MATERIALS OF NOBEL SYMPOSIUM 175: PRECISION MEDICINE TRANSFORMS HEALTHCARE: A NEW TRAJECTORY FOR RESEARCH AND INNOVATION SEPTEMBER 20-22, 2023, STOCKHOLM, SWEDEN.

Nobel Symposium 175 was organized by Professor Richard Rosenquist Brandell of Karolinska Institutet and was supported by the Royal Swedish Academy of Sciences. The focus of the symposium was a discussion on the development of precision medicine in infectious and rare diseases, cancer, and complex diseases. Presentations and discussions concerned new technologies, bioinformatics, and new diagnostic and therapeutic approaches based on findings in basic research. Organization of precision medicine models and their implementation in medical practice at the national and international levels were also on the agenda. 29 scientists from different fields of medicine presented their work during a three-day exciting trip into the future of patient' care. Panel discussions shed light on the development of precision medicine for better treatment of patients.

DIFFERENTIAL EXPRESSION PATTERN OF AIP, UCKL1, AND PKN1 GENES IN PROSTATE CANCER PATIENTS.

BACKGROUND: The evolution of research on the therapy of prostate cancer (PC) depends on a study of molecules that are involved in the progression of this disease. Nevertheless, there is a need for additional biomarkers that would help to refine the molecular profile of PC and propose the personalized therapeutic approach. AIM: To study differential expression patterns of the AIP, UCKL1, and PKN1 genes in blood sera and tumor tissue of patients with PC with different Gleason scores. MATERIALS AND METHODS: The total extracellular RNA was isolated from blood sera of 44 PC patients and 4 healthy donors. cDNAs were synthesized and quantitative polymerase chain reaction (qPCR) was performed. Immunohistochemical study of the UCKL, AIP and PKN1 proteins was performed on deparaffinized sections of tumors. The study was supplemented by a bioinformatic analysis of the publicly available databases. RESULTS: The UCKL1, AIP, PKN1 genes were overexpressed at the mRNA level in blood sera of PC patients, compared to healthy donors. Extracellular mRNA levels of AIP and UCKL-1 were 100-1000-fold increased in all PC samples compared to the healthy donors but without significant inequality between the groups of PC cases differing by the Gleason score. The highest levels were detected in the samples from PC patients with the Gleason score > 9. The PKN1 expression was higher in PC patients compared with healthy donors but without significant difference between the groups. CONCLUSIONS: From the three chosen genes, AIP and UCKL1 showed similar pattern of expression assessed either by extracellular mRNA levels in patient sera or the protein in PC tissues. AIP was up to 1000-fold increased in all PC samples, compared to the healthy donors, with the highest levels in PC cases with Gleason score > 9. Expression levels of the AIP and UCKL1 genes in the PC patient sera may be used as an additional criterion for prognosis of tumor progression.

IMPACT OF TUMOR MOLECULAR PHENOTYPE ON SURVIVAL OF PATIENTS WITH GASTRIC CANCER AFTER RADICAL SURGERY.

The strategy for the treatment of gastric cancer (GC), in particular the use of the extended surgical interventions in different countries varies. The different proportion of specific molecular GC subtypes in various populations frequently is not taken into account for comparing treatment outcomes. This pilot study analyzes the association of survival of GC patients after the extended combined surgical interventions depending on the molecular subtype of the tumors. An improved survival for patients with diffuse cancer types (p53-, VEGFR+, HER2/neu+, Ki-67+ phenotype) was demonstrated. The authors propose their point of view on the importance of recognizing GC molecular heterogeneity.Key Words: gastric cancer, molecular classification, tumor biology.

Differential expression patterns of AIP, UCKL1, and PKN1 genes in breast cancer of different molecular subtypes.

BACKGROUND: Classification of breast cancer (BC) in the molecular subtypes had the enormous impact on the development of the individualized therapy. Nevertheless, there is a need for additional biomarkers that would help to refine molecular subtypes of BC and propose the therapeutic approach for each patient. AIM: To study differential expression patterns of AIP, UCKL1, and PKN1 genes in blood sera and tumor tissue of patients with BC of different molecular subtypes. MATERIALS AND METHODS: The total extracellular RNA was isolated from serum of 26 BC patients. cDNAs was synthesized and quantitative polymerase chain reaction was performed. Also, immunohistochemical studies of UCKL, AIP and PKN1 were performed on deparaffined tissue sections. The study was supplemented by a bioinformatic analysis of the publicly available databases. RESULTS: AIP and UCKL-1 extracellular mRNA levels were 100-1000-fold increased in blood sera of all BC patients, compared to the healthy donors. The highest levels were detected in the luminal A and HER2 (ERRB2) BC subtypes. The highest levels of PKN1 were detected blood sera of the patients with luminal B and basal subtypes; its expression levels were just 10-100-fold higher in BC samples compared to healthy donors. CONCLUSIONS: The UCKL1, AIP, PKN1 genes are overexpressed at the mRNA level in blood sera of BC patients compared to the sera of healthy individuals. Among three genes under study, only for the AIP gene, the pattern of extracellular mRNA expression in sera paralleled to protein expression in BC tissues of each specified molecular subtype.

Expression pattern of MRPS18 family genes in gliomas.

AIM: To assess expression patterns of MRPS18 family genes in glioblastoma tissues and glioma cell lines. MATERIALS AND METHODS: Expression of MRPS18 family genes was analyzed by quantitative polymerase chain reaction in glioma cell lines and glioblastoma specimens. A bioinformatic analysis of the publicly available data on the expression of these genes was also provided. RESULTS: The genes of MRPS18 family show different expression patterns in glioblastomas and glioma cell lines. The highest levels of expression were found for MRPS18-2 at mRNA and protein levels in both glioblastomas and glioma cell lines; the lowest - for MRPS18-1 at mRNA level. CONCLUSIONS: The elevated levels of relative expression of the MRPS18-2 gene are characteristic for glioma tumor tissues and cell lines.

Expression pattern of MRPS18 family genes in malignantly transformed b-cells.

AIM: To compare expression patterns of proteins of a family of mitochondrial ribosomal protein S18 (MRPS18) in tumor cell lines of the B-cell origin. MATERIALS AND METHODS: The study has been performed on different subsets of tonsil B-cells and tumor cell lines of the B-cell origin using quantitative polymerase chain reaction analysis, western blot analysis, immunohistochemistry, bioinformatic analysis of the publicly available data bases on expression. RESULTS: We have found that genes of the MRPS18 family (1-3) show different expression patterns in tumor cell lines of the B-cell origin. The highest levels of expression were shown for MRPS18-3, the lowest - for MRPS18-1. MRPS18-2 was expressed at the highest levels in germinal center cells, Burkitt lymphoma and Hodgkin lymphoma cell lines. At the protein levels, MRPS18-2 showed the highest expression in Burkitt lymphoma and B-cell precursor acute lymphoblastic leukemia cell lines. In lymphoblastoid cell lines, and in germinal center B-cells MRPS18-2 levels were somewhat lower, but higher than in memory and plasma B-cells. CONCLUSIONS: The differential expression pattern of the MRPS18 family proteins suggests that they play various roles in cellular processes.

The TGF-beta - SMAD pathway is inactivated in cronic lymphocytic leukemia cells.

AIM: To study the status of the tumor growth factor beta (TGFB) pathway in chronic lymphocytic leukemia (CLL) cells and to uncover molecular details underlying CLL cell genesis. OBJECTS AND METHODS: The study was conducted on peripheral blood samples of patients with CLL using the following methods: RNA isolation, analysis of expression of transcription factors using RT2 profiler assay, bioinformatics analysis of publicly available data bases on expression. RESULTS: We have shown that the TGFB - SMAD canonical pathway is not active in CLL cells. SMAD-responsive genes, such as BCL2L1 (BCL-XL), CCND2 (Cyclin D2), and MYC, are down-regulated in CLL cells compared with peripheral blood B cells of healthy donors. CONCLUSIONS: The TGFB-mediated signaling is not active in CLL cells due to low (or absent) expression of SMAD1, -4, -5, -9, and ATF-3. Expression and phosphorylation status of SMAD2 and -3 should be further elucidated in the future studies.

CD150 and CD180 are involved in regulation of transcription factors expression in chronic lymphocytic leukemia cells.

BACKGROUND: Sequential stages of B-cell development is stringently coordinated by transcription factors (TFs) network that include B-lineage commitment TFs (Ikaros, Runx1/Cbfb, E2A, and FOXO1), B-lineage maintenance TFs (EBF1 and PAX5) and stage specific set of TFs (IRF4, IRF8, BCL6, BLIMP1). Deregulation of TFs expression and activity is often occurs in malignant B cells. The aim of this study was to evaluate TFs expression in chronic lymphocytic leukemia cells taking into consideration CD150 cell surface expression. From other side we attempted to regulate TFs expression via CD150 and CD180 cell surface receptors. MATERIALS AND METHODS: Studies were performed on normal peripheral blood B-cell subpopulations and chronic lymphocytic leukemia (CLL) cells isolated from peripheral blood of 67 primary untreated patients with CLL. Evaluation of TFs expression was performed on mRNA level using qRT-PCR and on protein level by western blot analysis. RESULTS: Median of PAX5 and EBF1 mRNA expression was higher in cell surface CD150 positive (csCD150+) compared to csCD150- CLL cases or normal CD19+ and CD19+CD5+ B-cell subsets. Differences in mRNA expression of IRF8, IRF4 and BLIMP1 between studied groups of CLL and normal B cells were not revealed. All CLL cases were characterized by downregulated expression of PU.1 and BCL6 mRNAs in comparison to normal B cells. At the same time elevated SPIB mRNA expression level was restricted to CLL cells. Protein expression of IRF4, IRF8 and BCL6 was uniformly distributed between csCD150- and csCD150+ CLL cases. PU.1 protein and CD20 that is direct PU.1 target gene positively correlated with CD150 cell surface expression on CLL cells. Ligation of CD150 and CD180 alone or in combination upregulated IRF8 and PU.1 while downregulated the IRF4 mRNA expression. Signaling via CD150 or CD180 alone elevated the level of BCL6 mRNA. Strong downregulation of IRF4 mRNA was observed after CD150, CD180 or CD150 andCD180 coligation on CLL cells. We found that in CLL cells CD150 is a negative regulator of SPIB while CD180 is involved in upregulation of EBF1 expression level. Moreover, CD180 ligation on CLL cells caused increase of CD150 mRNA level that is a one of the EBF1 target genes. CONCLUSIONS: Analysis of TFs expression profile revealed upregulated SPIB mRNA level and downregulated PU.1 in CLL cells. CD150 and CD180 receptors may modulate transcriptional program in CLL cells by regulating the TFs expression levels.

TGF-ß1 expression by glioma C6 cells in vitro.

The aim of the work was to study the impact of fetal rat brain cell supernatant (FRBCS) on the expression of transforming growth factor ß1 (TGF-ß1) and p53 in C6 cells of rat glioma in vitro. MATERIALS AND METHODS: FRBCS was obtained from suspensions of fetal rat brain cells on the 14th (E14) day of gestation. C6 glioma cells were cultured for 48 h in the presence of FRBCS or FRBCS + anti-TGF-ß1 monoclonal antibody. Immunocytochemical staining for TGF-ß1 and p53 was performed. RESULTS: The proportion of TGF-ß1-immunopositive tumor cells in C6 glioma cultures was statistically significantly higher than in the control cell cultures of normal fetal rat brain. FRBCS reduced the proportion of TGF-ß1-immunopositive tumor cells and increased the proportion of p53-immunopositive cells in C6 glioma cultures. In cells cultured with FRBCS + anti-TGF-ß1 monoclonal antibody, the above effects of FRBCS were abrogated. CONCLUSION: The obtained results suggest that TGF-ß1 seems to be responsible for decrease in TGF-ß1 expression and increase in p53 expression in C6 glioma cells treated with FRBCS.

Expression Levels of the Uridine-Cytidine Kinase Like-1 Protein As a Novel Prognostic Factor for Hepatitis C Virus-Associated Hepatocellular Carcinomas.

The expression levels of the two novel oncoproteins uridine-cytidine kinase like-1 (UCKL-1) and mitochondrial ribosomal protein S18-2 (MRPS18-2) were assessed in samples of hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) using immunohistochemistry. Tissue microarray (TMA) paraffin blocks were prepared from 42 HCC tumor samples with the corresponding peri-tumor tissues and from 11 tissues of a liver with HCV-induced cirrhosis. We found that the UCKL-1 signal in the liver tissues of the peri-tumor zone in the HCC samples was stronger than that in cirrhosis (50 ± 49.44 vs. 24.27 ± 14.53; p = 0.014). The MRPS18-2 expression was weak, and there was no differences between the groups (p = 0.26). Noteworthy, the UCKL-1 protein was expressed at higher levels in peri-tumor tissues in the cases of HCC recurrence; this was confirmed for 27 older patients (63.78 ± 9.22 vs. 53.53 ± 4.07 years, p < 0.001), in parallel with enhanced UCKL-1 staining in former HCC nodules (62.69 ± 50.4 vs. 26.0 ± 30.19, p = 0.006) and microvascular invasion (p = 0.02). A multivariate analysis of prognostic factors for HCC recurrence showed that the best predictive factors for these conditions were UCKL-1 expression in tumor, vascular invasion, and HCC treatment modality, other than liver transplantation (odds ratios: 1.029, 18.143 and 11.984, R2 = 0.633, p = 0.002). In conclusion, the high UCKL-1 expression might be a prognostic factor for HCC relapse, in combination with age and microvascular invasion. MRPS18-2 protein expression has no prognostic significance in the cases of HCV-associated HCC.

Differential expression of CD150/SLAMF1 in normal and malignant B cells on the different stages of maturation.

UNLABELLED: Within B-cell lineage cell surface receptor CD150/SLAMF1 is broadly expressed starting from pre-B cells with upregulation toward plasma cells. However, expression of CD150 is rather limited on the surface of malignant B cells with the block of differentiation at the different stages of maturation. The aim of our work was to explore CD150 expression both on protein and mRNA levels with the emphasis on CD150 isoforms in malignant B-cell lines at the different stages of maturation in comparison with their normal B cell counterparts. MATERIALS AND METHODS: Studies were performed on normal tonsillar B-cell subpopulations, B-lymphoblastoid cell lines, malignant B-cell lines of different origin, including pre-B acute lymphoblastic leukemia, Burkitt's lymphoma, Hodgkin's lymphoma, and multiple myeloma. Protein CD150 expression was assessed by western blot analysis and the expression level of CD150 isoforms was evaluated using qRT-PCR. RESULTS: Despite the similar CD150 expression both on mRNA and protein levels in normal B-cell subsets and B-lymphoblastoid cell lines, malignant B-cell lines demonstrated substantial heterogeneity in CD150 expression. Only Hodgkin's lymphoma cell lines, Burkitt's lymphoma cell lines BJAB and Raji, and also pre-B cell line BLIN-1 expressed CD150 protein. At the same time total CD150 and mCD150 mRNA was detected in all studied cell lines excluding pre-B cell line REH. The minor sCD150 isoform was found only in Hodgkin's lymphoma cell lines and Burkitt's lymphoma cell line Raji. The nCD150 isoform was broadly expressed in tested B cell lines with exception of REH and Daudi. CONCLUSION: Malignant B-cell lines at the different stages of maturation only partially resemble their normal counterparts by CD150 expression. In malignant B-cell lines, CD150 expression on mRNA level is much broader than on protein level. CD150 isoforms are differentially expressed in normal and malignant B cells with predominant expression of mCD150 isoform.

Intrinsic defect in B-lymphoblastoid cell lines from patients with X-linked lymphoproliferative disease type 1. II. receptor-mediated Akt/PKB and ERK1/2 activation and transcription factors expression profile.

BACKGROUND: X-linked lymphoproliferative disease type 1 (XLP1) belongs to genetically determined primary immunodeficiency syndromes with mutations in SH2D1A/DSHP/SAP gene. The dramatic increase of the risk of B-cell lymphoma development in XLP1 patients is linked not only to SAP deficiency of NK, NKT and T cells, but probably to the impairment of B cell differentiation. AIM: To analyze the receptor-mediated Akt/PKB and ERK1/2 phosphorylation and expression of transcription factors that are involved in B cell maturation in EBV-transformed B-lymphoblastoid cell lines (B-LCLs) from XLP1 patients (XLP B-LCLs) in comparison with conventional B-LCLs. METHODS: Studies were performed on EBV-transformed XLP B-LCLs IARC 739, SC-XLP and RP-XLP in comparison with SAP-negative B-LCL T5-1 and SAP-positive B-LCL MP-1. Western blot analysis was used for evaluation of Akt (Ser473) and ERK1/2 (Thr202/Tyr204) phosphorylation in response to ligation of CD150, CD40, and IgM cell surface receptors. The expression levels of transcription factors IRF4, IRF8, BCL6, BLIMP1, SPIB, PU.1 and MITF were assessed using quantitative RT-PCR. RESULTS: It was shown that SAP deficiency in XLP B-LCL did not abrogate CD150-mediated Akt and ERK1/2 phosphorylation. At the same time, ligation of CD150 or IgM affects kinetics and amplitude of ERK1/2 activation. In XLP B-LCL the CD150 signaling with IgM coligation play the dominant role in both Akt and ERK1/2 phosphorylation. We found that significantly reduced IRF4, IRF8 and PU.1 expression levels are the key features of XLP B-LCLs. CONCLUSION: XLP B-LCLs and conventional B-LCLs have differences in kinetics and amplitude of Akt and ERK1/2 phosphorylation. Analysis of transcription factors profile revealed the distinguishing features of XLP B-LCLs with SAP deficiency that may impair B cell differentiation.Key Words: B-lymphoblastoid cell lines, X-linked lymphoproliferative disease type 1, CD150, CD40, BCR, Akt/PKB, ERK1/2, transcription factors.

Intrinsic defect in B-lymphoblastoid cell lines from patients with X-linked lymphoproliferative disease type 1. I. Cell surface phenotype and functional studies.

BACKGROUND: Mutations in SH2D1A/DSHP/SAP gene are responsible for the onset of X-linked lymphoproliferative disease type 1 (XLP1) that have increased risk for B-cell lymphoma development. In XLP1 patients SAP deficient NK, NKT and CD8(+) cytotoxic T cells are inefficient in eliminating EBV-infected proliferating B cells that may partially contribute to the lymphoma development. However, little is known about impairment of B cell characteristics in XLP1. AIM: To analyze the cell surface phenotype and functional characteristics of EBV-transformed B-lymphoblastoid cell lines from XLP1 patients (XLP B-LCLs) in comparison with conventional B-lymphoblastoid cell lines (B-LCLs). METHODS: Studies were performed on SAP-negative B-LCLs T5-1, 6.16, RPMI 1788; SAP-positive B-LCL MP-1 and XLP B-LCLs IARC 739, XLP-D, XLP-8005. Cell surface immunophenotyping was performed using flow cytometry analysis. The level of apoptotic cells (Annexin V-binding), cell viability (MTT assay), and cell proliferation (trypan blue exclusion test) were evaluated in response to ligation of CD40, CD95, CD150 and IgM cell surface receptors. RESULTS: A cell surface phenotype and functional features that distinguish XLP B-LCLs from conventional B-LCLs were revealed. XLP B-LCLs showed the upregulated level of CD20, CD38 and CD86 cell surface expression and downregulation of CD40, CD80 and CD150 expression. The major functional differences of XLP B-LCLs from conventional B-LCLs concern the modulation of CD95 apoptosis via CD40 and CD150 receptors and unresponsiveness to proliferative signals triggered by CD40 or colligation of BCR with CD150. CONCLUSION: The data suggest that the B-LCL from XLP1 patients have an intrinsic defect that affects cell activation, apoptosis, and proliferation.

Differential expression of PKD1 and PKD2 in gastric cancer and analysis of PKD1 and PKD2 function in the model system.

AIM: To study the differential expression of PKD1 and PKD2 in primary gastric cancer samples and to examine the role of PKD1 and PKD2 protein kinases in regulation of gastric tumor cell biology in the model system. METHODS: Tumor samples of different histological variants of primary gastric cancer were analyzed. PKD1 and PKD2 expression levels in tumor samples were accessed by Western blot analysis and quantitative polymerase chain reaction (Q-PCR). As a model system we have used gastric adenocarcinoma сell line AGS sublines constitutively transfected by pcDNA3.1 coding PKD1 or PKD2, or empty pcDNA3.1 vector. These cell lines were analyzed by Western blot, Q-PCR, MTT and proliferation assays, in vitro scratch and Transwell assays, clonogenic assay. RESULTS: It was found that primary gastric tumors possess different levels of PKD1 and PKD2 expression on mRNA and protein levels. Low level of PKD1 expression on protein and mRNA level was detected in low differentiated adenocarcinoma and ring cell gastric cancer - disorders with poor clinical prognosis. The high level of PKD2 expression was also found in gastric tumors with poor prognosis: low differentiated adenocarcinoma and adenogen cancer. To find out whether differential expression of PKD1 and PKD2 could affect biology of gastric tumor cells in vitro, we used a model system based on AGS cell line that constitutively expressed PKD1 or overexpressed PKD2. PKD1 transfection led to the inhibition of cell proliferation, migration and colony formation, in the meanwhile, the PKD2 overexpression enhanced proliferation, migration and colony formation capacities of AGS cells. CONCLUSIONS: Our data suggest that both downregulation of PKD1 or upregulation of PKD2 expression may determine the behavior of gastric tumor cells, which promotes invasive phenotype and could result in general poor prognosis.

Immunohistochemical studies of protein kinase D (PKD) 2 expression in malignant human lymphomas.

AIM: To study the PKD2 expression, autophosphorylation and localization in reactive lymph nodes and tumors of lymphoid tissues. MATERIALS AND METHODS: Specific antibodies, which recognize PKD1/2 or PKD2 and autophosphorylated PKD1/2, were used for immunohistochemical and biochemical studies of tonsils, reactive lymph nodes, tumor samples of non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL). RESULTS: Immunohistochemical and biochemical analysis of PKD1 and PKD2 expression showed PKD2 expression in tonsils, reactive lymph nodes and tumor tissues from patients with NHL and HL. Furthermore, we were not able to reveal PKD1 expression in studied lymphoid tissues. In tonsils and reactive lymph nodes the PKD2 expression was detected in T and B cell zones with highest level in germinal centers of lymphoid follicles and the maximum level of autophosphorylation in the light zones of the germinal centers. We found that low level of PKD2 expression and autophosphorylation was characteristic feature for mantle cell lymphomas, Burkitt's lymphomas, and in 50% of CLL/small lymphocytic lymphomas. Lymphoma cells of germinal center origin and with activated B cell phenotype (diffuse large B cell lymphomas, HL) and anaplastic large cells lymphoma demonstrated the high level of PKD2 expression and autophosphorylation. CONCLUSIONS: The level of PKD2 expression and autophosphorylation in neoplastic cells corresponds to the expression pattern of this kinase in their normal analogs, and to the level of cell differentiation and activation.