Epigenetic alterations by methylation of RASSF1A and DAPK1 promoter sequences in mammary carcinoma detected in extracellular tumor DNA.

Novel strategies for early detection of breast cancer, the most common and second most lethal cancer in women, are urgently needed. Silencing tumor suppressor genes via DNA methylation has established hypermethylation as one of the most frequent molecular alterations that may initiate and drive many types of human neoplasia including breast cancer. Detecting such epigenetic changes in DNA derived not only from tumor tissue, but also from bodily fluids, may be a promising target for the molecular analysis of cancer. In this study we examined serum, a readily accessible bodily fluid known to contain neoplastic DNA, from individuals with breast carcinoma. Using sensitive methylation-specific polymerase chain reaction, we searched for aberrant promoter hypermethylation of two normally nonmethylated genes: RAS association domain family member 1A (RASSF1A) and death-associated protein kinase 1 (DAPK1) in 26 patients with breast cancer, 16 patients with benign breast diseases, and 12 age-matched healthy controls. Hypermethylation of at least one gene was detected in 25/26 (96%) cancer patients, in 7/16 (43%) cases with benign breast diseases, and only 1/12 (8%) control subjects. Furthermore, methylation of both genes was found to be associated with ductal type of breast carcinoma. RASSF1A was hypermethylated in 18/26 cases (69%) and DAPK1 in 23/26 (88%). However, DAPK1 promoter methylation was more pronounced, as 12/23 DAPK1 methylated cases (52%) were strongly methylated (>75%) compared to the weaker methylation of RASSF1A (none of the cases with methylation at the level of >75%). These findings, if confirmed in studies of extended cohorts, may lead to useful clinical application in early diagnosis of breast cancer and better management of the neoplastic disease.

Tissue inhibitor of matrix metalloproteinase-2 in nasopharyngeal carcinoma.

By regulating matrix metalloproteinase (MMP) activity and controlling the breakdown of extracellular matrix components, tissue inhibitors of metalloproteinases (TIMPs) play an important role in the process of tumor invasion and metastasis. The present study was designed to clarify the role of TIMP-2 in nasopharyngeal carcinoma (NPC) patients and to evaluate its importance relative to clinicopathologic parameters. It was carried out in 30 patients with NPC and 20 controls. Tissue biopsies were studied and graded pathologically, and Western blot analysis was performed to assess TIMP-2 protein expression. Clinically, in accordance with TNM classification (T: tumor size, N: lymph node involvement, M: distant metastasis), 8 cases were diagnosed as stage II, 12 as stage III, and 10 cases as stage IV; however, pathologic typing with use of the World Health Organization (WHO) classification revealed the presence of 9 specimens of squamous cell carcinoma (WHO type 1), 6 cases of nonkeratinizing carcinoma (WHO type 2), and 15 cases of undifferentiated carcinoma (WHO type 3). The difference in percentage of TIMP-2 positivity between NPC patients (76.6%) and normal controls (30%) was statistically highly significant (P < .01). In addition, there was a significant positive correlation between TIMP-2 protein positivity and either the clinical staging or the histopathologic typing (P < .01) using Chi-square test (x(2)), suggesting that TIMP-2 can be used as a marker of the severity of NPC.Accordingly, we can assume that TIMP-2 may play a role in regional lymph node and/or distant metastasis and in progression of squamous cell carcinoma. Further studies are needed to investigate the role of TIMP-2 as a marker for tumor progression and to evaluate its potential value in the follow-up of patients.

Comparison of cytokeratin 20 RNA and angiogenin in voided urine samples as diagnostic tools for bladder carcinoma.

BACKGROUND: We evaluated the diagnostic efficacy of urinary angiogenin (ANG) and cytokeratin 20 (CK-20) mRNA in comparison with voided urine cytology in the detection of bladder cancer patients. OBJECTIVES AND METHODS: A total of 97 Egyptian patients provided a single voided urine sample for ANG, CK-20 and cytology before cystoscopy. Of the 97 cases, 63 were histologically diagnosed as bladder cancer; 33 with transitional cell carcinoma (TCC) and 30 with squamous cell carcinoma (SCC), whereas the remaining 34 had benign urological disorders. A group of 46 healthy volunteers were also included in this study. Voided urine was centrifuged and the supernatant was used for estimation of ANG by EIA and confirmed by Western blotting (WB). The urine sediment was used for cytology and RNA extraction. CK-20 RNA was detected by RT-PCR. RESULTS: The best cutoff value for ANG was calculated by a ROC curve as 322.7 ng/mg protein. The median urinary ANG level in bladder carcinoma, benign urological disorders and healthy volunteer groups was: 802.7, 425 and 33 pg/mg protein, respectively. The positivity rate for urinary CK-20 mRNA of the control, benign and malignant groups was 0%, 2.9% and 82.3%, respectively (P = 0.000); while the rates for ANG were 11.6%, 54.8% and 75.4%, respectively (P = 0.000). There was no significant difference in positivity rates of CK-20 and ANG with respect to sex, smoking, schistosomiasis, urine cytology, tumor grade, tumor stage, hematuria or pus cells. The overall sensitivity and specificity were 71.4% and 90% for voided urine cytology, 75.4% and 70.3% for ANG, and 82.3% and 98.8% for CK-20. Combined sensitivity of voided urine cytology with ANG and CK-20 together (98.2%) was higher than either the combined sensitivity of voided urine cytology with ANG (96.5%) or with CK-20 (91.6%) or than that of the biomarker alone. We demonstrated significant positive correlation between CK-20 positivity with age (P = 0.043) and nodal involvement (P = 0.037); however, there was no significant correlation between CK-20 and ANG with the other clinicopathological parameters. CONCLUSIONS: Our data indicate that CK-20 and ANG in voided urine had higher sensitivities compared to voided urine cytology. However, when specificity was considered, CK-20 alone had superior sensitivity and specificity compared to ANG and voided urine cytology.