Intratumoral Heterogeneity of MicroRNA Expression in Rectal Cancer.
BACKGROUND
An increasing number of studies have investigated microRNAs (miRNAs) as potential markers of diagnosis, treatment and prognosis. So far, agreement between studies has been minimal, which may in part be explained by intratumoral heterogeneity of miRNA expression. The aim of the present study was to assess the heterogeneity of a panel of selected miRNAs in rectal cancer, using two different technical approaches.


METHODS
The expression of the investigated miRNAs was analysed by real-time quantitative polymerase chain reaction (RT-qPCR) and in situ hybridization (ISH) in tumour specimens from 27 patients with T3-4 rectal cancer. From each tumour, tissue from three different luminal localisations was examined. Inter- and intra-patient variability was assessed by calculating intraclass correlation coefficients (ICCs). Correlations between RT-qPCR and ISH were evaluated using Spearman's correlation.


RESULTS
ICCsingle (one sample from each patient) was higher than 50% for miRNA-21 and miRNA-31. For miRNA-125b, miRNA-145, and miRNA-630, ICCsingle was lower than 50%. The ICCmean (mean of three samples from each patient) was higher than 50% for miRNA-21(RT-qPCR and ISH), miRNA-125b (RT-qPCR and ISH), miRNA-145 (ISH), miRNA-630 (RT-qPCR), and miRNA-31 (RT-qPCR). For miRNA-145 (RT-qPCR) and miRNA-630 (ISH), ICCmean was lower than 50%. Spearman correlation coefficients, comparing results obtained by RT-qPCR and ISH, respectively, ranged from 0.084 to 0.325 for the mean value from each patient, and from -0.085 to 0.515 in the section including the deepest part of the tumour.


CONCLUSIONS
Intratumoral heterogeneity may influence the measurement of miRNA expression and consequently the number of samples needed for representative estimates. Our findings with two different methods suggest that one sample is sufficient for adequate assessment of miRNA-21 and miRNA-31, whereas more samples would improve the assessment of miRNA-125b, miRNA-145, and miRNA-630. Interestingly, we found a poor correlation between the expression estimates obtained by RT-qPCR and ISH, respectively.
Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark.|Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
Anne Haahr Mellergaard
http://orcid.org/0000-0003-0134-3374
AH
Eriksen
Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark.|Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
Rikke Fredslund
RF
Andersen
Bioneer A/S, Hørsholm, Denmark.
Boye Schnack
BS
Nielsen
Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark.|Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
Flemming Brandt
FB
Sørensen
Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark.|Department of Oncology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.
Ane Lindegaard
AL
Appelt
Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark.|Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
Anders
A
Jakobsen
Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark.|Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
Torben Frøstrup
TF
Hansen
D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D035683:MicroRNAs / Q000235:genetics*; D012004:Rectal Neoplasms / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
C541010:MIRN145 microRNA, human; C524940:MIRN21 microRNA, human; C548399:MIRN630 microRNA, human; D035683:MicroRNAs