Genomic alterations associated with mutational signatures, DNA damage repair and chromatin remodeling pathways in cervical carcinoma.

Despite recent advances in the prevention of cervical cancer, the disease remains a leading cause of cancer-related deaths in women worldwide. By applying the GISTIC2.0 and/or the MutSig2CV algorithms on 430 whole-exome-sequenced cervical carcinomas, we identified previously unreported significantly mutated genes (SMGs) (including MSN, GPX1, SPRED3, FAS, and KRT8), amplifications (including NFIA, GNL1, TGIF1, and WDR87) and deletions (including MIR562, PVRL1, and NTM). Subset analyses of 327 squamous cell carcinomas and 86 non-squamous cell carcinomas revealed previously unreported SMGs in BAP1 and IL28A, respectively. Distinctive copy number alterations related to tumors predominantly enriched for *CpG- and Tp*C mutations were observed. CD274, GRB2, KRAS, and EGFR were uniquely significantly amplified within the Tp*C-enriched tumors. A high frequency of aberrations within DNA damage repair and chromatin remodeling genes were detected. Facilitated by the large sample size derived from combining multiple datasets, this study reveals potential targets and prognostic markers for cervical cancer.

Dissection of PIK3CA Aberration for Cervical Adenocarcinoma Outcomes.

Personalized treatment of genetically stratified subgroups has the potential to improve outcomes in many malignant tumors. This study distills clinically meaningful prognostic/predictive genomic marker for cervical adenocarcinoma using signature genomic aberrations and single-point nonsynonymous mutation-specific droplet digital PCR (ddPCR). Mutations in PIK3CA E542K, E545K, or H1047R were detected in 41.7% of tumors. PIK3CA mutation detected in the patient's circulating DNA collected before treatment or during follow-up was significantly associated with decreased progression-free survival or overall survival. PIK3CA mutation in the circulating DNA during follow-up after treatment predicted recurrence with 100% sensitivity and 64.29% specificity. It is the first indication of the predictive power of PIK3CA mutations in cervical adenocarcinoma. The work contributes to the development of liquid biopsies for follow up surveillance and a possibility of tailoring management of this particular women's cancer.

Liquid biopsy of HPV DNA in cervical cancer.

BACKGROUND: A blood test to serve as a tumor marker for cervical cancer would be useful to clinicians to guide treatment and provide an early signal for recurrence. The development of droplet digital PCR has enabled the detection of HPV DNA in patient serum, providing a potential marker for cervical cancer. OBJECTIVES: To report on a blood-based test for HPV-specific E7 and L1 genes, which may serve as a tumor marker to guide treatment and detect early recurrence in cervical cancer. STUDY DESIGN: Pre-treatment plasma samples were investigated from 138 Hong Kong Chinese women with primary invasive squamous cell carcinoma and adenocarcinoma of the cervix with tumor samples expressing HPV16 or HPV18. Two genes specific to the human papillomavirus, E7 and L1, were measured in cell free DNA (cfDNA) extracted from plasma using droplet digital PCR. Analysis of detectable E7 and L1 levels was performed to investigate the potential of liquid biopsy of E7 and L1 as a clinically useful molecular biomarker. RESULTS: The majority of patients had HPV16 (71.7%), squamous cell carcinoma (78.3%) and stage IB-II disease (82.6%). HPV E7 and L1 sequences were detected in plasma cfDNA from 61.6% (85/138) of patients. Patients with high viral load (defined as ≥20 E7 or L1 copies per 20 µL reaction volume) had increased risk of recurrence and death at 5 years on univariate analysis but not multivariate analysis. CONCLUSIONS: HPV DNA can be quantitatively detected with the use of cfDNA. This has the potential to provide a clinically useful tumor marker for patients with cervical cancer that can aid in post-treatment surveillance and estimating the risk of disease relapse.

Liquid biopsy of PIK3CA mutations in cervical cancer in Hong Kong Chinese women.

INTRODUCTION: Cervical cancer is the fourth most common female cancer worldwide. The prognosis for women with advanced-stage or recurrent cervical cancer remains poor and response to treatment is variable. Standardized management protocols leave little room for individualization. We report on a novel blood-based liquid biopsy for specific PIK3CA mutations as a clinically useful biomarker in patients with invasive cervical cancer. METHODS: One hundred seventeen Hong Kong Chinese women with primary invasive cervical cancer and their pre-treatment plasma samples were investigated. Two PIK3CA mutations, p.E542K and p.E545K were measured in cell free DNA (cfDNA) extracted from plasma using droplet digital PCR. This liquid biopsy of PIK3CA in cervical cancer was correlated to clinico-pathological features to verify the potential of PIK3CA as a clinically useful molecular biomarker for predicting disease prognosis and monitoring for progression. RESULTS: PIK3CA mutations, either p.E542K or p.E545K, were detected in plasma cfDNA from 22.2% of the patients. PIK3CA mutation status was significantly correlated to median tumor size (p<0.01). PIK3CA mutations detected in the plasma were significantly associated with decreased disease-free survival and overall survival (p<0.05). CONCLUSIONS: As a liquid molecular biopsy, analysis of circulating PIK3CA mutations shows promise as a way to refine risk stratification of individual patients with cervical cancer, and provides a platform for further research to offer individualized therapy with the purpose of improving outcomes.

Osteopontin Fragments with Intact Thrombin-Sensitive Site Circulate in Cervical Cancer Patients.

We investigated whether circulating osteopontin (OPN) could be used as a biomarker for cervical cancer. We employed a monoclonal antibody (mAb 659) specific for the unique and intact thrombin-sensitive site in OPN using an inhibition ELISA. We found significantly higher levels of OPN in 33 cervical cancer patients in both the plasma (mean +/- SD, 612 +/- 106 ng/mL) and serum (424 +/- 121 ng/mL) compared to healthy subjects [409 +/- 56 ng/mL, from 31 plasma samples (P < 0.0001), and 314 +/- 98 ng/mL, from 32 serum samples (P = 0.0002), respectively]. Similar results were obtained when the plasma from a bigger group (147 individuals) of cervical cancer patients (560 +/- 211 ng/mL) were compared with the same plasma samples of the healthy individuals (P = 0.0014). More significantly, the OPN level was highest in stage III-IV disease (614 +/- 210 ng/mL, from 52 individuals; P = 0.0001) and least and non-discriminatory in stage I (473 +/- 110 ng/mL, from 40 individuals; P = 0.5318). No such discrimination was found when a mAb of a different specificity (mAb 446) was used in a similar inhibition ELISA to compare the two groups in the first study; a commercial capture ELISA also failed. The possibility that the target epitope recognized by the antibody probe in these assays was absent from the circulating OPN due to protein truncation was supported by gel fractionation of the OPN found in patients' plasma: 60-64 kDa fragments were found instead of the presumably full-length OPN (68 kDa) seen in healthy people. How these fragments are generated and what possible role they play in cancer biology remain interesting questions.

Genomic aberrations in cervical adenocarcinomas in Hong Kong Chinese women.

Although the rates of cervical squamous cell carcinoma have been declining, the rates of cervical adenocarcinoma are increasing in some countries. Outcomes for advanced cervical adenocarcinoma remain poor. Precision mapping of genetic alterations in cervical adenocarcinoma may enable better selection of therapies and deliver improved outcomes when combined with new sequencing diagnostics. We present whole-exome sequencing results from 15 cervical adenocarcinomas and paired normal samples from Hong Kong Chinese women. These data revealed a heterogeneous mutation spectrum and identified several frequently altered genes including FAT1, ARID1A, ERBB2 and PIK3CA. Exome sequencing identified human papillomavirus (HPV) sequences in 13 tumors in which the HPV genome might have integrated into and hence disrupted the functions of certain exons, raising the possibility that HPV integration can alter pathways other than p53 and pRb. Together, these provisionary data suggest the potential for individualized therapies for cervical adenocarcinoma based on genomic information.

An update on Mullerian-inhibiting substance: its potential application against ovarian cancer.

Each year, ∼25 000 women are newly diagnosed with ovarian cancer in the USA. The vast majority (>90%) of cases are of epithelial origin. This highly lethal cancer carries a mortality rate of >50% and a high risk of recurrence after conventional, first-line chemotherapy. Müllerian-inhibiting substance (MIS) is a gonadal hormone that causes regression of the Müllerian ducts. A series of studies have demonstrated that MIS also has multiple extra-Müllerian functions including inhibition of epithelial ovarian cancer cells in vitro and in vivo. Accumulating evidence has shown that many human cancers are organized hierarchically and contain a small population of cancer stem cells (CSCs) that are inherently resistant to common chemotherapy and radiation therapy. The effect of MIS on ovarian CSC seems to be particularly useful in rescuing ovarian cancer patients with resistance to conventional treatment. Based on recent studies evaluating MIS, this review updates our current understanding of the molecular genetic aspects of MIS, its pathophysiology, as well as its potential to treat chemoresistant epithelial ovarian cancer.

MicroRNA-182 plays an onco-miRNA role in cervical cancer.

OBJECTIVES: The purposes of this study were to identify aberrantly expressed miRNAs and investigate their pathogenic roles in cervical cancer. METHODS: miRNA expression was assessed in cervical cancer cell lines, micro-dissected normal cervical epithelium cells and primary cervical carcinoma by TaqMan RT-PCR. Spatial expression of miR-182 in cervical carcinoma and normal cervix was explored by in situ hybridization. HeLa xenograft mice model was used for evaluation of the effect on tumor growth of miR-182 inhibitor. Western blot, flow cytometry and gene expression analysis were used for identification of the functional role of miR-182 in HeLa cells. RESULTS: Two up-regulated (miR-182 and -183) and nine down-regulated (miR-211, 145, 223, 150, 142-5p, 328, 195, 199b, 142-3p) microRNAs were consistently identified in cervical cancer cell lines. Further investigation confirmed the most up-regulated miRNA (miR-182) was significantly elevated in primary cervical carcinoma and discovered a significant correlation between the increased expression of miR-182 and advanced stages of cervical cancer. In HeLa xenograft mouse model, we demonstrated that inhibition of the miR-182 could exert the effect of tumor growth regression. Western blot, flow cytometry and pathway analysis for the HeLa cells with miR-182 over/down-expression in vitro showed that miR-182 was involved in apoptosis and cell cycle pathways, it also associated with the regulation of FOXO1. CONCLUSIONS: Our findings indicated that miR-182 plays an onco-miRNA role in cervical cancer and its alteration is associated with cervical cancer pathogenesis by disrupting cell proliferation.

Dysregulated microRNAs in the pathogenesis and progression of cervical neoplasm.

MicroRNAs (miRNAs) play an important role in a variety of physiological as well as pathophysiological processes, including carcinogenesis. The aim of this study is to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to unveil individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues and compared to normal cervical epithelium. Unsupervised hierarchical clustering of the miRNA expression pattern displayed a distinct separation between the CIN and normal cervical epithelium samples. Supervised analysis identified 12 highly differentially regulated miRNAs, including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203, which distinguished the high-grade CIN specimens from normal cervical epithelium. This miRNA signature was further validated by an independent set of high-grade CIN cases. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma from normal controls. Target prediction analysis revealed that these dysregulated miRNAs mainly control apoptosis signaling pathways and cell cycle regulation. These findings contribute to understanding the role of microRNAs in the pathogenesis and progression of cervical neoplasm at the molecular level.

Frequent somatic demethylation of RAPGEF1/C3G intronic sequences in gastrointestinal and gynecological cancer.

RAPGEF1 (also known as C3G and GRF2) is a guanine nucleotide exchange factor that releases GDP from the inactive Rap1 protein, facilitating its subsequent activation by the binding of GTP. Rap1 plays regulatory roles in proliferation, differentiation and apoptosis. Amplification and overexpression of RAPGEF1 have been found in small cell lung cancers, suggesting an oncogenic role. In contrast, hypermethylation of a promoter CpG island (CGI-A) of RAPGEF1 has been reported in squamous cervical tumors, suggesting an anti-oncogenic role in these gynecological cancers. In our studies of DNA methylation alterations in gastrointestinal cancer we found somatic demethylation of a relaxed-criterion CpG island (CGI-B) located in the first intron of RAPGEF1 in 40% of colon cancers and 8% of gastric cancers relative to their matching normal tissues that were always methylated. We also found somatic demethylation in 47% of squamous cervical carcinomas as well as 33% of ovarian cancers. This somatic change in methylation, however, did not extend to the strict-criterion CpG island located in the promoter region (CGI-A) that was unmethylated in all normal and tumor tissues analyzed. Thus, promoter hypermethylation of RAPGEF1 seems insignificant in colorectal, cervical and ovarian cancers. In contrast, tumor-specific hypomethylation of the gene appears to be frequent in gastrointestinal and gynecological cancers.

Dysregulated microRNAs and their predicted targets associated with endometrioid endometrial adenocarcinoma in Hong Kong women.

The objective of this study, a parallel study to global gene expression profiling, was to identify dysregulated microRNAs (miRNAs) associated with endometrioid endometrial adenocarcinoma (EEC), examine their correlation with clinico-pathological characteristics and identify predicted target genes of the dysregulated miRNAs. Using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), profiling of miRNA expression was performed in 30 EECs and 22 normal counterparts in which genome-wide gene expression had been previously profiled and reported. Clustering analysis identified 30 miRNAs which were significantly dysregulated in EEC. The expression of a sub-group of miRNAs was significantly correlated with clinico-pathological characteristics including stage, myometrial invasion, recurrence and lymph node involvement. By searching for predicted miRNA targets that were linked to the dysregulated genes previously identified, 68 genes were predicted as candidate targets of these 30 dysregulated miRNAs. miR-205 was significantly overexpressed in EECs compared with normal controls. After transfection of a miR-205 inhibitor, the expression of miR-205 in endometrial cancer cell line RL95-2 cells decreased whereas its predicted target gene, JPH4, showed increased protein expression. JPH4 seems to be a real miR-205 target in vitro and in vivo, and a candidate tumor suppressor gene in EEC. Based on this study in EEC, miRNAs predicted to be involved in tumorigenesis and tumor progression have been identified and placed in the context of the transcriptome of EEC. This work provides a framework on which further research into novel diagnosis and treatment of EEC can be focused.

Expression of maspin in endometrioid adenocarcinoma of endometrium.

Maspin is a member of the serpin family, whose expression is altered in neoplasia and malignancies of many tissues. Underexpression of maspin has been reported in breast and prostatic cancers, but in some cancers such as ovarian, colorectal and pancreatic carcinoma, it was found to be up-regulated. This study aimed at demonstrating the expression of maspin in human endometrial tissue and searching for any altered expression in endometrioid adenocarcinoma of the endometrium compared to normal endometrium. The expression level of the maspin gene was studied using reverse transcriptase-polymerase chain reaction (RT-PCR) performed on RNA extracted from 34 endometrial cancer samples (including 24 with FIGO stage I disease and 10 with FIGO stage III disease) and 28 normal endometrium in proliferative or secretory phases. Immunohistochemical staining was also performed on 10 cases of endometrial cancer (6 FIGO stage I cases and 4 FIGO stage III cases) as well as 15 normal endometrium. Semi-quantitative RT-PCR revealed that the expression of maspin was significantly up-regulated in both stage I (p<0.01) and stage III (p<0.01) endometrial cancer compared with normal endometrium. However, no significant difference in maspin expression was demonstrated between stage I and stage III endometrial cancer. Immunostaining of all tissue sections revealed an immunopositive signal in the nuclei of the normal or cancerous endometrial glandular cells. In 60% of the cancer cases, cytoplasmic staining was also evident. Our results suggested that there is up-regulated expression of maspin in endometrioid endometrial adenocarcinoma. Cytoplasmic immuno-expression of maspin is common in endometrial cancer. It may play a role in the malignant transformation of human endometrial tissue.

DNA microchips to identify molecular signatures in cervical cancers.

Cervical cancer is still the leading cause of gynecological cancer deaths worldwide in spite of the advent of early diagnosis with the Pap smear. Ninety-five percent of cervical cancers are of squamous cell origin. Cervical carcinoma is almost always associated with infection from oncogenic subtypes of human papillomavirus (HPV). However, HPV infection alone is insufficient for malignant transformation; other genetic events independent or in conjunction with HPV infection are required. The early studies of genetics in cervical cancer were often hampered because only a few genes or genetic events could be evaluated at a time. Therefore, the interactions of multiple genes throughout the genome could not be evaluated. Gene-expression profiling utilizing microarrays allows quantitative measurement of the expression of thousands to all human expressed genes simultaneously. Here we describe how to obtain information on global genetic events in cervical cancer using oligonucleotide microarrays in combination with real-time reverse transcriptase polymerase chain reaction (RT-PCR). This facilitates understanding of the gene expression differences that underlie cervical neoplastic development and progression and can identify molecular signatures that can potentially be used in cervical cancer diagnosis and prognosis. This technology also represents a leap forward in the goal to eventually provide tailored therapy to individual patients and offers a genetic blueprint for gauging the potential effectiveness of all common cervical cancer treatments.

Detection of microsatellite instability in endometrial cancer: advantages of a panel of five mononucleotide repeats over the National Cancer Institute panel of markers.

The aim of this study was to find the optimal set of microsatellite markers for diagnosis of the microsatellite instability (MSI) phenotype in endometrial cancers. We compared the sensitivity, specificity and ease of use of a reference panel of five markers originally recommended by the National Cancer Institute (NCI) for colorectal cancer and a panel of five quasi-monomorphic mononucleotide repeat markers (pentaplex PCR system). We used these panels for establishing the MSI status of a series of 80 sporadic endometrial adenocarcinomas by comparing the allelic profiles of the markers between tumor and matching germline DNA. Both panels detected the same subset of 21 out of 80 (26%) endometrial MSI carcinomas. However, in the MSI cases, the mean instability of the five mononucleotide repeats was 96.1% as compared with a mean instability of 69.8% for the three dinucleotide repeats of the NCI panel, indicating a superiority of mononucleotide repeats over dinucleotide repeats in detecting MSI. The fact that the two panels of markers detect the same set of MSI tumors is due to the presence of two mononucleotide repeats within the NCI panel. As demonstrated previously in gastric and colon MSI cases, the pentaplex PCR reaction using mononucleotide repeats is thus an easier and more sensitive method than the NCI panel, for the screening of MSI status in endometrial tumors.

Biases in human papillomavirus genotype prevalence assessment associated with commonly used consensus primers.

Consensus primers targeting human papillomaviruses (HPVs) have biases in sensitivity toward certain HPV types. We applied 3 primer sets (GP5+/6+, MY09/11, PGMY09/11) in parallel on 120 Chinese cervical cancer specimens. GP5+/6+ exhibited a poor sensitivity for HPV52, for which the prevalence among squamous cell cervical cancer was underestimated from 14.6% to 0%. The fact that HPV52 should rank second in prevalence among squamous cell cervical carcinoma in Hong Kong could be missed if GP5+/6+, a worldwide commonly used primer set, was selected for HPV detection. Biases in HPV type-specific sensitivity may result in misprioritization of vaccine candidates.

Genome-wide gene expression profiling of cervical cancer in Hong Kong women by oligonucleotide microarray.

An analysis of gene expression profiles obtained from cervical cancers was performed to find those genes most aberrantly expressed. Total RNA was prepared from 29 samples of cervical squamous cell carcinoma and 18 control samples, and hybridized to Affymetrix oligonucleotide microarrays with probe sets complementary to over 20,000 transcripts. Unsupervised hierarchical clustering of the expression data readily distinguished normal cervix from cancer. Supervised analysis of gene expression data identified 98 and 139 genes that exhibited >2-fold upregulation and >2-fold downregulation, respectively, in cervical cancer compared to normal cervix. Several of the genes that were differentially regulated included SPP1 (Osteopontin), CDKN2A (p16), RPL39L, Clorf1, MAL, p11, ARS and NICE-1. These were validated by quantitative RT-PCR on an independent set of cancer and control specimens. Gene Ontology analysis showed that the list of differentially expressed genes included ones that were involved in multiple biological processes, including cell proliferation, cell cycle and protein catabolism. Immunohistochemical staining of cancer specimens further confirmed differential expression of SPP1 in cervical cancer cells vs. nontumor cells. In addition, 2 genes, CTGF and RGS1 were found to be upregulated in late stage cancer compared to early stage cancer, suggesting that they might be involved in cancer progression. The pathway analysis of expression data showed that the SPP1, VEGF, CDC2 and CKS2 genes were coordinately differentially regulated between cancer and normal. The present study is promising and provides potential new insights into the extent of expression differences underlying the development and progression of cervical squamous cell cancer. This study has also revealed several genes that may be highly attractive candidate molecular markers/targets for cervical cancer diagnosis, prognosis and therapy.

Multipopulation analysis of polymorphisms in five mononucleotide repeats used to determine the microsatellite instability status of human tumors.

PURPOSE: Human gastrointestinal tumors with inactivated DNA mismatch repair system (microsatellite instability [MSI] tumors) have distinct molecular and clinicopathologic profiles, and are associated with favorable prognosis. There is evidence suggesting that colorectal cancer patients with MSI tumors respond differently to adjuvant chemotherapy as compared with patients with non-MSI tumors. Finally, determination of the MSI status has clinical application for assisting in the diagnosis of suspected hereditary cases. It is thus becoming increasingly recognized that testing for MSI should be conducted systematically in all human cancers potentially of this type. We recently described a pentaplex polymerase chain reaction of five mononucleotide repeats to establish the MSI status of human tumors, and showed that this assay was 100% sensitive and specific. Moreover, these markers are quasimonomorphic in germline DNA of the white population (ie, individuals of Eurasian origin), and could be used for tumor MSI determination without the requirement for matching normal DNA in this group. PATIENTS AND METHODS: In this study, we analyzed a comparable panel of five mononucleotide markers in germline DNA from 1,206 individuals encompassing 55 different populations worldwide. Results With the exception of two Biaka Pygmies and one San individual for whom three markers showed variant alleles (three cases [0.2%]), the remaining 1,203 individuals showed no alleles of variant size (1,055 cases [87.5%]), or only one (122 cases [10.1%]) or two (26 cases [2.2%]) markers with variant alleles. All 60 MSI tumors investigated display instability in at least four of the five markers. CONCLUSION: We demonstrated that tumor MSI status can be determined using the pentaplex reaction for all human populations without the need for matching normal DNA.

Expression of maspin in gestational trophoblastic disease.

BACKGROUND: Maspin is a tumor suppressor gene whose expression is altered in neoplasia and malignancies of many tissues. In the human placenta, the maspin gene is expressed in trophoblastic cells and might act as an inhibitory regulator of trophoblastic invasion. Hence, in gestational trophoblastic disease (GTD), where there is increased propensity for invasion in the trophoblastic tissue, we hypothesized that maspin expression would be decreased. The present study aimed at investigating the expression of maspin in GTD and its prognostic significance. METHODS: Using immunohistochemical staining, we firstly studied the expression of maspin in hydatidiform moles, with gestational age-matched normal first trimester placenta used as control. A total of 38 cases of hydatidiform moles were studied, including 20 complete moles (CM) and 18 partial moles (PM). Among them, 10 cases of the CM group and 8 cases of the PM group subsequently developed gestational trophoblastic neoplasia (GTN). Immunostaining was also performed on tissue from 4 cases of choriocarcinoma and 5 cases of placental site trophoblastic tumor. Reverse transcriptase-polymerase chain reaction (RT-PCR) was further performed on RNA extracted from 10 hydatidiform moles (5 with GTN and 5 without) and 6 normal first-trimester placentae. RESULTS: In all tissue sections, nuclear expression of immunostaining signal was demonstrated, mainly in the cytotrophoblasts. The percentage of trophoblastic nuclei stained in both complete and partial moles was significantly lower than that in normal first-trimester placenta (P < 0.001). However, there was no significant difference in immunostaining between complete and partial moles (P > 0.05). There was also significantly lower expression of maspin in those cases subsequently developing GTN than those which did not (P = 0.01). Immunostaining on choriocarcinoma and placental site trophoblastic tumor showed reduced expression of maspin in all the tumor cells. Reverse transcriptase-polymerase chain reaction revealed that the expression of maspin was consistently down-regulated in all the hydatidiform mole samples. CONCLUSIONS: Our results suggest that there is down-regulated expression of maspin in gestational trophoblastic diseases, and the down-regulation is more prominent in cases developing gestational trophoblastic neoplasia. This may play a role with prognostic significance in the pathogenesis and malignant transformation of hydatidiform moles.

Quantitative analysis of human papillomavirus type 16 in cervical neoplasm: a study in Chinese population.

BACKGROUND: Human papillomavirus (HPV) infection was recognized as a major causal factor for the development and progression of squamous intraepithelial lesions (SIL). It is possible to use HPV test for the detection of cervical lesions as an adjunct to cervical cytology. OBJECTIVES: To evaluate the relation between HPV 16 viral load and the severity of cervical lesions in a Chinese population. METHODS: Study population was recruited from the colposcopy and general outpatient clinic. The presence of HPV 16 E6 and E7 in cytological specimens was detected using HPV 16 specific polymerase chain reaction (PCR). The viral load in the specimens that were positive for HPV 16 specific PCR, was quantified by using real-time PCR assay. RESULTS: The study recruited 394 women, in which 148 were high-grade SIL (HG-L), 121 were low-grade SIL (LG-L) and 125 were Normal. Sufficient DNA integrity was proven in 347 samples. Among 121 positive cases for HPV 16, 70 were HG-L, 34 were LG-L and 17 were Normal. Using quantitative real-time PCR, the percentages of samples with greater DNA copies were found to increase with the severity of diseases. There was also a significant difference in DNA copies among the three groups (HG-L versus Normal, p<0.001; HG-L versus LG-L, p<0.001). Area under receiver operating characteristic (ROC) curve of the HG-L versus LG-L and Normal was 0.836 indicating that quantitative PCR had a good diagnostic value in differentiating HG-L from the LG-L and Normal groups. CONCLUSIONS: Our data suggested HPV 16 viral load was significantly related to the severity of cervical lesions. Evaluation of viral burden could be a potential clinical tool in management of cervical lesions.

The role of viral integration in the development of cervical cancer.

The development of invasive cervical cancer is associated with human papillomavirus (HPV) infection and subsequent integration into the host epithelium. More than 99% of cervical cancers contain HPV sequences, and many of these contain a truncated HPV genome integrated into a single position within the host genome. Studies examining the role of viral integration in cervical cancer development have found that the sites of integration appear randomly distributed throughout the genome. This, and the observation that it frequently takes years after HPV infection for cervical cancer to develop, has led to the current paradigm that the site of HPV integrations is unimportant to the invasive cervical cancer that eventually develops. In our previous studies of HPV16 and HPV18 integration in cervical cancers, we also found integrations throughout the genome, but observed as well that more than half of the integrations occurred within common fragile site regions. To determine if HPV integration might play an important role in cervical cancer, we conducted two complementary studies. We first localized 40 new HPV16 integration sites from cervical tumors from women in Hong Kong; this, together with previous integration studies, provided a better picture of the distribution of integration sites throughout the genome. We then analyzed the sites of viral integration in an in vitro model of HPV integration. By comparing the sites of HPV integration in vivo (in multiple primary cervical tumors) to those obtained in vitro, the data can help to determine if HPV integrations observed in vivo are the result of random and nonselected integrations.