The discovery of oncogenic driver mutations rendering non-small cell lung cancer (NSCLC) targetable by small-molecule inhibitors, and the development of immunotherapies, have revolutionised NSCLC treatment. Today, instead of non-selective chemotherapies, all patients with advanced NSCLC eligible for treatment (and increasing numbers with earlier, less extensive disease) require fast and comprehensive screening of biomarkers for first-line patient selection for targeted therapy, chemotherapy, or immunotherapy (with or without chemotherapy). To avoid unnecessary re-biopsies, biomarker screening before first-line treatment should also include markers that are actionable from second-line onwards; PD-L1 expression testing is also mandatory before initiating treatment. Population differences exist in the frequency of oncogenic driver mutations: EGFR mutations are more frequent in Asia than Europe, whereas the converse is true for KRAS mutations. In addition to approved first-line therapies, a number of emerging therapies are being investigated in clinical trials. Guidelines for biomarker testing vary by country, with the number of actionable targets and the requirement for extensive molecular screening strategies expected to increase. To meet diagnostic demands, rapid screening technologies for single-driver mutations have been implemented. Improvements in DNA- and RNA-based next-generation sequencing technologies enable analysis of a group of genes in one assay; however, turnaround times remain relatively long. Consequently, rapid screening technologies are being implemented alongside next-generation sequencing. Further challenges in the evolving landscape of biomarker testing in NSCLC are actionable primary and secondary resistance mechanisms to targeted therapies. Therefore, comprehensive testing on re-biopsies, collected at the time of disease progression, in combination with testing of circulating tumour DNA may provide important information to guide second- or third-line therapies. Furthermore, longitudinal biomarker testing can provide insights into tumour evolution and heterogeneity during the course of the disease. We summarise best practice strategies for Europe in the changing landscape of biomarker testing at diagnosis and during treatment.

Introduction: The discovery of driver mutations in non-small cell lung cancer (NSCLC) has led to the development of genome-based personalized medicine. Fifteen to 20% of adenocarcinomas harbor an epidermal growth factor receptor (EGFR) activating mutation associated with responses to EGFR tyrosine kinase inhibitors (TKIs). Individual laboratories’ expertise and the availability of appropriate equipment are valuable assets in predictive molecular pathology, although the choice of methods should be determined by the nature of the samples to be tested and whether the detection of only well-characterized EGFR mutations or rather, of all detectable mutations, is required. Areas covered: The EGFR mutation testing landscape is manifold and includes both screening and targeted methods, each with their own pros and cons. Here we review one of these companion tests, the Roche cobas® EGFR mutation test v2, from a methodological point of view, also exploring its liquid-biopsy applications. Expert commentary: The Roche cobas® EGFR mutation test v2, based on real time RT-PCR, is a reliable option for testing EGFR mutations in clinical practice, either using tissue-derived DNA or plasma-derived cfDNA. This application will be valuable for laboratories with whose purpose is purely diagnostic and lacking high-throughput technologies.

Alterations in the tumor suppressor phosphatase and tensin homolog (PTEN) occur in a substantial proportion of solid tumors. These events drive tumorigenesis and tumor progression. Given its central role as a downregulator of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway, PTEN is deeply involved in cell growth, proliferation, and survival. This gene is also implicated in the modulation of the DNA damage response and in tumor immune microenvironment modeling. Despite the actionability of PTEN alterations, their role as biomarkers remains controversial in clinical practice. To date, there is still a substantial lack of validated guidelines and/or recommendations for PTEN testing. Here, we provide an update on the current state of knowledge on biologic and genetic alterations of PTEN across the most frequent solid tumors, as well as on their actual and/or possible clinical applications. We focus on possible tailored schemes for cancer patients’ clinical management, including risk assessment, diagnosis, prognostication, and treatment.

The CBX7 gene encodes a polycomb group protein that is known to be downregulated in many types of human cancers, although the role of this protein in carcinogenesis remains unclear. To shed light on this issue, we generated mice null for Cbx7. Mouse embryonic fibroblasts derived from these mice had a higher growth rate and reduced susceptibility to senescence compared with their WT counterparts. This was associated with upregulated expression of multiple cell cycle components, including cyclin E, which is known to play a key role in lung carcinogenesis in humans. Adult Cbx7-KO mice developed liver and lung adenomas and carcinomas. In in vivo and in vitro experiments, we demonstrated that CBX7 bound to the CCNE1 promoter in a complex that included HDAC2 and negatively regulated CCNE1 expression. Finally, we found that the lack of CBX7 protein expression in human lung carcinomas correlated with CCNE1 overexpression. These data suggest that CBX7 is a tumor suppressor and that its loss plays a key role in the pathogenesis of cancer.

Background: When tumour tissue is unavailable, cell-free DNA (cfDNA)can serve as a surrogate for genetic analyses. Because mutated alleles in cfDNA are usually below 1%, next-generation sequencing (NGS)must be narrowed to target only clinically relevant genes. In this proof-of-concept study, we developed a panel to use in ultra-deep sequencing to identify such mutations in cfDNA. Methods: Our panel (‘SiRe’) covers 568 mutations in six genes (EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα)involved in non-small-cell lung cancer (NSCLC), gastrointestinal stromal tumour, colorectal carcinoma and melanoma. We evaluated the panel performance in three steps. First, we analysed its analytical sensitivity on cell line DNA and by using an artificial reference standard with multiple mutations in different genes. Second, we analysed cfDNA from cancer patients at presentation (n=42), treatment response (n=12) and tumour progression (n=11); all patients had paired tumour tissue and cfDNA previously genotyped with a Taqman-derived assay (TDA). Third, we tested blood samples prospectively collected from NSCLC patients (n=79) to assess the performance of SiRe in clinical practice. Results: SiRe had a high analytical performance and a 0.01% lower limit of detection. In the retrospective series, SiRe detected 40 EGFR, 11 KRAS, 1 NRAS and 5 BRAF mutations (96.8% concordance with TDA). In the baseline samples, SiRe had 100% specificity and 79% sensitivity relative to tumour tissue. Finally, in the prospective series, SiRe detected 8.7% (4/46) of EGFR mutations at baseline and 42.9% (9/21) of EGFR p.T790M in patients at tumour progression. Conclusions: SiRe is a feasible NGS panel for cfDNA analysis in clinical practice.

Although appreciable attempts in screening and diagnostic approaches have been achieved, prostate cancer (PCa) remains a widespread malignancy, representing the second leading cause of cancer-related death in men. Drugs currently used in PCa therapy initially show a potent anti-tumor effect, but frequently induce resistance and PCa progresses toward metastatic castration-resistant forms (mCRPC), virtually incurable. Liquid biopsy has emerged as an attractive and promising strategy complementary to invasive tissue biopsy to guide PCa diagnosis and treatment. Liquid biopsy shows the ability to represent the tumor microenvironment, allow comprehensive information and follow-up the progression of the tumor, enabling the development of different treatment strategies as well as permitting the monitoring of therapy response. Liquid biopsy, indeed, is endowed with a significant potential to modify PCa management. Several blood biomarkers could be analyzed for diagnostic, prognostic and predictive purposes, including circulating tumor cells (CTCs), extracellular vesicles (EVs), circulating tumor DNA (ctDNA) and RNA (ctRNA). In addition, several other body fluids may be adopted (i.e., urine, sperm, etc.) beyond blood. This review dissects recent advancements and future perspectives of liquid biopsies, highlighting their strength and weaknesses in PCa management.