Integrity and quantity of salivary cell-free DNA as a potential molecular biomarker in oral cancer: a preliminary study

Background: differences in cell-free DNA (cfDNA) fragments have been described as a valuable tool to distinguish cancer patients from healthy individuals. We aim to investigate the concentration and integrity of cfDNA fragments in saliva from oral squamous cell carcinoma (OSCC) patients and healthy individuals in order to explore their value as diagnostic biomarkers. Methods: saliva samples were collected from a total of 34 subjects (19 OSCC patients and 15 healthy controls). The total concentration of salivary cfDNA (scfDNA) was determined using a fluorometry method and quantitative real-time polymerase chain reaction (qPCR). To evaluate the scfDNA quantity and integrity, qPCR targeting Arthobacter luteus (ALU) sequences at three amplicons of different lengths (60, 115, and 247 bp, respectively) was carried out. ScfDNA integrity indexes (ALU115/ALU60 and ALU247/ALU60) were calculated as the ratio between the absolute concentration of the longer amplicons 115 bp and 247 bp and the total scfDNA amount (amplicon 60 bp).Results: the total scfDNA concentration (ALU60) was higher in OSCC than in healthy donors, but this trend was not statistically significant. The medians of scfDNA integrity indexes, ALU115/ALU60 and ALU247/ALU60, were significantly higher in OSCC, showing area under the curve values of 0.8211 and 0.7018, respectively. Conclusion: our preliminary results suggest that scfDNA integrity indexes (ALU115/ALU60 and ALU247/ALU60) have potential as noninvasive diagnostic biomarkers for OSCCS

Analysis of a Real-World Cohort of Metastatic Breast Cancer Patients Shows Circulating Tumor Cell Clusters (CTC-clusters) as Predictors of Patient Outcomes

Circulating tumor cell (CTC) enumeration has emerged as a powerful biomarker for the assessment of prognosis and the response to treatment in metastatic breast cancer (MBC). Moreover, clinical evidences show that CTC-cluster counts add prognostic information to CTC enumeration, however, their significance is not well understood, and more clinical evidences are needed. We aim to evaluate the prognostic value of longitudinally collected single CTCs and CTC-clusters in a heterogeneous real-world cohort of 54 MBC patients. Blood samples were longitudinally collected at baseline and follow up. CTC and CTC-cluster enumeration was performed using the CellSearch® system. Associations with progression-free survival (PFS) and overall survival (OS) were evaluated using Cox proportional hazards modelling. Elevated CTC counts and CTC-clusters at baseline were significantly associated with a shorter survival time. In joint analysis, patients with high CTC counts and CTC-cluster at baseline were at a higher risk of progression and death, and longitudinal analysis showed that patients with CTC-clusters had significantly shorter survival compared to patients without clusters. Moreover, patients with CTC-cluster of a larger size were at a higher risk of death. A longitudinal analysis of a real-world cohort of MBC patients indicates that CTC-clusters analysis provides additional prognostic value to single CTC enumeration, and that CTC-cluster size correlates with patient outcomeThis research was supported by Roche-Chus Joint Unit (IN853B 2018/03), funded by Axencia Galega de Innovación (GAIN), Consellería de Economía, Emprego e Industria and by the Instituto de Salud Carlos III (ISCIII) and FEDER (PI13/01388). L.M.-R. is supported by Asociación Española Contra el Cáncer (AECC). I.M.-P. is funded by the Training Program for Academic Staff fellowship (FPU16/01018), from the Ministry of Education and Vocational Training, Spanish GovernmentS

Circulating Tumor Cells Characterization Revealed TIMP1 as a Potential Therapeutic Target in Ovarian Cancer

Background: Recent studies showed a relevant role of hematogenous spread in ovarian cancer and the interest of circulating tumor cells (CTCs) monitoring as a prognosis marker. The aim of the present study was the characterization of CTCs from ovarian cancer patients, paying special attention to cell plasticity characteristics to better understand the biology of these cells. Methods: CTCs isolation was carried out in 38 patients with advanced high-grade serous ovarian cancer using in parallel CellSearch and an alternative EpCAM-based immunoisolation followed by RT-qPCR analysis to characterize these cells. Results: Epithelial CTCs were found in 21% of patients, being their presence higher in patients with extraperitoneal metastasis. Importantly, this population was characterized by the expression of epithelial markers as MUC1 and CK19, but also by genes associated with mesenchymal and more malignant features as TIMP1, CXCR4 and the stem markers CD24 and CD44. In addition, we evidenced the relevance of TIMP1 expression to promote tumor proliferation, suggesting its interest as a therapeutic target. Conclusions: Overall, we evidenced the utility of the molecular characterization of EpCAM+ CTCs from advanced ovarian cancer patients to identify biomarkers with potential applicability for disseminated disease detection and as therapeutic targets such as TIMP1Part of this research was supported by CIBERONC funds (CB16/12/00328)S

Looking for a Better Characterization of Triple-Negative Breast Cancer by Means of Circulating Tumor Cells

Traditionally, studies to address the characterization of mechanisms promoting tumor aggressiveness and progression have been focused only on primary tumor analyses, which could provide relevant information but have limitations to really characterize the more aggressive tumor population. To overcome these limitations, circulating tumor cells (CTCs) represent a noninvasive and valuable tool for real-time profiling of disseminated tumor cells. Therefore, the aim of the present study was to explore the value of CTC enumeration and characterization to identify markers associated with the outcome and the aggressiveness of triple-negative breast cancer (TNBC). For that aim, the CTC population from 32 patients diagnosed with TNBC was isolated and characterized. This population showed important cell plasticity in terms of expression of epithelia/mesenchymal and stemness markers, suggesting the relevance of epithelial to mesenchymal transition (EMT) intermediate phenotypes for efficient tumor dissemination. Importantly, the CTC signature demonstrated prognostic value to predict the patients' outcome and pointed to a relevant role of tissue inhibitor of metalloproteinases 1 (TIMP1) and androgen receptor (AR) for TNBC biology. Furthermore, we also analyzed the usefulness of the AR and TIMP1 blockade to target TNBC proliferation and dissemination using in vitro and in vivo zebra fish and mouse models. Overall, the molecular characterization of CTCs from advanced TNBC patients identifies highly specific biomarkers with potential applicability as noninvasive prognostic markers and reinforced the value of TIMP1 and AR as potential therapeutic targets to tackle the most aggressive breast cancer

Noninvasive early detection of colorectal cancer by hypermethylation of the LINC00473 promoter in plasma cell-free DNA

Background Current noninvasive assays have limitations in the early detection of colorectal cancer. We evaluated the clinical utility of promoter methylation of the long noncoding RNA LINC00473 as a noninvasive biomarker to detect colorectal cancer and associated precancerous lesions. Methods We evaluated the epigenetic regulation of LINC00473 through promoter hypermethylation in colorectal cancer cell lines using bisulfite genomic sequencing and expression analyses. DNA methylation of LINC00473 was analyzed in primary colorectal tumors using 450K arrays and RNA-seq from The Cancer Genome Atlas (TCGA). Tissue-based findings were validated in several independent cohorts of colorectal cancer and advanced colorectal polyp patients by pyrosequencing. We explored the clinical utility of LINC00473 methylation for the early detection of colorectal cancer in plasma cell-free DNA by quantitative methylation-specific PCR and droplet digital PCR. Results LINC00473 showed transcriptionally silencing due to promoter hypermethylation in colorectal cancer cell lines and primary tumors. Methylation of the LINC00473 promoter accurately detected primary colorectal tumors in two independent clinical cohorts, with areas under the receiver operating characteristic curves (AUCs) of 0.94 and 0.89. This biomarker also identified advanced colorectal polyps from two other tissue-based clinical cohorts with high diagnostic accuracy (AUCs of 0.99 and 0.78). Finally, methylation analysis of the LINC00473 promoter in plasma cell-free DNA accurately identified patients with colorectal cancer and advanced colorectal polyps (AUCs of 0.88 and 0.84, respectively), which was confirmed in an independent cohort of patients. Conclusions Hypermethylation of the LINC00473 promoter is a new promising biomarker for noninvasive early detection of colorectal cancer and related precancerous lesions

Noninvasive early detection of colorectal cancer by hypermethylation of the LINC00473 promoter in plasma cell-free DNA

J.R.-B. has received honoraria for educational activities from Roche; honoraria for consultancies from Boehringer Ingelheim; institutional research funding from Roche; and travel, accommodations, expenses from Bristol-Myers Squibb, Merck Sharp & Dohme, Ipsen, PharmaMar, Merck, Pfizer, and Roche. E.B.-V. has received honoraria for consultancies from Leo, and Rovi; and travel, accommodations, expenses from Servier, Merck, Sanofi, Roche, Pierre Fabre, and Amgen. Y.V.-I. has received honoraria for consultancies from PharmaMar. F.V.-R. has received honoraria for consultancies from Roche, Eisai, and Servier; and travel, accommodations, expenses from Lilly, Merck, and Pierre Fabre. S.C.-F. has received honoraria for educational activities from Servier, and Pierre Fabre; and travel, accommodations, expenses from Lilly, and Merck. R.L.-L. has received honoraria for participation in Advisory Boards from Roche, AstraZeneca, Merck, Merck Sharp & Dohme, Bayer, Bristol-Myers Squibb, Novartis, Janssen, Lilly, Pfizer, and Leo; travel, accommodations, and expenses from PharmaMar, Roche, Bristol-Myers Squibb, and Pierre Fabre; research funding from Roche and Merck; and is co-founder and shareholder in Nasasbiotech, S.L., Mtrap Inc. A.R.-C., A.B.C., R.L.-L. and A.D.-L. are inventors in one patent application over these results licensed to Advanced Marker Discovery, S.L. (Amadix). L.C., A.C.M. and R.A. are employees of Advanced Marker Discovery, S.L. (Amadix). The rest of the authors declare no potential conflicts of interest.This research was co-funded by the ISCIII (PI18/00307) and the European Regional Development Fund (FEDER), PRIS3 grant from ACIS and Xunta de Galicia, 2015 Merck Serono research grant, all donors who participated in the Liquid Biopsy Crowdfunding campaign in 2017, and Advanced Marker Discovery S.L. (Amadix). J.R.-B. was supported by a Río Hortega fellowship from ISCIII (CM19/00087) and is supported by a Juan Rodés contract (JR21/00019) and a 2020 TTD Research Grant from the Spanish Cooperative Group for the Treatment of Digestive Tumors. A.R.-C. is supported by the Roche-Chus Joint Unit (IN853B 2018/03) funded by GAIN, "Consellería de Economía, Emprego e Industria." N.C.-F. is funded by a predoctoral contract from "Xunta de Galicia" (IN606A-2020/004). A.B.-C. is funded by a predoctoral contract PFIS from ISCIII (FI19/00240) co-funded by "Fondo Social Europeo" (FSE). L.M.-R. is supported by the AECC. ABC is a Miguel Servet researcher (ISCIII; CP17/0008). A.G. is funded by CA181572, CA184792, CA202797 and CA214254 grants from the National Cancer Institute, National Institutes of Health. A.D.-L. is funded by a contract Juan Rodés from ISCIII (JR17/00016). V.M. is funded by the Agency for Management of University and Research Grants (AGAUR) of the Catalan Government grant 2017SGR723, the Instituto de Salud Carlos III, co-funded by FEDER funds-a way to build Europe-grants PI17-0009, and the Spanish Association Against Cancer (AECC) Scientific Foundation grant GCTRA18022MORE.Background: Current noninvasive assays have limitations in the early detection of colorectal cancer. We evaluated the clinical utility of promoter methylation of the long noncoding RNA LINC00473 as a noninvasive biomarker to detect colorectal cancer and associated precancerous lesions. Methods: We evaluated the epigenetic regulation of LINC00473 through promoter hypermethylation in colorectal cancer cell lines using bisulfite genomic sequencing and expression analyses. DNA methylation of LINC00473 was analyzed in primary colorectal tumors using 450K arrays and RNA-seq from The Cancer Genome Atlas (TCGA). Tissue-based findings were validated in several independent cohorts of colorectal cancer and advanced colorectal polyp patients by pyrosequencing. We explored the clinical utility of LINC00473 methylation for the early detection of colorectal cancer in plasma cell-free DNA by quantitative methylation-specific PCR and droplet digital PCR. Results: LINC00473 showed transcriptionally silencing due to promoter hypermethylation in colorectal cancer cell lines and primary tumors. Methylation of the LINC00473 promoter accurately detected primary colorectal tumors in two independent clinical cohorts, with areas under the receiver operating characteristic curves (AUCs) of 0.94 and 0.89. This biomarker also identified advanced colorectal polyps from two other tissue-based clinical cohorts with high diagnostic accuracy (AUCs of 0.99 and 0.78). Finally, methylation analysis of the LINC00473 promoter in plasma cell-free DNA accurately identified patients with colorectal cancer and advanced colorectal polyps (AUCs of 0.88 and 0.84, respectively), which was confirmed in an independent cohort of patients. Conclusions: Hypermethylation of the LINC00473 promoter is a new promising biomarker for noninvasive early detection of colorectal cancer and related precancerous lesions