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.

Purpose: Resistance to tyrosine kinase inhibitors (TKI) of EGF receptor (EGFR) is often related to activation of other signaling pathways and evolution through a mesenchymal phenotype. Experimental Design: Because the Hedgehog (Hh) pathway has emerged as an important mediator of epithelial-to-mesenchymal transition (EMT), we studied the activation of Hh signaling in models of EGFR-TKIs intrinsic or acquired resistance from both EGFR-mutated and wild-type (WT) non–small cell lung cancer (NSCLC) cell lines. Results: Activation of the Hh pathway was found in both models of EGFR-mutated and EGFR-WT NSCLC cell line resistant to EGFR-TKIs. In EGFR-mutated HCC827-GR cells, we found SMO (the Hh receptor) gene amplification, MET activation, and the functional interaction of these two signaling pathways. In HCC827-GR cells, inhibition of SMO or downregulation of GLI1 (the most important Hh-induced transcription factor) expression in combination with MET inhibition exerted significant antitumor activity. In EGFR-WT NSCLC cell lines resistant to EGFR inhibitors, the combined inhibition of SMO and EGFR exerted a strong antiproliferative activity with a complete inhibition of PI3K/Akt and MAPK phosphorylation. In addition, the inhibition of SMO by the use of LDE225 sensitizes EGFR-WT NSCLC cells to standard chemotherapy. Conclusions:This result supports the role of the Hh pathway in mediating resistance to anti-EGFR-TKIs through the induction of EMT and suggests new opportunities to design new treatment strategies in lung cancer. Clin Cancer Res; 21(20); 4686–97. ©2015 AACR.

Aims To evaluate the accuracy, consumable cost and time around testing (TAT) of a next-generation sequencing (NGS) assay, the Ion Torrent AmpliSeq Colon and Lung Cancer Panel, as an alternative to Sanger sequencing to genotype KRAS, NRAS and BRAF in colorectal cancer patients. Methods The Ion Torrent panel was first verified on cell lines and on control samples and then prospectively applied to routine specimens (n=114), with Sanger sequencing as reference. Results The Ion Torrent panel detected mutant alleles at the 5% level on cell lines and correctly classified all control tissues. The Ion Torrent assay was successfully carried out on most (95.6%) routine diagnostic samples. Of these, 12 (11%) harboured mutations in the BRAF gene and 47 (43%) in either of the two RAS genes, in two cases with a low abundance of RAS mutant allele which was missed by Sanger sequencing. The mean TAT, from sample receipt to reporting, was 10.4 (Sanger) and 13.0 (Ion Torrent) working days. The consumable cost for genotyping KRAS, NRAS and BRAF was €196 (Sanger) and €187 (Ion Torrent). Conclusions Ion Torrent AmpliSeq Colon and Lung Cancer Panel sequencing is as robust as Sanger sequencing in routine diagnostics to select patients for anti-epidermal growth factor receptor (EGFR) therapy for metastatic colorectal cancer.

Aims: The incidence of RAS/RAF/PI3KA and TP53 gene mutations in colorectal cancer (CRC) is well established. Less information, however, is available on other components of the CRC genomic landscape, which are potential CRC prognostic/predictive markers. Methods: Following a previous validation study, ion-semiconductor next-generation sequencing (NGS) was employed to process 653 routine CRC samples by a multiplex PCR targeting 91 hotspot regions in 22 CRC significant genes. Results: A total of 796 somatic mutations in 499 (76.4%) tumours were detected. Besides RAS/RAF/PI3KA and TP53, other 12 genes showed at least one mutation including FBXW7 (6%), PTEN (2.8%), SMAD4 (2.1%), EGFR (1.2%), CTNNB1 (1.1%), AKT1 (0.9%), STK11 (0.8%), ERBB2 (0.6%), ERBB4 (0.6%), ALK (0.2%), MAP2K1 (0.2%) and NOTCH1 (0.2%). Conclusions: In a routine diagnostic setting, NGS had the potential to generate robust and comprehensive genetic information also including less frequently mutated genes potentially relevant for prognostic assessments or for actionable treatments.

Selection of lung cancer patients for therapy with tyrosine kinase inhibitors directed at EGFR requires the identification of specific EGFR mutations. In most patients with advanced, inoperable lung carcinoma limited tumor samples often represent the only material available for both histologic typing and molecular analysis. We defined a next generation sequencing protocol targeted to EGFR exons 18-21 suitable for the routine diagnosis of such clinical samples. The protocol was validated in an unselected series of 80 small biopsies (n=14) and cytology (n=66) specimens representative of the material ordinarily submitted for diagnostic evaluation to three referral medical centers in Italy. Specimens were systematically evaluated for tumor cell number and proportion relative to non-neoplastic cells. They were analyzed in batches of 100-150 amplicons per run, reaching an analytical sensitivity of 1% and obtaining an adequate number of reads, to cover all exons on all samples analyzed. Next generation sequencing was compared with Sanger sequencing. The latter identified 15 EGFR mutations in 14/80 cases (17.5%) but did not detected mutations when the proportion of neoplastic cells was below 40%. Next generation sequencing identified 31 EGFR mutations in 24/80 cases (30.0%). Mutations were detected with a proportion of neoplastic cells as low as 5%. All mutations identified by the Sanger method were confirmed. In 6 cases next generation sequencing identified exon 19 deletions or the L858R mutation not seen after Sanger sequencing, allowing the patient to be treated with tyrosine kinase inhibitors. In one additional case the R831H mutation associated with treatment resistance was identified in an EGFR wild type tumor after Sanger sequencing. Next generation sequencing is robust, cost-effective and greatly improves the detection of EGFR mutations. Its use should be promoted for the clinical diagnosis of mutations in specimens with unfavorable tumor cell content.