|Year : 2018 | Volume
| Issue : 4 | Page : 179-183
Prognostic significance of BCL6 and KI67 in patients with chronic lymphocytic leukemia
Maha Mohamed Adel Elgammal1, Nabil Ahmed El-Halawani2, Manal Abd El-Sattar El-Sorady2, Shereen Mohamed El-Maghraby1, Ghada Said Mahmoud Sallam1
1 Department of Hematology, Medical Research Institute, Faculty of Medicine, University of Alexandria, Alexandria, Egypt
2 Department of Hematology, Internal Medicine Department, Faculty of Medicine, University of Alexandria, Alexandria, Egypt
|Date of Submission||16-Jul-2018|
|Date of Acceptance||06-Aug-2018|
|Date of Web Publication||10-Apr-2019|
Maha Mohamed Adel Elgammal
Department of Hematology, Medical Research Institute, Alexandria University, Alexandria
Source of Support: None, Conflict of Interest: None
Background Chronic lymphocytic leukemia (CLL) is a heterogeneous disease; its prognosis depends on the disease stage at diagnosis as well as the presence or absence of high-risk markers to determine the treatment strategy.
Aim To evaluate the expression of BCL6 and Ki67 in patients with CLL and to assess their clinical prognostic significance in relation to other established prognostic markers such as Zeta-associated protein of 70-Kd (ZAP70) and β2 microglobulin (β2M).
Participants and methods BCL6 and Ki67 were measured using flow cytometry in thirty newly diagnosed CLL patients who presented to the Hematology units in Alexandria Main University Hospital and the Medical Research Institute, Alexandria University, they were also measured in 20 healthy age-matched and sex-matched controls.
Results A statistically significant difference was found between cases and controls for both BCL6 and Ki67 expressions (P<0.0001). Significantly positive correlations were found between both BCL6 and Ki67 expressions and the following parameters: Rai staging (P=0.000 and 0.000, respectively), serum marker lactate dehydrogenase (LDH) (P=0.001 and 0.000, respectively), β2M (P=0.005 and 0.000, respectively), and ZAP70 expression (P=0.000 and 0.000, respectively). Similarly, a positive significant correlation was found between BCL6 expression and Ki67 expression (P=0.000). Rai staging showed a significant positive correlation with β2M, LDH, and ZAP70 (P=0.000, 0.001, and 0.003, respectively).
Conclusion BCL6 expression and Ki67 expressions were positively correlated with the established prognostic markers (clinical staging, LDH, β2M, and ZAP70), so both of them may be considered as prognostic factors in patients with CLL.
Keywords: BCL6, chronic lymphocytic leukemia, Ki 67
|How to cite this article:|
Adel Elgammal MM, El-Halawani NA, El-Sorady MA, El-Maghraby SM, Sallam GS. Prognostic significance of BCL6 and KI67 in patients with chronic lymphocytic leukemia. Egypt J Haematol 2018;43:179-83
|How to cite this URL:|
Adel Elgammal MM, El-Halawani NA, El-Sorady MA, El-Maghraby SM, Sallam GS. Prognostic significance of BCL6 and KI67 in patients with chronic lymphocytic leukemia. Egypt J Haematol [serial online] 2018 [cited 2019 Aug 23];43:179-83. Available from: http://www.ehj.eg.net/text.asp?2018/43/4/179/255872
| Introduction|| |
Chronic lymphocytic leukemia (CLL) has been long thought to be a single disease, but recent studies have shown the heterogeneity of CLL, which differ in their cytogenesis, tendency toward disease progression, or response to therapy. The use of novel biological and genetic parameters combined with careful clinical evaluation allows us to distinguish patients with a mild-onset course, who often will not need any treatment, from those with an aggressive course (high-risk leukemia). On this background, it becomes increasingly challenging to select the suitable treatment strategy. Moreover, several promising new therapeutic modalities are being evaluated, involving novel drugs, monoclonal antibodies, stem cell transplantation, or gene therapy .
BCL6 functions as a transcriptional repressor and is necessary for germinal center (GC) formation. The gene is located on chromosome 3q27. It was confirmed that BCL6 suppresses the expression of the p53 gene and modulates DNA damage-induced apoptotic responses in GC B cells. The persistent expression of BCL6 may contribute to lymphomagenesis by blocking B-cell differentiation within the GC .
Ki67 protein is present during all active phases of the cell cycle (G1, S, G2, and mitosis), being absent from resting cells (G0), which makes it an excellent marker for determining the so-called growth fraction of a given cell population. It appears to provide valuable prognostic information particularly in lymphoproliferative diseases, as well as other malignancies .
The aim of the present work was to evaluate the expression of BCL6 and Ki67 in patients with CLL and assess their clinical prognostic significance in relation to other established prognostic markers such as ZAP70 and β2M.
| Participants and methods|| |
BCL6 and Ki67 were measured in 30 patients with newly diagnosed CLL who presented to the Hematology units in Alexandria Main University Hospital and the Medical Research Institute, Alexandria University. Twenty healthy participants with matched age and sex were recruited as a control group.
All participants enrolled in the study signed an informed written consent before enrollment in the study.The approval of Research Ethics Committee of faculty of medicine was obtained.
Full history taking, thorough clinical examination, Rai staging, complete blood count, morphological studies, Coombs test (direct and indirect), liver and renal functions, β2M, lactate dehydrogenase (LDH), imaging studies and flow cytometry were done for all cases. We used CD23-FITC (9P25), Beckman Coulter (Bd & Beckman Coulter, Alexandria); κ-FITC/λ-PE (polyclonal), DAKO; CD5 FITC (clone DK23)/CD20-PE (clone B-Ly1), DAKO; FMC7-FITC (F7110), DAKO; CD79b-PE (CB3-1) and PE Bcl6 (K112-91), BD Pharmingen; FITC − Ki67 (SOIA15), BD Pharmingen; and FITC-ZAP70 (1E7.2), BD Pharmingen.
Data were analyzed using the statistical package for social sciences (SPSS ver.20; SPSS Inc., Chicago, Illinois, USA). Comparison of quantitative variables between cases and controls was conducted using independent-sample t test. Pearson’s χ2-test was used to compare the two groups in qualitative variables. Pearson’s correlation test was used to test correlation between quantitative continuous variables. Spearman correlation test was used to test correlation between quantitative score variables and other quantitative variables.
| Results|| |
There was a statistically significant difference between patients and controls regarding BCL6 and Ki67 (P<0.0001). Significantly positive correlations were found between both BCL6 and Ki67 expressions on one side and Rai clinical staging, LDH, β2M, and ZAP70 on the other side. Moreover, there was a positive correlation between BCL6 expression and Ki67 expression (P=0.000). Rai staging showed a significant positive correlation with both β2M and LDH (P=0.000 and 0.001, respectively) ([Table 1], [Table 2] and [Figure 1] and [Figure 2]).
|Table 1 Correlation between Rai and both Ki67 and BCL6 in chronic lymphocytic leukemia cases|
Click here to view
|Figure 1 The correlations between both BCL6 and Ki67 and ZAP70 expression.|
Click here to view
|Figure 2 The relation between Rai staging and means of LDH, β2M) and (BCL6, Ki67and ZAP70. LDH is measured by U/l, and β2M is measured by mg/l, whereas BCL6, Ki67, and ZAP70 are measured by %. β2M, β2 microglobulin; LDH, lactate dehydrogenase.|
Click here to view
By receiver operating characteristic curve analysis, the diagnostic performance of BCL6 to diagnose cases from controls was statistically significant with area under the curve of 90.7%, P less than 0.01. The cases are considered positive if equal or more than the cutoff value of 46.65%, which had the highest sensitivity of 80%) and highest specificity of 85%). However, for Ki67, with area under the curve of 78%, P value of 0.01, the cases were considered positive if equal or more than the cut-off value of 6%, which had the highest sensitivity of 63% and highest specificity of 100% ([Figure 3] and [Figure 4]).
| Discussion|| |
Prognostic factors can be used to guide CLL management. Many researchers have evaluated the prognostic value of BCL6 gene rearrangements and mutations in chronic lymphoproliferative diseases. Their results were conflicting. Some studies reported a favorable prognosis especially in diffuse large B-cell lymphoma, but subsequent studies showed no effect or a poor outcome ,. In this study, BCL6 expression was assessed by flow cytometry on the peripheral blood, and a significant difference between cases and controls was found. There was a significant positive correlation between BCL6 expression and the prognostic markers Rai clinical staging, LDH, β2M and ZAP70. Moreover, it positively correlated with Ki67 expression, and these results may carry a worse prognostic effect in CLL. Same results were reported by Jantus Lewintre et al.  who studied BCL6 expression by real-time PCR and immunoblot in CLL cases of early stage, and found that CLL cases expressing high levels of BCL6 had significantly shorter treatment-free interval and worse prognosis. The hazard ratio demonstrated that patients with high expression of BCL6 have almost five times more risk of progression than those with lower levels of BCL6 expression. Previous studies performed by immunocytochemistry and/or immunohistochemistry failed to detect BCL6 expression in CLL. However, Capello et al.  could detect BCL6 expression in eight out of 19 CLL cases via flow cytometry performed on peripheral blood samples, which means that flow cytometry may be more sensitive than the usual immuno-staining of frozen sections.
For many years, CLL cells were defined as quiescent and non-proliferating cells in the G0 phase of the cell cycle. Now recent studies confirmed that some of the circulating CLL cells are proliferative. In the present study, Ki67 expression was estimated by flow cytometry on the peripheral blood showing significant difference between cases and controls for both (P<0.0001). A significantly positive correlation was found between Ki67 expression and the prognostic markers Rai clinical staging, LDH, β2M and ZAP70. Similar results were reported by Damle et al.  who studied the expressions of both Ki67 and ZAP70 on the peripheral blood of CLL cases by flow cytometry and found that Ki67 showed significantly higher percentage in cases than controls. Moreover, they showed that ZAP70 expression exhibited a strong positive correlation with Ki-67 expression. Another study done by Cordone et al.  confirmed the close correlation between the number of cells expressing Ki67 and advanced clinical stage. This was similar to Astsaturov et al.  who demonstrated that resistant CLL cases showed a higher percentage of Ki67-positive cells. Another study done by Khoudoleeva et al.  analyzed Ki67 expression in the peripheral blood, bone marrow, and lymph nodes (LNs) by flow cytometry in patients with CLL. They found the low levels of Ki67 in peripheral blood, which may represent a subpopulation of quiescent malignant B cells, as their real proliferation occurs in the lymph nodes and spleen.
Moreover, Giné et al.  studied lymph node tissue biopsies by Ki67 immunostaining in CLL cases, and they found that cells that had increased expression of Ki67 had higher serum LDH levels and more elevated ZAP70 expression, and predicted a poor outcome in such cases. Similarly, Naz et al.  studied Ki67 in tissue sections by immunostaining in CLL cases and showed a significant association between Ki67 expression and the presence of B symptoms and the clinical progression of the disease. ln addition, De Melo et al.  studied the expression of Ki67 in CLL cases and found that the highest proliferation rates were observed in patients who developed Richter’s syndrome. A similar study by Morabito et al.  recorded Ki67 by flow cytometry in previously untreated CLL cases and found that lower Ki67 expression had predicted significantly longer survival.
In our study, Rai staging showed a significant positive correlation with β2M, LDH and ZAP70 (P=0.000, 0.001, and 0.003, respectively). In agreement with our results, Di Giovanni et al.  also confirmed that staging correlated positively with LDH. The same results were reported by Di Giovanni and colleagues who measured β2M in patients with CLL and found a positive correlation between β2M and the clinical stage. In contrast to our study, Mozaheb et al.  found no correlation between serum level of LDH and the stage of the disease. Another study with similar results was done by Šoljić et al.  who demonstrated that ZAP-70 correlated with Rai staging.
| Conclusions and recommendations|| |
Ki67 and BCL6 expressions were positively correlated with other established prognostic markers (Rai clinical staging, LDH, β2M, and ZAP70) and can be considered as prognostic factors in patients with CLL. Rai staging was found to correlate with the prognostic parameters LDH, β2M, and ZAP70. This shows that Rai staging is still a prognostically reliable tool in CLL cases. It is recommended to study the effect of Ki67 and BCL6 expressions in Richter’s transformation and on a larger scale of patients. In addition to the study of BCL6 protein expression, mutational status of BCL6 gene is recommended to be analyzed. Assessment of ki67 in bone marrow and other lymphoid tissues is also recommended. Analysis of the expression of both BCL6 and Ki-67 in patients treated by different therapeutic protocols targeting Ki67 and BCL6 may be of value to be studied.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ghia P, Hallek M. Management of chronic lymphocytic leukemia. Haematologica
Phan RT, Dalla-Favera R. The BCL6 proto-oncogene suppresses p53 expression in germinal-centre B cells. Nature
Bruey J-M., Kantarjian H, Ma W, Estrov Z, Yeh C, Donahue A et al.
Circulating Ki-67 index in plasma as a biomarker and prognostic indicator in chronic lymphocytic leukemia. Leuk Res
Sarsotti E, Marugan I, Benet I, Terol M, Sanchez-Izquierdo D, Tormo M et al.
Bcl-6 mutation status provides clinically valuable information in early-stage B-cell chronic lymphocytic leukemia. Leukemia
De Leval L, Harris N. Variability in immunophenotype in diffuse large B‐cell lymphoma and its clinical relevance. Histopathology
Jantus Lewintre E, Reinoso Martín C, García Ballesteros C, Pendas J, Benet Campos C, Mayans Ferrer JR et al.
BCL6: somatic mutations and expression in early-stage chronic lymphocytic leukemia. Leuk Lymphoma
Capello D, Fais F, Vivenza D, Migliaretti G, Chiorazzi N, Gaidano G et al.
Identification of three subgroups of B cell chronic lymphocytic leukemia based upon mutations of BCL-6 and IgV genes. Leukemia
Damle RN, Temburni S, Calissano C, Yancopoulos S, Banapour T, Sison C et al.
CD38 expression labels an activated subset within chronic lymphocytic leukemia clones enriched in proliferating B cells. Blood
Cordone I, Matutes E, Catovsky D. Monoclonal antibody Ki-67 identifies B and T cells in cycle in chronic lymphocytic leukemia: correlation with disease activity. Leukemia
Astsaturov IA, Samoilova RS, Iakhnina EI, Pivnik AV, Vorobiov AI. The relevance of cytological studies and ki-67 reactivity to the clinical course of chronic lymphocytic leukemia. Leuk Lymphoma
Khoudoleeva O, Gretsov E, Barteneva N, Vorobjev I. Proliferative index and expression of CD38, Zap-70, and CD25 in different lymphoid compartments of chronic lymphocytic leukemia patients. Pathol Lab Med
Giné E, Martinez A, Villamor N, Lopez-Guillermo A, Camos M, Martinez D et al.
Expanded and highly active proliferation centers identify a histological subtype of chronic lymphocytic leukemia (‘accelerated‘ chronic lymphocytic leukemia) with aggressive clinical behavior. Haematologica
Naz E, Mirza T, Aziz S, Ali A, Danish F. Correlation of Ki 67 proliferative index with clinical and pathological features on tissue sections of non Hodgkins lymphoma by immunostaining. J Pak Med Assoc
De Melo N, Matutes E, Cordone I, Morilla R, Catovksy D. Expression of Ki-67 nuclear antigen in B and T cell lymphoproliferative disorders. J Clin Pathol
Morabito F, Cutrona G, Gentile M, Loiacono F, Matis S, Recchia AG et al.
More on the determination of Ki-67 as a novel potential prognostic marker in B-cell chronic lymphocytic leukemia. Leuk Res
Di Giovanni S, Valentini G, Carducci P, Giallonardo P. Beta-2-microglobulin is a reliable tumor marker in chronic lymphocytic leukemia. Acta Haematologica
Mozaheb Z, NazarAbadi MHH, Afzal M. Chronic lymphocytic leukemia and prognostic factors. Asian Pac J Cancer Prev
Šoljić V, Perak RB, Vukojević K, Saraga-Babić M, Bubalo P, Karan D et al.
ZAP-70 expression and proliferative activity in chronic lymphocytic leukemia. Leuk Lymphoma
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]