- Case report
- Open Access
A case of B-cell acute lymphoblastic leukemia in a child with Down syndrome bearing a t(2;12)(p12;p13) involving ETV6 and biallelic IGH@ rearrangements
- Carlos A. Tirado†1Email author,
- David Shabsovich†1,
- Yeun Kim1,
- Peter Traum1,
- Sheeja Pullarkat1,
- Michael Kallen1 and
- Nagesh Rao1
© Tirado et al. 2015
Received: 17 April 2015
Accepted: 26 May 2015
Published: 5 June 2015
The Erratum to this article has been published in Biomarker Research 2015 3:27
Rearrangements involving ETV6 (12p13) are among the most common structural abnormalities in pediatric B-cell acute lymphoblastic leukemia (B-ALL) and involve numerous partner genes. Additionally, the t(8;14)(q11.2;q32), which can result in the placement of CEBPD (8q11.2) near the regulatory regions of IGH@ (14q32) and consequent overexpression of CEPBD, occurs at a higher frequency in individuals with Down syndrome-associated ALL (DS-ALL) compared to both the general and pediatric population. The coexistence of cytogenetically detectable ETV6 abnormalities and t(8;14)(q11.2;q32) is a rare occurrence in B-ALL and has only been reported in a single case in the literature.
Herein, we present a case of B-ALL in a 9-year old male with Down syndrome in which conventional cytogenetic analysis revealed two reciprocal translocations: a t(8;14)(q11.2;q32) and a t(2;12)(p12;p13). Interphase and metaphase fluorescence in situ hybridization (FISH) analysis using break apart probes confirmed the involvement of IGH@ and ETV6 in these translocations, respectively. Additionally, interphase FISH revealed a clonal subpopulation bearing biallelic IGH@ rearrangements not observed by conventional cytogenetic analysis.
To the best of our knowledge, this is the first reported case of B-ALL bearing an ETV6 translocation with a partner gene on the short arm of chromosome 2 confirmed by FISH. Additionally, it is the second reported case of t(8;14)(q11.2;q32)-ALL bearing a concomitant, cytogenetically detectable abnormality involving ETV6. This case provides insight into a novel translocation involving ETV6 as well as potentially unique and understudied mechanisms of clonal evolution in pediatric B-ALL.
Pediatric B-acute lymphoblastic leukemia (B-ALL) often bears a range of numerical and structural cytogenetic abnormalities, including but not limited to the following, in decreasing frequency: t(12;21)(p13;q22) [ETV6/RUNX1], hyperdiploidy, t(1;19)(q23;p13.3) [PBX1/TCF3], t(4;11)(q21;q23) [AFF1/MLL], hypodiploidy, and t(9;22)(q34;q11.2) [BCR/ABL1] . Additionally, the t(8;14)(q11.2;q32), which can result in the placement of CEBPD (8q11.2) near the regulatory regions of IGH@ (14q32) and consequent overexpression of CEPBD, occurs at a much higher frequency in individuals with Down syndrome compared to both the general and pediatric population and is associated with an intermediate prognosis .
Rearrangements involving ETV6 (12p13) are among the most common structural abnormalities in pediatric B-ALL. The t(12;21)(p13;q22), the most common of these translocations, results in the production of a chimeric transcription factor bearing the DNA-binding domain of RUNX1 (21q22) and the transactivation domain of ETV6 (12p13), resulting in aberrant activation of genes regulated by RUNX1. Although the t(12;21) [ETV6/RUNX1] is the most common of these rearrangements, other translocations involving ETV6 with greater than 20 partner genes have been observed, including protein tyrosine kinases and transcription factors, many of which can act by distinct mechanisms to promote leukemogenesis . Additionally, other anomalies involving ETV6 have been observed in various hematological malignancies ranging from deletions, to point mutations, to alterations at the epigenetic level, to amplifications [3–5]. Although abnormalities involving ETV6 are a relatively common finding in B-ALL, the precise leukemogenic role of the gene in the context of some of the aforementioned aberrations remains understudied.
A 9-year old male with Down syndrome presented with persistent fever and fatigue. Complete blood count revealed pancytopenia (WBC 3.01×103/μL, RBC 2.87×106/μL, platelet count 43×103/μL) with a differential of 32 % lymphoblasts, 52 % lymphocytes, 7 % neutrophils, 4 % monocytes, 1 % metamyelocyte, and 1 % myelocyte. Flow cytometry on peripheral blood revealed excess abnormal blasts comprising 22 % of total cells, and expressing CD34, CD10, CD19, CD22, and HLA-DR. A bone marrow biopsy showed hypercellular marrow (~90 %) and 95 % replacement by sheets of lymphoblasts. These findings are consistent with a diagnosis of B-lymphoblastic leukemia, and thus, a diagnosis of B-ALL was rendered. Induction chemotherapy was immediately started with Vincristine and cytarabine. On day 29 post induction chemotherapy, a bone marrow biopsy showed variably lower cellular marrow with approximate overall cellularity of 80 %. A follow-up bone marrow biopsy showed minimal residual disease, displaying a favorable response to therapy.
Chromosome analysis was performed on 30 metaphase spreads from bone marrow and peripheral blood using standard cytogenetic techniques. Karyotypes were prepared using Applied Imaging CytoVision software (Applied Imaging, Genetix, Santa Clara, CA) and described according to the International System for Human Cytogenetic Nomenclature (ISCN) 2013 .
Fluorescence in situ hybridization (FISH)
FISH was performed on interphase nuclei and/or previously G-banded metaphase spreads using the following probes acquired from Abbott Molecular (Abbott Molecular, Des Plaines, Illinois 60018):
Vysis LSI ETV6/RUNX1 ES Dual Color Translocation Probe Set
Vysis LSI ETV6 Dual Color, Break Apart Probe Kit
Vysis LSI IGH Dual Color, Break Apart Rearrangement Probe
Vysis LSI BCR/ABL + 9q34 Tricolor, Dual Fusion Translocation Probe
Vysis LSI MLL Dual Color, Break Apart Rearrangement Probe
Vysis LSI PDGFRB (Cen) FISH Probe
Vysis LSI PDGFRB (Tel) SpectrumGreen FISH Probe
Vysis CEP4 Probe
Vysis CEP10 Probe
The karyotype of the bone marrow of this patient was described as: 47,XY,+21c/47,idem,t(2;12)(p12;p13), t(8;14)(q11.2;q32).
Fluorescence in situ hybridization (FISH)
Analysis using other probes did not reveal any additional abnormal signal patterns. This constellation of results was described as follows:
nuc ish(ETV6, RUNX1)x3[152/300]/(ETV6x2, RUNX1x3)[148/300].
nuc ish(IGH@x2)(3′IGH@ sep 5′IGH@x1)[269/300]/(IGH@x2)(3′IGH@ sep 5’IGH@x2)[20/300]
nuc ish(ASS1, ABL1, BCR)x2
nuc ish 4cen(CEP4x2)
The cytogenetic findings in this case highlight a unique combination of rearrangements that has not been previously described in B-ALL. The t(8;14)(q11.2;q32) is a recurrent translocation in B-ALL that generally causes exchange of the regulatory regions of the IGH@ gene and the CEBPD gene, placing CEBPD in close proximity to the regulatory regions of IGH@ and resulting in its overexpression . Although it is relatively rare in the general population, this translocation is more frequent in Down syndrome-associated ALL (DS-ALL) and is associated with a B-cell precursor immunophenotype and an intermediate prognosis [2, 8, 9]. It has also been shown to be associated with a number of secondary cytogenetic abnormalities, including der(14)t(8;14)(q11.2;q32), t(9;22)(q11.2;q34), +21, +X, and +14, although patients with Down syndrome and t(8;14)(q11.2;q32)-positive ALL generally do not bear a concomitant Philadelphia chromosome .
Cases of B-ALL bearing a t(2;12)(p11–p13;p12–p13) observed by conventional cytogenetic analysis
ETV6 is able to promote leukemogenesis via a variety of mechanisms. Most commonly, ETV6 is involved in translocations with partner genes that encode protein tyrosine kinases or transcription factors, leading to deregulation of signaling pathways essential to hematopoiesis via generation of fusion genes or aberrant activation of proto-oncogenes . Additionally, most evidence supports the notion that ETV6 functions as a tumor suppressor gene. For example, in cases where one allele of ETV6 is involved in a translocation, there is often a concomitant deletion of the other, non-rearranged allele . Additionally, decreased or absent ETV6 expression has been observed in cases that don’t bear a deletion of ETV6. Finally, point mutations leading to loss of function of ETV6 have also been observed . These lines of evidence suggest that the t(2;12)(p12; p13) in our case is likely a translocation involving ETV6 and an unknown partner gene on the short arm of chromosome 2 leading to aberrant activation of a protein tyrosine kinase pathway or a proto-oncogene encoded by that unknown partner gene. Alternatively, this rearrangement could result in loss of function of ETV6 without affecting or involving a partner gene, which has also been shown to contribute to leukemogenesis .
However, two reported cases – one case of myelodysplastic syndrome and one case of B-ALL – showed amplification of the ETV6 gene via the generation of homogeneously staining regions (hsr) on the short arm of chromosome 12 [4, 5]. In both of these cases, further molecular analysis confirmed that the homogeneously staining regions consisted primarily of ETV6 gene material, confirming the amplification, and immunohistochemical analysis confirmed overexpression of ETV6 compared to case-matched controls [4, 5]. Furthermore, no additional mutations involving ETV6 that are known or predicted to result in its loss of function were observed in these cases. These two cases provide evidence against the hypothesis that ETV6 functions exclusively as a tumor suppressor gene and suggests that in certain contexts, it can function as an oncogene via its overexpression, a rare phenomenon that has been observed in other genes such as the Wilms tumor 1 (WT1) gene . Because the t(2;12)(p12; p13) observed in MCL involves overexpression of CCND2 due to placement of the gene near the regulatory regions of IGK, it is possible that in our case, a similar mechanism could result in overexpression of ETV6 in the context of this translocation due to the placement of ETV6 near the regulatory regions of IGK. Additionally, Lu et al. reported the first case of B-ALL bearing a t(12;14)(p13;q32) involving ETV6 and IGH@ confirmed by metaphase FISH using probes specific to both genes, showing that translocations involving ETV6 and immunoglobulin genes can occur in B-cell neoplasias . However, such immunoglobulin translocations can also result in mutation of the partner gene in addition to aberrant expression, which can affect the wild-type function of a tumor suppressor gene . Unfortunately, due to limited sample material, we were not able to conduct further molecular or functional analysis of this case to determine the precise nature of the t(2;12) and the role of ETV6 in the evolution of this malignancy.
Among all reported cases of t(8;14)(q11.2; q32)-ALL, only one case, reported by Harrison et al., was found to bear a concomitant cytogenetic abnormality involving the 12p13 locus, described as follows: 47, XX, t(8;14)(q11; q32), del(12)(p12p13),+21c. Further FISH studies confirmed both a monoallelic IGH@ rearrangement and a monoallelic ETV6 deletion . It is evident that the involvement of cytogenetically detectable abnormalities involving ETV6 in the evolution of t(8;14)(q11.2; q32)-ALL is an extremely rare occurrence that presents a number of questions regarding molecular mechanisms and clinical implications that remain understudied. We emphasize the importance of utilizing both conventional cytogenetic and molecular genetic tools to elucidate relevant abnormalities and molecular mechanisms in such cases, ultimately to determine the clinical implications of rare cytogenetic abnormalities in pediatric ALL such as those presented in this case. In summary, this case provides insight into a novel translocation involving ETV6 as well as potentially unique and understudied mechanisms of clonal evolution in pediatric B-ALL that warrant further investigation.
Thank you to the UCLA Cytogenetics Laboratory.
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