Introduction

T-cell Acute Lymphoblastic Leukemia (T-ALL) is an aggressive hematological malignancy, with high tumor heterogeneity and a poor prognosis: it accounts for approximately 10–15% of pediatric ALL cases and 25% of adult ALL cases. Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL), which accounts for 15% of all T-ALL cases, has a poor prognosis with high rates of remission failure or relapse [1]. T-ALL is caused by the co-occurrence of multiple genetic abnormalities that are acquired over time and, once accumulated, lead to full-blown leukemia [2]. Although comprehensive cytogenetic and genomic landscape studies have revealed important molecular abnormalities [3], the drivers of genomic alterations in pediatric T-ALL cases remain unclear. In this study, we identified novel fusions in two pediatric T-ALLs cases diagnosed and treated at the Children’s Hospital of Soochow University using transcriptome sequencing (RNA-seq). In both cases, fusion genes related to the EVX family were identified, forming ETV6::EVX2 and EVX1::MSI2, which activated the HOXD and HOXA cluster genes. HOXA expression results in a subgroup of T-ALL with a wide spectrum of genomic abnormalities affecting HOXA, KMT2A, MLLT10, and NUP214 [3, 4]. However, HOXD activation in T-ALL has not been previously described.

Cases

In the first patient (CCCG140110), a 7-year-old boy was hospitalized for pallor and general weakness for one week. A complete blood count showed a white blood cell (WBC) count of 8.04 × 109/L, hemoglobin (Hb) of 79 g/L, a platelet count of 118 × 109/L, and 32% blast cells. Bone marrow aspiration smears showed the presence of 79% lymphoblasts. Flow cytometry (FCM) revealed that 79.9% of blast cells expressed T-lymphoid-related markers. The blasts were positive for CD7, CD34, and cCD3 and negative for CD3, CD5, and CD1a (Fig. 1A), which are characteristic criteria for an early T-cell precursor ALL (ETP-ALL) diagnosis. Conventional cytogenetic analyses failed, because no mitotic cells were observed. RNA-seq identified an in-frame fusion from ETV6 exon 2 on chromosome 12 to EVX2 exon 2 on chromosome 2 (Fig. 1B), which was further validated using Sanger sequencing and RT-PCR (Fig. 1C and D, Supplementary Table 1). The fusion product retained the DNA-binding domain of EVX2 but had no functional domain of ETV6, suggesting a potential transcription factor function of ETV6::EVX2. Meanwhile, the EVX2 gene is located adjacent to the HOXD gene cluster on chromosome 2. We observed aberrantly high transcription of EVX2 (FPKM = 23.3384), HOXD12 (FPKM = 28.2090), and HOXD13 (FPKM = 41.9178) in this case compared to other T-ALLs from the TARGET project, which do not carry this fusion [5] (Fig. 1E). We hypothesized that the activation of these transcription factors occurs through enhancer hijacking due to the disruption of the three-dimensional architecture of this genomic neighborhood by translocation. Other pathogenic or likely pathogenic variants detected in this case included NRAS G12S and SETD2 at P1571_Esplice and variants of uncertain significance (VUS) for CTNNB1, U2AF2, PHF12, PDS5A, BCR and SREBF2 (Supplementary Table 2). The patient was placed in the intermediate-risk (IR) group for treatment according to the Chinese Children’s Cancer Group (CCCG) ALL 2015 protocol [6]. Minimal residual disease (MRD) monitoring showed that the child responded well to treatment. The MRD was ˂ 0.025% and 0.01% on days 19 and 46, respectively. The patient had been treated for more than 19 months, and MRD remained negative as detected by FCM and RT-PCR.

Fig. 1
figure 1

The molecular data for CCCG140110 at diagnosis. A Immunophenotyping of bone marrow cells at diagnosis in CCCG140110. FCM analyses of leukemia cells revealed that the blast cells were positive for CD7, CD34, and cCD3 and negative for CD5, CD3, and CD1a. B RNA sequencing analysis revealed ETV6::EVX2 fusion with breakpoints in exon 2 of ETV6 and exon 2 of EVX2. C and D The ETV6::EVX2 fusion was validated by Sanger sequencing and RT-PCR in CCCG140110. E Transcription of EVX2, HOXD12, and HOXD13 in CCCG140110 and CCCG140093 together with 264 pediatric T-ALLs from the TARGET project. The Y-axis represents FPKM. The X-axis represents the rank of T-ALL cases

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The second patient (CCCG140093) was a 13-year-old boy with enlarged lymph nodes in multiple body parts, including the neck, armpit, and groin, with no pain and good mobility. Complete blood counts showed a WBC of 5.30 × 109/L, a hemoglobin level of 152 g/L, a platelet count of 209 × 109/L, and peripheral blast cells at 16%. Morphological examination of BM smears revealed 55% lymphoblast infiltration. FCM identified 74.9% of the blastic cells expressing the human leukocyte antigen-DR isotype (HLA-DR), CD7, CD2, and cCD3, whereas CD3 expression was negative, indicating early T-ALL (Fig. 2A). Karyotype was 46, XY. Total RNA-seq identified an out-of-frame fusion between the MSI2 locus on chromosome 17 and the EVX1/HOXA13 locus on chromosome 7 (Fig. 2B), which was confirmed by Sanger sequencing and RT-PCR (Fig. 2C and D, Supplementary Table 1). The breakpoint on chr 7 was 7731 bp upstream of HOXA13. Aberrant activation of HOXA13 was observed in this case (FPKM = 10.7565), comparable to other T-ALLs of the HOXA subtype from the TARGET project with a known mechanism activating HOXA genes [5] (Fig. 2E). Another likely pathogenic genomic variant detected in this case was the KDM6A H702fs mutation, whereas the VUS variants included SUZ12, BCR, and CHD4 (Supplementary Table 2). The patients was treated according to the Chinese Children’s Leukemia Group (CCLG) ALL 2008 protocol [7] and was classified into the high-risk (HR) group, because the MRD was 53.7% on day 15 and 8.1% on day 35. The patient underwent allogeneic hematopoietic stem cell transplantation for 14 months and MRD remained negative as detected by FCM and RT-PCR.

Fig. 2
figure 2

The molecular data for CCCG140093 at diagnosis. A Immunophenotyping of bone marrow cells at diagnosis in CCCG140093. FCM analyses of leukemia cells revealed that the blast cells expressed HLA-DR, CD7, CD2, and cCD3, while CD3 expression was negative. B RNA sequencing analysis showed the MSI2::EVX1/HOXA13 fusion between chr17 and chr7. C and D Validation of EVX1::MSI2 fusion using Sanger sequencing and PCR in CCCG140093. E Transcription of HOXA13 in CCCG140093 and CCCG140110 together with 264 pediatric T-ALLs from the TARGET project. The Y-axis represents FPKM. The X-axis represents the rank of T-ALL cases

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Discussion

In pediatric T-ALL, we observed two fusions, ETV6::EVX2 and MSI2::EXV1/HOXA13. Total RNA-seq was applied in this analysis, which contained the coding exons and non-transcribed regions of the genome like regulatory elements and led to the discovery of an out-of-frame rearrangement of MSI2::EVX1/HOXA13 in the second case. EVX1 and EVX2 are located at the 5′ end of HOXA13 and HOXD13, respectively [8]. These rearrangements activate the transcription of HOXD and HOXA cluster genes through enhancer hijacking, like previously reported [9, 10]. T-ALL is characterized by the dysregulation of key transcription factors involved in T-cell differentiation, including TAL1/2, LMO1/2, TLX1/3, NKX2-1/NKX2-5, LYL1 and HOXA cluster genes [5]. In the two cases reported in the current analysis, HOXA13 and HOXD12/HOXD13 were the only key transcription factors activated, suggesting a likely pathogenic role for these rearrangements. Immature, ETP, and near-ETP phenotypes are involved in 40–45% of HOXA-positive T-ALL [11,12,13]. The molecular findings of the two cases in this study were consistent with the ETP/near-ETP phenotype observed in multiple lines. First, both patients harbored activated HOX family genes. The first patient with the ETV6::EVX2 fusion gene had an ETP immunophenotype with high expression of HOXD13 and HOXD12 but not HOXA. The second patient with MSI2::EVX1/HOXA13 fusion showed high transcription of HOXA13 and had the characteristic immature immunophenotype of T-ALL (early T-ALL), which is between ETP and cortical T-ALL. Second, both cases reported here showed an expression pattern similar to that of ETP/near-ETP. As shown in the tSNE plot (Fig. 3), the two cases grouped with HOXA and LMO2/LYL1 T-ALLs from the TARGET project, representing T-ALL at an early T-cell differentiation stage [5]. Moreover, activating mutations in RAS signaling pathway genes and abnormalities in histone-modifying genes are common in ETP and early T-ALL [14]. Mutations in NRAS and SETD2 were observed in patient CCCG140110 and KDM6A mutations were observed in patient CCCG140093. Notably, EVX2 was activated in CCCG140110 cells by ETV6::EVX2 fusion. As a homeobox transcription factor, the role of EVX2 in leukemia remains largely unknown. EVX2 plays an important role in leukemogenesis in this case, together with HOXD12 and HOXD13. To our knowledge, a translocation involving the EVX1/2 locus has been reported in three T-ALLs, including RUNX1::EVX1 rearrangements in two cases [15, 16] and the ETV6::HOXD locus in one case [17]. The two cases reported here suggest likely pathogenic role for these recurrent translocations and expand our understanding of T-ALL leukemogenesis.

Fig. 3
figure 3

tSNE plot of CCCG140093, CCCG140110, and 264 pediatric T-ALLs from the TARGET project based on gene transcription. Each dot represents an individual T-ALL with molecular subgroups color coded as shown on the right. The two cases in this study, CCCG140093 and CCCG140110, are shown in red and labeled

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