Identification of a Proteomic Signature for Predicting Immunotherapy Response in Patients With Metastatic Non-Small Cell Lung Cancer.

Immunotherapy has improved survival rates in patients with cancer, but identifying those who will respond to treatment remains a challenge. Advances in proteomic technologies have enabled the identification and quantification of nearly all expressed proteins in a single experiment. Integrating mass spectrometry with high-throughput technologies has facilitated comprehensive analysis of the plasma proteome in cancer, facilitating early diagnosis and personalized treatment. In this context, our study aimed to investigate the predictive and prognostic value of plasma proteome analysis using the SWATH-MS (Sequential Window Acquisition of All Theoretical Mass Spectra) strategy in newly diagnosed patients with non-small cell lung cancer (NSCLC) receiving pembrolizumab therapy. We enrolled 64 newly diagnosed patients with advanced NSCLC treated with pembrolizumab. Blood samples were collected from all patients before and during therapy. A total of 171 blood samples were analyzed using the SWATH-MS strategy. Plasma protein expression in metastatic NSCLC patients prior to receiving pembrolizumab was analyzed. A first cohort (discovery cohort) was employed to identify a proteomic signature predicting immunotherapy response. Thus, 324 differentially expressed proteins between responder and non-responder patients were identified. In addition, we developed a predictive model and found a combination of seven proteins, including ATG9A, DCDC2, HPS5, FIL1L, LZTL1, PGTA, and SPTN2, with stronger predictive value than PD-L1 expression alone. Additionally, survival analyses showed an association between the levels of ATG9A, DCDC2, SPTN2 and HPS5 with progression-free survival (PFS) and/or overall survival (OS). Our findings highlight the potential of proteomic technologies to detect predictive biomarkers in blood samples from NSCLC patients, emphasizing the correlation between immunotherapy response and the idenfied protein set.

Comparative analysis of capture methods for genomic profiling of circulating tumor cells in colorectal cancer.

The genomic profiling of circulating tumor cells (CTCs) in the bloodstream should provide clinically relevant information on therapeutic efficacy and help predict cancer survival. Here, we contrasted the genomic profiles of CTC pools recovered from metastatic colorectal cancer (mCRC) patients using different enrichment strategies (CellSearch, Parsortix, and FACS). Mutations inferred in the CTC pools differed depending on the enrichment strategy and, in all cases, represented a subset of the mutations detected in the matched primary tumor samples. However, the CTC pools from Parsortix, and in part, CellSearch, showed diversity estimates, mutational signatures, and drug-suitability scores remarkably close to those found in matching primary tumor samples. In addition, FACS CTC pools were enriched in apparent sequencing artifacts, leading to much higher genomic diversity estimates. Our results highlight the utility of CTCs to assess the genomic heterogeneity of individual tumors and help clinicians prioritize drugs in mCRC.

Methods for the Detection of Circulating Biomarkers in Cancer Patients.

Liquid biopsy has emerged as one of the main pillars for personalized oncology. The term englobes body-fluid samples which contain tumor-derived material such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and circulating extracellular vesicles (cEVs). Potential clinical application of liquid biopsy analyses includes cancer screening, detection of minimal residual disease and recurrence, therapy selection, and evaluation of acquired resistance. Despite the great developments of technology focused on circulating biomarkers characterization only cfDNA testing is nowadays implemented for the therapy selection in some advanced tumors. This can be partially explained by the fact that there is still a lack of global standardization of procedures both in the pre-analytical and analytical steps. In the present chapter, we summarize the different strategies for addressing the study of liquid biopsy taking into account their pros and cons to be applied in a clinical context and we also discuss the main technical and clinical challenges in the field of circulating biomarkers and personalized oncology.

Immunotherapy in nonsmall-cell lung cancer: current status and future prospects for liquid biopsy.

Immunotherapy has been one of the great advances in the recent years for the treatment of advanced tumors, with nonsmall-cell lung cancer (NSCLC) being one of the cancers that has benefited most from this approach. Currently, the only validated companion diagnostic test for first-line immunotherapy in metastatic NSCLC patients is testing for programmed death ligand 1 (PD-L1) expression in tumor tissues. However, not all patients experience an effective response with the established selection criteria and immune checkpoint inhibitors (ICIs). Liquid biopsy offers a noninvasive opportunity to monitor disease in patients with cancer and identify those who would benefit the most from immunotherapy. This review focuses on the use of liquid biopsy in immunotherapy treatment of NSCLC patients. Circulating tumor cells (CTCs), cell-free DNA (cfDNA) and exosomes are promising tools for developing new biomarkers. We discuss the current application and future implementation of these parameters to improve therapeutic decision-making and identify the patients who will benefit most from immunotherapy.

Plasma cell-free DNA and circulating tumor cells as prognostic biomarkers in small cell lung cancer patients.

Background: Lack of biomarkers for treatment selection and monitoring in small cell lung cancer (SCLC) patients with the limited therapeutic options, result in poor outcomes. Therefore, new prognostic biomarkers are needed to improve their management. The prognostic value of cell-free DNA (cfDNA) and circulating tumor cells (CTCs) have been less explored in SCLC. Methods: We quantified cfDNA in 46 SCLC patients at different times during first-line of chemotherapy or chemo-immunotherapy. Moreover, CTCs were analyzed in 21 patients before therapy onset using CellSearch® system. The possible association between both biomarkers and patients' outcomes was investigated in order to develop a prognostic model. Results: High cfDNA levels before therapy were associated with shorter progression-free survival (PFS) and overall survival (OS). Furthermore, cfDNA levels at 3 weeks and at progression disease were also associated with patients' outcomes. Multivariate analyses confirmed the independence of cfDNA levels as a prognostic biomarker. Finally, the three-risk category prognostic model developed included Eastern Cooperative Oncology Group Performance Status (ECOG PS), gender and baseline cfDNA levels was associated with a higher risk of progression and death. Conclusions: We confirmed the prognostic utility of cfDNA quantitative analysis in SCLC patients before and during therapy. Our novel risk prognostic model in clinical practice will allow to identify patients who could benefit with actual therapies.

Circulating Free DNA and Its Emerging Role in Autoimmune Diseases.

Liquid biopsies can be used to analyse tissue-derived information, including cell-free DNA (cfDNA), circulating rare cells, and circulating extracellular vesicles in the blood or other bodily fluids, representing a new way to guide therapeutic decisions in cancer. Among the new challenges of liquid biopsy, we found clinical application in nontumour pathologies, including autoimmune diseases. Since the discovery of the presence of high levels of cfDNA in patients with systemic lupus erythaematosus (SLE) in the 1960s, cfDNA research in autoimmune diseases has mainly focused on the overall quantification of cfDNA and its association with disease activity. However, with technological advancements and the increasing understanding of the role of DNA sensing receptors in inflammation and autoimmunity, interest in cfDNA and autoimmune diseases has not expanded until recently. In this review, we provide an overview of the basic biology of cfDNA in the context of autoimmune diseases as a biomarker of disease activity, progression, and prediction of the treatment response. We discuss and integrate available information about these important aspects.

Current Status and Future Perspectives of Liquid Biopsy in Small Cell Lung Cancer.

Approximately 19% of all cancer-related deaths are due to lung cancer, which is the leading cause of mortality worldwide. Small cell lung cancer (SCLC) affects approximately 15% of patients diagnosed with lung cancer. SCLC is characterized by aggressiveness; the majority of SCLC patients present with metastatic disease, and less than 5% of patients are alive at 5 years. The gold standard of SCLC treatment is platinum and etoposide-based chemotherapy; however, its effects are short. In recent years, treatment for SCLC has changed; new drugs have been approved, and new biomarkers are needed for treatment selection. Liquid biopsy is a non-invasive, rapid, repeated and alternative tool to the traditional tumor biopsy that could allow the most personalized medicine into the management of SCLC patients. Circulating tumor cells (CTCs) and cell-free DNA (cfDNA) are the most commonly used liquid biopsy biomarkers. Some studies have reported the prognostic factors of CTCs and cfDNA in SCLC patients, independent of the stage. In this review, we summarize the recent SCLC studies of CTCs, cfDNA and other liquid biopsy biomarkers, and we discuss the future utility of liquid biopsy in the clinical management of SCLC.

Clinical potential of circulating free DNA and circulating tumour cells in patients with metastatic non-small-cell lung cancer treated with pembrolizumab.

Immune checkpoint inhibitors, such as pembrolizumab, are revolutionizing therapeutic strategies for different cancer types, including non-small-cell lung cancer (NSCLC). However, only a subset of patients benefits from this therapy, and new biomarkers are needed to select better candidates. In this study, we explored the value of liquid biopsy analyses, including circulating free DNA (cfDNA) and circulating tumour cells (CTCs), as a prognostic or predictive tool to guide pembrolizumab therapy. For this purpose, a total of 109 blood samples were collected from 50 patients with advanced NSCLC prior to treatment onset and at 6 and 12 weeks after the initiation of pembrolizumab. Plasma cfDNA was measured using hTERT quantitative PCR assay. The CTC levels at baseline were also analysed using two enrichment technologies (CellSearch® and Parsortix systems) to evaluate the efficacy of both approaches at detecting the presence of programmed cell death ligand 1 on CTCs. Notably, patients with high baseline hTERT cfDNA levels had significantly shorter progression-free survival (PFS) and overall survival (OS) than those with low baseline levels. Moreover, patients with unfavourable changes in the hTERT cfDNA levels from baseline to 12 weeks showed a higher risk of disease progression. Additionally, patients in whom CTCs were detected using the CellSearch® system had significantly shorter PFS and OS than patients who had no CTCs. Finally, multivariate regression analyses confirmed the value of the combination of CTCs and cfDNA levels as an early independent predictor of disease progression, identifying a subgroup of patients who were negative for CTCs, who presented low levels of cfDNA and who particularly benefited from the treatment.

Analysis of Killer Immunoglobulin-Like Receptor Genes in Colorectal Cancer.

Natural killer cells (NK cells) play a major role in the immune response to cancer. An important element of NK target recognition is the binding of human leucocyte antigen (HLA) class I molecules by killer immunoglobulin-like receptors (KIRs). Colorectal carcinoma (CRC) is one of the most common types of inflammation-based cancer. The purpose of the present study was to investigate the presence of KIR genes and HLA class I and II alleles in 1074 CRC patients and 1272 controls. We imputed data from single-nucleotide polymorphism (SNP) Illumina OncoArray to identify associations at HLA (HLA-A, B, C, DPB1, DQA1, DQB1, and DRB1) and KIRs (HIBAG and KIR*IMP, respectively). For association analysis, we used PLINK (v1.9), the PyHLA software, and R version 3.4.0. Only three SNP markers showed suggestive associations (p < 10-3; rs16896742, rs28367832, and rs9277952). The frequency of KIR2DS3 was significantly increased in the CRC patients compared to healthy controls (p < 0.005). Our results suggest that the implication of NK cells in CRC may not act through allele combinations in KIR and HLA genes. Much larger studies in ethnically homogeneous populations are needed to rule out the possible role of allelic combinations in KIR and HLA genes in CRC risk.

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 TIMP1.

Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients.

MET alterations may provide a potential biomarker to evaluate patients who will benefit from treatment with MET inhibitors. Therefore, the purpose of the present study is to investigate the utility of a liquid biopsy-based strategy to assess MET alterations in cancer patients. We analyzed MET amplification in circulating free DNA (cfDNA) from 174 patients with cancer and 49 healthy controls and demonstrated the accuracy of the analysis to detect its alteration in patients. Importantly, a significant correlation between cfDNA concentration and MET copy number (CN) in cancer patients (r = 0.57, p <10-10) was determined. Furthermore, we evaluated two approaches to detect the presence of MET on circulating tumor cells (CTCs), using the CellSearch® and Parsortix systems and monitored patients under anti-EGFR treatment (n = 30) combining both cfDNA and CTCs analyses. This follow-up provides evidence for the potential of MET CN assessment when patients develop resistance to anti-EGFR therapy and a significant association between the presence of CTCs MET+ and the Overall Survival (OS) in head and neck cancer patients (P = 0.05; HR = 6.66). In conclusion, we develop specific and noninvasive assays to monitor MET status in cfDNA/CTCs and demonstrate the utility of plasma MET CN determination as a biomarker for monitoring the appearance of resistance to anti-EGFR therapy.