Introduction

Acute lymphoblastic leukaemia (ALL) is the most common neoplasm in children, and B cell acute lymphoblastic leukaemia (B-ALL) accounts for 75–80% of all ALL cases [1]. According to the 2016 World Health Organization (WHO-2016) classification, B-ALL cases were classified into several subtypes: morphology, immunophenotype, cytogenetic, and molecular genetic characteristics [1, 2]. For instance, the immunophenotypes of B-ALL populations were classified as precursor B-ALL (pB-ALL) and mature B-ALL (BAL) by flow cytometry (FCM) [2].

The most recurrent ALL type is pB-ALL. The pB-ALL comprises 90% of B-ALL cases and is characterised by the morphologic type (French–American–British (FAB) classification systems) of ALL-L1 or ALL-L2; Flow cytometric analysis with combination of CD19, CyCD22, CyCD79a, TdT, HLA-DR and/or CD22, CD10, CD20, CyIgM, CD34 appear in pB-ALL [2,3,4]. Lysine methyltransferase 2A (KMT2A, previously called Mixed lineage leukaemia, MLL) gene rearrangements (KMT2Ar, previously called MLLr) are generally associated with ALL-L1/ALL-L2 pB-ALL and are present in 6% of paediatric ALL cases [2, 3]. KMT2A-AFF1 (also called MLL-AF4) and KMT2A-MLLT3 (previously called MLL-AF9) fusion gene have been reported in ALL cases, while the KMT2A-MLLT3 fusion gene is a reverse factor of ALL cases [4, 5].

BAL is uncommon for ALL patients and is characterised by FAB-L3 morphology; the presence of surface IgM (sIgM) with light-chain restriction and the absence of immature B cell antigens is typical of BAL cases [2, 6]. BAL is often associated with the translocation t(8;14)(q24;q32) or its variants; the molecular genetics of BAL is characterised by myelocytomatosis oncogene (MYC) rearrangements (MYCr), and it is considered the leukaemic phase of Burkitt lymphoma (BL) [6, 7]. MYCr is overexpressed in more than 95% of BL/BAL patients [2].

However, rare cases of BAL with KMT2Ar expression have been reported in children and adults [8, 9]. This study describes the clinical features, lab findings, treatment, and prognosis of three children with mature BAL with KMT2A-MLLT3 transcripts, and we also reviewed 24 case reports in the literature.

Patients and methods

Entry criteria

  1. 1.

    One month old to under 18 years old;

  2. 2.

    Bone marrow morphology diagnosed as acute mature B lymphocytic leukaemia;

  3. 3.

    Immunological classification is acute mature B lymphocytic leukaemia;

  4. 4.

    with KMT2Ar.

Exit criteria

  1. 1.

    Acute lymphoblastic changes in chronic myeloid leukaemia;

  2. 2.

    Trisomy 21, or congenital or genetic disease accompanied by organ dysfunction;

  3. 3.

    Other secondary leukaemia;

  4. 4.

    Congenital immunodeficiency or metabolic disease;

  5. 5.

    Those who used glucocorticoids for 14 days or more within one month before enrollment or had any history of chemotherapy or radiotherapy within three months.

Patients and samples collection

Three BAL patients with KMT2Ar were enrolled in our hospital study. Clinical data, including age, gender, laboratory findings, treatment, and prognosis, were obtained from the patient records and retrospectively analysed. This study was approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University (CHCMU) and Chengdu Women’s & Children’s Central Hospital (CWCCH), and written informed consent was obtained from all parents.

Bone marrow analysis

Three bone marrow (BM) samples were obtained at diagnosis and at different time points (TP) after chemotherapy. According to morphology, immunophenotype, cytogenetic and molecular genetics, classification was performed at diagnosis according to the WHO-2016 classification of tumours of the haematopoietic and lymphoid tissues [2]. The morphologic type was classified as ALL-L1, L2 or L3 by FAB subtyping. The immunophenotype was determined by FCM with monoclonal antibody markers consisting of B cell, T cell, myeloid and stem/progenitor cell markers, and minimal residential disease (MRD) markers screened by FCM at diagnosis and monitored at different TP [1]. Chromosomal karyotyping and fluorescence in situ hybridisation (FISH) of ETV6-RUNX1, BCR-ABL, KMT2Ar, PDGFRB and MYCr and other rearrangements were performed as reported in the literature; 29 common fusion genes (dupMLL, MLL/AF4, MLL/AF6, BCR/ABL1(p190), MLL/AF1P, MLL/AFX, ALL/ENL, BCR/ABL1(p210), TCF/PBX1, TEL/AML, SIL/TAL1, TLS/ERG, E2A/HLF, TEL/ABL1, HOX11, ETV6/ABL1, NUP214/ABL1, RANBP2/ABL1, SNX2/ABL1, ZMIZ1/ABL1, RCSD1/ABL1, RCSD1/ABL2, ZC3HAV1/ABL2, PAG/ABL2, SSBP2/CSF1R, SSBP2/PDGFRβ, TNIP1TNIP1/ PDGFRβ, ZEB2/PDGFRβ, EBF1/PDGFRβ) were assayed by multiplex nested reverse transcription-polymerase chain reaction (multiplex RT-PCR) and confirmed by split RT-PCR as reported in the literature [10].

Treatment

Patient 1 and Patient 2 were treated according to the B non-Hodgkin lymphoma 2009 (B-NHL-2009) protocol for risk group 3, modified according to the acute lymphoblastic leukaemia multicentre protocols (MCP-841, S1& S2). Additional file 1: S3, S4 and S5 provide the risk group and drug dosage details. Patient 3 received chemotherapy according to the protocol of the Chinese Children’s Cancer Group study ALL-2015 (CCCG-ALL-2015). Prophylactic intrathecal injections were administered for central nervous system (CNS) involvement (S6); BM samples were obtained and evaluated at different TP as protocols required. FCM monitored BM smears and MRD, and RT-PCR was utilised to verify the results.

Literature review

Mature BAL patients with KMT2Ar in the literature were retrieved from PubMed, the Web of Science and China national knowledge infrastructure (CNKI). The keyword is “mature lymphocytic leukaemia” or “lymphoma” and “paediatrics”. Data on clinical characteristics, laboratory findings, treatment, and prognosis were collected and analysed.

Results

Clinical and lab findings

A total of 198 newly diagnosed B-ALL patients, including 21 BAL patients, were admitted to CHCMU and CWCCH between January 2017 and November 2019, and 3 BAL patients with MLL transcripts were identified, accounting for 1.37% of the B-ALL population. The clinical and laboratory findings for the three reported patients are shown in Table 1. BM samples were obtained at diagnosis, and the results of the BM examinations are listed in Table 2, Figs. 1 and 2.

Table 1 Clinical and laboratory findings of reported patients
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Table 2 FCM, FISH, and PCR results for reported patients
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Fig. 1
figure 1

BM morphology

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Fig. 2
figure 2

BM examination by FCM and FISH

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Treatment and prognosis

Patient 1 and Patient 2 were treated with the B-NHL-2009 protocol, and Patient 3 was treated with the CCCG-ALL-2015 protocol. They achieved complete remission (CR) according to morphological, FCM and molecular examination after one chemotherapy course. Allogenic haematopoietic stem cell transplantation (allo-HSCT) was considered by clinicians and was refused by the patients’ parents. A total of six chemotherapy courses were completed for Patient 1 and Patient 2; as of the last follow-up in January 2020, these two patients had a CR status, and their event-free survival (EFS) times were 32 and 29 months, respectively. Chemotherapy was expected to continue for Patient 3, and the EFS time was three months.

Literature review

The literature, including Case reports and retrospective analysis, was searched in the abovementioned databases, and 12 articles involving 24 patients suffering from BAL with KMT2Ar were found. Clinical and laboratory findings, BM examination results, and the treatment and prognosis of these cases in the literature reports are listed in Tables 3, 4 and 5 [8, 9, 11,12,13,14,15,16,17,18,19,20,21].

Table 3 Clinical and laboratory findings of BAL with KMT2Ar according to the literature review
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Table 4 FAB type, FCM and chromosomal karyotyping of BAL with KMT2Ar according to the literature review
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Table 5 FISH, AB type, FCM and chromosomal karyotyping of BAL with KMT2Ar according to the literature review
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Data from our hospital and literature about 27 patients suffering from BAL with KMT2Ar were collected and analysed. 13 males and 14 females were enrolled, and the average and the median age at diagnosis were 19.5 ± 4.95 months old and 12 months old, respectively (ranging from 6 weeks to 9 years); 14 (51.85%) and 24 (88.89%) patients were ≤ 1 and ≤ 2 years of age, respectively. Renal, testicular, CNS and skin involvement at diagnosis were present in 6, 1, 4 and 3 patients, respectively. The average white blood cell (WBC) and platelet (PLT) counts and haemoglobin (Hb) levels were 87 ± 35.24 × 109/L, 69.96 ± 5.38 × 109/L and 65.12 ± 12.34 g/L, respectively.

Even though BAL is typically associated with the FAB-L3 morphology, in our research, 26 (96.30%) of the 27 patients showed non-ALL-L3 morphology, and one patient presented with ALL-L3 morphology. FCM confirmed the mature B-ALL phenotype in all 27 patients. Expression of CD19, CD22, and sIgM with light-chain restriction was detected, and TdT and CD34 did not exist in most cases. Expression of CD20 was found in 25 patients. Interestingly, negative or suspicious expression of CD20 was found in 16 (64%) patients, and positive expression of CD20 was detected with a monoclonal antibody in 9 (36%) patients. Although 2 of 3 patients in our report presented with positive CD20 expression, the expression level of CD20 was lower than 30%.

Chromosomal karyotype results were reported for 26 patients, while two patients had no metaphase chromosomes to be analysed; 11q23-related abnormal karyotypes were found in 11 patients. FISH of KMT2Ar was reported in 23 patients, and 22 (95.65%) cases were positive. The results of the detection of KMT2Ar transcripts were presented for 16 patients. Fourteen patients were positive and were identified as having KMT2A-MLLT3 (Formerly MLL-AF9, 6 cases), KMT2A-MLLT5 (Formerly MLL-AF1, 2 cases), KMT2A-MLLT10 (Formerly MLL-AF10, 2 cases), and KMT2A-MLLT1(also called MLL-ENL, 2 cases), while one patient did not have an exact result. FISH may be the most accurate tool for the detection of KMT2Ar. The t (8;14) and its variant were not detected by karyotyping; FISH detected MYCr in 17 patients, and the results were negative.

The 27 patients who received chemotherapy included patients treated with ALL-like (12 cases), BL (8 cases) or Interfant-99 [21] (7 cases) protocols. One patient succumbed to sepsis, one patient presented with refractory status, 25 patients achieved CR, and the CR rate was 92.59%. Besides, six patients received allo-HSCT, 1 (16.67%) patient relapsed six months later, and the prospective 2-yr EFS (pEFS) was 83.33%, as reported in the literature. Nineteen patients subsequently received chemotherapy according to the Interfant-99 (6 cases), BL (6 cases) or ALL-like (7 cases) protocols, and 9 (47.37%) of them relapsed. The 2-yr pEFS was 41.91% (Fig. 3). Four patients in the Interfant-99 and BL groups relapsed, and the 2-yr pEFS in these groups were 40% and 33.33%, respectively. One patient in the ALL-like group relapsed and died after five months of follow-up [22].

Fig. 3
figure 3

Survival curves of patients. Chemo, chemotherapy; HSCT, haematopoietic stem cell transplantation. Survival analysis by the Kaplan–Meier method and the survival curve of the whole group. Prospective event-free survival (pEFS) in patients who received allogeneic haematopoietic stem cell transplantation (allo-HSCT) was higher than that in patients who received chemotherapy alone (83.33% vs 41.91%)

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Discussion

BAL has been described as an uncommon subtype of B-ALL; it presents with a unique immunotype characterised by the expression of pan-B-cell markers (such as CD10, CD19, CD20, cCD79a.) and sIgM with light-chain restriction, whereas pB-ALL with surface light-chain immunoglobulin restriction has also been reported [22, 23]. The clinical features, biological characteristics, treatment and prognosis of BAL are similar to those of BL, since in the literature has BL, we compared BAL and BL [2, 7]. BAL patients often show an ALL-L3 FAB morphology; like BL, BAL is characterised by MYC translocations (chromosome 8q24) to an immunoglobulin gene locus, and the MYC gene overexpression was detected in most cases [2, 24]. For patients treated with an intensive short course of chemotherapy, the EFS of BAL and BL has exceeded 90% [24].

KMT2A genes, lysine-specific methyltransferase 2A -related genes, occur in 2.5–5% of paediatric ALL patients and 70% of infant ALL patients [2]. KMT2Ar genes’ presence is often correlated with the phenotype of pB-ALL and leads to a worse prognosis [1, 2], but the presence of KMT2Ar in BAL is a distinctive molecular biological feature, and patients’ prognosis of KMT2Ar is unclear.

We reviewed the literature in databases, and a total of 27 patients, including the three patients described in our manuscript, were found. These three BAL patients with KMT2Ar have unique clinical manifestations and laboratory findings compared with pB-ALL and BL patients. Infant leukaemia patients comprised half of these patients, and most of these patients (24/27) were ≤ two years of age at onset, whereas the median ages of pB-ALL and BL patients were 9 and 2–5 years of age, respectively. Renal, CNS and skin involvement at diagnosis were not unusual and were present in 6, 4, and 3 cases, respectively, in patients with BAL with KMT2Ar; however, renal involvement is not rare in BAL, and CNS or skin involvement is uncommon in both pB-ALL and MAL [1, 6]. Although the reported patients were classified as having a BAL phenotype by FCM, overexpression of CD19 was detected in most cases, and expression of CD20 was not detected; nevertheless, coexpression of CD20 and CD19 is common in BL patients [2]. It has been revealed that rituximab [25], an anti-CD20 monoclonal antibody resulting in the selective depletion of B lymphocytes, was unsuitable for treating these patients; any patient who appeared to be refractory or relapsed may benefit from chimeric antigen receptor T cell (CAR-T) immunotherapy targeting the overexpression of CD19 [26].

BL is often associated with t(8;14)(q24;q32) or its variants, and MYCr is detectable in more than 95% of the BL population [2]. Translocation is the essential driver of the MYC gene’s overexpression, and activation of the MYC gene leads to cell cycle progression, inhibition of differentiation, the promotion of cell proliferation and genomic instability and the activation of endogenous apoptotic programmes [25]. However, it is surprising that the MYC gene and its chromosomal translocation were undetectable in the MAL patients with KMT2Ar.

Conclusions

Standard treatment of BAL patients with KMT2Ar has not yet been established, and BAL patients seemed to be sensitive to chemotherapy, including chemotherapy administered according to the ALL, BL or Interfant-99 protocols (The details are in the Additional file 1). Most of these patients achieved CR after receiving one chemotherapy course, but the prognosis of patients subjected to different treatments was widely divergent. The pEFS was higher in the allo-HSCT group than in the chemotherapy group. Even though two patients described in our report received chemotherapy with the BL protocol and survived more than two years, the prognosis of patients treated with chemotherapy has remained poor, and allo-HSCT should be recommended for patients with CR1 status.