Introduction

Acute lymphoblastic leukemia (ALL) is a type of disease characterized by overproduction of malignant and immature lymphocytes. As a consequence of failure to produce mature blood cells and uncontrolled proliferation of lymphoblasts, it spreads to the blood and metastasizes other areas (1). Although the cause of ALL remains unknown, it is thought that various complex mechanisms such as chromosomal damage due to physical or chemical exposure are required for the development of the disease (2).

Conventional cytogenetic analysis (CCA) plays an essential role in the identification of structural and numerical chromosome aberrations that are useful prognostic indicators in patients with ALL. Chromosome aberrations are observed in 60-85% of patients with ALL (3). Hyperdiploidy, hypodiploidy, t(9;22)(q34;q11.2) [BCR-ABL1], t(v;11q23.3) MLL rearrangements, t(12;21)(p13;q22) [ETV6-RUNX1], t(1;19)(q23;p13.3) [TCF3-PBX1], t(5;14)(q31;q32) [IL3-IGH] and intrachromosomal amplification in chromosome 21 (iAMP21) are commonly observed and play significant role in the classification and prognosis of ALL (4). Inadequate specimens, low mitotic index and difficulty of obtaining high-quality metaphases in bone marrow (BM) are impeded or rendered the CCA impossible. Furthermore, some of the structural abnormalities, such as t(12;21) [ETV6-RUNX1] may exist cryptically and be undetectable by CCA. Since fluorescence in situ hybridization (FISH) allows determination of chromosomal changes at interphases besides metaphases with high specificity and sensitivity, it is advantageous for the examination of ALL related abnormalities in the patients with low mitotic activity or normal karyotype (5, 6). FISH panels using different probe combinations are available to detect common rearrangements for ALL simultaneously (7).

In our study, we aimed to investigate recurrent aberrations in patients with ALL with normal karyotype or unsuccessful karyotyping using the FISH method. We used a multiprobe panel carrying probes for t(12;21) [ETV6-RUNX1], t(9;22) [BCR-ABL1], deletion of 9p21.3 (CDKN2A), rearrangements of TCF3 located on 19p13.3, MLL located on 11q23.3, MYC located on 8q24.21, and IGH located on 14q32.33, also enumeration probes for chromosomes 4, 10 and 17.

Methods

Patients

Ten patients with normal karyotype (n=7) or karyotyping failure (n=3) were selected for this study from our patients with ALL whose BM samples were referred by hematology section for CCA. Equal patients of males and females were included in the study and three of them were patients with childhood ALL. Peripheral blood (PB) samples of healthy individuals (n=5) were used for establishing cutoff values. The median ages of patient and control groups were 24 and 23, respectively. The characteristics of the patients are given in Table 1. The informed consent forms were obtained in accordance with the Declaration of Helsinki and the study had the permission of our University’s Research Ethics Committee (approval number: 135385).

Conventional Cytogenetics Analysis

Twenty-four-hour and 48 h unstimulated BM cultures and 72 h unstimulated PB cultures were performed according to the standard protocols and banding was applied to slides using Giemsa-Trypsin-Leishman (GTL) method (8). To perform conventional karyotyping, at least 15 metaphases were analyzed per patient and karyotypes were defined according to the International System for Human Cytogenetic Nomenclature (ISCN 2016) (9).

Fluorescence In Situ Hybridization

For FISH assay, Chromoprobe multiprobe ALL panel (Cytocell Ltd, Cambridge, UK) consisted of 12p13.2 (ETV6)/21q22.12 (RUNX1), 22q11.22 (BCR)/9q34.11-q34.12 (ABL1), 9p21.3 (CDKN2A), 19p13.3 (TCF3), 11q23.3 (MLL), 8q24.21 (MYC), 14q32.33 (IGH), 10p11.1-q11.1 (centromere of chromosome 10), 17p11.1-q11.1 (centromere of chromosome 17) and 4q12 (CHIC2, chromosome 4) chromosomal regions were used. The experimental protocols were performed according to the previous study and manufacturer’s instructions (10). Slides were analyzed under the fluorescence microscope (Olympus BX51, Tokyo, Japan) with filter sets (TxRed, FITC, Aqua, DAPI). FISH scoring was performed independently by two investigators. The cutoff values were determined by examination of control subjects and calculated using inverse beta distribution (betainv) (11).

Results

The FISH assay revealed cytogenetically undetected chromosome rearrangements in target regions of the multiprobe panel in our patient group (Figure 1). The results are summarized in Table 1.

MYC

The MYC rearrangements were detected higher than cutoff values (13%) in two patients; Case No. P3 (25%), and Case No.P5 (25%).

CDKN2A

Deletions of the CDKN2A region were found in only Case No. P2 (28%) (Cutoff value: 10%).

TCF3

The rearrangements of TCF3 were detected in two patients; Case No. P2 (18%) and Case No. P8 (28%) (Cutoff value: 16%)

Chromosome 4

The cutoff values for gains and losses of the CHIC2 region of chromosome 4 were determined separately as 6% for gains and 5% for losses. All of the patients were negative for both losses and gains of the CHIC2 region.

Centromere 10

The cutoff values for gains of centromere 10 were 10% and 6% for the losses. Only case P2 was positive for the gain of chromosome 10 (32%).

Centromere 17

The cutoff values for gains of centromere 17 were 6% and 13% for the losses. While there was no patient with the gain of chromosome 17, two patients were positive for loss; case no. P5 (15%) and case no. P8 (14%).

ETV6/RUNX1

The cutoff value was 3% and none of the patients had ETV6/RUNX1 translocation.

MLL

The cutoff value was 9% and none of the patients had MLL rearrangements.

BCR/ABL1

The cutoff value was 3% and none of the patients had BCR/ABL1 translocation.

IGH

The rearrangements of IGH were detected in two patients; case no. P3 (19%) and case no. P8 (19%) (cutoff value 17%).

While Case P2 (TCF3, CDKN2A and gain of chromosome 10) and Case P8 (TCF3, IGH and loss of chromosome 17) had three abnormalities, Case P3 (MYC and IGH) and Case P5 (MYC and loss of chromosome 17) had two abnormalities. The other six patients had no abnormalities for the multiprobe panel. TCF3, MYC, IGH rearrangements, and loss of chromosome 17 were detected twice in the study while CDKN2A deletion was observed once. MLL rearrangements, translocations of ETV6/RUNX1 and BCR/ABL1, gains of chromosomes 4 and 17, losses of chromosomes 4 and 10 were not detected in this study.

Discussion

Multiprobe FISH panels provide an advantage to detect disease-specific genetic abnormalities that do not only have prognostic significance but also play roles in classification, follow-ups, and treatment of hematological malignancies (7). Previous studies showed that using FISH panels was effective to detect additional chromosomal abnormalities not detected by CCA in nearly 50% of patients with ALL (12,13). In this study, a FISH panel including probes for common abnormalities for ALL was applied to the 10 patients with ALL with normal karyotype or karyotyping failure. The reason for failure in conventional karyotyping in our 3 patients could either be culture failure, insufficient metaphase quality, or technical problems in trypsin digestion and staining stages, besides the known difficulty of obtaining chromosomes in ALL. However, in these patients, the FISH assay showed efficiency for identifying the chromosome aberrations. Chromosomal abnormalities were observed in 4 (40%) of the patients using FISH method. All of these patients had two or three abnormalities. Although adult and childhood patients with ALL were evaluated as separate groups generally, we discussed our adult and childhood patients altogether because of the smallness of our study group. Case P8 was our only childhood patient with positive FISH findings and had three abnormalities (TCF3 rearrangements, CDKN2A deletion, and hyperdiploidy).

The ETV6/RUNX1 translocation is the most frequent abnormality in childhood B-cell ALL (B-ALL) and associated with favorable outcome (4,14,15). It is difficult to detect this cryptic translocation by CCA (16, 17). Previous studies with FISH panels reported frequent occurrences of ETV6/RUNX1 translocation (10-44.3%) (6,12,18-21). However, there were no findings of ETV6/RUNX1 translocation in our patients. This was probably due to small number of patients, 3 of whom were in childhood.

The BCR/ABL1 fusion caused by t(9;22)(q34;q11) is present in 15-50% of adults and 3-5% of patients with childhood ALL and it is associated with poor outcome (4,16,22). CCA has relatively high (80%) sensitivity for detection of t(9;22)(q34;q11) (4,13,18). Similar to karyotypic results, we did not detect BCR/ABL1 translocation in any of the patients by FISH either.

The MYC rearrangements are usually found as translocations between MYC locus (8q24) and IGH heavy and light chain gene loci located on 14q32, 2p12, and 22q11, respectively. Rearrangements of MYC are characteristic in Burkitt lymphoma cytogenetics, also present in subtypes of mature B-cell neoplasms (less than 5% in both adults and children) (16,23,24). Kim BR et al. found gains of MYC in two (20%) patients with ALL using FISH panel including MYC rearrangement probe (18). In our study, MYC rearrangements were found in two patients (Cases P3 and P5) too. In Case P3, both MYC and IGH rearrangements were observed. The coexistence of these two rearrangements points out to the existence of t(8;14). The closeness of the ratios of MYC (25%) and IGH (19%) rearrangements also support this conclusion. The other patient (Case P5) with MYC rearrangement had no IGH rearrangement, but she had monosomy 17 meaning hypodiploidy. It was commonly assumed in previous studies that isolated MYC rearrangements were rare in B-ALL and we did not observe MYC rearrangement as sole abnormality either (23,24).

Study Limitations

Although IGH rearrangements are frequent in lymphomas and mature leukemias, several studies have revealed that these rearrangements account for 5% of patients with ALL with both B-cell and T-cell, mostly in adolescents and young adults. Multiple partner genes are involved in IGH translocations (4,25,26). We found that IGH rearrangements coexisted with TCF3 rearrangements and monosomy 17 in Case P8, and MYC rearrangements in Case P3. In previous studies, TCF3 has not been reported among partner genes of IGH translocations (25,26).

The TCF3 gene locus are involved in t(1;19)(q23;p13) and t(17;19)(q21;p13). While t(1;19)(q23;p13) has been reported in 2% of patients with childhood ALL and 6% of patients with adult ALL and associated with intermediate-risk, t(17;19)(q21;p13) is seen more rarely, in <0.1% of patients with  B-cell precursor ALL (BCP-ALL) (4,27). We observed rearrangements of TCF3 in combination with CDKN2A deletion and hyperdiploidy in one further patient (Case P2) apart from Case P8 discussed above. CDKN2A/2B deletions are frequent (30-50%) abnormalities in both patients with childhood ALL and patients with adult ALL and are associated with poor prognosis (28). Hyperdiplody is another frequent abnormality in childhood ALL, and high hyperdiploidy is considered a good prognostic factor (4,16,22). Case P8 was our childhood patient, and had CDKN2A deletions and hyperdiploidy.

Conclusion

In our study, despite the small number of patients, chromosomal abnormalities related to ALL were found in a significant amount of patients with normal karyotype or unsuccessful karyotyping. Using multiprobe FISH panels was effective in the detection of multiple chromosomal rearrangements with prognostic significance simultaneously. Of all the patients with ALL we analyzed, multiprobe FISH was able to detect MYC, TCF3 and IGH rearrangements, deletion of the CDKN2A, gains of centromere 10, losses of the centromere 17. Identification of these chromosome abnormalities in hematological malignancies, especially in ALL, may provide prognostic value for treatment planning, response or follow-up. Therefore, we suggest that FISH panels are needed to be used combining with conventional cytogenetics routinely to achieve optimum results for patients with ALL.

Ethics

Ethics Committee Approval: The informed consent forms were obtained in accordance with the Declaration of Helsinki and the study had the permission of our University’s Research Ethics Committee (approval number: 135385).

Informed Consent: Obtained.

Peer-review: Externally peer reviewed.

Authorship Contributions

Concept: B.G., S.A., A.Ç., Y.T.A., R.D.K., Ş.Y., Ş.Ö., A.D.,  Design: B.G., S.A., A.Ç., Y.T.A., R.D.K., Ş.Y., Ş.Ö., A.D., Data Collection or Processing: B.G., S.A., A.Ç., Y.T.A., R.D.K., Ş.Y., Ş.Ö., A.D., Analysis or Interpretation: B.G., S.A., A.Ç., Y.T.A., R.D.K., Ş.Y., Ş.Ö., A.D., Literature Search: B.G., S.A., A.Ç., Y.T.A., A.D., Writing: B.G., S.A., A.Ç., Y.T.A., R.D.K., Ş.Y., A.D.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This work was financially supported by İstanbul University Scientific Research Projects (project no: 57448).