Ann Rita Halvorsen

Pan-cancer analysis of somatic copy-number alterations implicates IRS4 and IGF2 in enhancer hijacking

Extensive prior research focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearrangement of cis-regulatory elements (CREs) remains unclear. Here we present a framework for inferring cancer-related gene overexpression resulting from CRE reorganization (e.g., enhancer hijacking) by integrating SCNAs, gene expression data and information on topologically associating domains (TADs). Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer is associated with recurrent deletions in cis, and we present evidence supporting a tumor-promoting role. We additionally pursued cancer-type-specific analyses and uncovered IGF2 as a target for enhancer hijacking in colorectal cancer. Recurrent tandem duplications intersecting with a TAD boundary mediate de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, resulting in high-level gene activation. Our framework enables systematic inference of CRE rearrangements mediating dysregulation in cancer.

A unique set of 6 circulating microRNAs for early detection of non-small cell lung cancer

Introduction Circulating microRNAs are promising biomarkers for diagnosis, predication and prognostication of diseases. Lung cancer is the cancer disease accountable for most cancer deaths, largely due to being diagnosed at late stages. Therefore, diagnosing lung cancer patients at an early stage is crucial for improving the outcome. The purpose of this study was to identify circulating microRNAs for detection of early stage lung cancer, capable of discriminating lung cancer patients from those with chronic obstructive pulmonary disease (COPD) and healthy volunteers. Results We identified 7 microRNAs separating lung cancer patients from controls. By using RT-qPCR, we validated 6 microRNAs (miR-429, miR-205, miR-200b, miR-203, miR-125b and miR-34b) with a significantly higher abundance in serum from NSCLC patients. Furthermore, the 6 miRNAs were validated in a different dataset, revealing an area under the receiver operating characteristic curve of 0.89 for stage I-IV and 0.88 for stage I/II. Materials and Methods We profiled the expression of 754 unique microRNAs by TaqMan Low Density Arrays, and analyzed serum from 38 patients with NSCLC, 16 patients suffering from COPD and 16 healthy volunteers from Norway, to explore their potential as diagnostic biomarkers. For validation, we analyzed serum collected from high-risk individuals enrolled in the Valencia branch of the International Early Lung Cancer Action Program screening trial (n=107) in addition to 51 lung cancer patients. Conclusion Considering the accessibility and stability of circulating miRNAs, these 6 microRNAs are promising biomarkers as a supplement in future screening studies.

Osteopontin is a prognostic biomarker in non-small cell lung cancer

BackgroundIn a previously published report we characterized the expression of the metastasis-associated proteins S100A4, osteopontin (OPN) and ephrin-A1 in a prospectively collected panel of non-small cell lung cancer (NSCLC) tumors. The aim of the present follow-up study was to investigate the prognostic impact of these potential biomarkers in the same patient cohort. In addition, circulating serum levels of OPN were measured and single nucleotide polymorphisms (SNP) in the -443 position of the OPN promoter were analyzed.MethodsAssociations between immunohistochemical expression of S100A4, OPN and ephrin-A1 and relapse free and overall survival were examined using univariate and multivariate analyses. Serum OPN was measured by ELISA, polymorphisms in the -443 position of the tumor OPN promoter were analyzed by PCR, and associations between OPN levels and promoter polymorphisms and clinicopathological parameters and patient outcome were investigated.ResultsHigh expression of OPN in NSCLC tumors was associated with poor patient outcome, and OPN was a strong, independent prognostic factor for both relapse free and overall survival. Serum OPN levels increased according to tumor pT classification and tumor size, and patients with OPN-expressing tumors had higher serum levels than patients with OPN-negative tumors. S100A4 was a negative prognostic factor in several subgroups of adenocarcinoma patients, but not in the overall patient cohort. There was no association between ephrin-A1 expression and patient outcome.ConclusionsOPN is a promising prognostic biomarker in NSCLC, and should be further explored in the selection of patients for adjuvant treatment following surgical resection.

Differential DNA methylation analysis of breast cancer reveals the impact of immune signaling in radiation therapy

Radiotherapy (RT) is a central treatment modality for breast cancer patients. The purpose of our study was to investigate the DNA methylation changes in tumors following RT, and to identify epigenetic markers predicting treatment outcome. Paired biopsies from patients with inoperable breast cancer were collected both before irradiation (n = 20) and after receiving 10–24 Gray (Gy) (n = 19). DNA methylation analysis was performed by using Illumina Infinium 27K arrays. Fourteen genes were selected for technical validation by pyrosequencing. Eighty‐two differentially methylated genes were identified in irradiated (n = 11) versus nonirradiated (n = 19) samples (false discovery rate, FDR = 1.1%). Methylation levels in pathways belonging to the immune system were most altered after RT. Based on methylation levels before irradiation, a panel of five genes (H2AFY, CTSA, LTC4S, IL5RA and RB1) were significantly associated with clinical response (p = 0.041). Furthermore, the degree of methylation changes for 2,516 probes correlated with the given radiation dose. Within the 2,516 probes, an enrichment for pathways involved in cellular immune response, proliferation and apoptosis was identified (FDR < 5%). Here, we observed clear differences in methylation levels induced by radiation, some associated with response to treatment. Our study adds knowledge on the molecular mechanisms behind radiation response.

Genome-wide DNA methylation analyses in lung adenocarcinomas: Association with EGFR, KRAS and TP53 mutation status, gene expression and prognosis

Background: DNA methylation alterations are early events in tumorigenesis and important in the regulation of gene expression in cancer cells. Lung cancer patients have in general a poor prognosis, and a deeper insight into the epigenetic landscape in lung adenocarcinoma tumors and its prognostic implications is needed.

Evaluation of Prognostic and Predictive Significance of Circulating MicroRNAs in Ovarian Cancer Patients

Ovarian cancer patients are recognized with poor prognosis. This study aimed to identify microRNAs in plasma for predicting response to treatment and outcome. We have investigated microRNAs in plasma from ovarian cancer patients enrolled in a large multicenter study (ICON7), investigating the effect of adding bevacizumab to standard chemotherapy in patients diagnosed with epithelial ovarian cancer. Patients with different histology, grade, and FIGO stages were included (n = 207) in this study. Screening of 754 unique microRNAs was performed in the discovery phase (n = 91) using TaqMan Low Density Arrays. The results were validated using single assays and RT-qPCR. Low levels of miR-200b, miR-1274A (tRNALys5), and miR-141 were significantly associated with better survival, confirmed with log-rank test in the validation set. The level of miR-1274A (tRNALys5) correlated with outcome was especially pronounced in the high-grade serous tumors. Interestingly, low level of miR-200c was associated with 5-month prolongation of PFS when treated with bevacizumab compared to standard chemotherapy. We found prognostic significance of miR-200b, miR-141, and miR-1274A (tRNALys5) in all histological types, where miR-1274A (tRNALys5) may be a specific marker in high-grade serous tumors. The level of miR-200c may be predictive of effect of treatment with bevacizumab. However, this needs further validation.

Human papilloma virus detection and typing in 334 lung cancer patients

Abstract Background. Unlike cervical, anogenital and oropharyngeal cancers, where high-risk human papillomavirus (hrHPV) has long been known to play a major role, a causative link between HPV and lung cancer has been investigated for decades with discrepant results. Methods. Lung cancer patients eligible for surgical treatment were tested for the presence of HPV-DNA in excised, fresh frozen lung tumor tissue. Patients that tested positive were further examined for the presence of HPV-DNA in adjacent normal lung parenchyma. HPV detection and genotyping was performed using a polymerase chain reaction (PCR)-based approach and allowed the typing of 13 “high-risk”-HPV-types and 2 “low-risk”-HPV-types. Results. Of the 334 tumor-DNA samples tested, 13 (3.9%) showed presence of HPV-DNA, of which 12 were of a high-risk HPV type (16, 33, 66). In those tested positive, HPV-DNA was not found in adjacent normal lung tissue. No correlation with smoking or EGFR/KRAS mutation status was seen, and only one of 84 squamous cell carcinomas was HPV-positive. Conclusion. We conclude that HPV is rarely associated with lung cancer in a Northern European population and in those tested positive, more functional studies are required to determine the role HPV plays in lung cancer oncogenesis.

TP53 Mutation Spectrum in Smokers and Never Smoking Lung Cancer Patients.

Background: TP53 mutations are among the most common mutations found in lung cancers, identified as an independent prognostic factor in many types of cancers. The purpose of this study was to investigate the frequency and prognostic impact of TP53 mutations in never-smokers and in different histological subtypes of lung cancer. Methods: We analyzed tumor tissue from 394 non-small cell carcinomas including adenocarcinomas (n = 229), squamous cell carcinomas (n = 112), large cell carcinomas (n = 30), and others (n = 23) for mutations in TP53 by the use of Sanger sequencing (n = 394) and next generation sequencing (n = 100). Results: TP53 mutations were identified in 47.2% of the samples, with the highest frequency (65%) of mutations among squamous cell carcinomas. Among never-smokers, 36% carried a TP53 mutation, identified as a significant independent negative prognostic factor in this subgroup. For large cell carcinomas, a significantly prolonged progression free survival was found for those carrying a TP53 mutation. In addition, the frequency of frameshift mutations was doubled in squamous cell carcinomas (20.3%) compared to adenocarcinomas (9.1%). Conclusion: TP53 mutation patterns differ between the histological subgroups of lung cancers, and are also influenced by smoking history. This indicates that the histological subtypes in lung cancer are genetically different, and that smoking-induced TP53 mutations may have a different biological impact than TP53 mutations occurring in never-smokers.

Novel Molecular Tumor Cell Markers in Regional Lymph Nodes and Blood Samples from Patients Undergoing Surgery for Non-Small Cell Lung Cancer

Introduction Recent evidence suggests that microscopic lymph node metastases and circulating tumor cells may have clinical importance in lung cancer. The purpose of this study was to identify new molecular markers for tumor cells in regional lymph nodes (LNs) and peripheral blood (PB) from patients with non-small cell lung cancer (NSCLC). Methods Candidate markers were selected based on digital transcript profiling and previous literature. KRT19, CEACAM5, EPCAM, DSG3, SFTPA, SFTPC and SFTPB mRNA levels were initially validated by real-time reverse transcription PCR-based quantification in 16 NSCLC tumors and 22 LNs and 12 PB samples from individuals without known cancer. Five of the candidate markers were selected for secondary validation by quantification in parallel tumor biopsies, regional LNs and PB samples from 55 patients undergoing surgery for NSCLC. LN and PB marker status were compared to clinicopathological patient data. Results All selected markers except DSG3 were present at high levels in the primary tumors and at very low or non-detectable levels in normal LNs and PB in the first round of validation, indicating a potential for detecting tumor cells in NSCLC patients. The expression profiles of KRT19, CEACAM5, DSG3, SFTPA and SFTPC mRNA were confirmed in the larger group during the secondary validation. Using the highest normal LN level of each marker as threshold, 39 (71%) of the 55 patients had elevated levels of at least one marker in regional LNs. Similarly, 26 (47%) patients had elevated levels of at least one marker in PB. A significantly higher number of patients with adenocarcinomas had positive LN status for these markers, compared with other histological types (P = 0.004). Conclusions Several promising molecular tumor cell markers in regional LNs and PB were identified, including the new SFTPA and SFTPC mRNAs. Clinical follow-up in a larger cohort is needed to elucidate their prognostic value.

Non-small cell lung cancer is characterised by a distinct inflammatory signature in serum compared with chronic obstructive pulmonary disease

Development of lung cancer is closely related to smoking in a majority of patients. Most smokers, however, do not develop lung cancer in spite of a high mutational load accumulating in the lung tissue. Here we investigate whether a cancer‐specific footprint can be revealed by investigating circulating inflammatory markers in patients with non‐small cell lung cancer (NSCLC) compared with patients with chronic obstructive pulmonary disease (COPD), both cohorts characterised by similar smoking history. Serum concentrations of 57 cytokines and matrix metalloproteinases (MMPs) from 43 patients with advanced NSCLC were evaluated by multiplex immunoassays and compared with serum samples from 35 patients with COPD. Unsupervised hierarchical clustering and non‐parametric analyses were performed. False discovery rate was used to adjust for multiple testing. Clustering of cytokine and MMP concentrations in the serum revealed a distinct separation of the NSCLC patients from the COPD group. Individual concentrations of thymus and activation‐regulated cytokine (C‐C motif chemokine ligand 17), Gro‐b (C‐X‐C motif chemokine ligand 2 (CXCL2)), CXCL13, interleukin (IL)‐1ra, IL‐6, IL‐8 (CXCL8), IL‐16, IL‐17A, macrophage migration inhibitory factor (MIF), granulocyte colony‐stimulating factor, platelet‐derived growth factor subunit B, MMP‐2, MMP‐8 and MMP‐12 were significantly different in serum from NSCLC and COPD patients. Moreover, the interferon‐γ/IL‐10 ratio was lower in cancer patients compared with COPD patients, consistent with a cytokine milieu favouring tumour tolerance. Our results suggest that NSCLC is characterised by a distinct inflammatory signature in serum. The different cytokine profiles in NSCLC and COPD patients may represent tumour‐promoting and tumour‐suppressing immune responses developing in response to mucosal inflammation and mutations induced by smoking.