MLLT10 and IL3 rearrangement together with a complex four-way translocation and trisomy 4 in a patient with early T-cell precursor acute lymphoblastic leukemia: A case report
- Authors:
- Published online on: November 25, 2014 https://doi.org/10.3892/or.2014.3624
- Pages: 625-630
Abstract
Introduction
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia derived from malignant transformation of T cell progenitors and is more common in males than in females. T-ALL affects mainly older children and adolescents and represents 10–15% of pediatric and 25% of young adult ALL cases (1). Hyperdiploidy (>46 chromosomes) is found in 30% of childhood and 10% of adulthood ALL cases. Notably, high hyperdiploidy (51–65 chromosomes) has been connected with high survival rates and excellent outcome (2,3), while low hyperdiploidy (47–50 chromosomes) has been associated with worse prognosis (4). The most commonly gained chromosomes in ALL are #4, #6, #10, #14, #17, #18, #21 and X (5). Trisomy 4 is rarely observed as a sole cytogenetic abnormality in T-ALL (6). However, the mechanism for chromosomal gains in ALL and their role in leukemogenesis are still ambiguous (7,8). In hyperdiploid karyotypes, the t(9;22)(q34;q11), 11q23 (MLL gene) rearrangements, t(12;21)(p13;q22), t(1;19)(q23;p13) and t(8;14)(q24;q32) are the most common structural cytogenetic abnormalities in ALL. However, in T-ALL, involvement of the T cell receptor (TCR) gene in 14q11 in rearrangements such as t(1;14)(p31;q11), t(10;14)(q24;q11) or t(8;14)(q24;q11) are frequently observed; also del(6)(q15) and del(1)(p32) have been described (3,9–11).
Still, cryptic structural chromosomal abnormalities were and are a challenge in the cytogenetics of T-ALL. For example, as the cryptic t(5;14)(q35;q32) is known to be present in ~20% of childhood and in 13% of adult T-ALL cases, this aberration is currently routinely tested by molecular (cyto)genetics, addressing the breakpoint on the TLX3 (HOX11L2) gene in 5q35 and to the promoter of the BCL11B gene in 14q32 (12). In addition, recent reports on newly detected cryptic chromosomal rearrangements such as the MLLT10 gene (previously AF10, in 10p13), and MLL (in11q23) or PICALM (in 11q14) highlight the necessity to further study clinical cases as detailed as possible (13,14). The goal of these studies must be, on the one hand, to provide the most accurate diagnosis to each individual patient and, on the other hand, to achieve insights into the biology and pathogenesis of T-ALL.
In the present study, an adolescent T-precursor cell ALL case with an MLLT10 and IL3 gene rearrangement together with trisomy 4 in complex four-way translocation is characterized in detail retrospectively using molecular cytogenetics and molecular genetics. This leukemia subtype would currently be classified as early T-cell precursor ALL (15–17).
Case report
Clinical description
A 16-year-old male presented in 1998 for diagnostics due to fever and unclear symptoms of malaise. Immunophenotypic analysis of bone marrow cells revealed the following results: HLA-DR+, TdT+, cyCD3+, CD5 weak, CD7+, CD8+, CD10+, CD13+, CD33+ and CD34+. This supported a diagnosis of early T-ALL; at present, it would be classified as early T-cell precursor ALL (Fig. 1A).
The patient was treated according to the ALL-BFM 95 protocol; the continuation therapy was completed 24 months after the initial diagnosis. Three months later an isolated bone marrow relapse with acute thrombocytopenia was diagnosed, and treatment according to the ALL-REZ-BFM protocol was initiated. One month later the patient died due to an Aspergillus sepsis and still with 100% blasts in the bone marrow.
Tests conducted at diagnosis
Banding cytogenetic analysis was performed on an unstimulated bone marrow aspirate according to standard procedures. A total of 20 metaphases were available for cytogenetic evaluation and analyzed on a banding level of 300 bands per haploid karyotype (22). GTG-banding revealed a normal male karyotype in our laboratory, and also a second cytogenetic analysis on 25 metaphases performed 4 months after the initial diagnosis in another laboratory confirmed this test result. Molecular diagnostic PCR tests for gene fusions BCR/ABL, MLL/AF4 and TEL/AML1 were negative (data not shown).
Test conducted in retrospect
Molecular cytogenetics
Fluorescence in situ hybridization (FISH) was performed according to standard procedures and/or according to the manufacturer’s instructions.
The following homemade probes and probe sets were used: i) 24-color-FISH using all human whole chromosome painting (WCP) probes (19); ii) FISH-banding probe sets as follows: genome-wide multitude multicolor banding (mMCB) and chromosome-specific high resolution array-proven multicolor banding (aMCB) (20–22); iii) DNA from bacterial artificial chromosome (BAC) probes (Table I) obtained from Resources Center (Oakland, CA, USA) were labeled by PCR with SpectrumGreen, SpectrumOrange or TexasRed-dUTP and applied in two- or three-color FISH approaches.
 | Table IResults of the locus-specific probes used for breakpoint analyses in metaphase FISH are listed. |
Additionally, the following commercially available probes were used: LSI EGR1/D5S23, D5S721 (EGR1 in 5q31; D5S23, D5S721 in 5p15.2; Abbott Molecular/Vysis, Mannheim, Germany), POSEIDON PDGFRB (5q33 Break probe; Kreatech Diagnostics, Amsterdam, The Netherlands), and subtelomeric probes for 3p, 5p, 5q and 10p (3p in D3S4559; 5p in C84c11/T3, 5q in D5S2907; 10p in Z96139; Abbott Molecular/Vysis).
A total of 10–15 metaphase spreads were analyzed, using a fluorescence microscope (Axio Imager Z1 mot; Carl Zeiss AG) equipped with appropriate filter sets to discriminate between a maximum of five fluorochromes and the counterstain DAPI (diaminophenylindol). Image capturing and processing were carried out using an ISIS imaging system (MetaSystems, Altlussheim, Germany).
DNA isolation
Genomic DNA was extracted from cells fixed in acetic acid-methonal (1:3) using the Puregene DNA purification kit (Gentra Systems, Inc., Minneapolis, MN, USA). DNA concentration was determined by a NanoDrop spectrophotometer. The quality of DNA was checked using agarose gel electrophoresis. DNA samples extracted from fixed cells of 2 healthy males and 2 healthy females by the same method were used as reference samples.
Multiplex ligation-dependent probe amplification (MLPA)
The P377-A1 hematologic malignancies probemix and SALSA reagents were used for the present study (MRC-Holland, Amsterdam, The Netherlands). Amplified probes and GeneScan 500 ROX standard were separated by capillary electrophoresis using a 4-capillary ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sizing of peaks and quantification of peak areas and heights were performed using the GeneMarker v1.9 software (Applied Biosystems). A minimum of 4 healthy control samples were included in each run.
High resolution array-comparative genomic hybridization (aCGH)
aCGH was performed using the Agilent SurePrint G3 Human Genome Microarray 180K (Agilent Technologies, Santa Clara, CA, USA), an oligonucleotide microarray containing ~180,000 probes 60-mer with a 17-kb average probe spacing. Genomic DNA of the patient was co-hybridized with a male control DNA (Agilent Technologies). Labeling was performed using the Agilent Genomic DNA Enzymatic Labeling kit (Agilent Technologies) according to the manufacturer’s instructions. After hybridization, the aCGH slide was scanned on an Agilent scanner, processed with the Feature Extraction software (v.10.7) and results were analyzed using Cytogenomics (v2.9.1.3) using ADM2 as aberration algorithm.
Results of the retrospective analyses
Genome-wide 24-color FISH using all human WCP probes and FISH-banding analysis using the mMCB probe set were applied as initial tests in this retrospective case. Thereby, a previously unrecognized numerical aberration, trisomy 4, and balanced translocations were identified between one chromosome 3 and 10, each, and both chromosomes 5. Overall, an abnormal karyotype was characterized as 47,XY,+4,der(3)t(3;5)(p23;q31.1),der(5) t(3;5)(p23;q35.3), der(5)t(5;10)(q31.1;p12.3),der(10)t(5;10) (q35.3;p12.3)[8]/46,XY[13] (Fig. 1B and C).
Chromosome-specific aMCB confirmed these results (Fig. 1D) and locus-specific probes narrowed down the breakpoints according to NCBI36/hg18 as follows (Table I). i) The breakpoint in 3p23 was determined between the positions 30,705,070 and 32,453,732; 6 OMIM genes are located there: STT3B, GPD1L, GADL1, OSBPL10, CMTM7 and CMTM8. ii) The breakpoint 5q31.1 locates between positions 130,460,728 and 131,817,004 and those flank the gene IL3 (interleukin 3 precursor) in 131,424,246-131,426,795. iii) The second breakpoint on chromosome 5 in subband q35.3 was mapped to positions 178,455,573 to 179,360,362; here the HNRNPH1 (heterogeneous nuclear ribonucleoprotein H1) gene is included in 178,973,785-178,983,328. iv) Finally, the breakpoint in 10p12.3 was narrowed down to localize between positions 21,495,264 and 22,399,352, where the MLLT10 (myeloid/lymphoid or mixed-lineage leukemia) gene has been mapped to 21,863,580-22,072,560.
No submicroscopic changes were detected by MLPA and aCGH; only the trisomy 4 was observed in aCGH (data not shown).
Discussion
Chromosomal translocations in ALL may be missed in banding karyotyping due to several reasons. They may be cryptic, as they are not resolvable due to a similar or identical GTG-banding pattern; an example is the t(12;21)(p13;q22) in childhood ALL (23). In addition, known aberrations may be masked in a complex karyotype (24). Finally, it may simply be difficult to obtain evaluable metaphases where chromosomes are well-spread and not clumsy or appearing as fuzzy with indistinct margins (25). In the present case the latter was the major problem. In the reanalyses, all well-spread metaphases were normal and all aberrant metaphases were clumsy and not evaluable in standard GTG-banding. Thus, cytogenetic analyses in two different laboratories missed the aberrations present in this case. Otherwise gross structural and a numerical aberration would not have been overlooked like in this case which were detected in retrospect by molecular cytogenetics.
Trisomy 4 as a sole abnormality is rare in acute myeloid leukemia (AML) (26) but is scarce in ALL and is not associated with a clear prognosis (6,27,28). In pediatric ALL, trisomy 4 has been reported to be associated with a favorable outcome suggesting that children who have trisomies of both chromosomes 4 and 10 may have a particularly low risk of treatment failure (3,5). Here, trisomy 4 was observed together with additional structural chromosomal aberrations. Most likely the oncogene MLLT10 in 10p12.31 was activated by the strong promoter of HNRNPH1 in 5q35.3. In addition, the translocation of 5q31.1 to 3p23 brought in close proximity the gene IL3, which has been shown to have an oncogenic effect on hematopoietic cells (29), to 6 OMIM genes listed in Table I, which could also potentially lead to overexpression of IL3.
MLLT10 gene
Rearrangements have previously been identified in both child and adulthood acute leukemia (30). The t(10;11) is a recurrent reciprocal translocation present in two common variants: t(10;11)(p12;q23) and t(10;11)(p12;q21); the latter tending to be more frequent in T-ALL patients (31). In addition, the t(10;11)(p12;q23) mainly found in childhood AML is rarely observed in B-ALL and T-ALL (32). The MLLT10 gene encodes a leucine zipper protein that functions as a transcription factor. MLLT10 gene rearrangements are associated with a poor outcome due to the poor response to therapy (33,34).
HNRNPH1 gene
While unbalanced structural aberration of chromosome 5 are common in myelodysplastic syndrome or AML (35,36), they are less common in ALL. Still Brandimarte et al (14) previously identified the HNRNPH1 gene as a new MLLT10 fusion partner in pediatric T-ALL, as we observed in our case of T-precursor cell ALL.
IL3 gene
Located in 5q31.1, the IL3 gene is a multipotent hematopoietic growth factor produced by activated T cells (37). Its involvement in malignancies was previously reported in B-ALL cases due to a t(5;14)(q31;q32). Overexpression of IL3 was associated with unfavorable outcome in such cases (38).
3p23 region
Six OMIM genes are located in the breakpoint region of chromosome 3 in subband p23. These include: STT3B (source of immunodominant MHC-associated), GPD1L (glycerol-3-phosphate dehydrogenase 1-like), GADL1, (glutamate decarboxylase-like 1), OSBPL10 (oxysterol-binding protein-like protein 10), CMTM7 (CKLF-like MARVEL transmembrane domain containing 7) and CMTM8 (CKLF-like MARVEL transmembrane domain containing 8). It is difficult to determine which one might have provided a strong promoter for IL3 gene expression.
In conclusion, the study in particular of ALL cases with unexpectedly adverse outcome in retrospect and in detail by high resolution molecular approaches is warranted. In the present case the combination of FISH-banding, FISH with locus-specific probes and aCGH revealed trisomy 4 but apart from that a balanced aberrant karyotype, explaining the severe course of the disease in this case with adverse outcome. Even if this complex karyotype would have been identified at the time of diagnosis most likely no additional therapy other than the applied protocol (ALL-BFM 95) would have been used. Yet, the recurrence may have been detected much earlier in the case of available cytogenetic markers. Thus, the most comprehensive molecular (cyto)genetic analyses should be offered to each individual ALL case. Even though aCGH would not have detected the balanced translocations, the detectable trisomy 4 would have hinted at the malignant clone missed by banding cytogenetics. In conclusion, the present case is the first one presenting with combined trisomy 4 with a four-way translocation activating IL3 together with MLLT10.
Acknowledgements
The present study was supported in part by the DAAD and KAAD.
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