Matthias Dürst

Heterogeneous Distribution of Human Papillomavirus (HPV) Integration Sites in Cervical Precancers Compromises the Diagnostic Accuracy of Integrant-Specific PCR

Human papillomavirus (HPV) DNA integration into the host genome is a frequent event in cervical carcinogenesis and may drive clonal expansion of the affected cells. Based on viral cellular junction (vcj) sequences, highly specific vcj-PCRs can be designed to detect viral integrants in DNA from cervical cell scrapes or tissue samples. In a recent study, such patient-specific vcj-PCR assays were employed for the detection of recurrent high-grade squamous intraepithelial lesions (HSIL) during postoperative surveillance. Although the specificity of vcj-PCR was 100%, only 50% of the recurrences were detected using this approach. The focus of the current study was to analyze the cause of this limited sensitivity. Using chemical microdissection and subsequent vcj-PCR analysis, we could demonstrate that the majority of lesions have a heterogeneous integrant pattern. Only 2 of 16 cones showed a homogeneous distribution of the respective integrants throughout the entire lesion. The other lesions displayed clonal outgrowths harboring the integrant in a background HPV16/18 DNA-positive HSIL tissue. In 4 cases, the respective integrant was undetectable in the lesion. These findings indicate that vcj-PCR has limited sensitivity for the detection of recurrent disease owing to intralesional heterogeneity. The observed heterogeneous integrant pattern may thus reflect the multifocal nature of most large HSIL. Alternatively, the possibility that HPV integration may be a late event in the carcinogenic process also needs to be considered.

Identification of a Locus Near ULK1 Associated With Progression-Free Survival in Ovarian Cancer

Abstract Background: Many loci have been found to be associated with risk of epithelial ovarian cancer (EOC). However, although there is considerable variation in progression-free survival (PFS), no loci have been found to be associated with outcome at genome-wide levels of significance. Methods: We carried out a genome-wide association study (GWAS) of PFS in 2,352 women with EOC who had undergone cytoreductive surgery and standard carboplatin/paclitaxel chemotherapy. Results: We found seven SNPs at 12q24.33 associated with PFS (P < 5 × 10–8), the top SNP being rs10794418 (HR = 1.24; 95% CI, 1.15–1.34; P = 1.47 × 10–8). High expression of a nearby gene, ULK1, is associated with shorter PFS in EOC, and with poor prognosis in other cancers. SNP rs10794418 is also associated with expression of ULK1 in ovarian tumors, with the allele associated with shorter PFS being associated with higher expression, and chromatin interactions were detected between the ULK1 promoter and associated SNPs in serous and endometrioid EOC cell lines. ULK1 knockout ovarian cancer cell lines showed significantly increased sensitivity to carboplatin in vitro. Conclusions: The locus at 12q24.33 represents one of the first genome-wide significant loci for survival for any cancer. ULK1 is a plausible candidate for the target of this association. Impact: This finding provides insight into genetic markers associated with EOC outcome and potential treatment options. See related commentary by Peres and Monteiro, p. 1604

Highly Cytotoxic Osmium(II) Compounds and Their Ruthenium(II) Analogues Targeting Ovarian Carcinoma Cell Lines and Evading Cisplatin Resistance Mechanisms

(1) Background: Ruthenium and osmium complexes attract increasing interest as next generation anticancer drugs. Focusing on structure-activity-relationships of this class of compounds, we report on 17 different ruthenium(II) complexes and four promising osmium(II) analogues with cinnamic acid derivatives as O,S bidentate ligands. The aim of this study was to determine the anticancer activity and the ability to evade platin resistance mechanisms for these compounds. (2) Methods: Structural characterizations and stability determinations have been carried out with standard techniques, including NMR spectroscopy and X-ray crystallography. All complexes and single ligands have been tested for cytotoxic activity on two ovarian cancer cell lines (A2780, SKOV3) and their cisplatin-resistant isogenic cell cultures, a lung carcinoma cell line (A549) as well as selected compounds on three non-cancerous cell cultures in vitro. FACS analyses and histone γH2AX staining were carried out for cell cycle distribution and cell death or DNA damage analyses, respectively. (3) Results: IC50 values show promising results, specifically a high cancer selective cytotoxicity and evasion of resistance mechanisms for Ru(II) and Os(II) compounds. Histone γH2AX foci and FACS experiments validated the high cytotoxicity but revealed diminished DNA damage-inducing activity and an absence of cell cycle disturbance thus pointing to another mode of action. (4) Conclusion: Ru(II) and Os(II) compounds with O,S-bidentate ligands show high cytotoxicity without strong effects on DNA damage and cell cycle, and this seems to be the basis to circumvent resistance mechanisms and for the high cancer cell specificity.

Triage of hrHPV-positive women: comparison of two commercial methylation-specific PCR assays

Abstract Aim High-risk human papillomavirus (hrHPV)-based screening is becoming increasingly important, either by supplementing or replacing the traditional cytology-based cervical Pap smear. However, hrHPV screening lacks specificity, because it cannot differentiate between transient virus infection and clinically relevant hrHPV-induced disease. Therefore, reliable triage methods are needed for the identification of HPV-positive women with cervical intraepithelial neoplasia (CIN) in need of treatment. Promising tools discussed for the triage of these patients are molecular diagnostic tests based on epigenetic markers. Here, we compare the performance of two commercially available DNA methylation-based diagnostic assays—GynTect® and the QIAsure Methylation Test—in physician-taken cervical scrapes from 195 subjects. Findings Both GynTect® and the QIAsure Methylation Test detected all cervical carcinoma and carcinoma in situ (CIS). The differences observed in the detection rates between both assays for the different grades of cervical lesions (QIAsure Methylation Test: CIN1 26.7%, CIN2 27.8% and CIN3 74.3%; GynTect®: CIN1 13.3%, CIN2 33.3% and CIN3 60%) were not significant. Concerning the false-positive rates, significant differences were evident. For the healthy (NILM) hrHPV-positive group, the false-positive rates were 5.7% for GynTect® and 26.4% for QIAsure Methylation Test (p = 0.003) and for the NILM hrHPV-negative group 2.2% vs. 23.9% (p = 0.006), respectively. When considering hrHPV-positive samples only for comparison (n = 149), GynTect® delivered significantly higher specificity compared to the QIAsure Methylation Test for CIN2 + (87.6% vs. 67.4% (p < 0.001)) and CIN3 + (84.1% vs. 68.2% (p = 0.002)). Overall our findings suggest that DNA methylation-based tests are suitable for the triage of hrHPV-positive women. With the goal to provide a triage test that complements the limited specificity of HPV testing in HPV-based screening, GynTect® may be preferable, due to its higher specificity for CIN2+ or CIN3+ .

Evidence for disseminated tumor cells in lymphatic vessels afferent to sentinel lymph nodes in patients diagnosed with cervical cancer

AbstractBackgroundIn patients diagnosed with cervical cancer, the purpose of lymphadenectomy is the removal of lymph nodes for diagnosis and potential treatment of metastasized tumor cells. It is unclear if afferent lymphatic vessels harbor tumor cells and, thus, may pose additional risk for recurrence or progression if not removed.AimIn this feasibility study, we analyzed the lymphatic vessels afferent to sentinel lymph node (SLN) using a highly sensitive and specific molecular marker for cervical cancer cells.Methods and ResultsTwenty patients diagnosed with cervical cancer of FIGO stage IA1 to IIB2 underwent laparoscopic SLN removal. Labeling was done using patent blue and the afferent lymphatic vessels were harvested from the parametric tissue and frozen at −80°C. HPV DNA type was evaluated in the primary tumor. Lymphatic vessels afferent to the sentinel lymph nodes were analyzed for the presence of viral oncogene transcripts of the respective HPV type. In one of 18 patients, all with tumor stage ≤IBI and pN0 by conventional histopathology, HPV mRNA could be detected in two of four lymphatic vessels, whereas at least one of the lymphatic vessel biopsies of both patients with tumors ˃4 cm and pN1 status was HPV mRNA positive. No clinical correlation with recurrence after a median follow‐up of 9 years was noticed.ConclusionHPV mRNA indicative of disseminated tumor cells could be detected in lymphatic vessels. The relevance of harvesting lymphatic vessels afferent to SLN in order to increase oncologic safety will have to be investigated in a future prospective study.