR, polymerase chain reaction; Ref., reference..

In conclusion, we report a rare case of AML with coexisting BCR-ABL1 and RUNX1-RUNX1T1 rearrangements, which was confirmed using RT-PCR and sequencing tests. The occurrence of de novo AML with concurrent BCR-ABL1 and RUNX1-RUNX1T1 is rare, and there is currently no consensus on the treatment guidelines. Therefore, we report an extremely rare case of AML with concurrent RUNX1-RUNX1T1 and BCR-ABL1 expression and poor prognosis.

No potential conflicts of interest relevant to this article were reported.