Identification of Recurrent Mutations in the microRNA-Binding Sites of B-Cell Lymphoma-Associated Genes in Follicular Lymphoma.

Follicular lymphoma (FL) is a common indolent B-cell lymphoma that can transform into the more aggressive transformed FL (tFL). However, the molecular process driving this transformation is uncertain. In this work, we aimed to identify microRNA (miRNA)-binding sites recurrently mutated in follicular lymphoma patients, as well as in transformed FL patients. Using whole-genome sequencing data from FL tumors, we discovered 544 mutations located in bioinformatically predicted microRNA-binding sites. We then studied these specific regions using targeted sequencing in a cohort of 55 FL patients, found 16 recurrent mutations, and identified a further 69 variants. After filtering for QC, we identified 21 genes with mutated miRNA-binding sites that were also enriched for B-cell-associated genes by Gene Ontology. Over 40% of mutations identified in these genes were present exclusively in tFL patients. We validated the predicted miRNA-binding sites of five of the genes by luciferase assay and demonstrated that the identified mutations in BCL2 and EZH2 genes impaired the binding efficiency of miR-5008 and miR-144 and regulated the endogenous levels of messenger RNA (mRNA).

Mutational profiling can identify laryngeal dysplasia at risk of progression to invasive carcinoma.

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.

Noncoding RNA Expression and Targeted Next-Generation Sequencing Distinguish Tubulocystic Renal Cell Carcinoma (TC-RCC) from Other Renal Neoplasms.

Tubulocystic renal cell carcinoma (TC-RCC) is a rare recently described renal neoplasm characterized by gross, microscopic, and immunohistochemical differences from other renal tumor types and was recently classified as a distinct entity. However, this distinction remains controversial particularly because some genetic studies suggest a close relationship with papillary RCC (PRCC). The molecular basis of this disease remains largely unexplored. We therefore performed noncoding (nc) RNA/miRNA expression analysis and targeted next-generation sequencing mutational profiling on 13 TC-RCC cases (11 pure, two mixed TC-RCC/PRCC) and compared with other renal neoplasms. The expression profile of miRNAs and other ncRNAs in TC-RCC was distinct and validated 10 differentially expressed miRNAs by quantitative RT-PCR, including miR-155 and miR-34a, that were significantly down-regulated compared with PRCC cases (n = 22). With the use of targeted next-generation sequencing we identified mutations in 14 different genes, most frequently (>60% of TC-RCC cases) in ABL1 and PDFGRA genes. These mutations were present in <5% of clear cell RCC, PRCC, or chromophobe RCC cases (n > 600) of The Cancer Genome Atlas database. In summary, this study is by far the largest molecular study of TC-RCC cases and the first to investigate either ncRNA expression or their genomic profile. These results add molecular evidence that TC-RCC is indeed a distinct entity from PRCC and other renal neoplasms.

miRNAs in B-cell lymphoma: Molecular mechanisms and biomarker potential.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate many human genes including those involved in normal B-cell development. When these miRNAs are aberrantly expressed in B-cells they play key pathogenetic roles in the development and maintenance of B-cell lymphomas and by association may serve as useful biomarkers. In this review, we provide an overview of the importance of miRNAs to B-cell lymphomagenesis, as well as considering their use as biomarkers, and their potential usefulness for the clinic.

Spatial intratumoural heterogeneity in the expression of GIT1 is associated with poor prognostic outcome in oestrogen receptor positive breast cancer patients with synchronous lymph node metastases.

Background: The outcome for oestrogen receptor positive (ER+) breast cancer patients has improved greatly in recent years largely due to targeted therapy. However, the presence of involved multiple synchronous lymph nodes remains associated with a poor outcome. Consequently, these patients would benefit from the identification of new prognostic biomarkers and therapeutic targets. The expression of G-protein-coupled receptor kinase-interacting protein 1 (GIT1) has recently been shown to be an indicator of advanced stage breast cancer. Therefore, we investigated its expression and prognostic value of GIT1 in a cohort of 140 ER+ breast cancer with synchronous lymph node involvement. Methods: Immunohistochemistry was employed to assess GIT1 expression in a tissue microarray (TMA) containing duplicate non-adjacent cores with matched primary tumour and lymph node tissue (n=140). GIT1 expression in tumour cells was scored and statistical correlation analyses were carried out. Results: The results revealed a sub-group of patients that displayed discordant expression of GIT1 between the primary tumour and the lymph nodes (i.e. spatial intratumoural heterogeneity). We observed that loss of GIT1 expression in the metastasis was associated with a shorter time to recurrence, poorer overall survival, and a shorter median survival time. Moreover, multivariate analysis demonstrated that GIT1 expression was an independent prognostic indicator. Conclusions: GIT1 expression enabled the identification of a sub-class of ER+ patients with lymph node metastasis that have a particularly poor prognostic outcome. We propose that this biomarker could be used to further stratify ER+ breast cancer patients with synchronous lymph node involvement and therefore facilitate adjuvant therapy decision making.

Aberrant expression of MicroRNAs in B-cell lymphomas.

B-cell lymphomas represent a heterogeneous collection of more than twenty-five different malignancies. Classification is often challenging as primarily based upon, sometimes subjective, histopathological criteria and misdiagnosis can result in inappropriate treatment decisions. MicroRNAs (miRNAs) hold great promise as novel biomarkers (diagnostic, prognostic and predictive) of B-cell lymphoma in addition to being potential therapeutic targets. The most promising of these miRNAs more often than not play key regulatory roles in lymphopoiesis (development of lymphocytes) when under physiological conditions, and in the pathology of lymphoid malignancies when aberrantly expressed. In this review we consider the identity and functional role of miRNAs in the most common forms of B-cell lymphomas, their role in lymphopoiesis and their potential as biomarkers for these malignancies.

New Concepts in Cancer Biomarkers: Circulating miRNAs in Liquid Biopsies.

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 circulating transcriptome as a source of non-invasive cancer biomarkers: concepts and controversies of non-coding and coding RNA in body fluids.

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 the circulating 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 are really to become part of future clinical practice.

Targeted next-generation sequencing and non-coding RNA expression analysis of clear cell papillary renal cell carcinoma suggests distinct pathological mechanisms from other renal tumour subtypes.

Clear cell tubulopapillary renal cell carcinoma (CCPRCC) is a recently described rare renal malignancy that displays characteristic gross, microscopic and immunohistochemical differences from other renal tumour types. However, CCPRCC remains a very poorly understood entity. We therefore sought to elucidate some of the molecular mechanisms involved in this neoplasm by carrying out targeted next-generation sequencing (NGS) to identify associated mutations, and in addition examined the expression of non-coding (nc) RNAs. We identified multiple somatic mutations in CCPRCC cases, including a recurrent [3/14 cases (21%)] non-synonymous T992I mutation in the MET proto-oncogene, a gene associated with epithelial-to-mesenchymal transition (EMT). Using a microarray approach, we found that the expression of mature (n = 1105) and pre-miRNAs (n = 1105), as well as snoRNA and scaRNAs (n = 2214), in CCPRCC cases differed from that of clear cell renal cell carcinoma (CCRCC) or papillary renal cell carcinoma (PRCC) tumours. Surprisingly, and unlike other renal tumour subtypes, we found that all five members of the miR-200 family were over-expressed in CCPRCC cases. As these miRNAs are intimately involved with EMT, we stained CCPRCC cases for E-cadherin, vimentin and ß-catenin and found that the tumour cells of all cases were positive for all three markers, a combination rarely reported in other renal tumours that could have diagnostic implications. Taken together with the mutational analysis, these data suggest that EMT in CCPRCC tumour cells is incomplete or blocked, consistent with the indolent clinical course typical of this malignancy. In summary, as well as describing a novel pathological mechanism in renal carcinomas, this study adds to the mounting evidence that CCPRCC should be formally considered a distinct entity. Microarray data have been deposited in the GEO database [GEO accession number (GSE51554)].