Irena Mlinarič-Raščan

A cytokine storm drives critical COVID-19, yet the determinants and long-term consequences of this exaggerated inflammatory response remain unclear. We investigated whether cytokine responsiveness remains altered 2 years after recovery from critical disease. In a cohort of 117 individuals with mild, severe, or critical COVID-19, acute-phase clinical and laboratory parameters were analyzed in moderate–severe and critical cases. Long-term immune function was assessed by ex vivo stimulation of whole blood with PMA/ionomycin, lipopolysaccharide (LPS), and phytohemagglutinin (PHA), followed by cytokine quantification and correlation with clinical parameters. A biobank of 76 patient-derived lymphoblastoid cell lines (LCL cells) was established to evaluate the stability of cytokine responses. During acute illness, critically ill patients exhibited marked inflammatory dysregulation, including lymphopenia (72%), neutrophilia (51%), elevated neutrophil-to-lymphocyte ratios, and increased C-reactive protein (median 91 mg/L) and ferritin (median 1,153 μg/L), consistent with a hyperinflammatory–hypercoagulable phenotype. Two years post-infection, individuals with prior critical disease showed more than threefold higher secretion of IL-1β, IL-6, TNF-α, and IL-8 following PMA/ionomycin (p < 0.0001) and LPS stimulation (p < 0.05) compared with younger mild cases, whereas PHA-induced responses remained comparable. PBMC counts were also higher in the moderate–severe and critical groups 2 years post-infection (p < 0.001), highlighting differences in cellular composition. LCL cells from critically ill donors secreted 79% more IL-8 and TNF-α than those from mild cases (p < 0.05). Collectively, these findings indicate differences in cytokine responsiveness 2 years after COVID-19 which correspond to prior disease severity and are associated particularly with age and innate immune activation.

Chronic lymphocytic leukemia (CLL) is an incurable B-cell malignancy characterized by the accumulation of mature but functionally impaired lymphocytes. Although targeted therapies such as venetoclax and ibrutinib have significantly improved patient outcomes, treatment resistance remains a major clinical challenge. The NOTCH signaling pathway is constitutively activated in CLL and contributes to cell survival and therapy resistance, making it a promising therapeutic target. Moreover, the NOTCH1 receptor was identified as strongly associated with CLL by the in silico disease‑association platform OpenTargets, underscoring the rationale for further investigation into its role in CLL pathogenesis. In this study, we evaluated the anti-leukemic potential of four inhibitors of the NOTCH pathway: limantrafin, RBPJ inhibitor-1 (RIN1), ZLDI-8, and FLI-06 in CLL cell lines MEC1 and HG3, established venetoclax- and ibrutinib-resistant cells as well as primary CLL cells obtained from patients after informed consent. All NOTCH inhibitors exhibited cytotoxic activity as determined by resazurin metabolic activity assay. Among the tested compounds, ZLDI-8 cytotoxic effects were most pronounced (EC₅₀ 3.2 μM) in MEC1 cells after 24 hours. To evaluate the therapeutic potential of combination strategies, MEC1 cells were treated with venetoclax and ibrutinib with NOTCH inhibitors. Among them, RIN1 exhibited the most pronounced synergistic effects, indicating its potential to augment current treatment regimens. Given the constitutive activation of NF-κB signaling in CLL, pathway crosstalk was investigated using the RamosBlue reporter cells, in which all four NOTCH inhibitors significantly attenuated NF-κB transcriptional activity. Flow cytometric analyses demonstrated that NOTCH pathway inhibition predominantly induces apoptosis and is associated with cell cycle arrest. These findings support the repurposing of NOTCH pathway inhibitors as promising therapeutic candidates for CLL.

Personalized thiopurine therapy is among the most established examples of pharmacogenomics translated into clinical practice. Variants in TPMT (rs1800462, rs1800460, rs1142345) and NUDT15 (rs116855232) are recognized clinical predictors of thiopurine efficacy and toxicity. Additional variants in genes such as PACSIN2 (rs2413739), ITPA (rs1127354) and MTHFR (rs1801133 and rs1801131), also contribute to variability in drug response. Here we characterize the frequency of the pharmacogenetic variants involved in thiopurine metabolism in a healthy population of Kosovo. We genotyped 299 healthy blood donors for polymorphisms. Among TPMT variant alleles, TPMT*3A was observed at a frequency of 2.0%, and the TPMT*3C at 0.1%. Notably, the MTHFR 677T variant (rs1801133) was significantly more frequent in the Kosovo population (49.8%) compared with the global and European frequencies. The minor allele frequency of MTHFR rs1801131 was 27.4%. Minor allele frequencies for PACSIN2 rs2413739 and ITPA rs1127354 variants were 48.8% and 4.0%, respectively. Sequencing of NUDT15 revealed six variants, with rs116855232 present at frequency of 0.8%. These findings provide important insights into the pharmacogenomic profile of the Kosovo population and support the implementation of pre-emptive genotyping to improve the safety and efficacy of thiopurine therapy in the region.

Determining variant TPMT alleles to predict patient response to thiopurine therapy represents one of the first successful implementations of pharmacogenomics in clinical practice. However, despite the TPMT-adjusted thiopurine dosing, some TPMT wild-type patients still exhibit toxicity at standard doses. Over the past decade, the pharmacogene NUDT15 has emerged as a significant co-modulator of thiopurine therapy. Initially, NUDT15 was considered important predominantly in Asian populations, but recent studies have highlighted its relevance in European populations as well.To evaluate the pharmacogenetic significance of NUDT15 in the Slovenian population, we sequenced extended regions of exon 1 and exon 3 in 109 healthy individuals and 37 patients with acute lymphoblastic leukemia.We identified eight variants, including one with established clinical significance (allele *3) and one extremely rare variant (Chr13 at 48045861; GRCh38, NC_000013.11). The frequencies of most previously described variants in both the general population and in the ALL cohort were consistent with those reported in other European populations, except for rs45465203, which was less frequent in the Slovenian population. None of the variants, except for NUDT15*3, were associated with cumulative thiopurine doses in ALL patients. However, these variants warrant further investigation in larger ALL cohorts.

Despite great therapeutic advances in the field of biologics, small synthetic molecules such as thiopurines, including azathioprine, mercaptopurine, and thioguanine, remain an important therapeutic pillar in the treatment of inflammatory bowel disease, other autoimmune disorders, and cancer. This review presents the latest guidelines for thiopurine administration, highlighting the importance of individualized therapy guided by pharmacogenomics. It emphasizes dose adjustment based on nudix hydrolase 15 ( NUDT15 ) and thiopurine S -methyltransferase ( TPMT ) genotype, along side thiopurine S -methyltransferase activity and thiopurine metabolic profile. In addition, the article takes a critical look at emerging research in the field of thiopurine pharmaco genomics featuring novel genetic markers and technological developments in genetic testing. Finally, the potential of integrated approaches that combine genetic, meta bolic, and clinical factors to further individualize thiopurine therapy is highlighted.

Advances in research, including novel biomarker identification and patient stratification, have significantly improved the therapy for childhood acute lymphoblastic leukemia (ALL), though access to improved healthcare services varies across geographical regions. In an effort to evaluate the advances in therapeutic approaches, we performed a retrospective analysis of childhood ALL in Kosovo. Our retrospective analysis included 225 cases diagnosed between 2008 and 2023, representing 52% of 429 diagnosed childhood cancers. The average annual incidence was 14, with a median age diagnosis of seven years, and a male predominance (59.54%). Patients were categorized into risk groups, with the majority (43%) in the standard-risk category. We identified five different treatment protocols for this study period. Over 61% of patients achieved remission after the first chemotherapy cycle and we observed a 20% mortality rate. Survival analysis showed that 55% and 40% of patients achieved 2-year and 5-year event-free survival (EFS), respectively, with significant differences across risk groups. Treatment advancements significantly correlated with improved survival rates, achieving a 5-year overall survival (OS) of 88% in the currently used standardized AIEOP-BFM-2009 protocol. Our study emphasizes the need for continued research and customized care strategies to enhance clinical outcomes.