Radiosensitization by Hyperthermia Critically Depends on the Time Interval.

PURPOSE: Hyperthermia is a potent sensitizer of radiation therapy that improves both tumor control and survival in women with locally advanced cervical cancer (LACC). The optimal sequence and interval between hyperthermia and radiation therapy are still under debate. METHODS AND MATERIALS: We investigated the interval and sequence in vitro in cervical cancer cell lines, patient-derived organoids, and SiHa cervical cancer hind leg xenografts in athymic nude mice and compared the results with retrospective results from 58 women with LACC treated with thermoradiotherapy. RESULTS: All 3 approaches confirmed that shortening the interval between hyperthermia and radiation therapy enhanced hyperthermic radiosensitization by 2 to 8 times more DNA double-strand breaks and apoptosis and 10 to 100 times lower cell survival, delayed tumor growth in mice, and increased the 5-year survival rate of women with LACC from 22% (interval ≥80 minutes) to 54% (interval <80 minutes). In vitro and in vivo results showed that the sequence of hyperthermia and radiation therapy did not affect the outcome. CONCLUSIONS: Shortening the interval between hyperthermia and radiation therapy significantly improves treatment outcomes. The sequence of hyperthermia and radiation therapy (before or after) does not seem to matter.

DNA methylation markers for cancer risk prediction of vulvar intraepithelial neoplasia.

Current clinical and histological classifications are unable to determine the risk of vulvar squamous cell carcinoma (VSCC) in high-grade vulvar intraepithelial neoplasia (VIN), making prognostic biomarkers highly needed. We studied host-cell DNA methylation markers in high-grade squamous intraepithelial lesion (HSIL) and differentiated VIN (dVIN) without VSCC, in HSIL and dVIN adjacent to VSCC and in human papillomavirus (HPV) positive and negative VSCC, relative to control vulvar tissues. A series of 192 formalin-fixed paraffin-embedded vulvar samples, including VSCC (n = 58), VIN adjacent to VSCC (n = 30), VIN without VSCC during follow-up (n = 41) and normal vulvar tissues (n = 63), were tested for 12 DNA methylation markers with quantitative multiplex methylation-specific PCR (qMSP). HPV status was determined by p16INK4A immunohistochemistry and high-risk HPV PCR analysis. Logistic regression analyses were used to determine methylation patterns and methylation marker performance for VIN and VSCC detection. Methylation markers showed significantly higher methylation levels with increasing severity of disease. VIN adjacent to VSCC showed a similar methylation-high pattern as VSCC, while VIN without VSCC displayed a heterogeneous methylation pattern. Vulvar carcinogenesis is associated with increased DNA methylation. Higher DNA methylation levels in VIN seem to reflect higher cancer risk, emphasizing the high potential of DNA methylation biomarkers in the diagnostic workup of VIN. As a next step, longitudinal studies are needed to verify the prognostic value of methylation biomarkers as a clinical tool for stratification of cancer risk in women with VIN.

Altered microRNA processing proteins in HPV-induced cancers.

High-risk human papilloma virus (hrHPV) infections are associated with the development of anogenital cancers, in particular cervical cancer, and a subset of head and neck cancers. Previous studies have shown that microRNAs (miRNAs) contribute to the development and progression of HPV-induced malignancies. miRNAs are small non-coding RNAs that exist as multiple length and sequence variants, termed isomiRs. Efficient processing of miRNAs and generation of isomiRs is accomplished by several processing proteins. Deregulation of Drosha, AGO2, and TENT2, among others, has been observed in HPV-induced cancers and was even found at the precancerous stage. This suggests that miRNA processing proteins may be involved during early cancer development and that the generated isomiRs could provide promising biomarkers for early cancer diagnosis.

Cervical cancer detection by DNA methylation analysis in urine.

Urine samples provide a potential alternative to physician-taken or self-collected cervical samples for cervical screening. Screening by primary hrHPV testing requires additional risk assessment (so-called triage) of hrHPV-positive women. Molecular markers, such as DNA methylation, have proven most valuable for triage when applied to cervical specimens. This study was set out to compare hrHPV and DNA methylation results in paired urine and cervical scrapes, and to evaluate the feasibility of DNA methylation analysis in urine to detect cervical cancer. Urine samples (n = 41; native and sediment) and paired cervical scrapes (n = 38) from cervical cancer patients, and urine from 44 female controls, were tested for hrHPV and 6 methylation markers. Results on native urine and sediment were highly comparable. A strong agreement was found between hrHPV testing on urine and scrapes (kappa = 0.79). Also, methylation levels in urine were moderately to strongly correlated to those detected in scrapes (r = 0.508-0.717). All markers were significantly increased in urine from cervical cancer patients compared to controls and showed a good discriminatory power for cervical cancer (AUC = 0.744-0.887). Our results show a good agreement of urine-based molecular analysis with reference cervical samples, and suggest that urine-based DNA methylation testing may provide a promising strategy for cervical cancer detection.

Genome-wide microRNA analysis of HPV-positive self-samples yields novel triage markers for early detection of cervical cancer.

Offering self-sampling for HPV testing improves the effectiveness of current cervical screening programs by increasing population coverage. Molecular markers directly applicable on self-samples are needed to stratify HPV-positive women at risk of cervical cancer (so-called triage) and to avoid over-referral and overtreatment. Deregulated microRNAs (miRNAs) have been implicated in the development of cervical cancer, and represent potential triage markers. However, it is unknown whether deregulated miRNA expression is reflected in self-samples. Our study is the first to establish genome-wide miRNA profiles in HPV-positive self-samples to identify miRNAs that can predict the presence of CIN3 and cervical cancer in self-samples. Small RNA sequencing (sRNA-Seq) was conducted to determine genome-wide miRNA expression profiles in 74 HPV-positive self-samples of women with and without cervical precancer (CIN3). The optimal miRNA marker panel for CIN3 detection was determined by GRridge, a penalized method on logistic regression. Six miRNAs were validated by qPCR in 191 independent HPV-positive self-samples. Classification of sRNA-Seq data yielded a 9-miRNA marker panel with a combined area under the curve (AUC) of 0.89 for CIN3 detection. Validation by qPCR resulted in a combined AUC of 0.78 for CIN3+ detection. Our study shows that deregulated miRNA expression associated with CIN3 and cervical cancer development can be detected by sRNA-Seq in HPV-positive self-samples. Validation by qPCR indicates that miRNA expression analysis offers a promising novel molecular triage strategy for CIN3 and cervical cancer detection applicable to self-samples.

Triage of high-risk HPV-positive women in population-based screening by miRNA expression analysis in cervical scrapes; a feasibility study.

Background: Primary testing for high-risk HPV (hrHPV) is increasingly implemented in cervical cancer screening programs. Many hrHPV-positive women, however, harbor clinically irrelevant infections, demanding additional disease markers to prevent over-referral and over-treatment. Most promising biomarkers reflect molecular events relevant to the disease process that can be measured objectively in small amounts of clinical material, such as miRNAs. We previously identified eight miRNAs with altered expression in cervical precancer and cancer due to either methylation-mediated silencing or chromosomal alterations. In this study, we evaluated the clinical value of these eight miRNAs on cervical scrapes to triage hrHPV-positive women in cervical screening. Results: Expression levels of the eight candidate miRNAs in cervical tissue samples (n = 58) and hrHPV-positive cervical scrapes from a screening population (n = 187) and cancer patients (n = 38) were verified by quantitative RT-PCR. In tissue samples, all miRNAs were significantly differentially expressed (p < 0.05) between normal, high-grade precancerous lesions (CIN3), and/or cancer. Expression patterns detected in cervical tissue samples were reflected in cervical scrapes, with five miRNAs showing significantly differential expression between controls and women with CIN3 and cancer. Using logistic regression analysis, a miRNA classifier was built for optimal detection of CIN3 in hrHPV-positive cervical scrapes from the screening population and its performance was evaluated using leave-one-out cross-validation. This miRNA classifier consisted of miR-15b-5p and miR-375 and detected a major subset of CIN3 as well as all carcinomas at a specificity of 70%. The CIN3 detection rate was further improved by combining the two miRNAs with HPV16/18 genotyping. Interestingly, both miRNAs affected the viability of cervical cancer cells in vitro. Conclusions: This study shows that miRNA expression analysis in cervical scrapes is feasible and enables the early detection of cervical cancer, thus underlining the potential of miRNA expression analysis for triage of hrHPV-positive women in cervical cancer screening.

Identification and Validation of a 3-Gene Methylation Classifier for HPV-Based Cervical Screening on Self-Samples.

Purpose: Offering self-sampling of cervico-vaginal material for high-risk human papillomavirus (hrHPV) testing is an effective method to increase the coverage in cervical screening programs. Molecular triage directly on hrHPV-positive self-samples for colposcopy referral opens the way to full molecular cervical screening. Here, we set out to identify a DNA methylation classifier for detection of cervical precancer (CIN3) and cancer, applicable to lavage and brush self-samples.Experimental Design: We determined genome-wide DNA methylation profiles of 72 hrHPV-positive self-samples, using the Infinium Methylation 450K Array. The selected DNA methylation markers were evaluated by multiplex quantitative methylation-specific PCR (qMSP) in both hrHPV-positive lavage (n = 245) and brush (n = 246) self-samples from screening cohorts. Subsequently, logistic regression analysis was performed to build a DNA methylation classifier for CIN3 detection applicable to self-samples of both devices. For validation, an independent set of hrHPV-positive lavage (n = 199) and brush (n = 287) self-samples was analyzed.Results: Genome-wide DNA methylation profiling revealed 12 DNA methylation markers for CIN3 detection. Multiplex qMSP analysis of these markers in large series of lavage and brush self-samples yielded a 3-gene methylation classifier (ASCL1, LHX8, and ST6GALNAC5). This classifier showed a very good clinical performance for CIN3 detection in both lavage (AUC = 0.88; sensitivity = 74%; specificity = 79%) and brush (AUC = 0.90; sensitivity = 88%; specificity = 81%) self-samples in the validation set. Importantly, all self-samples from women with cervical cancer scored DNA methylation-positive.Conclusions: By genome-wide DNA methylation profiling on self-samples, we identified a highly effective 3-gene methylation classifier for direct triage on hrHPV-positive self-samples, which is superior to currently available methods. Clin Cancer Res; 24(14); 3456-64. ©2018 AACR.

A Strategy to Find Suitable Reference Genes for miRNA Quantitative PCR Analysis and Its Application to Cervical Specimens.

miRNAs represent an emerging class of promising biomarkers for cancer diagnostics. To perform reliable miRNA expression analysis using quantitative PCR, adequate data normalization is essential to remove nonbiological, technical variations. Ideal reference genes should be biologically stable and reduce technical variability of miRNA expression analysis. Herein is a new strategy for the identification and evaluation of reference genes that can be applied for miRNA-based diagnostic tests without entailing excessive additional experiments. We analyzed the expression of 11 carefully selected candidate reference genes in different types of cervical specimens [ie, tissues, scrapes, and self-collected cervicovaginal specimens (self-samples)]. To identify the biologically most stable reference genes, three commonly used algorithms (GeNorm, NormFinder, and BestKeeper) were combined. Signal-to-noise ratios and P values between control and disease groups were calculated to validate the reduction in technical variability on expression analysis of two marker miRNAs. miR-423 was identified as a suitable reference gene for all sample types, to be used in combination with RNU24 in cervical tissues, RNU43 in scrapes, and miR-30b in self-samples. These findings demonstrate that the choice of reference genes may differ between different types of specimens, even when originating from the same anatomical source. More important, it is shown that adequate normalization increases the signal-to-noise ratio, which is not observed when normalizing to commonly used reference genes.

Better diagnostic signatures from RNAseq data through use of auxiliary co-data.

SUMMARY: Our aim is to improve omics based prediction and feature selection using multiple sources of auxiliary information: co-data. Adaptive group regularized ridge regression (GRridge) was proposed to achieve this by estimating additional group-based penalty parameters through an empirical Bayes method at a low computational cost. We illustrate the GRridge method and software on RNA sequencing datasets. The method boosts the performance of an ordinary ridge regression and outperforms other classifiers. Post-hoc feature selection maintains the predictive ability of the classifier with far fewer markers. AVAILABILITY AND IMPLEMENTATION: GRridge is an R package that includes a vignette. It is freely available at ( https://bioconductor.org/packages/GRridge/ ). All information and R scripts used in this study, including those on retrieval and processing of the co-data, are available from http://github.com/markvdwiel/GRridgeCodata . CONTACT: mark.vdwiel@vumc.nl. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Aberrant methylation-mediated silencing of microRNAs contributes to HPV-induced anchorage independence.

Cervical cancer and a subset of anogenital and head-and-neck carcinomas are caused by high-risk types of the human papillomavirus (hrHPV). During hrHPV-induced malignant transformation keratinocytes become able to grow anchorage independently, a tumorigenic trait at least partly associated with inactivation of tumor suppressor genes. We used hrHPV-containing keratinocytes to investigate the role of DNA methylation-mediated silencing of microRNAs (miRNAs) in the acquisition of anchorage independence.Anchorage dependent (n=11) and independent passages (n=19) of 4 hrHPV-immortalized keratinocyte cell lines were treated with 2'-deoxy-5-azacytidine (DAC). Genome-wide miRNA expression profiles before and after treatment were compared to identify miRNAs silenced by methylation. Bisulfite sequencing and methylation-specific PCR showed increased methylation of hsa-mir-129-2/-137/-935/-3663/-3665 and -4281 in anchorage independent HPV-transformed keratinocytes and cervical cancer cell lines. Mature miRNAs derived from hsa-mir-129-2/-137/-3663 and -3665 showed functional relevance as they decreased anchorage independence in cervical cancer cell lines. Cervical (pre)cancerous lesions demonstrated increased methylation of hsa-mir-129-2/-935/-3663/-3665 and -4281, underlining the clinical relevance of our findings.In conclusion, methylation-mediated silencing of tumor suppressive miRNAs contributes to acquisition of an anchorage independent phenotype. This study further substantiates the importance of miRNAs during early stages of carcinogenesis and underlines their potential as both disease markers and therapeutic targets.

Role of E3 ubiquitin ligases in lung cancer.

E3 ubiquitin ligases are a large family of proteins that catalyze the ubiquitination of many protein substrates for targeted degradation by the 26S proteasome. Therefore, E3 ubiquitin ligases play an essential role in a variety of biological processes including cell cycle regulation, proliferation and apoptosis. E3 ubiquitin ligases are often found overexpressed in human cancers, including lung cancer, and their deregulation has been shown to contribute to cancer development. However, the lack of specific inhibitors in clinical trials is a major issue in targeting E3 ubiquitin ligases with currently only one E3 ubiquitin ligase inhibitor being tested in the clinical setting. In this review, we focus on E3 ubiquitin ligases that have been found deregulated in lung cancer. Furthermore, we discuss the processes in which they are involved and evaluate them as potential anti-cancer targets. By better understanding the mechanisms by which E3 ubiquitin ligases regulate biological processes and their exact role in carcinogenesis, we can improve the development of specific E3 ubiquitin ligase inhibitors and pave the way for novel treatment strategies for cancer patients.