Limits...
Comparison of a healthy miRNome with melanoma patient miRNomes: are microRNAs suitable serum biomarkers for cancer?

Margue C, Reinsbach S, Philippidou D, Beaume N, Walters C, Schneider JG, Nashan D, Behrmann I, Kreis S - Oncotarget (2015)

Bottom Line: We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients.Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in serum (miR-3201 and miR-122-5p).Here we have compiled a healthy and widely applicable miRNome from serum samples and we provide strong evidence that levels of cell-free miRNAs only change significantly at later stages of melanoma progression, which has serious implications for miRNA biomarker studies in cancer.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Research Unit, University of Luxembourg, Luxembourg.

ABSTRACT
MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are profiled from tumor tissue (intracellular miRNAs) or serum/plasma samples (extracellular miRNAs). To improve detection of reliable biomarkers from blood samples, we first compiled a healthy reference miRNome and established a well-controlled analysis pipeline allowing for standardized quantification of circulating miRNAs. Using whole miRNome and custom qPCR arrays, miRNA expression profiles were analyzed in 126 serum, whole blood and tissue samples of healthy volunteers and melanoma patients and in primary melanocyte and keratinocyte cell lines. We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients. Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in serum (miR-3201 and miR-122-5p). Here we have compiled a healthy and widely applicable miRNome from serum samples and we provide strong evidence that levels of cell-free miRNAs only change significantly at later stages of melanoma progression, which has serious implications for miRNA biomarker studies in cancer.

No MeSH data available.


Related in: MedlinePlus

Biomarker studiesA) Heatmap and PCA showing the averages of samples belonging to different melanoma stages and healthy serum samples (based on Fig. 3B and 3C). S: stage; hy: healthy. B) ROC curves on pairwise comparisons between indicated sample groups (30 healthy, 4 stage 0, 11 stage I, 17 stage II, 11 stage III, 9 stage IV samples; early: stage 0+I+II; late: stage III+IV). Underlined miRNAs were upregulated while all others were downregulated compared to healthy or early stage samples. MiRNAs in grey shade should not be considered as potential biomarkers because they were highly expressed (Cq < 15) in whole blood samples and might therefore derive from contaminating blood cells (miR-1260a, miR-22-3p, miR-1280, miR-451a and miR-16-5p, also see Supplementary Table S3), or they were part of the unstable miRNAs (miR-432-3p, miR-373-5p; see Fig. 2A). ACC: accuracy value, AUC: area under the curve.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494926&req=5

Figure 4: Biomarker studiesA) Heatmap and PCA showing the averages of samples belonging to different melanoma stages and healthy serum samples (based on Fig. 3B and 3C). S: stage; hy: healthy. B) ROC curves on pairwise comparisons between indicated sample groups (30 healthy, 4 stage 0, 11 stage I, 17 stage II, 11 stage III, 9 stage IV samples; early: stage 0+I+II; late: stage III+IV). Underlined miRNAs were upregulated while all others were downregulated compared to healthy or early stage samples. MiRNAs in grey shade should not be considered as potential biomarkers because they were highly expressed (Cq < 15) in whole blood samples and might therefore derive from contaminating blood cells (miR-1260a, miR-22-3p, miR-1280, miR-451a and miR-16-5p, also see Supplementary Table S3), or they were part of the unstable miRNAs (miR-432-3p, miR-373-5p; see Fig. 2A). ACC: accuracy value, AUC: area under the curve.

Mentions: To nevertheless allow for identification of potential disease stage-specific miRNAs, we combined these samples according to their cancer stage and compared them to the average of the healthy samples (Fig. 4A). The different groups revealed distinct expression patterns, which prompted us to find signature miRNAs, potentially characteristic for the different stages of melanoma. First, accuracy values were computed to identify the optimal number of miRNAs required to make correct stage assignments. Then, AUC values were calculated for the different groups (Fig. 4B). Results from heatmaps and ROC curve analysis correlated well. Fig. 4B shows that upon comparison of healthy individuals with stage III or IV metastatic melanoma samples, biomarkers were found with excellent predictive scores (AUC 0.99/CI 0.96-1.00 for stage III and AUC 0.97/CI 0.89-1.00 for stage IV). Several miRNAs were profoundly down-regulated in late stage melanoma patients (e.g. miR-200c-3p, -204-5p, -182-5p, -301a-3p) while others were up-regulated (e.g. miR-211-5p, -193b-3p, -720, -205-5p). Six of these potential biomarkers were also described to be differentially regulated in secreted melanoma exosomes compared to healthy melanocytes [33]. Those identified miRNAs contain several new candidates as well as some miRNAs that have previously been connected to melanoma. MiR-211-5p is overexpressed in the plasma of late stage melanoma patients [15]. In this context, we have recently shown that miR-211-5p has no evident role in inhibiting invasion of melanoma cells [34] as was claimed before [35-37] and we describe here that indeed miR-211-5p is highly overexpressed in serum of stage IV melanoma patients when compared to healthy controls. In fact, the overexpression levels (68-fold) of this miRNA in serum samples of stage IV melanoma patients were the highest measured throughout the study (data not shown). MiR-211-5p has long been recognised as a marker miRNA for the melanocytic lineage [38, 39] and its high levels in sera of late stage patients might suggest that it is specifically secreted from melanoma cells.


Comparison of a healthy miRNome with melanoma patient miRNomes: are microRNAs suitable serum biomarkers for cancer?

Margue C, Reinsbach S, Philippidou D, Beaume N, Walters C, Schneider JG, Nashan D, Behrmann I, Kreis S - Oncotarget (2015)

Biomarker studiesA) Heatmap and PCA showing the averages of samples belonging to different melanoma stages and healthy serum samples (based on Fig. 3B and 3C). S: stage; hy: healthy. B) ROC curves on pairwise comparisons between indicated sample groups (30 healthy, 4 stage 0, 11 stage I, 17 stage II, 11 stage III, 9 stage IV samples; early: stage 0+I+II; late: stage III+IV). Underlined miRNAs were upregulated while all others were downregulated compared to healthy or early stage samples. MiRNAs in grey shade should not be considered as potential biomarkers because they were highly expressed (Cq < 15) in whole blood samples and might therefore derive from contaminating blood cells (miR-1260a, miR-22-3p, miR-1280, miR-451a and miR-16-5p, also see Supplementary Table S3), or they were part of the unstable miRNAs (miR-432-3p, miR-373-5p; see Fig. 2A). ACC: accuracy value, AUC: area under the curve.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494926&req=5

Figure 4: Biomarker studiesA) Heatmap and PCA showing the averages of samples belonging to different melanoma stages and healthy serum samples (based on Fig. 3B and 3C). S: stage; hy: healthy. B) ROC curves on pairwise comparisons between indicated sample groups (30 healthy, 4 stage 0, 11 stage I, 17 stage II, 11 stage III, 9 stage IV samples; early: stage 0+I+II; late: stage III+IV). Underlined miRNAs were upregulated while all others were downregulated compared to healthy or early stage samples. MiRNAs in grey shade should not be considered as potential biomarkers because they were highly expressed (Cq < 15) in whole blood samples and might therefore derive from contaminating blood cells (miR-1260a, miR-22-3p, miR-1280, miR-451a and miR-16-5p, also see Supplementary Table S3), or they were part of the unstable miRNAs (miR-432-3p, miR-373-5p; see Fig. 2A). ACC: accuracy value, AUC: area under the curve.
Mentions: To nevertheless allow for identification of potential disease stage-specific miRNAs, we combined these samples according to their cancer stage and compared them to the average of the healthy samples (Fig. 4A). The different groups revealed distinct expression patterns, which prompted us to find signature miRNAs, potentially characteristic for the different stages of melanoma. First, accuracy values were computed to identify the optimal number of miRNAs required to make correct stage assignments. Then, AUC values were calculated for the different groups (Fig. 4B). Results from heatmaps and ROC curve analysis correlated well. Fig. 4B shows that upon comparison of healthy individuals with stage III or IV metastatic melanoma samples, biomarkers were found with excellent predictive scores (AUC 0.99/CI 0.96-1.00 for stage III and AUC 0.97/CI 0.89-1.00 for stage IV). Several miRNAs were profoundly down-regulated in late stage melanoma patients (e.g. miR-200c-3p, -204-5p, -182-5p, -301a-3p) while others were up-regulated (e.g. miR-211-5p, -193b-3p, -720, -205-5p). Six of these potential biomarkers were also described to be differentially regulated in secreted melanoma exosomes compared to healthy melanocytes [33]. Those identified miRNAs contain several new candidates as well as some miRNAs that have previously been connected to melanoma. MiR-211-5p is overexpressed in the plasma of late stage melanoma patients [15]. In this context, we have recently shown that miR-211-5p has no evident role in inhibiting invasion of melanoma cells [34] as was claimed before [35-37] and we describe here that indeed miR-211-5p is highly overexpressed in serum of stage IV melanoma patients when compared to healthy controls. In fact, the overexpression levels (68-fold) of this miRNA in serum samples of stage IV melanoma patients were the highest measured throughout the study (data not shown). MiR-211-5p has long been recognised as a marker miRNA for the melanocytic lineage [38, 39] and its high levels in sera of late stage patients might suggest that it is specifically secreted from melanoma cells.

Bottom Line: We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients.Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in serum (miR-3201 and miR-122-5p).Here we have compiled a healthy and widely applicable miRNome from serum samples and we provide strong evidence that levels of cell-free miRNAs only change significantly at later stages of melanoma progression, which has serious implications for miRNA biomarker studies in cancer.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Research Unit, University of Luxembourg, Luxembourg.

ABSTRACT
MiRNAs are increasingly recognized as biomarkers for the diagnosis of cancers where they are profiled from tumor tissue (intracellular miRNAs) or serum/plasma samples (extracellular miRNAs). To improve detection of reliable biomarkers from blood samples, we first compiled a healthy reference miRNome and established a well-controlled analysis pipeline allowing for standardized quantification of circulating miRNAs. Using whole miRNome and custom qPCR arrays, miRNA expression profiles were analyzed in 126 serum, whole blood and tissue samples of healthy volunteers and melanoma patients and in primary melanocyte and keratinocyte cell lines. We found characteristic signatures with excellent prognostic scores only in late stage but not in early stage melanoma patients. Upon comparison of melanoma tissue miRNomes with matching serum samples, several miRNAs were identified to be exclusively tissue-derived (miR-30b-5p, miR-374a-5p and others) while others had higher expression levels in serum (miR-3201 and miR-122-5p). Here we have compiled a healthy and widely applicable miRNome from serum samples and we provide strong evidence that levels of cell-free miRNAs only change significantly at later stages of melanoma progression, which has serious implications for miRNA biomarker studies in cancer.

No MeSH data available.


Related in: MedlinePlus