Abstract
BackgroundWe aimed to better discriminate metastasized (lymphogen/occult/both combined) from non-metastasized seminoma based on post-transcriptional changes examined in the peripheral blood.MethodsTotal RNAs including small RNAs were isolated from the peripheral blood of patients suffering from metastasized testicular tumours (lymphogen, n = 5, clinical stage IIb/c; occult, n = 5, clinical stage I) and non-metastasized patients (n = 5, clinical stage I). Small RNA next generation sequencing (SOLID, Life Technologies) was employed to examine post-transcriptional changes. We searched for small RNAs showing at least 50 reads and a significant ≥ 2-fold difference using peripheral blood small RNAs of non-metastasized tumours as the reference group. Candidate small RNAs were examined in univariate logistic regression analysis and combinations of two small RNAs were further examined using support vector machines.ResultsOn average 1.3x107, 1.2x107 and 1.2x107 small RNA reads were detectable in non-metastasized, lymphogen and occult metastasized seminoma, respectively of which 73-76% remained after trimming. From these between 80-82% represented annotated reads and 7.2-7.8% (1.6-1.7x104) were annotated small RNA tags. Of them 137 small RNAs showed > 50 reads and a ≥ two-fold difference to the reference. In univariate analysis we detected 33-35 different small RNAs which significantly discriminated lymphogen/occult/combined metastasized from non-metastasized seminoma and among these different comparisons it were the same small RNAs in 44-79%. Many combinations of two of these small RNAs completely discriminated metastasized from non-metastasized seminoma irrespective of the metastasis subtype.ConclusionsMetastasized (either lymphogen or occult) seminoma can be completely discriminated from non-metastasized seminoma with a combination of two small RNAs measured in the peripheral blood.
Highlights
We aimed to better discriminate metastasized from non-metastasized seminoma based on post-transcriptional changes examined in the peripheral blood
Analysis of small RNA generation sequencing results 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)
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)
Summary
We aimed to better discriminate metastasized (lymphogen/occult/both combined) from non-metastasized seminoma based on post-transcriptional changes examined in the peripheral blood. Expression of miRNAs in testicular germ cell cancer is known to be associated with the histologic subtype [7] as well as cisplatin resistance [9,10]. Among seminoma bearing patients circulating tumour cells are already detected in the peripheral blood [12]. We wondered whether changes in microRNA expression in the peripheral blood might be able to discriminate metastasized (either lymphogen, occult or a combination of both subtypes) from non-metastasized seminoma. We utilized an agnostic approach investigating the whole genome for any kind of small RNA species suitable to discriminate metastatic stage in seminoma employing generation sequencing (NGS) on peripheral blood samples drawn at the time of the primary tumour’s diagnosis
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