MASARYK UNIVERSITY
FACULTY OF MEDICINE
HABILITATION THESIS
Brno 2022 Michal Fedorko, MD, Ph.D.
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MASARYK UNIVERSITY
FACULTY OF MEDICINE
DEPARTMENT OF UROLOGY
Habilitation thesis
Possibilities of diagnosis and prognosis of renal cell carcinoma using novel
biomarkers
(Collection of previously published scholarly works)
MUDr. Michal Fedorko, Ph.D.
Brno
2022
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Acknowledgement
My thanks go to prof. MUDr. Dalibor Pacík, CSc. for the support of my professional
development within the Department of Urology of the Faculty of Medicine of Masaryk
University and University Hospital Brno and his valuable comments and advice in my scientific
research work. Thanks to prof. RNDr. Ondřej Slabý, Ph.D. and his research group at CEITEC
MU Brno for inspiring cooperation on projects related to urological oncology, especially renal
cancer. Last, but not least, I want to thank my wife Beata for her long-term love, support and
patience during the many moments I devoted to my work and professional growth instead of
my family and my daughters Michalka and Karolínka for endless inspiration in my personal
and professional life.
……………………………..
MUDr. Michal Fedorko, Ph.D.
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Table of contents
1. Objectives of the habilitation thesis 4
2. Summary 5
3. Introduction 6
4. Molecular genetic mechanisms in the pathogenesis of renal cell carcinoma 17
5. Biomarkers of renal cell carcinoma 20
6. Non-coding RNAs and their use in clinical practice 28
6.1. Short non-coding RNAs 29
6.1.1. MicroRNAs 29
6.1.2. PIWI-interacting RNAs 75
6.2. Long non-coding RNAs 90
7. Future perspectives 102
8. Conclusion 104
9. List of figures and tables 105
10. List of abbreviations 106
11. Author’s papers included in the text 109
12. List of references in the text of the commentary 111
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1. Objectives of the habilitation thesis
The submitted habilitation thesis is a summary of published scientific works of which the
applicant is the author or co-author. These works represent the results of long-term scientific
research activities of the applicant related to renal cell carcinoma, especially in the search for
potential non-invasive biomarkers for diagnosis and follow-up. Outputs presented by the
applicant at international professional conferences are also included. The individual
publications are connected by an accompanying commentary, which puts them in context with
the current state of knowledge in the given issue. The work is divided into three thematically
related parts with the following goals:
1. Introduce the issue of renal cell carcinoma in terms of its occurrence and prognosis of
individual stages and reveal the complex molecular genetic mechanisms involved in its
pathogenesis (chapters Introduction and Molecular genetic mechanisms in the pathogenesis of
renal cell carcinoma).
2. To justify the need to search for biomarkers of this disease, to present the types and individual
groups of biomarkers and possible areas in which these biomarkers are useful in clinical practice
(chapter Biomarkers of renal cell carcinoma).
3. Describe in detail the individual groups of non-coding RNAs and their possible use in the
diagnosis and prognosis of renal cell carcinoma, critically evaluate their benefits and potential
direction of further research in this area (chapters Non-coding RNAs and their use in clinical
practice, Short non-coding RNAs, Long non-coding RNAs).
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2. Summary
Despite intensive research, there are currently no reliable biomarkers available for routine
practice for early detection, prognosis, or follow-up of renal cancer including the treatment
response, although it is a cancer with a significant proportion of advanced stages at the time of
diagnosis and thus with a considerable lethality. We therefore rely on the detection of the
disease by imaging examinations (very often incidentally) and we also use imaging practically
uniformly when monitoring patients after surgical or systemic treatment. Non-coding RNAs
whose levels can be determined in tissues and body fluids and which are able to differentiate
tumour tissue from non-tumour tissue and tumour patients from or healthy individuals represent
a significant shift forwards. Presented papers confirm the diagnostic and prognostic value of
many groups of these RNAs. A detailed analysis of the tumour tissue may thus reveal
biomarkers of potentially aggressive renal carcinomas, recurrent tumours or tumours poorly
responsive to treatment. The determination of circulating non-coding RNA levels as a
minimally invasive method of diagnosis fits into the attractive concept of so-called liquid
biopsy of cancer. The results are encouraging. The use of urine as a completely non-invasive
source of biomarkers is also attractive. Here, the data is still premature and insufficient. Last
but not least, in vitro and in vivo tests reveal the therapeutic potential of many of the RNAs
described, which, in combination with novel therapies for metastatic renal cancer, could lead to
prolonged overall and cancer-specific survival in this group of patients.
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3. Introduction
According to the latest data, renal cell carcinoma (RCC) accounts for 4.2% of all cancers in
men and 2.6% in women (1). Worldwide, the age standardized incidence (ASR) of RCC is 4.6
cases per 100 thousand in both sexes (16th
most common cancer), 6.1 per 100 thousand in males
(10th
most common cancer) and 3.2 per 100 thousand in females (14th
most common cancer).
Its current mortality is 1.8 per 100 thousand (2). In a long term, the highest incidence has been
reported in Northern America and European countries (Fig. 1). The Czech Republic has had
one of the highest incidences of RCC in world for several decades. The current ASR (14.4 per
100000) is the second highest after Lithuania (14.5 per 100000). In the Czech Republic, age
standardized incidence is 22.1 per 100 thousand for men and 9.9 per 100 thousand for women,
so it is 6th
and 10th
most common cancer, respectively (Fig. 2).
Fig. 1. Incidence and mortality (ASR) of RCC in different regions (2).
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Fig. 2. Age-standardized incidence (ASR) of ten major cancers in both sexes in the Czech
Republic (1).
According to the data of the National Cancer Registry (NOR), the age standardized incidence
and mortality of RCC in 2018 was 14.37 / 100 thousand and 4.01 / 100 thousand, respectively
(3). The development of incidence and mortality in the Czech Republic over time correlates
with the development in other industrialized countries, the highest incidence was reached in
2005, mortality has been slowly declining (or remains at a stationary level) since 2002 (Fig. 3,
4).
Fig. 3. RCC incidence growth index (1977 – 2018) (3)
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Fig. 4. Time evolution of incidence and mortality of RCC in the Czech Republic (3).
RCC is still one of the most lethal urological malignancies. Its five-year relative survival, i.e. a
statistical comparison of the survival of cancer patients and other populations of the same age,
race and sex without tumour (expressed as a percentage of patients who survive 5 years) is
74.8% (4). Of the urological malignancies, only malignant tumours of the penis and ureter reach
lower values, but their incidence is less than 1 case per 100 thousand (Table 1).
Malignancy 5-year relative survival
Prostate cancer 98%
Testicular cancer 95,2%
Bladder cancer 77,1%
Kidney cancer 74,8%
Penile cancer 66,7%
Ureteral cancer 46,5%
Tab. 1. Comparison of five-year relative survival of patients with tumours of the urogenital
tract (4).
The five-year relative survival of patients with RCC is gradually increasing (Fig. 5), but still a
relatively significant proportion of patients are diagnosed at an advanced stage of the disease,
i.e. with regional lymph node involvement or distant metastases (Fig. 6), when the percentage
of surviving patients decreases dramatically (Fig. 7). The increasing proportion of localized
tumours is mainly due to the use of imaging methods from another indication, which
incidentally detect a kidney tumour, which would otherwise manifest itself in the advanced
stage due to localization in the retroperitoneum (5).
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Fig. 5. Time evolution of 5-year relative survival of RCC patients (4).
Fig. 6. Percentage of RCC stages at the time of diagnosis (4).
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Fig. 7. Comparison of five-year relative survival according to the stage of RCC at the time of
diagnosis (4).
The proportion of clinical stages of RCC in the Czech Republic at the time of diagnosis is
similar, stage IV tumours make up to 20% (Fig. 8).
Fig .8. The proportion of clinical stages of RCC in the Czech Republic (3).
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In addition to these advanced stages, tumours that progress or recur after primary treatment
generally have a poor prognosis. Thus, it is clear that a prerequisite for effective treatment is
the early diagnosis of RCC and the identification of patients at risk of disease relapse or the
need for further treatment to achieve a better clinical effect.
Published work related to the topic:
Fedorko M, Kiss I, Pacík D. Nádory ledvin. In: Matějovská Kubešová H, Kiss I, eds.
Geriatrická onkologie. Praha, Česká republika: Mladá fronta; 2015: 120-124.
The chapter in the monography provides summary information on kidney tumours in terms of
epidemiology, diagnosis and treatment of localized and metastatic disease. It specifically targets
a group of patients older than 65 years, describes the benefits, risks and results of RCC treatment
in the geriatric population.
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4. Molecular genetic mechanisms in the pathogenesis of renal cell
carcinoma
The vast majority of renal cell carcinomas are sporadic tumours (96%), the remainder being
familial tumours manifested in known hereditary syndromes characterized by specific gene
mutations, the histological subtype of RCC and comorbidities. Research into hereditary forms
of RCC has led to the elucidation of the basic molecular genetic pathways of the pathogenesis
of RCC with a significant impact on new therapeutic approaches, especially the so-called
targeted treatment. These are the VHL / HIF cascade, PI3K / AKT / mTOR, Wnt / β-catenin,
HGF / MET, epithelial-mesenchymal transition and other genetic and epigenetic alterations (6).
In this chapter, these mechanisms are briefly described mainly for their significant influence by
short and long non-coding RNAs, which can be used as diagnostic, prognostic and therapeutic
biomarkers on the basis of these interactions (7).
VHL/HIF pathway
Molecular genetic studies in patients with von Hippel-Lindau (VHL) disease led in 1993 to the
identification of the VHL tumour suppressor gene, which is located on the short arm of
chromosome 3 (3p25-26). As with other solid tumours, RCCs are characterized by a state of
hypoxia due to an imbalance between oxygen supply and consumption. A critical regulator of
the hypoxic response is a mutation in the VHL gene. The VHL protein, together with other
factors, forms a complex that is responsible for the degradation of so-called hypoxia inducible
factors (HIF). These factors further regulate the expression of more than 200 genes whose target
proteins significantly affect angiogenesis, glucose metabolism, invasion, mitogenesis, cell
proliferation and survival and development of metastasis (VEGF, PDGF, EGFR, TGFα, Glut1,
MUC1 and others). Under hypoxic conditions in the absence of VHL, HIF-1α and HIF-2α are
stably expressed and induce the production of VEGF and other mentioned factors (Fig. 9). VHL
inactivation is responsible for virtually all familial clear cell RCC (ccRCC) and at least 2/3 of
sporadic ccRCC. Inactivation of both VHL gene alleles is necessary for RCC - in case of VHL
disease one allele is inherited mutated and mutation of the other leads to RCC, in case of
sporadic ccRCC both alleles are inactivated after birth, which leads to late RCC and unifocal
occurrence (8).
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Fig. 9. VHL/HIF pathway. Adapted from Bratslavsky et al. (9).
PI3K/AKT/mTOR
Protein kinase 3 (AKT) and mammalian target of rapamycin (mTOR) significantly affect cell
proliferation, cell survival, and angiogenesis. Binding of VEGF and PDGF to their receptors on
tumour cells activates PI3K and leads to overproduction of PIP3, thereby transferring
cytoplasmic AKT to the cell membrane, where it is activated. Activation of AKT results in
inhibition of apoptosis by both inactivation of proapoptotic proteins (such as procaspase 9) and
impaired degradation of proteins that promote cell cycle (e.g., cyclin D1) or proliferation (cmyc,
β-catenin). Stimulation of mTOR production positively regulates cell cycle protein
synthesis. The mechanism of AKT activation in tumours is probably through the decreased
expression of the tumour suppressor gene PTEN (10). In addition, mTOR activation further
stimulates HIF production and is therefore involved in the VHL / HIF cascade, too.
Wnt/β-catenin
Wnt is a family of glycoproteins that regulate cell proliferation, differentiation and migration.
The final effector of Wnt is β-catenin, a transcriptional co-activator that enters the nucleus and
activates the transcription of oncogenes, such as MYC. Increased β-catenin production is also
described in the case of VHL inactivation, which may be another way in which VHL is involved
in the pathogenesis of RCC. Wnt also stimulates the mTOR cascade by inhibiting GSK3.
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Epithelial – mesenchymal transition
The kidney is of mesenchymal origin and the process of mesenchymal-epithelial transition
results in the formation of epithelial structures, which further develop into nephrons. In the case
of ccRCC, this process is reversed, i.e. epithelial-mesenchymal transition (EMT), which is a
necessary prerequisite for the development of metastases. EMT requires activation of several
transcriptional regulators (ZEB1, SIP1, Snail, Slug), whose target protein is E-cadherin (crucial
for the epithelial phenotype). Loss of E-cadherin leads to dissociation of intercellular junctional
complexes. Furthermore, the repression of E-cadherin releases β-catenin, which activates the
transcription of mesenchymal markers (vimentin, fibroblast-specific protein 1). And, as with
the aforementioned signalling pathways, the absence of VHL with overproduction of HIF-1α
may be one of the factors leading to downregulation of E-cadherin. Interesting is the action of
TGF-β, a multifunctional cytokine involved in EMT, which in normal cells acts as a tumour
suppressor, but in the microenvironment of tumour cells acts as a promoter (11).
HGF/MET
Changes in the expression of hepatocyte growth factor (HGF) and its c-MET receptor are
associated with the development of hereditary papillary RCC (pRCC). Mutations in the MET
gene are also observed in 5-13% of sporadic pRCCs. MET phosphorylation stimulates the
growth and metastasis of RCC by activating the PI3K / AKT and Ras / MAPK cascades. In
addition, it induces the phosphorylation of β-catenin and subsequent transcription activation.
However, HGF is also related to VHL expression in RCC tumour cells: the higher VHL
expression, the more suppressed β-catenin production by HGF.
Genetic and epigenetic alterations in RCC
Genetic alterations are mainly gene mutations of various tumour suppressor genes, while
epigenetic changes (i.e. without changing the nucleotide sequence of DNA) are changes in
chromatin, the remodelling of which can activate regulatory factors, such as transcription
factors in chromatin DNA. These are usually changes in histones (methylation, demethylation,
acetylation) or ATP-dependent remodelling complexes. The most important chromatin
remodelling genes include PBRM1 (mutated in up to 41% of ccRCC), SETD2 or BAP1 (12).
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5. Biomarkers of renal cell carcinoma
RCC is considered a primary surgical disease because it can only be cured if it is localized and
thus surgically removable. The diagnosis is based on imaging, either specifically due to the
suspicion of a focal process (but the clinical suspicion usually indicates a locally advanced or
metastatic tumour), or the kidney lesion is diagnosed incidentally, by imaging from another
indication (more than half of all cases). Several studies have argued in favour of early diagnosis,
showing a lower stage of the disease and a better prognosis of accidentally detected tumours
compared to symptomatic tumours (13). In contrast, there is a group of small tumours (up to 4
cm) in which up to 20% are benign and clinically insignificant tumours for which early
detection would lead to unnecessary diagnosis and treatment. Imaging examinations may
therefore not reliably distinguish between benign and malignant tumours, tumour biopsy has its
exact indications and is not routinely recommended (14). The exclusive role of imaging
methods in the diagnosis and follow-up of RCC patients (as well as in the screening of selected
group of patients such as end-stage renal disease or hereditary forms of RCC) could be changed
by the identification of sufficiently accurate biomarkers.
In general, biomarkers of renal cell carcinoma can be divided into (i) biomarkers for early
detection of RCC, (ii) diagnostic biomarkers for individual RCC subtypes, (iii) prognostic
biomarkers, and (iv) biomarkers for prediction of treatment response (15).
In terms of early detection, there is currently no biomarker available in routine clinical practice
and this topic is therefore a relatively urgent challenge, as there is still a relatively significant
proportion of advanced forms of RCC at the time of diagnosis compared to other malignancies.
The detection of aggressive tumours with highly malignant potential among so-called small
tumours is also a challenge. Non-invasive detection assumes the presence of this biomarker
ideally in the blood (e.g. M2 pyruvate kinase, circulating VEGF, carbonic anhydrase 9, TATI,
M65) or urine of the patient (NMP22, AQP1, KIM-1, proteomic or metabolomics analysis).
Proteomics and metabolomics as an alternative way of identifying RCC have not yet found
wider application in clinical practice. Research on the urine proteome / metabolome is
particularly attractive. The main goal of proteomics is to detect quantitative and qualitative
differences between normal and pathological samples and to identify differentially expressed
proteins (16). Metabolomics examines endogenously produced metabolites in urine. In the case
of RCC, metabolic activity is characterized by disorders of energy metabolism (glycolysis,
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metabolism of amino acids and fatty acids), which are essential for cell growth and proliferation
(17).
The term “liquid biopsy” is generally used for the attractive non-invasive alternative to a
standard biopsy in order to obtain diagnostic tumour material (18). In relation to the early
detection of RCC, its purpose could be to detect a micro-cancer that cannot be revealed by
imaging. Although peripheral blood is usually mentioned as a fluid in this context, it can also
be urine or cerebrospinal fluid. In this way, circulating tumour cells (CTC), tumor-derived cellfree
DNA (ctDNA), tumor-derived cell-free miRNAs or extracellular vesicles (exosomes) can
be detected. However, in the case of localized RCC, the evaluation of ctDNA and CTCs is very
difficult compared to other solid tumors (19). Therefore, CTC detection tends to focus on
metastatic RCC, where baseline positivity may be a prognostic marker of progression-free
survival (20).
Diagnostic biomarkers to differentiate histological subtypes of RCC are used in
immunohistochemical examination of tumour tissue. One specific biomarker for a particular
subtype is not available, so the expression profiles of several proteins are determined. These
can be enzymes (carbonic anhydrase 9), cytoskeletal proteins (vimentin, keratins), adhesion
proteins (E-cadherin), CD proteins, transcription factors, glycoproteins or immunoglobulins.
Prognostic biomarkers are expected to correlate well with the clinicopathological
characteristics of the tumour and to be used in monitoring disease progression and patient
survival (Bcl-2, survivin, Ki-67). At the time of targeted biologic therapy with tyrosine kinase
inhibitors, mTOR inhibitors, and anti-growth factor antibodies, the use of predictive
biomarkers of treatment response (e.g. high expression of carbonic anhydrase 9 in ccRCC
associated with a favourable response to sorafenib and sunitinib, decreased VEGF levels during
treatment, blood neutrophil-to-lymphocyte ratio) is very appropriate. Anyway, in the case of
metastatic RCC, only MSKCC and IMDC risk scores have been validated as prognostic tools
and included in relevant international guidelines (21). That is why research in the field of renal
cancer biomarkers is increasingly focused on metastatic RCC.
One of the most important changes in the treatment of advanced stages of RCC in recent years
has been the introduction of immune checkpoint inhibitors, i.e. drugs that block the inhibitory
checkpoint molecules CTLA-4 and PD-1. The main well-studied biomarkers of immune
checkpoint blockade, PD-L1 expression and tumour mutational burden did not demonstrate
sufficient predictive ability (22, 23). Limitations on the use of PD-L1 expression include its
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intra-tumoral heterogeneity, heterogeneous expression between primary tumor and metastases,
unclear cut-off value defining positivity and variability of expression depending on sample age,
previous treatment, analysed cell population or assay choice (24). Therefore, the search for
other biomarkers is also fully justified here.
Published work related to the topic:
Fedorko M. Diagnostické možnosti časného zjištění nádorů ledvin, role nádorových
markerů při stanovení dalšího postupu. Urol List 2011; 9(4): 7-11.
The review describes the issue of screening and early detection of RCC, known molecular
biomarkers of renal cell carcinoma and their potential clinical applications based on the
knowledge of the time.
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6. Non-coding RNAs and their use in clinical practice
The central dogma of molecular biology is that RNA functions as an information intermediate
between DNA (gene) and the protein it encodes. Thus, the vast majority of genetic information
responsible for biological form and phenotype was thought to be expressed in proteins surprisingly,
the human genome encodes only about 20,000 proteins, which is less than 2% of
its total content. In the human transcriptome (it is important to note that up to 90% of the
genome is actively transcribed), in addition to the coding mRNA (which is actively translated),
there are a number of antisense transcripts and non-coding RNAs thought to be transcriptional
noise or evolutionary waste. However, current knowledge indicates that non-coding RNAs may
play a crucial role in cell development, physiology and pathology (25). These non-coding RNAs
can be divided into short and long (Fig. 10), the boundary between these groups being
approximately 200 nucleotides.
Fig. 10. Eukaryotic RNA - nomenclature and function. mRNA - mediator RNA, HOTAIR HOX
transcript antisense intergenic RNA, rRNA - ribosomal RNA, snRNA - small nuclear
RNA, snoRNA - small nucleolar RNA, tRNA - transfer RNA, miRNA - microRNA, siRNA small
interfering RNA, piRNA - PIWI-interacting RNA, tsRNA - tRNA-derived small RNA.
Adapted from Green et al (26).
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6.1 Short non-coding RNAs
6.1.1 MicroRNAs
MicroRNAs (miRNAs) are short single-stranded RNAs (usually 21-25 nucleotides in length)
that arise from a long primary transcript and from a precursor hairpin structure. The genes
encoding the miRNA are transcribed into the primary miRNA, from which the precursor
miRNA is formed in the nucleus by the action of the Drosha RNase. After transport to the
cytoplasm, a mature miRNA is formed, which by the action of another RNase Dicer and binding
to Argonaut protein forms an effector complex, the so-called RNA-induced silencing complex
– RISC (27). The mature miRNA binds to the 3 'untranslated end of the target mRNA, causing
its degradation or inhibition of translation (Fig. 11), thus regulating gene expression at the posttranscriptional
level (28).
Fig. 11. Schematic representation of miRNA function.
The fact that miRNA sequences are highly conserved across various organisms suggests their
role in basic biological processes such as development, differentiation, proliferation, apoptosis,
regulation of cell stem properties, the immune system, or tumour transformation. Abnormal
expression of miRNAs in tumours can have two basic pathogenetic consequences: miRNAs
can function as an oncogene or as a tumour suppressor. In the pathogenesis of RCC, miRNAs
affect fundamental mechanisms of carcinogenesis, such as hypoxia / VHL / HIF cascade, EMT,
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cell proliferation, invasion, apoptosis or angiogenesis. Aberrant expression in cancer and
different expression in tissues and body fluids make microRNAs suitable candidates for use as
biomarkers.
Published works related to the topic:
Fedorko M, Pacík D, Varga G, Wasserbauer R, Ghazal M, Nussir M. MikroRNA
v patogenezi renálního karcinomu a jejich využití pro stanovení diagnózy a prognózy
RCC. Urol List 2015; 13(1): 27-31
A review describing the relationship of miRNAs to the pathogenesis of RCC and summarizing
findings on miRNA deregulation in the comparison of tumour tissue, serum and urine of
patients with RCC.
Fedorko M, Pacik D, Wasserbauer R, Juracek J, Varga G, Ghazal M, Nussir MI.
MicroRNAs in the pathogenesis of renal cell carcinoma and their diagnostic and
prognostic utility as cancer biomarkers. Int J Biol Markers 2016; 31(1): e26-37.
IF 1,371
An extended review describing in detail the pathogenetic mechanisms in which miRNAs are
involved, supplemented by the latest knowledge regarding specific miRNAs as potential
diagnostic and prognostic biomarkers of RCC.
Number of timed cited (WoS, as of February 17th
2022): 16.
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Circulating miRNAs as diagnostic biomarkers
Several serum miRNAs have been shown to have relatively high accuracy in distinguishing
between tumour patients and healthy individuals. In a group of 68 patients with RCC, serum
miR-210 levels were significantly higher than in a control group of 42 healthy individuals (AUC
0.874 at a sensitivity of 81% and a specificity of 79%). One week after surgery, serum miR-
210 levels were significantly lower than before surgery (29). In a study of Czech authors, a
significantly higher level of miR-378 and a lower level of miR-451 were demonstrated. The
combination of both miRNAs identifies the serum of RCC patients with a sensitivity of 81%
and a specificity of 83%, AUC being 0.86 (30). Diagnostic accuracy can be increased by using
a panel of several miRNAs, e.g. a combination of miR-193a-3p, miR-362, miR-572, miR-28-
5p, miR-378 (31). Sensitivity of 100% and specificity of 73.3% for the diagnosis of ccRCC is
described for the combination of miR-141 and miR-1233 (32).
Published work related to the topic:
Fedorko M, Stanik M, Iliev R, Redova-Lojova M, Machackova T, Svoboda M, Pacik D,
Dolezel J, Slaby O. Combination of MiR-378 and MiR-210 Serum Levels Enables
Sensitive Detection of Renal Cell Carcinoma. Int J Mol Sci 2015; 16(10): 23382-23389.
IF 2,862
In this original work, we demonstrated significantly higher expression of miR-210 and miR-
378 in patients with ccRCC (serum sensitivity and specificity 80% and 76%, respectively, AUC
0.848) in a serum analysis of 195 patients with ccRCC and 100 healthy controls. At the same
time, a significant decrease in plasma levels of both miRNAs after surgical removal of the
tumour was demonstrated. A positive correlation between increased miR-378 levels and both
disease-free survival and the stage of the disease was confirmed.
Number of times cited (WoS, as of February 17th
2022): 52.
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The results of the work were presented by the applicant as a commented poster at the 30th
Annual Conference of the European Urological Association (EAU) in Madrid (March 20-24,
2015) and published as a conference abstract in European Urology Supplements (IF 3,370).
For details see: Fedorko M, Staník M, Iliev R, Mlčochová H, Macháčková T, Pacík D, Doležel
J, Slabý O. Circulating miRNA-378 and miRNA-210 in patients with renal cell carcinoma and
their levels after surgical removal of the tumour. Eur Urol Suppl 2015; 14(2): e861.
https://doi.org/10.1016/S1569-9056(15)60849-1
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Diagnostic miRNAs in urine
A great advantage for the use of miRNAs in urine as biomarkers is their stability under different
conditions. So far, however, data on their use in the diagnosis of RCC are insufficient. Recent
work describes miR-30c-5p as a potential biomarker of early stage RCC. Its increased
expression inhibits the growth of tumour cells by depleting the target heat-shock protein 5 (33).
MiR-15a expression is significantly increased in the urine of RCC patients compared to healthy
subjects, with no difference between ccRCC, papillary RCC, and chromophobic RCC. On the
eighth day after nephrectomy, a 99.53% reduction in miR-15a expression was noted (34). The
expression of miR-210, which was mentioned as a potential serum biomarker, was significantly
increased in the urine of patients with ccRCC in one study. Sensitivity and specificity for
differentiation of patients and healthy subjects were 57.8%, resp. 80%, AUC 0.76 (35).
Published work related to the topic:
Fedorko M, Juracek J, Stanik M, Svoboda M, Poprach A, Buchler T, Pacik D, Dolezel J,
Slaby O. Detection of let-7 miRNAs in urine supernatant as potential diagnostic approach
in non-metastatic clear-cell renal cell carcinoma. Biochem Med 2017; 27(2): 411-417.
IF 3,051
This original work can be considered as one of the pilot studies on the use of urinary miRNAs
as a tool for RCC detection and the first study demonstrating the diagnostic potential of let-7a,
which was the best of the six miRNAs studied to distinguish ccRCC patients from healthy
controls.
Number of times cited (WoS, as of February 17th
2022): 26.
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The results of the work were presented by the applicant in the form of a commented poster at
the 17th Central European Meeting (CEM) of the European Urological Association in Pilsen
(October 19 - 20, 2017) and published as a conference abstract in European Urology
Supplements (IF 3,462).
For details see: Fedorko M, Juracek J, Pacik D, Slaby O. Urinary let-7 miRNAs as possible
diagnostic biomarkers of renal cancer. Eur Urol Suppl 2017; 16(11); e2844.
https://doi.org/10.1016/S1569-9056(17)31986-3
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MiRNAs as prognostic biomarkers of RCC
Sufficient evidence can be found in the literature that aberrant miRNA expression affects the
survival of patients with RCC, so the use of these miRNAs as biomarkers to identify patients at
risk of disease relapse or progression can be considered. Most of the work concerns the
determination of miRNA expression in tumour tissue. The works of Czech authors have a
significant representation here. One of the pilot studies in this area was the study of miR-106b,
the expression of which is significantly lower in patients who have metastasized and may
therefore be a biomarker of early RCC metastasis after nephrectomy (36). Another of the Czech
papers describes the reduced expression of miR-143, miR-26a, miR-145, miR-10b, miR-195
and miR-126 in patients with RCC relapse and primary metastatic tumour. In addition, it
demonstrated a significant correlation with relapse-free survival for miR-127-3p and miR-126
(37). MiRNAs with increased expression and at the same time worse prognosis of RCC patients
also include miR-21, miR-1260b, miR-210, miR-100, miR-125b, miR-221, miR-630, and miR-
497, while lower expression and worse prognosis is described for miR-99a, miR-1826, miR-
215, miR-217, miR-187, miR-129-3p, miR-23b and miR-27b (38). The role of decreased miR-
126 expression as a biomarker of early relapse of ccRCC has been confirmed in recent research
(39). At the time of targeted biologic therapy, the potential role of miRNAs as biomarkers for
estimating therapeutic response in metastatic tumours appears. In patients treated with sunitinib,
there was a significantly longer time to progression in patients with reduced expression of miR-
155 and miR-484 in tumour tissue (40). Down-regulation of miR-141 is also associated with a
poor response to sunitinib treatment (41). Sunitinib-resistant patients show higher expression
of miR-942, miR-628-5p, miR-133a and miR-484 (42).
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Published work related to the topic:
Machackova T, Mlcochova H, Stanik M, Dolezel J, Fedorko M, Pacik D, Poprach A,
Svoboda M, Slaby O. MiR-429 in linked to metastasis and poor prognosis in renal cell
carcinoma by affecting epithelial-mesenchymal transition. Tumour Biol 2016; 37(11):
14653-14658.
IF 2,926
This work demonstrated significantly reduced expression of tumour suppressor miR-429 in
tumour tissue and ccRCC metastases with an effect on disease-free survival and overall patient
survival. Transfection of miR-429 into TGF-β-treated tumour cell lines inhibited the loss of Ecadherin
(caused by EMT) and thus reduced the migration capacity of tumour cells.
Number of times cited (WoS, as of February 17th
2022): 33.
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6.1.2. PIWI-interacting RNAs
As already mentioned in the miRNA characteristics, interaction at the RNA level, i.e. so-called
RNA silencing, is one of the key regulatory processes within eukaryotic organisms. PiRNAs
(PIWI-interacting RNAs) are short RNAs (24-32 nucleotides) that form RNA-induced
quenching complexes with PIWI proteins, belonging to the family of so-called Argonaut
proteins. PIWI proteins (P-element-induced wimpy testes) are expressed mainly in germ cells
in the gonads and pi-RNA regulation is important for maintaining normal gametogenesis and
reproduction (43). In addition to germ cells, piRNAs also perform regulatory functions in
somatic cells (chromatin modification, attenuation of transposons to maintain genomic
stability). In addition, however, they play an important role in the pathogenesis of various solid
tumours by influencing apoptosis, proliferation, gene stability, invasion and metastasis of
tumour cells (44).
Fig. 12. Schematic representation of the cellular functions of PIWI-piRNA. Adapted from
Litwin et al. (45).
PiRNAs as tumour biomarkers
Compared to non-tumour tissue, different expression of many piRNAs was demonstrated in
tumour cell lines. Different levels of some piRNAs in peripheral blood (piR-651, piR-823 in
gastric cancer, piR-5937, piR-28876 in colorectal cancer) were confirmed in studies on other
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solid tumours (46). Thus, they may be potential biomarkers for the diagnosis and prognosis of
cancer.
Published work related to the topic:
Iliev R, Fedorko M, Machackova T, Mlcochova H, Svoboda M, Pacik D, Dolezel J, Stanik
M, Slaby O. Expression Levels of PIWI-interacting RNA, piR-823, are Deregulated in
Tumor Tissue, Blood Serum and Urine of Patients with Renal Cell Carcinoma. Anticancer
Res 2016; 36: 6419-6424.
IF 1,895
The original work showed significantly lower expression of piR-823 in RCC tissue compared
to a healthy parenchyma and correlated with a worse prognosis of RCC patients. In contrast, in
the serum and urine of patients, the expression of piR-823 was significantly increased, which
can be explained by the active secretion of piR-823 by tumour cells.
Number of times cited (WoS, as of February 17th
2022): 47.
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PIWI proteins as tumour biomarkers
Four PIWI proteins are currently known - PIWI 1 to PIWI 4 (also referred to as PIWIL1 - 4).
Their aberrant expression is described in various types of cancer (Tab. 2).
PIWI Tumour Expression
PIWIL1 Lung cancer ↑
Gastric cancer ↑
Colorectal cancer ↑
Renal cancer ↓
Endometrial cancer ↑
Invasive ductal carcinoma ↑
PIWIL2 Glioma ↑
Cervical cancer ↑
Non-small cell lung cancer ↑
Renal cancer ↓
PIWIL3 Glioma ↓
Gastric cancer ↑
Multiple myeloma ↑
PIWIL4 Breast cancer ↑
Tab. 2. Deregulated PIWI proteins in tumour tissue. Adapted from Liu et al. (46).
83
Published work related to the topic:
Iliev R, Stanik M, Fedorko M, Poprach A, Vychytilova-Faltejskova P, Slaba K, Svoboda
M, Fabian P, Pacik D, Dolezel J, Slaby O. Decreased expression levels of PIWIL1,
PIWIL2, and PIWIL4 are associated with worse survival in renal cell carcinoma patients.
Onco Targets Ther 2016; 9: 217-222.
IF 2,311
In this original work, a significantly reduced expression of PIWIL1 in RCC tissue and a
correlation between decreasing expression of PIWIL1, PIWIL2 and PIWIL4 and a higher
clinical stage of the tumour and between the expression of PIWIL2 and PIWIL4 and a higher
nuclear grade was demonstrated. In the case of PIWIL1, PIWIL2 and PIWIL 4, low expression
in tumour tissue is significantly associated with poorer overall survival.
Number of times cited (WoS, as of February 17th
2022): 34.
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6.2. Long non-coding RNAs
Long non-coding RNAs (lncRNAs) are a group of non-coding RNAs longer than 200
nucleotides in length. They are actively involved in various cellular processes, such as
differentiation, proliferation, response to DNA damage or chromosomal imprinting (47). With
increasing knowledge of their biological functions, several of their basic properties crucial for
the pathogenesis of cancer can be characterized: maintenance of proliferation signals,
circumvention of growth suppressors, stimulation of cell replication, activation of invasion and
metastasis, induction of angiogenesis and resistance to cell death (48). Unlike miRNAs,
lncRNAs regulate gene expression at all levels. Nuclear lncRNAs are involved in epigenetic
regulation, such as various chromatin modifications (association with histone modifying
complexes, DNA methylation or chromatin remodeling), transcriptional regulation (in terms of
activation or repression) and at further level of gene expression in post-transcriptional splicing
of pre-mRNA (49-51). Cytoplasmic lncRNAs act at post-transcriptional (mRNA stability,
miRNA binding - "sponging"), translational (translation activation or suppression) and posttranslational
(e.g. influencing protein stability) level of gene expression (52).
Differential expression of lncRNA in the tissue (or serum) of RCC compared to non-tumour
samples and the relationship to the clinicopathological characteristics of the tumour suggest the
potential use of lncRNA as diagnostic and prognostic biomarkers of RCC, or as therapeutic
targets (53, 54). In addition to renal cell carcinoma, their use as biomarkers of other urological
malignancies, such as prostate or bladder cancer, can be expected (55).
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Published work related to the topic:
Fedorko M, Bohošová J, Poprach A, Pacík D. Dlouhé nekódující RNA a karcinom
z renálních buněk. Klin Onkol 2020; 33(5): 340-349.
The review describes in detail the importance of long non-coding RNAs in the pathogenesis of
renal cell carcinoma and the possibilities of their use in the diagnosis, prognosis and prediction
of treatment response in patients with renal cell carcinoma.
Number of times cited (Scopus, as of February 17th
2022): 2.
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7. Future perspectives
Short and long non-coding RNAs have been the target of intensive clinical research in the last
two decades in terms of potential biomarkers and therapeutic targets for cancer treatment.
Given the amount of preclinical evidence that have indicated the role of miRNAs in cancer
resistance, another goal of research is to utilize miRNAs to increase the sensitivity of tumour
cells to systemic treatment (56). Despite clinical evidence, miRNAs have not yet been used in
routine clinical practice. There are several possible explanations (57):
1. The amount of miRNA biomarkers have been studied in groups with limited number of
subjects and without validation cohorts.
2. It is difficult to overcome side effects of miRNA-based treatment, which is due to the
simultaneous regulation of multiple targets. An example is MRX34 (liposomal formulation of
miR-34a) halted due to severe immune-mediated adverse effects.
3. The need for a better delivery system and ensuring the highest possible stability of
oligonucleotides in vivo.
Further research of other groups of small non-coding RNAs (piRNA, tsRNA) may be the key
in terms of new therapeutic targets for RCC.
Similarly, there are several unresolved issues regarding lncRNAs, such as the exact
mechanism of abnormal lncRNA expression in the tumour or lack of information on the
various upstream regulatory mechanisms of lncRNA, such as histone status, DNA
methylation patterns, transcription factors or post-transcriptional mechanisms (58).
Recent research on genomic enhancers and non-coding RNAs generated from enhancers,
enhancer RNAs (eRNAs), has demonstrated their functional role in both normal and tumour
cells, especially gene activation and interactions with transcriptional activators and coactivators
(59). Due to their length, eRNAs are sometimes considered a subset of lncRNAs,
although their properties are more variable than lncRNAs. Thus, eRNAs can be expected to
become another target for the diagnosis and treatment of cancer.
An interesting area of research is exosomal lncRNA. Exosomes represent a medium for
lncRNA transmission among tumour cells and their content (including lncRNAs) is another
possible target of so-called liquid biopsy in tumour diagnosis (60). Engineered exosomes,
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capable of carrying anti-tumour drugs, proteins or therapeutic miRNAs are promising in terms
of treatment (61).
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8. Conclusion
The habilitation thesis presents the author's systematic work in the field of renal cell carcinoma
research in terms of suitable biomarkers for the diagnosis and monitoring of the disease. It
documents close and productive multidisciplinary cooperation of the clinical field (urology)
and molecular medicine, which results in the presented publication outputs in important
professional periodicals supported by active participation in national and international forums.
In the introductory part of the thesis presents the issue of renal cell carcinoma mainly in relation
to the clinical stage and prognosis of the disease, thus building a rational basis for efforts to
detect the disease early, while describing the main molecular genetic mechanisms involved in
its pathogenesis. The next part justifies the need to find biomarkers of this disease and presents
individual groups of biomarkers. The main part of the work is a detailed description of noncoding
RNAs, where the presented results clearly indicate their possible use in the diagnosis
and prognosis of renal cell carcinoma. It can be stated that the presented work fulfilled the goals
set by the author and in addition to a detailed description of the issue can also serve as an
impetus for further scientific work with the aim of implementing the acquired knowledge into
clinical practice and finding other modern diagnostic and therapeutic methods in clinical
oncology.
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9. List of figures and tables
Fig. 1. Incidence and mortality (ASR) of RCC in different regions 6
Fig. 2. Age-standardized incidence (ASR) of ten major cancers in both
sexes in the Czech Republic 7
Fig. 3. RCC incidence growth index (1977 – 2018) 7
Fig. 4. Time evolution of gross incidence and mortality of RCC
in the Czech Republic 8
Fig. 5. Time evolution of 5-year relative survival of RCC patients 9
Fig. 6. Percentage of RCC stages at the time of diagnosis 9
Fig. 7. Comparison of five-year relative survival according to the stage
of RCC at the time of diagnosis 10
Fig. 8. The proportion of clinical stages of RCC in the Czech Republic 10
Fig. 9. VHL/HIF pathway 18
Fig. 10. Eukaryotic RNA - nomenclature and function 28
Fig. 11. Schematic representation of miRNA function 29
Fig. 12. Schematic representation of the cellular functions
of PIWI-piRNA 75
Tab. 1. Comparison of five-year relative survival of patients
with tumours of the urogenital tract 8
Tab. 2. Deregulated PIWI proteins in tumour tissue 82
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10. List of abbreviations
AKT Protein kinase B
ASR Age standardized rate
AQP1 Aquaporin 1
ATP Adenosine triphosphate
AUC Area under the ROC curve
BAP1 BRCA1 associated protein 1
Bcl-2 B-cell lymphoma 2
ccRCC Clear-cell renal cell carcinoma
CD Cluster of differentiation
CEITEC Central European Institute of Technology
CTC Circulating tumour cell
ctDNA Circulating tumour DNA
CTLA4 Cytotoxic T-lymphocyte antigen 4
ČR Czech Republic
DNA Deoxyribonucleic acid
EAU European Association of Urology
EGFR Epidermal growth factor receptor
EMT Epithelial – mesenchymal transition
eRNA Enhancer RNA
FN University Hospital
Glut1 Glucose transporter 1
GSK3 Glycogen synthase kinase 3
HGF Hepatocyte growth factor
HIF Hypoxia inducible factor
HOTAIR HOX transcript antisense intergenic RNA
KIM-1 Kidney injury molecule 1
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LF MU Faculty of Medicine of Masaryk University
MAPK Mitogen-activated protein kinase
MET Hepatocyte growth factor receptor
miRNA MicroRNA
mRNA Mediator RNA
mTOR Mammalian target of rapamycin
MUC1 Mucin 1
MYC Myelocytomatosis viral oncogene homolog
NMP22 Nuclear matrix protein 22
NOR National Cancer Registry
PBRM1 Protein polybromo-1
PDGF Platelet-derived growth factor
PD-1 Programmed cell death protein 1
PD-L1 Programmed death-ligand 1
PFS Progression-free survival
PIP3 Phosphatidylinositol (3,4,5)-trisphosphate
piRNA PIWI-interacting RNA
PIWI P-element induced wimpy testis
PIWIL Piwi-like
PI3K Phosphoinositide 3-kinase
pRCC Papillary renal cell carcinoma
Pre-miRNA Precursor microRNA
PTEN Phosphatase and tensin homolog
RCC Renal cell carcinoma
RISC RNA-induced silencing complex
ROC Receiver operating characteristic
RNA Ribonucleic acid
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rRNA Ribosomal RNA
SETD2 SET domain containing 2
siRNA Small interfering RNA
snoRNA Small nucleolar RNA
snRNA Small nuclear RNA
TATI Tumour-associated trypsin inhibitor
TGFα Transforming growth factor alpha
TGFβ Transforming growth factor beta
tRNA Transfer RNA
tsRNA tRNA-derived small RNA
UTR Untranslated region
VEGF Vascular endothelial growth factor
VHL Von Hippel-Lindau
Wnt Wingless Int-1
WoS Web of Science
ZEB1 Zinc Finger E-Box Binding Homeobox 1
SIP1 Zinc finger E-box-binding Homeobox 2
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11. Author’s papers included in the text
1. Fedorko M, Kiss I, Pacík D. Nádory ledvin. In: Matějovská Kubešová H, Kiss I, eds.
Geriatrická onkologie. Praha, Česká republika: Mladá fronta; 2015: 120-124.
2. Fedorko M. Diagnostické možnosti časného zjištění nádorů ledvin, role nádorových
markerů při stanovení dalšího postupu. Urologické listy 2011; 9(4): 7-11.
3. Fedorko M, Pacík D, Varga G, Wasserbauer R, Ghazal M, Nussir M. MikroRNA
v patogenezi renálního karcinomu a jejich využití pro stanovení diagnózy a prognózy RCC.
Urol List 2015; 13(1): 27-31.
4. Fedorko M, Pacik D, Wasserbauer R, Juracek J, Varga G, Ghazal M, Nussir MI. MicroRNAs
in the pathogenesis of renal cell carcinoma and their diagnostic and prognostic utility as cancer
biomarkers. Int J Biol Markers 2016; 31(1): e26-37.
5. Fedorko M, Stanik M, Iliev R, Redova-Lojova M, Machackova T, Svoboda M, Pacik D,
Dolezel J, Slaby O. Combination of MiR-378 and MiR-210 Serum Levels Enables Sensitive
Detection of Renal Cell Carcinoma. Int J Mol Sci 2015; 16(10): 23382-23389.
6. Fedorko M, Juracek J, Stanik M, Svoboda M, Poprach A, Buchler T, Pacik D, Dolezel J,
Slaby O. Detection of let-7 miRNAs in urine supernatant as potential diagnostic approach in
non-metastatic clear-cell renal cell carcinoma. Biochem Med 2017; 27(2): 411-417.
7. Machackova T, Mlcochova H, Stanik M, Dolezel J, Fedorko M, Pacik D, Poprach A,
Svoboda M, Slaby O. MiR-429 in linked to metastasis and poor prognosis in renal cell
carcinoma by affecting epithelial-mesenchymal transition. Tumour Biol 2016; 37(11): 14653-
14658.
8. Iliev R, Fedorko M, Machackova T, Mlcochova H, Svoboda M, Pacik D, Dolezel J, Stanik
M, Slaby O. Expression Levels of PIWI-interacting RNA, piR-823, are Deregulated in Tumor
Tissue, Blood Serum and Urine of Patients with Renal Cell Carcinoma. Anticancer Res 2016;
36: 6419-6424.
110
9. Iliev R, Stanik M, Fedorko M, Poprach A, Vychytilova-Faltejskova P, Slaba K, Svoboda M,
Fabian P, Pacik D, Dolezel J, Slaby O. Decreased expression levels of PIWIL1, PIWIL2, and
PIWIL4 are associated with worse survival in renal cell carcinoma patients. Onco Targets Ther
2016; 9: 217-222.
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z renálních buněk. Klin Onkol 2020; 33(5): 340-349.
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