- Research article
- Open Access
Increased levels of cyclin D1 negatively impacts on acute lymphoblastic leukemia overall survival
- Received: 20 December 2017
- Accepted: 2 February 2018
- Published: 5 March 2018
Abstract
Background
Cyclin D1 is a protein essential for transition from G1 to S phase during cell cycle progression, which has an oncogenic potential and is highly expressed in several human malignancies. However, in view of the heterogeneity of the findings in the literature, the prognostic value of cyclin D1 expression still needs to be validated in different cohorts of adult acute lymphoblastic leukemia (ALL) patients.
Methods
Bone marrow samples from 13 healthy donors and 45 adult patients with acute lymphoblastic leukemia were included. Cyclin D1 gene expression was evaluated by quantitative PCR. For statistical analysis, Mann–Whitney test, Fisher’s exact test, Chi-squared test and Cox regression were used, as appropriate. All p values were two-sided with a significance level of 5%.
Results
Cyclin D1 mRNA levels were similar between primary cells from ALL patients and healthy donors. In ALL patients, high cyclin D1 expression was associated with older age at the diagnosis, presence of BCR-ABL1, and lower white blood cell counts. Importantly, increased cyclin D1 expression was an independent factor that predicted worse overall survival in our adult ALL cohort.
Conclusion
Increased levels of cyclin D1 negatively impacted on ALL survival outcome, suggesting that this gene is involved in the malignant phenotype of ALL.
Keywords
- Cyclin D1
- CCND1
- Acute lymphoblastic leukemia
- Prognosis
Background
Acute lymphoblastic leukemia (ALL) is a heterogeneous group of neoplasm characterized by aberrant clonal proliferation and accumulation of B or T lymphoid immature cells in hematopoietic tissues, which impairs the normal hematopoiesis [1]. Adult ALL is associated with higher rates of death and relapse [2]. Cyclin D1 (CCND1) is an essential protein in the transition from G1 to S phase during cell cycle progression [3], which has an oncogenic potential and is highly expressed in several human malignancies [3, 4]. In addition to its function in cell cycle control, cyclin D1 plays a role in the gene transcription regulation, cell migration, differentiation and energy balance that contributes to development and maintenance of cancer phenotype [3, 5].
The aim of the present study was to investigate the cyclin D1 (CCND1) mRNA expression and its association with clinical and laboratorial characteristics, and its impact on overall survival in a cohort of adult ALL patients.
Methods
Primary samples
Clinical and laboratory data from acute lymphoblastic leukemia patients
Characteristics | Total patients (n = 45) | Cyclin D1 a | p-value | |
|---|---|---|---|---|
Low expression (n = 20) | High expression (n = 20) | |||
Age, years; median (range) | 27 (18–79) | 22 (18–67) | 38 (18–79) | 0.022 |
Gender | ||||
Male, n (%) | 30 (66.7) | 14 (70) | 13 (65) | |
Female, n (%) | 15 (33.3) | 6 (30) | 7 (35) | 0.49 |
Immunophenotype | ||||
B-ALL, n (%) | 35 (79.5) | 14 (70) | 18 (90) | 0.114 |
T-ALL, n (%) | 9 (20.5) | 6 (30) | 2 (10) | |
BCR-ABL1 positive, n (%) | ||||
Positive, n (%) | 10 (22.2) | 1 (5) | 7 (35) | |
Negative, n (%) | 35 (77.8) | 19 (95) | 13 (65) | 0.018 |
Peripheral blood counts, median (range) | ||||
Hemoglobin, g/dL | 9.1 (5.2–15.8) | 7.9 (5.2–15.2) | 9.3 (5.3–15.8) | 0.235 |
WBC, × 109/L | 12.2 (0.8–549.5) | 23.8 (3.2–549.5) | 8.2 (0.8–61.8) | 0.013 |
Platelets, × 109/L | 38 (4–433) | 29 (4–162) | 38 (7–433) | 0.419 |
LDH, median (range); U/L | 1440 (245–13090) | 2625 (270–13090) | 1410 (245–6848) | 0.09 |
Quantitative PCR (qPCR) analysis
Total RNA was obtained from total nucleated bone marrow cells, after removal of erythrocytes by hemolysis, using TRIzol reagent (Thermo Fisher Scientific; Carlsbad, CA, USA). The cDNA was synthesized from 1 μg of RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative PCR was performed using PowerUp Sybr Green master mix (Thermo Fisher Scientific) in a 7500 Real Time PCR System (Thermo Fisher Scientific) with specific primers for cyclin D1 (CCND1; FW: CTGGGTGTCCTACAAATG; RV: AGCGGTCCAGGT AGTTCAT) and for the reference gene β-actin (ACTB; FW: AGGCCAACCGCAAGAAG; RV: ACAGCCTGGATA GCAACGTACA) using a total of 120 ng of cDNA for each replica for CCND1 and ACTB genes. ACTB was the reference gene. A negative ‘No Template Control’ was included for each primer pair. Two replicas were run on the same plate for each sample. The relative quantification value was calculated using the equation 2-ΔΔCT.[6].
Statistical analysis
Statistical analyses were performed using GraphPad Instat 5 (GraphPad Software Inc., San Diego, CA, USA), Stata Statistic/Data Analysis 14.1 (Stata corporation, USA) and statistical package for the social sciences (SPSS) 19.0. Mann–Whitney test was used for measured factors. Fisher’s exact test or Chi-squared test was used for categorical factors. Univariate and multivariate proportional hazard regression analysis was performed for potential prognostic factors for overall survival using Cox regression. Overall survival was defined from time of sampling to date of death or last seen. Patients included in the study at the time of diagnosis (n = 40) were included in the survival analysis. All p values were two-sided with a significance level of 5%.
Results
Cyclin D1 expression negatively impacts on ALL overall survival
Cyclin D1 expression in primary samples from adult acute lymphoblastic leukemia (ALL) and healthy donors. Quantitative RT-PCR analysis of cyclin D1 (CCND1) mRNA levels in samples from healthy donors and ALL patients (a) and in ALL patients stratified according to BCR-ABL1 status (b) and immunophenotype (c). The ACTB gene was used as the reference gene and a healthy donor was used as a calibrator sample. Horizontal lines indicate medians. The numbers of subjects studied are indicated in the graph. The p-values are indicated; Mann-Whitney test
Univariate and multivariate analyses of overall survival for patients with acute lymphoblastic leukemiaa
Factor | Univariate analysis | Multivariate analysis | ||||
|---|---|---|---|---|---|---|
HRb | (95% C.I.) | p | HRb | (95% C.I.) | p | |
Age | 1.04 | 1.02–1.07 | < 0.001 | 1.04 | 1.02–1.08 | < 0.001 |
BCR-ABL1 (positive vs. negative) | 1.09 | 0.44–2.73 | 0.852 | 0.68 | 0.26–1.76 | 0.431 |
White blood cell count (× 109/L) | 0.998 | 0.995–1.002 | 0.542 | 1.0007 | 0.997–1.004 | 0.702 |
Cyclin D1 expression | 1.07 | 1.01–1.13 | 0.019 | 1.07 | 1.01–1.15 | 0.026 |
Discussion
In the present study, we found no difference between the Cyclin D1 expression in primary cells from ALL patients and healthy donors. Using qPCR and Western blot assays, Wang et al. [7] observed an increased levels of cyclin D1 mRNA and protein expression in peripheral blood or bone marrow mononuclear cells of 60 ALL adult patients compared to 30 healthy donors. In contrast, Taniguchi et al. [8] did not find cyclin D1 overexpression in any of the 11 ALL patients evaluated.
We found that cyclin D1 expression independently predicted overall survival in our adult ALL cohort. Another study observed a positive correlation between cyclin D1 expression and the blast cell count in peripheral blood and bone marrow of ALL patients, but no correlation between cyclin D1 expression and WBC counts, hemoglobin level or platelet counts was found [9]. On the other hand, Elsayed et al. reported that cyclin D1 expression was not associated with response to chemotherapy and overall survival in 78 newly diagnosed adult and pediatric ALL patients [10]. High cyclin D1 mRNA levels had already been associated with poorer prognosis and relapse in childhood ALL [11, 12]. In view of the heterogeneity of the findings in the literature, the evaluation of the prognostic impact of cyclin D1 expression in ALL survival outcomes is still an open field for further investigation.
In our study, we found an association between the expression of cyclin D1 and the presence of BCR-ABL1: 8 out of 40 (20%) patients with ALL at diagnosis presented BCR-ABL1, among these, only one did not present high cyclin D1 expression. BCR-ABL1 is a potent oncogene that induces high rates of proliferation and cell cycle progression [13, 14]. Several studies have reported the relationship between BCR-ABL1 and cyclin D1 expression, mainly in chronic myeloid leukemia [14, 15]. Although BCR-ABL1 is recognized as a marker of poor prognosis in ALL [16, 17], BCR-ABL1 was not a prognostic factor in our cohort; the low number of BCR-ABL1 positive ALL cases probably interfered with the survival analysis.
Conclusion
In conclusion, our data indicates that cyclin D1 mRNA levels were similar between primary cells from ALL patients and healthy donors. Increased levels of cyclin D1 negatively impacted on adult ALL overall survival, suggesting the involvement of this gene in the malignant phenotype of ALL.
Abbreviations
- ACTB:
-
β-actin
- ALL:
-
Acute lymphoblastic leukemia
- B-ALL:
-
Precursor B-acute lymphoblastic leukemia
- BCR-ABL1:
-
Breakpoint cluster region-abelson 1
- CCND1:
-
Cyclin D1
- HR:
-
Hazard ratio
- LDH:
-
Lactic dehydrogenase
- qPCR:
-
Quantitative polymerase chain reaction
- RT-PCR:
-
Reverse transcription polimerase chain reaction
- T-ALL:
-
Precursor T-acute lymphoblastic leukemia
- WBC:
-
White blood cell
Declarations
Acknowledgments
The authors would like to thank Andy Cumming for English review, and Amélia Góes for her valuable technical assistance.
Funding
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo ; grant #2013/26213-0 and # 2013/08135-2, São Paulo Research Foundation (FAPESP).
Availability of data and materials
Please contact author for data requests.
Authors’ contributions
JCF designed the study and experiments, performed all the experiments, manuscript preparation, completion and final approval. APNRA contributed with sample selection, inputs on overall design of study, manuscript editing and final approval. JLC-S performed statistical analysis, contributed with inputs on overall design of study, manuscript editing and final approval. RS-R and BAF contributed with inputs on overall design of study, manuscript editing and final approval. BPS and EMR participated in the interpretation of manuscript data, in manuscript editing and final approval. JAM-N participated in overall design of study and experiments, statistical analyses, manuscript preparation, editing, completion and final approval. FT was the principal investigator and participated in overall design of study and experiments, statistical analyses, manuscript preparation, editing, completion and final approval. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved by the Ethics Committee of the University of São Paulo at Ribeirão Preto Medical School in accordance with the Helsinki Declaration. Written informed consent was obtained from all healthy donors and ALL patients who participated in this study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors’ Affiliations
References
- Paul S, Kantarjian H, Jabbour EJ. Adult acute lymphoblastic leukemia. Mayo Clin Proc. 2016;91(11):1645–66.View ArticlePubMedGoogle Scholar
- Pui CH, Robison LL, Look AT. Acute lymphoblastic leukaemia. Lancet. 2008;371(9617):1030–43.View ArticlePubMedGoogle Scholar
- Hydbring P, Malumbres M, Sicinski P. Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases. Nat Rev Mol Cell Biol. 2016;17(5):280–92.View ArticlePubMedPubMed CentralGoogle Scholar
- Asghar U, Witkiewicz AK, Turner NC, Knudsen ES. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat Rev Drug Discov. 2015;14(2):130–46.View ArticlePubMedPubMed CentralGoogle Scholar
- Casimiro MC, Velasco-Velázquez M, Aguirre-Alvarado C, Pestell RG. Overview of cyclins D1 function in cancer and the CDK inhibitor landscape: past and present. Expert Opin Investig Drugs. 2014;23(3):295–304.View ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25(4):402–8.View ArticlePubMedGoogle Scholar
- Wang CX, Wang X, Liu HB, Zhou ZH. Aberrant DNA methylation and epigenetic inactivation of hMSH2 decrease overall survival of acute lymphoblastic leukemia patients via modulating cell cycle and apoptosis. Asian Pac J Cancer Prev. 2014;15(1):355–62.View ArticlePubMedGoogle Scholar
- Taniguchi T, Fujita A, Takahashi S, Uchimaru K, Yoshikawa M, Asano S, et al. Cyclin D1 overexpression detected by a simple competitive reverse transcription-polymerase chain reaction assay for lymphoid malignancies. Jpn J Cancer Res. 1998;89(2):159–66.View ArticlePubMedGoogle Scholar
- Aref S, Mabed M, El-Sherbiny M, Selim T, Metwaly A. Cyclin D1 expression in acute leukemia. Hematology. 2006;11(1):31–4.View ArticlePubMedGoogle Scholar
- Elsayed GM, Ismail MM, Moneer MM. Expression of P-glycoprotein, Cyclin D1 and Ki-67 in acute lymphoblastic leukemia: relation with induction chemotherapy and overall survival. Indian J Hematol Blood Transfus. 2011;27(3):157–63.View ArticlePubMedPubMed CentralGoogle Scholar
- Sauerbrey A, Häfer R, Zintl F, Volm M. Analysis of cyclin D1 in de novo and relapsed childhood acute lymphoblastic leukemia. Anticancer Res. 1999;19(1B):645–9.PubMedGoogle Scholar
- Volm M, Koomägi R, Stammler G, Rittgen W, Zintl F, Sauerbrey A. Prognostic implications of cyclins (D1, E, A), cyclin-dependent kinases (CDK2, CDK4) and tumor-suppressor genes (pRB, p16INK4A) in childhood acute lymphoblastic leukemia. Int J Cancer. 1997;74(5):508–12.View ArticlePubMedGoogle Scholar
- Druker BJ. Translation of the Philadelphia chromosome into therapy for CML. Blood. 2008;112(13):4808–17.View ArticlePubMedGoogle Scholar
- Afar DE, McLaughlin J, Sherr CJ, Witte ON, Roussel MF. Signaling by ABL oncogenes through cyclin D1. Proc Natl Acad Sci U S A. 1995;92(21):9540–4.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu JH, Yen CC, Lin YC, Gau JP, Yang MH, Chao TC, et al. Overexpression of cyclin D1 in accelerated-phase chronic myeloid leukemia. Leuk Lymphoma. 2004;45(12):2419–25.View ArticlePubMedGoogle Scholar
- Gleissner B, Gokbuget N, Bartram CR, Janssen B, Rieder H, Janssen JW, et al. Leading prognostic relevance of the BCR-ABL translocation in adult acute B-lineage lymphoblastic leukemia: a prospective study of the German Multicenter Trial Group and confirmed polymerase chain reaction analysis. Blood. 2002;99(5):1536–43.View ArticlePubMedGoogle Scholar
- Short NJ, Kantarjian H, Jabbour E, Ravandi F. Which tyrosine kinase inhibitor should we use to treat Philadelphia chromosome-positive acute lymphoblastic leukemia? Best Pract Res Clin Haematol. 2017;30(3):193–200.View ArticlePubMedGoogle Scholar
