miRNA profiling of circulating EpCAM(+) extracellular vesicles:promising biomarkers of colorectal cancer

Cancer cells secrete small membranous extracellular vesicles (EVs) into their microenvironment and circulation. These contain biomolecules, including proteins and microRNAs (miRNAs). Both circulating EVs and miRNAs have received much attention as biomarker candidates for non-invasive diagnostics. Here we describe a sensitive analytical method for isolation and subsequent miRNA profiling of epithelial-derived EVs from blood samples of patients with colorectal cancer (CRC). The epithelial-derived EVs were isolated by immunoaffinity-capture using the epithelial cell adhesion molecule (EpCAM) as marker. This approach mitigates some of the specificity issues observed in earlier studies of circulating miRNAs, in particular the negative influence of miRNAs released by erythrocytes, platelets and non-epithelial cells. By applying this method to 2 small-scale patient cohorts, we showed that blood plasma isolated from CRC patients prior to surgery contained elevated levels of 13 EpCAM+-EV miRNAs compared with healthy individuals. Upon surgical tumour removal, the plasma levels of 8 of these were reduced (miR-16-5p, miR-23a-3p, miR-23b-3p, miR-27a-3p, miR-27b-3p, miR-30b-5p, miR-30c-5p and miR-222-3p). These findings indicate that the miRNAs are of tumour origin and may have potential as non-invasive biomarkers for detection of CRC. This work describes a non-invasive blood-based method for sensitive detection of cancer with potential for clinical use in relation to diagnosis and screening. We used the method to study CRC; however, it is not restricted to this disease. It may in principle be used to study any cancer that release epithelial-derived EVs into circulation

Evidence of No Association Between Human Papillomavirus and Breast Cancer

BackgroundGlobally, breast cancer is the most frequent cancer among women. Studies reported an increased risk of breast cancer among women with prior cervical dysplasia. This study aimed to describe the prevalence of human papillomavirus (HPV) in breast cancer and explore if women with prior cervical neoplasia carry an increased risk of HPV-positive breast cancer compared to women without.MethodsThis case–control study identified 193 Danish women diagnosed with breast cancer (1998–2012) at Aarhus University Hospital or Copenhagen University Hospital Herlev. Cases were 93 women with cervical intraepithelial neoplasia grade 3 or worse (CIN3+) prior to breast cancer. Controls were 100 women without prior cervical dysplasia. HPV testing and genotyping were done using SPF10 PCR-DEIA-LiPA25 and an in-house semi-Q-PCR assay.ResultsOverall HPV prevalence in breast cancer for the assays was 1.55% (95% CI 0.32–4.48) and 0.52% (95% CI 0.01–2.85). There was no difference in HPV prevalence between cases and controls (2.15 vs. 1.00%, p = 0.61 and 1.08 vs. 0.00%, p = 0.48). HPV prevalence in CIN3+ was 94.62% (95% CI 0.88–0.98). Concordance between the assays was 98.60%.ConclusionHPV prevalence in breast cancer is very low suggesting no etiological correlation between HPV and breast cancer

data_sheet_1_Evidence of No Association Between Human Papillomavirus and Breast Cancer.PDF

Background<p>Globally, breast cancer is the most frequent cancer among women. Studies reported an increased risk of breast cancer among women with prior cervical dysplasia. This study aimed to describe the prevalence of human papillomavirus (HPV) in breast cancer and explore if women with prior cervical neoplasia carry an increased risk of HPV-positive breast cancer compared to women without.</p>Methods<p>This case–control study identified 193 Danish women diagnosed with breast cancer (1998–2012) at Aarhus University Hospital or Copenhagen University Hospital Herlev. Cases were 93 women with cervical intraepithelial neoplasia grade 3 or worse (CIN3+) prior to breast cancer. Controls were 100 women without prior cervical dysplasia. HPV testing and genotyping were done using SPF₁₀ PCR-DEIA-LiPA₂₅ and an in-house semi-Q-PCR assay.</p>Results<p>Overall HPV prevalence in breast cancer for the assays was 1.55% (95% CI 0.32–4.48) and 0.52% (95% CI 0.01–2.85). There was no difference in HPV prevalence between cases and controls (2.15 vs. 1.00%, p = 0.61 and 1.08 vs. 0.00%, p = 0.48). HPV prevalence in CIN3+ was 94.62% (95% CI 0.88–0.98). Concordance between the assays was 98.60%.</p>Conclusion<p>HPV prevalence in breast cancer is very low suggesting no etiological correlation between HPV and breast cancer.</p

A new sensitive and fast assay for the detection of EGFR mutations in liquid biopsies

BACKGROUND: A major perspective for the use of circulating tumor DNA (ctDNA) in the clinical setting of non-small cell lung cancer (NSCLC) is expected as predictive factor for resistance and response to EGFR TKI therapy and, especially, as a non-invasive alternative to tissue biopsy. However, ctDNA is both highly fragmented and mostly low concentrated in plasma and serum. On this basis, it is important to use a platform characterized by high sensitivity and linear performance in the low concentration range. This motivated us to evaluate the newly developed and commercially available SensiScreen(®) EGFR Liquid assay platform (PentaBase) with regard to sensitivity, linearity, repeatability and accuracy and finally to compare it to our already implemented methods. The validation was made in three independent European laboratories using two cohorts on a total of 68 unique liquid biopsies. RESULTS: Using artificial samples containing 1600 copies of WT DNA spiked with 50% - 0.1% of mutant copies across a seven—log dilution scale, we assessed the sensitivity, linearity, repeatability and accuracy for the p.T790M, p.L858R and exon 19 deletion assays of the SensiScreen(®) EGFR Liquid assay platform. The lowest value detectable ranged from 0.5% to 0.1% with R(2)≥0,97 indicating good linearity. High PCR efficiency was shown for all three assays. In 102 single PCRs each containing theoretical one copy of the mutant at initiating, assays showed repeatable positivity in 75.5% - 80.4% of reactions. At low ctDNA levels, as in plasma, the SensiScreen(®) EGFR Liquid assay platform showed better sensitivity than the Therascreen(®) EGFR platform (Qiagen) and equal performance to the ctEGFR Mutation Detection Kit (EntroGen) and the IOT(®) Oncomine cell-free nucleic acids assay (Thermo Fisher Scientific) with 100% concordance at the sequence level. CONCLUSION: For profiling clinical plasma samples, characterized by low ctDNA abundance, the SensiScreen(®) EGFR Liquid assay is able to identify down to 1 copy of mutant alleles and with its high sensitivity, linearity and accuracy it may be a competitive platform of choice