Early alterations in cancer include the deregulation of epigenetic events such as changes in DNA methylation and abnormal levels of non-coding (nc)RNAs. Although these changes can be identified in tumors, alternative sources of samples may offer advantages over tissue biopsies. Because tumors shed DNA, RNA, and proteins, biological fluids containing these molecules can accurately reflect alterations found in cancer cells, not only coming from the primary tumor, but also from metastasis and from the tumor microenvironment (TME). Depending on the type of cancer, biological fluids encompass blood, urine, cerebrospinal fluid, and saliva, among others. Such samples are named with the general term “liquid biopsy” (LB). With the advent of ultrasensitive technologies during the last decade, the identification of actionable genetic alterations (i.e., mutations) in LB is a common practice to decide whether or not targeted therapy should be applied. Likewise, the analysis of global or specific epigenetic alterations may also be important as biomarkers for diagnosis, prognosis, and even for cancer drug response. Several commercial kits that assess the DNA promoter methylation of single genes or gene sets are available, with some of them being tested as biomarkers for diagnosis in clinical trials. From the tumors with highest incidence, we can stress the relevance of DNA methylation changes in the following genes found in LB: SHOX2 (for lung cancer); RASSF1A, RARB2, and GSTP1 (for lung, breast, genitourinary and colon cancers); and SEPT9 (for colon cancer). Moreover, multi-cancer high-throughput methylation-based tests are now commercially available. Increased levels of the microRNA miR21 and several miRNA- and long ncRNA-signatures can also be indicative biomarkers in LB. Therefore, epigenetic biomarkers are attractive and may have a clinical value in cancer. Nonetheless, validation, standardization, and demonstration of an added value over the common clinical practice are issues needed to be addressed in the transfer of this knowledge from “bench to bedside”.

Breast cancer constitutes the most common malignant neoplasm in women around the world. Approximately 12% of patients are diagnosed with metastatic stage, and between 5 and 30% of early or locally advanced BC patients will relapse, making it an incurable disease. PD-L1 ligation is an immune inhibitory molecule of the activation of T cells, playing a relevant role in numerous types of malignant tumors, including BC. The objective of the present review is to analyze the role of PD-L1 as a biomarker in the different BC subtypes, adding clinical trials with immune checkpoint inhibitors and their applicable results. Diverse trials using immunotherapy with anti-PD-1/PD-L1 in BC, as well as prospective or retrospective cohort studies about PD-L1 in BC, were included. Despite divergent results in the reviewed studies, PD-L1 seems to be correlated with worse prognosis in the hormone receptor positive subtype. Immune checkpoints inhibitors targeting the PD-1/PD-L1 axis have achieved great response rates in TNBC patients, especially in combination with chemotherapy, making immunotherapy a new treatment option in this scenario. However, the utility of PD-L1 as a predictive biomarker in the rest of BC subtypes remains unclear. In addition, predictive differences have been found in response to immunotherapy depending on the stage of the tumor disease. Therefore, a better understanding of tumor microenvironment, as well as identifying new potential biomarkers or combined index scores, is necessary in order to make a better selection of the subgroups of BC patients who will derive benefit from immune checkpoint inhibitors.

Introduction: Among non-small cell lung cancer (NSCLC) patients, there is one molecularly defined subgroup harboring activating mutations in the epidermal growth factor receptor gene (EGFR), which results in constitutive activation of its intrinsic kinase activity. Consistent data have demonstrated that these patients have a better outcome when treated with specific tyrosine-kinase inhibitors (EGFR-TKIs). Therefore, analysis of EGFR mutational status for treatment guidance is mandatory in this context. Areas covered: Herein we review the clinical development and technical features of cobas® EGFR Mutation Test v2 as a companion diagnostic test (CDx) for therapy with EGFR-TKIs, such as gefitinib, in advanced NSCLC. We also discuss the pros and cons of the current version of the CDx and its performance in both tissue and plasma samples. Expert opinion: The RT-PCR based cobas® EGFR Mutation Test v2 is a reliable and rapid solution for EGFR mutational status assessment at the time of diagnosis in advanced NSCLC that allows eligibility of patients for EGFR-TKI treatment. This test determines EGFR mutations with acceptable sensitivity in tissue or plasma samples. Pre-analytical considerations like tumor cell content, tumor burden or location of metastasis should be considered to better interpret results in the clinical contexture.

Lung cancer is a malignant disease with high mortality and poor prognosis, frequently diagnosed at advanced stages. Nowadays, immense progress in treatment has been achieved. However, the present scenario continues to be critical, and a full comprehension of tumor progression mechanisms is required, with exosomes being potentially relevant players. Exosomes are membranous vesicles that contain biological information, which can be transported cell-to-cell and modulate relevant processes in the hallmarks of cancer. The present research aims to characterize the exosomes’ cargo and study their role in NSCLC to identify biomarkers. We analyzed exosomes secreted by primary cultures and cell lines, grown in monolayer and tumorsphere formations. Exosomal DNA content showed molecular alterations, whereas RNA high-throughput analysis resulted in a pattern of differentially expressed genes depending on histology. The most significant differences were found in XAGE1B, CABYR, NKX2-1, SEPP1, CAPRIN1, and RIOK3 genes when samples from two independent cohorts of resected NSCLC patients were analyzed. We identified and validated biomarkers for adenocarcinoma and squamous cell carcinoma. Our results could represent a relevant contribution concerning exosomes in clinical practice, allowing for the identification of biomarkers that provide information regarding tumor features, prognosis and clinical behavior of the disease.

Despite the success of therapies in lung cancer, more studies of new biomarkers for patient selection are urgently needed. The present study aims to analyze the role of galectin‐3 (GAL‐3) in the lung tumor microenvironment (TME) using tumorspheres as a model and explore its potential role as a predictive and prognostic biomarker in non‐small cell lung cancer patients. For in vitro studies, lung adenocarcinoma (LUAD) and lung squamous carcinoma (LUSC) primary cultures from early‐stage patients and commercial cell lines were cultured, using tumorsphere‐forming assays and adherent conditions for the control counterparts. We analyzed the pattern of secretion and expression of GAL‐3 using reverse transcription–quantitative real‐time PCR (RTqPCR), immunoblot, immunofluorescence, flow cytometry, and immunoassay analysis. Our results using three‐dimensional (3D) models of lung tumor cells revealed that soluble GAL‐3 (sGAL‐3) is highly expressed and secreted. To more accurately mimic the TME, a co‐culture of tumorspheres and fibroblasts was used, revealing that GAL‐3 could be important as an immunomodulatory molecule expressed and secreted in the TME, modulating immunosuppression through regulatory T cells (T_REGS). In the translational phase, we confirmed that patients with high expression levels of GAL‐3 had more T_REGS, which suggests that tumors may be recruiting this population through GAL‐3. Next, we evaluated levels of sGAL‐3 before surgery in LUAD and LUSC patients, hypothesizing that sGAL‐3 could be used as an independent prognostic biomarker for overall survival and relapse‐free survival in early‐stage LUAD patients. Additionally, levels of sGAL‐3 at pretreatment and first response assessment from plasma to predict clinical outcomes in advanced LUAD and LUSC patients treated with first‐line pembrolizumab were evaluated, further supporting that sGAL‐3 has a high efficiency in predicting durable clinical response to pembrolizumab with an area under curve of 0.801 (P = 0.011). Moreover, high levels might predict decreased progression‐free survival and OS to anti‐PD‐1 therapy, with sGAL‐3 being a prognosis‐independent biomarker for advanced LUAD.

Immunotherapy has been proven a viable treatment option for non-small cell lung cancer (NSCLC) treatment in patients. However, some patients still do not benefit. Finding new predictive biomarkers for immunocheckpoint inhibitor (ICI) response will improve treatment management in the clinical routine. In this regard, liquid biopsy is a useful and noninvasive alternative to surgical biopsies. In the present study, we evaluated the potential diagnostic, prognostic, and predictive value of seven different soluble mediators involved in immunoregulation. Fifty-two plasma samples from advanced NSCLC treated in first-line with pembrolizumab at baseline (PRE) and at first response assessment (FR) were analyzed. In terms of diagnostic value, our results revealed that sFGL1, sGAL-3, and sGAL-1 allowed for optimal diagnostic efficacy for cancer patients. Additionally, the combination of sFGL1 and sGAL-3 significantly improved diagnostic accuracy. Regarding the predictive value to assess patients’ immune response, sCD276 levels at PRE were significantly higher in patients without tumor response (p = 0.035). Moreover, we observed that high levels of sMICB at PRE were associated with absence of clinical benefit (pembrolizumab treatment less than 6 months) (p = 0.049), and high levels of sMICB and sGAL-3 at FR are also related to a lack of clinical benefit (p = 0.027 and p = 0.03, respectively). Finally, in relation to prognosis significance, at PRE and FR, sMICB levels above the 75th percentile are related to poor progression-free survival (PFS) (p = 0.013 and p = 0.023, respectively) and overall survival (OS) (p = 0.001 and p = 0.011, respectively). An increase in sGAL3 levels at FR was associated with worse PFS (p = 0.037). Interestingly, high sGAL-3 at PRE was independently associated with PFS and OS with a hazard ratio (HR) of 2.45 (95% CI 1.14–5.25; p = 0.021) and 4.915 (95% CI 1.89–12.73; p = 0.001). In conclusion, plasma levels of sFGL1, sGAL-3, and sGAL-1 could serve as diagnostic indicators and sMICB, sCD276, and sGAL3 were linked to outcomes, suggesting their potential in assessing NSCLC under pembrolizumab treatment. Our results highlight the value of employing soluble immune biomarkers in advanced lung cancer patients treated with pembrolizumab at first-line.