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 Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 37  |  Issue : 4  |  Page : 268-273

Cyclin D1 gene amplification in multiple myeloma and its impact on the disease outcome and drug resistance


1 Internal Medicine Department, Assiut University Hospital, Assiut University, Assiut, Egypt
2 Cytogenetic and Immunohistochemistry Labs, Clinical Pathology Department, Assiut University, Assiut, Egypt
3 Cancer Institute, Assiut University, Assiut, Egypt
4 Medical Oncology Department, South Egypt Cancer Institute, Assiut University, Assiut, Egypt

Date of Submission10-Jan-2012
Date of Acceptance28-Jan-2012
Date of Web Publication21-Jun-2014

Correspondence Address:
Eman M. Sewify
MD, Internal Medicine Department, Assiut University Hospital, P.O. Box 71526, Assiut
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.7123/01.EJH.0000419285.02404.20

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  Abstract 

Background

Cyclin D1 is involved in the normal regulation of the cell cycle and in neoplasia. Inhibition of cyclin D1 function markedly attenuates the proliferation of fibroblasts of colon, esophageal, lung, and pancreatic cancer. However, the prognostic value of overexpression of the cyclin D1 gene in multiple myeloma (MM) is still a point of debate. This study aims at evaluating the prognostic significance of cyclin D1 gene amplification in MM and whether it is correlated with MDR1 expression as an indicator of chemoresistance.

Patients and methods

Twenty-five patients with MM were studied retrospectively. Cyclin D1 gene amplification was studied in bone marrow biopsies of these patients by fluorescence in-situ hybridization. Immunohistochemical study of the bone marrow biopsies was carried out to detect multidrug resistance (MDR1) gene expression. The correlations between the cyclin D1 gene amplification on the one hand and overall survival and MDR1 expression on the other were studied and analyzed statistically.

Results

Cyclin D1 gene amplification was found in 20% of patients with myeloma and was associated with a higher percentage of plasma cell infiltration of the bone marrow and increase liability for multiple osteolytic lesions. Cyclin D1-positive patients had a significantly lower progression-free and overall survival and higher levels of MDR1 compared with cyclin D1-negative patients. Cyclin D1 levels showed a statistically highly significant positive correlation with MDR1 levels (r: 0.802 and P<0.0001).

Conclusion

We suggest that there is an association between cyclin D1 gene amplification and higher disease severity, unfavorable prognosis, and increased expression of MDR1 in patients with MM.

Keywords: cyclin D1, fluorescence in-situ hybridization, immunohistochemistry, MDR1 gene expression, multiple myeloma


How to cite this article:
Sewify EM, Afifi OA, Mosad E, Zaki AH. Cyclin D1 gene amplification in multiple myeloma and its impact on the disease outcome and drug resistance. Egypt J Haematol 2012;37:268-73

How to cite this URL:
Sewify EM, Afifi OA, Mosad E, Zaki AH. Cyclin D1 gene amplification in multiple myeloma and its impact on the disease outcome and drug resistance. Egypt J Haematol [serial online] 2012 [cited 2019 Dec 9];37:268-73. Available from: http://www.ehj.eg.net/text.asp?2012/37/4/268/134976


  Introduction Top


Regulated progression through the cell cycle requires sequential expression of a family of proteins called cyclins 1. Cyclin D1 is a critical modulator of G1 progression 2. Cyclin D1 is encoded by the PRAD1, CCND1, or bcl-1 gene on chromosome 11q13, and is involved in normal regulation of the cell cycle and in neoplasia. Overexpression of cyclin D1 protein releases cells from their normal controls when they need to exit from the cell cycle, obstructs their maturation, and promotes transformation into a malignant phenotype 3. Inhibition of cyclin D1 function markedly attenuates the proliferation of fibroblasts of colon, esophageal, lung, and pancreatic cancer 2.

Dysregulation of at least one of the cyclin D genes (CCND1, CCND2, and/or CCND3) is a common unifying pathogenic event in multiple myeloma (MM). An increased level of cyclin D1 in myeloma has been reported by some research workers to be associated with a favorable prognosis. Other laboratories, however, have reported an unfavorable association with cyclin D1. Therefore, the clinical significance of cyclin D1 dysregulation in plasma cell myeloma has been uncertain 4.

Cyclin D1 has been correlated with high levels of glutathione-S-transferase (GST)-Л (a subtype of GST) in mantle cell lymphoma. An increased level of GST-Л may lead to resistance to chemotherapy 5. Clinical drug resistance has always been a major obstacle in the treatment of all types of cancer. The cell membrane is the major determinant of cancer drug penetration to subcellular targets. Cells have evolved complex chemical defense mechanisms to regulate the entry of foreign substances into and out of cells. Of the known pump mechanisms is p-glycoprotein (P-gp; MDR1; ABCB1) 6. A link between the expression of MDR1/P-gp and cellular apoptotic pathways has been shown. For example, cancer cells expressing MDR1/P-gp show resistance to anticancer drugs that trigger apoptosis 7. Clinical studies have established that MDR1 expression occurs in patients with MM and there is also clinical evidence of multidrug resistance 8.

There is some evidence that cyclin D1 may be involved in the process of drug resistance. Kamel et al. 9 reported that cyclooxygenase-2 (COX-2) upregulates MDR1/P-gp expression, which confers resistance to cancer drugs. COX-2 expression is also associated with inhibition of the apoptotic pathway in tumor cells and it induces cell proliferation by upregulation of cyclin D1. The former authors also reported that COX-2 inhibitors are associated with the downregulation of MDR1 and cyclin D1. Kornmann et al. 2 reported that suppression of cyclin D1 expression in human pancreatic cancer cells leads to enhanced chemosensitivity to drugs.

Aim of the work

In this work, we aimed to evaluate the prognostic significance of cyclin D1 gene amplification in MM and whether it is correlated with MDR1 expression as an indicator of chemoresistance.


  Patients and methods Top


This is a retrospective study of 25 patients of MM. The records were analyzed for the presenting symptoms, signs, and investigations. Special attention was focused on the following variables: age, clinical stage according to the International Staging System 10, presence of osteolytic lesions, bone marrow infiltration with plasma cells, serum protein electrophoresis, serum albumin, the presence of Bence Jones protein (BJP) in urine, blood urea, serum creatinine, calcium, lactate dehydrogenase (LDH), and serum B2-microglobulin levels. Patients received conventional chemotherapy (melphalan and prednisolone). Increased expression of MDR1 by immunohistochemistry was assessed as a marker of multidrug resistance. The overall survival (OS) was calculated from the time of diagnosis. The progression-free survival was calculated from the time of attaining plateau until disease progression.

Bone marrow biopsy was obtained for each patient and examined for the detection of cyclin D1 gene amplification using fluorescence in-situ hybridization and for MDR1 expression using immunohistochemical staining.

Fluorescence in-situ hybridization in paraffin-embedded tissue sections

Five micrometers of tissue section of bone marrow biopsies was placed on a coated slide (such as Fisher Superfrost, cat.# 12-550-15, Fisher Scientific, Ottawa, Canada). The section was deparaffinized in xylene 3× for 10 min. The slide was dehydrated in 100% ethanol 2× for 5 min at room temperature (RT) and air dried. The slide was incubated in 2× saline-sodium citrate (SSC), at 75°C for 10 min, and then the slide was incubated in 4 mg/ml pepsin [Sigma P-7012 (Sigma, St. Louis, Missouri, USA), in 0.9% NaCI pH 1.5] for 10 min at 37°C. The slide was dipped in H2O, and rinsed in 2× SSC for 2 min at RT. The slide was denatured in 70% formamide, 2× SSC at 75°C for 3–5 min. The slide was quenched in cold (2–8°C) 70% ethanol for 2 min.

The probe and hybridization solution (10 μl) were denatured for 5 min at 75°C (manufacturer’s recommended procedure). The Vysis LSI cyclin D1, CEP 11 probe was added to denatured tissue section, and then a cover slip and seal were added. The slide was incubated at 37°C in a humid chamber overnight. After hybridization, cover slips were removed gently and the slides were washed in a Coplin jar filled with 0.4 standard SSC (pH 7.0) at 72°C for 2 min. The slides were transferred to a Coplin jar filled with 2× standard SSC/N P-40 solutions (pH 7.0) at RT for 1 min. Nuclei were counterstained with a mixture of 1000 ng/ml of 4-,6-diamidino-2-phenylindole dihydrochloride and Vectashield antifade (Vector Laboratories Inc., Burlingame, California, USA) at a ratio of 1 : 10. A cover slip was then placed over the hybridization sites.

Microscopy

Microscopy and photography were carried out using a Zeiss Axiovert 200 fluorescence microscope (Zeiss, Jena, Germany) fitted with a high-resolution Leica CCD camera. Images were processed using the Leica CW4000 imaging system and software (Leica Microsystems GmbH, Wetzlar, Germany).

Fluorescence in-situ hybridization probe

The Vysis LSI cyclin D1 (11q13) SpectrumOrange/CEP 11 SpectrumGreen Probe is a mixture of two probes. The CCND1 probe is approximately 300 kb, contains the CCND1 gene, and is labeled in SpectrumOrange [Slide 1]. The second probe is specific to the D11Z1 alpha satellite centromeric repeat of chromosome 11 and is labeled in SpectrumGreen.

Results of hybridization

Hybridization of this probe to interphase nuclei of normal cells is expected to produce two orange and two green signals. The anticipated signal pattern in abnormal cells having a gain of copy number of the CCND1 target without a gain of the CEP 11 target is two green and multiple orange signals. Other patterns may be observed if additional genetic alterations are present. Two hundred nuclei were evaluated in every patient. All cases were examined for the presence of cyclin D1 amplification (defined as more cyclin D1 hybridization signals than copies of chromosome 11) and chromosome 11 duplication.

Immunohistochemistry

Immunostaining of P-gp (MDR1) P-gp expression was assessed by immunohistochemical staining on paraffin-embedded bone marrow biopsy [Slide 2] using the avidin–biotin complex method. Before staining, the sections were deparaffinized in xylene and rehydrated in graded alcohols, and then treated by heating in a microwave oven to enable antigen retrieval. Endogenous peroxidases were blocked with H2O2 and nonspecific sites were blocked using a serum-free protein in PBS. In order to study P-gp expression, we used the C494 antibody (a gift from Dako, Trappes, France), a monoclonal antibody that reacts with an internal epitope located on the C-terminal domain of the P-gp molecule. The antibody was used at 0.28 μg/ml (dilution 1 : 200) at RT for 15 min and P-gp expression was assessed by a catalyzed signal amplification system (CSA, Dako). One section from each specimen was stained as a negative control without the first antibody. Intestinal sections were used as positive controls. The criteria for membrane immunoreactivity included staining of the cell membrane.

Quantification of immunohistochemical staining

The slides were examined by two observers. Positive cells were counted on each slide in 10 fields at a magnification of ×200 and the percentages of positive cells were quantified semiquantitatively as follows: 0 (<1% positive cells); 1 (1–10%); 2 (11–50%); and 3 (51–100%).





Statistical analysis

To characterize patients in the study, we used descriptive statistics. The Mann–Whitney test was used for differences between patients with and without cyclin gene amplification. Correlations were studied using the Spearman test. Curves for OS were plotted according to Kaplan–Meier method. Receiver operator characteristic curve was used to determine the area under receiver operator characteristic of cyclin D1 level that can predict 6-month progression-free survival and 2-year OS. Results were considered significant when P was less than or equal to 0.05. All analyses were carried out using the SPSS software package (SPSS, Chicago, Illinois, USA).


  Results Top


Cyclin D1 amplification-positive cases represented 20% of all involved cases. [Table 1] shows the clinical and laboratory characteristics of cyclin D1-positive patients compared with cyclin D1-negative ones. There was a significantly higher percentage of bone marrow plasma cells and a higher chance of having multiple osteolytic lesions in cyclin D1-positive patients compared with cyclin D1-negative patients with myeloma.
Table 1: Clinical and laboratory characteristics of cyclin D1-positive patients compared with cyclin D1-negative patients (mean±SD)

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However, no statistically significant difference in the serum levels of LDH, B2-microglobulin, albumin, paraprotein, calcium, creatinine, the presence of BJP in urine, or the clinical stage (International Staging System) at presentation could be detected between cyclin D1-positive and cyclin D1-negative patients with MM. The progression-free survival and OS were significantly shorter in cyclin D1-positive patients (1.2±1.64 and 14.2±3.49 months, respectively) compared with cyclin D1-negative patients (4.95±2.11 and 22.55±7.07 months, respectively). [Figure 1] shows the Kaplan–Meier survival curves for cyclin-D1-positive and cyclin D1-negative patients.{Figure 1}

A significant positive correlation was found between the level of cyclin D1 on the one hand and the number of plasma cells in the bone marrow and the osteolytic lesion multiplicity at presentation on the other. A negative significant correlation was found between the level of cyclin D1 on the one hand and progression-free survival and OS on the other [Table 2].
Table 2: Correlation between the levels of cyclin D1 and disease parameters

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The MDR1-positive patients represent 40% of the myeloma group. The presence of MDR1 was associated with significantly higher levels of plasma cells in the bone marrow at presentation and shorter OS (14.7±4.67 months) compared with MDR1-negative patients (25±5.64 months), with a P<0.0001.

Determination of cyclin D1 and MDR1 levels was found to be helpful in predicting 6-month progression-free survival and 2-year OS [Figure 2] and [Figure 3].{Figure 2}
Figure 1: (a) Overall survival in cyclin D1-negative patients. (b) Overall survival in cyclin D1-positive patients.

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A level of 19.5% or less of MDR1 can predict 2-year survival with a sensitivity of 86.7% and a specificity of 90%. There was a highly significant positive correlation between the levels of cyclin D1 and MDR1 with an r: 0.802 and P<0.0001 [Table 3].
Table 3: Correlation between the level of cyclin D1 and MDR1 in multiple myeloma

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  Discussion Top


Cyclin D1 belongs to a family of protein kinases that have been implicated in normal cell cycle regulation and in neoplasia 2. The clinical significance of cyclin D1 dysregulation in plasma cell myeloma has been uncertain 4. This study assessed the prognostic value of cyclin D1 expression in patients with MM and its relationship with the expression of MDR1 as an indicator of chemoresistance.

The current study showed that cyclin D1 gene amplification was detected in 20% of patients with myeloma. This is in agreement with the findings of Athanasiou and colleagues, who reported a percentage of 24% for the presence of cyclin D1 protein in MM. Cook et al. 4, reported that 25% of patients with MM showed strong nuclear staining for cyclin D1 protein using immunohistochemical findings. The aforementioned authors added that approximately 15–20% of MM cases are associated with t (11,14)(q13;q32), which juxtaposes CCND1, leading to the expression of cyclin D1 protein.

The cyclin D1 amplification was associated with higher disease severity as shown by increased plasma cell infiltration of the bone marrow and increased numbers of osteolytic lesions. This is in agreement with Hoechtlen-Vollmar et al. 11, Athanasiou et al. 3, Soverini et al. 12, and Cook et al. 4, who reported the association between strong cyclin staining and the increased number of bone marrow plasma cells at diagnosis. Athanasiou et al. 3, also reported a positive correlation between cyclin D1 expression and higher histologic grade. Wang et al. 13 reported that cyclin D1 overexpression promoted cell proliferation, attenuated apoptosis, and enhanced the invasive capacity of glioblastoma. They also reported that cyclin D1 knockdown in human glioblastoma cells inhibited cell proliferation-induced apoptosis and decreased invasive capacity. The current study did not find any correlation between cyclin D1 positivity and LDH, B2-microglobulin, BJP, serum creatinine, or clinical stage. The lack of correlation with these parameters was supported by Hoechtlen-Vollmar et al. 11, Soverini et al. 12, Markovic et al. 14, and Cook et al. 4.

The present study showed that cyclin D1 amplification was associated with shorter OS. This is supported by the results reported by Hoechtlen-Vollmar et al. 11 and Athanasiou et al. 3, who concluded that cyclin D1 expression represents a marker of an unfavorable prognosis in MM. Sonoki et al. 15 reported a 1-year survival of 81.8% for cyclin D1-negative patients with MM and of 63.5% for cyclin D1-positive patients. In contrast, Cook et al. 4, found that cyclin D1-positive cases of myeloma had a significantly longer OS time. However, they reported that this finding did not reach statistical significance. Cook et al. 4 and Kelly et al. 16 concluded that cyclin D1 expression is associated with favorable prognosis in patients with MM. Soverini et al. 12 reported that patients with cyclin D1 rearrangement had a longer survival, both overall and progression free, than others, even though the difference was not statistically significant. Rasmussen et al. 17 and Markovic et al. 14 reported that there is no significant difference in survival between cyclin D1-positive and cyclin D1-negative MM. Kelly et al. 16, denied the poor prognostic effect associated with cyclin D1-negativepatients with myeloma.

Resistance of tumor cells to various cytotoxic drugs is a major impediment to cancer chemotherapy. It may occur through a number of mechanisms, including overexpression of the MDR1 and multidrug resistance protein (MRP) gene products. The present work showed that there is a highly significant positive correlation between the level of cyclin D1 and MDR1. This may raise the possibility that cyclin D1 plays a role in determining the response of myeloma cells to the chemotherapeutic agents. Dawson et al. 18, studied the effect of cyclin D1 expression in response to bortezomib in patients with myeloma and they reported that the expression of nuclear cyclin D1 was associated with a favorable response to bortezomib.

However, it was reported that tumor cell lines expressing higher levels of cyclin D1 showed resistance to cytotoxic drugs compared with cells expressing lower levels. Downregulation of the MDR1/P-gp expression in human breast cancer cells using COX-2 inhibitors was associated with downregulation of cyclin D1 9. Kornmann et al. 2, found that overexpression of cyclin D1 in a human fibrosarcoma cell line has been shown to confer resistance to methotrexate. Conversely, suppression of cyclin D1 levels has been shown to potentiate the response of human pancreatic cancer cells to cisplatinum. They also showed that cyclin D1 suppression decreased MDR1 and MRP mRNA levels. In the same context, Wang et al. 13, reported that cells underexpressing cyclin D1 showed decreased MRP (MDR1).

In conclusion, the present work suggests the association of cyclin D1 gene amplification with higher disease severity, unfavorable prognosis, and expression of MDR1. Downregulation of cyclin D1 expression may represent a new therapeutic modality for cases of MM that may affect the severity of the disease, its prognosis as well as response to chemotherapy. A wide-scale study is required to gain a more accurate and deeper understanding of the role of cyclin D1 amplification on the severity of disease and response to chemotherapy in MM. COX-2 inhibitor as an inhibitor of cyclin D1 that can potentially improve the response to chemotherapy in MM may be the point of interest for further study.[18]

 
  References Top

1.Klein E, Assoian R. Transcriptional regulation of the cyclin D1 gene at a glance. J Cell Sci. 2008;121:3853–3857  Back to cited text no. 1
    
2.Kornmann M, Danenberg KD, Arber N. Inhibition of cyclin D1 expression in human pancreatic cancer cells is associated with increased chemosensitivity and decreased expression of multiple chemoresistance genes. Cancer Res. 1999;59:3505–3511  Back to cited text no. 2
    
3.Athanasiou E, Kaloutsi V, Kotoula V, Hytiroglou P, Kostopoulos I, Zervas C, et al. Cyclin D1 overexpression in multiple myeloma. A morphologic, immunohistochemical, and in situ hybridization study of 71 paraffin-embedded bone marrow biopsy specimens. Am J Clin Pathol. 2001;116:535–542  Back to cited text no. 3
    
4.Cook J, His E, Worley S, Raymond R, Tubbs R, Hussein M. Immunohistochemical analysis identifies two cyclin D1+ subsets of plasma cell myeloma, each associated with favorable survival. Am J Clin Pathol. 2006;125:615–624  Back to cited text no. 4
    
5.Opornosci M, Nowotworow C. Mechanism of resistance to cancer chemotherapy. Adv Clin Exp Med. 2010;19:5–12  Back to cited text no. 5
    
6.Ooi MG Investigation of the intrinsic mechanism of drug resistance in multiple myeloma [thesis]. Dublin City University, Ireland; 2011. 2pp  Back to cited text no. 6
    
7.Kuan Y Effects of polyunsaturated fatty acids on multidrug resistance and DNA methylation in human cancer cell lines [thesis]. Clemson University, Clemson, South Carolina; 2009. 23pp  Back to cited text no. 7
    
8.Nakagawa Y, Abe S, Kurata M, Hasegawa M, Yamamoto K, Inoue M, et al. IAP family protein expression correlates with poor outcome of multiple myeloma patients in association with chemotherapy-induced overexpression of multidrug resistance genes. Am J Hematol. 2006;81:824–831  Back to cited text no. 8
    
9.Kamel M, Salama S, Saleh S, Osman A, Ahmed S, Al-Hendy A. Reversal of doxorubicin resistance by adenovirus-mediated transfer of cyclooxygenase-2 antisense in multidrug-resistant MCF-7 cells. Cancer Ther. 2007;5:1–10  Back to cited text no. 9
    
10.Greipp PR, San Miguel J, Durie BG. International staging system for multiple myeloma. J Clin Oncol. 2005;23:3412–3420  Back to cited text no. 10
    
11.Hoechtlen-Vollmar W, Menzel G, Bartl R, Lamerz R, Wick M, Seidel D. Amplification of cyclin D1 gene in multiple myeloma: clinical and prognostic relevance. Br J Haematol. 2000;109:30–38  Back to cited text no. 11
    
12.Soverini S, Cavo M, Cellini C, Terragna C, Zamagni E, Ruggeri D, et al. Cyclin D1 overexpression is a favorable prognostic variable for newly diagnosed multiple myeloma patients treated with high-dose chemotherapy and single or double autologous transplantation. Blood. 2003;102:1588–1594  Back to cited text no. 12
    
13.Wang J, Wang Q, Cui Y, Liu ZY, Zhao W, Wang CL, et al. Knockdown of cyclin D1 inhibits proliferation, induces apoptosis, and attenuates the invasive capacity of human glioblastoma cells. J Neurooncol. 2012;106:473–484  Back to cited text no. 13
    
14.Markovic O, Marisavaljevic D, Cemerikic V, Suvajdzic N, Milic N, Colovic M. Immunohistochemical analysis of cyclin D1 and p53 in multiple myeloma: relationship to proliferative activity and prognostic significance. Med Oncol. 2004;21:73–80  Back to cited text no. 14
    
15.Sonoki T, Hata H, Kuribayashi N, Yoshida M, Harada N, Nagasaki A, et al. Expression of PRAD1/cyclin D1 in plasma cell malignancy: incidence and prognostic aspects. Br J Haematol. 1999;104:614–617  Back to cited text no. 15
    
16.Kelly TW, Baz R, Hussein M, Karafa M, Cook JR. Clinical significance of cyclin D1, fibroblast growth factor receptor 3, and p53 immunohistochemistry in plasma cell myeloma treated with thalidomide-based regimen. Hum Pathol. 2009;40:405–412  Back to cited text no. 16
    
17.Rasmussen T, Knudsen LM, Johnsen HE. Frequency and prognostic relevance of cyclin D1 dysregulation in multiple myeloma. Eur J Haematol. 2001;67:293–301  Back to cited text no. 17
    
18.Dawson M, Opat S, Taouk Y, Donovan M, Zammit M, Monaghan K, et al. Clinical and Immunohistochemical features associated with a response to bortezomib in patients with multiple myeloma. Clin Cancer Res. 2009;15:714–722  Back to cited text no. 18
    


    Figures

  [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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