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A novel PAX5 rearrangement in TCF3-PBX1 acute lymphoblastic leukemia: a case report



Chromosome translocations are a hallmark of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Additional genomic aberrations are also crucial in both BCP-ALL leukemogenesis and treatment management. Herein, we report the phenotypic and molecular cytogenetic characterization of an extremely rare case of BCP-ALL harboring two concomitant leukemia-associated chromosome translocations: t(1;19)(q23;q13.3) and t(9;17)(p13;q11.2). Of note, we described a new rearrangement between exon 6 of PAX5 and a 17q11.2 region, where intron 3 of SPECC1 is located. This rearrangement seems to disrupt PAX5 similarly to a PAX5 deletion. Furthermore, a distinct karyotype between diagnosis and relapse samples was observed, disclosing a complex clonal evolution during leukemia progression.

Case presentation

A 16-year-old boy was admitted febrile with abdominal and joint pain. At clinical investigation, he presented with anemia, splenomegaly, low white blood cell count and 92% lymphoblast. He was diagnosed with pre-B ALL and treated according to high risk GBTLI-ALL2009. Twelve months after complete remission, he developed a relapse in consequence of a high central nervous system and bone marrow infiltration, and unfortunately died.


To our knowledge, this is the first report of a rearrangement between PAX5 and SPECC1. The presence of TCF3-PBX1 and PAX5-rearrangement at diagnosis and relapse indicates that both might have participated in the malignant transformation disease maintenance and dismal outcome.

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B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by recurrent chromosomal alterations including translocations, which result in aberrant fusion genes. TCF3-PBX1 is one of these fusions and is found in 3–6% of BCP-ALL patients; moreover, the acquisition of secondary genomic aberrations, such as loss of CDKN2A/B, PAX5 or RB1, is common and crucial to leukemogenesis in those cases [1, 2].

PAX5, a master regulator of B-cell differentiation, is frequently disrupted by deletions, point mutations and amplifications in BCP-ALL. These disruptions might result in potentially oncogenic proteins [3, 4]. For example, PAX5 amplifications (PAX5AMP) were identified in diagnostic-relapse matched samples of patients who lacked common cytogenetic abnormalities at relapse, indicating that PAX5AMP may be an important driver of leukemogenesis [5]. Notably, PAX5 can be labeled a “promiscuous” gene since over 16 partner genes have been identified in leukemia-associated rearrangements. These rearrangements result in fusion genes encoding chimeric proteins that modify PAX5 function and occur in 2–3% of pediatric BCP-ALL patients.

Here, we report an extremely rare case of BCP-ALL harboring two concomitant leukemia-associated alterations: TCF3-PBX1 and PAX5 rearrangement (PAX5-r). Additionally, to the best of our knowledge, this is the first report of PAX5 disruption caused by a rearrangement with a 17q11.2 region, where SPECC1 gene is located.

Case presentation

Clinical course

A 16-year-old boy was admitted to the Hospital das Clínicas de Porto Alegre, Porto Alegre, Brazil, febrile with abdominal and joint pain. At clinical investigation, he presented with anemia, splenomegaly and leukocytosis (white blood cells count 19.6 × 109 /L) with 72% lymphoblast. Bone marrow (BM) aspiration disclosed lymphoblast cells infiltration (92%). The central nervous system (CNS) was not infiltrated by blast cells. The immunophenotyping was characterized by nTdT, cCD10, CD20, CD22, CD38 and CD45(low) and cCD9, CD19, cCD79, and CD58(interm)-positive cells in 45% of blast cells. Myeloid and T-cell markers were negative. The patient was treated according to the GBTLI-ALL2009 at high-risk arm. He was a prednisone poor responder (at day 8; > 1000 circulating lymphoblasts), minimal residual disease at day 35 was negative, and he was considered in complete remission (CR). Twelve months after CR, he was hospitalized with a CNS infiltration and BM highly infiltrated with lymphoblasts. The laboratorial investigations demonstrated a similar immunophenotype profile and distinct karyotype. Despite undergoing the relapse treatment-rescue, the patient died due to complications from an opportunistic infection.

Molecular analysis

The diagnosis and characterization of leukemia were established by morphology, immunophenotyping, and molecular-cytogenetic analysis according to the World Health Organization classification [6].

Cytogenetic analysis of leukemic BM was performed using GTG-banding standard procedures, and the karyotype was described according to the International System for Human Cytogenetic Nomenclature (ISCN) of 2013 [7, 8]. The karyotype of the diagnostic sample showed evidence of two concomitant chromosomal translocations (48,XY,t(1;19)(q23;q13.3),del(4)(q27q35),der(9)t(9;17)(p13;q11.2),del(10)(q24q26),del(18)(q21q3,+ 8,+ 22,+marc[20]) (Fig. 1a). In addition to the two rearrangements observed at diagnosis, the karyotype of the BM at relapse also showed near-triploidy: 73,XX,t(1;19)(q23;p13),+ 1,+ 2,+ 3,+ 5,+ 5,+ 6,+ 7,+ 8,i(9q),+ 9,10,+ 12,+ 12,+ 13,+ 14,+ 14,+ 15,+ 15,+ 17,+ 18,+ 19,+ 20,+ 20,+ 20,+ 21,+ 22,+ 22,+ 22,+ 22,+mar [2] (Fig. 1b). The presence of TCF3-PBX1 was confirmed in both diagnostic and relapse samples by reverse transcription PCR (RT-PCR) followed by sequencing (Fig. 1c, d).

Fig. 1
figure 1

Karyotype and TCF3-PBX1 confirmation. a Representative GTG-banded metaphase of the leukemic clone at diagnosis and b at relapse. c RT-PCR to TCF3-PBX1 at diagnosis and relapse, respectively. M, marker (100pb); N, negative control; D, diagnosis sample; R, relapse sample. d Sequencing of truncated TCF3-PBX1 at diagnosis and relapse

Additional copy number alterations (CNA) were identified by multiplex ligation-dependent probe amplification (MLPA) using the SALSA MLPA P335-A4/B1 and P202-B1 kits (MRC-Holland, Amsterdam, the Netherlands) as previously described [9]. MLPA data were analyzed using Coffalyser.Net. The relative copy number was obtained after the normalization of peaks against controls. Values between 0.70 and 1.3 were considered to be within the normal range. Values below 0.70 or above 1.3 indicated deletion or gain, respectively. Values below 0.25 indicated homozygous deletion. Submicroscopic deletions were only identified at relapse moment affecting CDKN2A (exons 2 and 5), CDKN2B (exon 2), IKZF2 (exons 2 and 5) JAK2 (exon 23), and miR31 (exon 1), as well as a partial gain in RB1 (exons 14, 19, 24 and 26) (Fig. 2a, b).

Fig. 2
figure 2

Characterization of additional abnormalities identified. a, b MLPA profile of matched sample of case described using the P335 ALL SALSA MLPA kit at diagnosis and relapse sample, respectively. c 3’RACE-PCR PAX5 and sequencing of truncated PAX5 after 3’RACE-PCR with diagnosis sample. M, marker (1 kb); N, negative control; D, diagnosis sample; R, relapse sample. d RT-PCR to PAX5-SPECC1 and sequencing of PAX5 transcript at diagnosis and relapse. e Expected protein sequence of the truncated PAX5 transcript derived from the PAX5-SPECC1 head-to-head fusion. The translated protein refers to the PAX5–201 (NM_016734), showing its alternating exons (black and green) with splice acceptor amino acid residue (red), and amino acid residues coded from the fusion site on SPECC1 (green). f Schematic representation of the PAX5 wild-type protein and the fusion transcript detected

To investigate the presence of a putative gene fusion derived from the PAX5-r we performed a 3’ Rapid amplification of cDNA ends (3’RACE)-PCR based on Scotto-Lavino’s protocol [10]. After cDNA synthesis, including an oligonucleotide (dT)-tailed primer (QT), two rounds of PCR were performed to enrich the reaction for fragments containing the 3’end of PAX5 transcripts. The first round of amplification used the forward primer PAX5.E4.F1 (5’-AACCAACCAGTCCCAGCTTC-3′), while the second was performed using the PAX5.E5.F1 primer (5’-TACTCCATCAGCGGCATCC-3′). The results showed a head-to-head fusion between exon 6 of PAX5 and a 17q11.2 region, where intron 3 of SPECC1 is located (Fig. 2c).

Subsequently, we used SuperScript™ III One-Step RT-PCR System (Invitrogen, Carlsbad, EUA) and sequencing on ABI®3500 Genetic Analyzer (Applied Biosystems, Foster City, EUA) to confirm that PAX5-r was present at both time points diagnosis and relapse (Fig. 2d). Furthermore, the predicted truncated PAX5 protein consisted of 266 amino acid residues, preserving the paired domain, the octapeptide motif and the homeodomain of PAX5, and a tail of 6 amino acids coded by the contiguous intron 3 sequence of SPECC1, which does not correspond to any predictive functional domain (Fig. 2e, f).

Discussion and conclusions

In this case report, we describe the identification of a der(9)t(9;17)(p13;q11.2) translocation, resulting in PAX5-r, in a 16-year-old boy with TCF3-PBX1 positive BCP-ALL. To date, this is the first report of a rearrangement between PAX5-exon 6 and SPECC1-intron 3. Sixteen PAX5-partner genes had been previously described, composing a heterogeneous group of genes encoding proteins that play distinct roles in signaling pathways, transcription regulation, chromatin remodeling and cell structure [3, 4]. However, in this case report, due to the absence of amino acid residues from SPECC1 in the truncated protein formed, the effect of the new protein identified is likely to be similar to a PAX5 recurrent deletion [11,12,13].

The SPECC1 gene, also known as HCMOGT-1, has three different splicing variants and encodes the NSP5a3a protein, which belongs to the cytospin-A family. NSP5a3a is highly expressed in head and neck squamous cell carcinoma and in testis cell lines, but not in other normal cells [14]. Furthermore, NSP5a3a interacts with the nucleolar phosphoprotein B23, which plays multiple roles in cell growth and proliferation [15]. To the best of our knowledge, the only report of SPECC1 altered in the leukemic context was a case of juvenile myelomonocytic leukemia with PDGFRB-SPECC1 [16].

With regard to other CNAs and in concordance with previous findings in PAX5-rearranged leukemias, few deletions and/or gains are observed [3, 4]. Here, we detected a heterozygous deletions affecting CDKN2A/B, JAK2 and miR31, confirming the presence of an unbalanced t(9;17) with 9p13.2-pter deletion. CDKN2A/B deletions are common in ALL and frequently observed in concomitance with other PAX5 gene fusions, such as PAX5-C20orf112 and PAX5-ETV6 [4]. Regarding TCF3-PBX1 patients, the additional CNAs observed in this case report are in fact the most frequent alterations found in this cytogenetic-subtype. Furthermore, they are known to have a cumulative effect on the leukemic development [17]. Our analysis of CNAs at diagnosis and relapse revealed a high degree of clonal heterogeneity and a complex evolution between diagnosis and relapse samples, suggesting a clonal selection pattern during leukemia progression, which could have implications for the treatment efficiency. Moreover, based on the FMC characterization, we assume that the major clone harboring a TCF3-PBX1 fusion. Although there is no description of BCP-ALL subsets associated with PAX5- r, the presence of a second clone is not discharged and would be explored.

In conclusion, although the occurrence of near-triploidy has been found in BCP-ALL TCF3-PBX1 patients, the concomitant presence of PAX5-r should be revisited. The translocation PAX5-SPECC1 is a new report that requires further investigations. We also showed that both TCF3-PBX1 and PAX5-r were present at diagnosis and relapse, indicating that both might have participated in the malignant transformation disease maintenance and dismal outcome.



3’ Rapid amplification of cDNA ends


B-cell precursor acute lymphoblastic leukemia


Bone marrow


Copy number alterations


Central nervous system


Complete remission


International System for Human Cytogenetic Nomenclature


Multiplex ligation-dependent probe amplification


PAX5 amplifications


Reverse transcription PCR


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We are grateful to Dr. Archana Ganji for the commentaries on karyotype interpretation.


This study was supported by Brazilian National Counsel of Technological and Scientific Development-CNPq (CNPQ#301594/2015–5 and CNPQ-2017#305529/2017–0) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro-FAPERJ (E_26/201.539/2014, E_26/200.388/2016 and E_26/203.214/2017).

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The data sets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Authors and Affiliations



TCB, ME and MSPO conceived the study; TCB, BAL, CBB and MBM performed cytogenetic-molecular experiments, analyzed and interpreted data; TCB. and ME wrote the manuscript; ASVD provided leukemia samples and clinical data used in this study; and all authors contributed with writings and approved the final manuscript.

Corresponding author

Correspondence to Maria S. Pombo-de-Oliveira.

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Informed consent was obtained from the patient’s parent in accordance with the Declaration of Helsinki and ethics was approved by both participating center. The research ethics committee from INCA approved the sample and data collection (n° CAAE 33243214.7.0000.5274).

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Written informed consent was obtained from the patient’s parents for publication of patient’s clinical and molecular data.

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Barbosa, T.C., Lopes, B.A., Blunck, C.B. et al. A novel PAX5 rearrangement in TCF3-PBX1 acute lymphoblastic leukemia: a case report. BMC Med Genomics 11, 122 (2018).

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