- Email: [email protected]
Cyclin D1 Amplification in Multiple Myeloma Is Associated With Multidrug Resistance Expression
Original Study
Cyclin D1 Amplification in Multiple Myeloma Is Associated With Multidrug Resistance Expression Eman M. Sewify,1 Ola A. Afifi,2 Eman Mosad,3 Amen H. Zaki,4 Sahar A. El Gammal2 Abstract In this study, we evaluated cyclin D1 gene amplification using fluorescence in situ hybridization (FISH) and multidrug resistance (MDR1) protein using immunohistochemistry in bone marrow biopsies of 50 patients with multiple myeloma. Cyclin D1-positive patients had a significantly lower progression-free and overall survival and higher levels of MDR1 compared with cyclin D1-negative patients, suggesting an association between cyclin D1 gene amplification and MDR1 unfavorable prognosis. Background: Cyclin D1 is involved in 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 cyclin D1 in multiple myeloma is still a point of debate. This study aimed at evaluating the effect of cyclin D1 gene amplification in multiple myeloma on overall survival and response to therapy. Patients and Methods: Fifty patients with multiple myeloma were retrospectively studied. Cyclin D1 gene amplification was studied in bone marrow biopsies of these patients using FISH. An immunohistochemical study of the bone marrow biopsies was done to detect MDR1 protein expression. The correlations between the cyclin D1 gene amplification and overall survival and MDR1 expression were studied and analyzed statistically. Results: Cyclin D1 gene amplification was found in 20% of myeloma patients and was associated with higher percentage of plasma cell infiltration of the bone marrow and increased 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 highly statistically significant positive correlation with MDR1 levels (R, 0.8 and P < .0001). Conclusion: We suggest that there is an association between cyclin D1 gene amplification and disease severity, unfavorable prognosis, and increased expression of MDR1 in multiple myeloma patients. Clinical Lymphoma, Myeloma & Leukemia, Vol. -, No. -, --- ª 2013 Elsevier Inc. All rights reserved. Keywords: Chemoresistance, FISH, Hematologic malignancies, Immunohistochemistry, Overall survival
Introduction Regulated progression through the cell cycle requires sequential expression of a family of proteins called cyclins.1 Cyclin D1 (CCND1) is a critical modulator of G1 progression.2 Cyclin D1 is encoded by the parathyroid adenomatosis 1, CCND1, or bcl-1 gene on chromosome 11q13, and is involved in normal regulation of the cell cycle and in neoplasia. Overexpression of CCND1 protein 1
Internal Medicine Department, Faculty of Medicine Clinical Pathology Department, Faculty of Medicine Clinical Pathology Department, (Cytogenetic and immunohistochemistry labs), South Egypt Cancer Institute 4 Medical Oncology Department, South Egypt Cancer Institute, Assiut University, Assiut, Egypt 2 3
Submitted: Apr 7, 2013; Revised: Jul 28, 2013; Accepted: Jul 29, 2013 Address for correspondence: Eman Mosad, MD, PhD, Clinical Pathology Department, South Egypt Cancer Institute, Assiut University, Assiut, Egypt Fax: þ2-088-2333342; e-mail contact: [email protected]
2152-2650/$ - see frontmatter ª 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clml.2013.07.008
releases cells from their normal controls when they need to exit from the cell cycle, obstructs their maturation, and promotes transformation to malignant phenotypes.3 Inhibition of CCND1 function markedly attenuates the proliferation of fibroblasts of colon, esophageal, lung, and pancreatic cancer.2 Dysregulation of at least 1 of the cyclin D (CCND) genes (CCND1, CCND2, and/or CCND3) is a common unifying pathogenic event in multiple myeloma (MM). Increased levels of CCND1 in myeloma has been reported by some research workers to be associated with favorable prognosis. Other laboratories, however, have reported unfavorable association with CCND1. Therefore, the clinical significance of CCND1 dysregulation in plasma cell myeloma has been uncertain.4 Cyclin D1 has been correlated with high levels of a subtype of glutathione-S-transferase (GST) in mantle cell lymphoma. An increased level of GST-M might lead to resistance to chemotherapy.5 Clinical drug resistance has always been a major obstacle in the
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Cyclin-D1 and MDR1 in Multiple Myeloma 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 the cell. Of the known pump mechanisms is p-glycoprotein (P-gp; multidrug resistance [MDR1]; adenosine triphosphate binding cassette subfamily B (ABCB1).6 A link between expression of MDR1/P-gp and cellular apoptotic pathways has been shown. For example, cancer cells expressing MDR1/P-gp exhibit resistance to anticancer drugs that trigger apoptosis.7 Clinical studies have established that MDR1 expression occurs in MM patients and there is also clinical evidence of MDR1.8 There is some evidence that CCND1 might be involved in the process of drug resistance. Others said that cyclooxygenase-2 (COX-2) upregulates MDR1/P-gp expression, which confers the 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 CCND1. It was also reported that COX-2 inhibitors are associated with downregulation of MDR1 and CCND1.8 Others said that suppression of CCND1 expression in human pancreatic cancer cells leads to enhanced chemosensitivity to drugs.2 In this work, we aimed to evaluate the prognostic significance of CCND1 gene amplification in MM and if it is correlated with MDR1 expression.
Patients and Methods We analyzed the records of 50 patients with MM, for presenting symptoms, signs, and investigations, in this retrospective study. Special concern for the following variables was offered: age, and clinical stage according to the International Staging System (ISS).9 The presence of osteolytic lesions, bone marrow infiltration with plasma cells, serum protein electrophoresis, serum albumin, the presence of Bence Jones protein in urine, blood urea, serum creatinine, calcium, lactate dehydrogenase (LDH), and serum B2microglobulin levels. Patients received the conventional chemotherapy, MP (melphalan and prednisolone). MP consists of oral melphalan given at 8 mg/m2 on days 1 to 4 and oral prednisolone at 60 mg/m2 on days 1 to 4, every 4 weeks. In our routine laboratory, all patients with MM undergo quantification of serum M-protein and clonal bone marrow plasma cells, and a comprehensive evaluation for end organ damage using standard morphology and CD138 immunohistochemical staining on bone marrow biopsy samples. Increased expression of MDR1 using immunohistochemistry was assessed as a marker of resistance to chemotherapy. 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 detection of CCND1 gene amplification using fluorescence in situ hybridization and for MDR1 expression using immunohistochemical staining.
Fluorescence in Situ Hybridization in Paraffin-Embedded Tissue Sections A 5-mm tissue section of bone marrow biopsy was placed on a
2
-
coated slide (such as Fisher Superfrost, 12-550-15).The section was
Clinical Lymphoma, Myeloma & Leukemia Month 2013
deparaffinized in xylene 3 times for 10 minutes. The slide was dehydrated in 100% ethanol 2 times for 5 minutes at room temperature (RT), and air dried. The slide was incubated in 2 salinesodium citrate (SSC), at 75 C for 10 minutes, then the slide was incubated in 4 mg/mL pepsin (Sigma P-7012, in 0.9% NaCl pH 1.5) for 10 minutes at 37 C. The slide was dipped in H2O, and rinsed in 2 SSC for 2 minutes at RT. The slide was denatured in 70% formamide and 2 SSC at 75 C for 3 to 5 minutes. The slide was quenched in cold (2 C-8 C) 70% ethanol for 2 minutes. The probe and hybridization solution (10 mL) were denatured for 5 minutes at 75 C (manufacturer’s recommended procedure). The Vysis LSI CCND1, CEP 11 probe was added to the denatured tissue sections, then cover slip and seal were added. The slide was incubated at 37 C in a humid chamber overnight. After hybridization, cover slips were gently removed and the slides were washed in a Coplin jar filled with 0.4 standard SSC (pH 7.0) at 72 C for 2 minutes. The slides were transferred to a Coplin jar filled with 2_standard SSC/NP-40 solutions (pH 7.0) at RT for 1 minute. Nuclei were counterstained with a mixture of 1000 ng/mL of 4-,6diamidino-2-phenylindole dihydrochloride and Vectashield antifade (Vector Laboratories, Inc, Burlingame, CA) at a ratio of 1:10. A cover slip was then placed over the hybridization sites.
Microscopy The microscopy and photography were conducted using a Zeiss Axiovert 200 fluorescence microscope fitted with a high resolution Leica CCD camera. Images were processed using a Leica CW4000 imaging system and Karyotyping and FISH software 2003.
Fluorescence in Situ Hybridization Probe The Vysis LSI CCND1 (11q13) SpectrumOrange/CEP 11 SpectrumGreen Probe is a mixture of 2 probes. The CCND1 probe is approximately 300 kb, contains the CCND1 gene, and is labeled in SpectrumOrange. 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 2 orange and 2 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 2 green and multiple orange signals. Other patterns might be observed if additional genetic alterations are present. Two hundred nuclei were evaluated in every patient. All cases were examined for the presence of CCND1 amplification (defined as more CCND1 hybridization signals than copies of chromosome 11) and chromosome 11 duplication.
Immunohistochemistry Immunostaining of P-gp (MDR1) expression was assessed using immunohistochemical staining on paraffin-embedded bone marrow biopsy, using the avidin-biotin complex method. Before staining, the sections were deparaffinized in xylene and rehydrated in graded alcohols, 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
Eman M. Sewify et al phosphate buffer solution. To study P-gp expression, we used the C494 antibody (Dako, Trappes, France), a monoclonal antibody which reacts with an internal epitope located on the C-terminal domain of the P-gp molecule. The antibody was used at 0.28 mg/mL (dilution 1:200) at RT for 15 minutes and P-gp expression was revealed using a catalyzed signal amplification system (Dako). One section from each specimen was stained as a negative control without the first antibody. Intestinal sections were used as positive controls. Criteria for membrane immunoreactivity included staining of the cell membrane.
Quantification of Immunohistochemical Staining The slides were examined by 2 observers. Positive cells were counted on each slide in 10 fields at magnification 200 and the percentages of positive cells were semiquantitatively quantified as follows: 0 (< 1% positive cells); 1 (1%-10%); 2 (11%-50%); and 3 (51%-100%).
Table 1 Clinical and Laboratory Characteristics of Cyclin D1-Positive Patients Compared With Cyclin D1Negative Patients (Mean ± SD) Characteristic Number
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. Univariate analysis of the predictors of OS and progression-free survival was done using the Kaplan-Meier method. Multivariate survival analysis was done for the significant univariates using Cox regression analysis. Receiver operator characteristic (ROC) curve was used to determine the area under the ROC of CCND1 level that can predict 6-month progression-free survival and 2-year OS. Results were considered significant when P < .05. All analyses were performed using the SPSS version 16 software package (SPSS, Chicago, IL).
Results Cyclin D1 amplification-positive cases represented 20% of all involved cases. Table 1 shows the clinical and laboratory characteristics of CCND1-positive patients compared with CCND1negative ones. There was a significantly higher percentage of bone marrow plasma cells and higher chance of having multiple osteolyic lesions in CCND1-positive patients, compared with CCND1negative myeloma patients. However, no difference of statistical significance in the serum levels of LDH, B2-microglobulin, albumin, paraprotein, calcium, creatinine, the presence of Bence-Jones protein in urine or the clinical stage (ISS) at presentation could be detected between CCND1-positive and -negative MM patients. The MDR1-positive patients represented 40% of the myeloma group. The presence of MDR1 was associated with significantly higher levels of plasma cells in the bone marrow at presentation. Kaplan-Meier survival analysis showed that the univariate predictors of poor overall and progression-free survival were positive CCND1 amplification, positive MDR1, B2-microglobulin > 3 mg/L and high serum creatinine level (> 1.5 mg/dL). High serum uric acid was a significant univariate predictor of progression-free survival, but not OS. Bence Jones protein, serum LDH, serum calcium, and albumin levels failed to predict either (Table 2, Figs. 1 and 2). Multivariate Cox regression analysis showed that the independent predictors of OS and progression-free survival were high serum
Cyclin D1 Lve
P
10
40
NS
Cyclin D1, %
40.8 13.0
4.75 2.07
Age, Years
56.6 6.1
59.95 7.38
.003 NS < .0001
Bone Marrow Biopsy Plasma cells < 20%
0
8
Plasma cells 20%-50%
2
30
Plasma cells > 50%
8
2
0
20
< .0001
Osteolytic Lesion Single lesion
Statistical Analysis
Cyclin D1 Dve
Multiple lesions
10
20
520.2 66.76
533.7 168.7
NS
B2-Microglobulin, mg/dL
4.34 1.80
3.735 1.98
NS
Serum Albumin, g/dL
3.02 0.36
3.04 0.35
NS
Serum Calcium, mg/L
9.08 2.1
9.98 1.0
NS
Positive Bence Jones Protein
8 (80%)
14 (35%)
NS
Serum Paraprotein, g/dL
3.9 0.19
3.86 0.21
NS
Serum Creatinine, mg/dL
2.04 0.997
1.775 0.65
NS
LDH, IU/L
Stage
NS
I
0
4
II
6
26
III
4 44 12.94
MDR1, %
10 17.2 8.85
< .0001
P is significant if < .05. Abbreviations: þve ¼ positive; ve ¼ negative; LDH ¼ lactate dehydrogenase; MDR1 ¼ multidrug resistance; NS ¼ not significant.
creatinine (odds ratio [OR], 6.0; 95% confidence interval [CI], 1.328.5; P ¼ .03 and OR, 6.3; 95% CI, 1.2-32.3; P < .03, respectively) and MDR1 (OR, 0.4; 95% CI, 0.2-0.6; P < .0001 and OR, 0.024; 95% CI, 0.002-0.264; P ¼ .002, respectively). Studying CCND1 and MDR1 levels were shown to be helpful in predicting 6-month progression-free survival and 2-year OS (Figs. 3 and 4). There was a positive highly significant correlation between the levels of CCND1 and MDR1 (r ¼ 0.8; P < .0001).
Discussion Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation.2 This study assessed the prognostic value of CCND1 expression in patients with MM and its relationship with the expression of MDR1 as an indicator of chemoresistance.
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Cyclin-D1 and MDR1 in Multiple Myeloma Table 2 Univariate Predictors of OS and PFS Predictor
P
OS
Cyclin D1 Amplification
PFS
.02
Negative
34.3 2.8
Positive
19.8 2.5
6.8 0.8 1.8 0.8 < .0001
MDR1 (Immunohistochemistry)
< .0001
Negative
39.2 2.8
2.3 0.6
Positive
20.1 1.6
8.1 0.7
B2-Microglobulin
.03
.02
3 mg/L
37.4 3.3
7.9 0.8
> 3 mg/L
24.3 1.9
3.6 0.7
Bence Jones Protein
.09
Negative
35.2 2.7
Positive
27.7 3.5
S. Creatinine
.07 7.6 0.9 4.1 0.8
.009
.004
1.5 mg/dL
41.7 3.3
8.7 0.8
> 1.5 mg/dL
25.8 2.6
3.6 0.7
S. Uric Acid
.1
Normal (3.5-7.2 mg/dL)
36.2 3.5
High (> 7.2 mg mg/dL)
27.0 3.3
LDH
.03 7.2 0.9 3.6 0.9
.5
.8
Normal ( 450 IU/L)
35.8 3.5
5.9 0.6
High (> 450 IU/L)
27.1 1.9
6.1 0.9 .7
S. Calcium Normal (8-10.5 mg/L)
30.9 3.3
High (> 10.5 mg/L)
28.6 1.7
P .001
S. Albumin
.9 5.9 0.9 5.0 0.8
.4
.5
Normal (3.5-5.5 g/dL)
36.0 7.8
6.3 1.3
Low (< 3.5 g/dL)
31.7 2.6
5.7 0.8
Values are represented as mean survival SE (months). Abbreviations: LDH ¼ lactate dehydrogenase; MDR1 ¼ multidrug resistance; OS ¼ overall survival; PFS ¼ progression-free survival; S. ¼ serum.
The current study demonstrated that CCND1 gene amplification was detected in 20% of myeloma patients. This is compatible with the findings of Athanasiou et al, who reported a value of 24% for the presence of CCND1 protein in MM.3 Cook et al reported that 25% of MM patients showed strong nuclear staining for CCND1 protein using immunohistochemical findings.4 They added that approximately 15% to 20% of MM cases are associated with t(11,14)(q13;q32), which juxtaposes CCND1 leading to expression of CCND1 protein. The CCND1 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 keeping with other studies3,4,9,10 in which the association between strong cyclin staining and the increased number of bone marrow plasma cells at diagnosis was reported. Athanasiou et al also demonstrated a positive correlation between CCND1 expression and higher histologic grade.3 Others reported that CCND1 overexpression promoted cell proliferation, attenuated apoptosis, and enhanced invasive capacity of glioblastoma. They also demonstrated that CCND1 knockdown in human glioblastoma cells inhibited cell proliferation, induced
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apoptosis, and decreased invasive capacity.11 The current study did not show any correlation between CCND1 positivity and LDH, B2microglobulin, Bence Jones protein, serum creatinine, or clinical stage. The lack of correlation with these parameters was supported by many previous studies.4,9,10,12 The present study showed that CCND1 presentation was associated with shorter OS. This is supported by studies of others in which CCND1 expression represents a marker of unfavorable prognosis in MM.3,9 A 1-year survival of 81.8% for CCND1negative patients with MM and of 63.5% for CCND1-positive patients was reported.13 On the contrary, others found that CCND1-positive cases of myeloma display a significantly longer OS time.4 However, this finding did not reach statistical significance. Cook et al4 and Kelley et al14 concluded that CCND1 expression is associated with favorable prognosis in patients with MM. Soverini et al reported that patients with CCND1 rearrangement had a longer survival both overall and progression-free than others even though the difference was not statistically significant.10 Others suggested that there is no significant difference in survival between CCND1-positive and negative MM.14,15 Kelley et al denied the
Eman M. Sewify et al Figure 1 Kaplan-Meier Survival Curves for the Significant Univariate Predictors of Overall Survival. (A) Cyclin D1 Amplification Gene Using Fluorescence in situ Hybridization; (B) Multidrug Resistance 1 (MDR1) Protein Using Immunohistochemistry; (C) B2-microglobulin; and (D) Serum (S.) Creatinine
bad prognostic effect associated with CCND1-negative myeloma patients.14 Resistance of tumor cells to various cytotoxic drugs is a major impediment to cancer chemotherapy. It might occur via a number of mechanisms, including overexpression of the MDR1 and MDR1 protein (MRP) gene products. The present work showed that there is a highly significant positive correlation between the level of CCND1 and MDR1. This might raise the possibility that CCND1 has a role in determining the response of myeloma cells to the chemotherapeutic agents. Dawson et al studied the effect of CCND1 expression in response to bortezomib in myeloma patients and they reported that expression of nuclear CCND1 was associated with a favorable response to bortezomib.16 However, other studies reported that tumor cell lines expressing higher levels of CCND1 demonstrated resistance to cytotoxic drugs compared with cells expressing lower levels.17,18 Others reported that downregulation of the MDR1/P-gp expression in human breast cancer cells using COX-2 inhibitors was associated
with downregulation of CCND1.19 Kornmann et al showed that overexpression of CCND1 in a human fibrosarcoma cell line has been shown to confer resistance to methotrexate.2 Conversely, suppression of CCND1 levels has been shown to potentiate the response of human pancreatic cancer cells to cisplatinum. They also demonstrated that CCND1 suppression decreased MDR1 and MRP mRNA levels. In the same context,2 Wang et al reported that cells underexpressing CCND1 exhibited decreased MDR1 protein.11
Conclusion The present work suggests the association of CCND1 gene amplification with higher disease severity, unfavorable prognosis, and expression of MDR1. Downregulating CCND1 expression might provide a new therapeutic target for drug design in cases of MM that might affect the severity of the disease, its prognosis, and response to chemotherapy. A wide-scale study is required to achieve more accurate and deeper understanding of the role of CCND1
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Cyclin-D1 and MDR1 in Multiple Myeloma Figure 2 Kaplan-Meier Survival Curves for the Significant Univariate Predictors of Progression-Free Survival. (A) Cyclin D1 Amplification Gene Using Fluorescence in situ Hybridization; (B) Multidrug Resistance 1 (MDR1) Protein Using Immunohistochemistry; (C) B2-microglobulin; and (D) Serum (S.) Creatinine; and (E) Serum Uric Acid
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Eman M. Sewify et al Figure 3 ROC Curve Analysis for Predicting Progression Free Survival at 6 Months. (A) Left Curve Shows ROC Curve of Cyclin D1 Level in Predicting Progression-Free Survival of 6 Months or More (AUC [ 0.783). (B) Right Curve Shows ROC Curve for MDR1 Level in Predicting Progression-Free Survival of 6 Months or More (AUC [ 0.875)
Abbreviations: AUC ¼ area under the curve; MDR1 ¼ multidrug resistance; ROC ¼ receiver operator characteristic.
Figure 4 ROC Curve Analysis for Predicting Progression Free Survival at 2 Years. (A) Left Curve Shows ROC Curve for Cyclin D1 Level in Predicting Survival for More Than 2 Years (AUC [ 0.727). (B) Right Curve Shows ROC Curve for MDR1 Level in Predicting 2-Year Survival (AUC [ 0.87)
Abbreviations: AUC ¼ area under the curve; MDR1 ¼ multidrug resistance; ROC ¼ receiver operator characteristic.
amplification on disease severity and response to chemotherapy in MM. COX-2 inhibitor as an inhibitor of CCND1 that can potentially improve the response to chemotherapy in MM might be the point of interest for further study.
Clinical Practice Points It is already known that dysregulation of at least 1 of the cyclin D
genes (CCND1, CCND2, and/or CCND3) is a common unifying pathogenic event in MM. The clinical significance of CCND1 dysregulation in plasma cell myeloma has been uncertain.
CCND1 has been previously correlated with high levels of GST-
M in mantle cell lymphoma. An increased level of GST-M might lead to resistance to chemotherapy. Clinical drug resistance has always been a major obstacle in the treatment of all types of cancer. This study is the first to detect a significant relationship between the CCND1 amplification gene and MDR1 expression and to show their effect on poor prognosis in MM patients. The coexistence of the CCND1 amplification gene and MDR1 expression in MM patients can be used as a new poor prognostic
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Cyclin-D1 and MDR1 in Multiple Myeloma marker that might, in the future, provide a new therapeutic target for drug design in cases of MM.
Disclosure
9. 10.
The authors have stated that they have no conflicts of interest. 11.
References 1. Klein EA, Assoian RK. Transcriptional regulation of the cyclin D1 gene at a glance. J Cell Sci 2008; 121:3853-7. 2. Kornmann M, Danenberg KD, Arber N, et al. 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: 3505-11. 3. Athanasiou E, Kaloutsi V, Kotoula V, 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; 116:535-42. 4. Cook JR, Hsi ED, Worley S, et al. Immunohistochemical analysis identifies two cyclin D1þ subsets of plasma cell myeloma, each associated with favorable survival. Am J Clin Pathol 2006; 125:615-24. 5. Stefanko E, Wroel E. Mechanism of resistance to cancer chemotherapy. Adv Clin Exp Med 2010; 19:5-12. 6. Ooi MG, Hayden PJ, Kotoula V, et al. Interactions of the Hdm2/p53 and proteasome pathways may enhance the antitumor activity of bortezomib. Clin Cancer Res 2009; 15:7153-60. 7. Cole SP, Chanda ER, Dicke FP, et al. Non-P-glycoprotein-mediated multidrug resistance in a small cell lung cancer cell line: evidence for decreased susceptibility to drug-induced DNA damage and reduced levels of topoisomerase II. Cancer Res 1991; 51:3345-52. 8. Nakagawa Y, Abe S, Kurata M, et al. IAP family protein expression correlates with poor outcome of multiple myeloma patients in association with chemotherapy-
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12. 13. 14. 15. 16. 17.
18. 19.
induced overexpression of multidrug resistance genes. Am J Hematol 2006; 81: 824-31. Hoechtlen-Vollmar W, Menzel G, Bartl R, et al. Amplification of cyclin D1 gene in multiple myeloma: clinical and prognostic relevance. Br J Haematol 2000; 109: 30-8. Soverini S, Cavo M, Cellini C, 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; 102:1588-94. Wang J, Wang Q, Cui Y, et al. Knockdown of cyclin D1 inhibits proliferation, induces apoptosis, and attenuates the invasive capacity of human glioblastoma cells. J Neurooncol 2012; 106:473-84. Markovic O, Marisavljevic D, Cemerikic V, et al. Immunohistochemical analysis of cyclin D1 and p53 in multiple myeloma: relationship to proliferative activity and prognostic significance. Med Oncol 2004; 21:73-80. Sonoki T, Hata H, Kuribayashi N, et al. Expression of PRAD1/cyclin D1 in plasma cell malignancy: incidence and prognostic aspects. Br J Haematol 1999; 104:614-7. Kelley TW, Baz R, Hussein M, et al. Clinical significance of cyclin D1, fibroblast growth factor receptor 3, and p53 immunohistochemistry in plasma cell myeloma treated with a thalidomide-based regimen. Hum Pathol 2009; 40:405-12. Rasmussen T, Knudsen LM, Johnsen HE. Frequency and prognostic relevance of cyclin D1 dysregulation in multiple myeloma. Eur J Haematol 2001; 67:296-301. Dawson MA, Opat SS, Taouk Y, et al. Clinical and immunohistochemical features associated with a response to bortezomib in patients with multiple myeloma. Clin Cancer Res 2009; 15:714-22. Basnayake K, Cheung CK, Sheaff M, et al. Differential progression of renal scarring and determinants of late renal recovery in sustained dialysis dependent acute kidney injury secondary to myeloma kidney. J Clin Pathol 2010; 63: 884-7. Hochhauser D, Schnieders B, Ercikan-Abali E, et al. Effect of cyclin D1 overexpression on drug sensitivity in a human fibrosarcoma cell line. J Natl Cancer Inst 1996; 88:1269-75. Miller B, Patel VA, Sorokin A. Cyclooxygenase-2 rescues rat mesangial cells from apoptosis induced by adriamycin via upregulation of multidrug resistance protein 1 (P-glycoprotein). J Am Soc Nephrol 2006; 17:977-85.
Cyclin D1 Amplification in Multiple Myeloma Is Associated With Multidrug Resistance Expression Eman M. Sewify,1 Ola A. Afifi,2 Eman Mosad,3 Amen H. Zaki,4 Sahar A. El Gammal2 Abstract In this study, we evaluated cyclin D1 gene amplification using fluorescence in situ hybridization (FISH) and multidrug resistance (MDR1) protein using immunohistochemistry in bone marrow biopsies of 50 patients with multiple myeloma. Cyclin D1-positive patients had a significantly lower progression-free and overall survival and higher levels of MDR1 compared with cyclin D1-negative patients, suggesting an association between cyclin D1 gene amplification and MDR1 unfavorable prognosis. Background: Cyclin D1 is involved in 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 cyclin D1 in multiple myeloma is still a point of debate. This study aimed at evaluating the effect of cyclin D1 gene amplification in multiple myeloma on overall survival and response to therapy. Patients and Methods: Fifty patients with multiple myeloma were retrospectively studied. Cyclin D1 gene amplification was studied in bone marrow biopsies of these patients using FISH. An immunohistochemical study of the bone marrow biopsies was done to detect MDR1 protein expression. The correlations between the cyclin D1 gene amplification and overall survival and MDR1 expression were studied and analyzed statistically. Results: Cyclin D1 gene amplification was found in 20% of myeloma patients and was associated with higher percentage of plasma cell infiltration of the bone marrow and increased 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 highly statistically significant positive correlation with MDR1 levels (R, 0.8 and P < .0001). Conclusion: We suggest that there is an association between cyclin D1 gene amplification and disease severity, unfavorable prognosis, and increased expression of MDR1 in multiple myeloma patients. Clinical Lymphoma, Myeloma & Leukemia, Vol. -, No. -, --- ª 2013 Elsevier Inc. All rights reserved. Keywords: Chemoresistance, FISH, Hematologic malignancies, Immunohistochemistry, Overall survival
Introduction Regulated progression through the cell cycle requires sequential expression of a family of proteins called cyclins.1 Cyclin D1 (CCND1) is a critical modulator of G1 progression.2 Cyclin D1 is encoded by the parathyroid adenomatosis 1, CCND1, or bcl-1 gene on chromosome 11q13, and is involved in normal regulation of the cell cycle and in neoplasia. Overexpression of CCND1 protein 1
Internal Medicine Department, Faculty of Medicine Clinical Pathology Department, Faculty of Medicine Clinical Pathology Department, (Cytogenetic and immunohistochemistry labs), South Egypt Cancer Institute 4 Medical Oncology Department, South Egypt Cancer Institute, Assiut University, Assiut, Egypt 2 3
Submitted: Apr 7, 2013; Revised: Jul 28, 2013; Accepted: Jul 29, 2013 Address for correspondence: Eman Mosad, MD, PhD, Clinical Pathology Department, South Egypt Cancer Institute, Assiut University, Assiut, Egypt Fax: þ2-088-2333342; e-mail contact: [email protected]
2152-2650/$ - see frontmatter ª 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clml.2013.07.008
releases cells from their normal controls when they need to exit from the cell cycle, obstructs their maturation, and promotes transformation to malignant phenotypes.3 Inhibition of CCND1 function markedly attenuates the proliferation of fibroblasts of colon, esophageal, lung, and pancreatic cancer.2 Dysregulation of at least 1 of the cyclin D (CCND) genes (CCND1, CCND2, and/or CCND3) is a common unifying pathogenic event in multiple myeloma (MM). Increased levels of CCND1 in myeloma has been reported by some research workers to be associated with favorable prognosis. Other laboratories, however, have reported unfavorable association with CCND1. Therefore, the clinical significance of CCND1 dysregulation in plasma cell myeloma has been uncertain.4 Cyclin D1 has been correlated with high levels of a subtype of glutathione-S-transferase (GST) in mantle cell lymphoma. An increased level of GST-M might lead to resistance to chemotherapy.5 Clinical drug resistance has always been a major obstacle in the
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Cyclin-D1 and MDR1 in Multiple Myeloma 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 the cell. Of the known pump mechanisms is p-glycoprotein (P-gp; multidrug resistance [MDR1]; adenosine triphosphate binding cassette subfamily B (ABCB1).6 A link between expression of MDR1/P-gp and cellular apoptotic pathways has been shown. For example, cancer cells expressing MDR1/P-gp exhibit resistance to anticancer drugs that trigger apoptosis.7 Clinical studies have established that MDR1 expression occurs in MM patients and there is also clinical evidence of MDR1.8 There is some evidence that CCND1 might be involved in the process of drug resistance. Others said that cyclooxygenase-2 (COX-2) upregulates MDR1/P-gp expression, which confers the 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 CCND1. It was also reported that COX-2 inhibitors are associated with downregulation of MDR1 and CCND1.8 Others said that suppression of CCND1 expression in human pancreatic cancer cells leads to enhanced chemosensitivity to drugs.2 In this work, we aimed to evaluate the prognostic significance of CCND1 gene amplification in MM and if it is correlated with MDR1 expression.
Patients and Methods We analyzed the records of 50 patients with MM, for presenting symptoms, signs, and investigations, in this retrospective study. Special concern for the following variables was offered: age, and clinical stage according to the International Staging System (ISS).9 The presence of osteolytic lesions, bone marrow infiltration with plasma cells, serum protein electrophoresis, serum albumin, the presence of Bence Jones protein in urine, blood urea, serum creatinine, calcium, lactate dehydrogenase (LDH), and serum B2microglobulin levels. Patients received the conventional chemotherapy, MP (melphalan and prednisolone). MP consists of oral melphalan given at 8 mg/m2 on days 1 to 4 and oral prednisolone at 60 mg/m2 on days 1 to 4, every 4 weeks. In our routine laboratory, all patients with MM undergo quantification of serum M-protein and clonal bone marrow plasma cells, and a comprehensive evaluation for end organ damage using standard morphology and CD138 immunohistochemical staining on bone marrow biopsy samples. Increased expression of MDR1 using immunohistochemistry was assessed as a marker of resistance to chemotherapy. 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 detection of CCND1 gene amplification using fluorescence in situ hybridization and for MDR1 expression using immunohistochemical staining.
Fluorescence in Situ Hybridization in Paraffin-Embedded Tissue Sections A 5-mm tissue section of bone marrow biopsy was placed on a
2
-
coated slide (such as Fisher Superfrost, 12-550-15).The section was
Clinical Lymphoma, Myeloma & Leukemia Month 2013
deparaffinized in xylene 3 times for 10 minutes. The slide was dehydrated in 100% ethanol 2 times for 5 minutes at room temperature (RT), and air dried. The slide was incubated in 2 salinesodium citrate (SSC), at 75 C for 10 minutes, then the slide was incubated in 4 mg/mL pepsin (Sigma P-7012, in 0.9% NaCl pH 1.5) for 10 minutes at 37 C. The slide was dipped in H2O, and rinsed in 2 SSC for 2 minutes at RT. The slide was denatured in 70% formamide and 2 SSC at 75 C for 3 to 5 minutes. The slide was quenched in cold (2 C-8 C) 70% ethanol for 2 minutes. The probe and hybridization solution (10 mL) were denatured for 5 minutes at 75 C (manufacturer’s recommended procedure). The Vysis LSI CCND1, CEP 11 probe was added to the denatured tissue sections, then cover slip and seal were added. The slide was incubated at 37 C in a humid chamber overnight. After hybridization, cover slips were gently removed and the slides were washed in a Coplin jar filled with 0.4 standard SSC (pH 7.0) at 72 C for 2 minutes. The slides were transferred to a Coplin jar filled with 2_standard SSC/NP-40 solutions (pH 7.0) at RT for 1 minute. Nuclei were counterstained with a mixture of 1000 ng/mL of 4-,6diamidino-2-phenylindole dihydrochloride and Vectashield antifade (Vector Laboratories, Inc, Burlingame, CA) at a ratio of 1:10. A cover slip was then placed over the hybridization sites.
Microscopy The microscopy and photography were conducted using a Zeiss Axiovert 200 fluorescence microscope fitted with a high resolution Leica CCD camera. Images were processed using a Leica CW4000 imaging system and Karyotyping and FISH software 2003.
Fluorescence in Situ Hybridization Probe The Vysis LSI CCND1 (11q13) SpectrumOrange/CEP 11 SpectrumGreen Probe is a mixture of 2 probes. The CCND1 probe is approximately 300 kb, contains the CCND1 gene, and is labeled in SpectrumOrange. 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 2 orange and 2 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 2 green and multiple orange signals. Other patterns might be observed if additional genetic alterations are present. Two hundred nuclei were evaluated in every patient. All cases were examined for the presence of CCND1 amplification (defined as more CCND1 hybridization signals than copies of chromosome 11) and chromosome 11 duplication.
Immunohistochemistry Immunostaining of P-gp (MDR1) expression was assessed using immunohistochemical staining on paraffin-embedded bone marrow biopsy, using the avidin-biotin complex method. Before staining, the sections were deparaffinized in xylene and rehydrated in graded alcohols, 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
Eman M. Sewify et al phosphate buffer solution. To study P-gp expression, we used the C494 antibody (Dako, Trappes, France), a monoclonal antibody which reacts with an internal epitope located on the C-terminal domain of the P-gp molecule. The antibody was used at 0.28 mg/mL (dilution 1:200) at RT for 15 minutes and P-gp expression was revealed using a catalyzed signal amplification system (Dako). One section from each specimen was stained as a negative control without the first antibody. Intestinal sections were used as positive controls. Criteria for membrane immunoreactivity included staining of the cell membrane.
Quantification of Immunohistochemical Staining The slides were examined by 2 observers. Positive cells were counted on each slide in 10 fields at magnification 200 and the percentages of positive cells were semiquantitatively quantified as follows: 0 (< 1% positive cells); 1 (1%-10%); 2 (11%-50%); and 3 (51%-100%).
Table 1 Clinical and Laboratory Characteristics of Cyclin D1-Positive Patients Compared With Cyclin D1Negative Patients (Mean ± SD) Characteristic Number
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. Univariate analysis of the predictors of OS and progression-free survival was done using the Kaplan-Meier method. Multivariate survival analysis was done for the significant univariates using Cox regression analysis. Receiver operator characteristic (ROC) curve was used to determine the area under the ROC of CCND1 level that can predict 6-month progression-free survival and 2-year OS. Results were considered significant when P < .05. All analyses were performed using the SPSS version 16 software package (SPSS, Chicago, IL).
Results Cyclin D1 amplification-positive cases represented 20% of all involved cases. Table 1 shows the clinical and laboratory characteristics of CCND1-positive patients compared with CCND1negative ones. There was a significantly higher percentage of bone marrow plasma cells and higher chance of having multiple osteolyic lesions in CCND1-positive patients, compared with CCND1negative myeloma patients. However, no difference of statistical significance in the serum levels of LDH, B2-microglobulin, albumin, paraprotein, calcium, creatinine, the presence of Bence-Jones protein in urine or the clinical stage (ISS) at presentation could be detected between CCND1-positive and -negative MM patients. The MDR1-positive patients represented 40% of the myeloma group. The presence of MDR1 was associated with significantly higher levels of plasma cells in the bone marrow at presentation. Kaplan-Meier survival analysis showed that the univariate predictors of poor overall and progression-free survival were positive CCND1 amplification, positive MDR1, B2-microglobulin > 3 mg/L and high serum creatinine level (> 1.5 mg/dL). High serum uric acid was a significant univariate predictor of progression-free survival, but not OS. Bence Jones protein, serum LDH, serum calcium, and albumin levels failed to predict either (Table 2, Figs. 1 and 2). Multivariate Cox regression analysis showed that the independent predictors of OS and progression-free survival were high serum
Cyclin D1 Lve
P
10
40
NS
Cyclin D1, %
40.8 13.0
4.75 2.07
Age, Years
56.6 6.1
59.95 7.38
.003 NS < .0001
Bone Marrow Biopsy Plasma cells < 20%
0
8
Plasma cells 20%-50%
2
30
Plasma cells > 50%
8
2
0
20
< .0001
Osteolytic Lesion Single lesion
Statistical Analysis
Cyclin D1 Dve
Multiple lesions
10
20
520.2 66.76
533.7 168.7
NS
B2-Microglobulin, mg/dL
4.34 1.80
3.735 1.98
NS
Serum Albumin, g/dL
3.02 0.36
3.04 0.35
NS
Serum Calcium, mg/L
9.08 2.1
9.98 1.0
NS
Positive Bence Jones Protein
8 (80%)
14 (35%)
NS
Serum Paraprotein, g/dL
3.9 0.19
3.86 0.21
NS
Serum Creatinine, mg/dL
2.04 0.997
1.775 0.65
NS
LDH, IU/L
Stage
NS
I
0
4
II
6
26
III
4 44 12.94
MDR1, %
10 17.2 8.85
< .0001
P is significant if < .05. Abbreviations: þve ¼ positive; ve ¼ negative; LDH ¼ lactate dehydrogenase; MDR1 ¼ multidrug resistance; NS ¼ not significant.
creatinine (odds ratio [OR], 6.0; 95% confidence interval [CI], 1.328.5; P ¼ .03 and OR, 6.3; 95% CI, 1.2-32.3; P < .03, respectively) and MDR1 (OR, 0.4; 95% CI, 0.2-0.6; P < .0001 and OR, 0.024; 95% CI, 0.002-0.264; P ¼ .002, respectively). Studying CCND1 and MDR1 levels were shown to be helpful in predicting 6-month progression-free survival and 2-year OS (Figs. 3 and 4). There was a positive highly significant correlation between the levels of CCND1 and MDR1 (r ¼ 0.8; P < .0001).
Discussion Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation.2 This study assessed the prognostic value of CCND1 expression in patients with MM and its relationship with the expression of MDR1 as an indicator of chemoresistance.
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Cyclin-D1 and MDR1 in Multiple Myeloma Table 2 Univariate Predictors of OS and PFS Predictor
P
OS
Cyclin D1 Amplification
PFS
.02
Negative
34.3 2.8
Positive
19.8 2.5
6.8 0.8 1.8 0.8 < .0001
MDR1 (Immunohistochemistry)
< .0001
Negative
39.2 2.8
2.3 0.6
Positive
20.1 1.6
8.1 0.7
B2-Microglobulin
.03
.02
3 mg/L
37.4 3.3
7.9 0.8
> 3 mg/L
24.3 1.9
3.6 0.7
Bence Jones Protein
.09
Negative
35.2 2.7
Positive
27.7 3.5
S. Creatinine
.07 7.6 0.9 4.1 0.8
.009
.004
1.5 mg/dL
41.7 3.3
8.7 0.8
> 1.5 mg/dL
25.8 2.6
3.6 0.7
S. Uric Acid
.1
Normal (3.5-7.2 mg/dL)
36.2 3.5
High (> 7.2 mg mg/dL)
27.0 3.3
LDH
.03 7.2 0.9 3.6 0.9
.5
.8
Normal ( 450 IU/L)
35.8 3.5
5.9 0.6
High (> 450 IU/L)
27.1 1.9
6.1 0.9 .7
S. Calcium Normal (8-10.5 mg/L)
30.9 3.3
High (> 10.5 mg/L)
28.6 1.7
P .001
S. Albumin
.9 5.9 0.9 5.0 0.8
.4
.5
Normal (3.5-5.5 g/dL)
36.0 7.8
6.3 1.3
Low (< 3.5 g/dL)
31.7 2.6
5.7 0.8
Values are represented as mean survival SE (months). Abbreviations: LDH ¼ lactate dehydrogenase; MDR1 ¼ multidrug resistance; OS ¼ overall survival; PFS ¼ progression-free survival; S. ¼ serum.
The current study demonstrated that CCND1 gene amplification was detected in 20% of myeloma patients. This is compatible with the findings of Athanasiou et al, who reported a value of 24% for the presence of CCND1 protein in MM.3 Cook et al reported that 25% of MM patients showed strong nuclear staining for CCND1 protein using immunohistochemical findings.4 They added that approximately 15% to 20% of MM cases are associated with t(11,14)(q13;q32), which juxtaposes CCND1 leading to expression of CCND1 protein. The CCND1 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 keeping with other studies3,4,9,10 in which the association between strong cyclin staining and the increased number of bone marrow plasma cells at diagnosis was reported. Athanasiou et al also demonstrated a positive correlation between CCND1 expression and higher histologic grade.3 Others reported that CCND1 overexpression promoted cell proliferation, attenuated apoptosis, and enhanced invasive capacity of glioblastoma. They also demonstrated that CCND1 knockdown in human glioblastoma cells inhibited cell proliferation, induced
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apoptosis, and decreased invasive capacity.11 The current study did not show any correlation between CCND1 positivity and LDH, B2microglobulin, Bence Jones protein, serum creatinine, or clinical stage. The lack of correlation with these parameters was supported by many previous studies.4,9,10,12 The present study showed that CCND1 presentation was associated with shorter OS. This is supported by studies of others in which CCND1 expression represents a marker of unfavorable prognosis in MM.3,9 A 1-year survival of 81.8% for CCND1negative patients with MM and of 63.5% for CCND1-positive patients was reported.13 On the contrary, others found that CCND1-positive cases of myeloma display a significantly longer OS time.4 However, this finding did not reach statistical significance. Cook et al4 and Kelley et al14 concluded that CCND1 expression is associated with favorable prognosis in patients with MM. Soverini et al reported that patients with CCND1 rearrangement had a longer survival both overall and progression-free than others even though the difference was not statistically significant.10 Others suggested that there is no significant difference in survival between CCND1-positive and negative MM.14,15 Kelley et al denied the
Eman M. Sewify et al Figure 1 Kaplan-Meier Survival Curves for the Significant Univariate Predictors of Overall Survival. (A) Cyclin D1 Amplification Gene Using Fluorescence in situ Hybridization; (B) Multidrug Resistance 1 (MDR1) Protein Using Immunohistochemistry; (C) B2-microglobulin; and (D) Serum (S.) Creatinine
bad prognostic effect associated with CCND1-negative myeloma patients.14 Resistance of tumor cells to various cytotoxic drugs is a major impediment to cancer chemotherapy. It might occur via a number of mechanisms, including overexpression of the MDR1 and MDR1 protein (MRP) gene products. The present work showed that there is a highly significant positive correlation between the level of CCND1 and MDR1. This might raise the possibility that CCND1 has a role in determining the response of myeloma cells to the chemotherapeutic agents. Dawson et al studied the effect of CCND1 expression in response to bortezomib in myeloma patients and they reported that expression of nuclear CCND1 was associated with a favorable response to bortezomib.16 However, other studies reported that tumor cell lines expressing higher levels of CCND1 demonstrated resistance to cytotoxic drugs compared with cells expressing lower levels.17,18 Others reported that downregulation of the MDR1/P-gp expression in human breast cancer cells using COX-2 inhibitors was associated
with downregulation of CCND1.19 Kornmann et al showed that overexpression of CCND1 in a human fibrosarcoma cell line has been shown to confer resistance to methotrexate.2 Conversely, suppression of CCND1 levels has been shown to potentiate the response of human pancreatic cancer cells to cisplatinum. They also demonstrated that CCND1 suppression decreased MDR1 and MRP mRNA levels. In the same context,2 Wang et al reported that cells underexpressing CCND1 exhibited decreased MDR1 protein.11
Conclusion The present work suggests the association of CCND1 gene amplification with higher disease severity, unfavorable prognosis, and expression of MDR1. Downregulating CCND1 expression might provide a new therapeutic target for drug design in cases of MM that might affect the severity of the disease, its prognosis, and response to chemotherapy. A wide-scale study is required to achieve more accurate and deeper understanding of the role of CCND1
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Cyclin-D1 and MDR1 in Multiple Myeloma Figure 2 Kaplan-Meier Survival Curves for the Significant Univariate Predictors of Progression-Free Survival. (A) Cyclin D1 Amplification Gene Using Fluorescence in situ Hybridization; (B) Multidrug Resistance 1 (MDR1) Protein Using Immunohistochemistry; (C) B2-microglobulin; and (D) Serum (S.) Creatinine; and (E) Serum Uric Acid
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Eman M. Sewify et al Figure 3 ROC Curve Analysis for Predicting Progression Free Survival at 6 Months. (A) Left Curve Shows ROC Curve of Cyclin D1 Level in Predicting Progression-Free Survival of 6 Months or More (AUC [ 0.783). (B) Right Curve Shows ROC Curve for MDR1 Level in Predicting Progression-Free Survival of 6 Months or More (AUC [ 0.875)
Abbreviations: AUC ¼ area under the curve; MDR1 ¼ multidrug resistance; ROC ¼ receiver operator characteristic.
Figure 4 ROC Curve Analysis for Predicting Progression Free Survival at 2 Years. (A) Left Curve Shows ROC Curve for Cyclin D1 Level in Predicting Survival for More Than 2 Years (AUC [ 0.727). (B) Right Curve Shows ROC Curve for MDR1 Level in Predicting 2-Year Survival (AUC [ 0.87)
Abbreviations: AUC ¼ area under the curve; MDR1 ¼ multidrug resistance; ROC ¼ receiver operator characteristic.
amplification on disease severity and response to chemotherapy in MM. COX-2 inhibitor as an inhibitor of CCND1 that can potentially improve the response to chemotherapy in MM might be the point of interest for further study.
Clinical Practice Points It is already known that dysregulation of at least 1 of the cyclin D
genes (CCND1, CCND2, and/or CCND3) is a common unifying pathogenic event in MM. The clinical significance of CCND1 dysregulation in plasma cell myeloma has been uncertain.
CCND1 has been previously correlated with high levels of GST-
M in mantle cell lymphoma. An increased level of GST-M might lead to resistance to chemotherapy. Clinical drug resistance has always been a major obstacle in the treatment of all types of cancer. This study is the first to detect a significant relationship between the CCND1 amplification gene and MDR1 expression and to show their effect on poor prognosis in MM patients. The coexistence of the CCND1 amplification gene and MDR1 expression in MM patients can be used as a new poor prognostic
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Cyclin-D1 and MDR1 in Multiple Myeloma marker that might, in the future, provide a new therapeutic target for drug design in cases of MM.
Disclosure
9. 10.
The authors have stated that they have no conflicts of interest. 11.
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induced overexpression of multidrug resistance genes. Am J Hematol 2006; 81: 824-31. Hoechtlen-Vollmar W, Menzel G, Bartl R, et al. Amplification of cyclin D1 gene in multiple myeloma: clinical and prognostic relevance. Br J Haematol 2000; 109: 30-8. Soverini S, Cavo M, Cellini C, 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; 102:1588-94. Wang J, Wang Q, Cui Y, et al. Knockdown of cyclin D1 inhibits proliferation, induces apoptosis, and attenuates the invasive capacity of human glioblastoma cells. J Neurooncol 2012; 106:473-84. Markovic O, Marisavljevic D, Cemerikic V, et al. Immunohistochemical analysis of cyclin D1 and p53 in multiple myeloma: relationship to proliferative activity and prognostic significance. Med Oncol 2004; 21:73-80. Sonoki T, Hata H, Kuribayashi N, et al. Expression of PRAD1/cyclin D1 in plasma cell malignancy: incidence and prognostic aspects. Br J Haematol 1999; 104:614-7. Kelley TW, Baz R, Hussein M, et al. Clinical significance of cyclin D1, fibroblast growth factor receptor 3, and p53 immunohistochemistry in plasma cell myeloma treated with a thalidomide-based regimen. Hum Pathol 2009; 40:405-12. Rasmussen T, Knudsen LM, Johnsen HE. Frequency and prognostic relevance of cyclin D1 dysregulation in multiple myeloma. Eur J Haematol 2001; 67:296-301. Dawson MA, Opat SS, Taouk Y, et al. Clinical and immunohistochemical features associated with a response to bortezomib in patients with multiple myeloma. Clin Cancer Res 2009; 15:714-22. Basnayake K, Cheung CK, Sheaff M, et al. Differential progression of renal scarring and determinants of late renal recovery in sustained dialysis dependent acute kidney injury secondary to myeloma kidney. J Clin Pathol 2010; 63: 884-7. Hochhauser D, Schnieders B, Ercikan-Abali E, et al. Effect of cyclin D1 overexpression on drug sensitivity in a human fibrosarcoma cell line. J Natl Cancer Inst 1996; 88:1269-75. Miller B, Patel VA, Sorokin A. Cyclooxygenase-2 rescues rat mesangial cells from apoptosis induced by adriamycin via upregulation of multidrug resistance protein 1 (P-glycoprotein). J Am Soc Nephrol 2006; 17:977-85.