Human papillomavirus integration transforms chromatin to drive oncogenesis.

BACKGROUND: Human papillomavirus (HPV) drives almost all cervical cancers and up to 70% of head and neck cancers. Frequent integration into the host genome occurs predominantly in tumorigenic types of HPV. We hypothesize that changes in chromatin state at the location of integration can result in changes in gene expression that contribute to the tumorigenicity of HPV. RESULTS: We find that viral integration events often occur along with changes in chromatin state and expression of genes near the integration site. We investigate whether introduction of new transcription factor binding sites due to HPV integration could invoke these changes. Some regions within the HPV genome, particularly the position of a conserved CTCF binding site, show enriched chromatin accessibility signal. ChIP-seq reveals that the conserved CTCF binding site within the HPV genome binds CTCF in 4 HPV+ cancer cell lines. Significant changes in CTCF binding pattern and increases in chromatin accessibility occur exclusively within 100 kbp of HPV integration sites. The chromatin changes co-occur with out-sized changes in transcription and alternative splicing of local genes. Analysis of The Cancer Genome Atlas (TCGA) HPV+ tumors indicates that HPV integration upregulates genes which have significantly higher essentiality scores compared to randomly selected upregulated genes from the same tumors. CONCLUSIONS: Our results suggest that introduction of a new CTCF binding site due to HPV integration reorganizes chromatin state and upregulates genes essential for tumor viability in some HPV+ tumors. These findings emphasize a newly recognized role of HPV integration in oncogenesis.

Cell-free DNA topology depends on its subcellular and cellular origins in cancer.

Cancer cells release large quantities of cell-free DNA (cfDNA) into the surrounding tissue and circulation. As cfDNA is a common source of biomarkers for liquid biopsy and has been implicated as a functional mediator for intercellular communication, fundamental characterization of cfDNA topology has widespread biological and clinical ramifications. Whether the topology of cfDNA is such that it exists predominantly in membrane-bound extracellular vesicles (EVs) or in nonvesicular DNA-protein complexes remains poorly understood. Here, we employed a DNA-targeted approach to comprehensively assess total cfDNA topology in cancer. Using preclinical models and patient samples, we demonstrate that nuclear cfDNA is predominantly associated with nucleosomal particles and not EVs, while a substantial subset of mitochondrial cfDNA is membrane protected and disproportionately derived from nontumor cells. In addition, discrimination between membrane-protected and accessible mitochondrial cfDNA added diagnostic and prognostic value in a cohort of head and neck cancer patients. Our results support a revised model for cfDNA topology in cancer. Due to its abundance, nuclear cfDNA within nucleosomal particles is the most compelling liquid biopsy substrate, while EV-bound and accessible mitochondrial cfDNA represent distinct reservoirs of potential cancer biomarkers whose structural conformations may also influence their extracellular stability and propensity for uptake by recipient cells.

HPV Sequencing Facilitates Ultrasensitive Detection of HPV Circulating Tumor DNA.

PURPOSE: Human papillomavirus (HPV) DNA offers a convenient circulating tumor DNA (ctDNA) marker for HPV-associated malignancies, but current methods, such as digital PCR (dPCR), provide insufficient accuracy for clinical applications in patients with low disease burden. We asked whether a next-generation sequencing approach, HPV sequencing (HPV-seq), could provide quantitative and qualitative assessment of HPV ctDNA in low disease burden settings. EXPERIMENTAL DESIGN: We conducted preclinical technical validation studies on HPV-seq and applied it retrospectively to a prospective multicenter cohort of patients with locally advanced cervix cancer (NCT02388698) and a cohort of patients with oropharynx cancer. HPV-seq results were compared with dPCR. The primary outcome was progression-free survival (PFS) according to end-of-treatment HPV ctDNA detectability. RESULTS: HPV-seq achieved reproducible detection of HPV DNA at levels less than 0.6 copies in cell line data. HPV-seq and dPCR results for patients were highly correlated (R 2 = 0.95, P = 1.9 × 10-29) with HPV-seq detecting ctDNA at levels down to 0.03 copies/mL plasma in dPCR-negative posttreatment samples. Detectable HPV ctDNA at end-of-treatment was associated with inferior PFS with 100% sensitivity and 67% specificity for recurrence. Accurate HPV genotyping was successful from 100% of pretreatment samples. HPV ctDNA fragment sizes were consistently shorter than non-cancer-derived cell-free DNA (cfDNA) fragments, and stereotyped cfDNA fragmentomic patterns were observed across HPV genomes. CONCLUSIONS: HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR and reveals qualitative information about ctDNA. Our findings in this proof-of-principle study could have implications for treatment monitoring of disease burden in HPV-related cancers. Future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates.

Liquid Biopsy Goes Viral: Next-Generation Sequencing to Enhance HPV Detection.

Liquid biopsy approaches for the detection of viral DNA can provide important information for the diagnosis and identification of virally-associated cancers. Here we discuss the next-generation sequencing-based CaptHPV method for the detection and characterization of plasma human papillomavirus (HPV) DNA in HPV-associated cancers and its potential clinical utility. See related article by Sastre-Garau et al., p. 5307.

Serial Cell-free DNA Assessments in Preclinical Models.

Studying circulating cell-free DNA (cfDNA) release within preclinical model systems provides opportunities to investigate the mechanisms and kinetics underlying this process under various conditions. We present a detailed protocol for longitudinal evaluation of cfDNA release through (1) seeding of cancer cell lines and establishment of xenograft tumors, (2) treatment of cancer cells and xenograft tumors, (3) serial collection of cell line media and xenograft blood, and (4) processing and isolation of cfDNA for (5) quantification of cfDNA by quantitative PCR. For complete details on the use and execution of this protocol please refer to Rostami et al. (2020).

Senescence, Necrosis, and Apoptosis Govern Circulating Cell-free DNA Release Kinetics.

The kinetics of circulating cell-free DNA (cfDNA) release may provide a real-time assessment of induced cell death. However, there is a limited understanding of the underlying biological rationale for cfDNA release following distinct treatments and cell death mechanisms. Here, we uncover a complex interplay between apoptosis, necrosis, and senescence in determining cfDNA release kinetics. Utilizing multiple in vitro and in vivo preclinical models, we show how cfDNA release is modulated through a combination of apoptotic and senescent triggers and inhibitors. Interestingly, we identify treatment-induced senescence as a previously unrecognized determinant of cfDNA kinetics that can counteract its release. Necrosis is the predominant cell death mechanism that consistently contributes to cfDNA release in response to ionizing radiation, and, surprisingly, apoptosis plays a comparatively minor role in some tumors. Based on our results, we propose a model to explain cfDNA release from cells over time, with important implications for future studies.

The Utility of Liquid Biopsies in Radiation Oncology.

The use of therapeutic radiation is primarily guided by clinicopathologic factors and medical imaging, whereas molecular biomarkers currently play a comparatively minor role in most settings. Liquid biopsies provide a rich source of noninvasive tumor-specific biomarkers and are amenable to repeated and noninvasive assessment. Here, we review the current status of liquid biopsies and their potential impact on the field of radiation oncology. We focus on established and emerging approaches to analyze circulating tumor DNA and circulating tumor cells from peripheral blood. These promising classes of biomarkers could have an outsized impact on cancer management by meaningfully stratifying patients into risk groups, tracking radiation therapy efficacy during and after treatment, and identifying patients with radiosensitive or radioresistant disease. Finally, we highlight opportunities for future investigation including the need for prospective interventional studies employing liquid biopsies to guide the management of radiation therapy-treated patients.

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells.

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

Utilizing circulating tumour DNA in radiation oncology.

Emerging technologies for detection of circulating tumour DNA (ctDNA) are expanding the possibilities for clinical impact to patients with localized, potentially curable cancer. For such patients, ctDNA analysis could aid in prognostication, prediction of treatment response, longitudinal monitoring for adaptive treatment, and evaluation of minimal residual disease. Radiation oncologists currently have few tools at their disposal for predicting or rapidly assessing treatment efficacy. By reflecting the genetic and epigenetic makeup of tumours as well as dynamic changes with treatment, ctDNA as a biomarker for radiation response could enable new personalized treatment approaches. In this review, we will discuss recent advances in ctDNA technologies and potential clinical applications of ctDNA analysis throughout the therapeutic course. Furthermore, we will consider how ctDNA analysis could someday guide radiotherapy prescriptions by revealing differences in tumour radiophenotype.

Cell-free DNA as a post-treatment surveillance strategy: current status.

Circulating tumor DNA (ctDNA) consists of cell-free DNA (cfDNA) fragments that are released from tumor cells into the bloodstream. ctDNA harbors cancer-specific genetic and epigenetic alterations that allow its detection and quantification using a variety of emerging techniques. The promise of convenient non-invasive access to the complex and dynamic molecular features of cancer through peripheral blood has galvanized translational researchers around this topic with compelling routes to clinical implementation, particularly in the post-treatment surveillance setting. Although analysis methods must contend with the small quantities of ctDNA present in most patients, and the relative over-abundance of background cfDNA derived from normal tissues, recent technical innovations have led to dramatic improvements in the sensitivity of ctDNA detection. As a result, ever more studies are investigating the clinical utility of ctDNA for applications in (1) treatment response assessment, (2) identification of emerging resistance mechanisms, (3) minimal residual disease detection, and (4) characterization of clonal heterogeneity and selection. In this review, we describe the detection methods currently used in clinical studies to assess low fractions of ctDNA, as well as their utility in the applications previously described. Finally, we address current limitations that have hampered the clinical implementation of ctDNA analysis for post-treatment surveillance and propose steps that could be made to address them.

Diastereoselective Synthesis of C2'-Fluorinated Nucleoside Analogues Using an Acyclic Approach.

Nucleoside analogues bearing a fluorine in the C2'-position have been synthesized by SN2-like cyclizations of acyclic thioaminal precursors. This strategy provides access to two scaffolds, d-1',2'-cis-thiofuranosides and d-1',2'-trans-furanosides, which are difficult to generate using the standard approach for nucleoside synthesis. The addition of silylated nucleobases onto model C2-fluorinated dithioacetal substrates resulted in 1,2-syn diastereoselectivity, which is consistent with the C2-F and S-alkyl moiety being in close proximity. A new series of analogues bearing a C3' all-carbon quaternary center along with a C2'-F atom have also been synthesized using this approach and are being investigated as potential antimetabolites.