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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 6  |  Issue : 2  |  Page : 284-288

Biological implications of deletion p53 by fluorescence in situ hybridization in multiple myeloma


1 Department of Oncology, K.S Hegde Medical Academy, NITTE (Deemed To Be) University, Mangaluru, Karnataka, India
2 KSHEMA Centre for Genetic Services, KS Hegde Medical Academy, NITTE (Deemed to be) University, Mangaluru, Karnataka, India

Date of Submission04-Apr-2022
Date of Acceptance28-May-2022
Date of Web Publication17-Jun-2022

Correspondence Address:
Deyyenthody Prashanth Shetty
KSHEMA Centre for Genetic Services, K.S Hegde Medical Academy, NITTE (Deemed to be) University, Mangaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_84_22

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  Abstract 


Background: Multiple myeloma (MM) is a clonal plasma cell disorder characterized by heterogeneous complex genetic abnormalities. Due to the low proliferative index of plasma cells, conventional cytogenetic (CC) analysis is hampered in MM. Interphase fluorescence in situ hybridization (FISH) along with CC enhances the sensitivity of detection. The study aims to investigate the diagnostic yield and prevalence of P53 deletion in patients with MM. Materials and Methods: Cytogenetic analysis and FISH were performed on 41 MM patients. Results: Our study showed that 55–65 years of age range among all individuals, predominantly affected by the disease. The cytogenetic analysis detected abnormal karyotype in 12% (5/41), normal karyotype in 66% (27/41), and culture failure in 22% (9/41). Abnormal karyotype showed numerical abnormalities such as hyperdiploidy 5% (n = 2) and hypodiploidy 7% (n = 3%). Chromosomes 5, 9, 11, and 21 were common gains among hyperdiploid cases. Chromosome 7, 17, 22 and Y were the common missing chromosome in hypodiploid cases. P53 gene deletion is a rare genetic event and difficult to identify using CC. FISH analysis of deletion 17p was detected in 15% (6/41). Out of six cases, two cases showed deletion of 17p region, three cases showed monosomy 17, and one case showed amplification signals for chromosome 17. Conclusion: CC along with FISH increases the rate of detection of abnormality in MM cases. P53 being less frequent is uncommon at initial diagnosis; increasing its incidence with advanced stage is considered one of the important prognostic factors in MM.

Keywords: Conventional cytogenetics, fluorescence in situ hybridization, multiple myeloma


How to cite this article:
Shetty VV, Arumugam M, Shetty RA, Kalal AA, Kulkarni NV, Shetty DP. Biological implications of deletion p53 by fluorescence in situ hybridization in multiple myeloma. Biomed Biotechnol Res J 2022;6:284-8

How to cite this URL:
Shetty VV, Arumugam M, Shetty RA, Kalal AA, Kulkarni NV, Shetty DP. Biological implications of deletion p53 by fluorescence in situ hybridization in multiple myeloma. Biomed Biotechnol Res J [serial online] 2022 [cited 2022 Dec 9];6:284-8. Available from: https://www.bmbtrj.org/text.asp?2022/6/2/284/347726




  Introduction Top


Multiple myeloma (MM) is a hematological disorder characterized by clonal proliferation and accumulation of neoplastic plasma cells in the bone marrow. In MM, elevated urine or serum monoclonal paraprotein is associated with severe clinical manifestations such as renal impairment, lytic bone lesions, immunodeficiency, and anemia.[1],[2] The disease is characterized by significant heterogeneity at multiple levels such as interactions with bone marrow microenvironment, clinical presentation, treatment response, and clinical outcome. Molecular characteristics of tumor clones are mainly related to heterogeneity. Cytogenetic abnormalities are seen throughout the disease, some at early transformation, and some at the late course of the disease during the progression of malignancy.[3] Genetic aberrations displayed by the tumor clone are considered one of the most important hallmarks of the disease.[4] One powerful means of stratifying risk at diagnosis in MM patients includes cytogenetic profiling by metaphase cytogenetics and interphase fluorescence in situ hybridization (FISH).[5] Approximately 30%–50% of abnormal karyotypes can be identified by conventional cytogenetics (CC) in patients with MM. However, FISH being more sensitive, provide information on prespecified targeted abnormality.[6] Deletion 17p is a rare event at diagnosis in myeloma, increasing in incidence with disease progression. Patients with this abnormality show less response rate to therapy, therefore emphasizing the need for identifying this abnormality.

17p13 locus present on the chromosome codes for tumor suppressor gene p53, whose activation leads to cell cycle arrest, senescence, or apoptosis. It is also known as the “: Guardian of the genome,” acting as a regulator of DNA damage pathway by p53 protein, a transcription factor.[7] Deletion of this region is considered to be one of the most important prognostic factors. It is seen in 10% of newly diagnosed cases, and its prevalence is increasing with the disease progression.[8] Patients with involvement of the central nervous system (CNS) are said to harbor p53 deletion compared to non-CNS involved myeloma.[9] Analysis of 17p deletion by cytogenetics and through FISH is considered to be recommended risk stratification for MM. It is one of the late events occurring in the course of the disease. The identification of deletion (17p) or monosomy 17 indicates a high risk in patients with smoldering MM progressing into MM, and detection in newly diagnosed MM is considered an adverse prognostic factor.[10] Deletion 17p is a secondary cytogenetic abnormality that might coexist with other abnormalities influencing the clinical outcome.[11] MM patients with deletion 17p have aggressive disease courses with shorter progression-free survival and overall survival.[12],[13]

Prevalence and prognostic impact of deletion 17p vary significantly among different populations and detection methods. We aimed to investigate the diagnostic yield and prevalence of P53 deletion in patients with MM in Coastal Karnataka.


  Materials and Methods Top


Forty-one patients diagnosed with MM at the Department of Oncology, K. S. Hegde Charitable Hospital Mangalore, Karnataka, India, were taken for the study. The study was approved by the Institutional Ethics Committee; heparinized bone marrow samples were collected after the written informed consent of the patients. The sample size was finalized based on the prevalence of the patients in the hospital. The samples were processed for both CC and FISH simultaneously.

Conventional cytogenetics

Based on the white blood count (WBC) appropriate sample was added to 5ml of RPMI 1640 media (Gibco, USA) containing 20% fetal bovine serum (FBS, Gibco) for 24 - 48 hr. The culture was incubated in a CO2 incubator at 37°C. The culture was further treated with 100μl of KaryoMAX Colcemid (0.08 μg/mL, Gibco) for 20 min at 37°C and processed with hypotonic solution (KCL, 0.075 M). The cell pellet was fixed with Carnoy's fixative (methanol; acetic acid 3:1), then slides were prepared and aged overnight. Standard GTG banding was carried out using trypsin (0.05%) and 1% Giemsa stain. Twenty well-spread metaphases were analyzed using GENASIS software (Applied Spectral Imaging, Edingen-Neckarhausen, Germany). The results observed were interpreted based on the International System of Human Cytogenetic Nomenclature (ISCN–2013).[14]

Fluorescence In Situ hybridization

The interphase FISH was used to investigate all the samples. Fixed cells were dropped onto frosted microscopic slides and dried at 45°C. 10μl of the P53 gene detection probe (17p13.1) (Wuhan HealthCare Biotechnology) was applied to the target area and sealed with rubber cement. The sample and probe were codenatured and hybridized using a ThermoBrite denaturation/hybridization system with denaturation for 2 min at 88°C and overnight hybridization at 45°C. Posthybridization wash was carried out using 2× sodium saline citrate (SSC) at the room temperature for 1 min, followed by 0.3% NP-40/0.4XSSC solution for 2 min at 68°C and then in preheated deionized water for 1 min. Slides were then counterstained with 10μl of 4,6-diamidino-2-phenylindole (DAPI). A total of 100 interphase nuclei were scored. Visualized FISH signals using an OLYMPUS BX53 fluorescence microscope equipped with DAPI, fluorescein isothiocyanate, and Texas Red filters. The metaphases and interphase nuclei were scored, and the signals were captured using FISH view image acquisition (GENASIS, Applied Spectral Imaging) software (ISCN–2013).[14]

Ethical consideration

The present study was approved by Institutional Ethics Committee, NITTE (Deemed to be) University. Ref: INST. EC/EC/097/2018-2019.

Patients consent form

All patients' informed consent was obtained before the collection of samples.

Statistical analysis

Fisher's exact test, Chi-square, and nonparametric Wilcoxon test were used for statistical evaluation. The data were analyzed using Statistical Package for Social Sciences (SPSS 16.0, IBM, Chicago, IL, US). P < 0.05 is considered to be significant.


  Results Top


Age and sex distribution

A total of 41 MM patients were recruited under the study. Of these patients, 24 (59%) were male and 17 (41%) were female. The median age was 63 years, ranging from 44 to 75 years. About 44% of cases were present between the age group of 55 and 65 years, with females being predominant compared to males [Figure 1].
Figure 1: Age distribution in patients with multiple myeloma

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Cytogenetic analysis

Cytogenetic analysis was performed on the collected samples. Out of 41 cases, a normal karyotype was seen in 27 (66%) patients, 5 (12%) cases showed abnormal karyotype, and 9 (22%) subjects had no mitotic cells in the specimens that were unable to analyze. The clinical characteristics are summarized in [Table 1]. The patients with abnormal karyotypes are grouped into two subgroups based on the number of chromosomes present. Two cases showed hyperdiploid karyotype and the other three cases showed hypodiploid karyotype.
Table 1: Clinical characteristics of patients with multiple myeloma

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Hyperdiploidy and nonhyperdiploidy

Two patients displayed hyperdiploid karyotype with 46–49 chromosomes [Figure 2]a with a random gain of the odd number of chromosomes 5, 9, 11, and 21 and der (13).
Figure 2: Representative images of hyperdiploidy (a) and hypodiploidy (b)

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Three patients showed hypodiploid karyotype with 43–45 chromosomes [Figure 2]b. The most common monosomies observed were 7 (n = 2), 17 (n = 2), 22 (n = 2), and Y.

Fluorescence In Situ hybridization results

FISH was performed on the bone marrow of all the collected samples. FISH analysis revealed 6 out of 41 cases that is 15% of the total cases to be positive for deletion 17p. The frequencies of cytogenetic abnormalities are summarized in [Table 2]. FISH analysis in patients negative for deletion 17p showed two green and two red signals present on chromosome 17 [Figure 3]a. Two green and one red signal are seen in patients positive for deletion 17p [Figure 3]b, one green and one red signal are seen in monosomy 17 patients [Figure 3]c and amplification of signals [Figure 3]d.
Table 2: Association of clinical characteristics with del 17p

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Figure 3: FISH signal pattern illustrating (a) normal (two red and two green), (b) deletion 17p region (two green and one red), and (c) monosomy 17 (one green and one red) (d) Amplification signals. FISH: Fluorescence In Situ hybridization

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Further patients with deletion 17p were divided into two subgroups: patients with normal karyotype but with deletion 17p (NK/Del 17p) and those with complex karyotype 17p (CK/Del 17p). Among 41 cases, 3 showed NK/Del 17p, 2 cases with CF/Del 17p, and 1 case with AK/trisomy 17. No significant difference was seen between the patient with being positive and negative for Del 17p for age, WBC count, lymphocyte percentage, and karyotype [Table 2].

Correlation of fluorescence In Situ hybridization/karyotyping on detection of p53 deletion

Conventional karyotyping and FISH analysis were carried out on all the samples. Metaphase cytogenetics for the detection of 17p deletion was uninformative. Whereas deletion 17p was detected in 6 out of all the cases analyzed by FISH. Comparison of both the test results revealed the importance of FISH analysis for MM diagnosis.


  Discussion Top


MM is the second most common neoplasm-producing monoclonal protein, also called M protein, by malignant plasma cells. MM is a pool of mosaic heterogeneity displaying distinct genetic subclones which complete with each other showing different patterns of clonal evolution.[15] Cytogenetic changes in tumor cells play a pivotal role in its clinical features and predicting clinical outcomes in patients. Metaphase karyotyping helps encapsulate abnormal karyotypes such as hyperdiploidy and hypodiploidy cases, which are the most important prognostic features. However, this technique favors only minimal patients as it is often hindered by the low proliferative index of mitotic MM cells.[16] The percentage of abnormality screened in contrast to FISH is low. FISH is a highly sensitive and indispensable method to detect cytogenetic aberrations; as it applies to interphase cells, the frequency of detecting abnormality increases.[6] MM is considered to be the disease of the elderly with ages ranging from 65 to 75 years. The incidence is relatively low as compared with western literature.[17] In our study, the age group was younger by decade compared to the West, matching various Indian studies.[18] Our study showed males being predominantly affected with MM than females, which in contrary matched with the previously published data.[19] Plasma cell proliferation rate increases with diseased progression from monoclonal gammopathy of undetermined significance (MGUS) to newly diagnosed MM. In our study, we did not use any plasma enrichment technique, which is used to increase the accuracy and yield of testing. Complex and frequent genetic abnormalities displayed are results of tumorigenic events developing into MM.

We studied 41 MM patients screened for various cytogenetic abnormality and deletion 17p using metaphase cytogenetics and interphase FISH. Our study population showed less than one-third (12%) of anomaly, which is less than various other studies showing 30%–40% in patients with MM.[20] Cytogenetic abnormalities are classified into primary and secondary abnormalities. Primary genetic events occur during the early course of the disease and secondary during the pathogenesis of the disease. In our study, the incidence of genetic abnormality screened by metaphase cytogenetics was 12%. Abnormal karyotypes were classified into two groups based on the total number of chromosomes present as hyperdiploidy and hypodiploidy. The incidence of hyperdiploid was 40%, and hypodiploid was 60% among abnormal karyotypes. Hyperdiploid is characterized basically by the gain of the odd number of chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. The frequency obtained in our study is less than the study published by Jiangang Mei1.[21] Hyperdiploid is considered to be a favorable prognostic marker.[22] Hypodiploidy myeloma, also called nonhyperdiploidy myeloma (NH-M), is categorized in hypodiploid (≤44 chromosomes) and pseudodiploid (45–46 chromosomes).[23] The high frequency of IGH translocation involving the 14q32 region in NH-M is an early event. The incidence rate of abnormal karyotypes varies with other studies because of the number of samples taken for the study.

Deletion 17p region present on chromosome 17 involving p53 gene is one of the less recurrent abnormalities representing adverse poor prognostic factor. Our results demonstrated that the frequency of deletion 17p detected by interphase FISH was 15% analyzed in interphase cells. The frequency obtained was consistent with the Avet-Loiseau et al. and Fonseca et al.[24],[25]

In MM cytogenetic and FISH analysis, deletion 17p spanning the TP53 gene is considered part of recommended risk analysis. Deletion 17p is usually monoallelically associated with less favorable outcomes.[26] Our study stipulated that deletion 17p can be detected easily by interphase FISH rather than metaphase cytogenetics. Despite the finding of aneuploidy for deletion 17p by FISH, the band analysis was uninformative or normal in most cases. The failure to detect the abnormality in lymphoid malignancies is because the region of deletion on chromosome 17 is of submicroscopic level. The interstitial deletion is too tiny to be seen by band analysis on the metaphase chromosome. Few cases represented monosomy 17, revealing P53 gene deletion, and one case showed trisomy presenting gene amplification. Only one case had 17p13.1 region deletion. We could conclude that FISH is a more sensitive method to detect deletion 17p abnormality than CCs. Complex karyotype without del 17p is considered to be an adverse risk factor.[27] Studies suggest that patients with normal karyotype in association with del 17p do not have the same prognostic impact as patients with del 17p association and complex karyotype. Deletion of 17p is said to be a marker in the early evolution of the extramedullary disease and is considered in association with resistance to chemotherapy. Even after high-dose chemotherapy and autologous stem cell transplantation, it remains a negative prognostic marker.[28]


  Conclusion Top


In summary, we could conclude that metaphase cytogenetics and FISH are used together to provide comprehensive, inclusive cytogenetic information required for making a clinical decision in patients. Even though the treatment strategies for MM have increased yet, subsequent relapse remains a major challenge. The incidence of deletion 17p is said to be higher in relapsed myeloma than in newly diagnosed MM. The prevalence of p53 in our population was 15% due to different sample sizes and different stages of patients. Among all the genetic risk factors, deletion 17p is considered one of the most important prognostic factors. P53 deletions, one of the adverse prognostic factors, can be detected efficiently by FISH and karyotyping studies.

Limitation of the study

FISH analysis on CD138 enriched plasma cells may have improved the detection of cytogenetic diagnosis.

Acknowledgment

The authors are grateful to all study participants.

Financial support and sponsorship

This work was supported by NUFR2 grant (2018–2021) from NITTE (Deemed to be) University.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

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