Development and Analytical Validation of a One-Step Five-Plex RT-ddPCR Assay for the Quantification of SARS-CoV-2 Transcripts in Clinical Samples.

Highly sensitive methodologies for SARS-CoV-2 detection are essential for the control of COVID-19 pandemic. We developed and analytically validated a highly sensitive and specific five-plex one-step RT-ddPCR assay for SARS-CoV-2. We first designed in-silico novel primers and probes for the simultaneous absolute quantification of three different regions of the nucleoprotein (N) gene of SARS-CoV-2 (N1, N2, N3), a synthetic RNA as an external control (RNA-EC), and Beta-2-Microglobulin (B2M) as an endogenous RNA internal control (RNA-IC). The developed assay was analytically validated using synthetic DNA and RNA calibrator standards and then was applied to 100 clinical specimens previously analyzed with a commercially available CE-IVD RT-qPCR assay. The analytical validation of the developed assay resulted in very good performance characteristics in terms of analytical sensitivity, linearity, analytical specificity, and reproducibility and recovery rates even at very low viral concentrations. The simultaneous absolute quantification of the RNA-EC and RNA-IC provides the necessary metrics for quality control assessment. Direct comparison of the developed one-step five-plex RT-ddPCR assay with a CE-IVD RT-qPCR kit revealed a very high concordance and a higher sensitivity [concordance: 99/100 (99.0%, Spearman's correlation coefficient: -0.850, p < 0.001)]. The developed assay is highly sensitive, specific, and reproducible and has a broad linear dynamic range, providing absolute quantification of SARS-COV-2 transcripts. The inclusion of two RNA quality controls, an external and an internal, is highly important for standardization of SARS-COV-2 molecular testing in clinical and wastewater samples.

The value proposition of integrative diagnostics for (early) detection of cancer. On behalf of the EFLM interdisciplinary Task and Finish Group "CNAPS/CTC for early detection of cancer".

Disruptive imaging and laboratory technologies can improve clinical decision processes and outcomes in oncology. However, certain obstacles must be overcome before these technologies can be fully implemented as part of the standard for care. An integrative diagnostic approach represents a unique opportunity to unleash the full diagnostic potential and paves the way towards personalized cancer diagnostics. To meet this demand, an interdisciplinary Task Force of the EFLM was initiated as a consequence of an EFLM/ESR during the CELME 2019 meeting in order to evaluate the clinical value of CNAPS/CTC (circulating nucleic acids in plasma and serum/circulating tumor cells) in early detection of cancer. Here, an overview of current disruptive techniques, their clinical implications and potential value of an integrative diagnostic approach is provided. Furthermore, requirements such as the establishment of diagnostic tumor boards, development of adequate software solutions and a change of mindset towards a new generation of diagnosticians providing actionable health information are presented. This development has the potential to elevate the position and clinical recognition of diagnosticians.

SARS-CoV-2 wastewater surveillance data can predict hospitalizations and ICU admissions.

We measured SARS-CoV-2 RNA load in raw wastewater in Attica, Greece, by RT-qPCR for the environmental surveillance of COVID-19 for 6 months. The lag between RNA load and pandemic indicators (COVID-19 hospital and intensive care unit (ICU) admissions) was calculated using a grid search. Our results showed that RNA load in raw wastewater is a leading indicator of positive COVID-19 cases, new hospitalization and admission into ICUs by 5, 8 and 9 days, respectively. Modelling techniques based on distributed/fixed lag modelling, linear regression and artificial neural networks were utilized to build relationships between SARS-CoV-2 RNA load in wastewater and pandemic health indicators. SARS-CoV-2 mutation analysis in wastewater during the third pandemic wave revealed that the alpha-variant was dominant. Our results demonstrate that clinical and environmental surveillance data can be combined to create robust models to study the on-going COVID-19 infection dynamics and provide an early warning for increased hospital admissions.

USP44 Promoter Methylation in Plasma Cell-Free DNA in Prostate Cancer.

Liquid biopsy provides real-time monitoring of tumor evolution and response to therapy through analysis of circulating tumor cells (CTCs) and plasma-circulating tumor DNA (ctDNA). USP44 is a critical gene which plays an important role in cell proliferation; however, its accurate role in other cellular networks is under research. USP44 promoter methylation has been so far reported in colorectal neoplasia and metastatic breast cancer. In this study, we examined for the first time USP44 promoter methylation in plasma cell-free DNA (cfDNA) of patients with prostate cancer (early stage n = 32, metastatic n = 39) and 10 healthy donors (HD). USP44 promoter methylation was detected in plasma cell-free DNA by a newly developed highly specific and sensitive real-time MSP method. Our findings indicate that USP44 promoter is methylated in plasma cell-free DNA of metastatic prostate cancer patients and that detection of USP44 promoter methylation is significantly associated with overall survival (OS) (p = 0.008). We report for the first time that detection of USP44 promoter methylation in plasma cell free DNA provides significant prognostic information in metastatic prostate cancer.

A Comprehensive Molecular Analysis of in Vivo Isolated EpCAM-Positive Circulating Tumor Cells in Breast Cancer.

BACKGROUND: Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa). METHODS: In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining. RESULTS: All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection. CONCLUSIONS: In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs.

RNA-Based CTC Analysis Provides Prognostic Information in Metastatic Breast Cancer.

In metastatic breast cancer (MBC) the molecular characterization of circulating tumor cells (CTCs) provides a unique tool to understand metastasis-biology and therapy-resistance. We evaluated the prognostic significance of gene expression in EpCAM(+) CTCs in 46 MBC patients based on a long follow-up. We selected a panel consisting of stem cell markers (CD24, CD44, ALDH1), the mesenchymal marker TWIST1, receptors (ESR1, PGR, HER2, EGFR) and the epithelial marker CK-19. Singleplex RT-qPCR was used for TWIST1 and CK-19 and multiplex RT-qPCR for stem cell markers and receptors. A group of 19 healthy donors (HD) was used as control. Univariate (p = 0.001) and multivariate analysis (p = 0.002) revealed the prognostic value of combined gene expression of CK-19(+), CD44high/CD24low, ALDH1high/CD24low and HER2 over-expression for overall survival (OS). The Kaplan-Meier estimates of OS were significantly different in patients positive for CK-19 (p = 0.028), CD44high/CD24low (p = 0.002), ALDH1high/CD24low (p = 0.007) and HER2-positive (p = 0.022). Our results indicate that combined gene expression analysis in EpCAM(+) CTCs provides prognostic information in MBC.

Prognostic Significance of SLFN11 Methylation in Plasma Cell-Free DNA in Advanced High-Grade Serous Ovarian Cancer.

BACKGROUND: Epigenetic alterations in ctDNA are highly promising as a source of novel potential liquid biopsy biomarkers and comprise a very promising liquid biopsy approach in ovarian cancer, for early diagnosis, prognosis and response to treatment. METHODS: In the present study, we examined the methylation status of six gene promoters (BRCA1, CST6, MGMT, RASSF10, SLFN11 and USP44) in high-grade serous ovarian cancer (HGSOC). We evaluated the prognostic significance of DNA methylation of these six gene promoters in primary tumors (FFPEs) and plasma cfDNA samples from patients with early, advanced and metastatic HGSOC. RESULTS: We report for the first time that the DNA methylation of SLFN11 in plasma cfDNA was significantly correlated with worse PFS (p = 0.045) in advanced stage HGSOC. CONCLUSIONS: Our results strongly indicate that SLFN11 epigenetic inactivation could be a predictor of resistance to platinum drugs in ovarian cancer. Our results should be further validated in studies based on a larger cohort of patients, in order to further explore whether the DNA methylation of SLFN11 promoter could serve as a potential prognostic DNA methylation biomarker and a predictor of resistance to platinum-based chemotherapy in ovarian cancer.

Analytical methodologies for the detection of SARS-CoV-2 in wastewater: Protocols and future perspectives.

In March 2020 the World Health Organization announced a pandemic outbreak. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen for the coronavirus disease-19 (COVID-19) pandemic. The authorities worldwide use clinical science to identify infected people, but this approach is not able to track all symptomatic and asymptomatic cases due to limited sampling capacity of the testing laboratories. This drawback is eliminated by the Wastewater-Based Epidemiology (WBE) approach. In this review, we summarized the peer-reviewed published literature (available as of September 28, 2020), in the field of WBE. The commonly used steps (sampling, storage, concentration, isolation, detection) of the analytical protocols were identified. The potential limitations of each stage of the protocols and good practices were discussed. Finally, new methods for the efficient detection of SARS-CoV-2 were proposed.

Liquid biopsy in ovarian cancer.

Ovarian cancer has the worst survival rate because it is typically diagnosed at advanced stage. Despite treatment, the disease commonly recurs due to chemo-resistance. Liquid biopsy, based on minimally invasive blood tests, has the advantage of following tumor evolution in real time, offering novel insights on cancer prevention and treatment. Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating cell-free microRNAs (cfmiRNAs) and circulating exosomes represent the major components of liquid biopsy. In this chapter, we provide an overview of recent research on CTCs, ctDNA, cfmiRNAs and exosomes in ovarian cancer. We also focus on the clinical value of liquid biopsy in early diagnosis, prognosis, treatment response, as well as screening in the general population.

Direct comparison study between droplet digital PCR and a combination of allele-specific PCR, asymmetric rapid PCR and melting curve analysis for the detection of BRAF V600E mutation in plasma from melanoma patients.

Background In metastatic melanoma, 40%-50% of patients harbor a BRAF V600E mutation and are thereby eligible to receive a combined BRAF/MEK inhibitor therapy. Compared to standard-of-care tissue-based genetic testing, analysis of circulating tumor DNA (ctDNA) from blood enables a comprehensive assessment of tumor mutational status in real-time and can be used for monitoring response to therapy. The aim of our study was to directly compare the performance of two highly sensitive methodologies, droplet digital PCR (ddPCR) and a combination of ARMS/asymmetric-rapid PCR/melting curve analysis, for the detection of BRAF V600E in plasma from melanoma patients. Methods Cell-free DNA (cfDNA) was isolated from 120 plasma samples of stage I to IV melanoma patients. Identical plasma-cfDNA samples were subjected to BRAF V600E mutational analysis using in parallel, ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis. Results BRAF V600E mutation was detected in 9/117 (7.7%) ctDNA samples by ddPCR and in 22/117 (18.8%) ctDNA samples by the combination of ARMS/asymmetric- rapid PCR/melting curve analysis. The concordance between these two methodologies was 85.5% (100/117). The comparison of plasma-ctDNA analysis using ddPCR and tissue testing revealed an overall agreement of 79.4% (27/34), while the corresponding agreement using the combination of ARMS/asymmetric-rapid PCR/melting curve analysis was 73.5% (25/34). Moreover, comparing the detection of BRAF-mutant ctDNA with the clinics, overall agreement of 87.2% (48/55) for ddPCR and 79.2% (42/53) was demonstrated. Remarkably, the duration of sample storage was negatively correlated with correctness of genotyping results highlighting the importance of pre-analytical factors. Conclusions Our direct comparison study has shown a high level of concordance between ddPCR and the combination of ARMS/asymmetric-rapid PCR/melting curve analysis for the detection of BRAF V600E mutations in plasma.

Prognostic Significance of TWIST1, CD24, CD44, and ALDH1 Transcript Quantification in EpCAM-Positive Circulating Tumor Cells from Early Stage Breast Cancer Patients.

(1) Background: The aim of the study was to evaluate the prognostic significance of EMT-associated (TWIST1) and stem-cell (SC) transcript (CD24, CD44, ALDH1) quantification in EpCAM+ circulating tumor cells (CTCs) of early breast cancer patients. (2) Methods: 100 early stage breast cancer patients and 19 healthy donors were enrolled in the study. CD24, CD44, and ALDH1 transcripts of EpCAM+ cells were quantified using a novel highly sensitive and specific quadraplex RT-qPCR, while TWIST1 transcripts were quantified by single RT-qPCR. All patients were followed up for more than 5 years. (3) Results: A significant positive correlation between overexpression of TWIST1 and CD24-/low/CD44high profile was found. Kaplan-Meier analysis revealed that the ER/PR-negative (HR-) patients and those patients with more than 3 positive lymph nodes that overexpressed TWIST1 in EpCAM+ cells had a significant lower DFI (log rank test; p < 0.001, p < 0.001) and OS (log rank test; p = 0.006, p < 0.001). Univariate and multivariate analysis also revealed the prognostic value of TWIST1 overexpression and CD24-/low/CD44high and CD24-/low/ALDH1high profile for both DFI and OS. (4) Conclusions: Detection of TWIST1 overexpression and stem-cell (CD24, CD44, ALDH1) transcripts in EpCAM+ CTCs provides prognostic information in early stage breast cancer patients.

Development and Validation of Multiplex Liquid Bead Array Assay for the Simultaneous Expression of 14 Genes in Circulating Tumor Cells.

Liquid biopsy, based on the molecular information extracted from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), offers the possibility to characterize the evolution of a solid tumor in real time and is highly important for diagnostic and therapeutic purposes. The aim of the present study was the development and validation of a novel liquid bead array methodology for the molecular characterization of CTCs and its application in breast cancer. In the present study we developed and evaluated a multiplex polymerase chain reaction (PCR)-coupled liquid bead array (MLBA) assay for studying simultaneously the expression of 14 genes in CTCs. The 14-gene MLBA assay is characterized by high analytical specificity, sensitivity, and reproducibility. The analytical performance of the 14-gene MLBA assay was compared with a commercially available test (AdnaTest BreastCancer, Qiagen, Germany) and our previously described multiplex quantitative reverse transcription PCR (RT-qPCR) assays. The developed assay has the potential to be further expanded in order to include up to 100 gene targets. The assay is highly specific for each target gene and is not affected by the numerous primers and probes used for multiplexing; hence, it constitutes a sample-, cost-, and time-saving analysis.

Liquid biopsy in ovarian cancer: the potential of circulating miRNAs and exosomes.

Ovarian cancer still remains the most lethal female cancer, since in most cases it is diagnosed at an advanced stage. Usually after completion of primary treatment chemoresistance occurs, and recurrent disease is finally observed. Liquid biopsy, based on minimally invasive and serial blood tests, has the advantage of following tumor evolution in real time, offering novel insights on precision medicine. Circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating cell-free microRNAs (cfmiRNAs) and circulating exosomes represent the major components of liquid biopsy analysis. Liquid biopsy has been already implemented in ovarian cancer, and most studies so far are mainly focused on CTCs and ctDNA. This review is mainly focused on the clinical potential of circulating miRNAs and exosomes as a source of liquid biopsy biomarkers in ovarian cancer diagnosis, prognosis, and response to treatment.

ESR1 methylation in primary tumors and paired circulating tumor DNA of patients with high-grade serous ovarian cancer.

OBJECTIVE: Estrogen receptor, coded by the ESR1 gene, is highly expressed in epithelial ovarian cancer. ESR1 gene is frequently methylated in many types of gynecological malignancies. However, only a few studies attempted to investigate the role of ESR1 methylation and its clinical significance in ovarian cancer so far. The aim of our study was to examine ESR1 methylation status in primary tumors and corresponding circulating tumor DNA of patients with high-grade serous ovarian cancer (HGSC). METHODS: ESR1 methylation was detected by a highly specific and sensitive real-time methylation-specific PCR assay. Two groups of HGSC samples were analyzed: group A (n = 66 primary tumors) and group B (n = 53 primary tumors and 50 corresponding plasma samples). RESULTS: ESR1 was found methylated in both groups of primary tumors: in 32/66 (48.5%) of group A and in 15/53 (28.3%) of group B. 19/50 (38.0%) corresponding plasma samples of group B were also methylated for ESR1. A significant agreement for ESR1 methylation was observed between primary tumors and paired plasma ctDNA samples (P = 0.004). Interestingly, the presence of ESR1 methylation in primary tumor samples of group B was significantly correlated with a better overall survival (P = 0.027) and progression-free survival (P = 0.041). CONCLUSIONS: We report for the first time the presence of ESR1 methylation in plasma ctDNA of patients with HGSC. The agreement between ESR1 methylation in primary tumors and paired ctDNA is statistically significant. Our results indicate a correlation between the presence of ESR1 methylation and a better clinical outcome in HGSC patients.

Liquid biopsy in ovarian cancer: recent advances on circulating tumor cells and circulating tumor DNA.

Ovarian cancer remains the most lethal disease among gynecological malignancies despite the plethora of research studies during the last decades. The majority of patients are diagnosed in an advanced stage and exhibit resistance to standard chemotherapy. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) represent the main liquid biopsy approaches that offer a minimally invasive sample collection. Both have shown a diagnostic, prognostic and predictive value in many types of solid malignancies and recent studies attempted to shed light on their role in ovarian cancer. This review is mainly focused on the clinical value of both CTCs and ctDNA in ovarian cancer and, more specifically, on their potential as diagnostic, prognostic and predictive tumor biomarkers.

Multiplex Gene Expression Profiling of In Vivo Isolated Circulating Tumor Cells in High-Risk Prostate Cancer Patients.

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays-CellSearch®, PSA-EPISPOT, and immunofluorescence of CellCollector® in vivo-captured CTCs-using the same blood draw. METHODS: EpCAM-positive CTCs were isolated in vivo using the CellCollector from 108 high-risk PCa patients and 36 healthy volunteers. For 27 patients, samples were available before and after treatment. We developed highly sensitive multiplex RT-qPCR assays for 14 genes (KRT19, EpCAM, CDH1, HMBS, PSCA, ALDH1A1, PROM1, HPRT1, TWIST1, VIM, CDH2, B2M, PLS3, and PSA), including epithelial markers, stem cell markers, and epithelial-to-mesenchymal-transition (EMT) markers. RESULTS: We observed high heterogeneity in gene expression in the captured CTCs for each patient. At least 1 marker was detected in 74 of 105 patients (70.5%), 2 markers in 45 of 105 (40.9%), and 3 markers in 16 of 105 (15.2%). Epithelial markers were detected in 31 of 105 (29.5%) patients, EMT markers in 46 of 105 (43.8%), and stem cell markers in 15 of 105 (14.3%) patients. EMT-marker positivity was very low before therapy (2 of 27, 7.4%), but it increased after therapy (17 of 27, 63.0%), whereas epithelial markers tended to decrease after therapy (2 of 27, 7.4%) compared with before therapy (13 of 27, 48.1%). At least 2 markers were expressed in 40.9% of patients, whereas the positivity was 19.6% for CellSearch, 38.1% for EPISPOT, and 43.8% for CellCollector-based IF-staining. CONCLUSIONS: The combination of in vivo CTC isolation with downstream RNA analysis is highly promising as a high-throughput, specific, and ultrasensitive approach for multiplex liquid biopsy-based molecular diagnostics.

Exosomes: A Cancer Theranostics Road Map.

Interindividual variability is yet to be fully characterized, and for this, optimum patient stratification and companion diagnostics are still lacking. Especially when complex disease phenotypes and/or polygenic diseases are considered, patient monitoring and disease management become rather challenging, while acquired resistance to therapy and/or toxicity events are among the unmet needs in the clinic. No doubt, biomarkers are of great importance to disease management and tailor-made theranostics. Microfluidics has gathered great attention lately, mostly due to its low-invasive nature compared to tissue biopsies. Low invasiveness becomes greatly advantageous for microfluidics practices as the latter mirror cell biology revolutionizing cancer diagnostics and management. Recent advances in microfluidics hold the promise of robust clinical diagnostics after they have demonstrated effective exosome separation. We feel that microfluidics-based exosome isolation techniques, if cost-effective, could be implemented in the clinic and/or resource-scarce settings. This article (a) discusses exosomes, (b) comments on the first microfluidic advances in the field of cancer theranostics, (c) presents such advances in exosomes as complementary to liquid biopsies with an emphasis on circulating tumor cells, and (d) proposes a road map for future developments.

RASSF1A promoter methylation in high-grade serous ovarian cancer: A direct comparison study in primary tumors, adjacent morphologically tumor cell-free tissues and paired circulating tumor DNA.

The RASSF1A promoter is frequently methylated in high-grade serous ovarian cancer (HGSC). We examined RASSF1A promoter methylation in primary tumors, adjacent morphologically tumor cell-free tissues and corresponding circulating tumor DNA (ctDNA) samples of patients with HGSC, using a real-time methylation specific PCR (real-time MSP) and a methylation-sensitive high-resolution melting analysis (MS-HRMA) assay for the detection and semi-quantitative estimation of methylation, respectively. Two groups of primary HGSC tumor FFPE samples were recruited (Group A n=67 and Group B n=61), along with matched adjacent morphologically tumor cell-free tissues (n=58) and corresponding plasma samples (n=59) for group B. Using both assays, RASSF1A promoter was found highly methylated in primary tumors of both groups, and at lower percentages in the adjacent morphologically tumor cell-free tissues. Interestingly, RASSF1A promoter methylation was also observed in ctDNA by real-time MSP. Overall survival (OS) was significantly associated with RASSF1A promoter methylation in primary tumor samples using MS-HRMA (P=0.023). Our results clearly indicate that RASSF1A promoter is methylated in adjacent tissue surrounding the tumor in HGSC patients. We report for the first time that RASSF1A promoter methylation provides significant prognostic information in HGSC patients.

Gene expression profiling and DNA methylation analyses of CTCs.

A variety of molecular assays have been developed for CTCs detection and molecular characterization. Molecular assays are based on the nucleic acid analysis in CTCs and are based on total RNA isolation and subsequent mRNA quantification of specific genes, or isolation of genomic DNA that can be for DNA methylation studies and mutation analysis. This review is mainly focused on gene expression and methylation studies in CTCs in various types of cancer.

miRNA-21 as a novel therapeutic target in lung cancer.

Lung cancer is a leading cause of cancer death, and late diagnosis is one of the most important reasons for the high mortality rate. microRNAs (miRNAs) are key players in gene regulation and therefore in tumorigenesis. As far as lung carcinogenesis is concerned, miRNAs open novel fields in biomarker research, in diagnosis, and in therapy. In this review we focus on miR-21 in lung cancer and especially on how miR-21 is involved 1) as a biomarker in response or resistance to therapy or 2) as a therapeutic target.