|Year : 2018 | Volume
| Issue : 3 | Page : 81-84
B-Cell chronic lymphocytic leukemia fluorescence in situ hybridization panel findings at tertiary care hospital in Saudi Arabia
Wedian Mustafa Rawas1, Salem H Khalil1, Emad E Ghabashi2
1 Hematology Section, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
2 Adult Hematology/HSCT, Oncology Center, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia
|Date of Web Publication||31-Oct-2018|
Dr. Wedian Mustafa Rawas
Section of Hematopathology, Department Pathology and Laboratory Medicine, King Faisal Specialist Hospital And Research Center, Riyadh
Source of Support: None, Conflict of Interest: None
BACKGROUND/PURPOSE: B-cell-chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world and shows a remarkable heterogeneity in the clinical course. Understanding the genetic basis of B-cell-CLL may help in clarifying the molecular bases of this clinical heterogeneity. Recurrent chromosomal aberrations at 13q14, 12q15, 11q22-q23 and 17p13, and TP53 mutations are the first genetic lesions identified as drivers of the disease. While some of these lesions are associated with poor outcome (17p13 deletion, TP53 mutations and to a lesser extent, 11q22-q23 deletion), others are linked to a favorable course (13q14 deletion as sole aberration). This study evaluates the frequency of each chromosomal abnormality using fluorescence in situ hybridization (FISH) Panel at our institution with comparison to other international studies.
MATERIALS AND METHODS: We reviewed 147 peripheral blood and bone marrow samples which represent all B-cell-CLL cases diagnosed at our hospital from 2012 to 2016 by morphology and flow cytometry immunophenotyping followed by specific B-cell-CLL-FISH panel including MDM2/Cen12 for Trisomy 12, 13q14 (D13S319)/13q34 deletion, ATM (11q22.3) deletion, P53(17p13.1) deletion, and CCND1/IGH translocation (11;14).
RESULTS: Between 2012 and 2016, a total of 147 B-cell-CLL patients were investigated using B-cell-CLL-FISH panel at our institution King Faisal Specialist Hospital and Research Center-Riyadh, Kingdom of Saudi Arabia, with age ranging between 36 and 89 years and a median age of 62 years. The majority of the patients with the abnormal FISH pattern, 70% (73/105), had a single abnormality with the remaining 30% (32/105) showed more than one genetic abnormality including two cases where all five probes were positive.
CONCLUSION: The heterogeneous clinical course of B-CLL is likely explained by underlying molecular prognostic factors including data from FISH probes. Moving forward, analyzing these factors at diagnosis is recommended for better prognostication and outcome of the disease.
Keywords: B cell-chronic lymphocytic leukemia, flurescence in situ hybridization, cytogenetic
|How to cite this article:|
Rawas WM, Khalil SH, Ghabashi EE. B-Cell chronic lymphocytic leukemia fluorescence in situ hybridization panel findings at tertiary care hospital in Saudi Arabia. J Appl Hematol 2018;9:81-4
|How to cite this URL:|
Rawas WM, Khalil SH, Ghabashi EE. B-Cell chronic lymphocytic leukemia fluorescence in situ hybridization panel findings at tertiary care hospital in Saudi Arabia. J Appl Hematol [serial online] 2018 [cited 2020 Apr 4];9:81-4. Available from: http://www.jahjournal.org/text.asp?2018/9/3/81/244536
| Introduction|| |
B-cell-chronic lymphocytic leukemia (B-CLL) is the most common leukemia of adults in Western countries. The clinical course is highly variable, ranging from very indolent cases to patients with the aggressive and rapidly progressing disease. This heterogeneity has important consequences, which will impact on clinical approaches, treatment strategies, and finally, survival times from diagnosis.,,
Early genetic studies of B-CLL using conventional chromosome banding analysis detected chromosomal aberrations in 40%– 60% of cases., However, only dividing cells are evaluated by chromosome banding techniques. Because it has been difficult to stimulate B-CLL cells to divide, novel stimulation techniques have been reported to improve the detection of chromosomal aberrations, particularly translocations, in B-CLL lymphocytes.
Nevertheless, fluorescence in situ hybridization (FISH), which allows analysis of dividing and nondividing cells, is increasingly being offered as an alternative to conventional chromosome banding. With FISH, up to 80% of B-CLL cases demonstrate genetic alterations.
This retrospective study evaluates the frequency of each FISH probe abnormality in B-CLL patients at our institution (King Faisal Specialist Hospital and Research Center-Riyadh) between 2012 and 2016 with comparison to other international studies and reports.
| Materials and Methods|| |
A total of 147 peripheral blood (53) and bone marrow samples (94) were collected from B-CLL cases newly referred through our adult hematology/oncology clinics, at King Faisal Specialist Hospital (KFSH) between 2012 and 2016 without prior treatment. B-CLL was diagnosed by white cell count morphology and flow cytometry. FISH- B-CLL panel was done at the cytogenetics laboratory/Department of pathology and laboratory medicine, KFSH and Research Center-Riyadh. This retrospective study was conducted with ethics clearance from the Office of research affairs at our hospital.
Complete blood count and leukocyte differentials were performed on peripheral blood samples. Peripheral blood samples and/or bone marrow aspirate smears were stained with Wright–Giemsa for morphologic evaluation. Formalin-fixed, decalcified bone marrow trephine biopsy specimens were stained with H and E before histologic evaluation. The morphologic features of lymphocytes in blood and/or bone marrow were reviewed in all cases without the knowledge of immunophenotype or FISH results.
Immunophenotyping using flow cytometry was performed on blood or bone marrow aspirates specimens in all cases using previously published procedures and samples were analyzed using a flow cytometer (BD FACSCanto II, Becton Dickinson, Mountain View, CA) and the corresponding software (BD FACSDiva Software).
The following immunophenotype was interpreted as typical for B-CLL: CD5+, dim to moderate CD20+, and CD23+ monotypic B cells. Brightly positive staining for CD20, FMC7, and/or CD79b or negative staining for CD23 was regarded as an atypical immunophenotype for B-cell-CLL.
The FISH panel included five probes according to Laboratory Protocols which are CCND1/IGH translocation (11;14) [Figure 1]a, ATM (11q22.3) deletion [Figure 1]b, 13q14 (D13S319)/13q34 deletion [Figure 1]c, P53(17p13.1) deletion [Figure 1]d, MDM2/Cen12 for Trisomy 12 [Figure 1]e.
|Figure 1: (a) IGH/CCND1. (b) ATM/11 cen Chromosome 11q contains the ataxia telangiectasia mutated (ATM) gene. (c) 13q14. (d) P53 TP53 is a tumor-suppressor gene located on the short arm of chromosome 17; it is inactivated by deletion and/or point mutation in many human malignancies.|
Click here to view
Normal values, sensitivity, and specificity for each probe and sets of probes were established at Mayo Medical Laboratories and have been previously published.,
The Vysis B-CLL FISH Probe Kit from Abbott Diagnostics, USA uses FISH DNA probe technology to determine deletion status of probe targets for locus-specific identifier (LSI) TP53 (containing tumor protein p53 gene, located on chromosome 17p), LSI ATM (containing ataxia telangiectasia mutated gene, located on chromosome 11q), and LSI D13S319 (containing marker D13S319, located on chromosome 13q), as well as determining trisomy 12 with CEP12 (D12Z3 alpha satellite, located on chromosome 12). The Vysis B-CLL FISH Probe Kit includes LSI 13q34 (containing lysosome-associated membrane protein 1 gene, located on chromosome 13q) as a control probe.
| Results|| |
A total of 147 cases diagnosed with B-CLL with age ranging between 36 and 89 years and a median age of 62 years. [Table 1] shows the age break down with six patients are <40 years of age. In this study, the gender distribution of the patient was 97 males and 50 females.
All 147 B-CLL patients' samples investigated using B-CLL-FISH panel including 53 peripheral blood and 94 bone marrows were informative with an abnormality reaching 71% (105 abnormal cases). The majority of the patients with genetic alterations, 70% (73/105), had a single abnormality; however, the remaining 30% (32/105) showed more than one genetic abnormality including two cases with all 5 probes are positive.
Chromosome 13q deletion (13q–) was the most common aberration present in 61 samples (42.4%) [Table 2]. Trisomy 12 (MDM2) was the second most frequent abnormality (36.6%) and was the sole abnormality in 13 cases.
|Table 2: Chromosomal aberration frequency by fluorescence in situ hybridization for 147 B cell-chronic lymphocytic leukemia cases|
Click here to view
Deletion of chromosome 11q (ATM) was the third most common finding (10.7%). Abnormalities of chromosomes 17p (p53) occurred in <10% of B-cell-CLL cases, and an abnormal signal pattern for the IGH gene (CCND1/IGH) was present in 19 B-CLL cases (13.8%).
| Discussion|| |
Over the years, clinicians have used traditional prognostic factors that are easily obtained using staging, serum testing, and other accessible parameters to predict the progression risk of B-CLL and disease-related mortality. As newer prognostic factors became available, better ability to predict responses to therapy, duration of responses, time to first treatment, overall survival, and selections of therapy ensued.
Understanding these prognostic indicators allows for better design of future clinical trials that are tailored to the basic mechanism of the disease.,
Chromosomal FISH is the most frequently used approach for identifying chromosomal aberrations in B-CLL and is widely available for routine diagnostics. FISH uses specific probes targeted to regions of interest to search for chromosomal losses or gains in B-CLL cells. Interphase FISH can be successfully applied to nondividing cells such as B-CLL cells in bone marrow or peripheral blood samples. The classical FISH probe panel for B-CLL includes probes targeting 13q14, 11q22.3 and 17p13.1 deletions, as well as trisomy 12 abnormality.
A set of five prognostic categories were defined by Döhner et al., and were later confirmed in other studies showing that 82% of B-cell-CLL patients carry at least 1 genomic abnormality when FISH testing was used.
The worst prognosis of these categories was shown in patients with del17p13.1 involving the TP53 gene, followed by del11q22.3, trisomy 12q13 and those with a normal diploid karyotype, while patients with del (13q14) as the sole chromosomal abnormality had a good prognosis.,,,,,,,
In this study, a single probe abnormality was identified in up to 71% of bone marrow and peripheral blood samples, however; one-third showed more than one abnormality.
The frequency of these abnormalities is similar to published data from different international centers and confirms the usual common presentations and prognostic prediction of B-CLL patients [Table 3].
|Table 3: Chromosomal Aberration Frequency by fluorescence in situ hybridization for B cell-chronic lymphocytic leukemia at different centers|
Click here to view
| Conclusion|| |
Despite the lower median and average age of B-CLL patients referred to our institution, the incidence of all FISH probes abnormalities analyzed showed almost similar frequency compared to different international centers.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Howlader N, Noone A, Krapcho M, Miller D, Breast A, Yu M, et al
. SEER Cancer Statistics Review 1976–2009 (Vintage 2009 Populations). MD, USA: National Cancer Institute; 2012.
Zenz T, Mertens D, Küppers R, Döhner H, Stilgenbauer S. From pathogenesis to treatment of chronic lymphocytic leukaemia. Nat Rev Cancer 2010;10:37-50.
Clifford R, Schuh A. State-of-the-art management of patients suffering from chronic lymphocytic leukemia. Clin Med Insights Oncol 2012;6:165-78.
Peterson LC, Lindquist LL, Church S, Kay NE. Frequent clonal abnormalities of chromosome band 13q14 in B-cell chronic lymphocytic leukemia: Multiple clones, subclones, and nonclonal alterations in 82 Midwestern patients. Genes Chromosomes Cancer 1992;4:273-80.
Juliusson G, Oscier D, Juliusson G, Gahrton G, Oscier D, Fitchett M, et al.
Cytogenetic findings and survival in B-cell chronic lymphocytic leukemia. Second IWCCLL compilation of data on 662 patients. Leuk Lymphoma 1991;5 Suppl 1:21-5.
Fink SR, Paternoster SF, Smoley SA, Flynn HC, Geyer SM, Shanafelt TD, et al.
Fluorescent-labeled DNA probes applied to novel biological aspects of B-cell chronic lymphocytic leukemia. Leuk Res 2005;29:253-62.
Dewald GW, Brockman SR, Paternoster SF, Bone ND, O'Fallon JR, Allmer C, et al.
Chromosome anomalies detected by interphase fluorescence in situ
hybridization: Correlation with significant biological features of B-cell chronic lymphocytic leukaemia. Br J Haematol 2003;121:287-95.
Nelson BP, Gupta R, Dewald GW, Paternoster SF, Rosen ST, Peterson LC, et al.
Chronic lymphocytic leukemia FISH panel: Impact on diagnosis. Am J Clin Pathol 2007;128:323-32.
Shanafelt TD, Byrd JC, Call TG, Zent CS, Kay NE. Narrative review: Initial management of newly diagnosed, early-stage chronic lymphocytic leukemia. Ann Intern Med 2006;145:435-47.
Mayr C, Speicher MR, Kofler DM, Buhmann R, Strehl J, Busch R, et al.
Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia. Blood 2006;107:742-51.
Döhner H, Stilgenbauer S, Benner A, Leupolt E, Kröber A, Bullinger L, et al.
Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 2000;343:1910-6.
Dierlamm J, Michaux L, Criel A, Wlodarska I, Van den Berghe H, Hossfeld DK, et al.
Genetic abnormalities in chronic lymphocytic leukemia and their clinical and prognostic implications. Cancer Genet Cytogenet 1997;94:27-35.
Yoffe G, Howard-Peebles PN, Smith RG, Tucker PW, Buchanan GR. Childhood chronic lymphocytic leukemia with (2;14) translocation. J Pediatr 1990;116:114-7.
Shanafelt TD, Witzig TE, Fink SR, Jenkins RB, Paternoster SF, Smoley SA, et al.
Prospective evaluation of clonal evolution during long-term follow-up of patients with untreated early-stage chronic lymphocytic leukemia. J Clin Oncol 2006;24:4634-41.
Oscier DG, Gardiner AC, Mould SJ, Glide S, Davis ZA, Ibbotson RE, et al.
Multivariate analysis of prognostic factors in CLL: Clinical stage, IGVH gene mutational status, and loss or mutation of the p53 gene are independent prognostic factors. Blood 2002;100:1177-84.
Zenz T, Eichhorst B, Busch R, Denzel T, Häbe S, Winkler D, et al.
TP53 mutation and survival in chronic lymphocytic leukemia. J Clin Oncol 2010;28:4473-9.
Zenz T, Stilgenbauer S. Therapy with the FCR regimen does not overcome chronic lymphocytic leukemia biology: Aberrant p53 expression predicts response and survival. Leuk Lymphoma 2009;50:1559-61.
Lozanski G, Heerema NA, Flinn IW, Smith L, Harbison J, Webb J, et al.
Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions. Blood 2004;103:3278-81.
Schuh A, Becq J, Humphray S, Alexa A, Burns A, Clifford R, et al.
Monitoring chronic lymphocytic leukemia progression by whole genome sequencing reveals heterogeneous clonal evolution patterns. Blood 2012;120:4191-6.
Landau DA, Carter SL, Stojanov P, McKenna A, Stevenson K, Lawrence MS, et al.
Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell 2013;152:714-26.
[Table 1], [Table 2], [Table 3]