13q Deletions detected by fluorescence in situ hybridization for diagnosis and prognosis of chronic lymphoproliferative neoplasms

V. V. Sitko1, J. A. Misharina2, J. M. Minchenko1, L. O. Poluben1, O. O. Dmitrenko1, Y. O. Silaiev1, N. I. Kostyukova1, O. V. Tkachenko1, A. O. Tovstogan1, V. M. Polyanska1, L. O. Lyashenko1, V. G. Bebeshko1 1 State Institution «National Research Center for Radiation Medicine, NAMS of Ukraine» 53, Melnikova Str., Kyiv, Ukraine, 04050 2 Bogomolets National Medical University 13, T. Shevchenko Boul., Kyiv, Ukraine, 01601 Valentina-Sitko@yandex.ru


Introduction
Chromosomal abnormalities identifi ed by fl uorescence in situ hybridization (FISH) are known as prognostic factors for chronic lymphoproliferative neoplasms (CLPN) [1]. Among chromosomal abnormalities, deletion of the long arm of chromosome 13 (13q) is detected in more than 50 % of cases of chro nic lymphocytic leukemia (CLL) [2] as well as in other B-cell malignancies [3], including de novo and transformed diffuse large B-cell lymphomas (DLB-CL) [4,5] and multiple myeloma (MM) [6].
Deletion of 13q is the most common cytogenetic abnormality in CLL [2]. This deletion represents early clonal aberration and suggests the loss of a tumor suppressor gene. The loss or inactivation of the tumor suppressor gene may be crucial for the development of CLL [7]. Deletion of 13q as a single aberration is associated with a good prognosis but additional abnormalities neutralize this favorable effect in CLL [8].
DLBCL has heterogeneous biological and clinicopathological characteristics. 30 % of non-Hodgkin's lymphomas (NHL) and more than 80 % of aggressive lymphomas are presented by DLBCL [9]. The aberrations of chromosome 13q are shown in different NHLs including both indolent and aggressive B-NHLs. The data suggest that the loss of genetic ma-terial of the chromosome band 13q14 may play an important role in the formation or development of a wide variety of mature lymphoid malignancies [10].
Multiple myeloma (MM) is a type of cancer formed by malignant plasma cells which show a complex of cytogenetic and molecular genetic abnormalities that not only essentially contribute to the pathogenesis of this disease but also refl ect its prognostic heterogeneity [11,12]. The aberrations of chromosome 13 are found in approximately 50 % of cases [13]. They are associated with aggressive clinical condition, especially in combination with other genetic ab normalities. The recent studies suggest a crucial role of chromosome 13 deletions as a prerequisite for the clonal expansion of myeloma cells [14,15].
Our study was designed to evaluate the prevalence of deletion of the long arm of chromosome 13 in the patients with B-CLL, DLBCL and MM to provide prognostic assessments of the CLPN sub-variants progression, and the early detection of therapy resistant cases and relapses of CLPN.

Materials and Methods
Patients. 115 patients were included in the research: 30 patients with diffuse large B-cell lymphoma (DL-BCL), 25 patients with B-cell chronic lymphocytic leu kemia (B-CLL) and 60 patients with multiple mye loma (MM). All cases were diagnosed according to 2008 World Health Organization (Classifi cation of Lymphoid Neoplasms) criteria [16]. The age of patients at diagnosis ranged from 5 to 79 years (the median is 59 years ). Three patients with DLB-CL were children of 5, 10 and 11 years old. An average age for the patients with B-CLL was 59.60 ± ± 2.69, for the patients with DLBCL -47.30 ± 3.61, and 59.27 ± 1.03 for patients with MM. An average age of the patients with CLPN was 56.22 ± 1.31 years, among them 13 (11.3 %) were younger than 40 years. The substrate cells samples were obtained from 58 male and 57 female patients. The patients were informed about study objective and methods. Every patient has signed the informed consent.
Sample preparation and molecular cytogenetic stu dies. The research on cytogenetic abnormalities was performed on the 24-hour non-stimulated cul-tures of bone marrow cells. The cultivation of native bone marrow (0.5 ml) for 24 hours was performed in 5 ml culture medium RPMI-1640 («Sigma», USA) supplemented with 20 % fetal calf serum («Sigma», USA) and 20 ml of colchicines («Sigma», USA) for 2h before fi xation. The cell suspension was incubated in a thermostat at 37 °C for 24 h. Upon completion of the cultivation, the hypotonic treatment of cells was carried out with heated up to 38 °C and prepared ex tempore 0.075 M solution of potassium chloride for 20 min at 37 °C (1 ml of hypotonic solution to a precipitate obtained from 1 ml of culture). 8 ml of cooled holder (a mixture of methanol and glacial acetic acid in a ratio of 3:1) was added to the cell suspension. The samples were left at + 4 °C for 15 min. Replacement of clamp was performed three times.
Fluorescence in situ hybridization. FISH was performed on the samples from all patients (n = 115) with the Vysis LSI D13S319 (13q14.3) Spectrum Orange/ Vysis LSI 13q34 Spectrum Green FISH probe kit (Abbott Molecular, USA) according to the manufacturer's protocol. The slides (prepared from the 24-hour non-stimulated culture) and probes were codenatured at 75 °C with Vysis Hybrite and hybridized at 37 °C for 16 hours. In each case, at least 200 interphase nuclei with clear signals were analysed.
To determine the normal range for the test Vysis LSI D13S319 (13q14.3) Spectrum Orange/ Vysis LSI 13q34 Spectrum Green FISH probe kit at least 2000 interphase nucleus and 1000 metaphases of peripheral blood lymphocytes and bone marrow cells of ten healthy people of 16 to 67 years old (the average is 48.20 ± 4.88) were analysed. No 13q deletion was found in the nuclei from apparently healthy donors ( Fig. 1).
Cutoff levels for LSI D13S319/13q34 probe ran ged 5 % based on scoring of peripheral blood and bone marrow controls. Analysis was performed on the software and hardware complex CytoVision (Applied Imaging, UK) based on microscope Olympus BX51, Japan.
Statistical analysis. Statistical analysis of the results was performed using Statistica 6.0 and Microsoft Offi ce Excel 2007. Signifi cance of differences between groups, which were analysed, was assessed using χ2 criterion and the Fisher criterion point rec-ommended for the small group size. The difference was considered as statistically signifi cant at p < 0.05. Determined parameters: mean, standard deviation, mean error and measurement error, minimum, maximum and median values, the maximum level of cells with abnormal set of signals [17,18].

Results
Specifi c genomic abnormalities, such as loss of the 13q14 and 13q34 regions, provide clinically significant prognostic information. They are known to be associated with prognostic impact in CLPN patients, which is important for appropriate choice of therapeutic protocol.
In the present study, we describe the frequency of deletions involving D13S319(13q14)/13q34 detected by FISH analysis in 115 patients with CLPN. Among the tested patients, 71 (61.74 %) had normal results, while 44 (38.26 %) had at least one genetic aberration. The analysis of interphase nuclei of bone marrow cells from the patients with CLPN showed that a single anomaly was registered in 30 (68.18 %) patients, and coexistence of two aberrations was revealed in 14 (31.82 %) ones. The FISH results are given in Table 1. In molecular cytogenetic study of bone marrow cells of the patients with B-CLL for each sample from 200 to 300 interphase nuclei were analysed, the total number of nuclei was 5640 (in average, 225.60 ± ± 8.11), among them 5180 (91.84 %) of nuclei (in average, 207.20 ± 11.20) were with normal distribution of signals 2Ox2G (D13S319, Spectrum Orange x 13q34, Spectrum Green). According to the results, the percentage of abnormal cells in average was 9.12 ± ± 2.2. The number of nuclei with deletion of D13S319 varied in the range of 8 -94 with the average of 13.68 ± 4.31, the percentage of abnormal cells was 6.84 ± 2.16. The signals (1O), which characterize the gene D13S319 deletion, were determined in ten patients with B-CLL. In 100 nuclei (1.77 % of the total number of analysed cells), an abnormal distribution of signals (1G) was determined. Thus, in fi ve of 25 patients (20 %), the presence of 13q34 deletion has been found. Among 25 cases in the group with coexistence of two aberrations, three cases (12 %) had two aberrations. One of the patients had polysomy of chromosome 13 in 60 out of 200 analysed nuclei (30 %).
30 samples from the patients suffering from DL-BCL were studied. A normal pattern of signals (2Ox 2G) was registered in 6332 (in average, 211.07 ± ± 9.72) cells, presenting 93.39 %. In 374 nuclei (5.52 % of the total number of analysed cells) an ab-normal distribution of signals was determined. Overall, the aberrations of chromosome 13 were detected in 12 out of 30 (40 %) samples from the patients with DLBCL. Based on the interphase FISH analysis, six patients had D13S319 deletion; three of these patients had D13S319 deletion as a single abnormality, and three patients had additional delition (13q34). Thus, in eight out of 30 patients (26.67 %) Green hybridization signal was determined, indicating the presence of 13q34 deletions. The number of nuclei with 13q34 deletion varied in the range from 16 to 60 with the average of 9.20 ± 3.04. Therefore, the percentage of abnormal cells was 4.60 ± 1.52. One of the patients had also trisomy in 40 out of 200 analysed nuclei (20 %) and the other one had polysomy in 50 out of 300 cells (25 %).
We have evaluated 60 samples from the patients with MM. The interphase nuclei were analysed, a total amount of nuclei was 14170 (in average 236.17 ± ± 7.05), among them 12999 (91.74%) in average 216.65 ± 9.45 with a normal distribution of thr signals (2Ox2G). 19 patients (31.67%) had at least one genetic abnormality. Accordingly, the percentage of abnormal cells in average was 8.64 ± 2.24. 11 patients (57.89 %) had one abnormality; eight (42.11 %) had two abnormalities. The results showed the D13S319 deletion in 13 patients, abnormal distribu-  There were detected changes of chromosomes. The abnormalities of chromosomes 11, 13, 14, 16 and 17 may indicate unfavourable prognosis for the patient P.

Description of individual clinical case
After our studies, the cytoreductive treatment of the patient was intensifi ed with administration of immunotherapy (monoclonal antibodies such as Rituximab and Ibrutinib) in combination with chemotherapy. Despite the intensifi ed treatment , the patient achieved only short-lasting effects and the diseases continued to progress. The diseases prognosis is unfavourable.

Discussion
This study evaluated 115 CLPN patients with chromosomal abnormalities via interphase FISH. The genetic abnormalities at CLPN are typically complex and represent a hallmark of the disease, many chro-mosomes are impacted in both number and structure. The conventional cytogenetic methods detect abnormal chromosomes approximately in 26-40 % of cases due to a low proliferative activity in vitro of B-cells, whereas FISH enables the detection of specifi c abnormalities in up to 86-98 % of the cases [24,25]. The results of our research and the similar data of other authors are presented in Table 1.
In FISH analysis of the patients observed, the deletions of 13q are detected in almost 35 % of CLPN cases. Thus, the chromosome 13 deletions were found in 12 out of 25 patients with B-CLL, 10 out of 30 patients with DLBCL and 18 out of 60 patients with MM. The average percentage of deletion of 13q for the patients with CLPN was respectively 48 %, 33 % and 30 %, that was slightly lower than described in the literature. In general, the results presented in Table 1 show a slight divergence between our data and results of other researchers. At the same time, among 60 patients with MM, the deletion in chromosome 13 was registered in 18 patients, which is signifi cantly lower than Gao et al. [12] showed, they identifi ed the deletion of 13q in 38 out of 60 patients with MM. In our study, the deletion of D13S319 locus located in 13q14 was detected in 29 out of 115 patients with del(13q), while the del(13q14) was determined in 26 out of 115 patients with CLPN. In the patients with B-CLL the deletion of 13q14 was identifi ed in 10 out of 25 patients, while deletion of 13q34 was presented only in fi ve out of 25 patients. Conversely, testing 60 patients with MM showed the deletion of 13q in 18 patients: deletions of 13q14 and 13q34 were identifi ed in 13 analysed cases. In the patients with MM, the presence of deletion of 13q is a poor prognostic factor, while it is related to good prognosis in B-CLL.
Consequently, according to the analysed in detail research results, the following aspects are worth noting. In the case of B-CLL patients, our data are comparable with those received by Chang et al. [19], though they refer to a smaller number of patients examined. A little higher percentage of the chromosome 13 abnormalities is shown in the researches of Degheidy et al. [20]. Perhaps, it is connected with the fact that in this work peripheral blood cells were studied, whereas in our study -bone marrow cells. The work of Eid et al. [7] showed a signifi cant number of the patients with the chromosome 13 aberrations. However, in this work the FISH analyses were conducted following three chemotherapy courses. Our patients were examined to the background therapy, at the initial examination. Accordingly, the FISH analysis was carried out at different stages of diagnostics and treatment.
As for the MM patients, our data are consistent with the data received by Oh et al. [15], though a considerably larger number of patients were analysed in their work. In the work of Gao et al. [12], the authors used purifi ed plasma cells, we identifi ed the plasma cells using the monoclonal antibodies. In the work of Durak et al. [23], the FISH analysis was carried out on bone marrow cells with no plasma cell identifi cation. Therefore, considering this we and the authors of [12] were guided by recommendations of the European Myeloma Network in the abnormal cell counting, the differences in the frequency of chromosome 13 aberrations can be related to the use of different plasmocyte isolation methods.
Besides, we would like to notice that the differences may be of an ethnic nature as well since our work presented the data from various countries of the world.

Conclusion
The present study shows the signifi cance of identification of differential diagnostic markers of the disease for patients with CLPN, in particular, the deletion of critical regions 13q for optimizing the treatment of patients. The further FISH studies, which will involve more patients with B-CLL, DLBCL and MM, are required to fi nd out a more comprehensive pattern of genetic changes in the long arm of chromosome 13 for the diseases prognosis in the Ukrainian patients with CLPN.