T.K.H. Chung

Preferential integration of human papillomavirus type 18 near the c-myc locus in cervical carcinoma

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. Greater than 99% of all cervical tumors contain HPV DNA. Integration of high-risk HPV has been temporally associated with the acquisition of a malignant phenotype. Recent work from our lab has shown that HPV16, the most common high-risk HPV associated with cervical carcinoma, preferentially integrates at loci containing human common fragile sites (CFSs). CFSs are regions of genomic instability that have also been associated with deletions, translocations, and gene amplification during cancer development. The current work shows that HPV18, the second most prevalent high-risk HPV type found in cervical tumors, preferentially targets the CFSs. We identified 27 unique HPV18 integrations in cervical tumors, of which 63% (P<0.001) occur in CFSs. However, the distribution of HPV18 integrations found were profoundly different from those found for HPV16. Specifically, 30% of all HPV18 integrations occurred within the chromosomal band 8q24 near the c-myc proto-oncogene. None of the HPV16 integrations occurred in this region. Previous low-resolution mapping suggested that c-myc may be a target of HPV integration. Our data at nucleotide resolution confirm that in HPV18-positive cervical tumors, the region surrounding c-myc is indeed a hot spot of viral integration. These results demonstrate that CFSs are preferred sites of integration for HPV18 in cervical tumors. In addition, we have identified multiple cellular genes that have been disrupted by HPV18 integration in cervical tumors. Our results suggest that the sites of HPV18 integration are nonrandom and may play an important role in the development of cervical tumors.

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.

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.

Expression of p16INK4 and Rb Genes in Cervical Neoplasm.

Background. The molecular events surrounding the pathogenesis of human cervical cancer remain to be defined to a significant extent. The current study investigates the expression of two putative tumor suppressor genes p16INK4 and Rb in cervical neoplasm. Materials and Methods. Tissue was collected from 16 cervical intraepithelial neoplasia (CIN) patients and 91 cervical cancer patients prior to definitive treatment. Immunohistochemical staining for p16INK4 and Rb protein was performed in these cases to determine the relationship between the expression of these two genes in premalignant and malignant neoplasias of the cervix. Results. Eight (50%) CIN and 9 (10%) of cervical carcinoma cases lacked p16INK4 expression, while 4 CIN (25%) and 34 carcinoma (37%) cases did not display Rb expression. The prevalence of negative p16INK4 expression in CIN was significantly higher than in cervical carcinoma (p < .01). The prevalence of negative Rb expression was higher than was negative p16INK4 expression in cervical carcinoma (p < .01). Alterations of p16INK4 and Rb are relatively exclusive in both CIN and carcinoma (p < .01). No statistically significant relationship was found between either p16INK4 or Rb expression and tumor cell type, histological grade, or clinical stage in cervical carcinoma. Conclusions. Deficiency of either p16INK4 or Rb protein appears to be contributory to oncogenesis of cervical neoplasm in different subgroups of patients. Whether negative p16INK4 expression commonly is involved in the pathogenesis of CIN must be confirmed.

Ovarian epithelial tumour in gonadal dysgenesis. A case report and literature review.

A patient with gonadal dysgenesis was found to have a mucinous epithelial ovarian tumour on the left side and a streak gonad on the right. The preponderance of mucinous tumours (5 of 8) over other epithelial tumours in these patients is noted but the significance is not fully understood. Two models of pathogenesis (incessant ovulation and hypergonadotrophic hypogonadism) were proposed but neither satisfactorily explains the development of the tumours. Further ultrastructural, chromosomal and molecular biological study of these tumours may help to elucidate the underlying cause and pathogenesis.

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.

Abstract 4047: Mining of microRNA signatures in endometrial cancer

[Introduction] Endometrial cancer has now become the most common gynaecologic malignancy worldwide. About 80-90 % of endometrial cancer are endometrioid endometrial adenocarcinomas (EEC). MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression by inducing cleavage of the targeted messenger or by inhibiting translation. Our entire study was to mine miRNA signatures involved in EEC tumorigenesis and progression. Here we report a part of the results that is on miRNAs associated with endometrial cancer progression in vitro. [Methods] We examined the expression of 365 mature miRNAs by real-time RT-PCR based plate assays in 52 EEC including 30 advanced- and 22 early-stage tumors, and compared that to 30 normal counterparts. To know about the deregulated miRNAs associated with endometrial cell behavior, miRNA inhibitor and pre-miRNA were used to inhibit up-regulated miRNA and induce down-regulated miRNA in highly invasive endometrial cancer cell line HEC-1A cells. Quantitative TaqMan RT-PCR was performed to measure miRNA levels in cells before and after treatment, respectively. Migration and invasion assays were carried out to evaluate effects of those deregulated miRNAs on invasion of endometrial cancer cells in vitro. [Results] Supervised clustering analysis of global miRNA expression in endometrial caner and normal control identified 17 miRNAs up-regulated and 2 miRNAs down-regulated, respectively, in EEC. When the miRNA expression profiles in advanced stage tumors were compared to those in early stage tumors, 6 miRNAs were significantly over-expressed and one miRNA under-expressed in advanced stage tumors. Cell migration and invasion assay showed the ability of cell migration and invasion was significantly inhibited by anti-miR-7, anti-miR-194, anti-miR-449b and pre-miR-204 in HEC-1A cells in vitro. [Conclusion] Unique miRNA signatures seem to be existed in ECC. Deregulated miRNAs miR-7, miR-194, miR-204 and miR-449b might mediate endometrial cancer progress. These miRNAs are potentially to be developed as novel diagnostic and/or prognostic molecular markers as well as candidate therapeutic targets of EEC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4047.