Defining the role of gene deletion TP 53 in the diagnosis and prognosis of chronic lymphoproliferative neoplasms using the method of fluorescence in situ hybridization

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Defining the role of gene deletion TP 53 in the diagnosis and prognosis of chronic lymphoproliferative neoplasms using the method of fluorescence in situ hybridization

J.A. Misharina*, V.V. Sitko, A.I. Kurchenko*,

S.M. Kravchenko, N.I. Kostyukova, V. G. Bebeshko

SI "National Research Center for Radiation Medicine NAMS of Ukraine", Kyiv “National Medical University of O.O. Bohomolets, Kyiv

Summary

The results of molecular cytogenetic studies in 60 patients with chronic lymphoproliferative neoplasms (CLPN). Clonal deletions of TP 53 gene were detected in 20% of patients with CLPN: eight of 20 patients with B-cell chronic lymphocytic leukemia (B-CLL), six of 20 patients diffuse large B-cell lymphoma (DLBCL) and one of 20 patients with multiple myeloma (MM). Anomalies of TP 53 gene were registered significantly more frequent in patients on B-CLL with severe course of the disease compared with patients with MM (40% and 5%), respectively (p < 0,05).

Keywords: chronic lymphoproliferative neoplasms, B-cell chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, gene deletion TP 53, fluorescent in situ hybridization.

Резюме

ВИЗНАЧЕННЯ РОЛІ ДЕЛЕЦІЇ ГЕНУ ТР53 В ДІАГНОСТИЦІ ТА ПРОГНОЗІ ПЕРЕБІГУ ХРОНІЧНИХ ЛІМФОПРОЛІФЕРАТИВНИХ НЕОПЛАЗІЙ ЗА ДОПОМОГОЮ МЕТОДУ ФЛУОРЕСЦЕНТНОЇ IN SITU ГІБРИДИЗАЦІЇ

Ж.А. Мішаріна*, В.В. Сітько, А.І. Курченко*,

С.М. Кравченко, Н.І. Костюкова, В.Г. Бебешко

ДУ «Національний науковий центр радіаційної медицини НАМН України», Київ “Національний медичний університет імені О.О.Богомольця, Київ

Представлені результати молекулярно-цитогенетичних досліджень у 60 хворих на хронічні лімфопроліферативні неоплазії (ХЛПН). Клональні делеції гену ТР 53 були виявлені у 25% хворих на ХЛПН: у восьми із 20 хворих на хронічну B-клітинну лімфоцитарну лейкемію (В-ХЛЛ), у шести із 20 хворих на дифузну крупноклітинну В-лімфому (ДКВЛ) та одного із 20 хворих на множинну мієлому (ММ). Аномалії гена ТР53 достовірно частіше реєструвались у хворих на В-ХЛЛ з тяжким перебігом хвороби в порівнянні з групою хворих на ММ (40% та 5%), відповідно (p < 0,05).

Ключові слова: хронічні лімфопроліферативні неоплазії, хронічна B-клітинна лімфоцитарна лейкемія, множинна мієлома, дифузна крупноклітинна В-лімфома, делеція гену ТР 53, флуоресцентна in situ гібридизація.

Резюме

ОПРЕДЕЛЕНИЕ РОЛИ ДЕЛЕЦИИ ГЕНА ТР 53 В ДИАГНОСТИКЕ И ПРОГНОЗЕ ТЕЧЕНИЯ ХРОНИЧЕСКИХ ЛИМФОПРОЛИФЕРАТИВНЫХ НЕОПЛАЗИЙ С ПОМОЩЬЮ МЕТОДА ФЛУОРЕСЦЕНТНОЙ IN SITU ГИБРИДИЗАЦИИ

Ж.А. Мишарина*, В.В. Ситько, А.И. Курченко*,

С.М. Кравченко, Н.И. Костюкова, В.Г. Бебешко

ГУ "Национальный научный центр радиационной медицины НАМН Украины", Киев “Национальный медицинский университет имени А. А. Богомольца, Киев

Представлены результаты молекулярно-цитогенетических исследований у 60 больных хроническими лимфопролиферативными неоплазиями (ХЛПН). Клональные делеции гена ТР 53 были обнаружены в 25% больных ХЛПН: у восьми из 20 больных с хронической B-клеточной лимфоцитарной лейкемией (В- ХЛЛ), у шести из 20 больных с диффузной крупноклеточной В-лимфомой (ДКВЛ) и одного из 20 больных множественной миеломой (ММ). Аномалии гена ТР 53 достоверно чаще регистрировались у больных с тяжелым течением болезни В-ХЛЛ по сравнению с группой больных ММ (40% и 5%), соответственно (p < 0,05).

Ключевые слова: хронические лимфопролиферативные неоплазии, хроническая B-клеточная лимфоцитарная лейкемия, множественная миелома, диффузная крупноклеточныхВ-лимфома, делеция гена ТР 53, флуоресцентная in situ гибридизация.

Recent achievements in the research of mechanisms of development and progression of chronic lymphoproliferative neoplasms (CLPN) and analysis of the effectiveness of treatment depending on the type of therapeutic interventions are a valid basis for a new strategy in the diagnosis, prognosis and treatment of these patients.

The introduction into clinical practice of modern research methods, including cytogenetic and molecular cytogenetic, allows to determine the genomic reorganization that determine the development of the tumor, the degree of malignancy and metastatic potential level of sensitivity to anticancer drugs therapy. Unfortunately, cytogenetic studies of chronic lymph proliferative neoplasia are associated with certain difficulties. Thus, the substrate cells for this disease have very low spontaneous mitotic activity, and their response to used mitogens is very weak. Therefore, only in some CLPN patients we can get enough of metaphase in unstimulated cell culture for further study.

Today, opportunities of cytogenetic diagnosis of CLPN much expanded with the application of new technologies of molecular cytogenetics, based on fluorescent in situ hybridization (FISH) of nucleic acids. FISH method was developed to determine the localization of specific DNA sequences directly in cytological preparations. This method is based on the ability to form stable chromosomal DNA hybrid molecules of DNA (RNA) - tests that labeled fluorophore. The size of the DNA fragment under study can range from 60 to 1,500 kb [16].

For the diagnosis of chromosome abnormalities in patients with CLPN interphase fluorescence in situ hybridization (I-FISH) becomes most widely used technique, because it allows you to record the presence of abnormal clone in samples of tumor cells, determine amplification in it, translocations and deletions of certain genes, and evaluate the effectiveness of chemotherapy, performed to patients with / or without transplantation of stem cells (SC) of bone marrow and / or peripheral blood with B-cell chronic lymphocytic leukemia (B-CLL), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL).

Unlike standard methods of cytogenetic analysis when changes of karyotype are recorded only in 30-45% of patients with newly diagnosed CLPN, fluorescent in situ hybridization allows the identification of chromosomal aberrations in more than 80% of patients [8].

The aim was to determine the role of genomic disorders of chromosome 17 in the region 17p13.1, where suppressor gene of malignant transformation TP53 is localized in substrate cells of bone marrow and biopsy material of lymph nodes in patients with B-CLL, MM and DLBCL to provide prognostic assessments of the progress of CLPN sub-variants, and early detection of resistant to therapy cases and recurrence of CLPN.

Subjects and methods. Molecular cytogenetic studies were performed in molecular genetic laboratory of diagnosis and prognosis of radiation induced oncohematological pathology of department of Hematology and Transplantology in SI "National Research Centre of Radiation Medicine of NAMS of Ukraine". Bone marrow cells, peripheral blood samples of substrate cells of biopsy material were analyzed and a statistical analysis of results of 60 patients with diagnosis CLPN, including 20 people with B-cell chronic lymphocytic leukemia (B-CLL), 20 - with diffuse large B-cell lymphoma (DLBCL) and 20 - with multiple myeloma (MM) was conducted. The age of patients at diagnosis ranged from 21 to 75 years and averaged in patients with B-CLL - 60,30 ± 2,73, in patients with DLBCL - 48,95 ± 3,31 and 58,45 ± 1.83 - in patients with MM. The average age of patients with CLPN was 55,9 ± 1,66 years, 7 (11,67%) of patients were younger than 40 years. 34 (56,67%) analyzed samples of bone marrow cells were obtained from male patients and 26 (43,33%) - female.

Control group was formed from five healthy people aged 40 to 55 years (mean 48,00 ± 2,43). For each person in the control group nuclei and metaphase lymphocytes in peripheral blood and bone marrow were studied.

Patients and subjects were informed about the purpose and objectives of the study and consent from them was obtained.

Cytogenetic abnormalities research of gene TP 53 was performed on 24- hour unstimulated cultures of bone marrow cells that were received by sternal puncture. Cultivation of native bone marrow (0,5 ml) for 24 hours was carried out in 5 ml culture medium RPMI-1640 ("Sigma", USA) supplemented with 20% fetal calf serum ("Sigma", USA) and 20 ml colchicine ("Sigma", USA) for 2 hours before fixation. Cell suspension was incubated in a thermostat at 37,0 °C for 24 hours. Upon completion of the cultivation hypotonic treatment of cells was carried with heated to 38 °C and prepared ex tempore 0,075 M solution of potassium chloride for 20 min. at 37,0 °C (rate of 1 ml hypotonic solution to precipitate obtained from 1 ml of culture). To the cell suspension 8 ml of cooled holder (a mixture of methanol and glacial acetic acid in a ratio of 3: 1) was added. Samples left at + 4 °С 15 minutes. Replacement of clamp was performed three times.

FISH studies in interphase nuclei and metaphase that were received during cytogenetic studies were conducted using commercial test LSI TP53/CEP 17 FISH Probe Kit (Abbott Molecular, USA) according to manufacturer's instructions.

Analysis was performed on the software and hardware complex CytoVision (Applied Imaging, UK) based on microscope Olympus BX51, Japan. To visualize the signal samples Vysis TP53/CEP 17 FISH Probe Kit (17p13.1) filters used:

diagnosis chronic lymphoproliferative neoplasms

Figure 1 - Scheme of the sample Vysis TP 53/CEP 17 (Abbot Molecular Catalog 2011 for Oncology, Automation and Genetics).

CY3/FITC/DAPI (Fig. 1). In each case at least 200 interphase nuclei with clear signals were analyzed.

Statistical analysis of the results of studies was performed using Statistica 6.0 and Microsoft Office Excel 2007. Significance of differences between groups that were analyzed was assessed using X2 criterion and the Fisher criterion point recommended for the small group size. The difference was considered as statistically significant at p < 0,05. Determined parameters: mean, standard deviation, mean error and measurement error, minimum and maximum values, the maximum level of cells with abnormal set of signals [3; 16].

Cutoff level was calculated in the program Microsoft Office Excel 2007 by function BETAINV (A; B; C), where A - level of probability; B - number of false-positive nuclei +1; C - total number of nuclei analyzed.

Results and discussion. At present, the main clinical problem of treatment of B-CLL, MM and DLBCL is that substrate cells are resistant to modern, standard polychemotherapy that is given to patients, and recurrence of disease including molecular-genetic, which are associated with inactivation of the gene TP 53 due to deletions of chromosome 17 (17p13.1).

To verify the results and determine the characteristics of the sample Vysis TP 53/CEP 17 FISH Probe Kit (17p13.1), which was used, hybridization of the interphase nuclei of peripheral blood lymphocytes and bone marrow of five healthy people were performed. In addition, in all cases metaphase were studied - 100 per sample. Total number of analyzed metaphase and interphase nuclei is 2400. Pattern of signals 1O (TP 53, Orange)x2G(CEP 17, Green) was considered for positive. It should be noted that this test allows you to record not only changes in the number of signals corresponding to gene TP 53, but also take into account the quantitative changes of chromosome 17 as a whole (mono or polisomy). In chronic lymphoproliferative neoplasms dignosticly and prognostically for disease course and response to therapy significant is only deletion of gene TP 53.

Research of control group is associated with the need of imposing percentage of nuclei with false-positive signals, the main reason of which may be the imposition of locuses or their close spatial arrangement, resulting in normal nuclei pattern of signals (1O: 1G: 1F), (1O: 2G) or (2O: 1G) is observed. A similar pattern of signals can be observed in the abnormal nuclei, where as a result of deletion of the partner area translocation after hybridization is observed fewer on one signal. In addition, in some cases bodysized location of the site that is being analyzed, is the cause of the weak binding of sample - in the nuclei one signal is observed or signals are completely absent.

According the study of preparations of interphase nuclei of bone marrow cells and peripheral blood threshold level (cutoff level) of signals that corresponded to distribution 1O: 2G test for TP53/CEP 17, was 4,28% at mean values of 2,15%; and 3,93% (average - 2,10%) for pattern 3O: 3G and > At the same time in all metaphase a double yellow signal and a double green signal (2Ox2G pattern signals)were determined that testified about balance of genetic structures regarding gene TP 53 in healthy individuals and confirmed false-positive character of detected in the interphase nuclei changes (Table. 1).

Table 1 - Characteristics of changes of chromosome 17 and gene TP 53 in patients with chronic lymphoproliferative neoplasms

In substrate cells of patients with B-CLL number of cores with deletion of chromosome 17p13.1 varied in the range of 10-141 with an average 26,70 ± 8,48. Accordingly, the percentage of abnormal cells on average was 11,45 ± 3,43. Thus, in five of 20 patients (25%) the number of cores with deletions not exceeded 8%, and although these changes were not clonal nature, but far exceeded the limits of error of the method, which was about 2,15% (p < 0,05). Another eight patients (40%) had clonal deletion of gene TP 53 that is associated with a poor prognosis for the disease [11, 6], and in three of them the number of abnormal nuclei exceeded 40% (40%, 47% and 47%, respectively).

The results of analysis of samples of bone marrow and lymph nodes of 20 patients with DLBCL using FISH-method showed that the normal pattern of signals (2Ox2G) was registered in 3945 (an average of 197,25 ± 18,96) cells, which is 84,20%. In 498 nuclei (10,63% of the total number of analyzed cells) an abnormal distribution of signals (1Ox2G) was determined. The number of cells with deletion of chromosome 17p13.1 varied in the range of 10 - 180 with an average of 24,90 ± 9,67. Accordingly, the percentage of abnormal cells on average was 12,30 ± 4,84. Thus, in six of 20 patients (30%) a yellow hybridization signal was determined, indicating the presence of deletions of chromosome 17p13.1, including two patients with the number of abnormal nuclei was 10%; in other cases - 30%, 35% and 36%, indicating their clonal nature. One patient with DLBCL marked with especially high level - 90% nuclei 1Ox2G, which may indicate a lack of good response to treatment and require further review of cytostatic therapy [13].

Analysis of bone marrow cells of patients with MM showed that the normal pattern of signals (2Ox2G) was registered in 5326 (an average of 266,30 ± 10,30) cells, which is 98,63%. In 44 nuclei (0,81% of the total number of analyzed cells) an abnormal distribution of signals (1Ox2G) was determined. Average signals (1Ox2G), which are characteristic of gene TP 53 deletions, were determined in double with MM. In one patient the deletion 17p13 was recorded in 10% plasma cells, indicating its clonal nature. In another patient the number of abnormal nuclei (10 of 200) did not exceed 5%. Also in one of the patient's monosomy in 10 of 200 analyzed nuclei (5%) and polisomy 30 of 300 cells (10%) were registered. According to literature data gene TP 53 deletion may be an independent prognostic factor in MM. Patients with deletion17p13 have shorter progression-free remission period and overall survival, even after high-dose chemotherapy with autologous SC transplantation than patients without deletions of the gene [10].

Therefore, in this work we have studied the substrate cells of bone marrow and biopsy material of lymph nodes in patients with B-CLL, MM and DLBCL for the deletion of the gene TP 53.

A characteristic feature of these hematologic diseases is significant heterogeneity and individual variability of chromosomal abnormalities in tumor cells. Although each malignancy in this group the most frequently recurring changes in the genotype were identified, identification by methods of classical cytogenetics is significantly limited due to the low proliferative activity in vitro of B-cells. In addition, some submicroscopic adjustments, such as deletions of 11q, 13q and 17p, are almost impossible to determine on the chromosomal level. Therefore, in this study we used a more accurate method of analysis that allows you to record anomalies at the molecular level, namely interphase FISH. The results of our research and a number of similar data from other authors are presented in Table 2.

Table 2 - Results of studies of gene TP 53 deletions in bone marrow cells and biopsy material of lymph nodes in patients with B-CLL, MM and DLBCL