Circulating MicroRNAs as Cancer Biomarkers in Liquid Biopsies.

Biological fluids such as blood, saliva, and urine offer a rich source of biomarkers that have the potential to change the paradigm of cancer management. By allowing routine noninvasive sampling that can offer new insights into cancer progression and response to treatment so-called liquid biopsies can play an important role in personalized medicine. MicroRNAs (miRNAs) are a particularly attractive class of biomarkers as they are not only resistant to the high levels of RNases found in biological fluids, but also able to confer clinically useful information about the disease relating to diagnosis, prognosis, and the response to treatment. Circulating miRNAs are either associated with proteins or extracellular vesicles (EV) and although their origin and implied functions as intracellular messengers remain somewhat controversial, they are implicated in the progression and the establishment of metastatic niches. In this chapter, we review the rapidly emerging field of circulating miRNA cancer biomarkers, their origin and function, techniques to detect these molecules, and the use of bioinformatic tools to derive implied regulatory function, as well as the challenges that lie ahead for their clinical implementation.

MicroRNAs in Metastasis and the Tumour Microenvironment.

Metastasis is the process whereby cancer cells migrate from the primary tumour site to colonise the surrounding or distant tissue or organ. Metastasis is the primary cause of cancer-related mortality and approximately half of all cancer patients present at diagnosis with some form of metastasis. Consequently, there is a clear need to better understand metastasis in order to develop new tools to combat this process. MicroRNAs (miRNAs) regulate gene expression and play an important role in cancer development and progression including in the metastatic process. Particularly important are the roles that miRNAs play in the interaction between tumour cells and non-tumoral cells of the tumour microenvironment (TME), a process mediated largely by circulating miRNAs contained primarily in extracellular vesicles (EVs). In this review, we outline the accumulating evidence for the importance of miRNAs in the communication between tumour cells and the cells of the TME in the context of the pre-metastatic and metastatic niche.

The Use of circRNAs as Biomarkers of Cancer.

CircRNAs are a subclass of lncRNAs that have been found to be abundantly present in a wide range of species, including humans. CircRNAs are generally produced by a noncanonical splicing event called backsplicing that is dependent on the canonical splicing machinery, giving rise to circRNAs classified into three main categories: exonic circRNA, circular intronic RNA, and exon-intron circular RNA. Notably, circRNAs possess functional importance and display their functions through different mechanisms of action including sponging miRNAs, or even being translated into functional proteins. In addition, circRNAs also have great potential as biomarkers, particularly in cancer, thanks to their high stability, tissue type and developmental stage specificity, and their presence in biological fluids, which make them promising candidates as noninvasive biomarkers. In this chapter, we describe the most commonly used techniques for the study of circRNAs as cancer biomarkers, including high-throughput techniques such as RNA-Seq and microarrays, and other methods to analyze the presence of specific circRNAs in patient samples.

Circular RNAs and cancer: Opportunities and challenges.

Circular RNAs (circRNAs) are covalently circularized RNA moieties that despite being relatively abundant were only recently identified and have only begun to be investigated within the last couple of years. Even though there are many thousands of genes that appear capable of producing circRNAs, and the fact that many circRNAs appear to be highly evolutionarily conserved, the function of all but a few remain to be fully explored. What has been determined, however, is that circRNAs play key regulatory roles in many aspects of biology with focus being given to their function in cancer. Most of the studies to date have found that circRNAs act as master regulator of gene expression most often than not acting to regulate levels though sequestration or "sponging" of other gene expression regulators, particularly miRNAs. They can also function directly modulating transcription, or by interfering with splicing mechanisms. Some circRNAs can also be translated into functional proteins or peptides. A combination of tissue and developmental stage specific expression along with an innate resistance to RNAse activity means that circRNAs show perhaps their greatest potential as novel biomarkers of cancer. In this chapter we consider the current state of knowledge regarding these molecules, their synthesis, function, and association with cancer. We also consider some of the challenges that remain to be overcome to allow this emerging class of RNAs to fulfill their potential in clinical practice.

The Urinary Transcriptome as a Source of Biomarkers for Prostate Cancer.

Prostate cancer (PCa) is the second most common cancer of men and is typically slow-growing and asymptomatic. The use of blood PSA as a screening method has greatly improved PCa diagnosis, but high levels of false positives has raised much interest in alternative biomarkers. We used next-generation sequencing (NGS) to elucidate the urinary transcriptome of whole urine collected from high-stage and low-stage PCa patients as well as from patients with the confounding diagnosis of benign hyperplasia (BPH). We identified and validated five differentially expressed protein-coding genes (FTH1 BRPF1, OSBP, PHC3, and UACA) in an independent validation cohort of small-volume (1 mL) centrifuged urine (n = 94) and non-centrifuged urine (n = 84) by droplet digital (dd)PCR. These biomarkers were able to discriminate between BPH and PCa patients and healthy controls using either centrifuged or non-centrifuged whole urine samples, suggesting that the urinary transcriptome is a valuable source of non-invasive biomarkers for PCa that warrants further investigation.

The circulating transcriptome as a source of cancer liquid biopsy biomarkers.

Non-invasive biomarkers or liquid biopsies have the potential to revolutionise cancer patient management as repeated sampling allows real-time monitoring of disease progression and response to treatment. This allows for earlier intervention and dynamic treatment management; both cornerstones of personalised medicine. The circulating transcriptome represents a rich source of potential cancer biomarkers that includes many classes of RNA, both coding and non-coding, that are only now beginning to be explored. In particular the increasing power and availability of RNAseq techniques have pushed studies beyond circulating miRNAs, to other classes of RNA including mRNA, snRNA, snoRNA, piRNA, YRNA, lncRNA and circRNA. In this review we focus on the emerging potential for these different classes of RNA as cancer biomarkers, and in particular the barriers and limitations that remain to be overcome if these molecules are to become part of routine clinical practice.

The Circulating Transcriptome as a Source of Biomarkers for Melanoma.

The circulating transcriptome is a valuable source of cancer biomarkers, which, with the exception of microRNAs (miRNAs), remains relatively unexplored. To elucidate which RNAs are present in plasma from melanoma patients and which could be used to distinguish cancer patients from healthy individuals, we used next generation sequencing (NGS), and validation was carried out by qPCR and/or ddPCR. We identified 442 different microRNAs in samples, eleven of which were differentially expressed (p < 0.05). Levels of miR-134-5p and miR-320a-3p were significantly down-regulated (p < 0.001) in melanoma samples (n = 96) compared to healthy controls (n = 28). Differentially expressed protein-encoding mRNA 5'-fragments were enriched for the angiopoietin, p21-activated kinase (PAK), and EIF2 pathways. Levels of ATM1, AMFR, SOS1, and CD109 gene fragments were up-regulated (p < 0.001) in melanoma samples (n = 144) compared to healthy controls (n = 41) (AUC = 0.825). Over 40% of mapped reads were YRNAs, a class of non-coding RNAs that to date has been little explored. Expression levels of RNY3P1, RNY4P1, and RNY4P25 were significantly higher in patients with stage 0 disease than either healthy controls or more advanced stage disease (p < 0.001). In conclusion, we have identified a number of novel RNA biomarkers, which, most importantly, we validated in multi-center retrospective and prospective cohorts, suggesting potential diagnostic use of these RNA species.

MicroRNAs and Metastasis.

Metastasis, the development of secondary malignant growths at a distance from the primary site of a cancer, is associated with almost 90% of all cancer deaths, and half of all cancer patients present with some form of metastasis at the time of diagnosis. Consequently, there is a clear clinical need for a better understanding of metastasis. The role of miRNAs in the metastatic process is beginning to be explored. However, much is still to be understood. In this review, we present the accumulating evidence for the importance of miRNAs in metastasis as key regulators of this hallmark of cancer.

CircRNAs and cancer: Biomarkers and master regulators.

Circular RNAs (circRNAs) are a novel class of regulatory RNAs that despite being relatively abundant have only recently begun to be explored. There are many thousands of genes that appear capable of producing circRNAs, however the function of all but a handful remain to be determined. What is emerging about these highly conserved molecules is that they play important roles in biology and cancer biology in particular. The most explored function of circRNAs is as master regulators of gene expression that act to sequester or ´sponge´ other gene expression regulators, in particular miRNAs. They have also been demonstrated to function via direct modulation of transcription, and by interfering with splicing mechanisms. Although generally expressed in low abundance when compared to their linear counterparts, they are often expressed in a tissue- and developmental stage- specific manner. Coupled with their remarkable resistance to RNAse activity due to a covalent closed cyclic structure, circRNAs show great promise as novel biomarkers of cancer and other diseases. In this review we consider the current state of knowledge regarding these molecules, their synthesis, function, and association with cancer. We will also review some of the challenges that remain to be resolved if this emerging class of RNAs are really to become useful in the clinic.

MicroRNAs as Biomarkers of B-cell Lymphoma.

B-cell lymphomas represent a diverse group of neoplasms classified primarily by histopatholgy and are often challenging to accurately diagnose. Despite having been recognized less than 20 years ago, microRNAs (miRNAs) have emerged as one of the most promising class of cancer molecular biomarkers and are particularly attractive as they can be readily detected in formalin-fixed paraffin-embedded biopsy material and biological fluids such as blood. Many of the identified B-cell lymphoma miRNA biomarkers also play crucial regulatory roles in normal B-cell development. Below we consider the identity, function, and biomarker potential of miRNAs in B-cell lymphoma and most importantly the barriers that remain to be overcome if they are really to become part of routine clinical practice.

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.

In vivo intratumoral Epstein-Barr virus replication is associated with XBP1 activation and early-onset post-transplant lymphoproliferative disorders with prognostic implications.

Post-transplant lymphoproliferative disorders are life-threatening complications following hematopoietic or solid organ transplantation. They represent a spectrum of mostly EBV-driven lymphoplasmacytic proliferations. While the oncogenic effect of EBV is related to latent infection, lytic infection also has a role in lymphomagenesis. In vitro, EBV replication is linked to plasma cell differentiation and XBP1 activation, although this phenomenon has never been addressed in vivo. We analyzed for the first time latent and lytic intratumoral EBV infection in a series of 35 adult patients with a diagnosis of post-transplant lymphoproliferative disorder (26M/9F, median age 54 years). A complete EBV study was performed including the analysis of the latent EBER, latent membrane protein-11, and EBV nuclear antigens as well as the immediate-early BZLF1/ZEBRA and early BMRF1/EADE31 lytic genes. XBP1 activation was assessed by nuclear protein expression. EBV infection was observed in 28 (80%) cases being latency II and III the most frequently observed 22 (79%). Intratumoral EBV replication was detected in 17 (60%) cases. Among these, XBP1 activation was observed in 11/12 evaluable cases associated with strong cytoplasmic immunoglobulin expression consistent with plasma cell differentiation. Intriguingly, the combination of latency III infection and EBV replication identified a high-risk subgroup of patients with significantly shorter survival (overall survival at 1 year 18% vs 48%) and early-onset (median of 7 vs 26 months) post-transplant lymphoproliferative disorder. Moreover, these patients appear to be more heavily immunosuppressed, so they exhibit lower rates of rejection and graft vs host disease but higher rates of cytomegalovirus reactivation. In conclusion, EBV replication is associated with plasma cell differentiation and XBP1 activation with prognostic implications. Both latency III and lytic EBV infection are related to aggressive and early-onset post-transplant lymphoproliferative disorder. These results suggest that immunohistochemical study of latent and lytic EBV genes in the clinical practice may help to select higher-risk patients to new therapies including antiviral treatments.

Assessment of SOX11 expression in routine lymphoma tissue sections: characterization of new monoclonal antibodies for diagnosis of mantle cell lymphoma.

The diagnosis of mantle cell lymphoma (MCL) can be difficult, especially when no t(11;14) translocation and cyclin D1 overexpression can be detected. In such cases, the transcription factor SOX11 represents an important diagnostic marker, as it is expressed in most MCLs and, in particular, in all cyclin D1-negative MCLs reported so far. A reliable anti-SOX11 antibody is therefore a very useful tool for routine diagnosis. Here, we characterize the new monoclonal anti-SOX11 antibodies, suitable for Western blot assay and immunohistochemistry (IHC) on formalin-fixed paraffin-embedded tissue; we tested them on a large series of primary lymphoid tumors and compared these results with those of other routinely used antibodies. Moreover, we show that IHC results depend on transcription levels of SOX11, which suggests that posttranscriptional and posttranslational modifications do not significantly affect cutoff levels for IHC detection of SOX11.

Plasmablastic transformation of low-grade B-cell lymphomas: report on 6 cases.

Histologic transformation of low-grade B-cell lymphoma to diffuse large B-cell lymphoma is associated with poor prognosis. Although plasma cell differentiation is common in these lymphomas, an overt plasmablastic transformation (PBL-T) has been only rarely reported. We report 6 cases of PBL-T occurring in 3 chronic lymphocytic leukemias (CLL) and 3 follicular lymphomas. Five patients were men, and the mean age was 65 years (range, 52 to 72 y). None of them had history of immunodeficiency. In 3 cases the PBL-T occurred 34 to 85 months after the initial diagnosis, and in 3 it was detected simultaneously with the small cell component at diagnosis. All patients received chemotherapy after transformation, and 4 died 4 to 24 months after this diagnosis. In 3 cases, PBL-T occurred in an extranodal site. All PBL-Ts had immunoblastic morphology with admixed plasma cells, were CD20 and PAX5 negative, expressed λ light chain, and 5 were CD138 positive. All cases were negative for HHV8, and only 1 PBL-T was Epstein-Barr virus positive. Evidence of a clonal relationship between the small cell and PBL-T components was found in 5 cases. In 2 CLL cases, both components had 13q deletions, and in all follicular lymphoma cases both components harbored the t(14;18) translocation. MYC translocations were observed in 2 cases transformed from a CLL. In conclusion, PBL-T expands the clinicopathologic spectrum of the transformation of low-grade B-cell lymphomas. These transformed tumors are clinically, histologically, and phenotypically similar to primary plasmablastic lymphomas, but they are not associated with immunodeficiency and rarely have Epstein-Barr virus infection or MYC alterations.