Y.F. Wong

Methylation of p16INK4A in primary gynecologic malignancy

The p16INK4A gene mapped on band p21 of chromosome 9 can be inactivated via multiple mechanisms including homozygous deletion, point mutation and promoter hypermethylation in various human tumors. A polymerase chain reaction (PCR) based analysis was performed to examine methylation of the p16INK4A gene promoter in 196 primary gynecologic malignancies including 98 cervical, 49 endometrial and 49 ovarian carcinomas. Methylation of p16INK4A was detected in 31% of cervical, 20% of endometrial, and 4% of ovarian carcinomas, respectively. The incidence of p16INK4A methylation in patients with cervical and endometrial carcinomas at advanced stages (stages III-IV) was statistically higher than those at early stages (stages I-II). There were also significant differences in the incidence of p16INK4A methylation in both cancers between the patients who had died of their disease or were alive with evidence of disease, and those without evidence of disease. The results indicate that methylation of the p16INK4A gene is present in a proportion of primary gynecologic malignancies and this alteration may be associated with poor outcome in cervical and endometrial carcinomas.

Clinical and Prognostic Significance of Human Papillomavirus in a Chinese Population of Cervical Cancers

Objective: To investigate the clinical and prognostic significance of human papillomavirus (HPV) in a Chinese population of cervical cancers. Methods: We studied 121 cervical cancer tissue samples from patients treated at our hospital. Identification and typing of HPV were done by polymerase chain reaction (PCR) using consensus primers MY11 and MY09 followed by direct DNA sequencing. The results were correlated with various clinical and prognostic parameters. Results: We found HPV DNA in 95 (78.5%) cases, including HPV-16 in 59 (48.8%) and HPV-18 in 14 (11.6%) cases. χ2 analysis revealed no significant correlation between the presence of HPV DNA and age at diagnosis, clinical stage, histologic type, tumor grading, 2-year and 5-year survival rate. Of the factors evaluated, age at diagnosis and histologic type were found to have a statistically significant relationship with HPV type. The mean age of the HPV-18 group was 48.6 years compared to 57.1 years for the HPV-16 group (p = 0.045) and 58.2 years for the HPV-negative group (p = 0.04). HPV-18 was detected more often in adenocarcinomas (AC) than in squamous cell carcinomas (SCC). Conversely HPV-16 was detected significantly more often in SCC (p < 0.0001). The HPV-negative group also had a higher incidence of SCC (p = 0.007). HPV-18-positive patients seemed to have more nodal involvement than both HPV-16-positive patients (45.5 vs. 20.8%) and HPV-negative patients (45.5 vs. 18.2%); however, it did not reach statistical significance. Conclusions: These observations suggest that the presence of HPV DNA does not bear any clinical or prognostic significance in a Chinese population of cervical cancers. HPV-18 is found more often in younger patients and is associated with AC.

RAB32 hypermethylation and microsatellite instability in gastric and endometrial adenocarcinomas

The recently described gene, RAB32, is a ras proto‐oncogene family member that encodes an A‐kinase‐anchoring protein. RAB32 has been found to be frequently hypermethylated in microsatellite instability‐high (MSI‐H) colon cancers. We sought to determine the prevalence of RAB32 hypermethylation in gastric and endometrial adenocarcinomas, the 2 other major tumor types in which MSI‐H is common. Moreover, we delineated the association of RAB32 hypermethylation with microsatellite instability (MSI) and hMLH1 hypermethylation. MSI status and hypermethylation of the RAB32 and hMLH1 genes were studied in paired primary normal and tumor tissues from 48 patients with gastric cancer. An additional 80 endometrial cancer patients were studied for RAB32 methylation and MSI status. Thirteen (27%) of 48 gastric cancers demonstrated evidence of RAB32 hypermethylation. MSI status was determined in 46 of the tumors, with 7 (100%) of 7 MSI‐H tumors, 1 (33%) of 3 MSI‐low (MSI‐L) tumors and 4 (11%) of 36 microsatellite‐stable (MSS) tumors found to harbor RAB32 hypermethylation. RAB32 methylation was significantly associated with intestinal type histology and concomitant hMLH1 hypermethylation in gastric cancer. In contrast, RAB32 methylation occurred in only 1 of 80 endometrial cancers, including 20 MSI‐H, 8 MSI‐L and 52 MSS tumors. Hypermethylation of hMLH1 was noted in 16 (20%) of 80 endometrial tumors. We conclude that although RAB32 methylation is rare in endometrial cancers, it is strongly associated with hMLH1 hypermethylation and MSI in gastric adenocarcinomas. Given its similar involvement in colon cancer, RAB32 inactivation may represent a component of the oncogenic pathway of microsatellite‐unstable gastrointestinal adenocarcinomas.

Frequent loss of heterozygosity of chromosome 3 short arm detected by PCR-based microsatellite polymorphisms in cervical squamous cell carcinoma

Karyotypic studies have shown that genetic aberrations of the short arm of chromosome 3 (3p) may be involved in the pathogenesis of cervical carcinoma. In this study we analyzed nine polymorphic microsatellite repeats on 3p using a PCR-based assay for loss of heterozygosity (LOH) in 64 invasive squamous cell carcinomas of the cervix. These markers encompass chromosome region 3p13-25. LOH at one or more loci was detected in 46 (79%) out of the 58 informative cases. The incidence of LOH at locus D3S643 (3p13) was the highest among nine markers examined. The difference between the frequency of LOH at D3S643 in early stage (I-II) disease (43%) and those with advanced stage (stage III-IV) (79%) was statistically significant (P < 0.05). The results indicate that tumor suppressor gene(s) that play a role in cervical cancer may be located on the short arm of chromosome 3, likely near or at 3p13. The LOH at 3p13 appears to be a late event in tumor progression and may serve as an indicator for a less favorable clinical outcome.

HER‐2/neu Gene Amplification in Cervical Cancer in Chinese Women of Hong Kong and China

Objective: To determine the amplification of proto‐oncogene HER‐2/neu in invasive cervical cancer and its relationship with the stage of disease, grade of tumor and prognosis of patients.

Abstract A8: Deregulated microRNAs associated with cervical cancer metastasis

A8 Cervical cancer is one of the most common gynecologic malignancy in Hong Kong. The identification of pretreatment markers with predictive significance for the presence of lymph node metastasis in low stage of cervical cancer is clinically important. MicroRNAs (miRNAs) are noncoding RNA molecules of 21 to 24 nt that regulate the expression of target genes in a post-transcriptional manner. Recent evidence indicates that miRNAs play essential roles in tumorigenesis and cancer metastasis. This study was to evaluate the deregulated miRNAs associated with cervical cancer metastasis. A total of 20 cervical squamous cell carcinomas, in which 10 had pathologically confirmed lymph node metastasis and 10 no metastasis, were included in the study. RNAs were extracted from microdissected tumor tissue specimens. Real-time reverse transcription PCR technology-based miRNA array enabling quantitation of 365 human mature miRNAs, was used to profile global miRNA expression in cervical cancer. dChip software was used for biostatistical analysis of data. Global miRNA expression data obtained from all 20 arrays were normalized, and then the corresponding model-based expression index was calculated. Unsupervised hierarchical clustering analysis of data showed a distinct separation between cancers in the presence and absence of lymph node metastasis. Supervised hierarchical clustering analysis revealed that of 13 significantly differentially expressed miRNAs, 10 miRNAs including miR-137 (4.76-fold), miR-203 (3.82-fold), miR-594 (3.58-fold), miR-149 (3.05-fold), mir-365 (2.61-fold), miR-556 (2.43-fold), miR-33 (2.32-fold), miR-627 (2.12-fold), miR-20a (2.05-fold) and mir-653 (2.04-fold) were up-regulated and 3 miRNAs including miR-187 (-4.31), miR-4095p (-2.65-fold) and miR-615 (-2.1-fold) were down-regulated in the cervical squamous cell carcinomas with lymph node metastasis compared to that without metastasis. The results obtained from this preliminary study indicate that the deregulation of miRNAs appears to be involved in the cervical cancer progression, and miRNA expression signature can be of potential importance as predictive biomarker in cervical cancer metastasis. Further validation of such microRNA pattern by the testing set and an independent cohort of patients with cervical cancer as well as the functional study of these deregulated miRNAs are ongoing. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A8.

Dysregulation of the expression of miR204 mediates migration and invasion of endometrial cancer by regulating FOXC1

microRNAs (miRNAs) regulate gene expression and some have been shown to function as either oncogenes or tumor suppressors (oncomirs). Changes in miRNA expression have been observed in many human cancers but their role in endometrioid endometrial cancer (EEC) remains poorly understood. We studied the expression of 365 human miRNAs using Taqman low density arrays in 98 EECs as well as 60 normal endometria. Potentially interesting miRNAs were then validated by qRT-PCR. Expression of highly deregulated miRNAs on malignant cell properties was also examined in vitro using pre-/anti-miRNA transfection. Predicted mRNA targets for the miRNA of interest were also verified. We identified 16 significantly deregulated miRNAs in EEC and 7 of these are reported for the first time. Of these, miR-7, miR-194 and miR-449b knockdown and miR-204 induction repressed migration, invasion and extracellular matrix-adhesion in HEC1A endometrial cancer cells. For the cancer progression dysregulation of the expression of miR204 mediates migration and invasion of endometrial cancer by regulating FOXC1-related miR-204, FOXC1 was determined as its target gene possessing two miR-204-binding sites at the 3’-untranslated region. FOXC1 expression was inversely related to miR-204 expression in EEC. Functional analysis also revealed the involvement of FOXC1 in migration of HEC1A cells. We have identified dysfunctional miRNAs in endometrial cancer and elucidated the crucial role of miR-204-FOXC1 interaction in endometrial cancer progression. This work provides insight into miRNA-based diagnostic and therapeutic approaches for endometrial cancers.

CHD5 downregulation associated with poor prognosis in epithelial ovarian cancer in Hong Kong Chinese women

Background: CHD5 gene located on 1p36, encodes a non-histone chromosomal protein, chromodomain helicase DNA binding protein 5. Recently, CHD5 has been shown to be a tumor suppressor gene candidate. This study was to investigate the involvement of CHD5 in epithelial ovarian cancer and its clinico-pathological significance. Methods: CHD5 expression in ovarian cancer and its normal counterpart was determined by quantitative RT-PCR. The methylation of CHD5 promoter was evaluated by methylation specific PCR. The correlation of CHD5 expression to patient age, tumor histological type and grade, clinical stage and status, recurrence and patient survival time was analyzed statistically. Results: CHD5 expression was down at least two-fold in 32 of 72 (41%) invasive epithelial ovarian carcinomas when compared to 12 normal controls in Hong Kong Chinese women. Hypermethylation in two promoter regions of CHD5 was not detected in any of the 20 tumor samples available to be analyzed. CHD5 down-regulation was correlated to clinical status (P 0.05). Kaplan-Meier survival analysis showed that patients with CHD5 down-regulation in their tumors were associated with shorter disease-free and total survival times compared to those without CHD5 down-regulation (P Conclusion: This preliminary study shows that CHD5 s down-regulated in a certain part of ovarian cancers and appears to be an adverse predictor candidate of ovarian cancer disease-free and total survival in Hong Kong Chinese women. If this role of CHD5 dysregulation can be confirmed in more prospective studies with a large number of patients, it is also worthwhile to further evaluate if CHD5 can be as an individualized molecular therapeutic target for epithelial ovarian cancer.