GSTP1 methylation detection can serve an important role in prostate cancer managment. The meta-analysis clearly confirmed a link between tissue DNA hypermethylation of this and other genes and prostate cancer. Detection of DNA methylation in genes, including GSTP1, could serve an important role in clinical practice.
Background PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1 , APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence. Results An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1 , APC and RASSF1 genes, using methylation specific PCR (MSP). The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE) samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity. Conclusions The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.
Rapid and recurrent breakthroughs of new SARS-CoV-2 strains (variants) have prompted public health authorities worldwide to set up surveillance networks to monitor the circulation of variants of concern. The use of next-generation sequencing technologies has raised the need for quality control assessment as required in clinical laboratories. The present study is the first to propose a validation guide for SARS-CoV-2 typing using three different NGS methods fulfilling ISO15189 standards. These include the assessment of the risk, specificity, accuracy, reproducibility, and repeatability of the methods. Among the three methods used, two are amplicon-based involving reverse transcription polymerase chain reaction (Artic v3 and Midnight v1) on Oxford Nanopore Technologies while the third one is amplicon-based using reverse complement polymerase chain reaction (Nimagen) on Illumina technology. We found that all methods met the quality requirement (e.g., 100% concordant typing results for accuracy, reproducibility, and repeatability) for SARS-CoV-2 typing in clinical setting. Additionally, the typing results emerging from each of the three sequencing methods were compared using three widely known nomenclatures (WHO, Pangolineage, and Nextclade). They were also compared regarding single nucleotide variations. The outcomes showed that Artic v3 and Nimagen should be privileged for outbreak investigation as they provide higher quality results for samples that do not meet inclusion criteria for analysis in a clinical setting. This study is a first step towards validation of laboratory developed NGS tests in the context of the new European regulation for medical devices and in vitro diagnostics.
116 Background: Epigenetic changes through DNA methylation can serve as biomarkers for the presence of prostate cancer (PCa). Gene methylation in histologically benign tissue adjacent to PCa can provide “field effect" biomarkers to detect cancer missed due to sampling errors. Such errors may also result in underestimates of PCa stage or grade, complicating decisions about Active Surveillance. We previously showed increased field effect methylation in GS 7 over GS 6 subjects in a small patient cohort. Here, established methylation markers of PCa (GSTP1, APC and RASSF1) were tested in a larger cohort to compare histologically benign biopsy cores from patients diagnosed with no cancer, low volume GS 6 cancer and GS 7 cancer. The objective was to confirm that epigenetic field effects may be useful for detecting occult high grade malignancies in diagnostic and prognostic settings. Methods: DNA was extracted from prostate biopsy tissue prints and used for multiplex methylation-specific PCR (MSP) assays of the 3 genes. All testing was done blinded. Each of 12 tissue cores was tested from a series of 76 biopsy patients: 34 diagnosed with GS 6 PCa, 22 with GS 7 PCa, and 20 no-cancer controls. Results: In 19 of the 22 GS 7 cases (86%), epigenetic markers were positive for field effects in one or more adjacent benign cores; 12 (54%) were positive for all 3 epigenetic markers. In contrast, only 9 of 34 cases (26%) GS 6 cases were positive for all 3 markers. We again observed that robust marker expression in histologically benign cores from cases diagnosed with low volume GS 6 PCa was associated with higher grade cancer at radical prostatectomy (upgrading). APC showed the best performance as a single marker “field effect” test for adjacent high grade PCa. Conclusions: GSTP1, APC and/or RASSF1 gene promoter methylation was observed to be more prevalent in histologically benign cores from biopsy patients diagnosed with GS 7 PCa, as compared with low volume GS 6. This study confirms previous findings in a larger cohort of subjects that these “field effect” biomarkers can be useful for detecting cancer adjacent to histologically negative biopsies and may be indicative of occult aggressive PCa.
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