Publications by authors named "Marek Grega"

12 Publications

  • Page 1 of 1

ARG1 mRNA Level Is a Promising Prognostic Marker in Head and Neck Squamous Cell Carcinomas.

Diagnostics (Basel) 2021 Mar 31;11(4). Epub 2021 Mar 31.

Department of Genetics and Microbiology, Faculty of Science, Charles University, BIOCEV, 252 50 Vestec, Czech Republic.

Head and neck squamous cell carcinomas (HNSCC) can be induced by smoking or alcohol consumption, but a growing part of cases relate to a persistent high-risk papillomavirus (HPV) infection. Viral etiology has a beneficial impact on the prognosis, which may be explained by a specific immune response. Tumor associated macrophages (TAMs) represent the main immune population of the tumor microenvironment with a controversial influence on the prognosis. In this study, the level, phenotype, and spatial distribution of TAMs were evaluated, and the expression of TAM-associated markers was compared in HPV positive (HPV+) and HPV negative (HPV-) tumors. Seventy-three formalin and embedded in paraffin (FFPE) tumor specimens were examined using multispectral immunohistochemistry for the detection of TAM subpopulations in the tumor parenchyma and stroma. Moreover, the mRNA expression of TAM markers was evaluated using RT-qPCR. Results were compared with respect to tumor etiology, and the prognostic significance was evaluated. In HPV- tumors, we observed more pro-tumorigenic M2 in the stroma and a non-macrophage arginase 1 (ARG1)-expressing population in both compartments. Moreover, higher mRNA expression of M2 markers-cluster of differentiation 163 (CD163), ARG1, and prostaglandin-endoperoxide synthase 2 (PTGS2)-was detected in HPV- patients, and of M1 marker nitric oxide synthase 2 (NOS2) in HPV+ group. The expression of ARG1 mRNA was revealed as a negative prognostic factor for overall survival of HNSCC patients.
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http://dx.doi.org/10.3390/diagnostics11040628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065482PMC
March 2021

Sensitivity to Cisplatin in Head and Neck Cancer Cells Is Significantly Affected by Patient-Derived Cancer-Associated Fibroblasts.

Int J Mol Sci 2021 Feb 15;22(4). Epub 2021 Feb 15.

Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Kamenice 5, CZ-625 00 Brno, Czech Republic.

Cancer-associated fibroblasts (CAFs) are one of the most abundant and critical components of the tumor stroma. CAFs can impact many important steps of cancerogenesis and may also influence treatment resistance. Some of these effects need the direct contact of CAFs and cancer cells, while some involve paracrine signals. In this study, we investigated the ability of head and neck squamous cell carcinomas (HNSCC) patient-derived CAFs to promote or inhibit the colony-forming ability of HNSCC cells. The effect of cisplatin on this promoting or inhibiting influence was also studied. The subsequent analysis focused on changes in the expression of genes associated with cancer progression. We found that cisplatin response in model HNSCC cancer cells was modified by coculture with CAFs, was CAF-specific, and different patient-derived CAFs had a different "sensitizing ratio". Increased expression of , , , , and in cancer cells was characteristic for the increase of resistance. On the other hand, expression was associated with sensitizing effect. Significantly higher amounts of cisplatin were found in CAFs derived from patients who subsequently experienced a recurrence. In conclusion, our results showed that CAFs could promote and/or inhibit colony-forming capability and cisplatin resistance in HNSCC cells via paracrine effects and subsequent changes in gene expression of cancer-associated genes in cancer cells.
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http://dx.doi.org/10.3390/ijms22041912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918851PMC
February 2021

The cytokine milieu compromises functional capacity of tumor-infiltrating plasmacytoid dendritic cells in HPV-negative but not in HPV-positive HNSCC.

Cancer Immunol Immunother 2021 Sep 11;70(9):2545-2557. Epub 2021 Feb 11.

, Sotio, Prague, Czech Republic.

Plasmacytoid dendritic cells (pDCs) are the most potent type I interferon-producing cells and play an important role in antiviral immunity. Tumor-infiltrating pDCs were shown to be predominantly pro-tumorigenic, with reduced ability to produce interferon alpha (IFNα) and confirmed capacity to prime regulatory T cells (Tregs) by the ICOS/ICOS-L pathway. Because a significant number of HNSCCs are induced by human papillomaviruses and show markedly different immune profiles than non-virally induced tumors, we compared the phenotype and functional capacity of HNSCC-infiltrating pDCs to the HPV status of the tumor. We observed a reduced capacity of pDCs to produce IFNα upon toll-like receptor activation in HPV-negative samples and a rather uncompromised functionality in HPV-associated tumors. Additionally, supernatants from non-virally induced but not HPV-associated tumor cell suspensions significantly inhibited IFNα production by peripheral blood-derived pDCs. We identified IL-10 and TNFα as the soluble pDC-suppressive factors with the highest variability between HPV-negative and HPV-positive tumor-derived supernatants. Additionally, we observed a positive correlation of tumor-infiltrating pDCs with Tregs in HPV-negative samples but not in virally induced tumors. Overall, our study indicates that the immunosuppressive cytokine milieu rich in IL-10 and TNFα in HPV-negative but not in HPV-positive HNSCC significantly affects the functional capacity of tumor-infiltrating pDCs, and such dysfunctional pDCs may further support the immunosuppressive tumor microenvironment by promoting the expansion of Tregs in the tumor tissue.
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http://dx.doi.org/10.1007/s00262-021-02874-yDOI Listing
September 2021

Tumor-infiltrating B cells affect the progression of oropharyngeal squamous cell carcinoma via cell-to-cell interactions with CD8 T cells.

J Immunother Cancer 2019 10 17;7(1):261. Epub 2019 Oct 17.

SOTIO a.s, Jankovcova 1518/2, CZ-17000 Prague 7, Prague, Czech Republic.

Background: Standard treatment of oropharyngeal squamous cell carcinoma (OPSCC) is associated with high morbidity, whereas immunotherapeutic approaches using PD-1:PD-L1 checkpoint blockade only show moderate response rates in OPSCC patients. Therefore, a better stratification of patients and the development of novel therapeutic protocols are crucially needed. The importance of tumor-infiltrating B cells (TIL-Bs) in shaping antitumor immunity remains unclear; therefore, we analyzed frequency, phenotype, prognostic value and possible roles of TIL-Bs in OPSCC.

Methods: We utilized transcriptomic analysis of immune response-related genes in 18 OPSCC samples with respect to human papillomavirus (HPV) status. The density and localization of CD20, CD8 and DC-LAMP cells were subsequently analyzed in 72 tissue sections of primary OPSCC samples in relation to patients' prognosis. The immunohistochemical approach was supplemented by flow cytometry-based analysis of phenotype and functionality of TIL-Bs in freshly resected primary OPSCC tissues.

Results: We observed significantly higher expression of B cell-related genes and higher densities of CD20 B cells in HPV-associated OPSCC samples. Interestingly, CD20 TIL-Bs and CD8 T cells formed non-organized aggregates with interacting cells within the tumor tissue. The densities of both intraepithelial CD20 B cells and B cell/CD8 T cell interactions showed prognostic significance, which surpassed HPV positivity and CD8 TIL density in stratification of OPSCC patients. High density of TIL-Bs was associated with an activated B cell phenotype, high CXCL9 production and high levels of tumor-infiltrating CD8 T cells. Importantly, the abundance of direct B cell/CD8 T cell interactions positively correlated with the frequency of HPV16-specific CD8 T cells, whereas the absence of B cells in tumor-derived cell cultures markedly reduced CD8 T cell survival.

Conclusions: Our results indicate that high abundance of TIL-Bs and high density of direct B cell/CD8 T cell interactions can predict patients with excellent prognosis, who would benefit from less invasive treatment. We propose that in extensively infiltrated tumors, TIL-Bs might recruit CD8 T cells via CXCL9 and due to a highly activated phenotype contribute by secondary costimulation to the maintenance of CD8 T cells in the tumor microenvironment.
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http://dx.doi.org/10.1186/s40425-019-0726-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796441PMC
October 2019

Molecular Basis of Cisplatin Resistance in Testicular Germ Cell Tumors.

Cancers (Basel) 2019 Sep 6;11(9). Epub 2019 Sep 6.

Department of Pediatric Hematology and Oncology, CLIP, 2nd Faculty of Medicine, Charles University and University Hospital Motol, 150 00 Prague, Czech Republic.

The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients' samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains (chr 1, 17, 20, 21) and losses (chr X), with additional more CNVs found in CDDP-resistant cells (e.g., further losses on chr 1, 4, 18, and gain on chr 8). In the patients' samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g., , in one patient, and in another one). Among all primary tumor samples, the most commonly mutated gene was , affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development.
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http://dx.doi.org/10.3390/cancers11091316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769617PMC
September 2019

Dysfunction of HPV16-specific CD8+ T cells derived from oropharyngeal tumors is related to the expression of Tim-3 but not PD-1.

Oral Oncol 2018 07 26;82:75-82. Epub 2018 May 26.

Sotio, Jankovcova 1518/2, 170 00 Prague, Czech Republic. Electronic address:

Background: Human papillomavirus (HPV) type 16 infection is one of the most important etiological agents of oropharyngeal squamous cell carcinoma. Patients with HPV-associated carcinomas of the head and neck were reported to have a better clinical outcome than patients with HPV-negative tumors. Because HPV16 E6 and E7 oncoproteins are highly immunogenic and constitutively expressed, HPV-specific T cell immunity may play the key role in improving the prognosis of these patients.

Methods: Tumor-derived T cells were expanded in high levels of IL-2 and stimulated with HPV16 E6/E7 peptides in the presence or absence of anti-PD-1 monoclonal antibody nivolumab and soluble Tim-3.

Results: HPV16-specific tumor-infiltrating T cells were present in 73.1% of HPV-associated oropharyngeal tumors. HPV16 specific CD8+ TILs were able to produce IFNγ upon specific stimulation and predominantly expressed PD-1 but not Tim-3. Specific IFNγ production was further enhanced after a blockade of both PD-1 and Tim-3 pathways but not after a PD-1 blockade alone. Additionally, the specific stimulation of anti-HPV16 CD8+ T cells suppressed Tim-3 upregulation after the PD-1 blockade.

Conclusion: Our data provide the rationale for combination cancer immunotherapy approaches, including the dual blockade of PD-1 and Tim-3 and, potentially, the use of HPV16-directed therapeutic vaccines.
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http://dx.doi.org/10.1016/j.oraloncology.2018.05.010DOI Listing
July 2018

Comparison of the miRNA expression profiles in fresh frozen and formalin-fixed paraffin-embedded tonsillar tumors.

PLoS One 2017 23;12(6):e0179645. Epub 2017 Jun 23.

Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague, Czech Republic.

MicroRNAs are considered as promising prognostic and diagnostic biomarkers of human cancer since their profiles differ between tumor types. Most of the tumor profiling studies were performed on rarely available fresh frozen (FF) samples. Alternatively, archived formalin-fixed paraffin-embedded (FFPE) tissue samples are also well applicable to larger-scale retrospective miRNA profiling studies. The aim of this study was to perform systematic comparison of the miRNA expression profiles between FF and macrodissected FFPE tonsillar tumors using the TaqMan Low Density Array system, with the data processed by different software programs and two types of normalization methods. We observed a marked correlation between the miRNA expression profiles of paired FF and FFPE samples; however, only 27-38% of the differentially deregulated miRNAs overlapped between the two source systems. The comparison of the results with regard to the distinct modes of data normalization revealed an overlap in 58-67% of differentially expressed miRNAs, with no influence of the choice of software platform. Our study highlights the fact that for an accurate comparison of the miRNA expression profiles from published studies, it is important to use the same type of clinical material and to test and select the best-performing normalization method for data analysis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179645PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5482461PMC
October 2017

Comparison of the miRNA profiles in HPV-positive and HPV-negative tonsillar tumors and a model system of human keratinocyte clones.

BMC Cancer 2016 07 4;16:382. Epub 2016 Jul 4.

Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.

Background: Better insights into the molecular changes involved in virus-associated and -independent head and neck cancer may advance our knowledge of HNC carcinogenesis and identify critical disease biomarkers. Here we aimed to characterize the expression profiles in a matched set of well-characterized HPV-dependent and HPV-independent tonsillar tumors and equivalent immortalized keratinocyte clones to define potential and clinically relevant biomarkers of HNC of different etiology.

Methods: Fresh frozen tonsillar cancer tissues were analyzed together with non-malignant tonsillar tissues and compared with cervical tumors and normal cervical tissues. Furthermore, relative miRNAs abundance levels of primary and immortalized human keratinocyte clones were evaluated. The global quantitation of miRNA gene abundance was performed using a TaqMan Low Density Array system. The confirmation of differentially expressed miRNAs was performed on a set of formalin-fixed paraffin-embedded tumor samples enriched for the tumor cell fraction by macrodissection.

Results: We defined 46 upregulated and 31 downregulated miRNAs characteristic for the HPV-positive tonsillar tumors and 42 upregulated miRNAs and 42 downregulated miRNAs characteristic for HPV-independent tumors. In comparison with the expression profiles in cervical tumors, we defined miR-141-3p, miR-15b-5p, miR-200a-3p, miR-302c-3p, and miR-9-5p as specific for HPV induced malignancies. MiR-335-5p, miR-579-3p, and miR-126-5p were shared by the expression profiles of HPV-positive tonsillar tumors and of the HPV immortalized keratinocyte clones, whereas miR-328-3p, miR-34c-3p, and miR-885-5p were shared by the miRNA profiles of HPV-negative tonsillar tumors and the HPV-negative keratinocytes.

Conclusions: We identified the miRNAs characteristic for HPV-induced tumors and tonsillar tumors of different etiology, and the results were compared with those of the model system. Our report presents the basis for further investigations leading to the identification of clinically relevant diagnostic and/or therapeutic biomarkers for tumors of viral and non-viral etiology.
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http://dx.doi.org/10.1186/s12885-016-2430-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4932682PMC
July 2016

Analysis of the integration of human papillomaviruses in head and neck tumours in relation to patients' prognosis.

Int J Cancer 2016 Jan 13;138(2):386-95. Epub 2015 Aug 13.

Department of Immunology, Institute of Hematology and Blood Transfusion, Prague, Czech Republic.

Integration, which leads to the disruption of the circular HPV genome, is considered as a critical, albeit not obligatory, step in carcinogenic progression. Although cervical carcinomas with extrachromosomal HPV plasmid genomes have been described, the virus is integrated in 70% of HPV16-positive cervical tumours. Limited information is available about HPV integration in head and neck tumours (HNC). In this study, we have characterised the physical status of HPV in a set of tonsillar tumour samples using different methods--the mapping of E2 integration breakpoint at the mRNA level, the 3' RACE based Amplification of Papillomavirus Oncogene Transcripts (APOT) assay and Southern blot. Furthermore, the impact of HPV integration on patients' prognosis has been evaluated in a larger set of 186 patients with head and neck cancer. Based on the analysis of E2 mRNA, HPV was integrated in the host genome in 43% of the HPV-positive samples. Extrachromosomal or mixed form was present in 57%. In fresh frozen samples, the APOT and E2 mapping results were in agreement. The results were confirmed using Southern blotting. Furthermore, the type and exact site of integration were determined. The survival analysis of 186 patients revealed HPV positivity, tumour size and lymph node positivity as factors that influence disease specific survival. However, no statistically significant difference was found in disease specific survival between patients with HPV-positive integrated vs. extrachromosomal/mixed forms of the virus.
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http://dx.doi.org/10.1002/ijc.29712DOI Listing
January 2016

Distinct patterns of intratumoral immune cell infiltrates in patients with HPV-associated compared to non-virally induced head and neck squamous cell carcinoma.

Oncoimmunology 2015 Jan 30;4(1):e965570. Epub 2015 Jan 30.

Sotio, Prague , Czech Republic ; Department of Immunology; 2nd Faculty of Medicine; Charles University; Motol University Hospital ; Prague, Czech Republic.

Human papillomavirus (HPV) infection is one of the most important etiologic causes of oropharyngeal head and neck squamous cell carcinoma (HNSCC). Patients with HPV-positive HNSCC were reported to have a better clinical outcome than patients with HPV-negative cancers. However, little is known about the possible causes of different clinical outcomes. In this study, we analyzed a detailed immune profile of tumor samples from HNSCC patients with respect to their HPV status. We analyzed the characteristics of immune cell infiltrates, including the frequency and distribution of antigen-presenting cells and naïve, regulatory and effector T cells and the cytokine and chemokine levels in tumor tissue. There was a profound difference in the extent and characteristics of intratumoral immune cell infiltrates in HNSCC patients based on their HPV status. In contrast to HPV-negative tumor tissues, HPV-positive tumor samples showed significantly higher numbers of infiltrating IFNγ CD8 T lymphocytes, IL-17 CD8 T lymphocytes, myeloid dendritic cells and proinflammatory chemokines. Furthermore, HPV-positive tumors had significantly lower expression of mRNA and higher expression of PD1 mRNA compared to HPV-negative tumors. The presence of a high level of intratumoral immune cell infiltrates might play a crucial role in the significantly better response of HPV-positive patients to standard therapy and their favorable clinical outcome. Furthermore, characterization of the HNSCC immune profile might be a valuable prognostic tool in addition to HPV status and might help identify novel targets for therapeutic strategies, including cancer immunotherapy.
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http://dx.doi.org/10.4161/21624011.2014.965570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368144PMC
January 2015

[Soft tissue tumors - the view of the molecular biologist].

Cesk Patol 2014 Jul;50(3):132-40

Soft tissue tumors (SSTs) constitute a broad spectrum of neoplasms with diverse biological properties. Rare or unusual types are often difficult to classify. Recent studies show, that a significant subset of SSTs including many types of sarcomas are associated with specific genetic changes such as chromosomal translocations producing chimeric genes, which play a role in the pathogenesis of SSTs. Because SSTs represent a diagnostically challenging group of tumors, molecular-genetic techniques (FISH or PCR) are useful as supplementary and/or confirmatory diagnostic tools. In the present paper we demonstrate the usefulness of a combined diagnostic approach using the tools of classical histopathology and immunohistochemistry together with the molecular diagnostic approach in selected nosologic entites.
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July 2014
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