Journal List > Korean J Cytopathol > v.19(2) > 1006516

Korean J Cytopathol. 2008 Sep;19(2):126-135. English.
Published online September 30, 2008.
Copyright © 2008 The Korean Society for Cytopathology
Methylation Abnormality in Body Fluid Cytology: A Supplemental Molecular Marker for the Diagnosis of Malignant Mesothelioma
Joon Seon Song, M.D., Jin Kyung Jung, M.S., Ji Hye Kang, M.S., Ilseon Hwang, M.D. and Se Jin Jang, M.D., Ph.D.
Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea.
Received July 16, 2008; Accepted August 26, 2008.


Malignant mesothelioma (MM) is a highly lethal neoplasm arising in pleura and the peritoneum and a rapid and accurate diagnosis is crucial for treatment of the disease. However, the sensitivity of cytological analysis using pleural or ascitic fluid is relatively low, yielding an accurate diagnosis in only 32~79% of cases. We tested the diagnostic value of epigenetic alterations in body fluid cytology as a supplement to conventional methods. Paraffin-embedded tissue blocks from 21 MM patients and associated body fluid cytology slides considered no evidence of malignancy were used to test for epigenetic alteration. Using methylation-specific PCR, we detected methylation of RASSF1A and p16 in 47.6% (10/21) of both surgically resected tumor samples, respectively. Body fluid samples of MM also showed abnormal methylation of RASSF1A and p16INK4a genes in 38.1% (8/21) and 33.3% (7/21) of cases. The concordance in the rates of RASSF1A and p16INK4a gene-methylation abnormalities determined from cytology samples and tissue samples were 61.9% (13/21) and 66.7% (14/21), respectively. Combining both genes increases the sensitivity of the test to 57.1% (12 of 21) of cases. Our results suggest that testing for methylation abnormalities in selected individual genes or gene combinations has diagnostic value as an alternative or adjunct method to conventional cytological diagnosis.

Keywords: Mesothelioma; Methylation; p16 INK4a; RASSF1A; Body Fluid


Fig. 1
A. Epithelioid pleural malignant mesothelioma (MM) exhibits tubulopapillary profiles of polygonal cells (patient 8). B. Cytologic specimen of epithelioid pleural MM scored as negative for malignancy and reactive mesothelial hyperplasia. C. An example of sarcomatoid mesothelioma (patient 13) showing atypical spindle cells arranged in fascicles. D. Cytologic specimens from a sarcomatoid pleural MM patient show reactive mesothelial cells. (A,C : H&E, B,D : Papanicolaou stain).
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Fig. 2
Examples of representative MSP analyses of malignant mesothelioma patients, NSCLC controls and inflammatory non-neoplastic controls. Products were amplified with primers that recognize methylated and unmethylated sequences, respectively.
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Table 1
Clinocopathologic characteristics of patients with malignant mesothelioma and methylation patterns of p16 and RASSF1A genes
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Table 2
Clinocopathologic characteristics of patients with inflammatory/ non-cancer body fluid and Non-small cell lung cancer
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Table 3
PCR primer sequences and PCR product sizes used for MSP
Click for larger image


This work was supported by KOSEF research grant R01-2004-000-10670-0.

1. Antman KH, Schiff PB, Pass HI. In: Cancer: Principles and Practice of Oncology. 5th edition. Philadelphia: Lippincott; 1997. pp. 1853-1878.
2. DiBonito L, Falconieri G, Colautti I, Bonifacio Gori D, Dudine S, Giarelli L. Cytopathology of malignant mesothelioma: a study of its patterns and histological bases. Diagn Cytopathol 1993;9:25–31.
3. Renshaw AA, Dean BR, Antman KH, Sugarbaker DJ, Cibas ES. The role of cytologic evaluation of pleural fluid in the diagnosis of malignant mesothelioma. Chest 1997;111:106–109.
4. Teirstein AS. Diagnosing malignant pleural mesothelioma. Chest 1998;114:666–667.
5. Whitaker D, Shilkin KB. In: Gray W, editor. Mesotheliomas. 2nd ed. New York: Churchill Livingstone; 1995. pp. 195-224.
6. Whitaker D, Shilkin KB. In: Early diagnosis of malignant mesothelioma: the contribution of effusion and fine needle aspiration cytology and ancillary techniques. New York: Garland Law Publishing; 1989. pp. 71-115.
7. Tsou JA, Hagen JA, Carpenter CL, Laird-Offringa IA. DNA methylation analysis: a powerful new tool for lung cancer diagnosis. Oncogene 2002;21:5450–5461.
8. Marsit CJ, Houseman EA, Christensen BC, et al. Examination of a CpG island methylator phenotype and implications of methylation profiles in solid tumors. Cancer Res 2006;66:10621–10629.
9. Jair KW, Bachman KE, Suzuki H, et al. De novo CpG island methylation in human cancer cells. Cancer Res 2006;66:682–692.
10. Fujiwara K, Fujimoto N, Tabata M, et al. Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer. Clin Cancer Res 2005;11:1219–1225.
11. Wang J, Lee JJ, Wang L, et al. Value of p16INK4a and RASSF1A promoter hypermethylation in prognosis of patients with resectable non-small cell lung cancer. Clin Cancer Res 2004;10:6119–6125.
12. Toyooka S, Suzuki M, Maruyama R, et al. The relationship between aberrant methylation and survival in non-small-cell lung cancers. Br J Cancer 2004;91:771–774.
13. Hirao T, Bueno R, Chen CJ, Gordon GJ, Heilig E, Kelsey KT. Alterations of the p16(INK4) locus in human malignant mesothelial tumors. Carcinogenesis 2002;23:1127–1130.
14. Wong L, Zhou J, Anderson D, Kratzke RA. Inactivation of p16INK4a expression in malignant mesothelioma by methylation. Lung Cancer 2002;38:131–136.
15. Toyooka S, Carbone M, Toyooka KO, et al. Progressive aberrant methylation of the RASSF1A gene in simian virus 40 infected human mesothelial cells. Oncogene 2002;21:4340–4344.
16. Toyooka S, Pass HI, Shivapurkar N, et al. Aberrant methylation and simian virus 40 tag sequences in malignant mesothelioma. Cancer Res 2001;61:5727–5730.
17. Chan EC, Lam SY, Tsang KW, et al. Aberrant promoter methylation in Chinese patients with non-small cell lung cancer: patterns in primary tumors and potential diagnostic application in bronchoalevolar lavage. Clin Cancer Res 2002;8:3741–3746.
18. Toyooka S, Toyooka KO, Maruyama R, et al. DNA methylation profiles of lung tumors. Mol Cancer Ther 2001;1:61–67.
19. Yanagawa N, Tamura G, Oizumi H, Takahashi N, Shimazaki Y, Motoyama T. Promoter hypermethylation of tumor suppressor and tumor-related genes in non-small cell lung cancers. Cancer Sci 2003;94:589–592.
20. Harden SV, Tokumaru Y, Westra WH, et al. Gene promoter hypermethylation in tumors and lymph nodes of stage I lung cancer patients. Clin Cancer Res 2003;9:1370–1375.
21. Zhou JX, Niehans GA, Shar A, Rubins JB, Frizelle SP, Kratzke RA. Mechanisms of G1 checkpoint loss in resected early stage non-small cell lung cancer. Lung Cancer 2001;32:27–38.
22. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996;93:9821–9826.
23. Fackler MJ, McVeigh M, Evron E, et al. DNA methylation of RASSF1A, HIN-1, RAR-beta, Cyclin D2 and Twist in in situ and invasive lobular breast carcinoma. Int J Cancer 2003;107:970–975.
24. Pu RT, Laitala LE, Alli PM, Fackler MJ, Sukumar S, Clark DP. Methylation profiling of benign and malignant breast lesions and its application to cytopathology. Mod Pathol 2003;16:1095–1101.
25. Pu RT, Sheng ZM, Michael CW, Rhode MG, Clark DP, O'Leary TJ. 'Leary TJ. Methylation profiling of mesothelioma using real-time methylation-specific PCR: a pilot study. Diagn Cytopathol 2007;35:498–502.
26. Kobayashi N, Toyooka S, Yanai H, et al. Frequent p16 inactivation by homozygous deletion or methylation is associated with a poor prognosis in Japanese patients with pleural mesothelioma. Lung Cancer. 2008
[EPub ahead of print].
27. Kim H, Kwon YM, Kim JS, et al. Tumor-specific methylation in bronchial lavage for the early detection of non-small-cell lung cancer. J Clin Oncol 2004;22:2363–2370.
28. Topaloglu O, Hoque MO, Tokumaru Y, et al. Detection of promoter hypermethylation of multiple genes in the tumor and bronchoalveolar lavage of patients with lung cancer. Clin Cancer Res 2004;10:2284–2288.
29. Benlloch S, Galbis-Caravajal JM, Martin C, et al. Potential diagnostic value of methylation profile in pleural fluid and serum from cancer patients with pleural effusion. Cancer 2006;107:1859–1865.
30. Fischer JR, Ohnmacht U, Rieger N, et al. Promoter methylation of RASSF1A, RARbeta and DAPK predict poor prognosis of patients with malignant mesothelioma. Lung Cancer 2006;54:109–116.
31. Ramirez JL, Rosell R, Taron M, et al. 14-3-3sigma methylation in pretreatment serum circulating DNA of cisplatinplus-gemcitabine-treated advanced non-small-cell lung cancer patients predicts survival: The Spanish Lung Cancer Group. J Clin Oncol 2005;23:9105–9112.
32. Ramirez JL, Sarries C, de Castro PL, et al. Methylation patterns and K-ras mutations in tumor and paired serum of resected non-small-cell lung cancer patients. Cancer Lett 2003;193:207–216.
33. Esteller M, Sanchez-Cespedes M, Rosell R, Sidransky D, Baylin SB, Herman JG. Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients. Cancer Res 1999;59:67–70.