The effective and efficient management of cancer patients relies upon early diagnosis and/or the monitoring of treatment, something that is often difficult to achieve using standard tissue biopsy techniques. Biological fluids such as blood hold great possibilities as a source of non-invasive cancer biomarkers that can act as surrogate markers to biopsy-based sampling. The non-invasive nature of these “liquid biopsies” ultimately means that cancer detection may be earlier and that the ability to monitor disease progression and/or treatment response represents a paradigm shift in the treatment of cancer patients. Below, we review one of the most promising classes of circulating cancer biomarkers: microRNAs (miRNAs). In particular, we will consider their history, the controversy surrounding their origin and biology, and, most importantly, the hurdles that remain to be overcome if they are really to become part of future clinical practice.

The gold standard for cancer diagnosis remains the histological examination of affected tissue, obtained either by surgical excision, or radiologically guided biopsy. Such procedures however are expensive, not without risk to the patient, and require consistent evaluation by expert pathologists. Consequently, the search for non‐invasive tools for the diagnosis and management of cancer has led to great interest in the field of circulating nucleic acids in plasma and serum. An additional benefit of blood‐based testing is the ability to carry out screening and repeat sampling on patients undergoing therapy, or monitoring disease progression allowing for the development of a personalized approach to cancer patient management. Despite having been discovered over 60 years ago, the clear clinical potential of circulating nucleic acids, with the notable exception of prenatal diagnostic testing, has yet to translate into the clinic. The recent discovery of non‐coding (nc)RNA(in particular micro(mi)RNAs) in the blood has provided fresh impetuous for the field. In this review, we discuss the potential of thecirculating transcriptome(coding and ncRNA), as novel cancer biomarkers, the controversy surrounding their origin and biology, and most importantly the hurdles that remain to be overcome if they arereallyto become part of future clinical practice.

Bortezomib (PS-341; Velcade) is a reversible inhibitor of the 26S proteasome that produces significant clinical responses in both newly diagnosed and advanced multiple myeloma (MM). However, only 40% of patients respond to bortezomib as a single-agent therapy, and most of those will become resistant. Consequently, understanding the mechanism of action of bortezomib is of great interest. Although the inhibitory activity of bortezomib is clearly defined, the downstream mechanisms of cytotoxity remain poorly understood and at times controversial.1 There is now emerging evidence that microRNAs (miRNAs) are important factors in anticancer drug activity and resistance.2 We therefore decided to investigate the potential involvement of miRNAs in the action of bortezomib on MM cells. Three well-characterised MM cell lines (RPMI-8226, JJN3 and Thiel) were treated with bortezomib for 72 h (10 nM, LC laboratories, Woburn, MA, USA). Compared with diluent-only controls (0.008% dimethyl sulfoxide), levels of apoptosis (Cell Death Detection ELISAplus kit; Roche Diagnostics, Lewes, UK) in treated cell lines showed on average (n=3) an increase of 12.5- (JJN-3), 5.2- (Thiel) and 3.8-fold (RPMI-8226). Furthermore, cell proliferation (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega, WN, USA) levels were decreased 10.5- (JJN-3), 33.3- (Thiel) and 8.4-fold (RPMI-8226) (data not shown). Total RNA was extracted by Trizol (Invitrogen, Paisley, UK) from biological triplicate experiments and the RNA used for whole transcriptome (Affymetrix U133plus2.0) and miRnome (Affymetrix Genechip miRNA v.1.0) microarray analyses (Affymetix, Santa Clara, CA, USA). Differences in cell line identity followed by bortezomib treatment were discriminatory features for both mRNA and miRNA expression levels as demonstrated by unsupervised cluster analyses (Supplementary Figure S1). Each treated sample was normalised against its’ respective paired non-treated sample, and probes (mRNA or miRNA) filtered for a median±fold-change of >2 between triplicates. This resulted in 1516 and 4136 genes that were up- and downregulated, respectively, in JJN3 cells in response to bortezomib treatment; 824 and 1864 genes in RPMI-8226 cells; and 1292 and 5183 genes in Thiel cells (Supplementary Figure S2). There were 228 and 883 genes commonly upregulated and downregulated respectively in all three MM cell lines in response to treatment. Ontogeny analysis revealed that members of the protein ubiquitination canonical pathway were significantly enriched in the genes that were differentially expressed in response to bortezomib in each of the three MM cell lines individually, as well as for those genes that were commonly deregulated in all three lines (Supplementary Table S1). Interestingly, the most significant canonical pathway of differentially expressed genes in two of the cell lines (Thiel and JJN3) was the ‘role of BRCA1 in DNA damage response’; recently it has been suggested that bortezomib induces ‘BRCAness’ in MM cells.3 Many of the most deregulated genes that were common to all three cell lines (Supplementary Tables S2 and S3) had previously been associated with bortezomib treatment including upregulation of heat-shock proteins (HSP6, HSPA1), AP-1 complex (Jun), cell stress markers (ATF3),4 redox haemostasis genes (HMOX1, DDIT3),5 and the antiapoptotic protein BAG3,6 and downregulation of survivin (BIRC5), topoisomerase and MYB-oncogene.7 As with the gene expression data the miRNA microarray data were normalised between paired samples. This resulted in 37 and 75 miRNAs that were up- and downregulated, respectively, in JJN3 cells (median fold-change >2); 23 and 26 miRNAs in RPMI-8226 cells; and 30 and 98 miRNAs in Thiel cells (Supplementary Figure S3). Nine and 14 miRNAs were commonly upregulated and downregulated, respectively, in all three MM cell lines (Supplementary Figure S3 and Supplementary Table S4). Four of these miRNAs (miR-92a-5p, let-7f, miR-27a-5p and miR-188 (Xp11.23)) were validated by qualitative reverse transcriptase-PCR (Supplementary Figure S4). Interestingly, the most downregulated miRNA was mir-92a-5p, which along with miR-18a (also down-regulated), are encoded by the miR-17~92 cluster whose overexpression has been linked to tumorigenesis and poor prognosis in MM.8 Let-7f has been demonstrated to promote angiogenesis by targeting antiangiogenic genes.9 Administration of anti-Let-7f was found to increase apoptosis and decrease cell proliferation levels in MM cell lines and to significantly reduce size of tumours in an MM xenotransplant model.10 Four of the fourteen (29%) commonly downregulated miRNAs are encoded within a single cluster (Xp11.23) that contains a further four members, also downregulated in response to treatment (data not shown). The Xp11.23 cluster is encoded within intron 3 of the CLCN5 gene. Intriguingly, mutations in CLCN5 (including mutations involving intron 3) are frequently found in patients with Dent’s disease, a rare renal tubular disorder characterised by progressive renal failure, that share many clinical manifestations with MM including presentation of Fanconi syndrome.11 Recently cyclin-depedent kinase 5 (CDK5) was identified as being a major modulator of bortezomib sensitivity in both MM cell lines and patient tumour cells.12 We noted that miR-27a-5p, one of the miRNAs identified as being downregulated by all three MM cell lines in response to bortezomib treatment (Supplementary Table S4 and Supplementary Figure S4), was predicted to target CDK5 (Figure 1a). This putative interaction was tested by cloning the 3′-untranslated region sequence of CDK5 into a luciferase reporter vector. Transfection of miR-27a-5p significantly reduced luciferase output compared with a scrambled sequence (Figure 1b). Furthermore, we found that endogenous levels of CDK5 mRNA decreased significantly in MM cells transfected with miR-27a-5p (Figure 1c),...

Early diagnosis of laryngeal squamous cell carcinoma (LSCC) at the stage of dysplasia could greatly improve the outcome of affected patients. For the first time we compared the mutational landscape of non-progressing dysplasia (NPD; n = 42) with progressing dysplasia (PD; n = 24), along with patient-matched LSCC biopsies; a total of 90 samples. Using targeted next-generation sequencing identified non-synonymous mutations in six genes (PIK3CA, FGFR3, TP53, JAK3, MET, FBXW7), and mutations were validated by Sanger sequencing and/or qPCR. Analysis was extended in silico to 530 head and neck (HNSCC) cases using TCGA data. Mutations in PIK3CA and FGFR3 were detected in PD and LSCC cases, as well as other HNSCC cases, but absent in NPD cases. In contrast, mutations in JAK3, MET and FBXW7 were found in NPD cases but not PD, LSCC or other HNSCC cases. TP53 was the most frequently mutated gene in both PD and NPD cases. With the exception of R248W, mutations were mutually exclusive. Moreover, five of seven PD mutations were located in motif H2 of p53, whereas none of the NPD mutations were. In summary, we propose that the mutational profile of laryngeal dysplasia has utility for the early detection of patients at risk of progression.