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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 44  |  Issue : 4  |  Page : 254-259

Coexistence of (CCND1/PRAD-1/BCL1) juxtaposition and drug resistance gene is a marker for vincristine-resistant B-non-Hodgkin’s lymphoma


1 Department of Clinical Pathology, Faculty of Medicine, Assiut University, Asyut, Egypt
2 Assitant Professor of clinical pathology, Assiut University, Egypt

Date of Submission03-Aug-2019
Date of Acceptance06-Aug-2019
Date of Web Publication20-Jul-2020

Correspondence Address:
Sahar El Gammal
Clinical Pathology Department, Faculty of Medicine, Assiut University, Asyut
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_27_19

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  Abstract 


Background CCND1/PRAD-1/BCL1 juxtaposition activates immunoglobulin heavy chain (IgH) enhancing effect on cyclin D1 expression. This leads to overexpression of cyclin D1, a cell-cycle regulator. Cyclin D1 can function as an oncogene growth advantage for tumor cells by way of cell-cycle progression. Multidrug resistance (MDR)1 gene expression leads to overproduction of P-glycoprotein (P-gp) which leads to chemotherapy resistance causing poor prognosis and short survival [overall survival (OS) and disease free survival(DFS)] in B-non-Hodgkin’s lymphoma (NHL).
Objective To evaluate BCL1/IgH juxtaposition by fluorescent in-situ hybridization (FISH) and MDR1 protein expression by immunohistochemistry in B-non Hodgkin lymphoma (B-NHL) and to clarify if these genes have some relevance to developing vincristine treatment resistance of B-NHL and their correlation with clinical outcome.
Patients and methods Fifty-three bone marrow biopsy samples with B-NHL were included in the study for the expression of t(11;14) BCL1/IgH in various B-cell lymphomas by FISH and MDR1/P-gp by immunohistochemistry and its relation to the outcome and chemotherapy resistance.
Results In this study t(11;14) detected by FISH was positive in 64.2%. It was positive in (66.7%) of mantle cell lymphoma, in 64.3% of small cell lymphoma, and in 60% of large cell lymphoma. The detection frequency of MDR1/P-gp by immunohistochemistry in mantle cell lymphoma, small cell lymphoma and large cell lymphoma, is 80, 82, and 80%, respectively.
Conclusion In this study, the frequency of t(11;14) detection by FISH and MDR1/P-gp expression by immunohistochemistry in Egyptian patients were consistent with other studies and the coexistence of MDR1/P-gp and t(11;14) in B-NHL is important before treatment to allow the design of novel drug regimens.

Keywords: B-NHL, cyclin D1, vincristine, BCL1


How to cite this article:
El Gammal S, Mosad E. Coexistence of (CCND1/PRAD-1/BCL1) juxtaposition and drug resistance gene is a marker for vincristine-resistant B-non-Hodgkin’s lymphoma. Egypt J Haematol 2019;44:254-9

How to cite this URL:
El Gammal S, Mosad E. Coexistence of (CCND1/PRAD-1/BCL1) juxtaposition and drug resistance gene is a marker for vincristine-resistant B-non-Hodgkin’s lymphoma. Egypt J Haematol [serial online] 2019 [cited 2020 Aug 8];44:254-9. Available from: http://www.ehj.eg.net/text.asp?2019/44/4/254/290229




  Introduction Top


Cytogenetic analysis in non-Hodgkin’s lymphoma (NHL) can be difficult, given the problem of isolating neoplastic cells from tissue biopsy samples for karyotypic analysis. However, several large studies suggest that nearly all cases have a cytogenetic abnormality. It may be either numerical or structural but a number of translocations are particularly interesting because of their association with specific types of lymphomas and the insights they have provided into the process of lymphoma genesis [1].

The translocation t(11;14)(q13;q32) has been known to cause neoplasia for many years in a variety of B-cell malignancies including chronic lymphocytic leukemia (in 2–5% of the cases), B-cell prolymphocytic leukemia (10–30%), splenic lymphoma with villous lymphocytes, plasma cell leukemia, and multiple myeloma (5–25%) and Mantle cell lymphoma (MCL) vary widely from 50–70 to 100% [2]. Through this rearrangement, CCND1/PRAD-1/BCL1 on chromosome 11 becomes juxtaposed to the active immunoglobulin heavy chain (IgH) enhancer on chromosome. Cyclin D1 belongs to the G1 cyclins and plays a key role in cell-cycle regulation during the G1/S transition by cooperating with cyclin-dependent kinases [3]. Further evidence suggests that cyclin D1 can function as an oncogene, the overexpression of which may lead to growth advantage for tumor cells by way of cell-cycle progression [4].

Multidrug resistance (MDR) contributes to therapeutic failure in several hematological malignancies such as acute myeloid leukemia, multiple myeloma, and NHL, particularly when treated with vinca alkaloids [vincristine (VCR) and vinblastine]. The MDR phenotype of cells results in a pleiotropic drug resistance that leads to the development of a refractory disease. Overexpression of this P-glycoprotein (P-gp) 170 kDa protein, often developed after exposition to cytotoxic agents, is associated with cross-resistance to a wide range of compounds and consequently with drug-resistant diseases, especially in acute myeloid leukemia, acute lymphoblastic leukemia (ALL), and NHL patients. It is considered an independent prognostic variable for response and associated with a lower complete remission (CR) rate [3]. MDR1/P-gp driving chemotherapy resistance remains one of the hallmarks of poor outcomes in hematologic malignancies as B-non Hodgkin lymphoma (B-NHL) [5],[6].

In human VCR-resistant of leukemia/lymphoma cell lines, it was observed that MDR in VCR-resistant cell lines was mediated by either MDR1 or MDR-associated protein genes [7].

The aim of the study was to evaluate BCL1/IgH juxtaposition by fluorescent in situ hybridization (FISH) and MDR1 protein expression by immunohistochemistry in B-NHL to clarify if these genes have some relevance to developing VCR treatment resistance of B-NHL and their correlation with clinical outcome.


  Patients and methods Top


This is a retrospective study of patients with B-NHL who were referred to the South Egypt Cancer Institute, Assiut University during the period between 2002 and 2008. Fifty bone marrow biopsy samples (fixed and paraffin-embedded bone marrow biopsy) were included in this study diagnosed by: morphologic examination of bone marrow aspirate, immunophenotyping, immunostaining for bone marrow aspirate (BM) biopsy, and lymph node (LN) biopsy samples. Patients were divided into group A: VCR responder B-NHL patients (n=19 patients). Group B: VCR-resistant B-NHL patients (n=34 patients) and the control group: BM aspiration and biopsy (fixed and paraffin-embedded bone marrow biopsy) for nonmalignant disease patients (n=14 patient).

This patients’ criteria included newly diagnosed or relapsed cases of B-NHL by immunophenotyping and B-NHL stage IV, according to Ann Arbor staging.

t(11;14) by FISH on bone marrow biopsy (BMB), 5 μm thick paraffin sections were cut on positive charge slides and 3 μ thick paraffin sections were cut on slides for MDR1/P-gp by immunohistochemistry.

Methods

Fluorescent in situ hybridization

Poseidon (Kreatech, Leica Biosystem, Germany) Repeat BCL1/IgH t(11;14) fusion probe (product Nr/REF: KBI.10604) are direct-labeled DNA probes provided in a ready-to-use format. Apply 10 μl of the probe to a sample area of ∼22×22 mm. Briefly, pretreatment 4–6 μm paraffin-embedded tissue section on positively charged slides which fixed in formalin. Deparaffinized slides were obtained by soaking in xylene 3× for 10 min and were rehydrated by soaking in 100, 85, 70% ethanol for 3 min each, and washed with H2O for 3 min at room temperature. Finally, the slides were covered with proteinase K on a hot plate at 37°C for 11min. Then the denatured probe was mixed at 90°C for 10 min. Ten microliter aliquot of the probe solution was applied to the target area. The slide cover slip (22 mm×22 mm) was placed over the probe solution.

The slide was put in a Hybrite (vysis, Abbott Molecular, Germany) instrument programmed to raise the temperature of the slide first to 73°C for 1 min, then decreasing the temperature to 37°C for 4 h at least. The slides were washed in 2× SSC/0.1% NP-40 at room temperature for 5–60 s. Nuclei were then counterstained with DAPI counterstain cover slipped and at least 500 nuclei were examined using Axioskope 2 mot plus florescence Microscope Zeiss Microscopy GmbH, Germany (ZEISS) and diagnosed by the image system Leica Microsystem, GmbH, Germany (LEICA DG software) Germany 2003. The BCL1/IgH t(11;14) probe is designed as a dual-fusion probe to detect both rearranged chromosome der (11) and der (14) by two colocalized red/green or yellow and green (G) signals which will identify the normal chromosomes 14 and 11, respectively.

Signal patterns others than the above may indicate variant translocations, deletions on der (11) or der (14) or other rearrangements. The sample was considered positive at a cutoff value determined by the control group included in the study, for example, if the control case slide gives 2% positivity, it means that the cutoff is 2% and any case that gives more than 2% becomes positive and less than 2% becomes negative for the studied probe.

Immunohistochemistry

Deparaffinize 4–6 μm formalin-fixed paraffin-embedded tissue sections slides and rehydrate and then are pretreated and blocked by hydrogen peroxide (dual endogenous enzyme block). After that the primary antibody or negative control reagent was applied for 10 min, and then the specimen was rinsed. Apply enough labeled polymer to cover the specimen, incubate 30 min at RT and then the specimen was placed in a solution bath. Diaminobenzedine (substrate chromogen solution) was applied for 10 min, then rinsed. The specimen was counterstained by light green or hematoxylin for 1–2 min, and rinse gently in a distilled water bath. Finally, the specimen was mounted and coverslips were applied.


  Results Top


Fifty-three patients of B-NHL were included in this study. They were 28 (52.8%) men and 25 (47.2%) women. Control group: 14 cases apparently noninfiltrated normal BMB, there were 12 men (85.7%) and two women (14.3%). According to the pathology, the patients were classified to three groups. MCL: 15 (28.3%) patients, small cell lymphoma (SCL): 28 (52.8%) patients, and large cell lymphoma (LCL): 10 (18.9%) patients. Also, according to the course of the disease, the patients were divided into four groups: patients in CR1, 32/53 (60.4%), CR2, 4/53 (7.5%), CR3, 4/53 (7.5%), patients in resistance (R) 13/53 (24.5%).

In this study t(11;14) detected by FISH was positive in 34/53 (64.2%). It was positive in 66.7% of MCL, in 64.3% of SCL, and in 60% of LCL.

Qualitative detection of t(11;14) BCL1/IgH genes in B-NHL by FISH was significantly different from the control group (P<0.000). There was a significant relationship between t(11;14) by FISH in the VCR responder group as it was negative in 100% of the group compared with the VCR-resistant group which was positive in 34/34 (100%).

As regards the pathological group there was no significant difference between positive t(11;14) and negative t(11;14). But there was a highly significant difference between positive and negative t(11;14) as regards the disease course ([Table 1]). There was a high significant relationship between positive t(11;14) and the expression of MDR1 ([Table 2]).
Table 1 Relationship between t(11;14) and disease course

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Table 2 Relationship between t(11;14) and multidrug resistance 1

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There was a high significant difference between DFS of positive t(11;14) patients as its mean was 6.97±6.22 months compared with DFS of negative t(11;14) its mean was 23.68±16.22 months ([Figure 1]).
Figure 1 Relationship between overall survival in both positive and negative t(11;14).

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There was high significant correlation between OS of positive t(11;14); its mean was 16.41±11.15 compared with OS of negative t(11;14); its mean was 46.00±19.13 ([Figure 2]).
Figure 2 Relationship between disease-free survival in both positive and negative t(11;14).

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In the detection of MDR1 expression in BMB by immunohistochemistry the cutoff value in the control was 10% so the cases were considered positive when it was at least 10%. There was a significant difference between expression MDR1/P-gp protein in the VCR responder group compared with its expression in VCR-resistant group ([Table 3]). There was no significant difference between MDR1/P-gp expression and pathological classification of B-NHL. Also, there is a significant difference in the relationship of coexistence of both expression MDR1/P-gp protein and t(11;14) and the disease course in B-NHL ([Table 4]) and is significantly associated with VCR resistance ([Table 5]).
Table 3 Relation between multidrug resistance 1/P-glycoprotein and studied groups

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Table 4 Relationship between disease course and the presence of both multidrug resistance 1/P-glycoprotein and t(11;14)

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Table 5 Relationship between vincristine-resistant and the presence of both multidrug resistance 1/P-glycoprotein and t(11;14)

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In this study, there was significant negative correlation between the patients who are positive for both MDR1/P-gp protein expression and t(11;14) and the patients who are negative for both MDR1/P-gp protein expression and t(11;14) as regards DFS and OS ([Figure 3] and [Figure 4]).
Figure 3 Positive for both multidrug resistance (MDR)1/P-glycoprotein (P-gp) protein expression and t(11;14) and negative as regards DFS.

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Figure 4 Positive for both multidrug resistance (MDR)1/P-glycoprotein (P-gp) protein expression and t(11;14) and negative as regards OS.

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  Discussion Top


Is there a relationship between t(11;14) and prognosis in B-NHL (mantle cell lymphoma, small cell lymphoma and large cell lymphoma)?

There was a highly significant difference between t(11;14) in good VCR responder and resistance groups. This result is consistent with others. In most of the MCL patients, SCL and LCL with t(11;14) showed bad prognosis and are categorized as aggressive B-NHL neoplasm which is consistent with others [7],[8],[9].

Is there a relationship between t(11;14), protein cyclin D1 expression, and disease course B-NHL (mantle cell lymphoma, small cell lymphoma and large cell lymphoma)?

In this study, there was a significant difference between positive t(11;14) patients regarding the disease course in the form of CR1, CR2, and CR3 in comparing with the patients who were negative for translocation. Also, patients with positive t(11;14) and overexpression of cyclin D1 showed more resistance to chemotherapy than the patients who were negative for t(11;14) in B-NHL. These results were consistent with others [9],[10] who concluded that overexpression of cyclin D1/BCL1 play an important role in the disease course. In addition to inappropriate and constant expression of cyclin D1 positive t(11;14) shows high expression of p27, probably because the p27 is sequestered in complexes containing cyclin D1, therapy blocking its cell-cycle inhibitory function [11],[12].

Is there a relationship between coexistence of both multidrug resistance 1 expression and t(11;14) as regards DFS and OS in B-NHL?

Our results showed a significant difference between coexistence of both MDR1 expression and t(11;14) as regards DFS and OS of patients, whereas DFS and OS in positive t(11;14) are shorter than negative translocation, which is consistent with that reported by others [8],[13] who found that positive expression of BCL1 and overexpression of cyclin D1 due to t(11;14) in B-NHL is associated with bad prognosis, only temporary responses to chemotherapy, and a high recurrence rate resulting in a poor long-term prognosis and short survival because overexpression of P-gp contributes to antineoplastic resistance in at least two ways, active drug efflux and elevation of cellular apoptotic threshold [5].

Is there a relationship between coexistence of both multidrug resistance 1 expression and t(11;14) and the disease course in B-NHL patients?

In this study, there was a significant difference between coexistence of both MDR1 expression and t(11;14) and the disease course in B-NHL. This was consistent with others [14]. In this study, P-gp positive expression was associated with VCR resistance for chemotherapy in 30% of patients which was less than the study of Szendrei et al. [14], who reported that the resistance was in 89% of positive expression P-gp.

Tulpule et al. [15] reported that P-gp is one of the significant indicators to approach and regimen for treatment that lead to a difference in CR from regimen to another.

Is there a relationship between t(11;14) and multidrug resistance 1/P-glycoprotein in B-NHL?

This study showed a significant relationship between BCL1 gene expression and MDR1/P-gp in the patients studied. Most of the patients who expressed BCL1 were associated with MDR1/P-gp expression. This means that patients who expressed both MDR1/P-gp and t(11;14) were associated with aggressive chemotherapy resistance due to the production of P-gp which code by the MDR1 gene, where P-gp have different mechanisms in the resistance to chemotherapy in B-NHL.

Translocation (11;14) was one of the bad prognostic factors due to overexpression of cyclin D1 protein which leads to cell-cycle disorders. MDR1/P-gp expression is also one of the bad prognostic factors in B-NHL. Therefore, in this study the expression of both BCL1 and MDR1 P-gp in B-NHL was associated with poor outcome and chemotherapy resistance. This is consistent with Hirose et al. [16] who explained that the genes of G1/cyclin, such as cyclin D1 or P16, demonstrated a visible change when the cells obtained MDR1 expression in B-NHL. Ma et al. [17] explained that overexpression of cyclin D1 result of BCL1/IgH genes rearrangement leads to decreased cyclin A due to binding with cyclin-dependent kinase 2. Lower expression of cyclin A is an unfavorable prognostic factor in the neoplasm as B-NHL. Chen et al. [6] found that cyclin D1, a protein related to cell cycle, was demonstrated to be involved in the epidermal growth factor receptor-mediated effects since epidermal growth factor receptor increased the expression of cyclin D1, as well as t(11;14) promoted the MDR phenotypes in breast cancer cells and NHL via accelerating the G1/S phase transition. Therefore, cyclin D1 leads to MDR due to increased production of MDR1/P-gp in B-NHL and both expression in patients leads to poor prognosis and survival in addition to chemotherapy resistance.


  Conclusion Top


We concluded that (11;14) BCL1/IgH translocation is a poor prognostic factor as well as MDR1/P-gp expression in B-NHL. Determination of coexistence of MDR1/P-gp and t(11;14) in B-NHL is important before treatment to allow the design of novel drug regimens containing agents that reverse MDR function and to expect the development of VCR resistance. These results may provide a plausible diagnostic marker for the determination of drug sensitivity in NHL patients.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors declare that no conflict of interest.



 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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