Investigation of 13q14.3 deletion by cytogenetic analysis and FISH technique and miRNA-15a and miRNA-16-1 by real time PCR in chronic lymphocytic leukemia

Background The most frequent cytogenetic aberration is 13q14.3 deletion in Chronic Lymphocytic Leukemia (CLL). HsamiR-15a/hsa-miR-16-1 are tumor suppressor miRNAs encoded from 13q14.3 region. Objectives The aim of this study was to investigate the 13q14.3 deletion using molecular and cytogenetic techniques and association with miRNA-15a/miRNA-16-1. Materials And Methods We used peripheral blood samples of 30 CLL patients who were either induced and or non-induced with DSP30+IL-2 to determine 13q14.3 deletion by karyotyping and iFISH. Expression levels of hsa-miR-15a/miR-16-1 were measured using qRT PCR and compared with deletions. Results 13q14.3 deletion was detected in 8.6% of cases by karyotyping and in 65% by iFISH. Mosaic forms (monoallelic+biallelic) were observed in 50% of cases. Besides determining common chromosome abnormalities such as add(2)(q37), t(2;7) (p11.2;q22), del(6)(q13q21), del(6)(q25), add(9)(q21), del(11)(q23), t(11;14)(q13;q32), del(13)(q11q12), del(13)(q12q14), add(14) (q23), del(14)(q23), t(14;19)(q32;q13.1), del(15)(q23), del(17)(p12), t(18;22)(q21;q11.2), add(21)(p13) and t(17;21)(q11.2;122), we also determined t(1;13)(q32;q34), inv(2)(p25q21), del(13)(q22q32), t(14;19)(q24;q13), dup(17)(q21q23), der(21;21)(p13;p13) which have not been reported previously. Mitotic index data was found statistically significant and DSP30+IL-2 increased mitotic index by 2.5 folds. Association between decreased miR-16-1 expression and deletions was statistically significant. Conclusion We suggest that cytogenetic and iFISH analyses are complementary and use of DSP30+IL-2 is effective .in CLL. Decreased expression of hsa-miR-16-1 is remarkable.


Introduction
Chronic Lymphocytic Leukemia (CLL) is characterized by the gradual accumulation of functionally immature, small, monoclonal CD5 + and CD23 + B cells most of which are nonproliferating cells arrested at G0/G1 phase of the cell cycle 1 . CLL comprises about 30% of all cases of adult leukemia in Western World affecting both sexes and chromosomal abnormalities are widely used in CLL to prognose, treat, and follow the overall survival in patients 2 . These abnormalities include del(13)(q14), del (11) (q22), trisomy 12, and del(17)(p13) and they can be detected using conventional banding techniques. The most frequent abnormality is deletion of 13q14 which is associated with a favorable prognosis 3,4 .
Deletion of 13q14 is mostly monoallelic (in 76% of cases), but also it is detected in biallelic (24%) and mosaic forms by iFISH 4,5 . Conventional karyotyping and Interphase Fluorescence in Situ Hybridization (iFISH) techniques are widely used to detect cytogenetic abnormalities seen in CLL. However, approximately 50% of patients with CLL can be analyzed by conventional karyotyping due to the very low responsiveness of CLL cells to mitogenic stimuli in vitro 3 . Recently a new mitogen CpG-oligodinucleotide (CpD-ODN) called DSP30 was used in CLL cytogenetics which was reported to be the most effective CpG-ODN in stimulating human B cells 6 . MicroRNAs (miRNAs) are 20-22 nucleotide long small noncoding RNAs that can bind to untranslated regions (UTRs) of target mRNAs resulting in translational repression or mRNA degradation 7 . DLEU2 gene encoding miR-15a and miR-16-1 are located within the deletion region of 13q14. Both miR-15a and miR-16-1 have been shown to exhibit tumor-suppressing activities by inducing apoptosis and inhibiting proliferation. Generally speaking, levels of both miRNAs are decreased due to deletion 13q14 but the physical loss of 13q14 is not the only mechanism causing a decrease in miRNAs levels. Epigenetic regulations and defects in miRNA biogenesis can contribute to their dysregulation as well 8,9 . In this study, we investigated miR-15a and miR-16-1 levels by qRT-PCR and compared with 13q14.3 deletion detected by conventional karyotyping and iFISH techniques. We also investigated other possible cytogenetic abnormalities and effects of DSP30 + IL-2 combination using conventional karyotyping techniques in patients with CLL.

Materials And Methods
Peripheral blood was collected from 30 chronic lymphocytic leukemia patients. All patients were followed up in division of hematology. 18 (60%) of patients are males and 12 (40%) are females. 15 healthy control subjects (9 (60%) males and 6 (40%) females) were also included in the study. Expression levels of miR-15a and miR-16-1 were measured using qRT-PCR, while deletion of 13q14 region was assessed using conventional cytogenetic and iFISH techniques. Informed consent was obtained from mall patients prior to the study and all study was conducted following Declaration of Helsinki, 2013. The Ethical Committee of Istanbul University-Cerrahpasa, Cerrahpasa Medical Faculty approved the study (nr:83045809/604/0101/114304).
Conventional Cytogenetic Analysis Using DSP30 + IL-2 Conventional karyotyping analysis was performed in all patients using standard protocols. Peripheral blood samples (~1 ml) of patients were added in three separate conical tubes and suspended in 2.5 ml of 1640 RPMI (Biochrome, Berlin, Germany). We added 10 µl of CpG-ODN DSP30 (TıbMolBiol, Berlin, Germany) with a concentration of 1µM and 50 µl IL-2 (Roche, Mannheim, Germany) with a concentration of 100 U/mL for both first and second tubes. Third tubes were used as control which do not include DSP30 and IL-2. All samples were cultured at 37oC for 72 h. 50 µl of colcemid (KaryoMax, Thermo Fisher Scientific, Waltham, MA, USA) (with a final concentration of 10 µg/ml) was added prior to harvesting of the cells. Chromosome preparation and staining using G-Banding technique was performed following standard procedures in patients and control samples, as described elsewhere 10 . Karyotypes were scored according to the International System for Human Cytogenetic Nomenclature (ISCN) 2016 11 . Mitotic index was scored by counting metaphases on a slide containing 1000 cells per mitosis in induced by DSP30 and non-induced patient samples and compared to the controls.
iFISH Experiments iFISH analyses were performed in interphase cells using 13q14.3 locus specific probe (Cytocell LSI-D13S319 Plus, Cambridge, UK) in induced samples by DSP30 of all patients. Cultured samples were re-suspended with fresh Carnoy fixative (3 : 1 methanol : acetic acid) for two hours at room temperature (RT) , centrifuged, removed the supernatant, and then re-suspended in fresh fixative for a few more minutes before spreading and then Cytocell iFISH protocol was performed 12 . Three hundred interphase nuclei were scored in all samples. Same protocols were applied for the control samples.
qRT-PCR Experiments miRNA was isolated using Exiqon miRCURY RNA isolation kit (Exiqon A/S, Vedbaek, Denmark) following the manufacturer's instructions and cDNA was synthesized using miRCURY LNA™ mi-croRNA PCR, Polyadenylation and cDNA synthesis kit II (Exiqon A/S, Denmark) with an initial amount of 10 ng of RNA. cDNA synthesis was performed in T100 Thermal Cycler System (Bio-rad Laboratories, Hercules, CA, USA) with following conditions: 42°C for 60 min, 95°C for 5 min and 4 °C for cooling. The expression levels of miR-15a and miR-16-1 were determined with a quantitative system based on SYBR Green probe technology [miRCURY LNA™ microRNA PCR, ExiLENT SYBR® Green master mix (Exiqon S/A, Denmark)]. The assay was performed in a total volume of 10 μl and contained 5 μl 1xPCR master mix, 1 μl of each primer (miRCURY LNA PCR Primer mix for hsa-miR-15a-5p and hsa-miR-16-1-3p; Exiqon S/A, Denmark) and 4 μl of cDNA sample. The expression levels of miRNA-15a and miR-16-1 were measured in Light Cycler 1.5 System (Roche Diagnostics, Mannheim, Germany) under the following conditions: 95°C for 10 min initial denaturation, 40 cycles of 95°C 10 s and 60°C for 1 min for denaturation, 1 cycle of 95°C for 1min, 40°C for 2 min, 95°C for 1s for melting. Relative miRNA expression levels were calculated using the 2-ΔΔCT method 13 . The U6 housekeeping was used as a reference. All experiments were performed in triplicates.

Statistical Analysis
Statistical analyses were performed with the SPSS 21 software (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp). Wilcoxon test was used to confirmation of mitotic index in DSP30 + IL-2 induced and non-induced samples and mitotic index differences between patient and control samples (p<0.001). Cut-off values of deletion types in patients and control samples were calculated using ROC analysis. Association between miRNAs (hsa-miR-15a, hsa-miR-16-1) expression levels and 13q14.3 deletion types were analyzed using Mann-Whitney U test. Significance value was determined as (p < 0.05).

Results
In DSP30 + IL-2 induced samples, a sufficient number of metaphases suitable for cytogenetics analysis was obtained in 23/30 (76.6%) of the patients and clonal chromosomal aberration was detected in 16/23 (69.5%) of the patients. A normal karyotype was found in 7/23 (30.4%) of the patients. 13q14.3 deletion was detected only in 2/23 (8.6%) of the patients with conventional karyotyping techniques. In DSP30 + IL-2 non-induced samples metaphases suitable for cytogenetics analysis were observed in 7/30 (23.3%) of the patients and chromosomal aberrations were detected in 2/7 (28.5%) of the patients. 13q14.3 deletions were not observed in non-induced samples by conventional karyotyping. Cytogenetic aberrations of twenty-three successfully karyotyped patients are presented in Table 1. Also some different abnormalities we found in induced samples are shown in Figure 1. The difference in induced and non-induced samples was found statistically significant (p < 0.001) ( Table 2). Metaphase plaques (number of metaphases) and cell density in induced and non-induced samples are shown in Figure 2.  13q14.3 deletion was detected in 2/23 (8.6%) of DSP30 + IL-2 induced patients by conventional karyotyping whereas this deletion was detected by iFISH in 17/26 (65.3%) of the same patients. Monoallelic and mosaic de-letions were determined in 17/26 (65.3%) and in 13/26 (50%) of the patients, respectively. Statistically; cut-off values, performed by ROC analyses (p < 0.05) ( Table  3), were calculated as 1.88% and 0.13% for monoallelic (2G/1R) and biallelic (2G) deletions, respectively. that were detected in DSP30 + IL-2 induced samples are shown in Figure 3 and Figure 4, respectively.  Table 4. Statistical analyses showed that changes were not significantly correlated with deletions of 13q14 (Table 5) but decreased miRNA-16-1 expressions were found to be correlated (Table 6).

Discussion
CLL is a blood and bone marrow disease developing slowly over time resulting with over production of lymphocytes by bone marrow mostly affecting older adults. Many genetic and epigenetic factors contribute to the formation of the disease 14-16 . Therefore, detecting molecular and cytogenetic abnormalities has vital importance in diagnosis and treatment of the disease 17,18 . However, it is extremely difficult to diagnose the abnormalities using only conventional karyotyping techniques due to the low mitotic index of lymphocytes. Stimulating the CLL cells using various agents for cytogenetic analysis has major importance in diagnosis. One of these effective immunostimulatory agents is DSP30. Recent studies have shown the efficacy of DSP30 on CLL cells either alone or with another agent (IL-2) [17][18][19][20][21][22] .
In this study, we used conventional karyotyping, iFISH, and qRT-PCR techniques in 30 CLL patients and 15 healthy controls to detect the abnormalities developed in CLL. As karyotyping is considered as a low efficient method due to the low proliferation of CLL cells, iFISH technique and DSP30 have found a wider area of application to stimulate CLL lymphocytes in order to detect chromosomal abnormalities.
The main limitation of our study is small sample size. However, this did not affect the methodology or interpretation of the results. Nevertheless, it would be plausible to study a larger group of patients. In addition, other miRNAs need to be studied to understand the effect of these special molecules on disease progression. In conclusion, we didn't find any correlation between miR-15a expression levels and deletions but some patients with del(13q) had increased miRNA expression levels or some patients without del(13q) decreased expression levels. miR-16-1 expression was found to be decreased but it was not related to deletions. So these findings can suppose that expression of both miRNAs not only regulates chromosomal loss but also other mechanisms such as epigenetic changes. Also, increased expression levels of both miRs in del(13q) positive patients may be due to the presence of homologous gene loci miR-15b/miR-16-2 in the 3q25-26.1. Our results indicate that miR16-1 plays an effective role in CLL pathogenesis when compared with miR15a. According to our results, FISH method is more sensitive to detect deletion of 13q14.3 than conventional karyotyping methods. We also suggest that using DSP30 + IL-2 combination can be very useful for karyotyping methods of CLL. Also miR-16-1 expression levels can be used to investigate in CLL patients with del(13q).

Conflict of Interest
The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.

Funding
This study was supported by Scientific Research Project Coordination Unit (BAP) of Istanbul University with the project number 47123.