Background RAS assessment is mandatory for therapy decision in metastatic colorectal cancer (mCRC) patients. This determination is based on tumor tissue, however, genotyping of circulating tumor (ct)DNA offers clear advantages as a minimally invasive method that represents tumor heterogeneity. Our study aims to evaluate the use of ctDNA as an alternative for determining baseline RAS status and subsequent monitoring of RAS mutations during therapy as a component of routine clinical practice.Patients and methods RAS mutational status in plasma was evaluated in mCRC patients by OncoBEAM™ RAS CRC assay. Concordance of results in plasma and tissue was retrospectively evaluated. RAS mutations were also prospectively monitored in longitudinal plasma samples from selected patients.ResultsAnalysis of RAS in tissue and plasma samples from 115 mCRC patients showed a 93% overall agreement. Plasma/tissue RAS discrepancies were mainly explained by spatial and temporal tumor heterogeneity. Analysis of clinico-pathological features showed that the site of metastasis (i.e. peritoneal, lung), the histology of the tumor (i.e. mucinous) and administration of treatment previous to blood collection negatively impacted the detection of RAS in ctDNA. In patients with baseline mutant RAS tumors treated with chemotherapy/antiangiogenic, longitudinal analysis of RAS ctDNA mirrored response to treatment, being an early predictor of response. In patients RAS wt, longitudinal monitoring of RAS ctDNA revealed that OncoBEAM was useful to detect emergence of RAS mutations during anti-EGFR treatment.ConclusionThe high overall agreement in RAS mutational assessment between plasma and tissue supports blood-based testing with OncoBEAM™ as a viable alternative for genotyping RAS of mCRC patients in routine clinical practice. Our study describes practical clinico-pathological specifications to optimize RAS ctDNA determination. Moreover, OncoBEAM™ is useful to monitor RAS in patients undergoing systemic therapy to detect resistance and evaluate the efficacy of particular treatments.

Metastatic colorectal cancer (mCRC) relies on the detachment of aggressive malignant cells from the primary tumor into the bloodstream and, concordantly, the presence of these Circulating Tumor Cells (CTC) is associated with a poor prognosis. In this work, the molecular characterization of CTC from mCRC patients was approached, with the aim of understanding their biology and improving their clinical utility in the management of colorectal cancer patients. For this, EpCAM-based immunoisolation of CTC was combined with whole transcriptome amplification and hybridization onto cDNA microarrays. Gene expression data from mCRC patients, once the background of unspecific immunoisolation from a group of controls had been subtracted, resulted in 410 genes that characterized the CTC population. Bioinformatics were used for the biological interpretation of the data, revealing that CTC are characterized by genes related to cell movement and adhesion, cell death and proliferation, and cell signalling and interaction. RTqPCR on an independent series of mCRC patients and controls was used for the validation of a number of genes related to the main cellular functions characterizing the CTC population. Comparison between primary carcinomas and lung and liver metastases further involved the CTC-genes in the promotion of metastasis. Moreover, the correlation of CTC-gene expression with clinical parameters demonstrated detection and prognosis significance. In conclusion, the molecular characterization of CTC from mCRC patients and the identification of diagnostic and prognostic biomarkers represent an innovative and promising approach in the clinical management of this type of patients.

In the present study we investigated the prognostic value of Circulating Tumour Cells (CTC) and their utility for therapy monitoring in non-small cell lung cancer (NSCLC). A total of 43 patients newly diagnosed with NSCLC were prospectively enrolled. Blood samples were obtained before the 1st, 2nd and 5th cycles of chemotherapy and analyzed using CellSearch technology. Both CTC and CTC-related objects (not morphological standard or broken epithelial cells) were counted. At baseline 18 (41.9%) patients were positive for intact CTC count and 10 (23.2%) of them had ≥5 CTC, while CK positive events were found in 79.1% of patients. The group of patients with CTC ≥5 at baseline presented worse PFS and OS than those with <5 CTC (p = 0.034 and p = 0.008, respectively). Additionally, high levels of total CK positive events were associated with poor prognosis in the group of patients with <5 CTC. Regarding therapy monitoring, patients presenting increased levels of CTC during the treatment demonstrated lower OS and PFS rates. All these data supported the value of CTC as a prognostic biomarker and as a surrogate indicator of chemotherapy effectiveness in advanced NSCLC patients, with the additional value of analyzing other “objects” such as apoptotic CTC or CK fragments to guide the clinical management of these patients.

The incidence and mortality of endometrial cancer (EC) have risen in recent years, hence more precise management is needed. Therefore, we combined different types of liquid biopsies to better characterize the genetic landscape of EC in a non-invasive and dynamic manner. Uterine aspirates (UAs) from 60 patients with EC were obtained during surgery and analyzed by next-generation sequencing (NGS). Blood samples, collected at surgery, were used for cell-free DNA (cfDNA) and circulating tumor cell (CTC) analyses. Finally, personalized therapies were tested in patient-derived xenografts (PDXs) generated from the UAs. NGS analyses revealed the presence of genetic alterations in 93% of the tumors. Circulating tumor DNA (ctDNA) was present in 41.2% of cases, mainly in patients with high-risk tumors, thus indicating a clear association with a more aggressive disease. Accordingly, the results obtained during the post-surgery follow-up indicated the presence of ctDNA in three patients with progressive disease. Moreover, 38.9% of patients were positive for CTCs at surgery. Finally, the efficacy of targeted therapies based on the UA-specific mutational landscape was demonstrated in PDX models. Our study indicates the potential clinical applicability of a personalized strategy based on a combination of different liquid biopsies to characterize and monitor tumor evolution, and to identify targeted therapies.

Salivary microRNAs (miRNAs) are of high interest as diagnostic biomarkers for non-oral cancer. However, little is known about their value for colorectal cancer (CRC) detection. Our study aims to characterize salivary miRNAs in order to identify non-invasive markers for CRC diagnosis. The screening of 754 miRNAs was performed in saliva samples from 14 CRC and 10 healthy controls. The differential expressed miRNAs were validated by RT-qPCR in 51 CRC, 19 adenomas and 37 healthy controls. Receiver operating characteristic (ROC) curves and logistic regression models were performed to analyze the clinical value of these miRNAs. Twenty-two salivary miRNAs were significantly deregulated in CRC patients vs. healthy individuals (P < 0.05) in the discovery phase. From those, five upregulated miRNAs (miR-186-5p, miR-29a-3p, miR-29c-3p, miR-766-3p, and miR-491-5p) were confirmed to be significantly higher in the CRC vs. healthy group (P < 0.05). This five-miRNA signature showed diagnostic value (72% sensitivity, 66.67% specificity, AUC = 0.754) to detect CRC, which was even higher in combination with carcinoembryonic antigen (CEA) levels. Overall, after the first global characterization of salivary miRNAs in CRC, a five-miRNA panel was identified as a promising tool to diagnose this malignancy, representing a novel approach to detect cancer-associated epigenetic alterations using a non-invasive strategy.

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 outcome.

The accuracy in the diagnosis of metastatic colorectal cancer (mCRC) represents one of the challenges in the clinical management of patients. The detection of circulating tumour cells (CTC) is becoming a promising alternative to current detection techniques, as it focuses on one of the players of the metastatic disease and it should provide with more specific and sensitive detection rates. Here, we describe an improved method of detection of CTC from mCRC patients by combining immune-enrichment, optimal purification of RNA from very low cell numbers, and the selection of accurate PCR probes. As a result, we obtained a logistic model that combines GAPDH and VIL1 normalized to CD45 rendering powerful results in the detection of CTC from mCRC patients (AUROC value 0.8599). We further demonstrated the utility of this model at the clinical setting, as a reliable prognosis tool to determine progression-free survival in mCRC patients. Overall, we developed a strategy that ameliorates the specificity and sensitivity in the detection of CTC, resulting in a robust and promising logistic model for the clinical management of metastatic colorectal cancer patients.

Tumor-derived extracellular vesicles (EVs) are secreted in large amounts into biological fluids of cancer patients. The analysis of EVs cargoes has been associated with patient´s outcome and response to therapy. However, current technologies for EVs isolation are tedious and low cost-efficient for routine clinical implementation. To explore the clinical value of circulating EVs analysis we attempted a proof-of-concept in endometrial cancer (EC) with ExoGAG, an easy to use and highly efficient new technology to enrich EVs. Technical performance was first evaluated using EVs secreted by Hec1A cells. Then, the clinical value of this strategy was questioned by analyzing the levels of two well-known tissue biomarkers in EC, L1 cell adhesion molecule (L1CAM) and Annexin A2 (ANXA2), in EVs purified from plasma in a cohort of 41 EC patients and 20 healthy controls. The results demonstrated the specific content of ANXA2 in the purified EVs fraction, with an accurate sensitivity and specificity for EC diagnosis. Importantly, high ANXA2 levels in circulating EVs were associated with high risk of recurrence and non-endometrioid histology suggesting a potential value as a prognostic biomarker in EC. These results also confirmed ExoGAG technology as a robust technique for the clinical implementation of circulating EVs analyses.