Small RNAs in the peripheral blood discriminate metastasized from non-metastasized seminoma

The average age at diagnosis was 39.1 (+/- 7.2) years for non-metastasized, higher (45.2 years, +/- 10.8) for lymphogen metastasized and lower (32.1 years, +/- 5.4) for occult metastasized seminoma. Primary tumor size was comparable between lymphogen and occult metastasized seminoma (37.8 mm and 38.6 mm, respectively), but smaller (23.8 mm) in non-metastasized seminoma (Table 1).

Per 2.5 ml whole blood we isolated about the same amount (mean) of total RNA from seminoma patients without metastasis (7.7 μg, +/-1.9), lymphogen metastasis (4.9 μg, +/-2.0 μg) and occult metastasis (6.8 μg, +/-2.4, Table 1). The RNA integrity number (RIN) ranged between 7.0-8.6 per group. No DNA contamination could be detected in our samples.

The average total number of reads for lymphogen/occult metastasized and non-metastasized seminoma was 1.3x10⁷, 1.2x10⁷ and 1.2x10⁷ and on average 73-76% remained for further analysis after trimming of the reads (Table 2). From these reads 80-84% appeared annotated reads with 7.2-7.8% (1.6-1.7x10⁴) representing annotated small RNAs. Of them 137 small RNAs showed > 50 reads and a two-fold difference to the reference. In univariate analysis we identified 33/34 and 35 small RNA species which significantly discriminated lymphogen/occult metastasized and both metastasis subtypes combined from non-metastasized seminoma, respectively (Table 3). The overlap of small RNAs separating either lymphogen or occult metastasis from non-metastasized seminoma was 47-48% and increased up to 79% when comparing lymphogen and occult small RNAs candidates with the small RNAs based on the combined metastasis subtypes (Figure 1). We finally employed support vector machines which completely separated lymphogen, occult metastasis and the combined metastasis subtypes from non-metastasized seminoma using a combination of two small RNA species only (Table 4). Altogether, 891, 668 and 87 combinations allowed a complete separation of lymphogen (n = 5), occult metastasis (n = 5) and the combined metastasis subtypes (n = 10) from non-metastasized seminoma (n = 5), respectively.

Non-metastasized seminoma (n = 5) received no adjuvant treatment and were free of relapse/progress for at least two years of follow up. Occult metastasized patients (n = 5) presented without visible metastasis at primary staging, received no adjuvant treatment, and developed retroperitoneal tumour progress during the follow up of 12 month. For patients with detectable metastasis at primary staging (n = 5) we focused on clinical stage IIb and IIc, to include lymphogenic metastatic spread only and to provide a high level of diagnostic accuracy (avoiding doubtful lymph nodes). Lymphogen metastasis and non-metastasized patients were matched with occult metastasized patients considering demographic and histological parameters where applicable as well as quality criteria for isolated RNA (Table 1). All patients included in this study were treated between 2008 and 2010 in one testis cancer centre. These 15 patients were selected out of about 300 seminoma patients, according to the criteria mentioned above.

All testicular tumours (n = 15) were examined by an experienced pathologist for histological and TNM classification (Table 1) and pure seminoma was diagnosed in all cases.

Whole blood samples (2.5 ml) were taken intraoperative from cubital vein directly into the PAXgene Blood RNA system (BD Diagnostics, PreAnalytiX GmbH, Hombrechtikon, Switzerland). The tube was gently inverted (10 times), settled at room temperature overnight and stored at -20° until use. The Ethical Review Committee of the Medical Association Hamburg approved the study and all human samples were obtained with written informed consent.

After thawing the PAXGene tubes, and washing and centrifugation of the samples the cells became lysed (Proteinase K) followed by adding Lysis/Binding Solution taken from the mirVana Kit (Life Technologies, Darmstadt, Germany). With the mirVana kit total RNA including small RNA species was isolated by combining a Phenol-Chloroform RNA precipitation with further processing using a silica membrane. After several washing procedures DNA residuals became digested on the membrane (RNAse free DNAse Set, Qiagen, Hilden, Germany), washed, RNA was eluted in a collection tube and frozen at -80°C. Quality and quantity of isolated total RNA were measured spectrophotometrically (NanoDrop, PeqLab Biotechnology, Erlangen, Germany). RNA integrity was assessed by the 2100 Agilent Bioanalyser (Life Science Group, Penzberg, Germany) and DNA contamination was controlled by conventional PCR using actin primer.

For analysis, we used only RNA specimens with a ratio of A260/A280 ≥ 2.0 (Nanodrop) and RNA integrity number (RIN) between 6.0-9.3 for small RNA Next Generation Sequencing (IMGM Laboratories, Martinsried, Germany/CeGat, Tübingen, Germany). For RNA samples with RIN below 7.0 or questionable 28/18S bands (indications of RNA degradation) we performed additional checks to exclude the presence of RNAses and true RNA-degradation.

We performed a genome wide small RNA sequencing using the SOLiD5500xl Next Generation Sequencing Technology (Life Technologies, Penzberg, Germany). In brief, total RNA was purified (PureLink microRNA isolation Kit), enriched small RNAs were ligated to SOLiD adaptors, reverse transcribed (SOLiD RT primer and ArrayScript RT), cDNA was purified (MinElute PCR purification Kit, Qiagen), a cut-off size of 60-80 nt was selected (Novex pre-cast gel products, invitrogen), cDNA was in-gel amplified and samples were barcoded using SOLiD 3′Barcode primer at the same time. Amplified cDNA was purified (PureLink PCR Micro Kit, Life Technologies) and used in emulsion PCR (SOLiD EZ Bead). Thereafter, the emulsion was broken to recover enriched beads and the so-called di-base probes were used by the SOLiD system in the sequencing-by-ligation procedure. Beside the SOLiD5500xl inherent software (LifeScope) used for image and signal processing, software CLC Genomics Workbench 5.1 (CLC bio) was used for clustering, counting, and annotation of all generated 75 bp reads. After discarding reads without adaptor sequence and being shorter than 15 bp (trimming) reads were assigned to known microRNAs (Sanger miRBase release 18, http://​www.​mirbase.​org/​) and known non-coding RNAs (ensembl database Homo_sapiens.GRCh37.67.ncrna.fa, http://​www.​ensembl.​org). Small RNAs showing a significant and ≥ 2-fold differences in gene expression among groups and at least 50 reads were further analysed on their ability to separate both groups using univariate logistic regression analysis [21] and combinations of two small RNAs were examined employing support vector machines (linear Kermel).