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Background: Gliomas are aggressive malignant tumors, with poor prognosis. There is an unmet need for the discovery of new, non-invasive biomarkers for differential diagnosis, prognosis, and management of brain tumors. Our objective is to validate four plasma biomarkers - glial fibrillary acidic protein (GFAP), neurofilament light (NEFL), matrix metalloprotease 3 (MMP3) and fatty acid binding protein 4 (FABP4) - and compare them with established brain tumor molecular markers and survival., Methods: Our cohort consisted of patients with benign and malignant brain tumors (GBM = 77, Astrocytomas = 26, Oligodendrogliomas = 23, Secondary tumors = 35, Meningiomas = 70, Schwannomas = 15, Pituitary adenomas = 15, Normal individuals = 30). For measurements, we used ultrasensitive electrochemiluminescence multiplexed immunoassays., Results: High plasma GFAP concentration was associated with GBM, low GFAP and high FABP4 were associated with meningiomas, and low GFAP and low FABP4 were associated with astrocytomas and oligodendrogliomas. NEFL was associated with progression of disease. Several prognostic genetic alterations were significantly associated with all plasma biomarker levels. We found no independent associations between plasma GFAP, NEFL, FABP4 and MMP3, and overall survival. The candidate biomarkers could not reliably discriminate GBM from primary or secondary CNS lymphomas., Conclusions: GFAP, NEFL, FABP4 and MMP3 are useful for differential diagnosis and prognosis, and are associated with molecular changes in gliomas., (© 2024. The Author(s).)

Doxorubicin (Dox) is a widely utilized chemotherapeutic; however, it carries side effects, including drug-induced immune thrombocytopenia (DITP) and increased risk of venous thromboembolism (VTE). Currently, the mechanisms for Dox-associated DITP and VTE are poorly understood, and an effective inhibitor to relieve these complications remains to be developed. In this study, we found that Dox significantly induced platelet activation and enhanced platelet phagocytosis by macrophages and accelerated platelet clearance. Importantly, we determined that salvianolic acid C (SAC), a water-soluble compound derived from Danshen root traditionally used to treat cardiovascular diseases, inhibited Dox-induced platelet activation more effectively than current standard-of-care anti-platelet drugs aspirin and ticagrelor. Mechanism studies with tyrosine kinase inhibitors indicate contributions of phospholipase C, spleen tyrosine kinase, and protein kinase C signaling pathways in Dox-induced platelet activation. We further demonstrated that Dox enhanced platelet-cancer cell interaction, which was ameliorated by SAC. Taken together, these findings suggest SAC may be a promising therapy to reduce the risk of Dox-induced DITP, VTE, and the repercussions of amplified platelet-cancer interaction in the tumor microenvironment.

With sunitinib treatment of metastatic renal cell carcinoma, most patients end up developing resistance over time. Recent clinical trials have shown that individualizing treatment protocols could delay resistance and result in better outcomes. We developed an in vivo xenograft tumor model and compared tumor growth rate, morphological, and transcriptomic differences between alternative and traditional treatment schedules. Our results show that the alternative treatment regime could delay/postpone cancer progression. Additionally, we identified distinct morphological changes in the tumor with alternative and traditional treatments, likely due to the significantly dysregulated signaling pathways between the protocols. Further investigation of the signaling pathways underlying these morphological changes may lead potential therapeutic targets to be used in a combined treatment with sunitinib, which offers promise in postponing/reversing the resistance of sunitinib.

Renal cell carcinoma (RCC) is often diagnosed incidentally as a small renal mass (SRM; pT1a, ≤4 cm). Increasing concerns surrounding the overtreatment of patients with benign or clinically silent SRMs has resulted in a recent shift in treatment recommendations, especially in elderly and infirm patients. There are currently no biomarkers that can predict progression. We used a quantitative label-free liquid chromatography-tandem mass spectrometry peptidomics approach and targeted parallel-reaction monitoring to identify early, noninvasive diagnostic and prognostic biomarkers for early-stage RCC-SRMs. In total, 115 urine samples, including 33 renal oncocytoma (≤4 cm) cases, 30 progressive and 26 nonprogressive clear cell RCC-SRM cases, and 26 healthy controls were evaluated. Nine endogenous peptides that displayed significantly elevated expression in clear cell RCC-SRMs relative to healthy controls were identified. Peptides NVINGGSHAGNKLAMQEF, VNVDEVGGEALGRL, and VVAGVANALAHKYH showed significantly elevated expression in clear cell RCC-SRMs relative to renal oncocytoma. Additionally, peptides SHTSDSDVPSGVTEVVVKL and IVDNNILFLGKVNRP displayed significantly elevated expression in progressive relative to nonprogressive clear cell RCC-SRMs. Peptide SHTSDSDVPSGVTEVVVKL showed the most significant discriminatory utility (area under the curve, 0.76; 95% CI, 0.62-0.90; P = 0.0027). Patients with elevated SHTSDSDVPSGVTEVVVKL expression had significantly shorter overall survival (hazard ratio, 4.13; 95% CI, 1.09-15.65; P = 0.024) compared to patients with low expression. Pretreatment characterization of urinary peptides can provide insight into early RCC progression and may aid clinical decision-making and improve disease management., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Kallikrein-related peptidase 6 (KLK6) is a serine protease normally expressed in mammary tissue and aberrantly regulated in breast cancer. At physiological levels, KLK6 functions as a suppressor of breast cancer, while its aberrant overexpression (> 50-fold higher than normal) is characteristic of a subset of breast cancers and has been linked to accelerated growth of primary breast tumors in severe combined immunodeficiency mice (Pampalakis et al. Cancer Res 2009, 69, 3779). Here, we investigated the molecular mechanisms underlying the concentration-dependent functions of KLK6 by comparing MDA-MB-231 stable transfectants expressing increasing levels of KLK6 in in vitro and in vivo tumorigenicity assays (soft agar, xenograft growth, tail vein metastasis). Quantitative proteomics was applied to identify proteins that are altered upon re-expression of KLK6 in MDA-MB-231 at normal or constitutive levels. Overexpression of KLK6 is associated with increased metastatic ability of breast cancer cells into lungs, increased expression of certain S100 proteins (S100A4, S100A11) and keratins (KRT), and downregulation of the apoptosis-related proteases CASP7 and CASP8, and RABs. On the other hand, KLK6 re-expression at physiological levels leads to inhibition of lung metastases associated with suppression of S100 proteins (S100A4, S100A10, S100A13, S100A16) and induced CASP7 and CASP8 expression. As this is the first report that KLK6 expression is associated with S100 proteins, caspases, RABs, and KRTs, we validated this finding in clinical datasets. By integrating proteomics and microarray data from breast cancer patients, we generated two composite scores, KLK6 + S100B-S100A7 and KLK6 + S100B-S100A14-S100A16, to predict long-term survival of breast cancer patients. We present previously unknown pathways implicating KLK6 in breast cancer. The findings promise to aid our understanding of the functional roles of KLK6 in breast cancer and may yield new biomarkers for the cancer types in which KLK6 is known to be aberrantly upregulated., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Papillary renal cell carcinoma (PRCC) is the most common type of RCC in end-stage kidney disease (ESKD). Papillary adenoma (PA) is a small benign lesion morphologically similar to PRCC and is suggested to be its precursor. PA is also prevalent in ESKD. The evolution of PAs to PRCCs and their relationship to ESKD are poorly understood. A total of 140 PAs, normal kidneys, ESKDs, and PRCCs were analyzed. Previously described markers of renal tubular progenitor cells were analyzed using immunohistochemistry and quantified with digital analysis. Progenitor cells were significantly increased in ESKD (P < 0.0001) and PAs (P = 0.02) in comparison with the normal kidney. Pathway analysis using global miRNA and chromosomal copy number variations revealed a common developmental theme between PA and the PRCCs. Whole exome sequencing showed a KMT2C-specific pathogenic mutation among all PAs and PRCCs. KMT2C is a chromosome 7 epigenetic regulator implicated in development and oncogenesis. Collectively, results show possible connection of PRCCs to PA and the progenitor-like cell population, which are increased in response to renal tubular injury. In addition, each PRCC histologic subtype had its own set of mutational changes, indicating divergence from a common precursor. The study reports previously unknown biological aspects of PRCC development and could influence current surveillance criteria and early detection strategies of PRCC tumors., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Rapid advancements in next-generation sequencing (ngs) technology have created an unprecedented opportunity to decipher the molecular profile of tumours to more effectively prevent, diagnose, and treat cancer. Oncologists now have the option to order molecular tests that can guide treatment decisions. However, to date, most oncologists have received limited training in genomics, and they are now faced with the challenge of understanding how such tests and their interpretation align with patient management. Guidance on how to effectively use ngs technology is therefore needed to aid oncologists in applying the results of genomic tests. The Canadian guideline presented here describes best practices and unmet needs related to ngs-based testing for somatic variants in oncology, including clinical application, assay and sample selection, bioinformatics and interpretation of reports performed by laboratories, patient communication, and clinical trials., Competing Interests: CONFLICT OF INTEREST DISCLOSURES We have read and understood Current Oncology’s policy on disclosing conflicts of interest, and we declare the following interests: SY has received compensation from Bayer, Hoffmann–La Roche, and Pfizer for participating in advisory boards; AC has received funding from Hoffmann–La Roche, AstraZeneca, Pfizer, Illumina, and Thermo Fisher Scientific for medical writing services related to this paper; MRD has received compensation from Hoffman–La Roche and AstraZeneca for participating in advisory boards, and honoraria from AstraZeneca; II has received honoraria from Pfizer, AstraZeneca, Novartis, Hoffmann–La Roche, and Bayer for participating in advisory boards; JM has received honoraria from AstraZeneca; TS has received compensation from AstraZeneca, Bristol–Myers Squibb, and Janssen for participating in advisory boards, and has received research funding from AstraZeneca and Janssen; AS has participated in advisory boards for Merck, AstraZeneca, Janssen, Pfizer, Novartis, Roche, Bristol–Myers Squibb, and Myriad; he has also received research funding from Merck, AstraZeneca, Bristol–Myers Squibb, and Pfizer. The remaining authors have no conflicts to disclose.

Purpose: Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB , or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease., Experimental Design: We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models., Results: The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3-tRCC models was confirmed by significantly higher expression of phospho-S6 ( P < 0.0001) and phospho-4EBP1 ( P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro ( P < 0.0001) and in vivo ( P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation., Conclusions: These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3-tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3-tRCC., (©2018 American Association for Cancer Research.)

Papillary renal cell carcinoma (PRCC) is the most common nonclear cell RCCs and is known to comprise two histological subtypes. PRCC2 is more aggressive and is molecularly distinct from the other subtypes. Despite this, PRCCs are treated together as one entity, and they show poor response to the current therapies that do not target pathways implicated in their pathogenesis. We have previously detected ABCC2 (an ABC transporter), VEGF, and mTOR pathways to be enriched in PRCC2. In this study, we assess the therapeutic potential of targeting these pathways in PRCC2. Twenty RCC cell lines from the Cancer Cell Encyclopedia were compared to the Cancer Genome Atlas PRCC cohort (290), to identify representative PRCC2 cell lines. Cell lines were further validated in xenograft models. Selected cell lines were treated in vitro and in vivo (mice models) under five different conditions, untreated, anti-VEGF (sunitinib), ABCC2 blocker (MK571), mTOR inhibitor (everolimus) and sunitinib + MK571. Sunitinib +ABCC2 blocker group showed a significant response to therapy compared to the other treatment groups both in vitro (P ≤ 0.0001) and in vivo (P = 0.0132). ABCC2 blockage resulted in higher sunitinib uptake, both in vitro (P = 0.0016) and in vivo (P = 0.0031). Everolimus group demonstrated the second best response in vivo. The double-treatment group showed the highest apoptotic rate and lowest proliferation rate. There is an urgent need for individualized therapies of RCC subtypes that take into account their specific biology. Our results demonstrate that combined targeted therapy with sunitinib and ABCC2 blocker in PRCC2 has therapeutic potential. The results are likewise potentially significant for other ABCC2 high tumors. However, the results are preliminary and clinical trials are needed to confirm these effects in PRCC2 patients., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Platelets have long been recognized as key players in hemostasis and thrombosis; however, growing evidence suggests that they are also significantly involved in cancer, the second leading cause of mortality worldwide. Preclinical and clinical studies showed that tumorigenesis and metastasis can be promoted by platelets through a wide variety of crosstalk between platelets and cancer cells. For example, cancer changes platelet behavior by directly inducing tumor-platelet aggregates, triggering platelet granule and extracellular vesicle release, altering platelet phenotype and platelet RNA profiles, and enhancing thrombopoiesis. Reciprocally, platelets reinforce tumor growth with proliferation signals, antiapoptotic effect, and angiogenic factors. Platelets also activate tumor invasion and sustain metastasis via inducing an invasive epithelial-mesenchymal transition phenotype of tumor cells, promoting tumor survival in circulation, tumor arrest at the endothelium, and extravasation. Furthermore, platelets assist tumors in evading immune destruction. Hence, cancer cells and platelets maintain a complex, bidirectional communication. Recently, aspirin (acetylsalicylic acid) has been recognized as a promising cancer-preventive agent. It is recommended at daily low dose by the US Preventive Services Task Force for primary prevention of colorectal cancer. The exact mechanisms of action of aspirin in chemoprevention are not very clear, but evidence has emerged that suggests a platelet-mediated effect. In this article, we will introduce how cancer changes platelets to be more cancer-friendly and highlight advances in the modes of action for aspirin in cancer prevention. We also discuss the opportunities, challenges, and opposing viewpoints on applying aspirin and other antiplatelet agents for cancer prevention and treatment., (© 2018 by The American Society of Hematology.)

This corrects the article DOI: 10.1038/bjc.2016.367.

Upon sunitinib treatment of metastatic renal cell carcinoma patients eventually acquire resistance. Our aim was to investigate microRNAs behind sunitinib resistance. We developed an in vivo xenograft and an in vitro model and compared morphological, immunhistochemical, transcriptomical and miRNome data changes during sunitinib response and resistance by performing next-generation mRNA and miRNA sequencing. Complex bioinformatics (pathway, BioFunction and network) analysis were performed. Results were validated by in vitro functional assays. Our morphological, immunhistochemical, transcriptomical and miRNome data all pointed out that during sunitinib resistance tumor cells changed to migratory phenotype. We identified the downregulated miR-1 and miR-663a targeting FRAS1 (Fraser Extracellular Matrix Complex Subunit 1) and MDGA1 (MAM Domain Containing Glycosylphosphatidylinositol Anchor 1) in resistant tumors. We proved firstly miR-1-FRAS1 and miR-663a-MDGA1 interactions. We found that MDGA1 knockdown decreased renal cancer cell migration and proliferation similarly to restoration of levels of miR-1 and miR-663. Our results support the central role of cell migration as an adaptive mechanism to secure tumor survival behind sunitinib resistance. MDGA1, FRAS1 or the targeting miRNAs can be potential adjuvant therapeutic targets, through inhibition of cancer cell migration, thus eliminating the development of resistance and metastasis., Competing Interests: CONFLICTS OF INTEREST The authors declare no potential conflicts of interest.

Renal cell carcinoma (RCC) constitutes an array of morphologically and genetically distinct tumors the most prevalent of which are clear cell, papillary, and chromophobe RCC. Accurate distinction between the typically benign-behaving renal oncocytoma and RCC subtypes is a frequent challenge for pathologists. This is critical for clinical decision making. Subtypes also have different survival outcomes and responses to therapy. We extracted RNA from ninety formalin-fixed paraffin-embedded (FFPE) tissues (27 clear cell, 29 papillary, 19 chromophobe, 4 unclassified RCC and 11 oncocytomas). We quantified the expression of six miRNAs (miR-221, miR-222, miR-126, miR-182, miR-200b and miR-200c) by qRT-PCR, and by in situ hybridization in an independent set of tumors. We developed a two-step classifier. In the first step, it uses expression of either miR-221 or miR-222 to distinguish the clear cell and papillary subtypes from chromophobe RCC and oncocytoma (miR-221 AUC: 0.96, 95% CI: 0.9132-1.014, p < 0.0001 and miR-222 AUC: 0.91, 95% CI: 0.8478-0.9772, p < 0.0001). In the second step, it uses miR-126 to discriminate clear cell from papillary RCC (AUC: 1, p < 0.0001) and miR-200b to discriminate chromophobe RCC from oncocytoma (AUC: 0.95, 95% CI: 0.8933-1.021, p < 0.0001). In situ hybridization showed a nuclear staining pattern. miR-126, miR-222 and miR-200b were significantly differentially expressed between the subtypes by in situ hybridization. miRNA expression could distinguish RCC subtypes and oncocytoma. miRNA expression assessed by either PCR or in situ hybridization can be a clinically useful diagnostic tool to complement morphologic renal tumor classification, improving diagnosis and patient management., Competing Interests: CONFLICTS OF INTEREST The authors have declared no conflicts of interest.

The term "vasculogenic mimicry" (VM) refers to the phenomenon in which vascular-like channels, which are not lined by endothelial cells, are formed in tumors. Since its discovery in 1999, it has been observed in several tumor types and is proposed to provide blood perfusion to tumors in absence of co-apted or neo-angiogenic blood vessels. Pituitary tumors are generally slow growing, benign adenomas which are less vascularized than the normal pituitary gland. To date, VM in pituitary adenomas has not been described. In this histological study, we assessed the presence of VM in a series of surgically resected clinically non-functioning pituitary adenomas (NFPAs) using CD34 and Periodic Acid-Schiff (PAS) double staining. To identify VM, slides were assessed for the presence of CD34-negative and PAS-positive channels indicating that they were not lined by endothelial cells. The histological staining pattern suggestive of VM was noted in 22/49 (44.9%) of the specimens studied. VM was observed in both recurring and non-recurring NFPAs. The incidence of VM present varied from case to case and within groups. There was no association between the presence of VM and gender, tumor size, Ki-67 index, recurrence or cavernous sinus invasion. VM was not noted in cases of non-tumorous pituitaries. Our findings suggest the existence of a complementary perfusion system in pituitary adenomas, implying potential clinical implications with respect to response to therapy and clinical course. Further research is warranted to confirm the presence of VM in pituitary adenomas to elucidate its clinical relevance in patients diagnosed with a pituitary adenoma.

There is a growing trend towards exploring the use of a minimally invasive "liquid biopsy" to identify biomarkers in a number of cancers, including urologic malignancies. Multiple aspects can be assessed in circulating cell-free DNA, including cell-free DNA levels, integrity, methylation and mutations. Other prospective liquid biopsy markers include circulating tumor cells, circulating RNAs (miRNA, lncRNAs and mRNAs), cell-free proteins, peptides and exosomes have also emerged as non-invasive cancer biomarkers. These circulating molecules can be detected in various biological fluids, including blood, urine, saliva and seminal plasma. Liquid biopsies hold great promise for personalized medicine due to their ability to provide multiple non-invasive global snapshots of the primary and metastatic tumors. Molecular profiling of circulating molecules has been a stepping-stone to the successful introduction of several non-invasive multi-marker tests into the clinic. In this review, we provide an overview of the current state of cell-free DNA-based kidney, prostate and bladder cancer biomarker research and discuss the potential utility other circulating molecules. We will also discuss the challenges and limitations facing non-invasive cancer biomarker discovery and the benefits of this growing area of translational research.

Background: Our previous study found that dysregulated microRNA-146a-5p (miR-146a-5p) is involved in oesophageal squamous cell cancer (ESCC) proliferation. This article aimed to evaluate its detailed mechanisms in ESCC epithelial-mesenchymal transition (EMT) progression., Methods: Invasion assay, qRT-PCR and western blotting were used to validate the roles of miR-146a-5p and Notch2 in EMT progression. miRNA target gene prediction databases and dual-luciferase reporter assay were used to validate the target gene., Results: miR-146a-5p inhibitor led to increase of invaded ESCC cells, while miR-146a-5p mimics inhibited invasion ability of ESCC cells. Protein level of E-cadherin decreased, whereas those of Snail and Vimentin increased in the anti-miR-146a-5p group, which demonstrated that miR-146a-5p inhibits EMT progression of ESCC cells. miRNA target gene prediction databases indicated the potential of Notch2 as a direct target gene of miR-146a-5p and dual-luciferase reporter assay validated it. Importantly, shRNA-Notch2 restrained EMT and partially abrogated the inhibiting effects of miR-146a-5p on EMT progression of ESCC cells., Conclusions: miR-146a-5p functions as a tumour-suppressive miRNA targeting Notch2 and inhibits the EMT progression of ESCC.

Extracellular miRNAs are increasingly studied as markers for specific diseases. They are released in biological fluids in a remarkably stable form, and may play a role in intercellular communication. They are thought to be protected against degradation by either encapsulation within microparticles, or by binding to proteins (mostly AGO2). The particulate forms may be internalized by endocytosis or membrane fusion, but the protein-bound forms require a receptor mechanism for their uptake. A major question is whether there are natural cell-membrane receptors that capture and internalize protein-bound functional miRNAs. We examined neuropilin-1 (NRP1), in view of its properties as a receptor for many ligands, including growth factors such as vascular endothelial growth factor (VEGF), and efficiency at mediating ligand internalization. It is expressed by endothelial cells, many other normal cell types, and cancer cells. Here, we report that NRP1 binds miRNAs with high affinity, and promotes their entry into the cell. Furthermore, the internalized miRNAs remain functional, as they specifically regulate proliferation and migration of cancer cells, as well as tube formation by human endothelial cells. Anti-NRP1 antibodies or NRP1 siRNA knockdown block miRNA effects, further confirming NRP1-mediated uptake. VEGF does not compete with miRNAs for binding to NRP1. In addition, NRP1 binds extracellular AGO2 (carrying miRNA or not), and internalizes AGO2/miRNA complexes. Because miRNA bound to AGO2 appears to the most abundant form in body fluids, this may have important physiological and pathological effects.

Background: Prostate cancer is the second most frequently diagnosed cancer in men worldwide. Theranostics, a combination of diagnostics and therapeutics, is an emerging concept in the field of precision medicine, and microRNAs (miRNAs) are predictive pioneers in this area., Content: miRNAs are small endogenous noncoding RNA molecules that regulate gene expression posttranscriptionally by targeting messenger RNAs. More than 60% of all protein coding genes are controlled by miRNAs, which makes them powerful regulators of the different cellular processes involved in the pathogenesis of various types of cancer, including prostate cancer. Growing evidence indicates the differential expression of miRNAs in tumor tissues. In addition, miRNAs in body fluids, known as circulating miRNAs, are present in remarkably stable forms and their alteration in prostate cancer has been well documented. Circulating miRNAs are known to originate from tumor tissues, thereby enabling intercellular communication via carriers to promote tumorigenesis and malignancy. In addition, fueled by recent advances, the use of miRNA-based anticancer therapies has been proposed with the onset of early phase clinical trials to assess the therapeutic efficacy of miRNAs., Summary: In this review, we summarize the theranostic utility of miRNAs and outline their diagnostic and prognostic potential in prostate cancer. In addition, we discuss the current detection methodologies and emerging innovative strategies for the detection of miRNAs in body fluids and tumor tissues in the clinical setting. We also provide insight into the current and future therapeutic potential of miRNAs in prostate cancer., (© 2016 American Association for Clinical Chemistry.)

Urological malignancies are a major cause of morbidity and mortality worldwide. Advances in early detection, diagnosis, prognosis and prediction of treatment response can significantly improve patient care. Proteomic and peptidomic profiling studies are at the center of kidney, prostate and bladder cancer biomarker discovery and have shown great promise for improved clinical assessment. Mass spectrometry (MS) is the most widely employed method for proteomic and peptidomic analyses. A number of MS platforms have been developed to facilitate accurate identification of clinically relevant markers in various complex biological samples including tissue, urine and blood. Furthermore, protein profiling studies have been instrumental in the successful introduction of several diagnostic multimarker tests into the clinic. In this review, we will provide a brief overview of high-throughput technologies for protein and peptide based biomarker discovery. We will also examine the current state of kidney, prostate and bladder cancer biomarker research as well as review the journey toward successful clinical implementation., Competing Interests: CONFLICTS OF INTERESTS The authors declare no competing financial interest.

KLK6 is expressed in normal mammary tissues and is aberrantly regulated in breast cancer. At physiological levels of expression, i.e. those found in normal mammary tissues, KLK6 acts as a tumor suppressor in human breast cancer. However, aberrant overexpression of KLK6 (i.e. 50-100-fold higher than normal), a characteristic of a subset of human breast cancers is associated with increased tumorigenicity (Pampalakis et al. Cancer Res 69:3779-3787, 2009). Here, we stably transfected KLK6-non-expressing MDA-MB-231 breast cancer cells with the full-length KLK6 cDNA to overexpress KLK6 at levels comparable to those observed in patients, and investigated potential oncogenic miRNA networks regulated by these abnormally high KLK6 expression levels and increased activity of this serine protease. A number of miRNAs that are upregulated (e.g. miR-146a) or downregulated (e.g. miR-34a) via KLK6-induced alterations in the miRNA biogenesis machinery were identified. Integrated experimental and bioinformatics analyses identified convergent miRNA networks targeting the cell cycle, MYC, MAPK, and other signaling pathways. In large clinical datasets, significant correlations between KLK6 and downstream MAPK and MYC targets at both the RNA and protein levels was confirmed, as well as negative correlation with GATA3. It was also demonstrated that KLK6 overexpression and likely its proteolytic activity is associated with alterations in downstream miRNAs and their targets, and these differ with the molecular subtypes of breast cancer. The data partly explains the different characteristics of breast cancer subtypes. Importantly, we introduce a combined KLK6-CDKN1B+MYC+CDKN1C score for prediction of long-term patient survival outcomes, with higher scores indicating poor survival., (Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background: The anti-angiogenic Sorafenib is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC). However, acquired resistance limits its efficacy. An emerging theory to explain intrinsic resistance to other anti-angiogenic drugs is 'vessel co-option,' ie, the ability of tumors to hijack the existing vasculature in organs such as the lungs or liver, thus limiting the need for sprouting angiogenesis. Vessel co-option has not been evaluated as a potential mechanism for acquired resistance to anti-angiogenic agents., Methods: To study sorafenib resistance mechanisms, we used an orthotopic human HCC model (n = 4-11 per group), where tumor cells are tagged with a secreted protein biomarker to monitor disease burden and response to therapy. Histopathology, vessel perfusion assessed by contrast-enhanced ultrasound, and miRNA sequencing and quantitative real-time polymerase chain reaction were used to monitor changes in tumor biology., Results: While sorafenib initially inhibited angiogenesis and stabilized tumor growth, no angiogenic 'rebound' effect was observed during development of resistance unless therapy was stopped. Instead, resistant tumors became more locally infiltrative, which facilitated extensive incorporation of liver parenchyma and the co-option of liver-associated vessels. Up to 75% (±10.9%) of total vessels were provided by vessel co-option in resistant tumors relative to 23.3% (±10.3%) in untreated controls. miRNA sequencing implicated pro-invasive signaling and epithelial-to-mesenchymal-like transition during resistance development while functional imaging further supported a shift from angiogenesis to vessel co-option., Conclusions: This is the first documentation of vessel co-option as a mechanism of acquired resistance to anti-angiogenic therapy and could have important implications including the potential therapeutic benefits of targeting vessel co-option in conjunction with vascular endothelial growth factor receptor signaling., (© The Author 2016. Published by Oxford University Press.)

Clear cell renal cell carcinoma (ccRCC) is the most prevalent adult kidney cancer. Prognostic markers are needed to guide patient management toward aggressive versus more conservative approaches, especially for small tumors ≤4 cm. miR-194 was reported to be downregulated in several cancers and is involved in epithelial to mesenchymal transition. We evaluated miR-194 as a prognostic marker in ccRCC. In a cohort of 234 patients with primary ccRCC, we correlated miR-194 expression level with multiple clinicopathological features including disease-free and overall survival, tumor size, clinical stage, and histological grade. Our results shows a stepwise decrease in miR-194 expression from normal kidney to primary ccRCC (P = 0.0032) and a subsequent decrease from primary to metastatic lesions. Additionally, patients with higher miR-194 expression has significantly longer disease-free survival (P = 0.041) and overall survival (P = 0.031) compared to those with lower expression. In multivariate analysis, miR-194-positive tumors retain significance in disease-free survival and overall survival, suggesting miR-194 is an independent marker for good prognosis in ccRCC. Moreover, miR-194 is a marker for good prognosis for patients with small renal masses (P = 0.014). These findings were validated on an independent data set from The Cancer Genome Atlas. We also compared miR-194 expression between RCC subtypes. ccRCC had the highest levels, whereas chromophobe RCC and oncocytoma had comparable lower levels. Target prediction coupled with pathway analysis show that miR-194 is predicted to target key molecules and pathways involved in RCC progression. miR-194 represents a prognostic biomarker in ccRCC., (© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: It is anticipated that many licensing examination centres for pathology will begin fully digitizing the certification examinations. The objective of our study was to test the feasibility of a fully digital examination and to assess the needs, concerns and expectations of pathology residents in moving from a glass slide-based examination to a fully digital examination., Methods: We conducted a mixed methods study that compared, after randomization, the performance of senior residents (postgraduate years 4 and 5) in 7 accredited anatomical pathology training programs across Canada on a pathology examination using either glass slides or digital whole-slide scanned images of the slides. The pilot examination was followed by a post-test survey. In addition, pathology residents from all levels of training were invited to participate in an online survey., Results: A total of 100 residents participated in the pilot examination; 49 were given glass slides instead of digital images. We found no significant difference in examination results between the 2 groups of residents (estimated marginal mean 8.23/12, 95% confidence interval [CI] 7.72-8.87, for glass slides; 7.84/12, 95% CI 7.28-8.41, for digital slides). In the post-test survey, most of the respondents expressed concerns with the digital examination, including slowly functioning software, blurring and poor detail of images, particularly nuclear features. All of the respondents of the general survey (n = 179) agreed that additional training was required if the examination were to become fully digital., Interpretation: Although the performance of residents completing pathology examinations with glass slides was comparable to that of residents using digital images, our study showed that residents were not comfortable with the digital technology, especially given their current level of exposure to it. Additional training may be needed before implementing a fully digital examination, with consideration for a gradual transition.

Loss of the tumor suppressor gene AT-rich interactive domain-containing protein 1A (ARID1A) has been demonstrated in several cancers, but its prognostic role is unknown. We aimed to investigate the risk associated with loss of ARID1A (ARID1A-) for all-cause mortality, cancer-specific mortality and recurrence of disease in subjects with cancer. PubMed and SCOPUS search from database inception until 01/31/2015 without language restriction was conducted, contacting authors for unpublished data. Eligible were prospective studies reporting data on prognostic parameters in subjects with cancer, comparing participants with presence of ARID1A (ARID1A+) vs. ARID1A-, assessed either via immunohistochemistry (loss of expression) or with genetic testing (presence of mutation). Data were summarized using risk ratios (RR) for number of deaths/recurrences and hazard ratios (HR) for time-dependent risk related to ARID1A- adjusted for potential confounders. Of 136 hits, 25 studies with 5,651 participants (28 cohorts; ARID1A-: n = 1,701; ARID1A+: n = 3,950), with a mean follow-up period of 4.7 ± 1.8 years, were meta-analyzed. Compared to ARID1A+, ARID1A- significantly increased cancer-specific mortality (studies = 3; RR = 1.55, 95% confidence interval (CI) = 1.19-2.00, I(2) = 31%). Using HRs adjusted for potential confounders, ARID1A- was associated with a greater risk of cancer-specific mortality (studies = 2; HR = 2.55, 95%CI = 1.19-5.45, I(2) = 19%) and cancer recurrence (studies = 10; HR = 1.93, 95%CI = 1.22-3.05, I(2) = 76%). On the basis of these results, we have demonstrated that loss of ARID1A shortened time to cancer-specific mortality, and to recurrence of cancer when adjusting for potential confounders. For its role, this gene should be considered as an important potential target for personalized medicine in cancer treatment.

Sunitinib is a multitargeting tyrosine kinase inhibitor used for metastatic renal cancer. There are no biomarkers that can predict sunitinib response. Such markers are needed to avoid administration of costly medication with side effects to patients who would not benefit from it. We compared global miRNA expression between patients with a short (≤12 months) versus prolonged (>12 months) progression-free survival (PFS) under sunitinib as first-line therapy for metastatic renal cell carcinoma. We identified a number of differentially expressed miRNAs and developed miRNA statistical models that can accurately distinguish between the two groups. We validated our models in the discovery set and an independent set of 57 patients. Target prediction and pathway analysis showed that these miRNAs are involved in vascular endothelial growth factor (VEGF), TGFβ, and mammalian target of rapamycin (mTOR)-mediated signaling and cell-cell communication. We tested the effect of these miRNAs on cellular proliferation and angiogenesis. We validated the negative correlation between miR-221 and its target, VEGFR2.miR-221 overexpression was associated with a poor PFS while its target, VEGFR2 was associated with longer survival. Gain of function experiments showed that miR-221 and miR-222 decreased angiogenesis and cellular proliferation in human umbilical vein endothelial cells (HUVEC) while increasing cellular proliferation in ACHN cells. miRNAs represent potential predictive markers for sunitinib response.

Papillary renal cell carcinoma (pRCC) is the second most common RCC subtype and can be further classified as type 1 (pRCC1) or 2 (pRCC2). There is currently minimal understanding of pRCC1 pathogenesis, and treatment decisions are mostly empirical. The aim of this study was to identify biological pathways that are involved in pRCC1 pathogenesis using an integrated genomic approach. By microarray analysis, we identified a number of significantly dysregulated genes and microRNAs (miRNAs) that were unique to pRCC1. Integrated bioinformatics analyses showed enrichment of the focal adhesion and extracellular matrix (ECM) pathways. We experimentally validated that many members of these pathways are dysregulated in pRCC1. We identified and experimentally validated the downregulation of miR-199a-3p in pRCC1. Using cell line models, we showed that miR-199a-3p plays an important role in pRCC1 pathogenesis. Gain of function experiments showed that miR-199a-3p overexpression significantly decreased cell proliferation (p = 0.013). We also provide evidence that miR-199a-3p regulates the expression of genes linked to the focal adhesion and ECM pathways, such as caveolin 2 (CAV2), integrin beta 8 (ITGB8), MET proto-oncogene and mammalian target of rapamycin (MTOR). Using a luciferase reporter assay, we further provide evidence that miR-199a-3p overexpression decreases the expression of MET and MTOR. Using an integrated gene/miRNA approach, we provide evidence linking miRNAs to the focal adhesion and ECM pathways in pRCC1 pathogenesis. This novel information can contribute to the development of effective targeted therapies for pRCC1, for which there is none currently available in the clinic., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Clear cell renal cell carcinoma (ccRCC) is an aggressive tumor with frequent metastatic rate and poor survival. Integrated analyses allow understanding the interplay between different levels of molecular alterations.We integrated miRNA and gene expression data from 458 ccRCC and 254 normal kidney specimens to construct a miRNA-target interaction network.We identified the downregulated miR-124-3p, -30a-5p and -200c-3p as the most influential miRNAs in RCC pathogenesis.miR-124-3p and miR-200c-3p expression showed association with patient survival, miR-30a-5p was downregulated in metastases compared to primary tumors. We used an independent set of 87 matched samples for validation. We confirmed the functional impact of these miRNAs by in vitro assays. Restoration of these miRNAs reduced migration, invasion and proliferation. miR-124-3p decreased the S phase of cell cycle, as well. We compared transcriptome profiling before and after miRNA overexpression, and validated CAV1 and FLOT1 as miR-124-3p targets. Patients with higher CAV1 and FLOT1 had lower miR-124-3p expression and shorter overall survival.We hypothesize that these three miRNAs are fundamental contributing to ccRCC aggressive/metastatic behavior; and miR-124-3p especially has a key role through regulating CAV1 and FLOT1 expression. Restoration of the levels of these miRNAs could be considered as a potential therapeutic strategy for ccRCC.

Objective: Human kallikrein-like peptidase 10 (KLK10), a serine protease, plays an important role in the regulation of cell proliferation and tumor growth. In this work, we investigated KLK10 immunoexpression in various types of surgically removed human pituitary tumors., Design: Specimens were fixed in formalin and embedded in paraffin. Immunostaining was performed by the streptavidin-biotin-peroxidase complex protocol using the LSAB+ Kit and a KLK10-specific rabbit polyclonal antibody., Results: Results showed that both treated and untreated prolactin-producing pituitary adenomas and carcinomas as well as TSH-producing pituitary adenomas and carcinomas were conclusively immunopositive for KLK10. Immunostaining was mainly localized in the cytoplasm and was clearly visible in many adenoma cells. In various other tumor types (oncocytoma, gonadotroph, somatotroph adenomas, and carcinomas), cytoplasmic immunopositivity was mild to moderate and seen only in a few unevenly distributed adenoma cells. Immunopositivity in the nuclei of various tumor types, as well as dual cytoplasmic and nuclear localization of KLK10 in some of the tumor types, was an intriguing finding. Immunoexpression in GH-producing adenomas exposed to octreotide, a long-acting somatostatin analog, was significantly increased when compared to unexposed GH-producing tumors., Conclusion: More studies are needed to ascertain the role of KLK10 in pituitary tumor development, prognosis, and progression. The question of whether genetic abnormalities and the microenvironment can affect KLK10 immunoexpression should also be investigated.

Renal cell carcinoma (RCC) is an aggressive disease, with 35% chance of metastasis. The 'cancer stem cell' hypothesis suggests that a subset of cancer cells possess stem cell properties and is crucial in tumor initiation, metastasis and treatment resistance. We isolated RCC spheres and showed that they exhibit cancer stem cell/ tumor initiating cell-like properties including the formation of self-renewing spheres, high tumorigenicity and the ability to differentiate to cell types of the original tumor. Spheres showed increased expression of stem cell-related transcription factors and mesenchymal markers. miRNAs were differentially expressed between RCC spheres and their parental cells. Inhibition of miR-17 accelerated the formation of RCC spheres which shared molecular characteristics with the spontaneous RCC spheres. Target prediction pointed out TGFβ pathway activation as a possible mechanism to drive RCC sphere formation. We demonstrate that miR-17 overexpression interferes with the TGFβ-EMT axis and hinders RCC sphere formation; and validated TGFBR2 as a direct and biologically relevant target during this process. Thus, a single miRNA may have an impact on the formation of highly tumorigenic cancer spheres of kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is an aggressive tumor with unpredictable behavior. Clinical parameters are not always accurate for predicting prognosis. miR-126 is differentially expressed in many cancers, including RCC, and is down-regulated in metastatic versus primary ccRCC. We assessed the prognostic significance of miR-126 in 264 primary ccRCCs. We also compared its expression in normal kidney, primary and metastatic ccRCC, and RCC subtypes. We validated our results on an independent set of 481 ccRCCs. miR-126 was down-regulated in metastatic versus primary tumors and in tumors of higher stage (P = 0.005) or higher grade (P = 0.002). miR-126 up-regulation was associated with significantly prolonged disease-free survival (P < 0.001) and overall survival (P = 0.015). For larger tumors (>4 cm), patients with higher miR-126 expression had significantly longer survival. Restoration of miR-126 expression decreased cellular migration and proliferation in RCC cell lines. The ccRCCs exhibited the highest miR-126 expression, and papillary RCCs exhibited the lowest expression. We identified a number of miR-126 targets and pathways that are involved in carcinogenesis, including the apoptosis signaling pathway. miR-126 is a promising prognostic marker in ccRCC that can distinguish between clear cell and papillary subtypes. In addition, miR-126 has potential therapeutic applications., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Accurate assessment of prognosis of clear cell renal cell carcinoma (ccRCC) is key in optimizing management plans to fit individual patient needs. miRNAs are short noncoding single-stranded RNAs that control the expression of target genes and may act as cancer biomarkers. We analyzed the expression of miR-210 in 276 cases of primary ccRCC and compared its expression in 40 pairs of adjacent normal and cancerous tissues. We assessed its expression in primary and metastatic tumors, in the common RCC subtypes, and the benign oncocytoma. The results were validated with an independent data set from The Cancer Genome Atlas. miR-210 was significantly overexpressed in ccRCC compared with normal kidney. miR-210(+) patients had a statistically higher chance of disease recurrence [hazard ratio (HR), 1.82; P = 0.018] and shorter overall survival (HR, 2.46; P = 0.014). In multivariate analysis, miR-210 lost its statistically significant association with shorter disease-free survival and overall survival after adjusting for tumor size and tumor, node, metastasis stage. Papillary RCC showed comparable miR-210 overexpression, whereas decreased up-regulation was seen in chromophobe RCC and oncocytoma. A number of predicted targets that might be involved in carcinogenesis and aggressive tumor behavior were identified. miR-210 is a potential therapeutic target and independent marker of poor prognosis of ccRCC., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Kallikrein-related peptidase 5 (KLK5) displays aberrant expression in cancer. Recently, we showed KLK5 reconstitution in breast cancer cell lines suppresses malignancy. Present study aims to investigate the functional KLK5 mediated miRNA network on breast cancer progression, molecular subtype and survival. 28 miRNAs were up-regulated and 62 miRNAs were down-regulated upon KLK5 expression. Extracellular matrix (ECM) molecules and cell-adhesion pathways were the most significant KLK5-induced miRNA-mediated regulatory targets. Validation from The Cancer Genome Atlas (TCGA) database indicated KLK5 was specifically down-regulated in luminal B and basal-like breast cancer subtypes. There was a correlation between KLK5, miRNAs and their downstream ECM gene targets. Long-term patient survival correlated with dysregulation of KLK5 and interacting ECM target genes. It suggests biological differences between breast cancer molecular subtypes, patient survival, and their propensity for invasion and metastasis can be explained in part by altered miRNA networks induced by KLK5 dysregulation. We provide the first evidence that KLK5 can affect miRNA networks, which regulate MMPs and other novel ECM targets and a new compelling hypothesis of interplay between serine proteases and miRNAs. We developed a combined KLK5-(ITGB1+COL12A1) predictive score for recurrence-free survival that could be exploited in clinical applications.

Background: The outcome of clear cell renal cell carcinoma (ccRCC) is still unpredictable. Even with new targeted therapies, the average progression-free survival is dismal. Markers for early detection and progression could improve disease outcome., Methods: To identify efficient and hitherto unrecognized pathogenic factors of the disease, we performed a uniquely comprehensive pathway analysis and built a gene interaction network based on large publicly available data sets assembled from 28 publications, comprising a 3-prong approach with high-throughput mRNA, microRNA, and protein expression profiles of 593 ccRCC and 389 normal kidney samples. We validated our results on 2 different data sets of 882 ccRCC and 152 normal tissues. Functional analyses were done by proliferation, migration, and invasion assays following siRNA (small interfering RNA) knockdown., Results: After integration of multilevel data, we identified aryl-hydrocarbon receptor (AHR), grainyhead-like-2 (GRHL2), and KIAA0101 as new pathogenic factors. GRHL2 expression was associated with higher chances for disease relapse and retained prognostic utility after controlling for grade and stage [hazard ratio (HR), 3.47, P = 0.012]. Patients with KIAA0101-positive expression suffered worse disease-free survival (HR, 3.64, P < 0.001), and in multivariate analysis KIAA0101 retained its independent prognostic significance. Survival analysis showed that GRHL2- and KIAA0101-positive patients had significantly lower disease-free survival (P = 0.002 and P < 0.001). We also found that KIAA0101 silencing decreased kidney cancer cell migration and invasion in vitro., Conclusions: Using an integrative system biology approach, we identified 3 novel factors as potential biomarkers (AHR, GRHL2 and KIAA0101) involved in ccRCC pathogenesis and not linked to kidney cancer before., (© 2014 American Association for Clinical Chemistry.)

Background: Over 90% of cancer-related deaths in clear cell renal cell carcinoma (RCC) are caused by tumor relapse and metastasis. Thus, there is an urgent need for new molecular markers that can potentiate the efficacy of the current clinical-based models of prognosis assessment. The objective of this study is to evaluate the potential significance of lactate dehydrogenase A (LDHA), assessed by immunohistochemical staining, as a prognostic marker in clear cell renal cell carcinoma in relation to clinicopathological features and clinical outcome., Methods: We assessed the expression of LDHA at the protein level, by immunohistochemistry, and correlated its expression with multiple clinicopathological features including tumor size, clinical stage, histological grade, disease-free and overall survival in 385 patients with primary clear cell renal cell carcinoma. We also correlated the LDHA expression with overall survival, at mRNA level, in an independent data set of 170 clear cell renal cell carcinoma cases from The Cancer Genome Atlas databases. Cox proportional hazards models adjusted for the potential clinicopathological factors were used to test for associations between the LDHA expression and both disease-free survival and overall survival., Results: There is statistically significant positive correlation between LDHA level of expression and tumor size, clinical stage and histological grade. Moreover, LDHA expression shows significantly inverse correlation with both disease-free survival and overall survival in patients with clear cell renal cell carcinoma. Our results are validated by examining LDHA expression, at the mRNA level, in the independent data set of clear cell renal cell carcinoma cases from The Cancer Genome Atlas databases which also shows that higher lactate dehydrogenase A expression is associated with significantly shorter overall survival., Conclusion: Our results indicate that LDHA up-regulation can be a predictor of poor prognosis in clear cell renal cell carcinoma. Thus, it represents a potential prognostic biomarker that can boost the accuracy of other prognostic models in patients with clear cell renal cell carcinoma.

Prostate cancer (PCa) is one of the most commonly diagnosed cancers among men but has limited prognostic biomarkers available for follow up. MicroRNAs (miRNAs) are small non-coding RNAs that regulate expression of their target genes. Accumulating experimental evidence reports differential miRNA expression in PCa, and that miRNAs are actively involved in the pathogenesis and progression of PCa. miRNA and androgen receptor signaling cross-talk is an established factor in PCa pathogenesis. Differential miRNA expression was found between patients with high versus low Gleason scores, and was also observed in patients with biochemical failure, hormone-resistant cancer and in metastasis. Metastasis requires epithelial-mesenchymal transition which shares many cancer stem cell biological characteristics and both are associated with miRNA dysregulation. In the era of personalized medicine, there is a broad spectrum of potential clinical applications of miRNAs. These applications can significantly improve PCa management including their use as diagnostic and/or prognostic markers, or as predictive markers for treatment efficiency. Preliminary evidence demonstrates that miRNAs can also be used for risk stratification. Circulatory miRNAs can serve as non-invasive biomarkers in urine and/or serum of PCa patients. More recently, analysis of miRNAs and circulating tumor cells are gaining significant attention. Moreover, miRNAs represent an attractive new class of therapeutic targets for PCa. Here, we summarize the current knowledge and the future prospects of miRNAs in PCa, their advantages, and potential challenges as tissue and circulating biomarkers. Prostate cancer (PCa) is the most commonly diagnosed cancer among men in western populations. The American Cancer Society estimated 239, 590 new cases and 29, 720 expected deaths in the USA in 2013. One in every six men are at risk of developing PCa during their lifetime (1). Currently, the standard biomarker for PCa diagnosis is prostate-specific antigen (PSA), which has its limitations, leading to the risks of PCa over diagnosis and harmful overtreatment. The prognostic value of PSA is also questionable (2). Stepping into the new epoch of personalized medicine, molecular markers are urgently needed to improve the different aspects of PCa management (3). miRNAs represent an attractive class of emerging biomarkers that can help in this regard (4;5).

Background: Metastatic clear cell renal cell carcinoma (ccRCC) patients have <9% 5-year survival rate, do not respond well to targeted therapy and eventually develop resistance. A better understanding of molecular pathways of RCC metastasis is the basis for the discovery of novel prognostic markers and targeted therapies., Methods: We investigated the biological impact of galectin-1 (Gal-1) in RCC cell lines by migration and invasion assays. Effect of Gal-1 expression on the mitogen-activated protein kinase pathway was assessed by proteome array., Results: Increased expression of Gal-1 increased cell migration while knocking down Gal-1 expression by siRNA resulted in reduced cellular migration (P<0.001) and invasion (P<0.05). Gal-1 overexpression increased phosphorylation of Akt, mTOR and p70 kinase. Upon hypoxia and increased HIF-1α, Gal-1 increased in a dose-dependent manner. We also found miR-22 overexpression resulted in decreased Gal-1 and HIF-1α. Immunohistochemistry analysis showed that high Gal-1 protein expression was associated with larger size tumor (P=0.034), grades III/IV tumors (P<0.001) and shorter disease-free survival (P=0.0013). Using the Cancer Genome Atlas data set, we found that high Gal-1 mRNA expression was associated with shorter overall survival (41 vs 78 months; P<0.01)., Conclusions: Our data suggest Gal-1 mediates migration and invasion through the HIF-1α-mTOR signaling axis and is a potential prognostic marker and therapeutic target.

There are no serum biomarkers for the accurate diagnosis of clear cell renal cell carcinoma (ccRCC). Diagnosis and decision of nephrectomy rely on imaging which is not always accurate. Non-invasive diagnostic biomarkers are urgently required. In this study, we preformed quantitative proteomics analysis on a total of 199 patients including 30 matched pairs of normal kidney and ccRCC using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to identify differentially expressed proteins. We found 55 proteins significantly dysregulated in ccRCC compared to normal kidney tissue. 54 were previously reported to play a role in carcinogenesis, and 39 are secreted proteins. Dysregulation of alpha-enolase (ENO1), L-lactate dehydrogenase A chain (LDHA), heat shock protein beta-1 (HSPB1/Hsp27), and 10 kDa heat shock protein, mitochondrial (HSPE1) was confirmed in two independent sets of patients by western blot and immunohistochemistry. Pathway analysis, validated by PCR, showed glucose metabolism is altered in ccRCC compared to normal kidney tissue. In addition, we examined the utility of Hsp27 as biomarker in serum and urine. In ccRCC patients, Hsp27 was elevated in the urine and serum and high serum Hsp27 was associated with high grade (Grade 3-4) tumors. These data together identify potential diagnostic biomarkers for ccRCC and shed new light on the molecular mechanisms that are dysregulated and contribute to the pathogenesis of ccRCC. Hsp27 is a promising diagnostic marker for ccRCC although further large-scale studies are required. Also, molecular profiling may help pave the road to the discovery of new therapies.

Background: Stability of microRNAs (miRNAs) in formalin-fixed paraffin-embedded (FFPE) tissues enables their reliable analysis in archived FFPE tissue samples, which are an invaluable source for the evaluation of novel biomarkers. Especially in breast cancer, for which late relapses occur in many cases, analysis of miRNAs in FFPE tissues holds great potential, because it can lead to the discovery of novel biomarkers suitable for future routine clinical diagnostics for breast cancer. We investigated the prognostic significance of 6 metastasis-related miRNAs that can critically regulate various stages of migration and invasion and play critical roles in the multistep metastatic process., Methods: We quantified the expression of 6 mature miRNAs (namely miR-21, miR-205, miR-10b, miR-210, miR-335, and let-7a) by reverse-transcription quantitative PCR in FFPE tissues of 84 patients with early breast cancer and a long follow-up and 13 cancer-free breast tissue FFPE samples that were used as the control group. We further correlated individual miRNA over- or underexpression with the disease-free interval (DFI) and overall survival (OS)., Results: Univariate analysis revealed that both miR-21 and miR-205 were significantly associated with DFI and only miR-205 with OS. Multivariate analysis demonstrated that miR-205 and miR-21 were independent factors associated with early disease relapse, whereas only miR-205 overexpression was associated with OS., Conclusions: Our results clearly indicate that deregulation of metastasis-associated miRNAs in primary tumors is associated with clinical outcome in patients with early breast cancer and can differentiate patients with higher risk in well-characterized subgroups.

microRNAs (miRNAs) are small molecules that regulate gene expression posttranscriptionally. In a previous study, we identified miR-96 to be upregulated in prostate cancer specimens in comparison to normal adjacent tissue and to be an independent marker of biochemical relapse in a multivariate prediction model. Therefore, we investigated the functional role of miR-96 in prostate carcinogenesis. LNCaP and DU145 prostate cancer cells were transiently transfected with miR-96 precursors and phenotypic changes were analyzed. The miR-96 increased proliferation and impaired apoptosis induced by camptothecine in these cells. In silico target prediction analysis identified FOXO1 as potential pro-apoptotic miR-96 target. miR-96 was able to bind to both bindings sites in the FOXO1 3' UTR in a luciferase reporter gene assay. Overexpression of miR-96 in LNCaP cells resulted in a reduced FOXO1 expression. Overexpression of FOXO1 induced a strong apoptotic phenotype that was partially rescued by coexpression of miR-96. RT-qPCR and immunohistochemistry of 69 prostate cancer specimens revealed a downregulation of FOXO1 and an inverse correlation of miR-96 and FOXO1 protein expression. In conclusion, we show that miR-96 can regulate apoptosis in prostate cancer, by inhibiting the FOXO1 transcription factor.

Purpose: Prostate-specific antigen testing has led to overtreatment of prostate cancer (PCa). Only a small subset of PCa patients will have an aggressive disease that requires intensive therapy, and there is currently no biomarker to predict disease aggressiveness at the time of surgery. MicroRNAs (miRNAs) are reported to be involved in PCa pathogenesis., Methods: This study involved 105 participants. For the discovery phase, prostatectomy samples were dichotomized to high-risk (n = 27, biochemical failure <36 months after prostatectomy) and low-risk groups (n = 14, ≥ 36 months without biochemical failure). Expression of 754 mature miRNAs was compared between the 2 groups. Linear regression models were built to accurately predict biochemical failure risk. miRNA mimics were transfected into PCa model cell lines to test effects on proliferation and to deduce responding signaling pathways., Results: We identified 25 differentially expressed miRNAs between the biochemical failure risk groups. Based on the expression of 2-3 miRNAs, 3 logistic regression models were developed, each with a high positive predictive value. Candidate miRNAs and the best-performing model were also verified on an independent PCa set. miRNA-152, featured in the models, was further investigated by using cell line models and was shown to affect cell proliferation. Predicted interaction between miR-152 and (mRNA)ERBB3 (erythroblastic leukemia viral oncogene homolog 3) was experimentally validated in vitro., Conclusions: miRNAs can help to predict biochemical failure risk at the time of prostatectomy.

Background: Tumor tissue-associated KLKs (kallikrein-related peptidases) are clinically important biomarkers that may allow prognosis of the cancer disease and/or prediction of response/failure of cancer patients to cancer-directed drugs. Regarding the female/male reproductive tract, remarkably, all of the fifteen KLKs are expressed in the normal prostate, breast, cervix uteri, and the testis, whereas the uterus/endometrium and the ovary are expressing a limited number of KLKs only., Conclusions: Most of the information regarding elevated expression of KLKs in tumor-affected organs is available for ovarian cancer; depicting them as valuable biomarkers in the cancerous phenotype. In contrast, for breast cancer, a series of KLKs was found to be downregulated. However, in breast cancer, KLK4 is elevated which is also true for ovarian and prostate cancer. In such cases, selective synthetic KLK inhibitors that aim at blocking the proteolytic activities of certain KLKs may serve as future candidate therapeutic drugs to interfere with tumor progression and metastasis.

MicroRNAs (miRNAs) play a crucial role in tumor progression and metastasis. We, and others, recently identified a number of miRNAs that are dysregulated in metastatic renal cell carcinoma compared with primary renal cell carcinoma. Here, we investigated three miRNAs that are significantly downregulated in metastatic tumors: miR-192, miR-194 and miR-215. Gain-of-function analyses showed that restoration of their expression decreases cell migration and invasion in renal cell carcinoma cell line models, whereas knockdown of these miRNAs resulted in enhancing cellular migration and invasion abilities. We identified three targets of these miRNAs with potential role in tumor aggressiveness: murine double minute 2, thymidylate synthase, and Smad Interacting protein 1/zinc finger E-box binding homeobox 2. We observed a convergent effect (the same molecule can be targeted by all three miRNAs) and a divergent effect (the same miRNA can control multiple targets) for these miRNAs. We experimentally validated these miRNA-target interactions using three independent approaches. First, we observed that miRNA overexpression significantly reduces the mRNA and protein levels of their targets. In the second, we observed significant reduction of the luciferase signal of a vector containing the 3'UTR of the target upon miRNA overexpression. Finally, we show the presence of inverse correlation between miRNA changes and the expression levels of their targets in patient specimens. We also examined the prognostic significance of miR-215 in renal cell carcinoma. Lower expression of miR-215 is associated with significantly reduced disease-free survival time. These findings were validated on an independent data set from The Cancer Genome Atlas. These results can pave the way to the clinical use of miRNAs as prognostic markers and therapeutic targets.

A 64-year-old woman presented with a hemorrhagic perifollicular rash on her legs while taking warfarin. After biopsy, vitamin C deficiency was suggested as the diagnosis, which ascorbic acid assays later confirmed. Clinical resolution of the rash followed supplementation with vitamin C. Patients on a vitamin K limited diet may also be limiting their intake of vitamin C. Physicians should be aware of this possible correlation, and consider checking vitamin C levels in patients with a perifollicular hemorrhagic rash or other signs of vitamin C deficiency while on warfarin.