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.

• MeSH
• adjuvantní chemoradioterapie MeSH
• aktivace lymfocytů MeSH
• B-lymfocyty imunologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• dlaždicobuněčné karcinomy hlavy a krku imunologie   mortalita   terapie   virologie   MeSH
• dospělí MeSH
• infekce papilomavirem imunologie   mortalita   terapie   virologie   MeSH
• Kaplanův-Meierův odhad MeSH
• kohortové studie MeSH
• krční disekce MeSH
• lidé středního věku MeSH
• lidé MeSH
• mezibuněčná komunikace imunologie   MeSH
• nádorové mikroprostředí imunologie   MeSH
• nádory orofaryngu imunologie   mortalita   terapie   virologie   MeSH
• orofarynx patologie   chirurgie   MeSH
• Papillomaviridae imunologie   izolace a purifikace   MeSH
• prognóza MeSH
• progrese nemoci MeSH
• senioři MeSH
• tumor infiltrující lymfocyty imunologie   MeSH
• výběr pacientů MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Due to the close relationship between carcinogenesis and human papillomavirus (HPV), and since they are transmitted via huge number of asymptomatic carriers, the detection of HPV is really needed to reduce the risk of developing cancer. According to the best of our knowledge, our study provides the very first method for one-step detection of viral infection and if it has initiated the subsequent cancer proliferation. The proposed novel nanosystem consists of magnetic glass particles (MGPs), which were attached with DNA probe on their surface to hybridize with target DNAs. The MGP-probe-DNA hybrid was finally conjugated with CdTe/ZnSe core/shell quantum dots (QDs). The proposed detection system is based on a novel mechanism in which the MGPs separate out the target DNAs from different biological samples using external magnetic field for better and clear detection and the QDs give different fluorescent maxima for different target DNAs due to their ability to interact differently with different nucleotides. Firstly, the method was optimized using HPV genes cloned into synthetic plasmids. Then it was applied directly on the samples from normal and cancerous cells. After that, the real hospital samples of head and neck squamous cell carcinoma (HNSCC) with or without the infection of HPV were also analyzed. Our novel nano-system is proved successful in detecting and distinguishing between the patients suffering by HPV infection with or without subsequent cancer having detection limit estimated as 1.0 x 109 (GEq/mL). The proposed methodology is faster and cost-effective, which can be applied at the clinical level to help the doctors to decide the strategy of medication that may save the life of the patients with an early treatment.

• MeSH
• biosenzitivní techniky metody   MeSH
• dlaždicobuněčné karcinomy hlavy a krku virologie   MeSH
• DNA sondy chemie   genetika   MeSH
• DNA virů krev   chemie   genetika   MeSH
• dospělí MeSH
• fluorescenční mikroskopie metody   MeSH
• fluorescenční spektrometrie metody   MeSH
• hybridizace nukleových kyselin MeSH
• infekce papilomavirem diagnóza   MeSH
• kvantové tečky chemie   MeSH
• lidé MeSH
• limita detekce MeSH
• magnetické jevy MeSH
• nádorové buněčné linie MeSH
• Papillomaviridae chemie   MeSH
• senioři MeSH
• sklo chemie   MeSH
• sloučeniny kadmia chemie   MeSH
• telur chemie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH

Cíl studie: V rámci této studie jsme využili metody kvantitativní real-time PCR pro sledování úrovně relativní exprese miR-145-5p, miR-484 a miR-99a-5p v souboru HPV pozitivních a HPV negativních vzorků sinonasálního dlaždicobuněčného karcinomu. Typ studie: původní práce.Název a sídlo pracoviště: Ústav klinické biochemie a diagnostiky, Lékařská fakulta v Hradci Králové, Univerzita Karlova a Fakultní nemocnice Hradec Králové, Sokolská 581, 500 05 Hradec Králové.Materiál a metody: Metoda kvantitativní real-time PCR s TaqManTM Advanced miRNA Assays byla použita pro sledování relativní exprese vybraných mikroRNA (miR-145-5p, miR-484 a miR-99a-5p) v unikátním souboru vzorků fixovaných ve formalínu a archivovaných v parafinu získaných od 46 pacientů se sinonasálním dlaždicobuněčných karcinomem. Statistická analýza (Studentův t-test, jednofaktorová analýza rozptylu a regresní analýza) byla provedena s cílem porovnat úroveň relativní exprese mikroRNA se zaznamenanými klinickopatologickými daty jako je HPV status.Výsledky: Naše výsledky ukazují statisticky významnou downregulaci miR-145-5p (P < 0,001 a Fold change = -2,78) ve vzorcích sinonasálního skvamózního karcinomu. Přítomnost HPV infekce korelovala s mírou exprese všech tří studovaných mikroRNA. Exprese miR-145-5p byla nižší u HPV negativních vzorků (P = 0,019), miR-99a-5p byla upregulována u HPV pozitivních vzorků (P = 0,058) a miR-484 byla downregulována u HPV pozitivních vzorků (P = 0,016). Pacienti, kteří byli zařazeni do kategorie kuřáci nebo bývalí kuřáci, vykazovali downregulaci miR-99a-5p (P = 0,060). Perineurální šíření bylo spojeno s významnou downregulací miR-99a-5p (P = 0,0055). Významná downregulace miR-145-5p byla spojena s angioinvazí (P = 0,037) a regionálním šířením (P = 0,076).Závěr: V rámci naší studie jsme nalezli spojitost mezi expresí studovaných mikroRNA a přítomností HPV infekce v nádorových vzorcích. Naše výsledky naznačují, že miR-145-5p, miR-484 a miR-99a jsou součástí patogeneze HPV. Avšak přesná role miRNA spojených s HPV infekcí ve vývoji sinonasálních karcinomů není dosud známá.

Objective: In the present study, we used quantitative real-time PCR to investigate relative expression values of miR-145-5p, miR-484 and miR-99a-5p in HPV positive and HPV negative samples of sinonasal squamous cell carcinoma. Design: Original Article. Settings: Institute of Clinical Biochemistry and Diagnostics, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Sokolská 581, 500 05 Hradec Králové Materials and Methods: Quantitative real-time PCR with TaqManTM Advanced miRNA Assays was used to investigate relative expression values of selected microRNAs (miR-145-5p, miR-484 and miR-99a-5p) in a unique set of formalin-fixed paraffin-embedded tissue samples obtained from 46 patients with sinonasal squamous cell carcinoma. Statistical analysis (Student‘s t-test, one-way analysis of variance and regression analysis) was performed to compare relative expression miRNA values and recorded clinicopathological data such as HPV status. Results: Our results show statistically significant downregulation of miR-145-5p (P < 0.001 and Fold change = -2.78) in sinonasal squamous cell carcinoma samples. The presence of HPV infection correlated with the expression levels of all three studied miRNAs. The expression of miR-145-5p was lower in HPV negative samples (P = 0.019), miR-99a-5p was upregulated in HPV positive samples (P = 0.058) and miR-484 was downregulated in HPV positive samples (P = 0.016). Patients with recorded history of smoking or currents smokers showed downregulation of miR-99a-5p (P = 0.060). Perineural invasion was linked to significant downregulation of miR-99a-5p (P = 0.0055). Distinctive downregulation of miR-145-5p was linked to vascular invasion (P = 0.037) and regional recurrence (P = 0.076). Conclusion: We have found correlation between studied miRNA expression and presence of HPV infection in the patient samples. Our results suggest that miR-145, miR-484 and miR-99a are involved in HPV pathogenesis. However, the actual pathway of HPV related miRNA involvement in sinonasal tumor development is not yet known.

• Klíčová slova
• sinonasální karcinom,
• MeSH
• dlaždicobuněčné karcinomy hlavy a krku * patologie   virologie   MeSH
• infekce papilomavirem komplikace   MeSH
• klinická studie jako téma MeSH
• lidé MeSH
• mikro RNA MeSH
• nádory vedlejších dutin nosních patologie   virologie   MeSH
• Papillomaviridae * izolace a purifikace   MeSH
• Check Tag
• lidé MeSH

Although the role of high-risk human papillomaviruses (hrHPVs) as etiological agents in cancer development has been intensively studied during the last decades, there is still the necessity of understanding the impact of the HPV E6 and E7 oncogenes on host cells, ultimately leading to malignant transformation. Here, we used newly established immortalized human keratinocytes with a well-defined HPV16 E6E7 expression cassette to get a more complete and less biased overview of global changes induced by HPV16 by employing transcriptome sequencing (RNA-Seq) and stable isotope labeling by amino acids in cell culture (SILAC). This is the first study combining transcriptome and proteome data to characterize the impact of HPV oncogenes in human keratinocytes in comparison with their virus-negative counterparts. To enhance the informative value and accuracy of the RNA-Seq data, four different bioinformatic workflows were used. We identified potential novel upstream regulators (e.g., CNOT7, SPDEF, MITF, and PAX5) controlling distinct clusters of genes within the HPV-host cell network as well as distinct factors (e.g., CPPED1, LCP1, and TAGLN) with essential functions in cancer. Validated results in this study were compared to data sets from The Cancer Genome Atlas (TCGA), demonstrating that several identified factors were also differentially expressed in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and HPV-positive head and neck squamous cell carcinomas (HNSCs). This highly integrative approach allows the identification of novel HPV-induced cellular changes that are also reflected in cancer patients, providing a promising omics data set for future studies in both basic and translational research.IMPORTANCE Human papillomavirus (HPV)-associated cancers still remain a big health problem, especially in developing countries, despite the availability of prophylactic vaccines. Although HPV oncogenes have been intensively investigated for decades, a study applying recent advances in RNA-Seq and quantitative proteomic approaches to a precancerous model system with well-defined HPV oncogene expression alongside HPV-negative parental cells has been missing until now. Here, combined omics analyses reveal global changes caused by the viral oncogenes in a less biased way and allow the identification of novel factors and key cellular networks potentially promoting malignant transformation. In addition, this system also provides a basis for mechanistic research on novel key factors regulated by HPV oncogenes, especially those that are confirmed in vivo in cervical cancer as well as in head and neck cancer patient samples from TCGA data sets.

• MeSH
• adenokarcinom genetika   virologie   MeSH
• dlaždicobuněčné karcinomy hlavy a krku genetika   virologie   MeSH
• genové regulační sítě * MeSH
• karcinogeneze genetika   MeSH
• keratinocyty virologie   MeSH
• lidé MeSH
• lidský papilomavirus 16 genetika   MeSH
• nádorová transformace buněk MeSH
• nádory děložního čípku genetika   virologie   MeSH
• onkogenní proteiny virové genetika   MeSH
• proteom genetika   MeSH
• proteomika MeSH
• spinocelulární karcinom genetika   virologie   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom * MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• časové faktory MeSH
• cetuximab škodlivé účinky   terapeutické užití   MeSH
• dlaždicobuněčné karcinomy hlavy a krku farmakoterapie   mortalita   sekundární   virologie   MeSH
• infekce papilomavirem virologie   MeSH
• klinické rozhodování MeSH
• lidé MeSH
• lokální recidiva nádoru MeSH
• nádory hlavy a krku farmakoterapie   mortalita   patologie   virologie   MeSH
• protinádorové látky imunologicky aktivní škodlivé účinky   terapeutické užití   MeSH
• protokoly antitumorózní kombinované chemoterapie škodlivé účinky   terapeutické užití   MeSH
• rizikové faktory MeSH
• výsledek terapie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH