[Show abstract][Hide abstract] ABSTRACT: 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, such as mean, variance and percentiles, were used. In addition, texture analysis of Brix parameter maps was done by constructing gray level co-occurrence matrices (GLCM) from the maps, resulting in second order statistical features that captured spatial variations within the tumors. 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, which is stricter than the common leave-one-out cross-validation. In addition, a random value permutation test was used to evaluate model statistical 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 linked to tumor heterogeneity, were more suitable for outcome prediction than first order statistical features.
[Show abstract][Hide abstract] ABSTRACT: 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.
Trends in Microbiology 03/2014; · 8.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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).
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.
International journal of radiation oncology, biology, physics 09/2013; · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The pathogenetic role, including its target genes, of recurrent 3p12-p14 loss in cervical cancer has remains 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 downregulated 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 tumors with loss. Several partners were shared among the eight genes, indicating crosstalk in their signaling. Gene ontology analysis showed enrichment of biological processes like apoptosis, proliferation, and stress response in the network and suggested a relationship between downregulation of the eight genes and activation of tumorigenic 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 markers. 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 tumorigenic processes controlled by these targets.
The Journal of Pathology 01/2013; · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Radiotherapy and Oncology 01/2013; · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: To systematically screen the tumor contrast enhancement of locally advanced cervical cancers to assess the prognostic value of two descriptive parameters derived from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI).
This study included a prospectively collected cohort of 81 patients who underwent DCE-MRI with gadopentetate dimeglumine before chemoradiotherapy. The following descriptive DCE-MRI parameters were extracted voxel by voxel and presented as histograms for each time point in the dynamic series: normalized relative signal increase (nRSI) and normalized area under the curve (nAUC). The first to 100th percentiles of the histograms were included in a log-rank survival test, resulting in p value and relative risk maps of all percentile-time intervals for each DCE-MRI parameter. The maps were used to evaluate the robustness of the individual percentile-time pairs and to construct prognostic parameters. Clinical endpoints were locoregional control and progression-free survival. The study was approved by the institutional ethics committee.
The p value maps of nRSI and nAUC showed a large continuous region of percentile-time pairs that were significantly associated with locoregional control (p < 0.05). These parameters had prognostic impact independent of tumor stage, volume, and lymph node status on multivariate analysis. Only a small percentile-time interval of nRSI was associated with progression-free survival.
The percentile-time screening identified DCE-MRI parameters that predict long-term locoregional control after chemoradiotherapy of cervical cancer.
International journal of radiation oncology, biology, physics 03/2012; 82(3):e485-92. · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We compared the prognostic significance of ectodomain isoforms of the epidermal growth factor receptor (EGFR), which lack the tyrosine kinase (TK) domain, with that of the full-length receptor and its autophosphorylation status in cervical cancers treated with conventional chemoradiotherapy.
Expression of EGFR isoforms was assessed by immunohistochemistry in a prospectively collected cohort of 178 patients with squamous cell cervical carcinoma, and their detection was confirmed with Western blotting and reverse transcriptase PCR. A proximity ligation immunohistochemistry assay was used to assess EGFR-specific autophosphorylation. Pathways associated with the expression of ectodomain isoforms were studied by gene expression analysis with Illumina beadarrays in 110 patients and validated in an independent cohort of 41 patients.
Membranous expression of ectodomain isoforms alone, without the coexpression of the full-length receptor, showed correlations to poor clinical outcome that were highly significant for lymph node-negative patients (locoregional control, P = 0.0002; progression-free survival, P < 0.0001; disease-specific survival, P = 0.005 in the log-rank test) and independent of clinical variables. The ectodomain isoforms were primarily 60-kD products of alternative EGFR transcripts. Their membranous expression correlated with transcriptional regulation of oncogenic pathways including activation of MYC and MAX, which was significantly associated with poor outcome. This aggressive phenotype of ectodomain EGFR expressing tumors was confirmed in the independent cohort. Neither total nor full-length EGFR protein level, or autophosphorylation status, showed prognostic significance.
Membranous expression of ectodomain EGFR isoforms, and not TK activation, predicts poor outcome after chemoradiotherapy for patients with lymph node-negative cervical cancer.
Clinical Cancer Research 08/2011; 17(16):5501-12. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pharmacokinetic analysis of dynamic contrast enhanced magnetic resonance images (DCEMRI) allows for quantitative characterization of vascular properties of tumors. The aim of this study is twofold, first to determine if tumor regions with similar vascularization could be labeled by clustering methods, second to determine if the identified regions can be associated with local cancer relapse.
Eighty-one patients with locally advanced cervical cancer treated with chemoradiotherapy underwent DCEMRI with Gd-DTPA prior to external beam radiotherapy. The median follow-up time after treatment was four years, in which nine patients had primary tumor relapse. By fitting a pharmacokinetic two-compartment model function to the temporal contrast enhancement in the tumor, two pharmacokinetic parameters, K(trans) and ύ(e), were estimated voxel by voxel from the DCEMR-images. Intratumoral regions with similar vascularization were identified by k-means clustering of the two pharmacokinetic parameter estimates over all patients. The volume fraction of each cluster was used to evaluate the prognostic value of the clusters.
Three clusters provided a sufficient reduction of the cluster variance to label different vascular properties within the tumors. The corresponding median volume fraction of each cluster was 38%, 46% and 10%. The second cluster was significantly associated with primary tumor control in a log-rank survival test (p-value: 0.042), showing a decreased risk of treatment failure for patients with high volume fraction of voxels.
Intratumoral regions showing similar vascular properties could successfully be labeled in three distinct clusters and the volume fraction of one cluster region was associated with primary tumor control.
[Show abstract][Hide abstract] ABSTRACT: We have applied the sensitive and specific in situ proximity ligation assay (PLA) to characterize Tyr1068 phosphorylation of the epidermal growth factor receptor (EGFR) in cervical cancer in relation to the protein level and gene dosage.
Pretreatment tumor biopsies from 178 patients were analyzed. EGFR protein level was determined by immunohistochemistry, and Tyr1068 phosphorylation was detected with PLA in 97 EGFR positive tumors. EGFR gene dosage was derived from array comparative genomic hybridization of 86 cases.
EGFR was expressed in most tumors, whereas phosphorylation was seen in about half of the EGFR positive ones. A correlation was found between the expression of EGFR and phosphorylated EGFR (p=0.016, membrane; p=0.012, cytoplasm). However, tumor regions with high protein level without phosphorylation were occasionally seen and the percentage of EGFR positive cells was higher than the phosphorylated percentage (p<0.001). Moreover, an increase in the phosphorylation in both the membrane (p=0.014) and cytoplasm (p=0.002) was seen in 11 tumors with gain of EGFR. The protein level was not correlated with gene dosage.
In contrast to gain of the EGFR chromosomal region, high EGFR protein level may not necessarily indicate Tyr1068 phosphorylation and thereby receptor activation in cervical cancer.
Radiotherapy and Oncology 06/2011; 101(1):152-7. · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell cycle checkpoints ensure that eukaryotic cells do not enter mitosis after ionizing irradiation (IR). The G(2)-arrest after IR is the result of activation of multiple signalling pathways, the contributions of which vary with time after irradiation. We have studied the time evolution of the IR-induced G(2)-arrest in human B-lymphocyte cancer cell lines, as well as the molecular mechanisms responsible for the arrest. Cells that were in G(2) phase at the time of irradiation experienced a transient arrest that blocked entry into mitosis at 0-2 hours after IR (0.5 or 4 Gy). Activation of ATM and CHEK2 occurred at the same time as this early arrest and was, like the arrest, abrogated by the ATM-inhibitor KU-55933. A late, permanent and ATM-independent arrest (≥6 hours after IR) of cells that were in G(2)/S/G(1) at the time of irradiation (4 Gy) was inactivated by caffeine. This late G(2)-arrest could not be explained by down-regulation of genes with functions in G(2)/mitosis (e.g. PLK1, CCNB1/2), since the down-regulation was transient and not accompanied by reduced protein levels. However, the persistent phosphorylation of CHEK1 after 4 Gy suggested a role for CHEK1 in the late arrest, consistent with the abrogation of the arrest in CHEK1-depleted cells. TP53 was not necessary for the late G(2)-arrest, but mediated an intermediate arrest (2-10 hours after IR) independently of ATM and CHEK1. In conclusion, the IR-induced arrest in G(2) is mediated by ATM immediately after irradiation, with TP53 for independent and transient back-up, while CHEK1 is necessary for the late arrest.
[Show abstract][Hide abstract] ABSTRACT: The lasso is one of the most commonly used methods for high-dimensional regression, but can be unstable and lacks satisfactory asymptotic properties for variable selection. We propose to use weighted lasso with integrated relevant external information on the covariates to guide the selection towards more stable results. Weighting the penalties with external information gives each regression coefficient a covariate specific amount of penalization and can improve upon standard methods that do not use such information by borrowing knowledge from the external material. The method is applied to two cancer data sets, with gene expressions as covariates. We find interesting gene signatures, which we are able to validate. We discuss various ideas on how the weights should be defined and illustrate how different types of investigations can utilize our method exploiting different sources of external data. Through simulations, we show that our method outperforms the lasso and the adaptive lasso when the external information is from relevant to partly relevant, in terms of both variable selection and prediction.
Statistical Applications in Genetics and Molecular Biology 01/2011; 10(1):39-39. · 1.52 Impact Factor