Intratumoral Heterogeneity of MicroRNA Expression in Rectal Cancer

Anne Haahr Mellergaard Eriksen; Rikke Fredslund Andersen; Boye Schnack Nielsen; Flemming Brandt Sørensen; Ane Lindegaard Appelt; Anders Jakobsen; Torben Frøstrup Hansen

doi:10.1371/journal.pone.0156919

Intratumoral Heterogeneity of MicroRNA Expression in Rectal Cancer

PLoS One. 2016 Jun 3;11(6):e0156919. doi: 10.1371/journal.pone.0156919. eCollection 2016.

Authors

Anne Haahr Mellergaard Eriksen^{1

2}, Rikke Fredslund Andersen^{1

2}, Boye Schnack Nielsen³, Flemming Brandt Sørensen^{1

2}, Ane Lindegaard Appelt^{1

4}, Anders Jakobsen^{1

2}, Torben Frøstrup Hansen^{1

2}

Affiliations

¹ Danish Colorectal Cancer Center South, Vejle Hospital, Vejle, Denmark.
² Institute of Regional Health Research, University of Southern Denmark, Odense, Denmark.
³ Bioneer A/S, Hørsholm, Denmark.
⁴ Department of Oncology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.

Abstract

Introduction: 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.

Materials and 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.

Conclusion: 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.

MeSH terms

Gene Expression Profiling
Gene Expression Regulation, Neoplastic*
Humans
In Situ Hybridization
In Vitro Techniques
MicroRNAs / genetics*
Rectal Neoplasms / genetics*
Reverse Transcriptase Polymerase Chain Reaction

Substances

MIRN145 microRNA, human
MIRN21 microRNA, human
MIRN630 microRNA, human
MicroRNAs

Grants and funding

The project expenses regarding research materials were covered by Vejle Hospital Research Council. The funder did not provide support in the form of salaries for authors. The Agency of Research and Innovation provided funding for BSN (Bioneer A/S). The funding organizations did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.