One of the most common mechanisms of immune evasion in MSI colorectal cancers (CRCs) is loss of HLA class I expression due to mutations in B2M gene which can become a negative predictor for checkpoint blockade therapy. The aim of this study was the determination of prevalence of B2M somatic mutations in MSI CRC patients and relationship between B2M mutations and lymphocytes infiltration and other clinicopathological features as well as detection of methylation changes in B2M promoter region which can be another mechanism of immune escape. In our study, 37 MSI-H and 5 MSI-L patients were selected for screening of B2M mutational and methylation status. The characterization of patients was based on standard histopathological diagnosis and TNM classification; BRAF, KRAS mutations, tumor-infiltrating lymphocytes and peritumoral lymphoid reaction were also determined. MSI analysis was performed using fragment analysis. B2M mutations were identified by Sanger sequencing, and methylation of CpG islands in promoter region was detected by methylation-specific PCR. Heterozygous mutations in the B2M gene were detected in five MSI-H patients (13.5%), while the mutation c.45_48delTTCT was determined in four patients and mutation c.276delC was found in two patients. One of these five patients was compound heterozygote harboring both mutations. Methylation of the promoter region of the B2M gene was observed in one patient with MSI-H colorectal cancer. Detection of genetic and epigenetic changes in B2M gene could be important in personalized therapy for CRC patients as these changes may be one of the mechanisms of secondary resistance of MSI positive tumors to immunotherapy.

• Klíčová slova
• Beta-2-microglobulin, Cancer immunogenicity, Colorectal cancer, Microsatellite instability, Promoter methylation,
• MeSH
• beta-2-mikroglobulin genetika   MeSH
• CpG ostrůvky MeSH
• dospělí MeSH
• down regulace MeSH
• epigeneze genetická MeSH
• kolorektální nádory genetika   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA * MeSH
• mikrosatelitní nestabilita * MeSH
• mutace MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u nádorů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• staging nádorů MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• B2M protein, human MeSH Prohlížeč
• beta-2-mikroglobulin MeSH

In the majority of human tumors, downregulation of major histocompatibility complex class I (MHC‑I) expression contributes to the escape from the host immune system and resistance to immunotherapy. Relevant animal models are therefore needed to enhance the efficacy of cancer immunotherapy. As loss of β‑2 microglobulin expression results in irreversible downregulation of surface MHC‑I molecules in various human tumors, the β‑2 microglobulin gene (B2m) was deactivated in a mouse oncogenic TC‑1 cell line and a TC‑1/dB2m cell line that was negative for surface MHC‑I expression was derived. Following stimulation with interferon γ, MHC‑I heavy chains, particularly the H‑2Db molecules, were found to be expressed at low levels on the cell surface, but without β‑2 microglobulin. B2m deactivation in TC‑1/dB2m cells led to reduced proliferation and tumor growth. These cells were insensitive to DNA vaccination and only weakly responsive to combined immunotherapy with a DNA vaccine and the ODN1826 adjuvant. In vivo depletion demonstrated that NK1.1+ cells were involved in both reduced tumor growth and an antitumor effect of immunotherapy. The number of immune cells infiltrating TC‑1/dB2m‑induced tumors was comparable with that in tumors developing from TC‑1/A9 cells characterized by reversible MHC‑I downregulation. However, the composition of the cell infiltrate was different and, most importantly, infiltration with immune cells was not increased in TC‑1/dB2m tumors after immunotherapy. Therefore, the TC‑1/dB2m cell line represents a clinically relevant tumor model that may be used for enhancement of cancer immunotherapy.

• MeSH
• beta-2-mikroglobulin genetika   imunologie   MeSH
• cytotoxické T-lymfocyty imunologie   patologie   MeSH
• imunoterapie MeSH
• interferon gama imunologie   MeSH
• lidé MeSH
• MHC antigeny I. třídy genetika   imunologie   MeSH
• myši MeSH
• nádorové buněčné linie metabolismus   MeSH
• nádory genetika   imunologie   patologie   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• beta-2-mikroglobulin MeSH
• interferon gama MeSH
• MHC antigeny I. třídy MeSH

The selection of a suitable combination of reference genes (RGs) for data normalization is a crucial step for obtaining reliable and reproducible results from transcriptional response analysis using a reverse transcription-quantitative polymerase chain reaction. This is especially so if a three-dimensional multicellular model prepared from liver tissues originating from biologically diverse human individuals is used. The mRNA and miRNA RGs stability were studied in thirty-five human liver tissue samples and twelve precision-cut human liver slices (PCLS) treated for 24 h with dimethyl sulfoxide (controls) and PCLS treated with β-naphthoflavone (10 µM) or rifampicin (10 µM) as cytochrome P450 (CYP) inducers. Validation of RGs was performed by an expression analysis of CYP3A4 and CYP1A2 on rifampicin and β-naphthoflavone induction, respectively. Regarding mRNA, the best combination of RGs for the controls was YWHAZ and B2M, while YWHAZ and ACTB were selected for the liver samples and treated PCLS. Stability of all candidate miRNA RGs was comparable or better than that of generally used short non-coding RNA U6. The best combination for the control PCLS was miR-16-5p and miR-152-3p, in contrast to the miR-16-5b and miR-23b-3p selected for the treated PCLS. Our results showed that the candidate RGs were rather stable, especially for miRNA in human PCLS.

• Klíčová slova
• RT-qPCR, human liver, mRNA, miRNA, precision-cut liver slices, reference gene,
• MeSH
• beta-2-mikroglobulin genetika   metabolismus   MeSH
• beta-naftoflavon farmakologie   MeSH
• cytochrom P-450 CYP1A2 genetika   metabolismus   MeSH
• cytochrom P-450 CYP3A genetika   metabolismus   MeSH
• dimethylsulfoxid farmakologie   MeSH
• dospělí MeSH
• játra účinky léků   metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce normy   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mikro RNA genetika   metabolismus   MeSH
• proteiny 14-3-3 genetika   metabolismus   MeSH
• referenční standardy MeSH
• rifampin farmakologie   MeSH
• senioři MeSH
• stanovení celkové genové exprese normy   MeSH
• systém (enzymů) cytochromů P-450 farmakologie   MeSH
• transkriptom 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
• Názvy látek
• B2M protein, human MeSH Prohlížeč
• beta-2-mikroglobulin MeSH
• beta-naftoflavon MeSH
• CYP1A2 protein, human MeSH Prohlížeč
• CYP3A4 protein, human MeSH Prohlížeč
• cytochrom P-450 CYP1A2 MeSH
• cytochrom P-450 CYP3A MeSH
• dimethylsulfoxid MeSH
• messenger RNA MeSH
• mikro RNA MeSH
• proteiny 14-3-3 MeSH
• rifampin MeSH
• systém (enzymů) cytochromů P-450 MeSH
• YWHAZ protein, human MeSH Prohlížeč

Purpose Hodgkin Reed-Sternberg (HRS) cells evade antitumor immunity by multiple means, including gains of 9p24.1/ CD274(PD-L1)/ PDCD1LG2(PD-L2) and perturbed antigen presentation. Programmed death 1 (PD-1) receptor blockade is active in classic Hodgkin lymphoma (cHL) despite reported deficiencies of major histocompatibility complex (MHC) class I expression on HRS cells. Herein, we assess bases of sensitivity to PD-1 blockade in patients with relapsed/refractory cHL who were treated with nivolumab (anti-PD-1) in the CheckMate 205 trial. Methods HRS cells from archival tumor biopsies were evaluated for 9p24.1 alterations by fluorescence in situ hybridization and for expression of PD ligand 1 (PD-L1) and the antigen presentation pathway components-β2-microglobulin, MHC class I, and MHC class II-by immunohistochemistry. These parameters were correlated with clinical responses and progression-free survival (PFS) after PD-1 blockade. Results Patients with higher-level 9p24.1 copy gain and increased PD-L1 expression on HRS cells had superior PFS. HRS cell expression of β2-microglobulin/MHC class I was not predictive for complete remission or PFS after nivolumab therapy. In contrast, HRS cell expression of MHC class II was predictive for complete remission. In patients with a > 12-month interval between myeloablative autologous stem-cell transplantation and nivolumab therapy, HRS cell expression of MHC class II was associated with prolonged PFS. Conclusion Genetically driven PD-L1 expression and MHC class II positivity on HRS cells are potential predictors of favorable outcome after PD-1 blockade. In cHL, clinical responses to nivolumab were not dependent on HRS cell expression of MHC class I.

• MeSH
• antigeny CD274 antagonisté a inhibitory   biosyntéza   genetika   imunologie   MeSH
• antigeny CD279 antagonisté a inhibitory   biosyntéza   genetika   imunologie   MeSH
• beta-2-mikroglobulin biosyntéza   genetika   imunologie   MeSH
• buňky Reedové-Sternberga účinky léků   imunologie   patologie   MeSH
• doba přežití bez progrese choroby MeSH
• Hodgkinova nemoc farmakoterapie   genetika   imunologie   patologie   MeSH
• kohortové studie MeSH
• lidé MeSH
• lidské chromozomy, pár 9 MeSH
• MHC antigeny II. třídy biosyntéza   genetika   imunologie   MeSH
• nivolumab terapeutické užití   MeSH
• prediktivní hodnota testů MeSH
• prezentace antigenu MeSH
• protinádorové látky imunologicky aktivní terapeutické užití   MeSH
• výsledek terapie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky, fáze II MeSH
• multicentrická studie MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• antigeny CD274 MeSH
• antigeny CD279 MeSH
• B2M protein, human MeSH Prohlížeč
• beta-2-mikroglobulin MeSH
• CD274 protein, human MeSH Prohlížeč
• MHC antigeny II. třídy MeSH
• nivolumab MeSH
• PDCD1 protein, human MeSH Prohlížeč
• protinádorové látky imunologicky aktivní MeSH

We compared the effect of control genes (CG): total Abelson (total-ABL), beta-2-microglobulin (B2M) and beta-glucuronidase (GUS), recommended in the Europe Against Cancer (EAC) program, on real-time BCR-ABL monitoring in patients with chronic myeloid leukemia (CML). We focused on the stability of CG expressions during therapy and the effect of the CGs on BCR-ABL ability to characterize the disease status and disease prognosis, issues that have not been addressed yet. The results showed B2M as a very convenient CG for BCR-ABL monitoring. On the contrary, the widely used total-ABL was not confirmed as appropriate for normalization of gene expression in CML.

• MeSH
• bcr-abl fúzní proteiny genetika   MeSH
• beta-2-mikroglobulin genetika   MeSH
• chronická myeloidní leukemie genetika   terapie   MeSH
• dospělí MeSH
• glukuronidasa genetika   MeSH
• komplementární DNA genetika   MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protoonkogenní proteiny c-abl genetika   MeSH
• regulace genové exprese u leukemie MeSH
• RNA nádorová analýza   MeSH
• senioři 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
• Názvy látek
• bcr-abl fúzní proteiny MeSH
• beta-2-mikroglobulin MeSH
• glukuronidasa MeSH
• komplementární DNA MeSH
• messenger RNA MeSH
• protoonkogenní proteiny c-abl MeSH
• RNA nádorová MeSH

• MeSH
• analýza selhání vybavení MeSH
• beta-2-mikroglobulin genetika   MeSH
• DNA analýza   MeSH
• nádorový supresorový protein p53 genetika   MeSH
• pohyb vzduchu MeSH
• polymerázová řetězová reakce přístrojové vybavení   metody   MeSH
• reprodukovatelnost výsledků MeSH
• řízení kvality MeSH
• senzitivita a specificita MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-2-mikroglobulin MeSH
• DNA MeSH
• nádorový supresorový protein p53 MeSH

Monoclonal antibody TG1 recognizes specifically antigens HLA-B27, B7, B22 and B17 on cell surface in cytotoxicity and cytofluorometry tests. When cell detergent extracts were subjected to SDS PAGE under mild conditions (no heating and no reduction of the sample) followed by Western blotting, TG1 detected exclusively a complex of B27 heavy chains with beta(2)-microglobulin (as a 50 kDa band) whereas the other B-locus antigens (B7, B22, B17) were detected as free 43 kDa heavy chains under the same conditions. When the samples were boiled prior to SDS PAGE, TG1 detected again the 43 kDa free heavy chains of B7, B22 and B17 but no zone corresponding to B27 could be detected indicating that the epitope in free B27 chains is more sensitive to denaturation by SDS. Thus, our main finding is that the interaction of HLA-B27 heavy chain with beta(2)-microglobulin appears to be stronger than that of the other HLA-B chains. The resistance of the HLA-B27/beta(2)-microglobulin complex to the SDS dissociation is strikingly similar to the behavior of MHC class II molecules under similar conditions. Thus, it may be speculated that HLA-B27 complexes can be also more stable than other MHC class I molecules under more physiological dissociative conditions (e.g. in endosomal compartments). This feature might potentially influence antigen presentation by HLA-B27 and contribute to the well known disease linkage of HLA-B27.

• MeSH
• beta-2-mikroglobulin genetika   izolace a purifikace   metabolismus   MeSH
• buněčné linie MeSH
• dodecylsíran sodný MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• HLA-B27 antigen genetika   izolace a purifikace   metabolismus   MeSH
• lidé MeSH
• lymfocyty chemie   MeSH
• molekulová hmotnost MeSH
• monoklonální protilátky metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• transformované buněčné linie MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-2-mikroglobulin MeSH
• dodecylsíran sodný MeSH
• HLA-B27 antigen MeSH
• monoklonální protilátky MeSH

• MeSH
• beta-2-mikroglobulin genetika   metabolismus   MeSH
• beta-globuliny metabolismus   MeSH
• buněčné linie MeSH
• fenotyp * MeSH
• kultivované buňky MeSH
• leukemie metabolismus   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• Názvy látek
• beta-2-mikroglobulin MeSH
• beta-globuliny MeSH
• membránové proteiny MeSH