Publications by authors named "Heidi Lyng"

50 Publications

Amino acid transporter expression and 18F-FACBC uptake at PET in primary prostate cancer.

Am J Nucl Med Mol Imaging 2021 15;11(4):250-259. Epub 2021 Aug 15.

Division of Radiology and Nuclear Medicine, Oslo University Hospital Oslo, Norway.

Little is known about the transport mechanism of -3-18F-fluorocyclobutane-1-carboxylic acid (FACBC) into prostate tumors. Because of the structural similarity to natural amino acids, FACBC is anticipated to cross the cell membrane via amino acid transporters, and preclinical studies have suggested that ASCT2, LAT1 and SNAT2 are involved. In 16 patients with intermediate or high-risk prostate cancer we matched the FACBC uptake from clinical PET to the location of punch biopsies from resected prostatectomy specimens and compared maximum standardized uptake value (SUVmax) with the gene expression of 40 amino acid transporters. The study also included immunohistochemistry for the three amino acid transporters ASCT2, LAT1 and SNAT2. Furthermore, we performed global gene expression analysis of the biopsies to investigate biological processes associated with FACBC uptake. Several amino acid transporters had a higher gene expression level than the others, but we found no significant correlations between SUVmax and the gene expression levels of any of 40 different amino acid transporters. In the immunohistochemical analyses, ASCT2 and SNAT2 were highly expressed, but not correlated to SUVmax. LAT1 had low gene- and protein expression. Global gene expression analyses identified 153 unique genes that were positively correlated to SUVmax. These genes were found to be associated with gene sets reflecting intracellular transport and high metabolic activity. Based on the study findings we propose that the uptake mechanism of FACBC is more complex than mediated by a few amino acid transporters.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8414398PMC
August 2021

Tumor Hypoxia as a Barrier in Cancer Therapy: Why Levels Matter.

Cancers (Basel) 2021 Jan 28;13(3). Epub 2021 Jan 28.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, 0424 Oslo, Norway.

Hypoxia arises in tumor regions with insufficient oxygen supply and is a major barrier in cancer treatment. The distribution of hypoxia levels is highly heterogeneous, ranging from mild, almost non-hypoxic, to severe and anoxic levels. The individual hypoxia levels induce a variety of biological responses that impair the treatment effect. A stronger focus on hypoxia levels rather than the absence or presence of hypoxia in our investigations will help development of improved strategies to treat patients with hypoxic tumors. Current knowledge on how hypoxia levels are sensed by cancer cells and mediate cellular responses that promote treatment resistance is comprehensive. Recently, it has become evident that hypoxia also has an important, more unexplored role in the interaction between cancer cells, stroma and immune cells, influencing the composition and structure of the tumor microenvironment. Establishment of how such processes depend on the hypoxia level requires more advanced tumor models and methodology. In this review, we describe promising model systems and tools for investigations of hypoxia levels in tumors. We further present current knowledge and emerging research on cellular responses to individual levels, and discuss their impact in novel therapeutic approaches to overcome the hypoxia barrier.
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http://dx.doi.org/10.3390/cancers13030499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866096PMC
January 2021

MRI Distinguishes Tumor Hypoxia Levels of Different Prognostic and Biological Significance in Cervical Cancer.

Cancer Res 2020 09 30;80(18):3993-4003. Epub 2020 Jun 30.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

Tumor hypoxia levels range from mild to severe and have different biological and therapeutical consequences but are not easily assessable in patients. Here we present a method based on diagnostic dynamic contrast enhanced (DCE) MRI that reflects a continuous range of hypoxia levels in patients with tumors of cervical cancer. Hypoxia images were generated using an established approach based on pixel-wise combination of DCE-MRI parameters and , representing oxygen consumption and supply, respectively. Using two tumor models, an algorithm to retrieve surrogate measures of hypoxia levels from the images was developed and validated by comparing the MRI-defined levels with hypoxia levels reflected in pimonidazole-stained histologic sections. An additional indicator of hypoxia levels in patient tumors was established on the basis of expression of nine hypoxia-responsive genes; a strong correlation was found between these indicator values and MRI-defined hypoxia levels in 63 patients. Chemoradiotherapy outcome of 74 patients was most strongly predicted by moderate hypoxia levels, whereas more severe or milder levels were less predictive. By combining gene expression profiles and MRI-defined hypoxia levels in cancer hallmark analysis, we identified a distribution of levels associated with each hallmark; oxidative phosphorylation and G-M checkpoint were associated with moderate hypoxia, epithelial-to-mesenchymal transition, and inflammatory responses with significantly more severe levels. At the mildest levels, IFN response hallmarks together with HIF1A protein expression by IHC appeared significant. Thus, our method visualizes the distribution of hypoxia levels within patient tumors and has potential to distinguish levels of different prognostic and biological significance. SIGNIFICANCE: These findings present an approach to image a continuous range of hypoxia levels in tumors and demonstrate the combination of imaging with molecular data to better understand the biology behind these different levels.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0950DOI Listing
September 2020

Combining imaging- and gene-based hypoxia biomarkers in cervical cancer improves prediction of chemoradiotherapy failure independent of intratumour heterogeneity.

EBioMedicine 2020 Jul 21;57:102841. Epub 2020 Jun 21.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Ullernchausseen 70, 0379 Oslo, Norway; Department of Physics, University of Oslo, Sem Sælands vei 24, 0371 Oslo, Norway. Electronic address:

Background: Emerging biomarkers from medical imaging or molecular characterization of tumour biopsies open up for combining the two and exploiting their synergy in treatment planning of cancer patients. We generated a paired data set of imaging- and gene-based hypoxia biomarkers in cervical cancer, appraised the influence of intratumour heterogeneity in patient classification, and investigated the benefit of combining the methodologies in prediction of chemoradiotherapy failure.

Methods: Hypoxic fraction from dynamic contrast enhanced (DCE)-MR images and an expression signature of six hypoxia-responsive genes were assessed as imaging- and gene-based biomarker, respectively in 118 patients.

Findings: Dichotomous biomarker cutoff to yield similar hypoxia status by imaging and genes was defined in 41 patients, and the association was validated in the remaining 77 patients. The two biomarkers classified 75% of 118 patients with the same hypoxia status, and inconsistent classification was not related to imaging-defined intratumour heterogeneity in hypoxia. Gene-based hypoxia was independent on tumour cell fraction in the biopsies and showed minor heterogeneity across multiple samples in 9 tumours. Combining imaging- and gene-based classification gave a significantly better prediction of PFS than one biomarker alone. A combined dichotomous biomarker optimized in 77 patients showed a large separation in PFS between more and less hypoxic tumours, and separated the remaining 41 patients with different PFS. The combined biomarker showed prognostic value together with tumour stage in multivariate analysis.

Interpretation: Combining imaging- and gene-based biomarkers may enable more precise and informative assessment of hypoxia-related chemoradiotherapy resistance in cervical cancer.

Funding: Norwegian Cancer Society, South-Eastern Norway Regional Health Authority, and Norwegian Research Council.
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http://dx.doi.org/10.1016/j.ebiom.2020.102841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317686PMC
July 2020

Influx rate of F-fluoroaminosuberic acid reflects cystine/glutamate antiporter expression in tumour xenografts.

Eur J Nucl Med Mol Imaging 2019 Sep 1;46(10):2190-2198. Epub 2019 Jul 1.

Department of Physics, University of Oslo, P.O. Box 1048 Blindern, 0316, Oslo, Norway.

Purpose: F-fluoroaminosuberic acid (F-FASu) is a recently developed amino acid tracer for positron emission tomography (PET) of oxidative stress that may offer improved tumour assessment over the conventional tracer F-fluorodeoxyglucose (F-FDG). Our aim was to evaluate and relate dynamic F-FASu and F-FDG uptake with pharmacokinetic modelling to transporter protein expression levels in a panel of diverse tumour xenograft lines.

Methods: Four different tumour xenograft lines were implanted in female athymic nude mice: MAS98.12 and HBCx3 (breast), TPMX (osteosarcoma) and A549 (lung). Dynamic PET over 60 min was performed on a small animal unit. The time-activity curves (TACs) for F-FASu and F-FDG in individual tumours were used to extract early (SUV; 2 min p.i.) and late (SUV; 55 min p.i.) standardised uptake values. Pharmacokinetic two-tissue compartment models were applied to the TACs to estimate rate constants K-k and blood volume fraction v. Relative levels of cystine/glutamate antiporter subunit xCT were assessed by western blotting, and expression of GLUT1 and CD31 by immunohistochemistry.

Results: F-FASu showed higher SUV, whilst F-FDG exhibited higher SUV. Influx rate K for F-FASu was significantly correlated with xCT levels (p = 0.001) and was significantly higher than K for F-FDG (p < 0.001). K for F-FDG was significantly correlated with GLUT1 levels (p = 0.002). v estimated from F-FASu and F-FDG TACs was highly consistent and significantly correlated (r = 0.85, p < 0.001). Two qualitatively different F-FASu uptake profiles were identified: type α with low xCT expression and low K (A549 and HBCx3), and type β with high xCT expression and high K (MAS98.12 and TPMX).

Conclusion: The influx rate of F-FASu reflects xCT activity in tumour xenografts. Dynamic PET with pharmacokinetic modelling is needed to fully appraise F-FASu distribution routes.
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http://dx.doi.org/10.1007/s00259-019-04375-8DOI Listing
September 2019

PTEN Expression in Prostate Cancer: Relationship With Clinicopathologic Features and Multiparametric MRI Findings.

AJR Am J Roentgenol 2019 Mar 19:1-9. Epub 2019 Mar 19.

6 Department for Research and Development, Division for Radiology and Nuclear Medicine, Oslo University Hospital, Oslo, Norway.

Objective: The objective of our study was to investigate whether phosphatase and tensin homolog (PTEN) expression is associated with clinicopathologic features and multiparametric MRI findings in prostate cancer.

Materials And Methods: Forty-three patients with prostate cancer who underwent radical prostatectomy were included. Index tumor was identified on pretreatment MRI and delineated in the area that correlated best with histopathology results. The apparent diffusion coefficient (ADC) from DWI and pharmacokinetic parameters derived from dynamic contrast-enhanced MRI (DCE-MRI) using the extended Tofts model (K, k, v, and v) within the tumor were estimated. The following clinicopathologic parameters were assessed: pretreatment serum levels of prostate-specific antigen, disseminated tumor cell status, age, Gleason score, tumor size, extraprostatic extension (EPE), tumor location, and lymph node metastases. Gene expression profiles were acquired in biopsies from the tumor using bead arrays, and validated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) on a different part of the biopsy.

Results: Based on bead arrays (p = 0.006) and RT-qPCR (p = 0.03) data, a significantly lower ADC was found in tumors with low PTEN expression. Moreover, PTEN expression was negatively associated with lymph node metastases (bead arrays, p = 0.008; RT-qPCR, p < 0.001). A weak but significant association between PTEN expression, EPE (p = 0.048), and Gleason score (p = 0.028) was revealed on bead arrays. ADC was negatively correlated with Gleason score (p = 0.001) and tumor size (p = 0.023). No association among DCE parameters, PTEN expression, and clinicopathologic features was found.

Conclusion: ADC derived from DWI may be useful in selecting patients with potentially aggressive tumor caused by PTEN deficiency.
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http://dx.doi.org/10.2214/AJR.18.20743DOI Listing
March 2019

Mitochondrial Function of CKS2 Oncoprotein Links Oxidative Phosphorylation with Cell Division in Chemoradioresistant Cervical Cancer.

Neoplasia 2019 04 8;21(4):353-362. Epub 2019 Mar 8.

Department of Radiation Biology, Oslo University Hospital, Oslo, Norway. Electronic address:

CDK regulatory subunit 2 (CKS2) has a nuclear function that promotes cell division and is a candidate biomarker of chemoradioresistance in cervical cancer. The underlying mechanisms are, however, not completely understood. We investigated whether CKS2 also has a mitochondrial function that augments tumor aggressiveness. Based on global gene expression data of two cervical cancer cohorts of 150 and 135 patients, we identified a set of genes correlated with CKS2 expression. Gene set enrichment analysis showed enrichment of mitochondrial cellular compartments, and the hallmarks oxidative phosphorylation (OXPHOS) and targets of the MYC oncogene in the gene set. By in situ proximity ligation assay, we showed that CKS2 formed complex with the positively correlated MYC target, mitochondrial single-stranded DNA binding protein SSBP1, in the mitochondrion of cervix tumor samples and HeLa and SiHa cervical cancer cell lines, indicating a role in mitochondrial DNA (mtDNA) replication and thereby OXPHOS. CDK1 was found to be part of the complex. Flow cytometry analyses of HeLa cells showed cell cycle regulation of the CKS2-SSBP1 complex consistent with mtDNA replication activity. Moreover, repression of mtDNA replication and OXPHOS by acute hypoxia decreased CKS2-SSBP1 complex abundance and expression of MYC targets. By immunohistochemistry, cytoplasmic CKS2 expression was found to add to the prognostic impact of nuclear CKS2 expression in patients, suggesting that the mitochondrial function promotes tumor aggressiveness. Our study uncovers a novel link between regulation of cell division by nuclear pathways and OXPHOS in the mitochondrion that involves CKS2 and promotes chemoradioresistance of cervical cancer.
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http://dx.doi.org/10.1016/j.neo.2019.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6411633PMC
April 2019

Reference MicroRNAs for RT-qPCR Assays in Cervical Cancer Patients and Their Application to Studies of HPV16 and Hypoxia Biomarkers.

Transl Oncol 2019 Mar 17;12(3):576-584. Epub 2019 Jan 17.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway. Electronic address:

MicroRNA (miRNA) expressions in tumor biopsies have shown potential as biomarkers in cervical cancer, but suitable reference RNAs for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays in patient cohorts with different clinicopathological characteristics are not available. We aimed to identify the optimal reference miRNAs and apply these to investigate the potential of miR-9-5p as human papilloma virus (HPV) 16 biomarker and miR-210-3p as hypoxia biomarker in cervical cancer. Candidate reference miRNAs were preselected in sequencing data of 90 patients and ranked in a stability analysis by RefFinder. A selection of the most stable miRNAs was evaluated by geNorm and NormFinder analyses of RT-qPCR data of 29 patients. U6 small nuclear RNA (RNU6) was also included in the evaluation. MiR-9-5p and miR-210-3p expression was assessed by RT-qPCR in 45 and 65 patients, respectively. Nine candidates were preselected in the sequencing data after excluding those associated with clinical markers, HPV type, hypoxia status, suboptimal expression levels, and low stability. In RT-qPCR assays, the combination of miR-151-5p, miR-152-3p, and miR-423-3p was identified as the most stable normalization factor across clinical markers, HPV type, and hypoxia status. RNU6 showed poor stability. By applying the optimal reference miRNAs, higher miR-9-5p expression in HPV16- than HPV18-positive tumors and higher miR-210-3p expression in more hypoxic than less hypoxic tumors were found in accordance with the sequencing data. MiR-210-3p was associated with poor outcome by both sequencing and RT-qPCR assays. In conclusion, miR-151-5p, miR-152-3p, and miR-423-3p are suitable reference miRNAs in cervical cancer. MiR-9-5p and miR-210-3p are promising HPV16 and hypoxia biomarkers, respectively.
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http://dx.doi.org/10.1016/j.tranon.2018.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349320PMC
March 2019

Potentials and challenges of diffusion-weighted magnetic resonance imaging in radiotherapy.

Clin Transl Radiat Oncol 2018 Nov 20;13:29-37. Epub 2018 Sep 20.

Section for Biomedical Physics, Department of Radiation Oncology, University Hospital Tübingen, Germany.

Purpose: To review the potential and challenges of integrating diffusion weighted magnetic resonance imaging (DWI) into radiotherapy (RT).

Content: Details related to image acquisition of DWI for RT purposes are discussed, along with the challenges with respect to geometric accuracy and the robustness of quantitative parameter extraction. An overview of diffusion- and perfusion-related parameters derived from mono- and bi-exponential models is provided, and their role as potential RT biomarkers is discussed. Recent studies demonstrating potential of DWI in different tumor sites such as the head and neck, rectum, cervix, prostate, and brain, are reviewed in detail.

Conclusion: DWI has shown promise for RT outcome prediction, response assessment, as well as for tumor delineation and characterization in several cancer types. Geometric and quantification robustness is challenging and has to be addressed adequately. Evaluation in larger clinical trials with well designed imaging protocol and advanced analysis models is needed to develop the optimal strategy for integrating DWI in RT.
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http://dx.doi.org/10.1016/j.ctro.2018.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169338PMC
November 2018

Transkingdom network reveals bacterial players associated with cervical cancer gene expression program.

PeerJ 2018 19;6:e5590. Epub 2018 Sep 19.

College of Pharmacy, Oregon State University, Corvallis, OR, USA.

Cervical cancer is the fourth most common cancer in women worldwide with human papillomavirus (HPV) being the main cause the disease. Chromosomal amplifications have been identified as a source of upregulation for cervical cancer driver genes but cannot fully explain increased expression of immune genes in invasive carcinoma. Insight into additional factors that may tip the balance from immune tolerance of HPV to the elimination of the virus may lead to better diagnosis markers. We investigated whether microbiota affect molecular pathways in cervical carcinogenesis by performing microbiome analysis via sequencing 16S rRNA in tumor biopsies from 121 patients. While we detected a large number of intra-tumor taxa (289 operational taxonomic units (OTUs)), we focused on the 38 most abundantly represented microbes. To search for microbes and host genes potentially involved in the interaction, we reconstructed a transkingdom network by integrating a previously discovered cervical cancer gene expression network with our bacterial co-abundance network and employed bipartite betweenness centrality. The top ranked microbes were represented by the families , , and . While we could not define the first two families to the species level, was assigned to . By co-culturing a cervical cancer cell line with , we confirmed that three out of the ten top predicted genes in the transkingdom network (lysosomal associated membrane protein 3 (LAMP3), STAT1, TAP1), all regulators of immunological pathways, were upregulated by this microorganism. Therefore, we propose that intra-tumor microbiota may contribute to cervical carcinogenesis through the induction of immune response drivers, including the well-known cancer gene LAMP3.
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http://dx.doi.org/10.7717/peerj.5590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6170155PMC
September 2018

Combined MR Imaging of Oxygen Consumption and Supply Reveals Tumor Hypoxia and Aggressiveness in Prostate Cancer Patients.

Cancer Res 2018 08 26;78(16):4774-4785. Epub 2018 Jun 26.

Department of Radiation Biology, Radiumhospitalet, Oslo University Hospital, Oslo, Norway.

The established role of hypoxia-induced signaling in prostate cancer growth, metastasis, and response to treatment suggests that a method to image hypoxia in tumors could aid treatment decisions. Here, we present consumption and supply-based hypoxia (CSH) imaging, an approach that integrates images related to oxygen consumption and supply into a single image. This integration algorithm was developed in patients with prostate cancer receiving hypoxia marker pimonidazole prior to prostatectomy. We exploited the intravoxel incoherent motion (IVIM) signal in diagnostic diffusion-weighted (DW) magnetic resonance (MR) images to generate separate images of the apparent diffusion coefficient (ADC) and fractional blood volume (fBV). ADC and fBV correlated with cell density (CD) and blood vessel density (BVD) in histology and whole-mount sections from 35 patients, thus linking ADC to oxygen consumption and fBV to oxygen supply. Pixel-wise plots of ADC versus fBV were utilized to predict the hypoxia status of each pixel in a tumor and to visualize the predicted value in a single image. The hypoxic fraction (HF) of CSH images correlated strongly ( = 0.66; = 41) with pimonidazole immunoscore (HS); this relationship was validated in a second pimonidazole cohort ( = 0.54; = 54). We observed good agreement between CSH images and pimonidazole staining in whole-mount sections. HF correlated with tumor stage and lymph node status, consistent with findings for HS Moreover, CSH imaging could be applied on histologic CD and BVD images, demonstrating transferability to a histopathology assay. Thus, CSH represents a robust approach for hypoxia imaging in prostate cancer that could easily be translated into clinical practice. These findings present a novel imaging strategy that indirectly measures tumor hypoxia and has potential application in a wide variety of solid tumors and other imaging modalities. http://cancerres.aacrjournals.org/content/canres/78/16/4774/F1.large.jpg .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-3806DOI Listing
August 2018

Low dose-rate irradiation with [H]-labelled valine to selectively target hypoxic cells in a human colorectal cancer xenograft model.

Acta Oncol 2018 Sep 9;57(9):1216-1224. Epub 2018 Apr 9.

a Department of Physics , University of Oslo , Oslo , Norway.

Background: Earlier in vitro studies show that irradiation with an ultra-low dose-rate of 15 mGy/h delivered with [H]-valine leads to loss of clonogenicity in hypoxic T-47D cells. Here, the aim was to determine if [H]-valine could be used to deliver low dose-rate irradiation in a colorectal cancer model.

Methods: Clonogenicity was measured in cultured cancer cell line HT29 irradiated with 15 mGy/h combined with intermittent hypoxia. Mice with HT29 xenografts were irradiated by repeated injections of [H]-valine intravenously. The activity in the tumor tissue was measured by scintillation counting and tumor growth, hypoxic fraction and tritium distribution within tumors were assessed by pimonidazole staining and autoradiography.

Results: Ultra-low dose-rate irradiation decreased clonogenicity in hypoxic colorectal cancer cells. In vivo, the tumor growth, hypoxic fraction and weight of the mice were similar between the treated and untreated group. Autoradiography showed no [H]-valine uptake in hypoxic tumor regions in contrast to aerobic tissue.

Conclusion: Continuous low-dose-rate irradiation was well tolerated by aerobic tissue. This indicates a potential use of low dose-rate irradiation to target hypoxic tumor cells in combination with high dose-rate irradiation to eradicate the well oxygenated tumor regions. However, [H]-valine is not the appropriate method to deliver ultra-low dose-rate irradiation in vivo.
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http://dx.doi.org/10.1080/0284186X.2018.1457223DOI Listing
September 2018

Hypoxia in cervical cancer: from biology to imaging.

Clin Transl Imaging 2017 10;5(4):373-388. Epub 2017 Jul 10.

Department of Medical Physics, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

Purpose: Hypoxia imaging may improve identification of cervical cancer patients at risk of treatment failure and be utilized in treatment planning and monitoring, but its clinical potential is far from fully realized. Here, we briefly describe the biology of hypoxia in cervix tumors of relevance for imaging, and evaluate positron emission tomography (PET) and magnetic resonance imaging (MRI) techniques that have shown promise for assessing hypoxia in a clinical setting. We further discuss emerging imaging approaches, and how imaging can play a role in future treatment strategies to target hypoxia.

Methods: We performed a PubMed literature search, using keywords related to imaging and hypoxia in cervical cancer, with a particular emphasis on studies correlating imaging with other hypoxia measures and treatment outcome.

Results: Only a few and rather small studies have utilized PET with tracers specific for hypoxia, and no firm conclusions regarding preferred tracer or clinical potential can be drawn so far. Most studies address indirect hypoxia imaging with dynamic contrast-enhanced techniques. Strong evidences for a role of these techniques in hypoxia imaging have been presented. Pre-treatment images have shown significant association to outcome in several studies, and images acquired during fractionated radiotherapy may further improve risk stratification. Multiparametric MRI and multimodality PET/MRI enable combined imaging of factors of relevance for tumor hypoxia and warrant further investigation.

Conclusions: Several imaging approaches have shown promise for hypoxia imaging in cervical cancer. Evaluation in large clinical trials is required to decide upon the optimal modality and approach.
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http://dx.doi.org/10.1007/s40336-017-0238-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532411PMC
July 2017

Autodelineation of cervical cancers using multiparametric magnetic resonance imaging and machine learning.

Acta Oncol 2017 Jun 8;56(6):806-812. Epub 2017 Feb 8.

a Faculty of Science and Technology , Norwegian University of Life Sciences , Ås , Norway.

Background: Tumour delineation is a challenging, time-consuming and complex part of radiotherapy planning. In this study, an automatic method for delineating locally advanced cervical cancers was developed using a machine learning approach.

Materials And Methods: A method for tumour segmentation based on image voxel classification using Fisher?s Linear Discriminant Analysis (LDA) was developed. This was applied to magnetic resonance (MR) images of 78 patients with locally advanced cervical cancer. The segmentation was based on multiparametric MRI consisting of T2- weighted (T2w), T1-weighted (T1w) and dynamic contrast-enhanced (DCE) sequences, and included intensity and spatial information from the images. The model was trained and assessed using delineations made by two radiologists.

Results: Segmentation based on T2w or T1w images resulted in mean sensitivity and specificity of 94% and 52%, respectively. Including DCE-MR images improved the segmentation model?s performance significantly, giving mean sensitivity and specificity of 85?93%. Comparisons with radiologists? tumour delineations gave Dice similarity coefficients of up to 0.44.

Conclusion: Voxel classification using a machine learning approach is a flexible and fully automatic method for tumour delineation. Combining all relevant MR image series resulted in high sensitivity and specificity. Moreover, the presented method can be extended to include additional imaging modalities.
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http://dx.doi.org/10.1080/0284186X.2017.1285499DOI Listing
June 2017

Hypoxia-independent gene expression signature associated with radiosensitisation of prostate cancer cell lines by histone deacetylase inhibition.

Br J Cancer 2016 10 6;115(8):929-939. Epub 2016 Sep 6.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Pb 4950, Nydalen, 0424 Oslo, Norway.

Background: Histone deacetylase inhibitors (HDACis) like vorinostat are promising radiosensitisers in prostate cancer, but their effect under hypoxia is not known. We investigated gene expression associated with radiosensitisation of normoxic and hypoxic prostate cancer cells by vorinostat.

Methods: Cells were exposed to vorinostat under normoxia or hypoxia and subjected to gene expression profiling before irradiation and clonogenic survival analysis.

Results: Pretreatment with vorinostat led to radiosensitisation of the intrinsically radioresistant DU 145 cells, but not the radiosensitive PC-3 and 22Rv1 cells, and was independent of hypoxia status. Knockdown experiments showed that the sensitisation was not caused by repression of hypoxia-inducible factor HIF1 or tumour protein TP53. Global deregulation of DNA repair and chromatin organisation genes was associated with radiosensitisation under both normoxia and hypoxia. A radiosensitisation signature with expression changes of 56 genes was generated and valid for both conditions. For eight signature genes, baseline expression also correlated with sensitisation, showing potential as pretreatment biomarker. The hypoxia independence of the signature was confirmed in a clinical data set.

Conclusions: Pretreatment with HDACi may overcome radioresistance of hypoxic prostate tumours by similar mechanisms as under normoxia. We propose a gene signature to predict radiosensitising effects independent of hypoxia status.
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http://dx.doi.org/10.1038/bjc.2016.278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061908PMC
October 2016

Cluster analysis of dynamic contrast enhanced MRI reveals tumor subregions related to locoregional relapse for cervical cancer patients.

Acta Oncol 2016 Nov 26;55(11):1294-1298. Epub 2016 Aug 26.

a Department of Mathematical Sciences and Technology , Norwegian University of Life Sciences , Ås , Norway.

Background: Solid tumors are known to be spatially heterogeneous. Detection of treatment-resistant tumor regions can improve clinical outcome, by enabling implementation of strategies targeting such regions. In this study, K-means clustering was used to group voxels in dynamic contrast enhanced magnetic resonance images (DCE-MRI) of cervical cancers. The aim was to identify clusters reflecting treatment resistance that could be used for targeted radiotherapy with a dose-painting approach.

Material And Methods: Eighty-one patients with locally advanced cervical cancer underwent DCE-MRI prior to chemoradiotherapy. The resulting image time series were fitted to two pharmacokinetic models, the Tofts model (yielding parameters K and ν) and the Brix model (A, k and k). K-means clustering was used to group similar voxels based on either the pharmacokinetic parameter maps or the relative signal increase (RSI) time series. The associations between voxel clusters and treatment outcome (measured as locoregional control) were evaluated using the volume fraction or the spatial distribution of each cluster.

Results: One voxel cluster based on the RSI time series was significantly related to locoregional control (adjusted p-value 0.048). This cluster consisted of low-enhancing voxels. We found that tumors with poor prognosis had this RSI-based cluster gathered into few patches, making this cluster a potential candidate for targeted radiotherapy. None of the voxels clusters based on Tofts or Brix parameter maps were significantly related to treatment outcome.

Conclusion: We identified one group of tumor voxels significantly associated with locoregional relapse that could potentially be used for dose painting. This tumor voxel cluster was identified using the raw MRI time series rather than the pharmacokinetic maps.
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http://dx.doi.org/10.1080/0284186X.2016.1189091DOI Listing
November 2016

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

Oncotarget 2016 Jul;7(28):43805-43819

Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands.

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.
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http://dx.doi.org/10.18632/oncotarget.9698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5190061PMC
July 2016

Identification and Validation of Reference Genes for RT-qPCR Studies of Hypoxia in Squamous Cervical Cancer Patients.

PLoS One 2016 31;11(5):e0156259. Epub 2016 May 31.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

Hypoxia is an adverse factor in cervical cancer, and hypoxia-related gene expression could be a powerful biomarker for identifying the aggressive hypoxic tumors. Reverse transcription quantitative PCR (RT-qPCR) is a valuable method for gene expression studies, but suitable reference genes for data normalization that are independent of hypoxia status and clinical parameters of cervical tumors are lacking. In the present work, we aimed to identify reference genes for RT-qPCR studies of hypoxia in squamous cervical cancer. From 422 candidate reference genes selected from the literature, we used Illumina array-based expression profiles to identify 182 genes not affected by hypoxia in cervical cancer, i.e. genes regulated by hypoxia in eight cervical cancer cell lines or correlating with the hypoxia-associated dynamic contrast-enhanced magnetic resonance imaging parameter ABrix in 42 patients, were excluded. Among the 182 genes, nine candidates (CHCHD1, GNB2L1, IPO8, LASP1, RPL27A, RPS12, SOD1, SRSF9, TMBIM6) that were not associated with tumor volume, stage, lymph node involvement or disease progression in array data of 150 patients, were selected for further testing by RT-qPCR. geNorm and NormFinder analyses of RT-qPCR data of 74 patients identified CHCHD1, SRSF9 and TMBIM6 as the optimal set of reference genes, with stable expression both overall and across patient subgroups with different hypoxia status (ABrix) and clinical parameters. The suitability of the three reference genes were validated in studies of the hypoxia-induced genes DDIT3, ERO1A, and STC2. After normalization, the RT-qPCR data of these genes showed a significant correlation with Illumina expression (P<0.001, n = 74) and ABrix (P<0.05, n = 32), and the STC2 data were associated with clinical outcome, in accordance with the Illumina data. Thus, CHCHD1, SRSF9 and TMBIM6 seem to be suitable reference genes for studying hypoxia-related gene expression in squamous cervical cancer samples by RT-qPCR. Moreover, STC2 is a promising prognostic hypoxia biomarker in cervical cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156259PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887009PMC
July 2017

Integrative Analysis of DCE-MRI and Gene Expression Profiles in Construction of a Gene Classifier for Assessment of Hypoxia-Related Risk of Chemoradiotherapy Failure in Cervical Cancer.

Clin Cancer Res 2016 Aug 24;22(16):4067-76. Epub 2016 Mar 24.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

Purpose: A 31-gene expression signature reflected in dynamic contrast enhanced (DCE)-MR images and correlated with hypoxia-related aggressiveness in cervical cancer was identified in previous work. We here aimed to construct a dichotomous classifier with key signature genes and a predefined classification threshold that separated cervical cancer patients into a more and less hypoxic group with different outcome to chemoradiotherapy.

Experimental Design: A training cohort of 42 patients and two independent cohorts of 108 and 131 patients were included. Gene expression data were generated from tumor biopsies by two Illumina array generations (WG-6, HT-12). Technical transfer of the classifier to a reverse transcription quantitative PCR (RT-qPCR) platform was performed for 74 patients. The amplitude ABrix in the Brix pharmacokinetic model was extracted from DCE-MR images of 64 patients and used as an indicator of hypoxia.

Results: Classifier candidates were constructed by integrative analysis of ABrix and gene expression profiles in the training cohort and evaluated by a leave-one-out cross-validation approach. On the basis of their ability to separate patients correctly according to hypoxia status, a 6-gene classifier was identified. The classifier separated the patients into two groups with different progression-free survival probability. The robustness of the classifier was demonstrated by successful validation of hypoxia association and prognostic value across cohorts, array generations, and assay platforms. The prognostic value was independent of existing clinical markers, regardless of clinical endpoints.

Conclusions: A robust DCE-MRI-associated gene classifier has been constructed that may be used to achieve an early indication of patients' risk of hypoxia-related chemoradiotherapy failure. Clin Cancer Res; 22(16); 4067-76. ©2016 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2322DOI Listing
August 2016

Hypoxia-induced alterations of G2 checkpoint regulators.

Mol Oncol 2016 05 8;10(5):764-73. Epub 2016 Jan 8.

Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, 0310 Oslo, Norway. Electronic address:

Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, genome stability is also surveilled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can influence the DNA damage-induced cell cycle checkpoints. Here, we show that hypoxia (24 h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, the levels of several G2 checkpoint regulators, in particular Cyclin B, were reduced in G2 phase cells after hypoxic exposure, as shown by flow cytometric barcoding analysis of individual cells. These effects were accompanied by decreased phosphorylation of a Cyclin dependent kinase (CDK) target in G2 phase cells after hypoxia, suggesting decreased CDK activity. Furthermore, cells pre-exposed to hypoxia showed increased G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (∼0.03% O2 20 h and 0.2% O2 72 h). These results demonstrate that the DNA damage-induced G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors.
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http://dx.doi.org/10.1016/j.molonc.2015.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423158PMC
May 2016

Interplay between promoter methylation and chromosomal loss in gene silencing at 3p11-p14 in cervical cancer.

Epigenetics 2015 ;10(10):970-80

a Department of Radiation Biology ; Norwegian Radium Hospital; Oslo University Hospital ; Oslo , Norway.

Loss of 3p11-p14 is a frequent event in epithelial cancer and a candidate prognostic biomarker in cervical cancer. In addition to loss, promoter methylation can participate in gene silencing and promote tumor aggressiveness. We have performed a complete mapping of promoter methylation at 3p11-p14 in two independent cohorts of cervical cancer patients (n = 149, n = 121), using Illumina 450K methylation arrays. The aim was to investigate whether hyperm-ethylation was frequent and could contribute to gene silencing and disease aggressiveness either alone or combined with loss. By comparing the methylation level of individual CpG sites with corresponding data of normal cervical tissue, 26 out of 41 genes were found to be hypermethylated in both cohorts. The frequency of patients with hypermethylation of these genes was found to be higher at tumor stages of 3 and 4 than in stage 1 tumors. Seventeen of the 26 genes were transcriptionally downregulated in cancer compared to normal tissue, whereof 6 genes showed a significant correlation between methylation and expression. Integrated analysis of methylation, gene dosage, and expression of the 26 hypermethylated genes identified 3 regulation patterns encompassing 8 hypermethylated genes; a methylation driven pattern (C3orf14, GPR27, ZNF717), a gene dosage driven pattern (THOC7, PSMD6), and a combined methylation and gene dosage driven pattern (FHIT, ADAMTS9, LRIG1). In survival analysis, patients with both hypermethylation and loss of LRIG1 had a worse outcome compared to those harboring only hypermethylation or none of the events. C3orf14 emerged as a novel methylation regulated suppressor gene, for which knockdown was found to promote invasive growth in human papilloma virus (HPV)-transformed keratinocytes. In conclusion, hypermethylation at 3p11-p14 is common in cervical cancer and may exert a selection pressure during carcinogenesis alone or combined with loss. Information on both events could lead to improved prognostic markers.
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http://dx.doi.org/10.1080/15592294.2015.1085140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4844207PMC
August 2016

Targeting tumour hypoxia to prevent cancer metastasis. From biology, biosensing and technology to drug development: the METOXIA consortium.

J Enzyme Inhib Med Chem 2015 27;30(5):689-721. Epub 2014 Oct 27.

a Department of Physics , University of Oslo , Oslo , Norway .

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.
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http://dx.doi.org/10.3109/14756366.2014.966704DOI Listing
May 2016

Classification of dynamic contrast enhanced MR images of cervical cancers using texture analysis and support vector machines.

IEEE Trans Med Imaging 2014 Aug 29;33(8):1648-56. Epub 2014 Apr 29.

Dynamic contrast enhanced MRI (DCE-MRI) provides insight into the vascular properties of tissue. Pharmacokinetic models may be fitted to DCE-MRI uptake patterns, enabling biologically relevant interpretations. The aim of our study was to determine whether treatment outcome for 81 patients with locally advanced cervical cancer could be predicted from parameters of the Brix pharmacokinetic model derived from pre-chemoradiotherapy DCE-MRI. First-order statistical features of the Brix parameters were used. In addition, texture analysis of Brix parameter maps was done by constructing gray level co-occurrence matrices (GLCM) from the maps. Clinical factors and first- and second-order features were used as explanatory variables for support vector machine (SVM) classification, with treatment outcome as response. Classification models were validated using leave-one-out cross-model validation. A random value permutation test was used to evaluate model significance. Features derived from first-order statistics could not discriminate between cured and relapsed patients (specificity 0%-20%, p-values close to unity). However, second-order GLCM features could significantly predict treatment outcome with accuracies (~70%) similar to the clinical factors tumor volume and stage (69%). The results indicate that the spatial relations within the tumor, quantified by texture features, were more suitable for outcome prediction than first-order features.
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http://dx.doi.org/10.1109/TMI.2014.2321024DOI Listing
August 2014

Ménage à trois: an evolutionary interplay between human papillomavirus, a tumor, and a woman.

Trends Microbiol 2014 Jun 24;22(6):345-53. Epub 2014 Mar 24.

College of Pharmacy, Oregon State University, Corvallis, OR, USA. Electronic address:

Cervical cancer is the third most common cancer in women with human papillomavirus (HPV) being a key etiologic factor of this devastating disease. In this article, we describe modern advances in the genomics and transcriptomics of cervical cancer that led to uncovering the key gene drivers. We also introduce, herein, a model of cervical carcinogenesis that explains how the interplay between virus, tumor, and woman results in the selection of clones that simultaneously harbor genomic amplifications for genes that drive cell cycle, antiviral response, and inhibit cell differentiation. The new model may help researchers understand the controversies in antiviral therapy and immunogenetics of this cancer and may provide a basis for future research directions in early diagnostics and personalization of therapy.
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http://dx.doi.org/10.1016/j.tim.2014.02.009DOI Listing
June 2014

Hypoxia-independent downregulation of hypoxia-inducible factor 1 targets by androgen deprivation therapy in prostate cancer.

Int J Radiat Oncol Biol Phys 2013 Nov 10;87(4):753-60. Epub 2013 Sep 10.

Department of Radiation Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

Purpose: We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred.

Methods And Materials: Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry.

Results: A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001).

Conclusions: AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to tumor regression. HIF1α expression is probably not a useful hypoxia biomarker during ADT in prostate cancer.
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http://dx.doi.org/10.1016/j.ijrobp.2013.07.023DOI Listing
November 2013

Gene network reconstruction reveals cell cycle and antiviral genes as major drivers of cervical cancer.

Nat Commun 2013 ;4:1806

Instituto de Imunogenética-Associação Fundo de Incentivo à Pesquisa (IGEN-AFIP), São Paulo, São Paulo, Brazil.

Although human papillomavirus was identified as an aetiological factor in cervical cancer, the key human gene drivers of this disease remain unknown. Here we apply an unbiased approach integrating gene expression and chromosomal aberration data. In an independent group of patients, we reconstruct and validate a gene regulatory meta-network, and identify cell cycle and antiviral genes that constitute two major subnetworks upregulated in tumour samples. These genes are located within the same regions as chromosomal amplifications, most frequently on 3q. We propose a model in which selected chromosomal gains drive activation of antiviral genes contributing to episomal virus elimination, which synergizes with cell cycle dysregulation. These findings may help to explain the paradox of episomal human papillomavirus decline in women with invasive cancer who were previously unable to clear the virus.
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http://dx.doi.org/10.1038/ncomms2693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4237593PMC
December 2013

Identification of eight candidate target genes of the recurrent 3p12-p14 loss in cervical cancer by integrative genomic profiling.

J Pathol 2013 May 14;230(1):59-69. Epub 2013 Mar 14.

Department of Radiation Biology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

The pathogenetic role, including its target genes, of the recurrent 3p12-p14 loss in cervical cancer has remained unclear. To determine the onset of the event during carcinogenesis, we used microarray techniques and found that the loss was the most frequent 3p event, occurring in 61% of 92 invasive carcinomas, in only 2% of 43 high-grade intraepithelial lesions (CIN2/3), and in 33% of 6 CIN3 lesions adjacent to invasive carcinomas, suggesting a role in acquisition of invasiveness or early during the invasive phase. We performed an integrative DNA copy number and expression analysis of 77 invasive carcinomas, where all genes within the recurrent region were included. We selected eight genes, THOC7, PSMD6, SLC25A26, TMF1, RYBP, SHQ1, EBLN2, and GBE1, which were highly down-regulated in cases with loss, as confirmed at the protein level for RYBP and TMF1 by immunohistochemistry. The eight genes were subjected to network analysis based on the expression profiles, revealing interaction partners of proteins encoded by the genes that were coordinately regulated in tumours with loss. Several partners were shared among the eight genes, indicating crosstalk in their signalling. Gene ontology analysis showed enrichment of biological processes such as apoptosis, proliferation, and stress response in the network and suggested a relationship between down-regulation of the eight genes and activation of tumourigenic pathways. Survival analysis showed prognostic impact of the eight-gene signature that was confirmed in a validation cohort of 74 patients and was independent of clinical parameters. These results support the role of the eight candidate genes as targets of the 3p12-p14 loss in cervical cancer and suggest that the strong selection advantage of the loss during carcinogenesis might be caused by a synergetic effect of several tumourigenic processes controlled by these targets.
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http://dx.doi.org/10.1002/path.4168DOI Listing
May 2013

Pharmacokinetic parameters derived from dynamic contrast enhanced MRI of cervical cancers predict chemoradiotherapy outcome.

Radiother Oncol 2013 Apr 17;107(1):117-22. Epub 2013 Jan 17.

Department of Medical Physics, Oslo University Hospital, Olso, Norway.

Purpose: To assess the prognostic value of pharmacokinetic parameters derived from pre-chemoradiotherapy dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of cervical cancer patients.

Materials And Methods: Seventy-eight patients with locally advanced cervical cancer underwent DCE-MRI with Gd-DTPA before chemoradiotherapy. The pharmacokinetic Brix and Tofts models were fitted to contrast enhancement curves in all tumor voxels, providing histograms of several pharmacokinetic parameters (Brix: A(Brix), k(ep), k(el), Tofts: K(trans), ν(e)). A percentile screening approach including log-rank survival tests was undertaken to identify the clinically most relevant part of the intratumoral parameter distribution. Clinical endpoints were progression-free survival (PFS) and locoregional control (LRC). Multivariate analysis including FIGO stage and tumor volume was used to assess the prognostic significance of the imaging parameters.

Results: A(Brix), k(el), and K(trans) were significantly (P<0.05) positively associated with both clinical LRC and PFS, while ν(e) was significantly positively correlated with PFS only. k(ep) showed no association with any endpoint. A(Brix) was positively correlated with K(trans) and ν(e), and showed the strongest association with endpoint in the log-rank testing. k(el) and K(trans) were independent prognostic factors in multivariate analysis with LRC as endpoint.

Conclusions: Parameters estimated by pharmacokinetic analysis of DCE-MR images obtained prior to chemoradiotherapy may be used for identifying patients at risk of treatment failure.
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http://dx.doi.org/10.1016/j.radonc.2012.11.007DOI Listing
April 2013

Hypoxia-induced gene expression in chemoradioresistant cervical cancer revealed by dynamic contrast-enhanced MRI.

Cancer Res 2012 Oct 13;72(20):5285-95. Epub 2012 Aug 13.

Department of Radiation Biology, The Norwegian Radium Hospital, Nydalen, Norway.

Knowledge of the molecular background of functional magnetic resonance (MR) images is required to fully exploit their potential in cancer management. We explored the prognostic impact of dynamic contrast-enhanced MR imaging (DCE-MRI) parameters in cervical cancer combined with global gene expression data to reveal their underlying molecular phenotype and construct a representative gene signature for the relevant parameter. On the basis of 78 patients with cervical cancer subjected to curative chemoradiotherapy, we identified the prognostic DCE-MRI parameter A(Brix) by pharmacokinetic analysis of pretreatment images based on the Brix model, in which tumors with low A(Brix) appeared to be most aggressive. Gene set analysis of 46 tumors with pairwise DCE-MRI and gene expression data showed a significant correlation between A(Brix) and the hypoxia gene sets, whereas gene sets related to other tumor phenotypes were not significant. Hypoxia gene sets specific for cervical cancer created in cell culture experiments, including both targets of the hypoxia inducible factor (HIF1α) and the unfolded protein response, were the most significant. In the remaining 32 tumors, low A(Brix) was associated with upregulation of HIF1α protein expression, as assessed by immunohistochemistry, consistent with increased hypoxia. On the basis of the hypoxia gene sets, a signature of 31 genes that were upregulated in tumors with low A(Brix) was constructed. This DCE-MRI hypoxia gene signature showed prognostic impact in an independent validation cohort of 109 patients. Our findings reveal the molecular basis of an aggressive hypoxic phenotype and suggest the use of DCE-MRI to noninvasively identify patients with hypoxia-related chemoradioresistance.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-1085DOI Listing
October 2012

Vascular responses to radiotherapy and androgen-deprivation therapy in experimental prostate cancer.

Radiat Oncol 2012 May 23;7:75. Epub 2012 May 23.

Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, PO Box 4953, Nydalen, 0424 Oslo, Norway.

Background: Radiotherapy (RT) and androgen-deprivation therapy (ADT) are standard treatments for advanced prostate cancer (PC). Tumor vascularization is recognized as an important physiological feature likely to impact on both RT and ADT response, and this study therefore aimed to characterize the vascular responses to RT and ADT in experimental PC.

Methods: Using mice implanted with CWR22 PC xenografts, vascular responses to RT and ADT by castration were visualized in vivo by DCE MRI, before contrast-enhancement curves were analyzed both semi-quantitatively and by pharmacokinetic modeling. Extracted image parameters were correlated to the results from ex vivo quantitative fluorescent immunohistochemical analysis (qIHC) of tumor vascularization (9 F1), perfusion (Hoechst 33342), and hypoxia (pimonidazole), performed on tissue sections made from tumors excised directly after DCE MRI.

Results: Compared to untreated (Ctrl) tumors, an improved and highly functional vascularization was detected in androgen-deprived (AD) tumors, reflected by increases in DCE MRI parameters and by increased number of vessels (VN), vessel density (VD), and vessel area fraction (VF) from qIHC. Although total hypoxic fractions ( HF) did not change, estimated acute hypoxia scores (AHS)--the proportion of hypoxia staining within 50 μm from perfusion staining--were increased in AD tumors compared to in Ctrl tumors. Five to six months after ADT renewed castration-resistant (CR) tumor growth appeared with an even further enhanced tumor vascularization. Compared to the large vascular changes induced by ADT, RT induced minor vascular changes. Correlating DCE MRI and qIHC parameters unveiled the semi-quantitative parameters area under curve (AUC) from initial time-points to strongly correlate with VD and VF, whereas estimation of vessel size (VS) by DCE MRI required pharmacokinetic modeling. HF was not correlated to any DCE MRI parameter, however, AHS may be estimated after pharmacokinetic modeling. Interestingly, such modeling also detected tumor necrosis very strongly.

Conclusions: DCE MRI reliably allows non-invasive assessment of tumors' vascular function. The findings of increased tumor vascularization after ADT encourage further studies into whether these changes are beneficial for combined RT, or if treatment with anti-angiogenic therapy may be a strategy to improve the therapeutic efficacy of ADT in advanced PC.
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http://dx.doi.org/10.1186/1748-717X-7-75DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441216PMC
May 2012
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