Cancer Res Treat.  2017 Jul;49(3):790-797. 10.4143/crt.2016.108.

p15(Ink4b) Loss of Expression by Promoter Hypermethylation Adds to Leukemogenesis and Confers a Poor Prognosis in Acute Promyelocytic Leukemia Patients

• Baba SM
• Azad NA
• Shah ZA
• Dil-Afroze
• Pandith AA
• Jan A
• Aziz SA

• KMID: 2388324
• DOI: http://doi.org/10.4143/crt.2016.108

Abstract

PURPOSE
The p15(Ink4b) gene exerts its influence as an inhibitor of cyclin-dependent kinases and is frequently associated with hematological malignancies. Inactivation of this gene through DNA methylation has been found to be the most prevalent epigenetic alteration reported, with a high frequency in all French-American-British subtypes of acute myeloid leukemias, including acute promyelocytic leukemia (APL). In this study,we investigated the prognostic significance of p15 gene promoter hypermethylation and its expression in APL patients of Kashmir (North India).
MATERIALS AND METHODS
p15 gene promoter hypermethylation was conducted by methylation-specific polymerase chain reaction, while its subsequent expression analysis was carried out by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR).
RESULTS
Of the 37 patients, 16 (43.2%) were found to have methylated p15 genes. Of these 16 cases, seven (43.8%) were methylated partially and nine (56.2%) were found to have complete methylation. Moreover, nine of the 37 patients (24.3%) who presented with leukocytosis at their baseline had complete p15 gene methylation as well (p < 0.05). Semiquantitative RT-PCR showed a complete loss of p15 expression in nine patients with complete methylation coupled with leukocytosis (p=0.031), while seven patients with partial methylation showed decreased p15 expression. Six patients relapsed during the maintenance phase of treatment and were found to have a completely methylated p15 gene and no p15 mRNA.
CONCLUSION
Complete methylation and loss of p15 gene expression causes susceptibility to relapse and decreased survival in APL patients. Thus, p15 promoter hypermethylation is a prospective prognostic indicator and a reliable clinical aid in assessment of patients with APL.

Keyword

p15(Ink4b); Acute promyelocytic leukemia; Arsenic tri-oxide; Leukocytosis; Promoter hypermethylation; Kashmir

MeSH Terms

Cyclin-Dependent Kinases
DNA Methylation
Epigenomics
Gene Expression
Hematologic Neoplasms
Humans
Leukemia, Myeloid, Acute
Leukemia, Promyelocytic, Acute*
Leukocytosis
Methylation
Polymerase Chain Reaction
Prognosis*
Prospective Studies
Recurrence
Reverse Transcriptase Polymerase Chain Reaction
RNA, Messenger
Cyclin-Dependent Kinases
RNA, Messenger

Figure

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  Fig. 1. Representative pic of methylation-specific polymerase chain reaction showing different patterns of p15 gene methylation in APL patients. Lane 1, molecular weight markers (MW, 50 bp); lanes 2 and 3, unmethylated p15 gene (U, 154 bp); lanes 4 and 5, partial methylated p15 showing both 154 and 148 bp bands (U/M); lanes 6 and 7, methylated p15 gene (M, 148 bp); lane 8, positive control for methylated DNA (PC, 148 bp); lane 9, negative control without template DNA (NC).

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  Fig. 2. Kaplan-Meier overall survival (A) and disease-free survival (B) plots of acute promyelocytic leukemia patients according to the methylation status of the p15 promoter region.

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  Fig. 3. Kaplan-Meier overall survival (A) and disease-free survival (B) plots of acute promyelocytic leukemia patients according to p15 mRNA expression.

Reference

References

1. Di Croce L, Raker VA, Corsaro M, Fazi F, Fanelli M, Faretta M, et al. Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science. 2002; 295:1079–82.

2. Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature. 1994; 368:753–6.

3. Teofili L, Martini M, Di Mario A, Rutella S, Urbano R, Luongo M, et al. Expression of p15(ink4b) gene during megakaryocytic differentiation of normal and myelodysplastic hematopoietic progenitors. Blood. 2001; 98:495–7.

4. Bergh G, Ehinger M, Olsson I, Jacobsen SE, Gullberg U. Involvement of the retinoblastoma protein in monocytic and neutrophilic lineage commitment of human bone marrow progenitor cells. Blood. 1999; 94:1971–8.

5. Haviernik P, Schmidt M, Hu X, Wolff L. Consistent inactivation of p19(Arf) but not p15(Ink4b) in murine myeloid cells transformed in vivo by deregulated c-Myc. Oncogene. 2003; 22:1600–10.

6. Teofili L, Morosetti R, Martini M, Urbano R, Putzulu R, Rutella S, et al. Expression of cyclin-dependent kinase inhibitor p15(INK4B) during normal and leukemic myeloid differentiation. Exp Hematol. 2000; 28:519–26.

7. Aggerholm A, Guldberg P, Hokland M, Hokland P. Extensive intra- and interindividual heterogeneity of p15INK4B methylation in acute myeloid leukemia. Cancer Res. 1999; 59:436–41.

8. Rush LJ, Plass C. Alterations of DNA methylation in hematologic malignancies. Cancer Lett. 2002; 185:1–12.

9. Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al. Proposed revised criteria for the classification of acute myeloid leukemia: a report of the French-American-British Cooperative Group. Ann Intern Med. 1985; 103:620–5.

10. Chim CS, Wong AS, Kwong YL. Epigenetic inactivation of INK4/CDK/RB cell cycle pathway in acute leukemias. Ann Hematol. 2003; 82:738–42.

11. Drexler HG. Review of alterations of the cyclin-dependent kinase inhibitor INK4 family genes p15, p16, p18 and p19 in human leukemia-lymphoma cells. Leukemia. 1998; 12:845–59.

12. Herman JG, Jen J, Merlo A, Baylin SB. Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B. Cancer Res. 1996; 56:722–7.

13. Issa JP, Baylin SB, Herman JG. DNA methylation changes in hematologic malignancies: biologic and clinical implications. Leukemia. 1997; 11 Suppl 1:S7–11.

14. Teofili L, Martini M, Luongo M, Diverio D, Capelli G, Breccia M, et al. Hypermethylation of GpG islands in the promoter region of p15(INK4b) in acute promyelocytic leukemia represses p15(INK4b) expression and correlates with poor prognosis. Leukemia. 2003; 17:919–24.

15. Chim CS, Wong SY, Kwong YL. Aberrant gene promoter methylation in acute promyelocytic leukaemia: profile and prognostic significance. Br J Haematol. 2003; 122:571–8.

16. Vardiman JW, Harris NL, Brunning RD. The World Health Organization (WHO) classification of the myeloid neoplasms. Blood. 2002; 100:2292–302.

17. Trost D, Hildebrandt B, Beier M, Muller N, Germing U, Royer-Pokora B. Molecular cytogenetic profiling of complex karyotypes in primary myelodysplastic syndromes and acute myeloid leukemia. Cancer Genet Cytogenet. 2006; 165:51–63.

18. Asou N, Adachi K, Tamura J, Kanamaru A, Kageyama S, Hiraoka A, et al. Analysis of prognostic factors in newly diagnosed acute promyelocytic leukemia treated with all-trans retinoic acid and chemotherapy. Japan Adult Leukemia Study Group. J Clin Oncol. 1998; 16:78–85.

19. Ferrara F, Morabito F, Martino B, Specchia G, Liso V, Nobile F, et al. CD56 expression is an indicator of poor clinical outcome in patients with acute promyelocytic leukemia treated with simultaneous all-trans-retinoic acid and chemotherapy. J Clin Oncol. 2000; 18:1295–300.

20. Preisler HD, Li B, Chen H, Fisher L, Nayini J, Raza A, et al. P15INK4B gene methylation and expression in normal, myelodysplastic, and acute myelogenous leukemia cells and in the marrow cells of cured lymphoma patients. Leukemia. 2001; 15:1589–95.

21. Shimamoto T, Ohyashiki JH, Ohyashiki K. Methylation of p15(INK4b) and E-cadherin genes is independently correlated with poor prognosis in acute myeloid leukemia. Leuk Res. 2005; 29:653–9.

22. Deneberg S, Grovdal M, Karimi M, Jansson M, Nahi H, Corbacioglu A, et al. Gene-specific and global methylation patterns predict outcome in patients with acute myeloid leukemia. Leukemia. 2010; 24:932–41.