Urological research is facing future challenges, the most difficult one is the fast and meaningful transfer of the massive amount of data from research basic to clinical practice. Between the most important issues that research should focus in the next years are targeting of tumor stem cells, clinical application of biomarkers, and wide application of genetic profiling of urological neoplasms. Several clinical implications are expected, from diagnosis to selection of candidates for different treatment modalities, to modulation of sequential treatment plans, to prognosis. A number of clinical trials based on research data from the hottest issues are in the pipeline. In this review, we will focus on new insights from recent work worlwide in urological research, with particular attention to high-risk nonmuscle-invasive and muscle-invasive bladder cancer, prostate cancer, and kidney cancer. Cancer care is moving towards a personalized approach in patient management. The most important issues in urological research point strongly in this direction and show an enormous potential for the rapid landing of Urology in the era of personalized medicine.
Urologia 2016; 83(1): 4 - 13
Article Type: REVIEW
AuthorsMariangela Mancini, Michele Zazzara, Filiberto Zattoni
- • Accepted on 05/02/2016
- • Available online on 26/02/2016
- • Published in print on 05/03/2016
This article is available as full text PDF.
Research in Urology is facing urgent challenges. The most critical one is the fast transfer of knowledge from basic scientific findings into clinical practice. Focusing on getting ready for this difficult task, we would like to underline the most promising fields for the next future: stem cells, genetic profiling, and biomarkers. The key point is not so much to improve diagnostic power of clinical tests or prognostic stratification of patients, but to design personalized treatment plans. Each tumor is going to be considered as an individual unique disease, and treatment plans will have to be tailored to such individuality (1, 2). Individualized therapy is not quite the same as targeted therapy, itself underdeveloped. In lung cancer, for example the main cause of death for cancer at present, differences between different chemotherapeutical protocols are negligeble (3).
The concept of individualized or personalized medicine is rapidly going to be extended to Urology. In particular, for each single urological cancer, modulation of clinical management sequences will be fine-tuned through genetic profiling of the tumor and of the patient. This innovative platform in clinical urology is in an advanced phase in basic research, currently being evaluated in animal models, and starting to be utilized in the clinic. Possible utilization of biomarkers and genetic profiling is under way, for example in active surveillance (AS) in prostate cancer, wherein new parameters can guide management and trigger a switch for surveillance to active treatment. Moreover, epigenetic biomarkers are under evaluation for diagnosis and prognostic definition of urological cancers (4). Epigenetic alterations can be useful for early diagnosis, as they usually happen before the development of the malignant phenotype. Early diagnosis of neoplastic relapse or fine prognostic tuning can be powered by epigenetic-based biomarkers, such as aberrant DNA methylations, or deregulated expression of chromatin structure proteins and miRNAs. This review is going to focus on these topics, with novel unpublished data and recent references, providing a synthesis of current knowledge, with a step already in the next future.
High-risk nonmuscle-invasive and muscle-invasive bladder cancer
The use of biomarkers for diagnosis of high-risk nonmuscle-invasive bladder cancer (NMIBC) is probably the most relevant issue in bladder cancer research. Early diagnosis of a high-risk NMIBC is a life-saving event for the patient. At least 25% of patients who die for bladder cancer had been diagnosed at first with nonmuscle-invasive disease (75% of patients are diagnosed with nonmuscle-invasive disease). Despite these data, worldwide mortality for bladder cancer is not decreased in the last 30 years (5). It is mandatory to identify reliable progression markers for bladder cancer, able to achieve an improvement in bladder cancer prognosis. Molecular classification has been shown to be useful in this regard, as reported at the 29th Annual EAU Meeting in Stockholm 2014, where subtypes of T1 bladder carcinomas at a high risk of progression have been identified (Best Abstract EAU 2014-Oncology: Patschan et al: 40 Molecular classification of T1 urothelial bladder cancer identifies high-risk subtypes) (6). Moreover, as for muscle-invasive bladder cancer (MIBC), treatment strategies did not change from chemotherapy and surgery in the last 30 years (7). No new drug has been approved and no studies on targeted therapies have been published for bladder cancer, except one case report (8). But something is changing. Projects are being carried out on predictive markers of response to neoadjuvant chemotherapy in high-risk patients (EUSP-European Urological Scholarship Program-Best Scholar Award 2014: Neuzillet Y: Predictive markers of response to neoadjuvant chemotherapy before cistectomy for bladder cancer, and the molecular characterization of non responder tumors) (9). Promising projects on this issue have been discussed at the last ESUR meetings in Glasgow 2014 and Nijmegen 2015. During the Glasgow meeting, Seth Lerner (Baylor College of Medicine Medical Center, Houston, Texas, USA) gave a talk about first results of a breakthrough study on molecular characterization of bladder cancer (Lerner, S.: Insights from TCGA-The Cancer Genome Atlas Project on Muscle Invasive Bladder Cancer, 22nd ESUR Meeting, Glasgow, Oct. 2014), recently published in
Efforts in biomarkers research in the last 10 years have included all levels of cellular molecular mechanisms, chromosomes, genes, transcriptoms, proteoms/peptidomes, and metaboloms (11). This significant mass of data has lead to a better understanding of cancer biology, but large-scale clinical transfer of this impressive work is still lacking, as well as a significant improvement in treatment strategies, or prognostic impact. Each year, the prestigious Dominique Chopin Award for Significant Achievement in Urological Research is awarded during the Annual ESUR Meeting. This prize is dedicated to the memory of the French urologist and scientist Dominique Chopin, a leader in European urological research in the last decades, recently deceased. Ellen Zwarthoff, Erasmus University, Rotterdam, NL, received the prize in 2014. The title of her talk at the meeting was “Development and validation of biomarkers for bladder cancer diagnosis and prognosis” (
Prof. Ellen Zwarthoff (Erasmus University, Rotterdam, NL), second from left, receiving the Dominique Chopin Award for Significant Achievements in Urological Research, (22nd ESUR Meeting, Glasgow, 2014). The prize motivation was Ellen’s pioneer work on biomarkers in clinical management of bladder cancer.
The problem of clinical relevance of biomarkers in bladder cancer needs to be fully elucidated: thousands of papers on biomarkers in cancer are published each year (13).
Despite this huge mass of information, only a few biomarkers have been transferred to current clinical practice. The next decade will have to be much more scientifically and clinically efficient than the previous one in biomarkers research. Studies will have to be rigorous, with an adequate number of tumors, and independent validations. Scientific journals will have to publish only biomarkers studies with high scientific standards, and results that can be clinically applied, reproduced, validated in different groups, and utilized as therapeutic targets. In receiving the Dominique Chopin Prize, Ellen Zwarkoff outlined the importance of collaboration between different medical specialties in order to achieve results rapidly transferable from the bench to the bedside (14). She finally underlined a very important and often forgotten issue: the inadequacy of bladder cancer research funding. In the US, the annual funding for bladder cancer research is 20 million dollars/year, while in other epithelial neoplasms, such as lung or breast cancer, funding is around 300-600 million dollars/year (
A particular issue of biomarker research is chemo-resistance and the discovery of novel mechanisms able to induce it. For this reason, paired cisplatin-resistant or sensitive cell lines have been created (18). According to the miRNA profile of these cells, it has been possible to identify a panel of RNAs able to stratify the cells for drug response. These RNAs target a glutamate-cystine carrier, implied in glutathion synthesis. As a consequence, the increased amount of glutathione binds to cisplatin, preventing DNA damage. Transfecting the resistant cell lines with miRNA and reversing this targeting, it is possible to restore cisplatin sensitivity in cisplatin-resistant cells. Accordingly, in human tumor samples, high expression of the glutamate-cystine transporter is associated with resistance to cisplatin, suggesting that this transporter can be utilized as a prognostic-therapeutic biomarker, usable to design individualized treatment strategies.
Circulating tumor cells
Circulating tumor cells (CTCs) are responsible for the majority of bladder cancer deaths (19-20-21). Molecular and immunological methods to evaluate early dissemination of CTCs have been discovered, (22) such as the FDA-approved CellSearch® System (23). The majority of studies published so far did not show a clear association between CTS and clinical parameters in bladder cancer. However, it has been recently reported that the presence of CTCs before surgery is associated with survival, disease-free survival, and cancer-specific death in nonmetastatic MIBC patients treated with cystectomy (22). Moreover, CTCs can be phenotypically and molecularly characterized, in order to identify novel therapeutic targets, to monitor clinical response to therapy, and to stratify patients for individualized management plans. Clinical trials are under way at the moment to test the potential of CTCs as key actors in the decision-making process of multimodality therapies.
Final remarks on bladder cancer research
It is time to launch a public campaign to promote funding for bladder cancer research. New treatment strategies are needed to improve the survival of bladder cancer patients. Surgical techniques for bladder cancer treatment are currently well codified regarding the open, laparoscopic or, more recently, robotic approach. On the contrary, research on new drugs, new therapeutic strategies, and new individualized management plans is highly needed. Bladder cancer is a very heterogeneous disease, not only between different patients but also between different lesions in the same patient or between primary tumor and metastases. The TCGA (The Cancer Genome Atlas (10, 24)) is now providing data on all mutations, alterations, methylation patterns, and genetic expression in MIBC. New potential therapeutic targets are being identified. It is now possible to envision what the future approach to bladder cancer treatment will be: multiple sampling from different tumor zones or “liquid biopsy” for CTCs, genetic profiling and identification of driving mutations that will drive decisions and will be targeted by adjuvant therapies. Moreover, signaling pathways, cross-talk mechanisms, and feedback loops will be characterized, and will be selectively blocked with specific inhibitors. A translational approach to bladder cancer treatment is being clearly defined (25). The main challenges are early identification of potentially progressing or relapsing tumors, simplification of follow-up plans, and reduction of cystoscopies and, in case of MIBC, identification of which tumors will benefit from cystectomy and other therapies and in which temporal sequence. A large amount of work currently ongoing on biomarkers for bladder cancer surveillance will most probably lead to clinical significant findings. A large number of genetic data has appeared in the last 15 years. Papillary tumors, often noninvasive and harboring oncogenes mutations and limited additional mutations, show a relatively good prognosis. In contrast, solid invasive tumors show a complex genetic mutational pattern and have a poor prognosis. Classical pathological factors and genetic signatures can be used to stratify patients in prognostic subgroups in NMIBC. Moreover, in MIBC, pathological features and molecular changes can help to predict prognosis and response to therapy. Various different pathological subtypes requiring distinct therapeutical approaches have been described. A real breakthrough is represented by the TGCA project, which demonstrates that bladder cancer is a very complex genetic disease, suitable for targeted therapy. The whole genome mutation analysis will open the way to new targeted treatments on the basis of molecular stratification of bladder cancer. Important innovations are on the way.
Biomarkers in prostate cancer
Biomarkers research is very active in prostate cancer. A recent important study shows the importance of inhibitors of mitochondrial autophagy, associated with poor prognosis (26). Mitochondrial autophagy is regulated by the Pten gene and by its target gene Pink1. The deletion of the Pten gene is associated with the growth of aggressive prostatic carcinoma, as shown in animal models (27). In addition, proteins involved in mitochondrial autophagy could be associated with carcinogenesis. LRPPRC (leucine-rich pentatricopetide repeat motif-containing protein) is a protein, which stabilizes the Bcl-2 oncogene and blocks autophagy, disrupting the cell death regulatory mechanisms. Immunohystochemistry of expression of LRPPRC in 112 samples of prostate cancer was compared with the expression of the same protein in 38 samples of BPH. Tumor levels of the protein were significantly higher than BPH levels, and a significant correlation was found with other prognostic factors in tumors, such as Gleason score, PSA, metastatic state, hormone resistance within 2 years, and poor survival. This study identifies LRPPRC as a new prognostic biomarker in prostate cancer and the mitochiondria as a new potential therapeutic target. Another example of prostatic biomarkers is PIAS1, a key factor for survival of prostatic cancer cells and a possible target in Docetaxel resistance (28). Finally, the inhibition of mcl-1 during hormonal therapy has been demostrated (29).
Pathobiology of prostate cancer, an important factor for efficient molecular prognostic definition and risk stratification, is only partially defined. An epigenome-wide discovery approach has been applied to discover epigenetic events in prostate cancer, such as DNA methylation or microRNA expression, during the transition from normal cell to precancerous lesions, to primary tumor, and to metastatic disease. It is now possible to compare epigenomes of indolent and aggressive tumors, in order to identify early potentially aggressive tumors (30). Finally, with a lipodomic approach based on mass spectrometry, it is now possible to characterize important alterations in fospholipidic profiles of several urological neoplasms, including prostate and kidney cancers. These changes are driven by alterations in key metabolic pathways, such as fatty acids synthesis and elongation. Phospholipids are functional and structural elements of cellular membranes. Their alterations are implied in various cancer-specific functions, including cell survival and response to therapy. These data suggest that lipidomic analysis plays an important role in personalized treatment strategies, and can have a potential implication in the development of biomarkers for prostate cancer. Finally, it is worth mentioning the Marie Curie Initial Training Network PROSENSE (31) “Cancer diagnosis: Parallel Sensing of Prostate Cancer Biomarkers,” one of the most relevant innovations in biosensor research in prostate cancer. This project’s main task is to train a new generation of scientist in interdisciplinary techniques and methods required for development of innovative diagnostic tools in prostate cancer. New platforms “lab-on-a-chip” are being designed, based on electrochemical sensors, nanotecnology, memristors and novel optical techniques. This tool will allow simultaneous screening of panels of protein biomarkers, glycosylation processes, or miRNAs for early diagnosis of prostate cancer and for biochemical follow-up of the evolution of the disease, in real time and in a personalized fashion.
Genetic profiling of prostate cancer
Decisions in Oncology are based currently on histo-pathological features of the tumor, rather than on genetic or molecular alterations. Tumors are categorized traditionally on the basis of morphological aspects, and distinct according to static, histological and structural criteria. Morphological analysis alone, even in its most sophisticated forms, such as electron microscopy, is being currently integrated by genomic or molecular data (
More clinical trials are needed on this promising topic, some of which are ongoing at the moment.
Stem cells and the epithelial-mesenchimal transition (EMT)
Two of the most important characteristics of aggressive prostate cancer are (1) loss of cellular differentiation and of prostate architecture, with an expansion of the stem cells, and (2) the ability of prostate cells to acquire mobility and to grow outside the prostate, through the epithelial-to-mesenchymal transition, EMT. The development of therapies able to block cellular de-differentiation and the EMT is highly needed. The role of stromal cells in promoting the EMT is currently under investigation. There is a growing interest in noncoding miRNA in the stem cells of prostatic carcinoma. A large number of miRNAs are involved in stem cells regulation. Prostate stem cells and EMT are very difficult to be demonstrated. Recent studies have shown the presence of stem cells in the prostate, and their possible key-role in disease progression and resistance to treatment. There is certainly an association between stem cells and resistance to therapy. Indeed, any human tumor contains a population of quiescent stem cells resistant to therapy (42). This population is extremely important. Stem cells, malignant as well as benign, are equipped with multiple resistance mechanisms. Therapies currently in use, such as hormonal therapy, can promote an expansion of the resistant stem cells population. The EMT is mandatory for the development of the malignant phenotype of tumor cells (43, 44). Transformed epithelial cells can activate embryonary programs of behavior and epithelial plasticity, moving from a sessile epithelial phenotype, to a mesenchimal mobile one. Interactions between the supporting stromal microenvironment and the neoplastic tissue, which lead to the oncogenic EMT, are essential for tumor growth, progression, and resistance to therapy. EMT induction can be regulated by several cytochines and growth factors, such as transforming growth factor (TGF)-beta. Studies on animal models of prostate cancer have shown that a subset of tumor cells with a staminal phenotype is more tumorigenic and able to metastasize. Osteotropic prostate cancer cells colonize the hematopoietic niches in the system bone-bone marrow. Molecular characterization of the stromal reaction in bone metastasis shows the expansion of the hematopoietic niches and of the stem cells (44). The stroma is able to produce factors that regulates the EMT (such as TGF-beta, DV-integrines, and miR25). A new small molecule, OCD155, has been recently identified, able to efficiently antagonize the EMT and the acquisition of the aggressive phenotype. This compound is well tolerated and inhibits the growth of the metastasis in preclinical models of prostate cancer
A stem cells population in the luminal layer of the prostate has been recently discovered (46). The project has been supported by a grant of the EAU Research Foundation (EAU-RF). Tumor growth seems to start from stem cells, which had been identified so far only in basal layer of the prostate. In contrast, luminal stem cells, slowly replicating, have been demonstrated in the luminal differentiated layer in a mouse model. These stem cells express surface receptors and the nuclear AR, a fact that opens the possibility that AR-expressing luminal stem cells could function as oncogenic cells. This is the first paper to report that stem cells can be found in the prostatic luminal layer (46). Some of the biomarkers linked to stem cells could in the future be used as an aid in the personalized approach to prostate cancer therapy. Products of these biomarkers could be used as targets. Work is being carried out on identification of proteins activated in stem cells that could be useful for prognostic definition, for patient stratification for therapy or therapeutical sequential plans (47, 48). There is an urgent need to characterize better human stem cells and their related biomarkers. It is extremely relevant to validate the results on human tumor samples. The next challenge is to move from the murine model to human patients.
Genetic signatures for prevention of overdiagnosis and overtreatment of prostate cancer
The problem of overdiagnosis of prostate cancer is a current difficult challenge. In the past, the focus was on
The figure represents a whole-mount section of a prostate after radical prostatectomy and shows the risks of patients selection for AS. Two foci of Gleason 8 tumor had been missed by the pre-operative biopsy, which had revealed only a small Gleason 6 tumor (marked with the dotted yellow line; slide courtesy of Dr. Judd Moul. Duke Cancer Institute, Durham, NC, Annual Meeting ESOU, Munich, 2015).
Despite the doubts and risks of AS, overdiagnosis still represents an important challenge in prostate cancer management, especially in case of PSA increase based diagnosis. AS strategies have risen in frequency in the last 10 years, in order to avoid overtreatment. It is known that after 10 years, more than 50% of patients switch to active treatment, in the majority of cases for progression of T stage, or grade, or PSA increase. About 10-20% of these “deferred treatment patients” are not curable, which means that for some patients, the switch has been too late. There are currently no evidence-based triggers to guide the switch from AS to active treatment. In protocols currently in use, PSA is checked every 3-6 months, and a re-biopsy performed every 1-2 years. Re-biopsy is associated with an increased number of severe complications with time and number of biopsies (65). mpMRI is getting increased attention as a noninvasive modality, well accepted by the patients. A positive MRI makes the identification of a high-risk cancer more probable. Until ongoing studies are completed, it is necessary to repeat the biopsies, in order not to loose the window of opportunity to treat prostate cancer patients. Genetic profiling and tumor biomarkers can fill the dangerous void still present in AS protocols. The future will move in the direction of an integrated approach between genetic profiling, mpMRI, and re-biopsy, with a primary endpoint to optimize the switch and eliminate the disturbing 10-20% of patients who seem currently to miss the window of opportunity for treatment.
Genetic signatures and biomarkers in renal carcinoma
Renal cell carcinoma (RCC) is the most frequent kidney tumor (more than 90%). It is subdivided into histologically distinct subtypes. The most frequent are clear cell RCC (ccRCC, 70-75%), papillary carcinoma (10-15%), chromophobic carcinoma (5%), and benign oncocytoma. Despite the vast amount of molecular data now available, the nomograms for prognostic risk in renal cancer are currently based only on clinical and pathological parameters. High throughput technologies have made possible a fine molecular mapping of tumors. Integration of expression analysis, methylome analysisis, and genome-wide association analysis could lead to redefinition of nomograms and to a real personalized approach to renal cancer management. Genetic analysis has shown that RCC subtypes are characterized by distinct chromosomal alterations, suggesting that each subtype is a distinct oncological entity, with a different biological behavior. Genetic analysis, in this regard, can provide innovative and essential data. Subtypes can show a different expression pattern of biomarkers as well. The most promising biomarkers are those derived from miRNAs. A number of studies have recently shown distinct miRNA profiles for each cancer subtype. Biomarkers and genetic profiling are possible not only on the primary or metastatic tumor tissue but also on circulating tumor fragments. The presence of circulating tumor fragments in ccRCC has been demonstrated (66). Laminin-coated cellular aggregates with metastatic potential have been found. Moreover, ccRCC expresses large amounts of endothelial and vascular growth factors (in particular VEGF) correlated with tumor angiogenic ability, a key factor in distant metastatic potential (66). This angiogenic factor is often overexpressed in ccRCC (as a result of mutations of the Von Hippel Lindau gene,
Regarding novel imaging modalities, molecular analysis has demonstrated a direct link between molecular defects in ccRRC and expression of carbonic anhydrase IX (CAIX) (81). CAIX is a reliable biomarker for the differential diagnosis
In conclusion, biomarkers and genetic signatures are useful for prognostic individual definition. It is necessary to define individual risk of metastatic potential in confined tumors, and to develop effective systemic therapies, not only to treat metastatic patients but also as adjuvant strategies. Genetic analysis of primary tumors has shown that ccRCC, either metastatic or synchronous or metachronous, harbors specific chromosomal alterations when compared with a primary nonmetastatic tumor. The mRNA expression profiling also shows differences between primary nonmetastatic and metastatic tumors. Complex analysis of molecular alterations at the genetic, epigenetic, and expression level, as the one provided by the TCGA (The Cancer Genome Atlas Project), (24) offers novel data on important characteristics of each subtype. We can foresee a new era for the personalized approach to renal cancer management.
The “PRIME-approach” in oncological urology
The PRIME-approach (AppRoach to IMprovE the outcome of cancer treatment in uro-oncology) was recently introduced in the program of the ESOU Annual Meeting (Munich, 2015); this innovative approach’s prime goal is to improve management of cancer in oncological Urology. The aim is to identify the most appropriate single intervention or multimodal treatment strategy of a specific tumor, considering all parameters, both cancer-specific and noncancer-specific. A personalized cancer treatment plan can be therefore designed, specific for each patient and for each single tumor. New challenges in urological research, such as biomarkers, stem cells, and genetic signatures are part of this picture, and open the way to the next future in Urology. It is key to be aware of this process and ready for the task.