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.

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.

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.

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 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.

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.

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 Gene Expression and DNA Methylation Markers in Circulating Tumor Cells and Paired Plasma Derived Exosomes in Metastatic Castration Resistant Prostate Cancer.

Liquid biopsy, based on the analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), provides non-invasive real-time monitoring of tumor evolution and therapeutic efficacy. We performed for the first time a direct comparison study on gene expression and DNA methylation markers in CTCs and paired plasma-derived exosomes and evaluated their prognostic significance in metastatic castration resistant prostate cancer. This prospective liquid biopsy (LB) study was based on a group of 62 metastatic castration resistant prostate cancer (mCRPC) patients and 10 healthy donors (HD) as controls. Identical blood draws were used to: (a) enumerate CTC and tumor-derived extracellular vesicles (tdEVs) using CellSearch (CS) and (b) analyze CTCs and paired plasma-derived exosomes at the gene expression and DNA methylation level. CTCs were enumerated using CellSearch in 57/62 patients, with values ranging from 5 to 854 cells/7.5 mL PB. Our results revealed for the first time a significantly higher positivity of gene expression markers (CK-8, CK-18, TWIST1, PSMA, AR-FL, AR-V7, AR-567 and PD-L1 mRNA) in EpCAM-positive CTCs compared to plasma-derived exosomes. GSTP1, RASSF1A and SCHLAFEN were methylated both in CTC and exosomes. In CTCs, Kaplan-Meier analysis revealed that CK-19 (p = 0.009), PSMA (p = 0.001), TWIST1 (p = 0.001) expression and GSTP1 (p = 0.001) methylation were correlated with OS, while in exosomes GSTP1 (p = 0.007) and RASSF1A (p = 0.001) methylation was correlated with OS. Our direct comparison study of CTCs and exosomes at gene expression and DNA methylation level, revealed for the first time a significantly higher positivity in EpCAM-positive CTCs compared to plasma-derived exosomes. Future perspective of this study should be the evaluation of clinical utility of molecular biomarkers in CTCs and exosomes on independent multicentric cohorts with mCRPC patients.

Androgen Receptor and PIM1 Expression in Tumor Tissue of Patients With Triple-negative Breast Cancer.

BACKGROUND/AIM: Effective targeted therapies for triple-negative breast cancer (TNBC) are limited. In a subset of TNBC, androgen receptor (AR) plays an important role, while the human proviral integration site for Moloney murine leukemia virus-1 (PIM1) overexpression is also implicated. PIM1 kinases phosphorylate AR, thus regulating its transcriptional activity, regardless of the presence or not of androgens. We evaluated the expression of AR and PIM1 and their prognostic significance in TNBC. MATERIALS AND METHODS: AR and PIM1 transcripts were quantified by quantitative reverse transcription polymerase chain reaction in formalin-fixed paraffin-embedded tumor from 141 patients with TNBC. RESULTS: AR was expressed in 38.3%, PIM1 in 10.6%, while co-expression of AR and PIM1 was detected in 7/141 cases (5.0%). No prognostic significance of AR or PIM1 was reached for overall or disease-free survival. CONCLUSION: Co-expression of AR and PIM1 exists in only in a small percentage of patients with TNBC. The implications of this finding in the therapeutic management of patients with TNBC should be investigated in larger patient cohorts.

Gene expression in circulating tumor cells reveals a dynamic role of EMT and PD-L1 during osimertinib treatment in NSCLC patients.

Liquid biopsy is a tool to unveil resistance mechanisms in NSCLC. We studied changes in gene expression in CTC-enriched fractions of EGFR-mutant NSCLC patients under osimertinib. Peripheral blood from 30 NSCLC patients before, after 1 cycle of osimertinib and at progression of disease (PD) was analyzed by size-based CTC enrichment combined with RT-qPCR for gene expression of epithelial (CK-8, CK-18, CK-19), mesenchymal/EMT (VIM, TWIST-1, AXL), stem cell (ALDH-1) markers, PD-L1 and PIM-1. CTCs were also analyzed by triple immunofluorescence for 45 identical blood samples. Epithelial and stem cell profile (p = 0.043) and mesenchymal/EMT and stem cell profile (p = 0.014) at PD were correlated. There was a strong positive correlation of VIM expression with PIM-1 expression at baseline and increased PD-L1 expression levels at PD. AXL overexpression varied among patients and high levels of PIM-1 transcripts were detected. PD-L1 expression was significantly increased at PD compared to baseline (p = 0.016). The high prevalence of VIM positive CTCs suggest a dynamic role of EMT during osimertinib treatment, while increased expression of PD-L1 at PD suggests a theoretical background for immunotherapy in EGFR-mutant NSCLC patients that develop resistance to osimertinib. This observation merits to be further evaluated in a prospective immunotherapy trial.

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.

PIM-1 Is Overexpressed at a High Frequency in Circulating Tumor Cells from Metastatic Castration-Resistant Prostate Cancer Patients.

PIM-1 is an oncogene involved in cell cycle progression, cell growth, cell survival and therapy resistance, activated in many types of cancer, and is now considered as a very promising target for cancer therapy. We report for the first time that PIM-1 is overexpressed in circulating tumor cells (CTCs) from metastatic castration-resistant prostate cancer patients (mCRPC). We first developed and validated a highly sensitive RT-qPCR assay for quantification of PIM-1 transcripts. We further applied this assay to study PIM-1 expression in EpCAM(+) CTC fraction isolated from 64 peripheral blood samples of 50 mCRPC patients. CTC enumeration in all samples was performed using the FDA-cleared CellSearch® system. PIM-1 overexpression was detected in 24/64 (37.5%) cases, while in 20/24 (83.3%) cases that were positive for PIM-1 expression, at least one CTC/7.5 mL PB was detected in the CellSearch®. Our data indicate that PIM-1 overexpression is observed at high frequency in CTCs from mCRPC patients and this finding, in combination with androgen receptor splice variant 7 (AR-V7) expression in CTCs, suggest its potential role as a very promising target for cancer therapy. We strongly believe that PIM-1 overexpression in EpCAM(+) CTC fraction merits to be further evaluated and validated as a non-invasive circulating tumor biomarker in a large and well-defined patient cohort with mCRPC.

Prognostic impact of indoleamine 2,3-dioxygenase 1 (IDO1) mRNA expression on circulating tumour cells of patients with head and neck squamous cell carcinoma.

BACKGROUND: We sought to determine the prognostic role of indoleamine 2,3-dioxygenase 1 (IDO1) by evaluating IDO1 expression in circulating tumour cells (CTCs) at baseline and after completion of chemoradiotherapy in patients with locally advanced (LA) head and neck squamous cell carcinoma (HNSCC) treated with curative intent. METHODS: In a prospective cohort of 113 patients with LA HNSCC, we evaluated expression of IDO1 in the EpCAM+ CTC fraction at baseline and after cisplatin chemoradiation. The prognostic value of combined programmed cell death ligand-1 (PDL-1) and IDO1 expression was assessed. RESULTS: IDO1 was significantly overexpressed at baseline compared with the post-treatment counterparts (p=0.007). IDO1 messenger RNA (mRNA) expression at baseline was associated with better survival in terms of progression-free survival (PFS) (HR=0.19, p=0.017). Post-treatment IDO1 mRNA levels were correlated with unfavourable prognosis in terms of overall survival (OS) (HR=3.27, p=0.008). Patients with combined decreased expression levels of PDL-1 and IDO1 after treatment exhibited superior PFS (p=0.043) and OS (p=0.021). CONCLUSIONS: Our results strongly suggest that IDO1 mRNA expression is an independent prognostic factor for clinical outcome. Our study provides useful information for future trials combining chemoradiation with immune checkpoint inhibitors and IDO1 inhibitors.

Direct comparison of size-dependent versus EpCAM-dependent CTC enrichment at the gene expression and DNA methylation level in head and neck squamous cell carcinoma.

We directly compared two different approaches used for Circulating Tumor Cell (CTC) isolation, a size-dependent microfluidic system versus an EpCAM-dependent positive selection for downstream molecular characterization of CTC both at the gene expression and DNA methylation level in Head and Neck Squamous Cell Carcinoma (HNSCC). A size-dependent microfluidic device (Parsortix, ANGLE) and an EpCAM-dependent positive immune-magnetic isolation procedure were applied in parallel, using 10 mL PB from 50 HNSCC patients and 18 healthy donors. Total RNA was isolated from enriched CTCs and RT-qPCR was used to study the expression levels of CK-19, PD-L1, EGFR, TWIST1, CDH2 and B2M (reference gene). Real time methylation specific PCR (MSP) was used to study the methylation status of RASSF1A and MLL3 genes. In identical blood draws, the label-free size-dependent CTC-isolation system was superior in terms of sensitivity when compared to the EpCAM-dependent CTC enrichment, since a significantly higher percentage of identical PB samples was found positive at the gene expression and DNA methylation level, while the specificity was not affected. Our results indicate that future studies focused on the evaluation of clinical utility of CTC molecular characterization in HNSCC should be based on size-dependent enrichment approaches.

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.