title: Investigating intra-tumor heterogeneity and expression gradients of miR-21, miR-92a and miR-200c and their potential of predicting lymph node metastases in early colorectal cancer.
abstract: BACKGROUND
  miR-21, miR-92a and miR-200c are regulators of pathways involved in migration, intravasation and metastasis, and their tumor expression levels have been proposed as potential prognostic markers in colorectal cancer (CRC). In two parallel cohorts we examine intra-tumor expression levels in early stage CRC tissue in order to determine intra-tumor heterogeneity, potential systematic intra-tumor expression gradients of the miRNAs and to investigate the association to metastatic disease in early stage CRC.
  
  
  METHODS
  Two parallel studies on archived formalin-fixed paraffin-embedded (FFPE) CRC tissue. Intra-tumor and inter-patient variances were analyzed in 9 early metastatic CRCs by measuring expression levels by qRT-PCR on isolated tissue samples from luminal, central and invasive border zones. Associations between miRNA expression levels and early metastasizing tumors was investigated in FFPE tissue from invasive border and central tumor zones from 47 early metastatic CRCs matched with 47 non-metastatic CRCs. Intra-tumor expression gradients were analyzed on both cohorts.
  
  
  RESULTS
  Mean intra-tumor coefficient of variation in the heterogeneity cohort was 38.5% (range: 33.1-49.0%) only slightly less than variation between patients (45.1%, range 37.0-49.5%). We demonstrated systematic expression gradients between tumor zones equal to a 3.23 (p=0.003) and 1.36 (p=0.014) fold lower expression in invasive areas for miR-200c, 1.52 (p<0.001) and 1.27 (p=0.021) fold lower expression in invasive areas for miR-92a. For miR-21 we found a 1.75 (p<0.001) and 1.21 (p=0.064) fold higher expression in invasive areas compared to luminal and central zones, respectively. No significant difference in expression levels between metastatic and non-metastatic tumors was demonstrated, nor a difference in intra-tumor gradients between metastatic and non-metastatic tumors.
  
  
  CONCLUSIONS
  This study provides evidence for moderate intra-tumor and inter-patient heterogeneities of three well-described potential prognostic markers in CRC. We demonstrate intra-tumor expression gradients indicating a differentiated expression of the target miRNAs between functional tumor zones, but the potential role as markers of early metastatic disease is still not fully clarified.
authors: Department of Pathology and Molecular Unit, University of Copenhagen, Herlev and Gentofte Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark. Electronic address: rikke.karlin.jepsen.01@regionh.dk.
authors: Rikke Karlin
authors: RK
authors: Jepsen
authors: Department of Pathology and Molecular Unit, University of Copenhagen, Herlev and Gentofte Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark.
authors: Guy Wayne
authors: GW
authors: Novotny
authors: Department of Pathology and Molecular Unit, University of Copenhagen, Herlev and Gentofte Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark.
authors: Louise Laurberg
authors: LL
authors: Klarskov
authors: Department of Pathology and Molecular Unit, University of Copenhagen, Herlev and Gentofte Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark.
authors: Ib Jarle
authors: IJ
authors: Christensen
authors: Department of Pathology and Molecular Unit, University of Copenhagen, Herlev and Gentofte Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark.
authors: Estrid
authors: E
authors: Høgdall
authors: Department of Pathology and Molecular Unit, University of Copenhagen, Herlev and Gentofte Hospital, Herlev Ringvej 75, DK-2730 Herlev, Denmark.
authors: Lene Buhl
authors: LB
authors: Riis
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D015331:Cohort Studies; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology*; D018450:Disease Progression; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D018740:Genetic Heterogeneity; D006801:Humans; D008207:Lymphatic Metastasis / Q000235:genetics*; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D011379:Prognosis; D012015:Reference Standards; D012307:Risk Factors; D016019:Survival Analysis
chemical_list: C546783:MIRN192 microRNA, human; C533896:MIRN200 microRNA, human; C524940:MIRN21 microRNA, human; D035683:MicroRNAs
keywords: Early colorectal cancer; Expression gradients; Metastases; microRNA-200c; microRNA-21; microRNA-92a