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1.  The Kidney Transcriptome and Proteome Defined by Transcriptomics and Antibody-Based Profiling 
PLoS ONE  2014;9(12):e116125.
To understand renal functions and disease, it is important to define the molecular constituents of the various compartments of the kidney. Here, we used comparative transcriptomic analysis of all major organs and tissues in the human body, in combination with kidney tissue micro array based immunohistochemistry, to generate a comprehensive description of the kidney-specific transcriptome and proteome. A special emphasis was placed on the identification of genes and proteins that were elevated in specific kidney subcompartments. Our analysis identified close to 400 genes that had elevated expression in the kidney, as compared to the other analysed tissues, and these were further subdivided, depending on expression levels, into tissue enriched, group enriched or tissue enhanced. Immunohistochemistry allowed us to identify proteins with distinct localisation to the glomeruli (n = 11), proximal tubules (n = 120), distal tubules (n = 9) or collecting ducts (n = 8). Among the identified kidney elevated transcripts, we found several proteins not previously characterised or identified as elevated in kidney. This description of the kidney specific transcriptome and proteome provides a resource for basic and clinical research to facilitate studies to understand kidney biology and disease.
PMCID: PMC4281243  PMID: 25551756
2.  The Transcriptomic and Proteomic Landscapes of Bone Marrow and Secondary Lymphoid Tissues 
PLoS ONE  2014;9(12):e115911.
The sequencing of the human genome has opened doors for global gene expression profiling, and the immense amount of data will lay an important ground for future studies of normal and diseased tissues. The Human Protein Atlas project aims to systematically map the human gene and protein expression landscape in a multitude of normal healthy tissues as well as cancers, enabling the characterization of both housekeeping genes and genes that display a tissue-specific expression pattern. This article focuses on identifying and describing genes with an elevated expression in four lymphohematopoietic tissue types (bone marrow, lymph node, spleen and appendix), based on the Human Protein Atlas-strategy that combines high throughput transcriptomics with affinity-based proteomics.
An enriched or enhanced expression in one or more of the lymphohematopoietic tissues, compared to other tissue-types, was seen for 693 out of 20,050 genes, and the highest levels of expression were found in bone marrow for neutrophilic and erythrocytic genes. A majority of these genes were found to constitute well-characterized genes with known functions in lymphatic or hematopoietic cells, while others are not previously studied, as exemplified by C19ORF59.
In this paper we present a strategy of combining next generation RNA-sequencing with in situ affinity-based proteomics in order to identify and describe new gene targets for further research on lymphatic or hematopoietic cells and tissues. The results constitute lists of genes with enriched or enhanced expression in the four lymphohematopoietic tissues, exemplified also on protein level with immunohistochemical images.
PMCID: PMC4277406  PMID: 25541736
3.  1p36 deletion is a marker for tumour dissemination in microsatellite stable stage II-III colon cancer 
BMC Cancer  2014;14(1):872.
The clinical behaviour of colon cancer is heterogeneous. Five-year overall survival is 50-65% with all stages included. Recurring somatic chromosomal alterations have been identified and some have shown potential as markers for dissemination of the tumour, which is responsible for most colon cancer deaths. We investigated 115 selected stage II-IV primary colon cancers for associations between chromosomal alterations and tumour dissemination.
Follow-up was at least 5 years for stage II-III patients without distant recurrence. Affymetrix SNP 6.0 microarrays and allele-specific copy number analysis were used to identify chromosomal alterations. Fisher’s exact test was used to associate alterations with tumour dissemination, detected at diagnosis (stage IV) or later as recurrent disease (stage II-III).
Loss of 1p36.11-21 was associated with tumour dissemination in microsatellite stable tumours of stage II-IV (odds ratio = 5.5). It was enriched to a similar extent in tumours with distant recurrence within stage II and stage III subgroups, and may therefore be used as a prognostic marker at diagnosis. Loss of 1p36.11-21 relative to average copy number of the genome showed similar prognostic value compared to absolute loss of copies. Therefore, the use of relative loss as a prognostic marker would benefit more patients by applying also to hyperploid cancer genomes. The association with tumour dissemination was supported by independent data from the The Cancer Genome Atlas.
Deletions on 1p36 may be used to guide adjuvant treatment decisions in microsatellite stable colon cancer of stages II and III.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-872) contains supplementary material, which is available to authorized users.
PMCID: PMC4251789  PMID: 25420937
Colon cancer; Prognostic marker; Allele-specific copy number analysis; Genome duplication; 1p36; Metastasis; Tumour dissemination
4.  Prognostic Influence of Pre-Operative C-Reactive Protein in Node-Negative Breast Cancer Patients 
PLoS ONE  2014;9(10):e111306.
The importance of inflammation is increasingly noticed in cancer. The aim of this study was to analyze the prognostic influence of pre-operative serum C-reactive protein (CRP) in a cohort of 148 lymph node-negative breast cancer patients. The prognostic significance of CRP level for disease-free survival (DFS), metastasis-free survival (MFS) and overall survival (OS) was evaluated using univariate and multivariate Cox regression, also including information on age at diagnosis, tumor size, tumor grade, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status, proliferation index (Ki67) and molecular subtype, as well as an assessment of the presence of necrosis and inflammation in the tumor tissue. Univariate analysis showed that CRP, as a continuous variable, was significantly associated with DFS (P = 0.002, hazard ratio [HR]  = 1.04, 95% confidence interval [CI]  = 1.02–1.07) and OS (P = 0.036, HR  = 1.03, 95% CI  = 1.00–1.06), whereas a trend was observed for MFS (P = 0.111). In the multivariate analysis, CRP retained its significance for DFS (P = 0.033, HR  = 1.01, 95% CI  = 1.00–1.07) as well as OS (P = 0.023, HR  = 1.03, 95% CI  = 1.00–1.06), independent of established prognostic factors. Furthermore, large-scale gene expression analysis by Affymetrix HG-U133A arrays was performed for 72 (48.6%) patients. The correlations between serum CRP and gene expression levels in the corresponding carcinoma of the breast were assessed using Spearman's rank correlation, controlled for false-discovery rate. No significant correlation was observed between CRP level and gene expression indicative of an ongoing local inflammatory process. In summary, pre-operatively elevated CRP levels at the time of diagnosis were associated with shorter DFS and OS independent of established prognostic factors in node-negative breast cancer, supporting a possible link between inflammation and prognosis in breast cancer.
PMCID: PMC4207815  PMID: 25340395
5.  In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics 
Oncotarget  2013;4(12):2407-2418.
Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.
PMCID: PMC3926836  PMID: 24280411
Padlock probes; RCA; in situ; KRAS; cancer diagnostics
6.  Genomic and Transcriptional Alterations in Lung Adenocarcinoma in Relation to EGFR and KRAS Mutation Status 
PLoS ONE  2013;8(10):e78614.
In lung adenocarcinoma, the mutational spectrum is dominated by EGFR and KRAS mutations. Improved knowledge about genomic and transcriptional alterations in and between mutation-defined subgroups may identify genes involved in disease development or progression.
Genomic profiles from 457 adenocarcinomas, including 113 EGFR-mutated, 134 KRAS-mutated and 210 EGFR and KRAS-wild type tumors (EGFRwt/KRASwt), and gene expression profiles from 914 adenocarcinomas, including 309 EGFR-mutated, 192 KRAS-mutated, and 413 EGFRwt/KRASwt tumors, were assembled from different repositories. Genomic and transcriptional differences between the three mutational groups were analyzed by both supervised and unsupervised methods.
EGFR-mutated adenocarcinomas displayed a larger number of copy number alterations and recurrent amplifications, a higher fraction of total loss-of-heterozygosity, higher genomic complexity, and a more distinct expression pattern than EGFR-wild type adenocarcinomas. Several of these differences were also consistent when the three mutational groups were stratified by stage, gender and smoking status. Specific copy number alterations were associated with mutation status, predominantly including regions of gain with the highest frequency in EGFR-mutated tumors. Differential regions included both large and small regions of gain on 1p, 5q34-q35.3, 7p, 7q11.21, 12p12.1, 16p, and 21q, and losses on 6q16.3-q21, 8p, and 9p, with 20-40% frequency differences between the mutational groups. Supervised gene expression analyses identified 96 consistently differentially expressed genes between the mutational groups, and together with unsupervised analyses these analyses highlighted the difficulty in broadly resolving the three mutational groups into distinct transcriptional entities.
We provide a comprehensive overview of the genomic and transcriptional landscape in lung adenocarcinoma stratified by EGFR and KRAS mutations. Our analyses suggest that the overall genomic and transcriptional landscape of lung adenocarcinoma is affected, but only to a minor extent, by EGFR and KRAS mutation status.
PMCID: PMC3812039  PMID: 24205279
7.  KRAS analysis in colorectal carcinoma: Analytical aspects of Pyrosequencing and allele-specific PCR in clinical practice 
BMC Cancer  2010;10:660.
Epidermal growth factor receptor inhibitor therapy is now approved for treatment of metastatic colorectal carcinomas (CRC) in patients with tumors lacking KRAS mutations. Several procedures to detect KRAS mutations have been developed. However, the analytical sensitivity and specificity of these assays on routine clinical samples are not yet fully characterised.
The practical aspects and clinical applicability of a KRAS-assay based on Pyrosequencing were evaluated in a series of 314 consecutive CRC cases submitted for diagnostic KRAS analysis. The performance of Pyrosequencing compared to allele-specific, real-time PCR was then explored by a direct comparison of CE-IVD-marked versions of Pyrosequencing and TheraScreen (DxS) KRAS assays for a consecutive subset (n = 100) of the 314 clinical CRC samples.
Using Pyrosequencing, 39% of the 314 CRC samples were found KRAS-mutated and several of the mutations (8%) were located in codon 61. To explore the analytical sensitivity of the Pyrosequencing assay, mutated patient DNA was serially diluted with wild-type patient DNA. Dilutions corresponding to 1.25-2.5% tumor cells still revealed detectable mutation signals. In clinical practice, our algorithm for KRAS analysis includes a reanalysis of samples with low tumor cell content (< 10%, n = 56) using an independent assay (allele-specific PCR, DxS). All mutations identified by Pyrosequencing were then confirmed and, in addition, one more mutated sample was identified in this subset of 56 samples. Finally, a direct comparison of the two technologies was done by re-analysis of a subset (n = 100) of the clinical samples using CE-IVD-marked versions of Pyrosequencing and TheraScreen KRAS assays in a single blinded fashion. The number of samples for which the KRAS codon 12/13 mutation status could be defined using the Pyrosequencing or the TheraScreen assay was 94 and 91, respectively, and both assays detected the same number of codon 12 and 13 mutations.
KRAS mutation detection using Pyrosequencing was evaluated on a consecutive set of clinical CRC samples. Pyrosequencing provided sufficient analytical sensitivity and specificity to assess the mutation status in routine formalin-fixed CRC samples, even in tissues with a low tumor cell content.
PMCID: PMC3002357  PMID: 21122130
8.  Quantification of Normal Cell Fraction and Copy Number Neutral LOH in Clinical Lung Cancer Samples Using SNP Array Data 
PLoS ONE  2009;4(6):e6057.
Technologies based on DNA microarrays have the potential to provide detailed information on genomic aberrations in tumor cells. In practice a major obstacle for quantitative detection of aberrations is the heterogeneity of clinical tumor tissue. Since tumor tissue invariably contains genetically normal stromal cells, this may lead to a failure to detect aberrations in the tumor cells.
Principal Finding
Using SNP array data from 44 non-small cell lung cancer samples we have developed a bioinformatic algorithm that accurately models the fractions of normal and tumor cells in clinical tumor samples. The proportion of normal cells in combination with SNP array data can be used to detect and quantify copy number neutral loss-of-heterozygosity (CNNLOH) in the tumor cells both in crude tumor tissue and in samples enriched for tumor cells by laser capture microdissection.
Genome-wide quantitative analysis of CNNLOH using the CNNLOH Quantifier method can help to identify recurrent aberrations contributing to tumor development in clinical tumor samples. In addition, SNP-array based analysis of CNNLOH may become important for detection of aberrations that can be used for diagnostic and prognostic purposes.
PMCID: PMC2699026  PMID: 19557126

Results 1-8 (8)