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1.  Cancer suspicion in general practice, urgent referral and time to diagnosis: a population-based GP survey and registry study 
BMC Cancer  2014;14(1):636.
Many countries have implemented standardised cancer patient pathways (CPPs) to ensure fast diagnosis of patients suspected of having cancer. Yet, studies are sparse on the impact of such CPPs, and few have distinguished between referral routes. For incident cancer patients, we aimed to determine how often GPs suspected cancer at the time of first presentation of symptoms in general practice and to describe the routes of referral for further investigation. In addition, we aimed to analyse if the GP’s suspicion of cancer could predict the choice of referral to a CPP. Finally, we aimed to analyse associations between not only cancer suspicion and time to cancer diagnosis, but also between choice of referral route and time to cancer diagnosis.
We conducted a population-based, cross-sectional study of incident cancer patients in Denmark who had attended general practice prior to their diagnosis of cancer. Data were collected from GP questionnaires and national registers. We estimated the patients’ chance of being referred to a CPP (prevalence ratio (PR)) using Poisson regression. Associations between the GP’s symptom interpretation, use of CPP and time to diagnosis were estimated using quantile regression.
5,581 questionnaires were returned (response rate: 73.8%). A GP was involved in diagnosing the cancer in 4,101 (73.5%) cases (3,823 cases analysed). In 48.2% of these cases, the GP interpreted the patient’s symptoms as ‘alarm’ symptoms suggestive of cancer. The GP used CPPs in 1,426 (37.3%) cases. Patients, who had symptoms interpreted as ‘vague’ had a lower chance of being referred to a CPP than when interpreted as ‘alarm’ symptoms (PR = 0.53 (95%CI: 0.48;0.60)). Patients with ‘vague’ symptoms had a 34 (95% CI: 28;41) days longer median time to diagnosis than patients with ‘alarm’ symptoms.
GPs suspect cancer more often than they initiate a CPP, and patients were less likely to be referred to a CPP when their symptoms were not interpreted as alarm symptoms of cancer. The GP’s choice of referral route was a strong predictor of the duration of the diagnostic interval, but the GP’s symptom interpretation was approximately twice as strong an indicator of a longer diagnostic interval.
PMCID: PMC4164756  PMID: 25175155
Fast-track; Neoplasm; (Early) diagnosis; General practice; Delay; Cancer suspicion; Denmark
2.  Hypomethylation and increased expression of the putative oncogene ELMO3 are associated with lung cancer development and metastases formation 
Oncoscience  2014;1(5):367-374.
Numerous genetic and epigenetic events driving tumorigenesis have been characterized. However, knowledge is lacking on the particular events required for the metastatic spread of cancer cells. The engulfment and cell motility 3 (ELMO3) gene plays an important role for the migratory potential of cells, but have not previously been studied in primary samples from cancer patients. We collected material from primary non-small cell lung cancer (NSCLC) tumors and paired brain or adrenal gland metastases from 26 patients and from 26 primary tumor samples from metastasis-free patients matched for age, gender, histology, T-stage, smoking status, and proportion of tumor cells. Using reverse transcriptase–quantitative PCR (RT-qPCR) ELMO3 was shown to be overexpressed in primary tumors from patients with distant metastases compared to normal lung tissue (p<0.001), and compared to primary tumors from metastasis-free patients (p<0.001). The increased expression coincided with decreased methylation levels of the ELMO3 promoter region. High expression and hypomethylation of ELMO3 were also observed when studying the paired brain and adrenal gland metastases. In conclusion, the putative oncogene, ELMO3, is overexpressed in NSCLC in combination with hypomethylation of its promoter and these cancer-specific events are associated with the formation of metastases.
PMCID: PMC4278312  PMID: 25594031
Non-Small Cell Lung Cancer; metastases; DNA methylation; oncogenes; microRNA
3.  Identification and characterization of locus-specific methylation patterns within novel loci undergoing hypermethylation during breast cancer pathogenesis 
Despite similar clinical and pathological features, large numbers of breast cancer patients experience different outcomes of the disease. This, together with the fact that the incidence of breast cancer is growing worldwide, emphasizes an urgent need for identification of new biomarkers for early cancer detection and stratification of patients.
We used ultrahigh-resolution microarrays to compare genomewide methylation patterns of breast carcinomas (n = 20) and nonmalignant breast tissue (n = 5). Biomarker properties of a subset of discovered differentially methylated regions (DMRs) were validated using methylation-sensitive high-resolution melting (MS-HRM) in a case–control study on a panel of breast carcinomas (n = 275) and non-malignant controls (n = 74).
On the basis of microarray results, we selected 19 DMRs for large-scale screening of cases and controls. Analysis of the screening results showed that all DMRs tested displayed significant gains of methylation in the cancer tissue compared to the levels in control tissue. Interestingly, we observed two types of locus-specific methylation, with loci undergoing either predominantly full or heterogeneous methylation during carcinogenesis. Almost all tested DMRs (17 of 19) displayed low-level methylation in nonmalignant breast tissue, independently of locus-specific methylation patterns in cases.
Specific loci can undergo either heterogeneous or full methylation during carcinogenesis, and loci hypermethylated in cancer frequently show low-level methylation in nonmalignant tissue.
PMCID: PMC3978461  PMID: 24490656
4.  Individual and combined effects of DNA methylation and copy number alterations on miRNA expression in breast tumors 
Genome Biology  2013;14(11):R126.
The global effect of copy number and epigenetic alterations on miRNA expression in cancer is poorly understood. In the present study, we integrate genome-wide DNA methylation, copy number and miRNA expression and identify genetic mechanisms underlying miRNA dysregulation in breast cancer.
We identify 70 miRNAs whose expression was associated with alterations in copy number or methylation, or both. Among these, five miRNA families are represented. Interestingly, the members of these families are encoded on different chromosomes and are complementarily altered by gain or hypomethylation across the patients. In an independent breast cancer cohort of 123 patients, 41 of the 70 miRNAs were confirmed with respect to aberration pattern and association to expression. In vitro functional experiments were performed in breast cancer cell lines with miRNA mimics to evaluate the phenotype of the replicated miRNAs. let-7e-3p, which in tumors is found associated with hypermethylation, is shown to induce apoptosis and reduce cell viability, and low let-7e-3p expression is associated with poorer prognosis. The overexpression of three other miRNAs associated with copy number gain, miR-21-3p, miR-148b-3p and miR-151a-5p, increases proliferation of breast cancer cell lines. In addition, miR-151a-5p enhances the levels of phosphorylated AKT protein.
Our data provide novel evidence of the mechanisms behind miRNA dysregulation in breast cancer. The study contributes to the understanding of how methylation and copy number alterations influence miRNA expression, emphasizing miRNA functionality through redundant encoding, and suggests novel miRNAs important in breast cancer.
PMCID: PMC4053776  PMID: 24257477
5.  Should Postmastectomy Radiotherapy to the Chest Wall and Regional Lymph Nodes Be Standard for Patients with 1–3 Positive Lymph Nodes? 
Breast Care  2011;6(5):347-351.
The indication for adjuvant postmastectomy radiotherapy (PMRT) in breast cancer patients with small tumors and 1–3 macrometastases in the axilla remains a controversial issue, despite the recommendation that PMRT should be applied in these patients in the most recent overview by the Early Breast Cancer Trialists’ Collaborative Group. In this report, we discuss the available data on the benefit from PMRT in patients diagnosed with N1 breast cancer. Based on this, we recommend adjuvant PMRT to the chest wall and regional lymph nodes in patients diagnosed with early node-positive breast cancer.
PMCID: PMC3357155  PMID: 22619643
Postmastectomy radiotherapy; Breast cancer; Positive lymph nodes; Randomized clinical trial
6.  A Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers 
Clinical proteomics  2010;6(4):129-151.
The lack of large panels of validated antibodies, tissue handling variability, and intratumoral heterogeneity potentially hamper comprehensive study of the functional proteome in non-microdissected solid tumors. The purpose of this study was to address these concerns and to demonstrate clinical utility for the functional analysis of proteins in non-microdissected breast tumors using reverse phase protein arrays (RPPA).
Herein, 82 antibodies that recognize kinase and steroid signaling proteins and effectors were validated for RPPA. Intraslide and interslide coefficients of variability were <15%. Multiple sites in non-microdissected breast tumors were analyzed using RPPA after intervals of up to 24 h on the benchtop at room temperature following surgical resection.
Twenty-one of 82 total and phosphoproteins demonstrated time-dependent instability at room temperature with most variability occurring at later time points between 6 and 24 h. However, the 82-protein functional proteomic “fingerprint” was robust in most tumors even when maintained at room temperature for 24 h before freezing. In repeat samples from each tumor, intratumoral protein levels were markedly less variable than intertumoral levels. Indeed, an independent analysis of prognostic biomarkers in tissue from multiple tumor sites accurately and reproducibly predicted patient outcomes. Significant correlations were observed between RPPA and immunohistochemistry. However, RPPA demonstrated a superior dynamic range. Classification of 128 breast cancers using RPPA identified six subgroups with markedly different patient outcomes that demonstrated a significant correlation with breast cancer subtypes identified by transcriptional profiling.
Thus, the robustness of RPPA and stability of the functional proteomic “fingerprint” facilitate the study of the functional proteome in non-microdissected breast tumors.
PMCID: PMC3116520  PMID: 21691416
Functional proteome; RPPA; Breast cancer; Kinase signaling; Steroid signaling
7.  Methylation of cancer related genes in tumor and peripheral blood DNA from the same breast cancer patient as two independent events 
Diagnostic Pathology  2011;6:116.
Recently it has been suggested that acquisition of methylation of the BRCA1 promoter detectable in peripheral blood (PB) DNA, could give raise to development of breast cancer. In this study, we aimed to investigate a relationship between methylation of three breast cancer related genes in PB DNA, and tumor specific (somatic) methylation of these genes in the same individual.
We have examined methylation status of the BRCA1, APC and RASSF1A promoter regions in a panel of 75 breast tumor and PB DNA samples from the same individual. In our study group, 4.0% of the patients displayed methylation of BRCA1 and APC in both tumor and the corresponding PB DNA. At the same time despite of marked methylation in tumor DNA, no methylation of BRCA1 and APC was seen in PB DNA of 4.3% and 2.7% of the patients respectively. The RASSF1A promoter did not show methylation in PB DNA.
Our results show that for at least a subset of cancer patients methylation of certain cancer related genes in PB DNA does not seem to be directly linked to somatic methylation of the same genes in tumor DNA, and therefore may only be specific to PB DNA.
PMCID: PMC3253685  PMID: 22129206
Methylation; cancer predisposition; BRCA1; APC; RASSF1A.
8.  Functional proteomics can define prognosis and predict pathologic complete response in patients with breast cancer 
Clinical proteomics  2011;8(1):11.
To determine whether functional proteomics improves breast cancer classification and prognostication and can predict pathological complete response (pCR) in patients receiving neoadjuvant taxane and anthracycline-taxane-based systemic therapy (NST).
Reverse phase protein array (RPPA) using 146 antibodies to proteins relevant to breast cancer was applied to three independent tumor sets. Supervised clustering to identify subgroups and prognosis in surgical excision specimens from a training set (n = 712) was validated on a test set (n = 168) in two cohorts of patients with primary breast cancer. A score was constructed using ordinal logistic regression to quantify the probability of recurrence in the training set and tested in the test set. The score was then evaluated on 132 FNA biopsies of patients treated with NST to determine ability to predict pCR.
Six breast cancer subgroups were identified by a 10-protein biomarker panel in the 712 tumor training set. They were associated with different recurrence-free survival (RFS) (log-rank p = 8.8 E-10). The structure and ability of the six subgroups to predict RFS was confirmed in the test set (log-rank p = 0.0013). A prognosis score constructed using the 10 proteins in the training set was associated with RFS in both training and test sets (p = 3.2E-13, for test set). There was a significant association between the prognostic score and likelihood of pCR to NST in the FNA set (p = 0.0021).
We developed a 10-protein biomarker panel that classifies breast cancer into prognostic groups that may have potential utility in the management of patients who receive anthracycline-taxane-based NST.
PMCID: PMC3170272  PMID: 21906370
Breast Cancer; Functional Proteomics; Prognosis; Prediction
9.  In vivo Identification and Specificity assessment of mRNA markers of hypoxia in human and mouse tumors 
BMC Cancer  2011;11:63.
Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers.
Mice carrying human (FaDudd) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared.
In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDudd. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker.
The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among tested genes, our study identified CA9, GLUT1 and possibly LOX as highly specific biomarkers of tumor hypoxia in vivo.
PMCID: PMC3042974  PMID: 21306648
10.  In Silico Ascription of Gene Expression Differences to Tumor and Stromal Cells in a Model to Study Impact on Breast Cancer Outcome 
PLoS ONE  2010;5(11):e14002.
Breast tumors consist of several different tissue components. Despite the heterogeneity, most gene expression analyses have traditionally been performed without prior microdissection of the tissue sample. Thus, the gene expression profiles obtained reflect the mRNA contribution from the various tissue components. We utilized histopathological estimations of area fractions of tumor and stromal tissue components in 198 fresh-frozen breast tumor tissue samples for a cell type-associated gene expression analysis associated with distant metastasis. Sets of differentially expressed gene-probes were identified in tumors from patients who developed distant metastasis compared with those who did not, by weighing the contribution from each tumor with the relative content of stromal and tumor epithelial cells in their individual tumor specimen. The analyses were performed under various assumptions of mRNA transcription level from tumor epithelial cells compared with stromal cells. A set of 30 differentially expressed gene-probes was ascribed solely to carcinoma cells. Furthermore, two sets of 38 and five differentially expressed gene-probes were mostly associated to tumor epithelial and stromal cells, respectively. Finally, a set of 26 differentially expressed gene-probes was identified independently of cell type focus. The differentially expressed genes were validated in independent gene expression data from a set of laser capture microdissected invasive ductal carcinomas. We present a method for identifying and ascribing differentially expressed genes to tumor epithelial and/or stromal cells, by utilizing pathologic information and weighted t-statistics. Although a transcriptional contribution from the stromal cell fraction is detectable in microarray experiments performed on bulk tumor, the gene expression differences between the distant metastasis and no distant metastasis group were mostly ascribed to the tumor epithelial cells of the primary breast tumors. However, the gene PIP5K2A was found significantly elevated in stroma cells in distant metastasis group, compared to stroma in no distant metastasis group. These findings were confirmed in gene expression data from the representative compartments from microdissected breast tissue. The method described was also found to be robust to different histopathological procedures.
PMCID: PMC2988804  PMID: 21124964
11.  A simple way to evaluate self-designed probes for tumor specific Multiplex Ligation-dependent Probe Amplification (MLPA) 
BMC Research Notes  2010;3:179.
The Multiplex Ligation-dependent Probe Amplification (MLPA) is widely used for analysis of copy number variations (CNVs) in single or multiple loci. MLPA is a versatile methodology and important tool in cancer research; it provides precise information on increased or decreased copy number at specific loci as opposed to loss of heterozygosity (LOH) studies based upon microsatellite analysis. Pre-designed MLPA kits and software are commercially available to analyze multiple exons, genes, and genomic regions. However, an increasing demand for new gene specific assays makes it necessary to self-design new MLPA probes for which the available software may not be applicable. During evaluation of new self-designed reference probes, we encountered a number of problems, especially when applying the MLPA methodology to tumor samples.
DNA samples from 48 unaffected individuals and 145 breast cancer patients were used to evaluate 11 self-designed MLPA probes and determine the cut-off values for CNV, before applying the MLPA probes to normalize the target probes in a cohort of affected individuals. To test the calculation strategy, three probes were designed to cover regions in Regulator of G-protein Signaling 8 (RGS8), which we previously have identified as being affected by allelic imbalance by LOH analysis across RGS8 in the cohort comprising 145 breast tumors. Agreement between the LOH results and the results obtained by each of the three MLPA probes in RGS8 was found for 64%, 73%, and 91%, of the analyzed samples, respectively.
Here, we present a straightforward method, based upon the normalization pattern in both unaffected and affected individuals, to evaluate self-designed reference probes and to calculate CNV for the MLPA assay with specific focus on the difficulties when analyzing tumor DNA.
PMCID: PMC2913922  PMID: 20579386
12.  Integrative Analysis of Cyclin Protein Levels Identifies Cyclin B1 as a Classifier and Predictor of Outcomes in Breast Cancer 
We studied the expression levels of cyclins B1, D1, and E1 and the implications of cyclin overexpression for patient outcomes in distinct breast cancer subtypes defined by clinical variables and transcriptional profiling.
Experimental Design
The expression levels of cyclins B1, D1, and E1 were quantified in 779 breast tumors and 53 cell lines using reverse phase protein arrays and/or transcriptional profiling.
Whereas cyclin E1 overexpression was a specific marker of triple-negative and basal-like tumors, cyclin B1 overexpression occurred in poor prognosis hormone receptor–positive, luminal B and basal-like breast cancers. Cyclin D1 overexpression occurred in luminal and normal-like cancers. Breast cancer subgroups defined by integrated expression of cyclins B1, D1, and E1 correlated significantly (P < 0.000001) with tumor subtypes defined by transcriptional profiling and clinical criteria. Across three hormone receptor–positive data sets, cyclin B1 was the dominant cyclin associated with poor prognosis in univariate and multivariate analyses. Although CCNE1 was present in significantly higher copy numbers in basal-like versus other subtypes (ANOVA P < 0.001), CCNB1 gene copy number did not show gain in breast cancer. Instead, cyclin B1 expression was increased in tumors with co-occurrence of TP53 mutations and MYC amplification, a combination that seems to characterize basal-like and luminal B tumors. CCNB1 gene expression was significantly correlated with PLK, CENPE, and AURKB gene expression.
Cyclins B1, D1, and E1 have distinct expressions in different breast cancer subtypes. Novel PLK, CENPE, and AURKB inhibitors should be assessed for therapeutic utility in poor prognosis cyclin B1–overexpressing breast cancers.
PMCID: PMC2887710  PMID: 19470724
13.  Germline Mutation in RNASEL Predicts Increased Risk of Head and Neck, Uterine Cervix and Breast Cancer 
PLoS ONE  2008;3(6):e2492.
The Background
Ribonuclease L (RNASEL), encoding the 2′-5′-oligoadenylate (2-5A)-dependent RNase L, is a key enzyme in the interferon induced antiviral and anti-proliferate pathway. Mutations in RNASEL segregate with the disease in prostate cancer families and specific genotypes are associated with an increased risk of prostate cancer.
Infection by human papillomavirus (HPV) is the major risk factor for uterine cervix cancer and for a subset of head and neck squamous cell carcinomas (HNSCC). HPV, Epstein Barr virus (EBV) and sequences from mouse mammary tumor virus (MMTV) have been detected in breast tumors, and the presence of integrated SV40 T/t antigen in breast carcinomas correlates with an aggressive phenotype and poor prognosis.
A genetic predisposition could explain why some viral infections persist and induce cancer, while others disappear spontaneously. This points at RNASEL as a strong susceptibility gene.
Methodology/Principal Findings
To evaluate the implication of an abnormal activity of RNase L in the onset and development of viral induced cancers, the study was initiated by searching for germline mutations in patients diagnosed with uterine cervix cancer. The rationale behind is that close to 100% of the cervix cancer patients have a persistent HPV infection, and if a defective RNase L were responsible for the lack of ability to clear the HPV infection, we would expect to find a wide spectrum of mutations in these patients, leading to a decreased RNase L activity. The HPV genotype was established in tumor DNA from 42 patients diagnosed with carcinoma of the uterine cervix and somatic tissue from these patients was analyzed for mutations by direct sequencing of all coding and regulatory regions of RNASEL. Fifteen mutations, including still uncharacterized, were identified. The genotype frequencies of selected single nucleotide polymorphisms (SNPs) established in the cervix cancer patients were compared between 382 patients with head and neck squamous cell carcinomas (HNSCC), 199 patients with primary unilateral breast cancer and 502 healthy Danish control individuals. We found that the genotype frequencies of only one of the 15 mutations, the yet uncharacterized 5′UTR mutation rs3738579 differed significantly between cancer patients and control individuals (P-value: 4.43×10−5).
In conclusion, we have discovered an increased risk, a heterozygous advantage and thereby a protective effect linked to the RNASEL SNP rs3738579. This effect is found for patients diagnosed with carcinoma of the uterine cervix, HNSCC, and breast cancer thus pointing at RNASEL as a general marker for cancer risk and not restricted to familial prostate cancer.
PMCID: PMC2424240  PMID: 18575592
14.  Carbonic anhydrase IX and response to postmastectomy radiotherapy in high-risk breast cancer: a subgroup analysis of the DBCG82 b and c trials 
A significant survival improvement after postmastectomy radiotherapy was identified in the Danish Breast Cancer Cooperative Group (DBCG82) b and c studies and in the British Columbia Randomized Radiation Trial. Recently, potential predictive value regarding response to postmastectomy radiotherapy was reported for carbonic anhydrase (CA) IX in a study (reported in abstract form) that included 160 patients. The purpose of the present study was to examine the importance of CA IX to response to postmastectomy radiotherapy in the larger scaled DBCG82 b and c studies.
The DBCG82 b and c studies included 3,083 high-risk Danish breast cancer patients. The women were randomly assigned to postmastectomy radiotherapy plus systemic therapy (cyclophosfamide, methotrexate and fluorouracil in premenopausal women; and tamoxifen in postmenopausal women) or to systemic therapy alone. Cores from invasive tumour-containing paraffin blocks from 1,000 patients (more than seven nodes surgically removed) were transferred to tissue microarrays. Tissue microarray sections were stained immunohistochemically for CA IX (M75). The median follow up for patients remaining alive was 17 years. Clinical end-points were loco-regional recurrence, distant metastases, disease-specific survival and overall survival. Statistical analyses included κ statistics, χ2 or exact tests, Kaplan-Meier probability plots, Log-rank test and Cox regression analyses.
CA IX was assessable in 945 cores. The percentage of tumours positive for CA IX was 16% (≥ 10% invasive tumour staining). CA IX was not an independent prognostic marker for survival, distant metastases, or locoregional recurrence in the subgroup of 945 patients or within either of the two randomization arms. In subgroup analyses, however, CA IX was an independent prognostic marker for overall survival among postmenopausal women (P = 0.001), women with one to three positive nodes (P = 0.02) and hormone receptor positive women (P = 0.001). Fifteen-year probabilities of overall survival were improved by 9% and 7% after postmastectomy radiotherapy for the subgroups of CA IX negative and CA IX positive patients, respectively.
Within this series of 945 high-risk premenopausal and postmenopausal women, positivity for CA IX was not overall an independent prognostic marker for survival; only in subgroup analyses was it found to have prognostic value. The improvement in 15-year survival after postmastectomy radiotherapy was of similar magnitude in the two subgroups of CA IX positive and CA IX negative patients.
PMCID: PMC2397523  PMID: 18355402

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