A Novel Cryptic Three-Way Translocation t(2;9;18)(p23.2;p21.3;q21.33) with Deletion of Tumor Suppressor Genes in 9p21.3 and 13q14 in a T-Cell Acute Lymphoblastic Leukemia

Acute leukemia often presents with pure chromosomal resolution; thus, aberrations may not be detected by banding cytogenetics. Here, a case of 26-year-old male diagnosed with T-cell acute lymphoblastic leukemia (T-ALL) and a normal karyotype after standard GTG-banding was studied retrospectively in detail by molecular cytogenetic and molecular approaches. Besides fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and high resolution array-comparative genomic hybridization (aCGH) were applied. Thus, cryptic chromosomal aberrations not observed before were detected: three chromosomes were involved in a cytogenetically balanced occurring translocation t(2;9;18)(p23.2;p21.3;q21.33). Besides a translocation t(10;14)(q24;q11) was identified, an aberration known to be common in T-ALL. Due to the three-way translocation deletion of tumor suppressor genes CDKN2A/INK4A/p16, CDKN2B/INK4B/p15, and MTAP/ARF/p14 in 9p21.3 took place. Additionally RB1 in 13q14 was deleted. This patient, considered to have a normal karyotype after low resolution banding cytogenetics, was treated according to general protocol of anticancer therapy (ALL-BFM 95).


Introduction
T-cell acute lymphoblastic leukemia (T-ALL) is a quite rare and heterogeneous disease, more common in males than in females. It accounts for 15% of childhood and 25% of adult ALL cases [1]. Underlying genetic causes of T-ALL are poorly understood and this is highlighted by the fact that T-ALL is associated with a normal karyotype in 30-50% of the cases [2,3]. In abnormal karyotypes recurrent chromosomal aberrations are reported [4]. Regularly, promoter and enhancer elements of genes involved in T-cell development are juxtaposed with translocations in close proximity of oncogenes [5,6]. The most common structural chromosomal abnormalities in T-ALL are TCR (T-cell receptor) loci rearrangements. Breakpoints in 14q11 (TCRA/D) and 7q34 (TCR ) are observed frequently. Besides, deletions in the long arm of chromosome 6 may be found; the common deleted region involves mainly subband 6q16; however, candidate gene(s) have not been formally identified yet [7,8]. Also tumor suppressor genes have been seen to be involved in T-ALL [9].
Cryptic structural chromosomal abnormalities are still a challenge in cytogenetic standard diagnostics of acute 2 Leukemia Research and Treatment leukemia. However, many cryptic aberrations have been identified by molecular cytogenetics already. Examples in T-ALL are cryptic deletions in 9p21 involving the genes CDKN2A/INK4A/p16, CDKN2B/INK4B/p15, and MTAP/ ARF/p14 leading to loss of G1 checkpoint control of the cell cycle or the RB1 locus in 13q14, which also plays a role as tumor suppressor gene in cell cycle regulation [9].
Here, a case of a young adult T-ALL patient with a novel cryptic three-way translocation, a reciprocal translocation, and submicroscopic deletions is reported.

Test Done at
Diagnosis. GTG-banding was done according to standard procedures. A total of 7 metaphases were available for cytogenetic evolution derived from unstimulated bone marrow of the patient and were analyzed on a banding level of 180-250 bands per haploid karyotype [11] and determined as 46,XY [7,12]. RT-PCR performed for TEL/AML1 and BCR/ABL fusion genes was reported to be negative and fluorescence in situ hybridization (FISH) analysis carried out according to manufacturer's instructions for the same loci was negative (probes used: LSI BCR/ABL and LSI TEL/AML1, Abbott Molecular/Vysis, Mannheim, Germany). Whole chromosome painting (WCP) probe for chromosomes 2, 9, 10, 14, and 18 and bacterial artificial chromosome probes (BACs) for chromosomes 2 and 9 (Table 1) were homemade [13]. The homemade multitude multicolorbanding (mMCB) and chromosome specific high resolution array-proven multicolor-banding (aMCB) probe sets were also applied as previously reported [10,14,15].

Test Done in
A total of 10-15 metaphase spreads were analyzed, using a fluorescence microscope (AxioImager.Z1 mot, Zeiss) 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 : methanol (1 : 3) by Puregene DNA Purification Kit (Gentra Systems, 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 this 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, USA). Sizing of peaks and quantification of peak areas and heights were performed using 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 Agilent Sure-Print G3 Human Genome microarray 180 K (Agilent Technologies, Santa Clara, CA, USA), an oligonucleotide microarray containing approximately 180,000 probes 60-mer with a 17 kb average probe spacing. Genomic DNA of patient was cohybridized with a male control DNA (Agilent Technologies, Santa Clara, CA, USA). Labeling was performed using Agilent Genomic DNA enzymatic labeling kit (Agilent) according to the manufacturers' instructions. After hybridization, the aCGH slide was scanned on an Agilent scanner and processed with Feature Extraction software (v10.7) and results were analyzed using Cytogenomics (v2.9.1.3) using ADM2 as aberration algorithm.

Results of Retrospective Analysis
As an initial test of retrospective analysis a genome wide FISH-banding applying mMCB was performed. Thereby, a previously unrecognized reciprocal and apparently balanced translocation between the three chromosomes 2, 9, and 18 was identified. Besides a known recurrent translocation of chromosomes 10 and 14 was recognized and the karyotype was suggested as 46,XY,t(2;9;18)(p23.2;p21.3;q21.33), t(10;14)(q24;q11) ( Figure 1). aMCB and WCP probes as shown in Figure 2 confirmed these suggestions. Locus specific probes narrowed down the breakpoints as shown in  Figure 3). FISH showed a mosaic condition of mixed heterozygous and homozygous deletion of 9p21.3 and 13q14.2 (Table 1(b)).

Discussion
Chromosomal translocations are considered to be the primary cause of leukemia for both acute and chronic phase. In this study, we retrospectively identified previously undetected balanced and unbalanced chromosomal and subchromosomal changes by application of molecular cytogenetics including FISH-banding, locus-specific FISH-probes, and aCGH plus MLPA. FISH-banding, especially mMCB, allows the identification of balanced and unbalanced inter-and intrachromosomal rearrangements of the whole human karyotype Evaluation was done as previously reported [10] using all 6 color channels and pseudocoloring. Breakpoints were determined as 2p23.2, 9p21.3, 10q24, 14q11, and 18q21. 33. in one single experiment [10]. It might be indicated to apply mMCB or comparable FISH-banding approaches routinely in T-ALL cases exhibiting poor quality of the metaphase, that is, not well spreading ones with chromosomes appearing as fuzzy with indistinct margins [16,17].
Data from the literature confirmed that the oncogenes tested and located nearby the chromosomal breakpoints of the three-way translocation were not yet found to be involved in T-ALL: ALK located in 2p23.2 was previously detected in a variety of B-and T-cell lymphomas and nonhematopoietic solid tumors [18][19][20][21][22][23], the BCL2 gene is overexpressed in lymphomas [24,25], and the MLLT3 gene was one of the most highly upregulated transcripts and the most common fusion partner of MLL in de novo acute myeloid leukemia (AML) subtype M5 and therapy-related AML [26][27][28]; however, Meyer et al. [29] found that MLLT3 also plays a role in pediatric rather than adult ALL.
In the present case, an additional chromosomal translocation t(10;14)(q24;q11), known as sole abnormality in 10% of T-ALL patients, was identified. Also it is present in 5% of pediatric and 30% of adult T-ALL [20,30,31]. The TLX1 gene at 10q24 is a transcription factor becoming overexpressed as oncogene due to its juxtaposition to a strong promoter and enhancer elements of the TCR loci at 14q11 [5,[32][33][34]. A favorable outcome was reported in pediatric and adult T-ALL to be associated with the t(10;14) or TLX1 gene overexpression [5,20,35]. Even though balanced rearrangements are known to be typical for hematopoietic malignancies to date, only a limited number of studies have used whole genome directed FISH approaches to identify cryptic chromosomal abnormalities in ALL patients [36][37][38]. Still, in ALL it is uncommon to see three-way translocations. However, due to low metaphase resolution in ALL the real incidence of three-way translocations is currently unknown.
The present report highlights that after identification of apparently balanced chromosomal aberrations, it is still necessary to screen for further unbalanced submicroscopic abnormalities by molecular approaches such as MLPA and aCGH. However, also a confirmation of the results by molecular cytogenetics is necessary, as aCGH was partially misclassified a mix of homo-and heterozygote deletions as pure homozygote ones. 9p21.3 deletions, which lead to the loss of CDKN2A/ INK4A/p16, CDKN2B/INK4B/p15, and MTAP/ARF/p14 tumor suppressor genes expression, are the most predominant aberrations seen in precursor B-cell ALL (∼20% of the cases) and T-ALL (>60% of the case) [39][40][41][42]. Besides also a deletion of RB1 gene resulting in inactivation of another tumor suppressor gene expression was identified. RB1 is rarely reported to be deleted in T-ALL. In contrast, deletion of RB1 has been detected in 30% of B-ALL and nearly to 60% in B-CLL cases [43,44]. Thus, RB1 pathway was identified as potential targets for therapy of ALL [45,46].

Conclusion
In conclusion, we report a case of T-ALL with complex chromosomal aberrations. Even if at time of diagnosis the deletion on 9p21.3 would have been detected and accordingly treated, it remains unclear what influence the other tumor suppressors and oncogenes (possibly) activated by the complex rearrangements would have had for the clinical outcome. Overall, the present case stresses the necessity to study hematological malignancies by different means to get a comprehensive picture of the genetic changes in connection with the acquired disease, as aCGH or MLPA alone would only have identified the imbalanced rearrangements, while molecular cytogenetics predominantly gave hints on the presence of balanced rearrangements.