Characterization of Stem-Like and Differentiated Urothelial Carcinoma Cells Using GFP/FACS Separation and Transcriptomic Profiling | Claudia Maria Clara Sbiroli
Characterization of Bladder Cancer Stem-Like Cells
Authors: Claudia Maria Clara Sbiroli (1), Kateřina Houfková (1), Michaela Kripnerová (1), Martin Pešta
(1), Jiří Hatina (1)
Supervisor: Kateřina Houfková (1)
(1) Department of Biology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Invasive urothelial carcinoma of the bladder is treated with radical cystectomy
combined with neoadjuvant cisplatin-based chemotherapy to improve survival or adjuvant
chemotherapy for high-risk patients. Despite these treatments, local recurrence and metastasis
remain common and clinically significant. Recurrence is driven by tumor cell heterogeneity,
which includes chemosensitive differentiated cells and stem-like, chemoresistant cells. This intrinsic
heterogeneity limits efficacy of chemotherapy, and stem-like cells are associated with
aggressive, high-grade tumors. These findings highlight the need to characterize the molecular
and genetic profiles of stem-like cells as a basis for potential targeted therapies.
Objective: This study aimed to characterize stem-like and well-differentiated bladder cancer
cells using a stem-cell–specific GFP reporter and fluorescence-activated cell sorting (FACS), and
subsequently analyze gene expression profiles of the sorted populations.
Material and Methods: Urothelial carcinoma cell lines derived from both low-grade and highgrade
tumors were included and maintained under standard culture conditions.
To distinguish stem-like from differentiated tumor cell populations, cells were labeled with a
doxorubicin-responsive GFP reporter and physically separated using FACS into GFP-positive
(GFP⁺) and GFPlow fractions. The stem-like GFP pattern was validated by mutually exclusive GFP
and CK-14 expression.
Transcriptomic profiling was performed using DNA microarray analysis to assess global gene
expression patterns and identify genes associated with each cell population.
Subsequent immunofluorescence analysis was performed using a primary antibody against the
pluripotency factor DPPA3 (Stella) and a secondary TRITC-conjugated antibody.
Results & Discussion: Putative stem cells show constitutive ABC-efflux pump activity, preventing
doxorubicin-activated GFP expression; GFPlow cells therefore exhibited stem-like characteristics,
whereas GFP⁺ cells represented differentiated populations. Upon doxorubicin treatment,
GFP⁺ cells were mostly eliminated, while GFPlow cells survived, expanded, and partially differentiated,
restoring the original GFP pattern and demonstrating stem-like plasticity.
Transcriptomic analysis revealed higher DPPA3 expression in GFPlow cells and higher GRHL3 expression
in GFP⁺ cells, suggesting DPPA3 as a stemness marker. Future work will quantify DPPA3
expression in both populations by RT-qPCR to clarify its role in stemness and plasticity. This may
aid strategies to overcome chemoresistance and limit tumor relapse.
Conclusion: We successfully distinguished stem-like (GFPlow) and differentiated (GFP⁺) bladder
cancer cells. Microarray analysis identified distinct gene expression signatures in each population,
including DPPA3 in GFPlow cells and GRHL3 in GFP⁺ cells, suggesting targets for preventing
tumor recurrence.
Funding: This work was supported by the Specific University Research program: SVV 261773.
Automatic AI-powered analysis of experimental and clinical progression of urothelial
carcinoma based on cell and nuclear morphometry | Kristián Hordynskyj
AI-Powered Analysis of Urocarcinoma Progression
Authors: Kristián Hordynskyj (1), Michaela Kripnerová (1), Martin Pešta (1), Petra Vohradská (2),
Kristýna Pivovarčíková (3,5), Lenka Červenková (4),Jiří Hatina (1)
Supervisor: Michaela Kripnerová (1)
(1) Department of Biology, Faculty of Medicine in Pilsen, Charles University (2) Institute of Medical
Genetics, University Hospital Pilsen, Czech Republic (3) Department of Pathology, Faculty of
Medicine in Pilsen, Charles University (4) Biomedical Center, Faculty of Medicine in Pilsen, Czech
Republic (5) Bioptical Laboratory, Ltd., Pilsen, Czech Republic
State-of-the-Art: The progression of bladder cancer, both in clinic and experimental, is characterized
by substantial morphological alterations, with tumor cells transitioning from early,
well-differentiated states to advanced, poorly differentiated, and invasive phenotypes. Conventional
histopathological grading predominantly rely on subjective visual assessment performed
by pathologists. Recent advances in digital pathology and AI–based image analysis offer new
opportunities to integrate objective and quantitative metrics into morphological evaluation.
Establishing a robust link between traditional qualitative assessment and quantitative morphological
characterization is therefore critical for improving diagnostic accuracy, reproducibility,
and the broader clinical implementation of morphometric data.
Objective: The aim of this study is to develop and validate an objective, AI–driven methodology
for the morphometric analysis of bladder cancer cells in experimental and clinical progression.
This approach is evaluated using both theoretical models and clinical samples to assess its general
applicability.
Material and Methods: Our experimental model comprised five human bladder cancer cell
lines: RT4, RT112, RT112H, RT112D21, and UMUC-3. RT4 and RT112 represent low grade urothelial
carcinoma, RT112H represents grade- and potentinally also stage-progression, RT112D21
is high-grade chemoresistant derivative of RT112H, and UMUC-3 corresponds to high-grade
advanced stage cancer cell line. The clinical model comprised bladder carcinoma pathological
slides and urine cytologies of tumours of defined grades and stages; samples were stained with
Papanicolaou and hematoxylin–eosin (H&E) and analyzed from microscopic images and wholeslide
scans. Image processing and quantitative analysis of morphological parameters were performed
in QuPath with AI-based advanced cell detection based on custom Python script and
Cellpose.
Results & Discussion: Our complex and robust approach allows for highly accurate cell detection
and morphometric meassurements, even within the complex microenvironment of clinical
samples. The resulting objective data revealed clear differences in morphometric parameters
among cancer cell lines representing distinct levels of differentiation and aggressiveness. Quantifiable
cellular morphological profiles were also found to correlate reliably with tumor grading
and staging of clinical samples. Our findings thus demonstrate that this advanced and optimized
AI-based detection approach can be successfully applied not only in controlled in vitro environments
using established cell lines but is also fully functional and applicable to real-world solid
tumor samples derived from clinical practice.
Conclusion: The integration of QuPath with advanced AI-based cell detection provides a robust platform for objective morphometric analysis of bladder cancer cells. Quantitative morphometric
profiling can thus significantly contribute to accurate pathological diagnosis in both experimental
and clinical setting.
Funding: This study was supported by SVV-2025-260773 and SVV-2026-206773, Academic Minigrant
4EU+ MA/25/F1/0/018 and by the League Against Cancer Prague.
Immunoexpression and Amplification of MDM2 gene as a Potential Modifier of Behavior in PLAG1::LIFR-Fused Salivary Gland Tumors (SGT) | Constantina Constantinou
MDM2 in PLAG1::LIFR-Fused SGTs
Authors: Constantina Constantinou (1)
Supervisor: Alena Skálová (1,2)
(1) Department of Pathology, Faculty of Medicine in Pilsen, Charles University and University
Hospital, Pilsen (2) Bioptical Laboratory, Ltd, Pilsen, Czech Republic
State-of-the-Art: Recently, immunohistochemical (IHC) expression of MDM2, including MDM2
amplification, was proposed to play a role in malignant progression in the group of HMGA2-altered
pleomorphic adenomas. The MDM2 is associated with tumor aggressiveness in various
neoplasms, often through amplification but also via protein overexpression. Its clinicopathologic
relevance in PLAG1::LIFR fusion-positive SGTs has not been yet systematically investigated.
Objective: To explore the potential impact of recurrent MDM2 expression and amplification on
tumor behavior in different subtypes of PLAG1::LIFR fusion-positive SGTs.
Material and Methods: A retrospective search in the authors’ registry identified 12 cases of
SGTs with PLAG1::LIFR fusion. All tumors were stained for anti-MDM2 antibodies. All slides were
scanned and IHC positivity was assessed using semi-quantitative scoring system, incorporating
both nuclear staining intensity and the extend of positive tumor nuclei.
Results & Discussion: 12 PLAG1::LIFR-fused SGTs were analyzed (2 males and 10 females;
ages 35-79). The anatomical distribution showed a predominance of tumors arising in palate
(7/12), followed by the parotid gland (2/12), submandibular gland (2/12) and jaw (1/12). Diagnoses
varied, including pleomorphic adenoma (PA) and its variants (n=5), myoepithelioma (n=1)
and malignant myoepithelial neoplasms (n=6). Weak immunopositivity was seen in four tumors
comprising both benign and malignant SGTs. Moderate immunoexpression was detected in
four cases, predominantly in malignant myoepithelial tumors. Negative staining was observed
in three cases including benign PA-related tumors and one carcinoma ex-PA. One case was not
analyzed due to insufficient material.
Conclusion: MDM2 IHC was expressed in eight PLAG1::LIFR-fused SGTs, while FISH did not reveal
a MDM2 gene amplification in any case. These findings suggest that MDM2 alterations do
not play a significant role in malignant progression, and that the PLAG1::LIFR fusion itself is the
major oncogenic driver.
Diagnostic Significance of HRAS gene mutation in Salivary Gland Tumors: A Histological, Molecular and Immunohistochemical Correlation | Sarkis Tsinarian
Significance of HRAS gene in SGTs
Authors: Sarkis Tsinarian (1), Bacem Othman (2,3)
Supervisor: Alena Skalova (1,3)
(1) Department of Pathology, Faculty of Medicine in Pilsen, Charles University, Czech Republic
(2) Department of Anatomy, Faculty of Medicine in Pilsen, Charles University, Czech Republic
(3) Bioptical Laboratory, Ltd., Pilsen, Czech Republic
State-of-the-Art: H RAS g ene m utation h as b een d iscovered a s p rimary d river e vent i n e pithelial-
myoepithelial carcinoma (EMC). We propose that HRAS-driven salivary neoplasia is not
confined to EMC, but may possibly relate to multiple lines of ductal and acinar differentiation in
Salivary Gland Tumors (SGTs).
Objective: SGTs show tumor type-specific recurrent fusions and/or mutations. HRAS mutation
was defined as a diagnostic marker of EMC. We aimed to (a) examine the specificity of HRAS
mutation in EMC and (b) correlate an HRAS gene mutation to immunohistochemical (IHC) expression.
Material and Methods: A retrospective search in the authors’ registry identified 16 cases of
SGTs with HRAS mutation. All tumors were stained for anti-RAS Q61R antibodies. All slides were
scanned and IHC positivity was assessed.
Results & Discussion: 16 HRAS-mutated SGTs were analyzed (9 males and 7 females; age 43–
92). Most arose in parotid gland (N=15); one involved a lymph node with metastatic salivary
ductal carcinoma (SDC). All cases showed HRAS codon 182A mutation on next generation sequencing
(NGS). RAS Q61R was positive in 13 cases (81%). Two major phenotypes of SGTs were
encountered: biphasic ductal–myoepithelial neoplasms diagnosed as EMC (N=10) and apocrine
ductal carcinomas with tubular/cribriform and solid architecture characterized by androgen receptor-
positive and myoepithelial markers-negative, diagnosed as SDC (N=4). Two additional
cases showed variable phenotypes, including metastatic squamous cell carcinoma (mSCC) and
intraductal carcinoma (IDC) arising in sclerosing polycystic adenoma (SPA).
Conclusion: RAS Q61R was expressed in 13 HRAS-mutated carcinomas including strong staining
in 9/10 cases of EMC, mild or focal staining in all four SDCs, while IDC ex SPA and mSCC were
negative. Our short seriescompiled immunophenotypic and genotypic assessment of HRAS,
which was strongly correlated.
Funding: The study was partly supported by the grant SVV 262773 from Ministry of Education,
Youth and Sports of the Czech Republic, (ST).
Salivary Gland-like Tumors (SGT) of the Breast: Histopathologic and Genetic features | Barbora Jandová
Salivary Gland-like Tumors of the Breast
Significance of HRAS gene in SGTs
Authors: Barbora Jandová (1)
Supervisor: Alena Skalova (1,2)
(1) Department of Pathology, Faculty of Medicine in Pilsen, Charles University and University
Hospital, Pilsen (2) Bioptical Laboratory, Ltd, Pilsen, Czech Republic
State-of-the-Art: There are several special types of breast carcinomas that share morphologic,
immunophenotypic, and molecular genetic features of salivary gland tumors (SGT). These
breast carcinomas are typically negative for estrogen receptor (ER), progesterone receptor (PR),
and human epidermal growth factor receptor 2 (HER2/neu), referred to as “triple-negative,” yet
generally have a much better prognosis than triple-negative breast carcinomas of no special
type.
Objective: To discuss the histologic, immunophenotypic, molecular, and clinical features of salivary
gland–like carcinomas of the breast.
Material and Methods: A retrospective search in the authors’ registry identified 71 cases of
SGT of the breast. Most of the tumors were stained for ER, PR, and HER-2/neu. All slides were
scanned, and IHC positivity was assessed. Molecular testing was performed in 28 cases using
next-generation sequencing (NGS) and/or fluorescence in situ hybridization (FISH).
Results & Discussion: 50 SGTs of breast were analyzed (3 males and 47 females; age 3-87). All
tested cases were triple negative (ER/PR and HER2/neu). Three major phenotypes of SGTs of
the breast were encountered: adenoid cystic carcinoma (AdCC) (n = 17), secretory carcinoma
(SC) (n = 21), and mucoepidermoid carcinoma (n = 1), characterized by MYB::NFIB, ETV6::NTRK3,
and CRTC1::MAML2 gene fusion, respectively. The other SGT of the breast included acinic cell
carcinoma (AciCC) (n = 2), polymorphous adenocarcinoma (n = 2), and myoepithelial carcinoma
(n = 7).
Conclusion: Salivary gland-like breast tumors represent a subset of triple-negative breast carcinomas.
Identifying characteristic genetic alterations and/or immunohistochemical surrogates
in these tumors has practical applications for establishing an accurate diagnosis and directing
clinical management.
Evaluation of the therapeutic potential of lipophosphonoxins DR-6180 & DR-7072 in
biodegradable nanofibrous carrier systems for treatment of murine skin wounds experimentally infected with S. aureus | Elizabeth Tsibouki
Lipophosphonoxin in Nanofibers for S. aureus Wounds
Authors: Elizabeth Tsibouki (1), Pavel Klein (1,2), Marek Kindermann (1), Ivana Kleinová (3), Věra
Jenčová (4), Dana Králová (1,5), Vendula Pecková (1,5) , Eva Kuželová – Košťáková (4), Maxim Lisenko
(4), Kristýna Havlíčková (4), Šárka Hauzerová (5), David Lukáš (4), Kateřina Chudějová (1,5), Dominik
Rejman (6)
Supervisor: Pavel Klein (1,2)
(1) Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Czech Republic
(2) Department of Pathological Physiology, Faculty of Medicine in Pilsen, Charles University,
Czech Republic (3) Faculty of Agriculture and Technology, University of South Bohemia, Czech
Republic (4) Faculty of Science, Humanities and Education, Technical University of Liberec, Czech
Republic (5) Department of Microbiology, Faculty of Medicine in Pilsen, Charles University,
Czech Republic (6) Institute of Organic Chemistry and Biochemistry of the Czech Academy of
Sciences, Prague, Czech Republic
State-of-the-Art: The presence of infection in wounds is a common cause of complications
of healing and in most cases antimicrobial therapy is necessary. In view of the development of
resistance, it is desirable to develop new antimicrobial agents useful for the control of localised
bacterial infections of soft tissues. Lipophosphonoxins (LPPOs), unique molecules synthesized
at the Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, appear
to be a very promising group of agents suitable for such an indication. The aim of the study
was to test the efficacy of LPPOs (molecules DR-6180 and DR-7072) in a biodegradable nanofibrous
sandwich carrier system consisting of PCL and PVA layers for antimicrobial therapy of the
wounds experimentally infected with Staphylococcus aureus.
Objective: To evaluate the antimicrobial efficacy of lipophosphonoxins DR-6180 and DR-7072
incorporated in biodegradable nanofibrous carrier systems for the treatment of murine skin
wounds experimentally infected with Staphylococcus aureus.
Material and Methods: An in vivo murine wound infection model was used. Male CB6F1 mice
immunosuppressed with cyclophosphamide were inoculated with Staphylococcus aureus in
two full-thickness dorsal skin wounds. After infection establishment, wounds were covered
with experimental biodegradable nanofibrous dressings consisting of PCL/PVA layers incorporating
lipophosphonoxins DR-6180 or DR-7072. Control groups included nanofibers without
active compound and standard antimicrobial treatment. Wounds were covered with an occlusive
membrane dressing. Treatment efficacy was evaluated after four days using microbiological
examination of wound tissue and visual wound scoring assessing infection severity and healing
Results & Discussion: Both tested lipophosphonoxins demonstrated antimicrobial activity
against S. aureus in infected wounds. The biodegradable PCL/PVA nanofibrous sandwich
structure proved to be a suitable carrier system enabling local delivery of the compounds. In
several cases, substantial reduction of bacterial burden and improvement of wound healing
were observed. However, variability in treatment efficacy was noted, suggesting that the current
concentration of lipophosphonoxins in the nanofibrous dressing may be close to the minimal
effective dose. These findings indicate that optimization of drug loading and release kinetics will
likely improve therapeutic performance
Conclusion: Lipophosphonoxins incorporated in biodegradable nanofibrous dressings show promising antimicrobial activity in a murine model of S. aureus wound infection. Further optimization
of drug concentration and release properties is required to enhance therapeutic efficacy
HPLC-Guided Purification of Antibacterial Metabolites from UV-Stressed Agaricus bisporus | Robert Miller
Mushroom Bioactive Compounds
Authors: Robert Miller (1), Lucy Bludovská (1), Zdeněk Tůma (2), Eliška Fousková (1)
Supervisor: Shashank Pandey (1)
(1) Department of Pharmacology and Toxicology, Faculty of Medicine in Pilsen, Charles University
(2) Biomedical Center, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Ultraviolet (UV) irradiation modulates the metabolisms of certain mushrooms
by inducing oxidative stress, which in turn stimulates the production of secondary „stress-response“
metabolites, such as of ergosterol into Vitamin D2, or anti-oxidants like flavinoids and
ascorbic acid. Although this exposure to UV radiation is known to enhance antioxidant capacities
and alter the metabolite profiles of many mushrooms, the isolation of UV-induced compounds
with antibacterial properties in Agaricus bisporus remains limited. In this study, an HPLC-guided
bioassay fractionation approach was employed in order to purify metabolites from UV-stressed
Agaricus bisporus mushrooms, which were then screened for their antibacterial activity.
Objective: To purify and concentrate the bioactive metabolites of Agaricus bisporus responsible
for antibacterial activity against Gram-positive and Gram-negative bacteria.
Material and Methods: For this study, we developed a method of extracting and purifying
antibacterial compounds from UV-stressed Agaricus bisporus through an HPLC system. Mushroom
samples were acquired through AGARICUS, spol s.r.o. (Czech Republic), which were then
washed, lyophilized, and stored at -80°C. 1 gram of sample was dissolved in 15ml of distilled
water along with 0,1 mM PMSF. The sample was then sonicated for 30 seconds, followed by
30 seconds of rest on ice. After sonication, the sample was centrifuged at 10 000 RPM for 20
minutes at 4°C. Debris was removed and the supernatant collected. Prior to HPLC, the sample
was ultrafiltrated using a Amicon Ultra 3 kDa filter. A C18 reversed-phase column was used to
separate the bioactive compound from the Agaricus bisporus extract.
Results & Discussion: HPLC purification of the aqueous extract derived from UV-stressed Agaricus
bisporus allowed for the collection of multiple fractions, each of which were individually
evaluated for antibacterial activity. One of these fractions exhibited significant inhibitory activity
against E. coli and S. aureus, while the other fractions showed none. The zone of inhibition of
this active fraction is equivalent to that of the pre-HPLC purified extract, about 11mm, indicating
successful isolation of its antibacterial metabolites. The metabolite‘s activity is broad spectrum,
acting against both Gram positive and negative bacteria, and should further be tested against
clinical isolates. Further goals are to determine its minimum inhibitory concentration, and to
characterise the HPLC-purified compound.
Conclusion: In conclusion, this study demonstrates that HPLC-guided bioassay fractionation
has enabled us to identify and purify the antibacterial metabolites of UV-stressed Agaricus bisporus.
Future work will be focused on the characterisation of the HPLC-purified molecule using
NMR and mass specrometry.
Susceptible but Not Eradicated: Persistence in Low-MIC OXA-48-Producing Escherichia coli and Klebsiella pneumoniae | Markéta Brejníková
Persistence in Low-MIC OXA-48 isolates
Authors: Markéta Brejníková (1), Regina Tokárová (1)
Supervisor: Kateřina Chudějová (1)
(1) Department of Microbiology, Faculty of Medicine in Pilsen, Charles University, Czech Republic
State-of-the-Art: Bacterial persistence represents a reversible phenotypic state in which a
small subpopulation of cells survives antibiotic exposure without apparent resistance mechanisms.
Unlike resistant mutants, persister cells remain genetically susceptible but enter a
transient dormant or slow-growing state that reduces the efficacy of antibiotics targeting active
cellular processes. This phenomenon is particularly relevant in chronic and relapsing infections,
where persisters can repopulate the niche once antimicrobial pressure is removed.
Objective: The aim of the study was to identify low-MIC carbapenemase-producing isolates,
particularly those harboring OXA-48-like enzymes, and to assess their persistence levels.
Material and Methods: The detection of bacterial persisters was performed using the Tolerance
Disk (TD) test, a modified version of the standard disk diffusion assay. In this method,
bacteria are first exposed to an antibiotic disk (meropenem or amikacin) under routine susceptibility
testing conditions. Following overnight incubation, susceptibility is evaluated, and the
assay is continued only with isolates categorized as susceptible (S) or susceptible, increased
exposure (I). In these cases, the antibiotic disk is replaced with a glucose disk. In the presence
of persister cells, glucose supplementation induces metabolic resuscitation, resulting in visible
colony growth within the original inhibition zone.
Results & Discussion: Total 185 bacterial strains were screened, of which 141 were susceptible
to at least one of the antibiotics – meropenem and/or amikacin. In 49 strains, we detected persistent
subpopulations, with strains susceptible to meropenem showing persistent subpopulations
in 34.8% of cases, while for amikacin it was less than 1%. Our study shows that a significant
proportion of antibiotic-susceptible clinical isolates exhibit persistent subpopulations, despite
the absence of inherited resistance mechanisms. Persistence was significantly more common
in meropenem-susceptible strains than in amikacin-susceptible strains, suggesting that the induction
or survival of persistent cells may depend on the class of antibiotic and its mechanism
of action.
Conclusion: Persistence in OXA-48-producing E. coli and K. pneumoniae is clinically relevant,
especially during carbapenem therapy and relapse risk. Distinguishing persistence from resistance
and understanding its mechanisms is essential to improve treatment and prevent recurrent
infections.
Neuron-Specific Enolase as a Prognostic Marker in Patients with Metastatic Castration-Resistant Prostate Cancer Treated with Androgen Receptor Signaling Inhibitors | Zuzana Váchalová
NSE in mCRPC patients
Authors: Zuzana Váchalová (1)
Supervisor: Ondřej Fiala (1)
(1) Department of Oncology and Radiotherapeutics, Faculty of Medicine in Pilsen, Charles University
and University Hospital, Pilsen
State-of-the-Art: Prostate cancer is the most commonly diagnosed cancer in men. Its progressive
form, metastatic castration-resistant prostate cancer (mCRPC), is characterized by resistance
to androgen-deprivation therapy. Androgen receptor signaling inhibitors (ARSIs) are used
in this advanced stage, and the response to treatment varies according to the biology of the
tumor. Currently, the most widely used prognostic marker is prostatic-specific antigen (PSA).
However, its role in patients with mCRPC is limited. NSE is an enzyme produced as a result of
neuroendocrine differentiation of prostate cancer and may therefore play a significant role in
predicting the behavior of mCRPC.
Objective: The aim of this retrospective study was to evaluate the prognostic significance of
serum NSE at baseline and its early on-treatment dynamics in patients with mCRPC treated with
ARSI.
Material and Methods: W e r etrospectively s tudied A RSI-treated m CRPC p atients w ith a ssessed
baseline serum NSE (n=120). Radiographic progression-free survival (rPFS) and overall
survival (OS) were estimated with Kaplan–Meier curves and compared using the log-rank test.
Multivariable Cox models adjusted for age, baseline PSA, therapy line, Gleason score, metastasis
timing, and visceral involvement. NSE dynamics were assessed at a 28-day landmark in
patients with baseline and week-4 measurements, modeled as log2(NSE4w/NSE0) per doubling
(rPFS n=47; OS n=48).
Results & Discussion: The median follow-up duration was 44.7 months (95% CI 35.7–48.3).
Baseline NSE levels above the upper limit of normal (ULN) were associated with to shorter rPFS
(9.4 vs 20.8 months) and overall survival (OS; 23.5 vs 38.2 months). In the multivariable analysis,
baseline NSE >ULN remained an independent predictor of poor survival outcomes (rPFS HR 1.81,
95% CI 1.15–2.85, p=0.011; OS HR 1.85, 95% CI 1.12–3.05, p=0.017).
Conclusion: The results of this study suggest that the serum NSE levels are associated with
treatment outcomes in patients with mCRPC undergoing ARSI therapy.