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.

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.

Clinical use of laparoscopic ultrasonography in detecting nodal metastasis in advanced-stage cervical carcinoma.

OBJECTIVE: To assess the clinical use of a laparoscopic ultrasound scan (LUS) to identify pelvic and para-aortic node metastasis in patients with advanced-stage cervical cancer. METHODS: After examination under general anesthesia and cystoscopy, LUS was used to examine the pelvic nodes of patients with advanced-stage cervical cancer. Abnormal nodes were excised before definitive treatment to confirm the nodal status. Patients without abnormal para-aortic nodes on preoperative computer tomography/magnetic resonance imaging in the past 3 years were surgically staged via laparoscopic extraperitoneal aortic node sampling, and the findings were correlated with LUS findings. The predictive values of abnormal pelvic nodes on LUS for pelvic and aortic node metastasis were determined. RESULTS: A total of 119 advanced-stage cervical cancer patients underwent LUS of pelvic nodes. Abnormal pelvic nodes were found in 62 (52.1%) patients, and metastasis was confirmed by histology in 38 (31.9%) patients. Three patients had micro-metastasis in para-aortic nodes, and all of these patients had abnormal pelvic lymph nodes on LUS. CONCLUSION: Abnormal pelvic nodes are commonly found on LUS in patients with advanced-stage cervical cancer, and selective excision biopsy is needed to confirm pelvic node metastasis. Surgical staging of aortic nodes might be considered for patients with abnormal pelvic nodes on LUS.

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.

Profile of viral load, integration, and E2 gene disruption of HPV58 in normal cervix and cervical neoplasia.

The clinical utility of viral-load and integration status of human papillomavirus (HPV) infection remains uncertain. We examined 75 women infected with HPV58, a worldwide rare type found to be prevalent in cervical cancers in eastern Asia. Viral load was significantly higher for cervical intraepithelial neoplasia (CIN) 1/2, but those for a normal control group and for CIN 3 or cancer overlapped substantially. A pure integrated genome was found for all lesion grades, giving a poor positive predictive value (23.1%) for cancer. The pure episomal form's negative predictive value for cancer was only 76.3%. Mixed patterns of E2 gene disruption were common and often involved the amino-terminal and hinge regions. Disruption of the whole E2 gene was rare and was restricted to high-grade lesions. The HPV58 variant E67-HK-2 was more likely to exist in the pure episomal form. Routinely collected cervical samples contain a heterogeneous population of viruses, hampering the application of viral load and integration testing in clinical settings.

Association between HLA-DRB1 polymorphism, high-risk HPV infection and cervical neoplasia in southern Chinese.

Multiple determinants are involved in the progression of human papillomavirus (HPV)-infected cervical lesion to invasive cancer. Human leukocyte antigen (HLA) polymorphism seems to play a role. This study examined the association between HLA-DRB1 polymorphism, high-risk HPV infection, and the development of cervical neoplasia in southern Chinese. Three hundred and seventy women with cervical neoplasia (43 cervical intraepithelial neoplasia grade II, 154 grade III, and 173 invasive cancers) and 323 controls were recruited for HLA-DRB1 typing by a sequence-based approach. Cervical specimens were collected for HPV detection by a consensus primer-based polymerase chain reaction, and with the type of HPV identified by hybridization with type-specific oligonucleotide probes. A protective effect of HLA-DRB1*12 for cervical neoplasia was observed, and with stronger associations when subgroup analyses were carried out for patients infected with HPV16 and HPV58. The protective effect of HLA-DRB1*13 that had been reported from other populations was not observed. The data obtained in this study showed that HLA-DRB1*03 conferred a higher risk for HPV18-infected, but not for HPV16-, HPV52-, or HPV58-infected cervical lesions. Although, HPV52 was reported as uncommon worldwide, it was found to be the second most prevalent type in the southern Chinese population. However, no additional risk association was observed when subgroup analyses were performed for HPV52-infected patients. The current study shows that, among southern Chinese, the outcome of HPV-infected cervical lesions is associated with HLA-DRB1 polymorphism. These associations often vary with the type of HPV infection.

The role of laparoscopic surgery in the management of tubal prolapse: a 7 case series and literature review.

Fallopian tube prolapse is an unusual complication after hysterectomy. Different surgical options have been proposed, including abdominal, vaginal, and combined laparoscopic approaches, with partial or complete salpingectomy. This article presents experience in the management of 7 cases of fallopian tube prolapse with different surgical approaches according to the characteristics of the case. Additionally, 6 cases were reported in the English literature between 1960 and 2006 that required a second procedure after vaginal partial salpingectomy, and the causes of failure were reviewed. It is suggested that the choice between abdominal, vaginal, and combined laparoscopic approach with partial or complete salpingectomy in the management of prolapsed tubes must be decided individually, according to the patient's characteristics and the presenting symptoms. Laparoscopic surgery has a role in cases with dense intraperitoneal adhesions. It safely enables the mobilization of the prolapsed tube, allowing complete removal of the structure and resolution of symptoms.

HLA-DQB1 polymorphisms and risk for cervical cancer: a case-control study in a southern Chinese population.

OBJECTIVES AND METHODS: HLA II DQB1 polymorphisms have been shown to associate with cervical cancer risk, but results varied among different populations. In this study, the HLA DQB1 alleles among 221 southern Chinese women with cervical intraepithelial neoplasia grade III (CIN III)/invasive cervical carcinoma (ICC) were compared to 191 controls. RESULTS: The frequency of DQB1*03 was significantly lower among ICC overall as compared to controls (65.4% vs. 79.1%, odds ratio [95% confidence interval]: 0.50 [0.28-0.88], corrected p-value: 0.04). The protective association of DQB1*03 remained significant for human papillomavirus (HPV) 16-positive ICC, but not for HPV16-negative cases. This is in contrast to studies on European populations where DQB1*03 was associated with an increased risk for ICC. In the current study, 70.1% of the HPV16 isolates were Asian variants, and 28.0% were European variants. However, no significant association between HPV16 variant and DQB1*03 distribution was observed. HPV52 and HPV58 were found respectively in 16.3% and 10.0% of CIN III/ICC, which were higher compared to that of Europe and North America. Further analyses revealed a positive risk association between DQB1*06 and HPV58-positive CIN III/ICC (3.68 [1.37-9.92], corrected p-value: 0.012). CONCLUSION: The host genetics and the distribution of HPV types/variants may account for the observed differences among southern Chinese and other populations.

Viral load, E2 gene disruption status, and lineage of human papillomavirus type 16 infection in cervical neoplasia.

The clinical utility of human papillomavirus (HPV) load and integration status remains unclear. We applied refined methods to delineate the viral load, integration status, and lineage of 104 women with HPV-16 monotype infection, including 19 with normal cervices, 9 with histologically proven cervical intraepithelial neoplasia (CIN) 1, 24 with CIN 2, 27 with CIN 3, and 25 with squamous cell carcinoma (SCC). Higher crude viral load, as determined by real-time polymerase chain reaction (PCR) targeting the E7 gene, was observed for SCC but became insignificant after normalization for cell content. Integration was located and quantified by real-time PCRs targeting, respectively, the carboxyl, amino, and hinge domains of the E2 gene. Pure episomal, integrated, and mixed forms were observed in all disease groups. Most E2 gene disruptions involved the amino-terminal, but sparing the hinge region that has been frequently used as a surrogate marker of integration. Large-fragment disruption involving all 3 E2 regions was observed only in the CIN 3 and SCC groups. Altogether, 33.3% of the CIN 3 group and 28.0% of the SCC group harbored pure episomal genomes. The Asian lineage was associated with a higher risk for CIN 3/SCC than the European lineage, and 6 of the 7 large-fragment E2 disruptions were from Asian lineage. The link between viral lineage, integration pattern, and oncogenesis deserves further study.

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.

HLA-B alleles, high-risk HPV infection and risk for cervical neoplasia in southern Chinese women.

A population-based study was conducted on 256 southern Chinese with cervical intraepithelial neoplasia grade III (CIN III) or invasive cervical cancer (ICC) and on 258 controls to examine the associations between HLA-B alleles, infection with high-risk human papillomaviruses (HPVs) and the development of cervical neoplasia. HLA-B15 was found to be protective for CIN III/ICC overall (p(corrected) = 0.003), and for HPV52-positive CIN III/ICC (p(corrected) = 0.003). A marginal protective effect of B15 was observed for HPV16-positive CIN III/ICC, but no significant associations were revealed for HPV18- or HPV58-positive cases. None of the HLA-B alleles were found to confer an increased risk for cervical neoplasia. HLA-B15 is common among Asian for whom HPV52, a worldwide uncommon HPV type, also exists in a relatively high prevalence. It would also be worthwhile to assess the association between HLA-B15, HPV52 and cervical cancer in other Asian populations.

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.

Risk association between human leukocyte antigen-A allele and high-risk human papillomavirus infection for cervical neoplasia in Chinese women.

To examine the association between human leukocyte antigen-A (HLA-A) allele polymorphism, human papillomavirus (HPV) infection, and risk for cervical neoplasia in Chinese women, 263 patients (155 with cervical intraepithelial neoplasia [CIN] II/III and 108 with invasive cervical cancer [ICC]) were compared with 572 controls. Overall, regardless of HPV status, a decreased risk for ICC was observed for patients with A*0207/0215N or A*2402, and an increased risk was observed for patients with A*1104. The protective association of A*0207/0215N was reproduced in HPV-16-positive patients with ICC, but not in subgroups infected with other HPV types. The risk association between A*1104 and both HPV-16 and HPV-18 was reproduced in the subgroups with CIN III/ICC. The protective association between A*2402 and HPV-16, HPV-18, HPV-52, and HPV-58 was consistently observed in all subgroups with CIN III/ICC, suggesting a linkage with a general antioncogenic genetic factor. The results of the present study indicate that HLA-A polymorphism is one of the host genetic factors that alter the risk for the development of cervical cancer in Chinese women.

Detection and quantitation of human papillomavirus DNA in primary tumour and lymph nodes of patients with early stage cervical carcinoma.

Fifteen Chinese women with early stage cervical squamous cell carcinoma (14 stage IB, one stage IIA) were retrospectively analysed for the correlation between human papillomavirus (HPV) load in primary tumour and the presence of HPV DNA in histologically tumour-free pelvic lymph nodes. HPV16 DNA was detected from majority (12/15) of primary tumours, with a viral load ranging from 12 to 1800 copies per cell. Of the 156 histologically tumour-free pelvic lymph nodes, 41 (26.3%) were positive for HPV DNA. The levels of viral load detected in histologically tumour-free lymph nodes were low and most were not detectable by the less sensitive consensus PCR GP5+/6+. Among patients without histological evidence of nodal involvement, the presence of HPV DNA in lymph nodes was associated with a significantly higher viral load in primary tumour (mean [interquartile range]=800 [600-1450] versus 40 [19-70] copies per cell, P=0.016). Three of the four patients with recurrence had histological evidence of lymph node metastases. In contrast, none of the seven patients with HPV DNA-positive lymph nodes but without histologically evidence of nodal involvement developed recurrence. The results of this study suggest that the presence of HPV DNA in histologically tumour-free lymph nodes do not have prognostic significance. The HPV DNA detected from lymph nodes may have originated from circulating necrotic tumour cells or those internalized by scavengers, which was easier to be detected when the viral load per tumour cell was high.

La histeroscopia en el tratamiento del cáncer endometrial: su valor y seguridad / Hysteroscopy in the treatment of endometrial cancer: its value and safety

Abnormal uterine bleeding is one of the most common problems presented to gynecologists. It frequently requires more than clinical assessment to rule out intrauterine pathology or endometrial histopathology. Diagnostic hysteroscopy allows direct visualization of the endometrial cavity under magnified view and makes assessment possible. It is now generally considered the gold standard for evaluating abnormal uterine bleeding.1 Hysteroscopic examination of the uterine cavity and the endocervical canal also plays an important role in both the diagnosis and the staging of endometrial cancer.2 However, its value and safety in the management of endometrial cancer is always a contention. We have reported our results of diagnostic hysteroscopy and evaluated the role of such a procedure in identifying endometrial cancer.3 We have also studied the efficacy of hysteroscopy, using different distension medium, in assessing tumour invasion of the uterine cervix by endometrial cancer.4 As disseminating tumour cells into the peritoneal cavity during hysteroscopy became a concern, we therefore reviewed our cases and determined whether dissemination represented a genuine problem, especially in relation to the choice of distension medium.5 Recently, we also looked into the possible way to prevent hysteroscopic dissemination in endometrial cancer.

El sangrado uterino anómalo constituye uno de los problemas más comunes que enfrentan los ginecólogos. Este a menudo requiere más que una evaluación clínica para descartar patología intrauterina o histopatología endometrial. La histeroscopia diagnóstica permite la visualización directa de la cavidad endometrial bajo visión magnificada y hace posible la evaluación de la patología intrauterina. Se la considera actualmente como el gold standard para la evaluación del sangrado uterino anormal.1 El examen histeroscópico de la cavidad uterina y del canal endocervical también juega un importante papel, tanto en el diagnóstico como en la estadificación del cáncer endometrial.2 Sin embargo, su valor y seguridad en el tratamiento del cáncer endometrial motivan siempre controversias. Publicamos nuestros resultados respecto de las histeroscopias diagnósticas, evaluando el papel de tal procedimiento en la identificación del cáncer endometrial.3 Estudiamos ­además­ la eficacia de la histeroscopia mediante el empleo de diferentes medios de distensión para evaluar la invasión tumoral en el cérvix uterino del cáncer endometrial.4 Dado que la diseminación de células tumorales en la cavidad peritoneal durante la histeroscopia constituye una preocupación, revisamos nuestros casos determinado si la diseminación representa un problema genuino, especialmente en cuanto a la elección del medio de distensión.5 Además, recientemente investigamos el modo de prevenir la diseminación histeroscópica en el cáncer endometrial.

Nodal detection in malignant melanoma of the vagina using laparoscopic ultrasonography.

BACKGROUND: Primary malignant melanoma of the vagina is a rare variant of melanoma. It has worse prognosis compared to nongenital melanomas or other vaginal malignant neoplasms. CASE: A 40-year-old Chinese was diagnosed vaginal melanoma. Laparoscopic ultrasonography (USG) was used to search for abnormal pelvic and abdominal lymph nodes. Two metastatic pelvic lymph nodes were detected and excised. The vaginal tumour was removed by hysterectomy and partial vaginectomy. Despite a clear surgical margin and adjuvant radiotherapy, the patient died shortly after the operation. CONCLUSION: Patient with vaginal melanoma has grave prognosis, especially when metastatic disease presents. Radical surgery appears unjustified as a routine, it is essential to exclude lymphatic and distant metastases before embark to radical surgery. This report presents the first case of laparoscopic ultrasonographic detection of metastatic pelvic lymph nodes in patient with vaginal melanoma.

Intravenous leiomyomatosis: two cases with different routes of tumor extension.

Intravenous leiomyomatosis is a rare smooth muscle tumor. We report two cases of intravenous leiomyomatosis that grew along different routes of the venous system into the inferior vena cava and the right atrium. The different route of extension makes a difference in the ease of excision of tumor masses. Using MEDLINE together with the references in each publication, we identified all cases of intracardiac leiomyomatosis reported in the English literature in the period between 1980 and 2003 and performed a brief review on this potentially lethal disease entity.

Human papillomavirus type 16 intratypic variant infection and risk for cervical neoplasia in southern China.

A case-control study was conducted on 1986 Hong Kong women to assess the risk of human papillomavirus (HPV) type 16 variants for cervical neoplasia. In total, 255 women were HPV-16 positive and were analyzed for E6 and E7 sequence variation. Two novel substitutions at E6 (T86I and Q116E) and 1 at E7 (R66W) were found. Most HPV-16 variants were of Asian (50.6%) or European (44.3%) lineage, and both lineages showed similar risk associations for high-grade and invasive cervical neoplasia. No increased risk was observed for the subclasses European variant and European 350G, which carry a higher risk for invasive cancer in some Western populations. The E7 N29S substitution, reported to have a higher risk in Korean women, was found equally distributed among normal and various degrees of neoplasia. The epidemiology and risk implication of HPV-16 variant infection in Hong Kong differ markedly from other parts of the world.