Prostate cancer radiogenomics reveals proliferative gene expression programs associated with distinct MRI-based hypoxia levels.

BACKGROUND AND PURPOSE: The biology behind individual hypoxia levels in patient tumors is poorly understood. Here, we used radiogenomics to identify associations between magnetic resonance imaging (MRI)-based hypoxia levels and biological processes derived from gene expression data in prostate cancer. MATERIALS AND METHODS: For 85 prostate cancer patients, MRI-based hypoxia images were constructed by combining diffusion-weighted images reflecting oxygen consumption and supply. The ability to differentiate hypoxia levels in these images was verified by comparison with matched biopsy sections stained for the hypoxia marker pimonidazole. For MRI-defined hypoxia levels, corresponding hypoxic fractions were calculated and correlated with biopsy gene expression profiles. Biological processes were predicted by gene set enrichment analysis (GSEA) and validated by immunohistochemistry (Ki67 proliferation marker, reactive stroma grade) and RT-PCR (MYC). RESULTS: Genes with correlation between expression level and hypoxic fraction were identified for 56 MRI-based hypoxia levels. At all levels, GSEA identified proliferation as the predominant biological process enriched among the correlating genes. Two independent proliferative gene signatures were developed. The Peak1 signature, upregulated at moderate/severe hypoxia, reflected MYC upregulation and high Ki67-proliferation index of cancer cells in pimonidazole-positive regions. The Peak2 signature, upregulated at mild to non-hypoxic levels, was associated with fibroblast gene signature and reactive stroma grade. High scores of both Peak1 and Peak2 indicated elevated risk of biochemical recurrence in multiple cohorts. CONCLUSION: Radiogenomics identified two gene expression programs activated at different hypoxia levels, reflecting proliferation of cancer cells and stroma cells. Genes involved in these programs could be candidate targets for intervention.

miR-200a/b/-429 downregulation is a candidate biomarker of tumor radioresistance and independent of hypoxia in locally advanced cervical cancer.

Many patients with locally advanced cervical cancer experience recurrence within the radiation field after chemoradiotherapy. Biomarkers of tumor radioresistance are required to identify patients in need of intensified treatment. Here, the biomarker potential of miR-200 family members was investigated in this disease. Also, involvement of tumor hypoxia in the radioresistance mechanism was determined, using a previously defined 6-gene hypoxia classifier. miR-200 expression was measured in pretreatment tumor biopsies of an explorative cohort (n = 90) and validation cohort 1 (n = 110) by RNA sequencing. Publicly available miR-200 data of 79 patients were included for the validation of prognostic significance. A score based on expression of the miR-200a/b/-429 (miR-200a, miR-200b, and miR-429) cluster showed prognostic significance in all cohorts. The score was significant in multivariate analysis of central pelvic recurrence. No association with distant recurrence or hypoxia status was found. Potential miRNA target genes were identified from gene expression profiles and showed enrichment of genes in extracellular matrix organization and cell adhesion. miR-200a/b/-429 overexpression had a pronounced radiosensitizing effect in tumor xenografts, whereas the effect was minor in vitro. In conclusion, miR-200a/b/-429 downregulation is a candidate biomarker of central pelvic recurrence and seems to predict cell adhesion-mediated tumor radioresistance independent of clinical markers and hypoxia.

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

Little is known about the transport mechanism of anti-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.

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

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 ν e and K trans, 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 G2-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.

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

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.

Influx rate of 18F-fluoroaminosuberic acid reflects cystine/glutamate antiporter expression in tumour xenografts.

PURPOSE: 18F-fluoroaminosuberic acid (18F-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 18F-fluorodeoxyglucose (18F-FDG). Our aim was to evaluate and relate dynamic 18F-FASu and 18F-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 18F-FASu and 18F-FDG in individual tumours were used to extract early (SUVE; 2 min p.i.) and late (SUVL; 55 min p.i.) standardised uptake values. Pharmacokinetic two-tissue compartment models were applied to the TACs to estimate rate constants K1-k4 and blood volume fraction vB. Relative levels of cystine/glutamate antiporter subunit xCT were assessed by western blotting, and expression of GLUT1 and CD31 by immunohistochemistry. RESULTS: 18F-FASu showed higher SUVE, whilst 18F-FDG exhibited higher SUVL. Influx rate K1 for 18F-FASu was significantly correlated with xCT levels (p = 0.001) and was significantly higher than K1 for 18F-FDG (p < 0.001). K1 for 18F-FDG was significantly correlated with GLUT1 levels (p = 0.002). vB estimated from 18F-FASu and 18F-FDG TACs was highly consistent and significantly correlated (r = 0.85, p < 0.001). Two qualitatively different 18F-FASu uptake profiles were identified: type α with low xCT expression and low K1 (A549 and HBCx3), and type ß with high xCT expression and high K1 (MAS98.12 and TPMX). CONCLUSION: The influx rate of 18F-FASu reflects xCT activity in tumour xenografts. Dynamic PET with pharmacokinetic modelling is needed to fully appraise 18F-FASu distribution routes.

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

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.

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

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 Bacillaceae, Halobacteriaceae, and Prevotellaceae. While we could not define the first two families to the species level, Prevotellaceae was assigned to Prevotella bivia. By co-culturing a cervical cancer cell line with P. bivia, 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.

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

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 (HFDWI) of CSH images correlated strongly (R2 = 0.66; n = 41) with pimonidazole immunoscore (HSPimo); this relationship was validated in a second pimonidazole cohort (R2 = 0.54; n = 54). We observed good agreement between CSH images and pimonidazole staining in whole-mount sections. HFDWI correlated with tumor stage and lymph node status, consistent with findings for HSPimo 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.Significance: 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.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4774/F1.large.jpg Cancer Res; 78(16); 4774-85. ©2018 AACR.

Low dose-rate irradiation with [3H]-labelled valine to selectively target hypoxic cells in a human colorectal cancer xenograft model.

BACKGROUND: Earlier in vitro studies show that irradiation with an ultra-low dose-rate of 15 mGy/h delivered with [3H]-valine leads to loss of clonogenicity in hypoxic T-47D cells. Here, the aim was to determine if [3H]-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 [3H]-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 [3H]-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, [3H]-valine is not the appropriate method to deliver ultra-low dose-rate irradiation in vivo.

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

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.

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.

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.

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

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.

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

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.

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

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.

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

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.

Antiapoptotic intervention in repeated blood cardioplegia: a porcine study of myocardial function.

BACKGROUND: Lethal reperfusion injury has been associated with apoptotic cell death. Insulin and insulin-like growth factors (IGF-I/IGF-II) may modulate this cell death when administered at the onset of reperfusion after ischemia. We explored if antiapoptotic treatment with IGF-II could influence left ventricular function in an experimental model with cardiopulmonary bypass and repeated oxygenated blood cardioplegia. METHODS: Twenty pigs underwent cardiopulmonary bypass with aortic cross-clamping for 60 minutes. In controls, hearts were arrested with cold, oxygenated blood cardioplegia repeated after 20 and 40 minutes. In the intervention group IGF-II was added to the cardioplegic solution at 20 and 40 minutes. After declamping and weaning from cardiopulmonary bypass, left ventricular global and local function was evaluated with a conductance catheter and tissue velocity imaging. Three hours after declamping the anterior left ventricle wall was divided in three layers and studied for blood flow rate with microspheres, Akt phosphorylation, and caspase-3 cleavage. Troponin-T levels were measured at baseline and after 3 hours of reperfusion. RESULTS: A reduction of myocardial levels of cleaved caspase-3 (p < 0.001) was found in the subendocardial wall layer and serum troponin-T was reduced (p < 0.025) in the IGF-II group 3 hours after declamping. In the IGF-II treated animals, left ventricular preload recruitable stroke work was low 1 hour after declamping and increased to levels higher than in controls (p < 0.025) 3 hours after declamping. Other cardiac variables did not differ between groups. CONCLUSIONS: When added to repeated cold blood cardioplegia, IGF-II reduces apoptosis and ischemia-reperfusion injury with minor effects on cardiac function.

Normalization strategy is critical for the outcome of miRNA expression analyses in the rat heart.

Since normalization strategies plays a pivotal role for obtaining reliable results when performing quantitative PCR (qPCR) analyses, this study investigated several miRNA normalization candidates in regards to their efficiency as normalization standards in the ischemic reperfused ex vivo rat heart, with special reference to regulation of the miRNAs miR-1 and miR-101b. The possibility of including primers for several miRNAs in one reverse transcription (RT) reaction was also investigated. Langendorff perfused rat hearts were subjected to 30 min regional ischemia and 0, 1, 5, 15, or 120 min reperfusion. Total RNA was isolated and reverse transcribed for miRNA qPCR analysis. Normalization candidates were evaluated by the NormFinder and geNorm algorithms and the following stability expression rank order was obtained: sno202 < U6B < U87 < snoRNA < 4.5S RNA A < Y1 < 4.5S RNA B < GAPDH. Applying U6B as a normalizer it was found that miR-1 and miR-101b was downregulated in the ischemic reperfused myocardium. Furthermore, up to three primers could be included in one RT reaction by replacing RNase-free water with two supplemental sets of primers in the TaqMan MicroRNA assay protocol. This study demonstrates the importance of validating normalization standards when performing miRNA expression analyses by qPCR, and that miR-1 and miR-101b may play an important role during early reperfusion of the ischemic rat heart.