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.

EXPRESSION PROFILE OF miR-145, -182, -21, -27a, -29b, and -34a IN BREAST CANCER PATIENTS OF YOUNG AGE.

BACKGROUND: Breast cancer (BC) in young women remains a significant public health concern. While progress has been made in understanding the etiology, diagnosis, and treatment of BC in this population, challenges persist. The identification and utilization of prognostic biomarkers offer valuable tools for tailoring treatment strategies and improving outcomes for BC patients. AIM: To evaluate the relationship between the expression of tumor-associated microRNAs and the clinical and pathological features of BC in young patients. MATERIALS AND METHODS: The work is based on the results of the examination and treatment of 50 women younger than 45 years with stage I-II BC. miR-145, -182, -21, -27a, -29b, and -34a expression in tumor samples was analyzed by the real-time reverse transcription polymerase chain reaction. RESULTS: Higher expression of miR-182, -21, and -29b and lower levels of miR-27a were associated with tumor stage in young BC patients. Patients without lymph node metastases (N0) had significantly higher levels of miR-182, -27a, and -34a and lower levels of miR-29b compared to N1 cases (p < 0.05). Expression of miR-145, -182, -21, -27a, and -29b was associated with molecular BC subtypes. CONCLUSION: Obtained results show that a high malignancy degree of BC in young women is associated with an increase in the miR-182, -21, -29b, and -34a expressions and a decrease in the miR-27a level in the tumor tissue, which indicates the prospects of the use of them for predicting the aggressiveness of the disease.

FEATURES OF COL1A1 EXPRESSION IN BREAST CANCER TISSUE OF YOUNG PATIENTS.

BACKGROUND: In the last decades, the incidence of breast cancer (BCa) in young women has been increasing steadily. The quantitative indicators of expression of collagen, which play important role in stromal microenvironment, and their association with the age and survival rates of BCa patients have not been yet definitively clarified. AIM: To investigate the relationship between the COL1A1 gene expression at the mRNA and protein levels in BCa tissue and the clinicopatological features and survival rates of BCa patients of different age groups. MATERIALS AND METHODS: The study was conducted on the clinical material of 50 patients with stage I-III BCa. COL1A1 gene expression at the mRNA and protein levels in BCa tissue were studied using the real-time PCR and immunohistochemical methods, as well as the bioinformatic analysis (UALCAN and Kaplan - Meier Plotter databases). RESULTS: The bioinformatic analysis showed that BCa tissue is characterized by 6.0 times (p < 0.05) higher level of COL1A1 mRNA compared to normal breast tissue. The correlation of COL1A1 expression at the mRNA and protein levels with the molecular subtype of neoplasms was demonstrated. According to Kaplan - Meier Plotter database, a low level of expression of COL1A1 protein level in BCa tissue is associated with lower rates of relapse-free survival of patients. The ex vivo study of the clinical material revealed a decrease in COL1A1 protein expression in tumor tissue of young patients with BCa of T3 category (p < 0.0374), low differentiation grade (p < 0.0163) and basal molecular subtype (p < 0.0001). A correlation between the expression of COL1A1 at the mRNA and protein levels and the expression status of estrogen receptors (p < 0.0001) and progesterone receptors (p < 0.0040) was established. The relapse-free 3-year survival rate of young BCa patients is significantly lower in the presence of a low COL1A1 optical density index in the tumor tissue. CONCLUSIONS: The identified relationship between COL1A1 expression and such indicators of BCa malignancy as tumor size, differentiation grade, molecular subtype, receptor status, and the recurrencefree survival of patients indicates the prospects of its use to predict the aggressiveness of the BCa course in young 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.

EXPRESSION OF MARKERS OF BONE TISSUE REMODELING IN BREAST CANCER AND PROSTATE CANCER CELLS IN VITRO.

The aim of the study was to compare the expression of markers of bone remodeling in vitro in breast cancer (BCa) cells and prostate cancer (PCa) cells varying in their malignancy phenotype. MATERIALS AND METHODS: The study was performed on human BCa cells (MCF-7 and MDA-MB-231 lines) and PCa cells (LNCaP and DU-145 lines). Expression levels of bone tissue remodeling proteins (osteopontin (OPN), osteonectin (ON) and bone morphogenetic protein 7 (BMP-7) were determined immunocytochemically. The mRNA levels of bone tissue remodeling proteins OPN (SPP1), ON (SPARC), BMP-7 (BMP7)) and miRNA-10b, -27a, -29b, -145, -146a were assessed by quantitative reverse transcription polymerase chain reaction. To search for miRNAs involved in the regulation of target genes, miRNet v. 2.0 resource was used. RESULTS: We have shown that highly malignant MDA-MB-231 cells are characterized by significantly higher expression of OPN and ON on the background of decreased SPARC and BMP7 mRNA expression. In highly malignant DU-145 cells, ON and SPP1, SPARC, and BMP7 mRNA expression was significantly higher compared with low malignant LNCaP cells. MDA-MB-231 line was characterized by significantly higher expression of miRNA-10b, -27a, -29b, -145 and -146a. In DU-145 cells, significantly lower levels of expression of miRNAs-27a and -145 against the background of increasing levels of miRNAs-29b and -146a were recorded. CONCLUSION: High malignancy phenotype of the BCa and PCa cells is characterized by high levels of expression of bone remodeling proteins, which may be caused by impaired regulation of their expression at the epigenetic level.

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.

[Immunological changes in old-age group children with primary and secondary tuberculous infection].

In modern society, there is a rise in the incidence of tuberculosis in all age groups, including children and adolescents. In old age group, a specific inflammation is detectable from Mantoux test results only in every four children. Tuberculous infection is diagnosed in half of cases when they turn to physicians for complains. Disseminated and complicated forms of tuberculosis are more frequently identified in these situations. The immune system has a particular emphasis on the course and outcome of the disease. The authors have established that caseous masses actively form, followed by the stimulation of the adequate cell pathway promoting the limitation of specific inflammation in old-age group children with primary tuberculosis. In secondary forms of tuberculous infection, there is an increase in the level of monocytes where the persistence and multiplication of the causative microorganism, as well as the activation of the humoral pathway inadequate for tuberculous infection are likely to occur, i.e. the infectious agent may be inhibited until activation of the Th-2 pathway of an immune response takes place.

[Tuberculin sensitivity in old children with new-onset active tuberculosis].

In recent years, there have been reports on the reduced diagnostic value of a tuberculin test with 2 TE PPD-L. Tuberculin diagnosis is a ground for suspecting tuberculosis only in 25.9 +/- 0.06% of cases in senior pupils and in 10.7 +/- 0.03% amongst adolescents. This is associated with the higher proportion tuberculin-positive individuals and it is very difficult to make the diagnosis in children with monotonous tuberculin sensitivity. A contact with a bacterium-discharging person undoubtedly increases a risk for the disease, but fails to affect the true outcome of delayed hypersensitivity. According to the date of this study, comorbidity and helminthic invasion have no significant impact on tuberculin sensitivity. Reactivation impairs immune homeostasis towards inadequate immune system performance.

[Predictors of the progress of latent tuberculosis to clinically active one].

The question recently arises as to the diagnosis of tuberculous infection at early stages of its development. Tuberculin diagnosis ranks below due to the rise ofcomorbidity, including allergic diseases. There is increasing evidence for the leading role in the development of this or that type of tuberculous infection. A mathematical model was used to identify 5 major of the 35 laboratory parameters characterizing the immune profile. The equations of classifying discriminant functions derived by the chosen immunological parameters make it possible to formalize data strictly and to organize an immunological screening with a high degree of accuracy in children with unchanged tuberculin sensitivity, without making compound statistical calculations. This fact can meet the requirements of immunological prediction of the development of tuberculosis in children.

Overexpression of the mitochondrial ribosomal protein S18-2 in the invasive breast carcinomas.

BACKGROUND: Recent studies allow to consider the mitochondrial ribosomal protein S18-2 (MRPS18-2, S18-2) as a potential oncoprotein, which suggests the need for further characterization of its expression in tumors of different genesis including breast cancer (BC). The aim of the study was to analyze the expression of the S18-2 protein in BC of luminal A and basal subtypes. MATERIALS AND METHODS: Operational material of BC patients stage І-ІІ (luminal A subtype, n = 30, and basal subtype, n = 10) was studied with the use of morphological, immunohistochemical, statistical and bioinformatic methods. RESULTS: Using the immunohistochemical analysis, we found that the S18-2 protein showed the nuclear signal in 66.7% of luminal A subtype BC samples and 80.0% of basal subtype BC samples. The variability of the S18-2 expression in both the luminal A and basal subtypes of BC was revealed. Noteworthy, the number of cells expressing S18-2 in high-proliferating tumors of luminal A and basal subtype is significantly higher than in tumors with a low proliferative potential (p < 0.05). In 10 samples of luminal A subtype, the nuclear S18-2 signal was higher than median value. Moreover, the S18-2 protein was overexpressed in 4 out of such 10 samples. Metastases in the lymph nodes were found in 3 out of 4 patients with the stage II BC, low differentiation grade of the tumor and high proliferative activity. The bioinformatic analysis confirms our preliminary findings that the trend for increasing expression of the S18-2 protein in tumors correlates with the aggressiveness of malignant BC. CONCLUSION: The S18-2 protein may be a marker of cancer aggressiveness in BC patients.

[Proliferativeactivity biorhythms of museum and hospital strains of microorganisms].

In series of experiments intraday dynamics of proliferative activity of museum and hospital strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa was revealed. Statistically significant circadian and ultradian biorhythms in these strains were detected. Chronoinfrastructure of hospital strains differed from museum ones. This finding allows to detect hospital strains of these bacteria.

Do MRPS18-2 and RB proteins cooperate to control cell stemness and differentiation, preventing cancer development?

In childhood tumors, including retinoblastoma, osteosarcoma, and neuroblastoma, the RB-E2F1 pathway is inactivated, as a rule. These tumors arise from precursor cells that fail to undergo the terminal differentiation. Noteworthy, the RB1-encoded protein (RB) does not control the cell cycle in embryonic stem cells. It has not been yet well understood how RB controls cell stemness and differentiation. The question arises why "inactive" RB is required for the survival and stemness of cells? Recently, we have found that overexpression of the RB-binding protein MRPS18-2 (S18-2) in primary fibroblasts leads to their immortalization, which is accompanied by the induction of embryonic stem cell markers and, eventually, malignant transformation. We suggest that cell stemness may be associated with high expression levels of both proteins, RB and S18-2. There must be a strict regulation of the expression levels of S18-2 and RB during embryogenesis. Disturbances in the expression of these proteins would lead to the abnormalities in development. We think that the S18-2 protein, together with the RB, plays a crucial role in the control on cell stemness and differentiation. We hope to uncover the new mechanisms of the cell fate determination. The S18-2 may serve as a new target for anticancer medicines, which will help to improve human health.

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.

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.

Role of the RB-Interacting Proteins in Stem Cell Biology.

Human retinoblastoma gene RB1 is the first tumor suppressor gene (TSG) isolated by positional cloning in 1986. RB is extensively studied for its ability to regulate cell cycle by binding to E2F1 and inhibiting the transcriptional activity of the latter. In human embryonic stem cells (ESCs), only a minute trace of RB is found in complex with E2F1. Increased activity of RB triggers differentiation, cell cycle arrest, and cell death. On the other hand, inactivation of the entire RB family (RB1, RBL1, and RBL2) in human ESC induces G2/M arrest and cell death. These observations indicate that both loss and overactivity of RB could be lethal for the stemness of cells. A question arises why inactive RB is required for the survival and stemness of cells? To shed some light on this question, we analyzed the RB-binding proteins. In this review we have focused on 27 RB-binding partners that may have potential roles in different aspects of stem cell biology.

Epstein-Barr virus encoded nuclear protein EBNA-3 binds XAP-2, a protein associated with Hepatitis B virus X antigen.

EBNA-3 (also called EBNA-3A) is one of the EBV encoded nuclear antigens that are necessary for B-cell transformation. EBNA-3 is known to target RBPs, nuclear proteins that also interacts with EBNA-2, EBNA-4 and EBNA-6. In order to identify additional EBNA-3 targets, an EBV-transformed human lymphocyte cDNA library was screened in the yeast two-hybrid system with N-terminus truncated EBNA-3 that cannot interact with RBP-Jkappa. A clone, encoding Xap-2 protein, a cellular partner of Hepatitis B virus X-antigen was isolated. This protein is also known as the p38 subunit of the aryl hydrocarbon receptor complex (ARA9). The specific binding to EBNA-3 was confirmed by showing that the GST-Xap-2 precipitated EBNA-3 from CV1 cells that were infected with recombinant vaccinia virus expressing EBNA-3. Deletion of the C-terminus of Xap-2 eliminated the binding. Fusion with green fluorescent protein showed that Xap-2 is preferentially cytoplasmic but translocates to the nucleus upon expression of EBNA-3.

[Comparative analysis of indicators of the immune status of population of the Eastern regions of the USSR]. / Sravnitel'nyi analiz pokazatelei immunnogo statusa naseleniia vostochnykh regionov SSSR.

Altogether 1,500 healthy residents of seven cities situated in the Asian part of the USSR were examined. In Novosibirsk, Tomsk, Tyumen, Norilsk, Magadan, Yakutsk and Ussuriisk, the people were examined for the blood levels of T and B lymphocytes, the ratio of regulatory subclasses of T lymphocytes, the concentration of IgG, IgM and IgA, and for the content of immune complexes. Analysis was made of the general and regional regularities in changes seen in the immune system depending on climatic and geographic factors. Parameters of the populations similarity in the regions with different environmental conditions were delineated.

[The formation of host-parasite relationships in an experimental mixed pathology of opisthorchiasis-tuberculosis as dependent on the phase of the Opisthorchis infestation]. / Formirovanie otnoshenii khoziain--parazit pri éksperimental'noi mikstpatologii opistorkhoz--tuberkulez v zavisimosti ot fazy opistorkhoznoi invazii.

Superinfection of animals having Opisthorchis invasion with Mycobacterium tuberculosis leads to destabilization of host-parasite relationships in opisthorchiasis and formation of new host-parasitocenotic interrelations whose manifestation depends on the phase of the invasive process. At the acute invasive phase of mixed pathology (2 weeks) the activity of the host's immune system increases, while the biological activity of helminths and the number of M. tuberculosis colonies decrease. And on the contrary, at the subacute phase of mixed pathology (2.5 months) the activity of the host's immune system decreases, while the reproductive activity of helminths and the isolation rate of M. tuberculosis from pulmonary tissue increases in comparison with the groups of animals with monoinvasion and monoinfection.