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.

Development and Analytical Validation of a 6-Plex Reverse Transcription Droplet Digital PCR Assay for the Absolute Quantification of Prostate Cancer Biomarkers in Circulating Tumor Cells of Patients with Metastatic Castration-Resistant Prostate Cancer.

BACKGROUND: Gene expression in circulating tumor cells (CTCs) can be used as a predictive liquid biopsy test in metastatic castration-resistant prostate cancer (mCRPC). We developed a novel 6-plex reverse transcription droplet digital PCR (RT-ddPCR) assay for the absolute quantification of 4 prostate cancer biomarkers, a reference gene, and a synthetic DNA external control (DNA-EC) in CTCs isolated from mCRPC patients. METHODS: A novel 6-plex RT-ddPCR assay was developed for the simultaneous absolute quantification of AR-FL, AR-V7, PSA, and PSMA, HPRT (used as a reference gene), and a synthetic DNA-EC that was included for quality control. The assay was optimized and analytically validated using DNA synthetic standards for each transcript as positive controls. Epithelial cellular adhesion molecule (EpCAM)-positive CTC fractions isolated from 90 mCRPC patients and 11 healthy male donors were analyzed, and results were directly compared with reverse transcription quantitative PCR (RT-qPCR) for all markers in all samples. RESULTS: Linear dynamic range, limit of detection, limit of quantification, intra- and interassay precision, and analytical specificity were determined for each marker. Application of the assay in EpCAM-positive CTC showed positivity for AR-FL (71/90; 78.9%), AR-V7 (28/90; 31.1%), PSA (41/90; 45.6%), PSMA (38/90; 42.2%), and HPRT (90/90; 100%); DNA-EC concentration was constant across all samples. Direct comparison with RT-qPCR for the same markers in the same samples revealed RT-ddPCR to have superior diagnostic sensitivity. CONCLUSIONS: Our 6-plex RT-ddPCR assay was highly sensitive, specific, and reproducible, and enabled simultaneous and absolute quantification of 5 gene transcripts in minute amounts of CTC-derived cDNA. Application of this assay in clinical samples gave diagnostic sensitivity and specificity comparable to, or better than, RT-qPCR.

Development and Analytical Validation of a Reverse Transcription Droplet Digital PCR (RT-ddPCR) Assay for PD-L1 Transcripts in Circulating Tumor Cells.

BACKGROUND: PD-L1, an immune checkpoint protein, is an important biomarker for monitoring cancer patients during the administration of cancer immunotherapy. Droplet digital PCR (ddPCR), is a highly sensitive and accurate tool for the quantification of cancer biomarkers in liquid biopsy. We report the development and analytical validation of a novel duplex RT-ddPCR assay for the simultaneous quantification of PD-L1 and hypoxanthine phosphoribosyltransferase (HPRT) (used as reference gene) transcripts in circulating tumor cells (CTCs). METHODS: RT-ddPCR experimental conditions were first optimized and the assay was analytically validated using synthetic standards and the BB49 and SCC47 cancer cell lines. The developed assay was further applied in 71 peripheral blood (PB) samples from head and neck squamous cell carcinoma (HNSCC) patients and 20 PB samples from healthy donors. PD-L1 and HPRT transcripts were quantified in cDNAs derived from CTCs isolated by a size-dependent microfluidic device. The developed RT-ddPCR assay was directly compared to RT-qPCR using 71 identical patient cDNA samples. RESULTS: Analytical sensitivity was 0.64 copies/µL, while estimation of intra- and interassay variation revealed a high reproducibility (within-run CV%:4.7-23%; between-run CV%:13%). Using the developed RT-ddPCR assay 33/71(46.5%) HNSCC patients' samples were found positive for PD-L1 expression in CTCs, while by using RT-qPCR fewer samples (23/71, 32.4%) were positive (concordance: 55/71, 77.5%). CONCLUSIONS: The developed RT-ddPCR assay for PD-L1 in CTCs is highly sensitive, specific, and reproducible; additionally, it offers improved diagnostic sensitivity over RT-qPCR. The clinical utility of the assay should be prospectively evaluated for the real-time monitoring of CTCs of cancer patients under immunotherapy.

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.

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.

Expression pattern of androgen receptors, AR-V7 and AR-567es, in circulating tumor cells and paired plasma-derived extracellular vesicles in metastatic castration resistant prostate cancer.

Androgen-receptor splice variant 7 (AR-V7) is a highly promising liquid biopsy predictive biomarker showing primary or acquired resistance to novel androgen receptor signaling inhibitors in metastatic castration resistant prostate cancer (mCRPC). We present for the first time the expression pattern of AR-FL, AR-V7, and AR-567es at a quantitative level in circulating tumor cells (CTCs) and paired plasma-derived extracellular vesicles in mCRPC. We first developed and analytically validated a novel multiplex RT-qPCR assay for AR full length (AR-FL), AR-V7, AR-567es and AR-total. We then quantified the expression levels of AR-splice variants, CK-19 (epithelial marker) and B2M (reference gene) in EpCAM+ CTCs, and paired plasma-derived extracellular vesicles isolated from peripheral blood (20 mL) of 62 mCRPC patients and 10 healthy donors. CTCs were enumerated using the FDA-cleared CellSearch® system. In CTCs AR-FL was detected in 64/69 (92.3%), AR-V7 in 34/69 (49.3%), AR-567es in 16/69 (23.2%) and AR-total in 62/69 (89.9%). In 52 out of 69 samples, paired plasma-derived extracellular vesicles were analyzed. AR-FL was detected in 40/52 (76.9%), AR-V7 in 4/52 (7.7%), AR-567 in 2/52 (3.8%) and AR total in 39/52 (75.0%). In all cases AR splice variants were expressed in higher levels in CTCs than in paired extracellular vesicles, while AR-V7 was detected in higher percentages than in AR-567es. Using CellSearch®, CTCs were detected in 52/69 (75.4%) mCRPC patient samples; 27/52 (51.9%) of these samples were CTC+/AR-V7+ and 14/52 (26.9%) were CTC+/AR-567es+, while 7/17 (41.2%) were CTC-/AR-V7+ and 2/17 (11.8%) were CTC-/AR-567es+. Our results reveal for the first time a remarkable heterogeneity in the expression levels of AR-FL, AR-V7 and AR-567es in EpCAM+ CTCs and paired extracellular vesicles between individual mCRPC patients. The clinical significance of this finding will be further investigated in a large patient cohort with respect to therapy response.

Evaluation of Preanalytical Conditions and Implementation of Quality Control Steps for Reliable Gene Expression and DNA Methylation Analyses in Liquid Biopsies.

BACKGROUND: Liquid biopsy provides important information for the prognosis and treatment of cancer patients. In this study, we evaluated the effects of preanalytical conditions on gene expression and DNA methylation analyses in liquid biopsies. METHODS: We tested the stability of circulating tumor cell (CTC) messenger RNA by spiking MCF-7 cells in healthy donor peripheral blood (PB) drawn into 6 collection-tube types with various storage conditions. CTCs were enriched based on epithelial cell adhesion molecule positivity, and RNA was isolated followed by cDNA synthesis. Gene expression was quantified using RT-quantitative PCR for CK19 and B2M. We evaluated the stability of DNA methylation in plasma under different storage conditions by spiking DNA isolated from MCF-7 cells in healthy donor plasma. Two commercially available sodium bisulfite (SB)-conversion kits were compared, in combination with whole genome amplification (WGA), to evaluate the stability of SB-converted DNA. SB-converted DNA samples were analyzed by real-time methylation-specific PCR (MSP) for ACTB, SOX17, and BRMS1. Quality control was assessed using Levey-Jennings graphs. RESULTS: RNA-based analysis in CTCs is severely impeded by the preservatives used in many PB collection tubes (except for EDTA), as well as by time to analysis. Plasma and SB-converted DNA samples are stable and can be used safely for MSP when kept at -80 °C. Downstream WGA of SB-converted DNA compensated for the limited amount of available sample in liquid biopsies. CONCLUSIONS: Standardization of preanalytical conditions and implementation of quality control steps is extremely important for reliable liquid biopsy analysis, and a prerequisite for routine applications in the clinic.

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.

A Comparison of Three Methods for the Detection of Circulating Tumor Cells in Patients with Early and Metastatic Breast Cancer.

BACKGROUND: We directly compared CTC detection rates and prognostic significance, using three different methods in patients with breast cancer (BC). METHODS: Early (n=200) and metastatic (n=164) patients were evaluated before initiating adjuvant or first-line chemotherapy, using the CellSearchTM System, an RT-qPCR for CK-19 mRNA detection and by double immunofluorescence (IF) microscopy using A45-B/B3 and CD45 antibodies. RESULTS: Using the CellSearchTM System, 37% and 16.5% of early BC patients were CTC-positive (at ≥1 and ≥2 CTCs/23 ml of blood), 18.0% by RT-qPCR and 16.9% by IF; no agreement was observed between methods. By the CellSearchTM 34.8% and 53.7% (at≥ 5 and ≥ 2 CTCs/7.5 ml) of metastatic patients were CTC-positive, 37.8% by RT-qPCR and 28.5% by IF. A significant agreement existed only between the CellSearchTM and RT-qPCR. In 60.8% of cases, differential EpCAM and CK-19 expression on CTCs by IF could explain the discrepancies between the CellSearchTM and RT-qPCR. CTC-positivity by either method was associated with decreased overall survival in metastatic patients. CONCLUSION: A significant concordance was observed between the CellSearchTM and RT-qPCR in metastatic but not in early BC. Discordant results could be explained in part by CTC heterogeneity. CTC detection by all methods evaluated had prognostic relevance in metastatic patients.

The Role of CTCs as Tumor Biomarkers.

Detection of Circulating Tumor Cells (CTCs) in peripheral blood can serve as a "liquid biopsy" approach and as a source of valuable tumor markers. CTCs are rare, and thus their detection, enumeration and molecular characterization are very challenging. CTCs have the unique characteristic to be non-invasively isolated from blood and used to follow patients over time, since these cells can provide significant information for better understanding tumour biology and tumour cell dissemination. CTCs molecular characterization offers the unique potential to understand better the biology of metastasis and resistance to established therapies and their analysis presents nowadays a promising field for both advanced and early stage patients. In this chapter we focus on the latest findings concerning the clinical relevance of CTC detection and enumeration, and discuss their potential as tumor biomarkers in various types of solid cancers. We also highlight the importance of performing comparison studies between these different methodologies and external quality control systems for establishing CTCs as tumor biomarkers in the routine clinical setting.

Circulating tumor cells as promising novel biomarkers in solid cancers.

The presence of circulating tumor cells (CTCs) in peripheral blood can serve as a "liquid biopsy" approach and has thus emerged lately as one of the hottest fields in cancer research. CTCs can be isolated from blood in a non-invasive approach, and can be used to follow patients over time since these cells can provide significant information for a better understanding of tumor biology and tumor cell dissemination. CTC molecular characterization offers the unique potential to better understand the biology of metastasis and resistance to established therapies, and analysis of these cells presents a promising field for both advanced and early-stage patients. CTC detection, enumeration, and molecular characterization are very challenging since CTCs are rare, and the amount of available sample is very limited. Since detection of CTCs has been shown to be of considerable utility in the clinical management of patients with solid cancers, various analytical systems for their isolation and detection have been developed. New areas of research are directed towards developing novel assays for single-CTC isolation and molecular characterization. The clinical significance of CTCs has been evaluated in many types of solid cancers, and the CTC enumeration test in metastatic breast, colorectal, and prostate cancer was cleared by the FDA almost a decade ago. This review is mainly focused on the clinical potential of CTCs as novel biomarkers in 10 different types of solid cancers: breast, ovarian, prostate, lung, colorectal, hepatocellular carcinoma, pancreatic, head and neck, bladder cancer and melanoma.

Comprehensive Analysis of CXCR4, JUNB, and PD-L1 Expression in Circulating Tumor Cells (CTCs) from Prostate Cancer Patients.

CXCR4, JUNB and PD-L1 are implicated in cancer progression and metastasis. The current study investigated these biomarkers in CTCs isolated from metastatic prostate cancer (mPCa) patients at the RNA and protein levels. CTCs were isolated from 48 mPCa patients using the Ficoll density gradient and ISET system (17 out of 48). The (CK/PD-L1/CD45) and (CK/CXCR4/JUNB) phenotypes were identified using two triple immunofluorescence stainings followed by VyCAP platform analysis. Molecular analysis was conducted with an EpCAM-dependent method for 25/48 patients. CK-8, CK-18, CK-19, JUNB, CXCR4, PD-L1, and B2M (reference gene) were analyzed with RT-qPCR. The (CK+/PD-L1+/CD45-) and the (CK+/CXCR4+/JUNB+) were the most frequent phenotypes (61.1% and 62.5%, respectively). Furthermore, the (CK+/CXCR4+/JUNB-) phenotype was correlated with poorer progression-free survival [(PFS), HR: 2.5, p = 0.049], while the (CK+/PD-L1+/CD45-) phenotype was linked to decreased overall survival [(OS), HR: 262.7, p = 0.007]. Molecular analysis revealed that 76.0% of the samples were positive for CK-8,18, and 19, while 28.0% were positive for JUNB, 44.0% for CXCR4, and 48.0% for PD-L1. Conclusively, CXCR4, JUNB, and PD-L1 were highly expressed in CTCs from mPCa patients. The CXCR4 protein expression was associated with poorer PFS, while PD-L1 was correlated with decreased OS, providing new biomarkers with potential clinical relevance.

Comparison of three molecular assays for the detection and molecular characterization of circulating tumor cells in breast cancer.

INTRODUCTION: Comparison studies between different analytical methodologies for circulating tumor cells (CTC) detection and molecular characterization are urgently needed, since standardization of assays is essential before their use in clinical practice. METHODS: We compared three different CTC molecular assays. To avoid discrepancies due to pre-analytical errors we used the same cDNAs throughout our study. CTC were isolated using anti-EpCAM and anti-MUC1 coated magnetic beads from 2 × 5 ml of peripheral blood of 254 early and 51 metastatic breast cancer patients and 30 healthy individuals. The same cDNAs were analyzed by: a) singleplex RT-qPCR assay for CK-19; b) multiplex RT-qPCR for CK-19, HER-2, MAGE- A3, and PBGD; and c) a commercially available molecular assay (AdnaTest BreastCancer) for GA733-2, MUC-1, HER-2 and beta-actin. RESULTS: In early breast cancer, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest, were positive for the presence of CTC in 14.2%, 22.8% and 16.5% subjects, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 72.4% while between the AdnaTest and multiplex RT-qPCR was 64.6%. In patients with overt metastasis, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest were positive in 41.2%, 39.2% and 54.9% patients, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 70.6% while between the AdnaTest and multiplex RT-qPCR was 68.6%. CONCLUSIONS: All CTC assays gave similar results in about 70% of cases. Better agreement was found in the metastatic setting, possibly explained by the higher tumor load in this group. Discordances could be attributed to the different gene transcripts used to evaluate CTC positivity. Our results indicate the importance of CTC heterogeneity for their detection by different analytical methodologies.

Molecular characterization of circulating tumor cells in breast cancer: challenges and promises for individualized cancer treatment.

Blood testing using Circulating Tumor Cells (CTCs) has emerged as one of the hottest fields in cancer diagnosis. Research on CTCs present nowadays a challenge, as these cells are well defined targets for understanding tumour biology and improving cancer treatment. The presence of tumor cells in patient's bone marrow or peripheral blood is an early indicator of metastasis and may signal tumor spread sooner than clinical symptoms appear and imaging results confirm a poor prognosis. CTC enumeration can serve as a "liquid biopsy" and an early marker to assess response to systemic therapy. Definition of biomarkers based on comprehensive characterization of CTCs has a strong potential to be translated to individualized targeted treatments and spare breast cancer patients unnecessary and ineffective therapies but also to reduce the costs for the health system and to downsize the extent and length of clinical studies. In this review, we briefly summarize recent studies on the molecular characterization of circulating tumor cells in breast cancer and discuss challenges and promises of CTCs for individualized cancer treatment.

SOX17 promoter methylation in circulating tumor cells and matched cell-free DNA isolated from plasma of patients with breast cancer.

INTRODUCTION: Detection of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in the peripheral blood of patients with solid tumors has been widely studied for the early detection of metastatic spread. We evaluated whether there was an association between the origin of cfDNA and CTCs. We investigated whether SRY (sex determining region Y)-box 17 (SOX17) promoter methylation in CTCs was associated with the methylation pattern of this gene in matched cfDNA isolated from plasma of patients with breast cancer. METHODS: We examined SOX17 methylation in 79 primary breast tumors, in 114 paired samples of DNA isolated from CTCs and cfDNA, and in 60 healthy individuals. Isolated DNA was modified by sodium bisulfite and subjected to methylation specific PCR. RESULTS: The SOX17 promoter was methylated in 68 (86.0%) of 79 of primary breast tumors. In CTCs, SOX17 was methylated in 19 (34.5%) of 55 patients with early breast cancer, 27 (45.8%) of 59 patients with metastatic cancer, and 1 (4.3%) of 23 healthy individuals, whereas in matched cfDNA SOX17 was methylated in 19 (34.5%) of 55, 24 (40.7%) of 59, and 1 (2.0%) of 49 of these same groups, respectively. There was a significant correlation between SOX17 methylation in cfDNA and CTCs in patients with early breast cancer (P = 0.008), but not in patients with verified metastasis (P = 0.283). CONCLUSIONS: The SOX17 promoter is highly methylated in primary breast tumors, in CTCs isolated from patients with breast cancer, and in corresponding cfDNA samples. Our findings indicate a direct connection between the presence of CTCs and cfDNA in patients with operable breast cancer, after surgical removal of the primary tumor.

Clinical Significance of PD-L1 Status in Circulating Tumor Cells for Cancer Management during Immunotherapy.

The approval of monoclonal antibodies against programmed death-ligand 1 (PD-L1) and programmed cell death protein (PD1) has changed the landscape of cancer treatment. To date, many immune checkpoint inhibitors (ICIs) have been approved by the FDA for the treatment of metastatic cancer as well as locally recurrent advanced cancer. However, immune-related adverse events (irAEs) of ICIs highlight the need for biomarker analysis with strong predictive value. Liquid biopsy is an important tool for clinical oncologists to monitor cancer patients and administer or change appropriate therapy. CTCs frequently express PD-L1, and this constitutes a clinically useful and non-invasive method to assess PD-L1 status in real-time. This review summarizes all the latest findings about the clinical significance of CTC for the management of cancer patients during the administration of immunotherapy and mainly focuses on the assessment of PD-L1 expression in CTCs.

Detection and Molecular Characterization of Circulating Tumour Cells: Challenges for the Clinical Setting.

Over the last decade, liquid biopsy has gained much attention as a powerful tool in personalized medicine since it enables monitoring cancer evolution and follow-up of cancer patients in real time. Through minimally invasive procedures, liquid biopsy provides important information through the analysis of circulating tumour cells (CTCs) and circulating tumour-derived material, such as circulating tumour DNA (ctDNA), circulating miRNAs (cfmiRNAs) and extracellular vehicles (EVs). CTC analysis has already had an important impact on the prognosis, detection of minimal residual disease (MRD), treatment selection and monitoring of cancer patients. Numerous clinical trials nowadays include a liquid biopsy arm. CTC analysis is now an exponentially expanding field in almost all types of solid cancers. Functional studies, mainly based on CTC-derived cell-lines and CTC-derived explants (CDx), provide important insights into the metastatic process. The purpose of this review is to summarize the latest findings on the clinical significance of CTCs for the management of cancer patients, covering the last four years. This review focuses on providing a comprehensive overview of CTC analysis in breast, prostate and non-small-cell lung cancer. The unique potential of CTC single-cell analysis for understanding metastasis biology, and the importance of quality control and standardization of methodologies used in this field, is also discussed.

Comprehensive liquid biopsy analysis as a tool for the early detection of minimal residual disease in breast cancer.

Liquid biopsy (LB) provides a unique minimally invasive tool to follow-up cancer patients over time, to detect minimal residual disease (MRD), to study metastasis-biology and mechanisms of therapy-resistance. Molecular characterization of CTCs offers additionally the potential to understand resistance to therapy and implement individualized targeted treatments which can be modified during the disease evolution and follow-up period of a patient. In this study, we present a long-term follow-up of operable breast cancer patients based on a comprehensive liquid biopsy analysis. We performed a comprehensive liquid biopsy analysis in peripheral blood of 13 patients with early-stage operable breast cancer at several time points for a period of ten years, consisting of: (a) CTC enumeration using the CellSearch system, (b) phenotypic analysis of CTCs using Immunofluorescence, (c) gene expression analysis, in EpCAM(+) CTCs for CK-19, CD24,CD44, ALDH1, and TWIST1, (d) analysis of PIK3CA and ESR1 mutations in EpCAM(+) CTCs and corresponding plasma ctDNA and (e) DNA methylation of ESR1 in CTCs. 10/13 (77%) patients were found negative for LB markers in PB during the whole follow-up period, and these patients did not relapse during the follow-up. However, 3/13(18%) patients that were positive for at least one LB marker relapsed within the follow-up period. The molecular characteristics of CTCs were highly different even for the same patient at different time points, and always increased before the clinical relapse. Our results indicate that liquid biopsy can reveal the presence of MRD at least 4 years before the appearance of clinically detectable metastatic disease demonstrating that a comprehensive liquid biopsy analysis provides highly important information for the therapeutic management of breast cancer patients.

Phase II Window Study of Olaparib Alone or with Cisplatin or Durvalumab in Operable Head and Neck Cancer.

Purpose: We conducted a phase II randomized noncomparative window of opportunity (WOO) trial to evaluate the inhibition of cellular proliferation and the modulation of immune microenvironment after treatment with olaparib alone or in combination with cisplatin or durvalumab in patients with operable head and neck squamous cell carcinoma (HNSCC). Experimental Design: Forty-one patients with HNSCC were randomized to cisplatin plus olaparib (arm A), olaparib alone (arm B), no treatment (arm C) or durvalumab plus olaparib (arm D). The primary endpoint was to evaluate the percentage of patients in each arm that achieved a reduction of at least 25% in Ki67. Secondary endpoints included objective response rate (ORR), safety, and pathologic complete response (pCR) rate. Paired baseline and resection tumor biopsies and blood samples were evaluated for prespecified biomarkers. Results: A decrease in Ki67 of at least 25% was observed in 44.8% of treated patients, as measured by quantitative immunofluorescence. The ORR among treated patients was 12.1%. pCR was observed in 2 patients. Two serious adverse events occurred in 2 patients.Programmed death ligand 1 (PD-L1) levels [combined positive score (CPS)] were significantly higher after treatment in arms A and D. Expression of CD163 and colony-stimulating factor 1 receptor (CSF1R) genes, markers of M2 macrophages, increased significantly posttreatment whereas the expression of CD80, a marker of M1 macrophages, decreased. Conclusion: Preoperative olaparib with cisplatin or alone or with durvalumab was safe in the preoperative setting and led to decrease in Ki67 of at least 25% in 44.8% of treated patients. Olaparib-based treatment modulates the tumor microenvironment leading to upregulation of PD-L1 and induction of protumor features of macrophages. Significance: HNSCC is characterized by defective DNA repair pathways and immunosuppressive tumor microenvironment. PARP inhibitors, which promote DNA damage and "reset" the inflammatory tumor microenvironment, can establish an effective antitumor response. This phase II WOO trial in HNSCC demonstrated the immunomodulatory effects of PARP inhibitor-induced DNA damage. In this chemo-naïve population, PARP inhibitor-based treatment, reduced tumor cell proliferation and modulated tumor microenvironment. After olaparib upregulation of PD-L1 and macrophages, suggests that combinatorial treatment might be beneficial. Synopsis: Our WOO study demonstrates that preoperative olaparib results in a reduction in Ki67, upregulation of PD-L1 CPS, and induction of protumor features of macrophages in HNSCC.

Evaluation of viral concentration and extraction methods for SARS-CoV-2 recovery from wastewater using droplet digital and quantitative RT-PCR.

The ongoing pandemic caused by the emergence of SARS-CoV-2 has resulted in millions of deaths worldwide despite the various measures announced by the authorities. Wastewater-based epidemiology has the ability to provide a day-to-day estimation of the number of infected people in a fast and cost-effective manner. However, owing to the complex nature of wastewater, wastewater monitoring for viral genome copies is affected by the extensive viral fragmentation that takes place all the way to the sewage and the analytical lab. The aim of this study was to evaluate different methodologies for the concentration and extraction of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. We compare 5 different concentration methods and 4 commercially available kits for the RNA extraction. To evaluate the performance and the recovery of these, SARS-CoV-2 isolated from patients was used as a spike control. Additionally, the presence of SARS-CoV-2 in all wastewater samples was determined using reverse transcription quantitative PCR (RT-qPCR) and reverse transcription droplet digital PCR (RT-ddPCR), targeting three genetic markers (N1, N2 and N3). Using spiked samples, recoveries were estimated 2.1-37.6% using different extraction kits and 0.1-2.1% using different concentration kits. It was found that a direct capture-based method, evaluated against a variety of concentration methods, is the best in terms of recovery, time and cost. Interestingly, we noticed a good agreement between the results provided by RT-qPCR and RT-ddPCR in terms of recovery. This evaluation can serve as a guide for laboratories establishing a protocol to perform wastewater monitoring of SARS-CoV-2. Overall, data presented here reinforces the validity of WBE for SARS-CoV-2 surveillance, uncovers potential caveats in the selection of concentration and extraction protocols and points towards optimal solutions to maximize its potential.