Background

Abnormalities of chromosome 16 are found in about 5–8% of acute myeloid leukemia (AML) and are one of the three AML defining chromosomal aberrations regardless of blast percentage under the World Health Organization (WHO) classification [1]. The AML with inv(16)(p13.1q22) or t (16;16)(p13.1;q22) is associated with a high rate of complete remission (CR) and favorable overall survival (OS) when treated with high-dose Cytarabine [2, 3]. The inv(16) results in a leukemogenic CBFB/MYH11 gene fusion [4, 5]. However, additional chromosome changes and/or gene mutations, such as + 22, + 8, deletion of 7q, and the CBL, FLT3, KIT gene mutations, are frequently found in AML with inv(16). These additional changes/mutations may influence the OS positively or negatively [6, 7]. For example, gain of an additional chromosome 22 in AML with inv(16) may predict an improved outcome [6, 8], whereas KIT mutations appear to have an increased risk of relapse and shorter survival in adult patients [6, 9]. Less commonly, deletions, especially deletion of 3’CBFB, may occur at the inversion breakpoints. Fourteen cases with inv(16) and deletion of 3’CBFB have been reported in literature [10,11,12,13,14,15,16,17,18,19]. However, only three of them were studied by microarray analysis, and only one was reported with unambiguous breakpoint coordinates [10]. The genomic characteristics of the vast majority of the reported 3’CBFB deletions were undefined.

Here we reported two AML with inv(16) cases, both carried an additional deletion at one inversion breakpoint involving the fusion between 5’MYH11 at 16p13.1 and 3’CBFB at 16q22. The genomic characteristics of both cases were characterized by chromosome, FISH and SNP microarray analyses. We also reviewed similar cases reported in literature to investigate possible clinical significance of the deletions at the inv(16) breakpoints.

Case presentation

Case 1

A 24-year-old male presented with intermittent fever and sore throat. A complete blood examination demonstrated a hemoglobin (Hb) count of 70 g/L, a white blood cell (WBC) count of 170 × 109/L with 80% blasts and a platelet count of 25 × 109/L. He had no hepatosplenomegaly. His bone marrow (BM) aspirate exhibited greater than 90% myelomonoblastic cells, with increased maturing eosinophils. Cytochemical staining was positive for peroxidase and esterase. Flow cytometry showed 76% positive CD34 and 5% positive CD64, but negative CD14, consistent with a diagnosis of AML. The patient started induction chemotherapy and achieved a complete hematological recovery on day 21. He then received autologous stem cell transplant (ASCT) after two cycles of consolidation chemotherapy and has remained in CR for three years.

Case 2

A 47-year-old male presented with intermittent low-grade fever and progressive fatigue. A complete blood examination demonstrated a Hb count of 92 g/L, a WBC count of 3.5 × 109/L and a platelet count of 43 × 109/L. This patient had splenomegaly. His BM aspirate showed 50% myeloid blasts. Flow cytometry showed 50% blasts expressed CD34, CD117, CD13, CD33, HLA-DR, and myeloperoxidase (MPO), consistent with a diagnosis of AML. The patient started induction chemotherapy and achieved a complete hematological recovery. He then received two cycles of consolidation chemotherapy. The patient has been in CR for four years.

Methods

Chromosome and FISH analyses

Chromosome G-banding was performed following standard techniques on BM aspirate. FISH was carried out with the commercial CBFB dual-color break-apart probe kit (Abbott Laboratories, Lake Bluff, IL) following the manufacturer’s protocol. The results were analyzed using the Leica CytoVision system (Leica Biosystems, San Jose, CA).

SNP microarray analysis

Genomic DNA was extracted from BM using QIAGEN EZ1 kit (Qiagen, Hilden, Germany). SNP microarray was set up using Illumina CytoSNP-850 K v1.1 BeadChip (Illumina, San Diego, CA) and analyzed using BlueFuse Multi (Illumina), based on human genome build GRCh37/hg19.

The nomenclature for the chromosome, FISH and array findings is based on the International System for Human Cytogenomic Nomenclature (ISCN) 2016 [20].

Results

In case 1, chromosome analysis detected an abnormal karyotype with a pericentric inversion of chromosome 16 in all 20 cells analyzed (Fig. 1a), consistent with the diagnosis of AML with inv(16)(p13.1q22); CBFB-MYH11. In addition, 4 of these cells showed an extra copy of chromosome 22, which is a common secondary change in this disease. FISH analysis confirmed the inversion but also detected a deletion of 3’CBFB in approximately 95.5% of the interphase cells examined (Fig. 1a). SNP microarray confirmed the gain of chromosome 22 and a 1.1 Mb deletion involving 3’CBFB on the long arm of chromosome 16 within 16q22.1, and further detected a 416 Kb deletion involving the 5’MYH11 gene on the short arm of chromosome 16 within 16p13.11. The 16q22.1 deletion involved 53 known genes including 3’CBFB, and the 16p13.11 deletion involved 7 known genes including 5’MYH11 (Fig. 1b). The nomenclature of the cytogenomic findings in case 1 can be described as: 46,XY,inv(16)(p13.1q22)[16]/47,idem,+ 22[4].nuc ish (5’CBFBx2,3’CBFB×1)(5’CBFB con 3’CBFB×1)[191/200].arr[GRCh37] 16p13.11(15875744_16291983)×1[0.9],16q22.1(67128019_68214140)×1[0.9],(22) ×3[0.8].

Fig. 1
figure 1

a Chromosome 16 with inv(16)(p13.1q22), 3’CBFB deletion detected by interphase FISH (5’CBFB, red; 3’CBFB, green), and two small deletions on chromosome 16 detected by SNP microarray. b Genes in the deleted regions (black bars), adopted from UCSC genome browser (https://www.genome.ucsc.edu). Case 3 was from Dawson et al. [10], and COSMIC cancer genes were underlined

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In case 2, a similar inversion of 16 with gain of additional chromosomes 9 and 22 was detected in all 20 metaphases analyzed by chromosome analysis (Fig. 1a). FISH revealed a similar abnormal signal pattern with a deletion of 3’CBFB in approximately 83.5% of the interphase cells examined (Fig. 1a). Similar to case 1, SNP microarray confirmed the chromosome and FISH findings and detected an additional deletion on the short arm of chromosome 16 (Fig. 1a). The 16p13.11p12.3 deletion is 1.1 Mb in size involving 14 known genes including 5’MYH11, and the 16q22.1 deletion is 986 kb in size involving 52 genes including 3’CBFB (Fig. 1b). The cytogenomic findings of case 2 can be described as: 48,XY,+ 9,inv(16)(p13.1q22),+ 22[20].nuc ish(5’CBFBx2,3’CBFB×1)(5’CBFB con 3’CBFB×1) [167/200].arr[GRCh37] (9)×3[0.7],16p13.11p12.3(15817490_16869754)×1[0.7],16q22.1(67113418_68099821)×1[0.7],(22)×3[0.7].

Discussion and conclusion

AML with inv(16) and deletion of 3’CBFB has been reported in at least 14 cases and most of the deletions were detected by FISH using CBFB break-apart probes [10,11,12,13,14,15,16,17,18,19]. To our knowledge, only three cases were analyzed using microarray, and of these cases, only one reported unambiguous genomic coordinates of the deletion [10, 11]. Noteworthily, all the cases analyzed with microarray, including the ones in this report, showed an additional deletion of 5’MYH11, suggesting that the 3’CBFB and 5’MYH11 were most likely deleted together as a fusion product of inv(16) instead of being deleted separately. More than 10 CBFB-MYH11 fusion transcripts with different sizes due to various genomic breakpoints have been reported [21, 22]. In this study, CBFB exons 1–5 fused with MYH11 exons 8–42 in case 1 and CBFB exons 1–4 fused with MYH11 exons 33–42 in case 2. Both are typical gene fusions in AML-associated inv(16), and the 5’CBFB/3’MYH11 fusion genes are believed to be responsible for the disease of the patients [23]. However, there is no evidence of additional phenotypical effects of deletion of the 5’MYH11/3’CBFB fusion genes. Both patients in this study responded to chemotherapy well and achieved CR for multiple years. Three cancer-related genes listed in the Cancer Gene Census (CGC) in the Catalogue of Somatic Mutations in Cancer (COSMIC) were involved in the deleted regions, including MYH11 in 16p, CBFB and CTCF in 16q (Fig. 1b) [24]. CBFB/MYH11 gene fusion caused by inv(16) or t (16;16) results in two fusion genes, 5’CBFB/3’MYH11 and 5’MYH11/3’CBFB. The former is a known pathogenic cause of AML, but the pathogenic effect of the latter is uncertain. We further reviewed reported AML cases with inv(16) and deletion of 3’CBFB (Table 1) and did not find significant phenotypic differences caused by the deletion of 3’CBFB. Of the 14 cases with clinical data available, 11 (79%) were known to achieve CR with known survival time up to 48 months at the time of report. This small cohort data appears to be in line with the CR rate (86–88%) and the five-year OS rate (50%) in AML with inv(16) [8, 25]. Taken together, the findings suggest that the 5’MYH11/3’CBFB fusion gene may play minimal roles in AML pathogenesis. CTCF is thought to be a tumor suppresser gene [26]. Kemp et al. reported CTCF haploinsufficiency destabilized DNA methylation and predisposed mice to cancer [27]. However, deletion of this gene in the current cases apparently did not result in additional phenotypic changes. Such changes, if there are any, may be less significant or overlapped with that of the AML with inv(16). Nevertheless, the significance of deletion of CTCF in AML with inv(16) needs to be further investigated. There are five cases with inv(16) and deletion of 3’CBFB, which either did not achieve CR or relapsed (Table 1). Since the deletion sizes and other genomic characteristics of these cases were undefined, it is unclear whether additional genes or other genomic elements were affected. Array analysis of similar cases may provide more insights on genomic changes and corresponding pathogenic effects in these cases.

Table 1 The previously reported AML cases with inv(16) and 3’CBFB deletion
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Deletion of the 3’CBFB gene is a recurrent finding in AML with inv(16). It most likely represents deletions of the 5’MYH11/3’CBFB gene fusion from the inv(16) as a secondary change following the inversion. The 5’CBFB/3’MYH11 fusion resulting from the inv(16) is a known pathogenic cause of AML, but the 5’MYH11/3’CBFB fusion may play a minimal role in AML pathogenesis. The CTCF gene adjacent to the 3’CBFB was deleted in the current cases, suggesting that deletions of CTCF in AML with inv(16) may not have prognostic significance either. However, potential pathogenic effects of other genes involved in extended deletion regions may not be excluded.