Malin Lando

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.

Gene Dosage, Expression, and Ontology Analysis Identifies Driver Genes in the Carcinogenesis and Chemoradioresistance of Cervical Cancer

Integrative analysis of gene dosage, expression, and ontology (GO) data was performed to discover driver genes in the carcinogenesis and chemoradioresistance of cervical cancers. Gene dosage and expression profiles of 102 locally advanced cervical cancers were generated by microarray techniques. Fifty-two of these patients were also analyzed with the Illumina expression method to confirm the gene expression results. An independent cohort of 41 patients was used for validation of gene expressions associated with clinical outcome. Statistical analysis identified 29 recurrent gains and losses and 3 losses (on 3p, 13q, 21q) associated with poor outcome after chemoradiotherapy. The intratumor heterogeneity, assessed from the gene dosage profiles, was low for these alterations, showing that they had emerged prior to many other alterations and probably were early events in carcinogenesis. Integration of the alterations with gene expression and GO data identified genes that were regulated by the alterations and revealed five biological processes that were significantly overrepresented among the affected genes: apoptosis, metabolism, macromolecule localization, translation, and transcription. Four genes on 3p (RYBP, GBE1) and 13q (FAM48A, MED4) correlated with outcome at both the gene dosage and expression level and were satisfactorily validated in the independent cohort. These integrated analyses yielded 57 candidate drivers of 24 genetic events, including novel loci responsible for chemoradioresistance. Further mapping of the connections among genetic events, drivers, and biological processes suggested that each individual event stimulates specific processes in carcinogenesis through the coordinated control of multiple genes. The present results may provide novel therapeutic opportunities of both early and advanced stage cervical cancers.

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. Copyright

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.

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers.

Membranous Expression of Ectodomain Isoforms of the Epidermal Growth Factor Receptor Predicts Outcome after Chemoradiotherapy of Lymph Node–Negative Cervical Cancer

Purpose: 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. Experimental Design: 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. Results: 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. Conclusion: Membranous expression of ectodomain EGFR isoforms, and not TK activation, predicts poor outcome after chemoradiotherapy for patients with lymph node–negative cervical cancer. Clin Cancer Res; 17(16); 5501–12. ©2011 AACR.

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

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

Phosphorylation of EGFR measured with in situ proximity ligation assay: Relationship to EGFR protein level and gene dosage in cervical cancer

PURPOSE 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSION 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.

Hypoxia-induced alterations of G2 checkpoint regulators

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.

Abstract 807: Eight candidate target genes of the recurrent 3p12-p14 loss in cervical cancer.

Genetic gains and losses, i.e. gene dosage alterations, influence gene expressions and thereby promote tumor development and progression. Identification and functional assessment of such alterations may help to understand the biology of the disease and be useful for the discovery of diagnostic and therapeutic biomarkers. The recurrent 3p12-p14 loss in cervical cancer has been proposed as a prognostic biomarker, however, its pathogenetic role, including its target genes, remains to be clarified. The purpose of this work was to identify 3p target genes in cervical cancer, and explore their role in the development of chemoradioresistance. Cox regression analysis of the 3p gene dosages showed that loss of 3p11.2-p14.2 was significantly associated with clinical outcome. To depict candidate target genes of the loss, pairwise gene dosage and expression profiling was performed on 77 patients by array comparative genomic hybridization and Illumina gene expression beadarrays, respectively, including all genes within the 3p11.2–p14.2 region. The expression of eight of the 147 genes within the region; i.e., THOC7, PSMD6, SLC25A26, TMF1, RYBP, SHQ1, EBLN2, and GBE1, were highly downregulated in cases with loss. This was confirmed at the protein level for RYBP and TMF1 in 150 patients by immunohistochemistry. A gene signature with the expression values of the eight candidate genes was constructed to explore the prognostic impact of all genes combined. Unsupervised hierarchical clustering divided the patients into two groups, for which the group with downregulation of the genes had the highest frequency of 3p loss and a poor outcome compared to the other. A 3p target gene score was calculated for each tumor, and was shown to be lower for patients with 3p loss and associated with clinical outcome. The prognostic impact of the eight gene signature was confirmed in a validation cohort of 74 patients, showing a worse outcome for patients with a low 3p target gene score, confirming the clinical significance of the signature. To assess the importance of the signature in comparison to existing clinical markers, we merged the two cohorts to a group of 151 patients. In univariate Cox regression analysis, the 3p target gene score was associated with outcome. All eight genes showed a significant or clear tendency towards a relationship to outcome in a single gene analysis, and therefore seemed to contribute to the univariate result. In multivariate analysis, the score emerged as a prognostic factor independent of lymph node status, tumor size, and stage. These results support a role of the eight candidate genes as targets of the 3p12–p14 loss in cervical cancer and propose their use as biomarker in the clinical decision-making. Citation Format: Malin Lando, Marit Holden, Ruth Holm, Gunnar B. Kristensen, Heidi Lyng. Eight candidate target genes of the recurrent 3p12-p14 loss in cervical cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 807. doi:10.1158/1538-7445.AM2013-807