Diagnostic Utility of STAT5 Immunohistochemistry | Inka Kovářková
Diagnostic Utility of Phosphorylated Signal Transducer and Activator of Transcription 5 Immunostaining in the Diagnosis of Secretory Carcinoma of the Salivary Gland: A Comparative Study
Author: Inka Kovářová (1)
Supervisor: Alena Skálová (1,2)
(1) Department of Pathology, Faculty of Medicine in Pilsen, Charles University and University Hospital, Pilsen (2) Bioptická laboratoř, s.r.o., Pilsen
State-of-the-Art: Salivary secretory carcinoma (SC) is a relatively newly described neoplasm, characterized by ETV6::NTRK3 or ETV6::RET gene fusion. Immunohistochemical (IHC) expression of phosphorylated signal transducer and activator of transcription 5 (STAT5) has been proposed as diagnostic marker specific for SC. We have recently encountered a case of sclerosing polycystic adenoma (SPA) with strong STAT5 immunoexpression.
Objective: In the present study, the STAT5 immunohistochemistry was performed in selected cases of salivary gland tumors in order to clarify its potential differential diagnostic utility.
Material and Methods: A retrospective analysis of total of 44 salivary gland neoplasms retrieved from the salivary gland tumor registry consultation files, including 10 cases formerly diagnosed as acinic cell carcinoma (AciCC), and a spectrum of salivary neoplasms harboring an intercalated duct differentiation, including SPA (10 cases), SC (15 cases) and intraductal carcinoma (IC) (9 cases) was conducted. Formalin fixed, paraffin embedded tissue samples were used for hematoxylin and eosin and IHC staining using STAT5, DOG1, mammaglobin, S100 protein and SOX10 antibodies.
Results & Discussion: SCs harboring an ETV6::NTRK3 gene fusion were all positive for STAT5 in almost 100% of the cells. In 5 cases of SC harboring an ETV6::RET, the STAT5 was diffusely positive in only 3 cases, in one case 10% of cells showed nuclear positivity, and one case was negative. In 9 cases of the IC (intercalated type confirmed by NCOA4::RET fusion), 6 cases showed nuclear positivity in 10-50% of cells, one case was negative, and 2 cases showed only granular cytoplasmic immunoreaction. AciCCs showed nuclear positivity for STAT5 in 10-20% of cells, while 3 cases were completely negative and one case showed only a cytoplasmic reaction. SPA displayed nuclear staining for STAT5 in foci with intercalated type dysplasia in 5 cases, while 4 other showed only focal granular cytoplasmic reaction.
Conclusion: Even though all ETV6::NTRK3 translocated SCs were strongly positive for STAT5, this IHC marker is not entirely specific for SC, as nuclear staining has also been observed in NCOA4::RET translocated IC (intercalated type), in AciCC, and in 5 cases of SPA in up to 10-20% of the cells.
Study program: Master´s degree – General Medicine | Year of study: 6
ID: 1081
PBMC as Assay Controls in the Comet Assay | Jakob Steer
Expanding Assay Controls: Using Multi-Species PBMC and Whole Blood in the Enzyme-Modified Comet Assay Following Potassium Bromate Exposure
Authors: Jakob Steer (1), Dagmar Jarkovská (1,2), Milan Štengl (1,2), Kristýna Tomášová (3), Soňa
Vodenková (2,3)
Supervisor: Milan Štengl (1,2)
(1) Department of Physiology, Faculty of Medicine in Pilsen, Charles University(2) Biomedical
Center, Faculty of Medicine in Pilsen, Charles University (3) Department of Molecular Biology of
Cancer, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague
State-of-the-Art: Background: The enzyme-modified comet assay is a widely used tool to assess oxidative DNA damage. OECD guidelines and consensus statements emphasize the importance of assay controls to ensure experimental validity. A multi-laboratory study identified potassium bromate (KBrO3) as a suitable agent to produce assay controls using monocytic THP-1 cells. However, the high cost of cell cultures and limited resources in handling cultured cells may hinder widespread adoption, especially in hospital-based laboratories. Peripheral blood mononuclear cells (PBMC) and whole blood (WB), commonly used in comet assays, could offer a practical alternative.
Objective: This study evaluates whether cryopreserved PBMC and WB from different species
are suitable cells for KBrO3-induced oxidative DNA damage, enabling their use as standardized
assay controls in the FPG-modified comet assay.
Material and Methods: WB was collected from humans (venous), pigs (arterial), and rabbits/
mice (cardiac) using K2EDTA tubes. PBMC were isolated via density gradient centrifugation,
washed, and divided into three experimental sets. Cells were incubated with increasing KBrO3
concentrations (0mM, 3mM, 6mM, 9mM, 12mM) and cryopreserved at –80°C. Within three
months, the FPG-modified comet assay was performed, using Ro19-8022-treated HCT116 cells
and untreated cells as assay controls. Tail intensity was measured via semi-automated scoring
software (Lucia Comet Assay).
Results & Discussion: Linear regression analysis of porcine and rat PBMC showed slopes ranging
from 7.9 (±1.0) to 8.5 (±0.4) with p < 0.004 and R² values between 0.95 to 0.99. Porcine control
PBMC (RPMI-1640 only) exhibited mean Fpg-sensitive sites of 0.20 (±0.7) and 63.67 (±0.76) after
12 mM KBrO3 exposure. Rat PBMC from one experiment showed Fpg-sensitive sites of 3.59 in
RPMI-1640 and 67.78 after 12mM KBrO3 exposure.
Conclusion: These preliminary findings suggest porcine and rat PBMC are suitable for KBrO3-
based assay controls. Further validation, including additional experimental replicates and testing
of human and rabbit PBMC, is ongoing to confirm their broader applicability.
Funding: This work was supported by the Cooperation Program, research area Medical Diagnostics
and Basic Medical Sciences and by the Ministry of Health of the Czech Republic, grant nr.
NU22J-03-00033.
Study program: Master´s degree – General Medicine | Year of study: 6
ID: 1109
Flubendazole Affects Pancreatic Cancer Cells | Petr Kučera
EFFECT OF FLUBENDAZOLE ON PANCREATIC CANCER CELLS WITH AN EMPHASIS ON THE MICROTUBULAR CYTOSKELETON.
Author: Petr Kučera (1)
Supervisor: Barbora Vítovcová (1)
(1) Department of Biology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Pancreatic adenocarcinoma is a malignant tumor arising from the exocrine part of the pancreas, characterized by a very poor prognosis and limited therapeutic options. The unfavorable prognosis is further exacerbated by the typically late diagnosis due to the tumor’s initially asymptomatic growth, as well as by its high degree of chemoresistance. The inadequacy of current treatment strategies highlights the urgent need for innovative approaches. One such possibility is the use of flubendazole (FLU), originally an anthelmintic developed for veterinary use, which has been shown in previous studies to destabilize microtubules and induce apoptosis in various cancer cell types.
Objective: The main aim of this study is to analyze the effect of flubendazole (FLU) on the viability, proliferation, and morphology of pancreatic cancer cells, as well as to observe its impact on the microtubular cytoskeleton of these cells.
Material and Methods: Two established pancreatic cancer cell lines, BxPC3 and MIA PaCa-2, were used in this project. Cell proliferation was monitored in real time using the IncuCyte live-cell imaging system. Morphological changes were also evaluated using phase-contrast microscopy. Changes in the microtubular cytoskeleton were then observed via fluorescence microscopy following immunofluorescent staining of tubulins.
Results & Discussion: FLU effectively inhibited the growth of pancreatic cancer cells, and this effect was observed even after using very low concentrations of the drug. Furthermore, FLU profoundly affected the morphology of the cancer cells, which was particularly evident in BxPC3 cells, where FLU treatment led to the formation of giant multinucleated cells. In addition, FLU significantly disrupted the organization and structure of the microtubular network, with our findings also suggesting an impact on the expression of specific tubulin markers.
Conclusion: Our results indicate FLU as a promising anticancer agent targeting pancreatic tumor cells and their microtubular cytoskeleton even at low concentrations of treatment. Given the poor prognosis of pancreatic cancer and the potential of FLU effect on pancreatic cancer, further investigation is needed.
Study program: Master´s degree – General Medicine | Year of study: 2
ID: 1117
Validation Study of HiTAIC | Natálie Šimonová
Deciphering the Origin of Cancer of Unknown Primary Using Epigenetic Tools: A Validation Study of The DNA Methylation-based Hierarchical Tumor Artificial Intelligence Classifier (HiTAIC )
Authors: Natálie Šimonová (1), Pavel Toman (1), Petr Martinek (2), Andrea Straková Peteříková
(1,2), Zdenek Kinkor (1, 2), Kvetoslava Michalova (1,2), Natálie Klubíčková (1,2), Michal Michal
(1,2), Marian Svajdler (1,2)
Supervisor: Michael Michal (1,2)
(1) Department of Pathology, Faculty of Medicine in Pilsen, Charles University and University
Hospital, Pilsen (2) Bioptická laboratoř, s.r.o., Pilsen, Czech Republic
State-of-the-Art: Cancer of unknown origin (CUO) represents 1-4% of malignancies of Czech
oncological patients and has a high mortality rate due to limited treatment options. Accurately
identifying the primary tissue/organ of origin of CUO may significantly improve survival thanks
to potentially more tailored therapeutical options. Since DNA methylation pattern of neoplastic
cells reflects their tissue of origin as well as changes that occurred during oncogenesis, most
tumor types have distinct methylation patterns. HiTAIC, a recently developed web-based application
was designed using AI algorithms to trace tissue of origin and tumor type of CUO by utilizing DNA methylation data from 7735 tumors (obtained from publicly available sources, e.g. TCGA) to recognize 27 cancer types from 23 tissue sites.
Objective: We hypothesized that most of the HiTAIC training data were obtained from well differentiated
tumors. Therefore, we aimed to perform an independent validation study using highly undifferentiated cases which are more likely to benefit from adjunct diagnostic tools such as HiTAIC in clinical practice.
Material and Methods: 42 samples were divided into two groups. Group 1 consisted of 15 well-differentiated malignancies, whose origin was established based on their morphological and immunohistochemical (IHC) features only. Group 2 consisted of 27 undifferentiated cancers
whose primary origin could be correctly established only after incorporation of patient‘s clinical
history or through further investigations (imaging, subsequent biopsy, molecular studies). Formalin-
fixed paraffin-embedded tissue blocks from all cases were collected and in selected cases
macrodissected to achieve at least 70% of tumor content. The methylation data were obtained
using Infinium MethylationEPIC v2.0 platform (Illumina). The raw data files were uploaded to
hitaic.herokuapp.com and results were recorded.
Results & Discussion: In Group 1, 14 out of 15 cases (93%) were diagnosed correctly, whereas
in Group 2, it was 15 out of 27 samples (56%). HiTAIC achieved an overall accuracy of 69% (29 out
of 42). The original study of HiTAIC reported accuracy 93 and 99% in the test and validation sets,
respectively (PMID: 37089814). During tumor progression, undifferentiated neoplasms tend to
lose their characteristic morphologic and IHC features and the methylome may be slightly altered
as well (PMID: 37098294) which probably explains the lower diagnostic accuracy in group. While indeed showing excellent performance for well-differentiated neoplasms, these can be usually readily diagnosed in clinical practice using microscopy and immunohistochemistry which are significantly less costly than methylome sequencing.
Conclusion: Although HiTAIC correctly classified only 56% of undifferentiated malignancies, it
still represents a useful ancillary diagnostic tool for these challenging cases. However, its users
should be aware of its limitations and the results always need to be correlated with other diagnostic
modalities.
Study program: Master´s degree – General Medicine | Year of study: 4 | ID: 1108