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Background
Circulating cell-free DNA (ccfDNA), released from normal and cancerous cells, is a promising biomarker for cancer detection as in neoplastic patients it is enriched in tumor-derived DNA (ctDNA). ctDNA contains cancer-specific mutations and epigenetic modifications, which can have diagnostic/prognostic value. However, in primary tumors, and in particular in localized prostate cancer (PCa), the fraction of ctDNA is very low and conventional strategies to study ccfDNA are unsuccessful.
Methods
ccfDNA was isolated from plasma before and after prostate biopsy by using the Maxwell RSC ccfDNA Plasma Kit and quantitatively and qualitatively analyzed by a fluorometer and by the Agilent High Sensitivity D5000 ScreenTape System. Somatic mutations were searched for by targeted RNAseq on prostate biopsies, sequencing libraries were prepared using the TruSight RNA Pan-Cancer Panel from Illumina. Detection of selected somatic mutations in ccfDNA was performed by ultra-deep sequencing of amplicons, using the NEBNext® Ultra™ II DNA Library Prep Kit for library preparation. All libraries were sequenced on the NextSeq550 platform.
Results
Here we demonstrate that prostate biopsy, by causing multiple injuries to the organ, leads to a significant increase in plasma concentration of ccfDNA (P < 0.0024) in primary PCa patients. By calculating the minor allele fraction at patient-specific somatic mutations pre- and post-biopsy, we show that ctDNA is significantly enriched (from 3.9 to 164 fold) after biopsy, representing a transient “molecular window” to access and analyze ctDNA. Moreover, we show that newly released ccfDNA contains a larger fraction of di-, tri- and multi-nucleosome associated DNA fragments. This feature could be exploited to further enrich prostate-derived ccfDNA and to analyze epigenetic markers.
Conclusion
Our data represent a proof-of-concept that liquid tumor profiling from peripheral blood performed just after the biopsy procedure can open a “valuable molecular metastatic window” giving access to the tumor genetic asset, thus providing an opportunity for early cancer detection and individual genomic profiling in the view of PCa precision medicine.
Keywords
circulating tumor DNA, circulating cell-free DNA, primary prostate cancer, biopsy, biomarker
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A new preprint design is coming!
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 19 Jan, 2021
No community comments so far
Metrics
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PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Circulating cell-free DNA (ccfDNA), released from normal and cancerous cells, is a promising biomarker for cancer detection as in neoplastic patients it is enriched in tumor-derived DNA (ctDNA). ctDNA contains cancer-specific mutations and epigenetic modifications, which can have diagnostic/prognostic value. However, in primary tumors, and in particular in localized prostate cancer (PCa), the fraction of ctDNA is very low and conventional strategies to study ccfDNA are unsuccessful.
Methods
ccfDNA was isolated from plasma before and after prostate biopsy by using the Maxwell RSC ccfDNA Plasma Kit and quantitatively and qualitatively analyzed by a fluorometer and by the Agilent High Sensitivity D5000 ScreenTape System. Somatic mutations were searched for by targeted RNAseq on prostate biopsies, sequencing libraries were prepared using the TruSight RNA Pan-Cancer Panel from Illumina. Detection of selected somatic mutations in ccfDNA was performed by ultra-deep sequencing of amplicons, using the NEBNext® Ultra™ II DNA Library Prep Kit for library preparation. All libraries were sequenced on the NextSeq550 platform.
Results
Here we demonstrate that prostate biopsy, by causing multiple injuries to the organ, leads to a significant increase in plasma concentration of ccfDNA (P < 0.0024) in primary PCa patients. By calculating the minor allele fraction at patient-specific somatic mutations pre- and post-biopsy, we show that ctDNA is significantly enriched (from 3.9 to 164 fold) after biopsy, representing a transient “molecular window” to access and analyze ctDNA. Moreover, we show that newly released ccfDNA contains a larger fraction of di-, tri- and multi-nucleosome associated DNA fragments. This feature could be exploited to further enrich prostate-derived ccfDNA and to analyze epigenetic markers.
Conclusion
Our data represent a proof-of-concept that liquid tumor profiling from peripheral blood performed just after the biopsy procedure can open a “valuable molecular metastatic window” giving access to the tumor genetic asset, thus providing an opportunity for early cancer detection and individual genomic profiling in the view of PCa precision medicine.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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