Data-Independent Acquisition Mass Spectrometry Analysis of FFPE Rectal Cancer Samples Offers In-Depth Proteomics Characterization of the Response to Neoadjuvant Chemoradiotherapy.

Locally advanced rectal cancer (LARC) presents a challenge in identifying molecular markers linked to the response to neoadjuvant chemoradiotherapy (nCRT). This study aimed to utilize a sensitive proteomic method, data-independent mass spectrometry (DIA-MS), to extensively analyze the LARC proteome, seeking individuals with favorable initial responses suitable for a watch-and-wait approach. This research addresses the unmet need to understand the response to treatment, potentially guiding personalized strategies for LARC patients. Post-treatment assessment included MRI scans and proctoscopy. This research involved 97 LARC patients treated with intense chemoradiotherapy, comprising radiation and chemotherapy. Out of 97 LARC included in this study, we selected 20 samples with the most different responses to nCRT for proteome profiling (responders vs. non-responders). This proteomic approach shows extensive proteome coverage in LARC samples. The analysis identified a significant number of proteins compared to a prior study. A total of 915 proteins exhibited differential expression between the two groups, with certain signaling pathways associated with response mechanisms, while top candidates had good predictive potential. Proteins encoded by genes SMPDL3A, PCTP, LGMN, SYNJ2, NHLRC3, GLB1, and RAB43 showed high predictive potential of unfavorable treatment outcome, while RPA2, SARNP, PCBP2, SF3B2, HNRNPF, RBBP4, MAGOHB, DUT, ERG28, and BUB3 were good predictive biomarkers of favorable treatment outcome. The identified proteins and related biological processes provide promising insights that could enhance the management and care of LARC patients.

A new approach for activation of the kiwifruit cysteine protease for usage in in-vitro testing.

Actinidin (Act d 1), a highly abundant cysteine protease from kiwifruit, is one of the major contributors to the development of kiwifruit allergy. Many studies have focused on the optimization of Act d 1 purification and its role in the development of food allergies. Testing on cell culture monolayers is a common step in the elucidation of food allergen sensitization. In the case of cysteine proteases, an additional activation step with L-cysteine is required before the testing. Hence, we aimed to evaluate whether L-cysteine already present in commonly used cell culture media would suffice for Act d 1 activation. Successfully activated Act d 1 (98.1% of proteolytic activity, as compared to L-cysteine activated Act d 1) was further tested in two commonly used 2D model systems (Caco-2 and HEK293 cells) to evaluate its role on the mRNA expression of cytokines involved in the innate immunity (IL-1ß, IL-6, TNFα, TSLP). Furthermore, the contribution of Act d 1 in the promotion of inflammation through regulation of inducible nitric oxide synthase (iNOS) mRNA expression was also examined. These results demonstrate that activation of cysteine proteases can be achieved without previous enzyme incubation in L-cysteine -containing solution. Act d 1 incubated in cell culture medium was able to modulate gene expression of pro-inflammatory cytokines when tested on two model systems of the epithelial barrier.

Functional Fine-Tuning of Metabolic Pathways by the Endocannabinoid System-Implications for Health and Disease.

The endocannabinoid system (ECS) employs a huge network of molecules (receptors, ligands, and enzymatic machinery molecules) whose interactions with other cellular networks have still not been fully elucidated. Endogenous cannabinoids are molecules with the primary function of control of multiple metabolic pathways. Maintenance of tissue and cellular homeostasis by functional fine-tuning of essential metabolic pathways is one of the key characteristics of the ECS. It is implicated in a variety of physiological and pathological states and an attractive pharmacological target yet to reach its full potential. This review will focus on the involvement of ECS in glucose and lipid metabolism, food intake regulation, immune homeostasis, respiratory health, inflammation, cancer and other physiological and pathological states will be substantiated using freely available data from open-access databases, experimental data and literature review. Future directions should envision capturing its diversity and exploiting pharmacological options beyond the classical ECS suspects (exogenous cannabinoids and cannabinoid receptor monomers) as signaling through cannabinoid receptor heteromers offers new possibilities for different biochemical outcomes in the cell.

Prognostic Factors for Post-Recurrence Survival in Stage II and III Colorectal Carcinoma Patients.

Background and objectives: This study aimed to evaluate prognostic factors for post-recurrence survival in local and locally advanced colorectal cancer patients. Materials and Methods: A total of 273 patients with stage III and high-risk stage II colorectal cancer were prospectively enrolled. All patients underwent operative treatment of the primary tumor and adjuvant fluorouracil-based chemotherapy. Results: Over the three-year period (2008-2010), a cohort of 273 patients with stage III and high-risk stage II colorectal cancer had been screened. During follow up, 105 (38.5%) patients had disease recurrence. Survival rates 1-, 3- and 5-year after recurrence were 53.9, 18.2 and 6.5%, respectively, and the median post-recurrence survival time was 13 months. Survival analysis showed that age at diagnosis (p < 0.01), gender (p < 0.05), elevated postoperative Ca19-9 (p < 0.01), tumor histology (adenocarcinoma vs. mucinous vs. signet ring tumors, p < 0.01) and tumor stage (II vs. III, p < 0.05) had a significant influence on post-recurrence survival. Recurrence interval and metastatic site were not related to survival following recurrence. Multivariate analysis showed that older age (HR 2.43), mucinous tumors (HR 1.51) and tumors expressing Ca19-9 at baseline (HR 3.51) were independently associated with survival following recurrence. Conclusions: Baseline patient and tumor characteristics largely predicted patient outcomes after disease recurrence. Recurrence intervals in local and locally advanced colorectal cancer were not found to be prognostic factors for post-recurrence survival. Older age, male gender, stage III and mucinous histology were poor prognostic factors after the disease had recurred. Stage II patients had remarkable post-recurrence survival compared to stage III patients.

Kiwifruit cysteine protease actinidin compromises the intestinal barrier by disrupting tight junctions.

BACKGROUND: The intestinal epithelium forms a barrier that food allergens must cross in order to induce sensitization. The aim of this study was to evaluate the impact of the plant-derived food cysteine protease--actinidin (Act d1) on the integrity of intestinal epithelium tight junctions (TJs). METHODS: Effects of Act d1 on the intestinal epithelium were evaluated in Caco-2 monolayers and in a mouse model by measuring transepithelial resistance and in vivo permeability. Integrity of the tight junctions was analyzed by confocal microscopy. Proteolysis of TJ protein occludin was evaluated by mass spectrometry. RESULTS: Actinidin (1 mg/mL) reduced the transepithelial resistance of the cell monolayer by 18.1% (after 1 h) and 25.6% (after 4 h). This loss of barrier function was associated with Act d 1 disruption of the occludin and zonula occludens (ZO)-1 network. The effect on intestinal permeability in vivo was demonstrated by the significantly higher concentration of 40 kDa FITC-dextran (2.33 µg/mL) that passed from the intestine into the serum of Act d1 treated mice in comparison to the control group (0.5 µg/mL). Human occludin was fragmented, and putative Act d1 cleavage sites were identified in extracellular loops of human occludin. CONCLUSION: Act d1 caused protease-dependent disruption of tight junctions in confluent Caco-2 cells and increased intestinal permeability in mice. GENERAL SIGNIFICANCE: In line with the observed effects of food cysteine proteases in occupational allergy, these results suggest that disruption of tight junctions by food cysteine proteases may contribute to the process of sensitization in food allergy.

Effect of malondialdehyde on the ovalbumin structure and its interactions with T84 epithelial cells.

BACKGROUND: Protein oxidation can occur as a consequence of lipid peroxidation during food processing. The aim of this work was to explore the effect of malondialdehyde (MDA) modification of ovalbumin (OVA) on its interaction with T84 intestinal cells. METHODS: Molecular dynamics simulation was employed for the prediction of MDA modification in the OVA, while introduced structural changes were evaluated by measurement of carbonyl group content, fluorescence spectra, MS/MS analysis, and IgE reactivity. Effects of MDA modified OVA on T84 epithelial cells were analyzed by gene expression for pro-inflammatory cytokines and protein secretion. RESULTS: Out of 9 predicted, five modified Lys residues were confirmed by MS/MS analysis: 51TQINKVVR58, 85DILNQITKPNDVYSFSLASR104, 111YPILPEYLQCVKELYR126, 187AFKDEDTQAMPFR199, 277KIKVYLPR284, and 278IKVYLPR284. The introduced MDA modifications influenced profile of IgE reactivity to OVA. Treatment of T84 epithelial cells with OVA and OVA modified with 1mM MDA, induced up-regulation of pro-inflammatory cytokines (IL-1ß, IL-25, IL-33, TSLP and TNFα), while OVA modification with 10mM MDA induced down regulation of the cytokine expression profile, except for IL-1ß. OVA and OVA modified with 1mM MDA induced secretion of epithelial cells specific cytokine IL-33. CONCLUSIONS: This finding indicated that OVA and its MDA modified form have the potential to trigger the innate immunity by inducing up-regulation and secretion of pro-allergenic IL-33 in T84 intestinal epithelial cells. GENERAL SIGNIFICANCE: Interactions of ovalbumin and its MDA modified form with intestinal epithelial cells can induce a specific immunological priming necessary for the downstream activation of innate immunity.

Sensitization of K562 Leukemia Cells to Doxorubicin by the Viscum album Extract.

Toxicity of conventional chemotherapeutics highlights the requirement for complementary or alternative medicines that would reduce side effects and improve their anticancer effectiveness. European mistletoe (Viscum album) has long been used as a complementary and alternative medicine supporting cancer therapy. The aim of this study was to investigate synergistic antitumor action of V. album extract and doxorubicin during co-treatment of chemoresistant chronic myelogenic leukemia K562 cells. Combined treatment of leukemia cells led to inhibitory synergism at sub-apoptotic doxorubicin concentrations and multifold reduction of cytotoxic effects in healthy control cells. Prolonged co-treatment was associated with reduced G2/M accumulation and increased expression of early and late apoptotic markers. Our data indicate that V. album extract increases antileukemic effectiveness of doxorubicin against resistant K562 cells by preventing G2/M arrest and inducing apoptosis.

Pharmacogenetics in cancer therapy - 8 years of experience at the Institute for Oncology and Radiology of Serbia.

PURPOSE: Pharmacogenetics is a study of possible mechanism by which an individual's response to drugs is genetically determined by variations in their DNA sequence. The aim of pharmacogenetics is to identify the optimal drug and dose for each individual based on their genetic constitution, i.e. to individualize drug treatment. This leads to achieving the maximal therapeutic response for each patient, while reducing adverse side effects of therapy and the cost of treatment. A centralized pharmacogenetics service was formed at the Institute for Oncology and Radiology of Serbia (IORS) with the aim to provide a personalized approach to cancer treatment of Serbian patients. METHODS: Analyses of KRAS mutations in metastatic colorectal cancer, EGFR mutations in advanced non-small cell lung cancer, CYP2D6 polymorphism in breast cancer, DPD polymorphism in colorectal cancer and MTHFR polymorphism in osteosarcoma have been performed by real time polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). RESULTS: Mutation testing analyses were successful for 1694 KRAS samples and 1821 EGFR samples, while polymorphism testing was successful for 9 CYP2D6 samples, 65 DPD samples and 35 MTHFR samples. CONCLUSIONS: Pharmacogenetic methods presented in this paper provide cancer patients in Serbia the best possible choice of treatment at the moment.

Overall human papilloma virus and types 16/18 prevalence in women with normal cervical cytology in Serbia: is it time for human papillomavirus testing and/or vaccination?

PURPOSE: Infection with high-risk human papilloma viruses (HR-HPV), especially types 16/18, is the main factor in cervical carcinogenesis. Although the incidence of cervical cancer in Serbia is among the highest ones in Europe, data about HPV infection are insufficient. The aim of this study was to investigate the presence of overall and HPV16/18 infections in women with healthy appearance and cytologically (Pap) normal cervix. METHODS: The study was performed on women who participated in this cervical cancer screening pilot study. Cervical HPV infection was detected by GP5+/6+ PCR. HPV16/18 were detected by amplification of E7/E1 viral gene, respectively. RESULTS: In 350 women we got the following results: cytological abnormalities (10.3%); visible cervical changes (20.3%); previous precancerous lesion (2.3%); normal Pap and speculum finding without history of precancerous lesion (67.1%). In the last group overall HPV prevalence was 41.3%, with 10.5% HPV16 and 23.7% HPV18. The rate of multiple HPV16 plus HPV18 infections was 2.6%. HR-HPV16/18 comprised 31.6% of the total HPV positive participants. CONCLUSION: Owing to the high prevalence of overall and HPV16/18 infections in women with healthy appearance and cytologically normal cervix, we postulate that testing/ prophylaxis for these HR-HPV types could be introduced in cervical cancer screening and preventive programmes in Serbia.

The influence of methylenetetrahydrofolate reductase and thymidylate synthetase gene polymorphisms on lung adenocarcinoma occurrence.

PURPOSE: Methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthetase (TYMS) are suggested as risk factors for lung cancer. The purpose of this study was to analyze the association of MTHFR C677T polymorphism and variable number tandem repeat 2R/3R and single nucleotide polymorphism G>C in the 3R allele of the TYMS gene with lung adenocarcinoma. METHODS: A case-control study including lung adenocarcinoma patients and healthy subjects was performed. Restriction fragment length polymorphism analysis was used for genotyping. Descriptive analyses included genotype and allelic frequencies; the odds ratio and 95% confidence interval were calculated as an estimate of relative risk. Significance was set at p<0.05. RESULTS: A significant difference in CC vs TT+CT MTHFR genotype distribution was observed between patients and controls. There was no significant association between the TYMS polymorphisms and the risk of lung adenocarcinoma. CONCLUSIONS: The MTHFR 677T allele is likely to have a protective effect against lung adenocarcinoma development.

Perspective on Management of Low-Dose Computed Tomography Findings on Low-Dose Computed Tomography Examinations for Lung Cancer Screening. From the International Association for the Study of Lung Cancer Early Detection and Screening Committee.

Lung cancer screening using low-dose computed tomography (LDCT) carefully implemented has been found to reduce deaths from lung cancer. Optimal management starts with selection of eligibility criteria, counseling of screenees, smoking cessation, selection of the regimen of screening which specifies the imaging protocol, and workup of LDCT findings. Coordination of clinical, radiologic, and interventional teams and ultimately treatment of diagnosed lung cancers under screening determine the benefit of LDCT screening. Ethical considerations of who should be eligible for LDCT screening programs are important to provide the benefit to as many people at risk of lung cancer as possible. Unanticipated diseases identified on LDCT may offer important benefits through early detection of leading global causes of death, such as cardiovascular diseases and chronic obstructive pulmonary disease, as the latter may result from conditions such as emphysema and bronchiectasis, which can be identified early on LDCT. This report identifies the key components of the regimen of LDCT screening for lung cancer which include the need for a management system to provide data for continuous updating of the regimen and provides quality assurance assessment of actual screenings. Multidisciplinary clinical management is needed to maximize the benefit of early detection, diagnosis, and treatment of lung cancer. Different regimens have been evolving throughout the world as the resources and needs may be different, for countries with limited resources. Sharing of results, further knowledge, and incorporation of technologic advances will continue to accelerate worldwide improvements in the diagnostic and treatment approaches.

Performance and Dimensionality of Pretreatment MRI Radiomics in Rectal Carcinoma Chemoradiotherapy Prediction.

(1) Background: This study aimed to develop a machine learning model based on radiomics of pretreatment magnetic resonance imaging (MRI) 3D T2W contrast sequence scans combined with clinical parameters (CP) to predict neoadjuvant chemoradiotherapy (nCRT) response in patients with locally advanced rectal carcinoma (LARC). The study also assessed the impact of radiomics dimensionality on predictive performance. (2) Methods: Seventy-five patients were prospectively enrolled with clinicopathologically confirmed LARC and nCRT before surgery. Tumor properties were assessed by calculating 2141 radiomics features. Least absolute shrinkage selection operator (LASSO) and multivariate regression were used for feature selection. (3) Results: Two predictive models were constructed, one starting from 72 CP and 107 radiomics features, and the other from 72 CP and 1862 radiomics features. The models revealed moderately advantageous impact of increased dimensionality, with their predictive respective AUCs of 0.86 and 0.90 in the entire cohort and 0.84 within validation folds. Both models outperformed the CP-only model (AUC = 0.80) which served as the benchmark for predictive performance without radiomics. (4) Conclusions: Predictive models developed in this study combining pretreatment MRI radiomics and clinicopathological features may potentially provide a routine clinical predictor of chemoradiotherapy responders, enabling clinicians to personalize treatment strategies for rectal carcinoma.

From the International Association for the Study of Lung Cancer Early Detection and Screening Committee: Terminology Issues in Screening and Early Detection of Lung Cancer-International Association for the Study of Lung Cancer Early Detection and Screening Committee Expert Group Recommendations.

INTRODUCTION: To facilitate global implementation of lung cancer (LC) screening and early detection in a quality assured and consistent manner, common terminology is needed. Researchers and clinicians within different specialties may use the same terms but with different meanings or different terms for the same intended meanings. METHODS: The Diagnostics Working Group of the International Association for the Study of Lung Cancer Early Detection and Screening Committee has analyzed and discussed relevant terms used on a regular basis and suggests recommendations for consensus definitions of terminology applicable in this setting. We explored how to reach consensus to define relevant and unambiguous terminology for use by health care providers, researchers, patients, screening participants, and family. RESULTS: Terms and definitions for epidemiologic and health-economical purposes included the following: standardized incidence and mortality rates, LC-specific survival, long-term survival and cure rates, overdiagnosis, overtreatment, and undertreatment. Terms and definitions for defining screening findings included the following: positive, false-positive, negative, false-negative, and indeterminate findings and additional and incidental findings. Terms and definitions for describing parameters in screening programs included the following: opportunistic versus programmatic screening, screening rounds, interval or interim diagnoses, and invasive and minimally invasive procedures. Terms and definitions for shared decision-making included the following: LC screening-possible harms and risks and LC risk and modifiers prior and posterior to a measure. CONCLUSIONS: A common set of terminology with standard definitions is recommended for describing clinical LC screening programs, the discussion about effectiveness and outcomes, or the clinical setting. The use of the terms should be clearly defined and explained.

The usability of allele-specific PCR and reverse-hybridization assays for KRAS genotyping in Serbian colorectal cancer patients.

BACKGROUND: Colorectal cancers (CRCs) with wild-type KRAS respond to EGFR-targeted antibody treatment. Analysis of the hotspot clustered mutations in codons 12 and 13 is compulsory before therapy and no standardized methodology for that purpose has been established so far. Since these mutations may have different biological effects and clinical outcome, reliable frequency and types of KRAS mutations need to be determined for individual therapy. AIMS: The purpose of this study was to describe the KRAS mutation spectrum in a group of 481 Serbian mCRC patients and to compare the general performances of allele-specific PCR and reverse-hybridization assays. METHODS: KRAS testing was performed with two diagnostic analyses, DxS TheraScreen K-RAS PCR Kit and KRAS StripAssay™. RESULTS: KRAS mutations in codons 12 and 13 were present in 37.6 % of analyzed formalin-fixed paraffin-embedded (FFPE) DNA samples. The seven most frequent mutation types were observed with both assays: p.G12D 34.6 %, p.G12V 24.9 %, p.G12A 10.3 %, p.G12C 8.1 %, p.G12S 5.4 %, p.G12R 1.6 %, and p.G13D 15.1 %. Regarding double mutants, 0.8 % of them were present among all tested samples and 2.2 % among KRAS mutated ones. CONCLUSIONS: Two screening approaches that were used in this study have been shown as suitable tests for detecting KRAS mutations in diagnostic settings. In addition, they appear to be good alternatives to methods presently in use. In our experience, both methods showed capacity to detect and identify double mutations which may be important for potential further subgrouping of CRC patients.

EGFR mutation testing from pleural effusions of non-small cell lung cancer patients at the institute for oncology and radiology of Serbia.

BACKGROUND: The use tumor-derived cell-free DNA extracted from body fluids is being evaluated for genetic testing in lung cancer. The aim of this study was to explore the feasibility and utility of implementation of EGFR molecular testing from pleural effusions in non-small cell lung cancer in the clinical diagnostics workflow. PATIENTS AND METHODS: This study included patients diagnosed with primary lung adenocarcinoma in the period July 2016 to June 2023. EGFR mutation testing was performed by qPCR (Cobas®) and dPCR. Testing was performed from 211 plasma samples when tissue was unavailable at diagnosis, and from 301 plasma samples and 18 pleural effusions at progression on first/second generation of EGFR TKIs. Descriptive methods of statistical analysis were used to summarize the sample data. Fisher's exact test, McNemar's test, Cohen's kappa tests were used for statistical analyses. Two-sided p-values <0.05 were considered statistically significant. RESULTS: A significantly higher detection rate of the T790M mutation in pleural effusion was obtained compared to blood (50% and 20%, p=0.047). When comparing the detection success rate of the resistant T790M mutation in pleural effusion and blood, a statistically significant difference was obtained in favor of pleural effusion (50% vs. 21.87%, p=0.01). CONCLUSIONS: Superior performance of pleural effusions compared to blood plasma was shown both in the analysis of success rate and in the detection of the resistant T790M mutation, at progression on EGFR TKIs. Pleural effusion should be considered in this setting whenever available, especially in countries with limited health resources.

Nischarin expression may have differing roles in male and female melanoma patients.

Due to the development of resistance to previously effective therapies, there is a constant need for novel treatment modalities for metastatic melanoma. Nischarin (NISCH) is a druggable scaffolding protein reported as a tumor suppressor and a positive prognostic marker in breast and ovarian cancers through regulation of cancer cell survival, motility and invasion. The aim of this study was to examine the expression and potential role of nischarin in melanoma. We found that nischarin expression was decreased in melanoma tissues compared to the uninvolved skin, and this was attributed to the presence of microdeletions and hyper-methylation of the NISCH promoter in the tumor tissue. In addition to the previously reported cytoplasmic and membranous localization, we observed nischarin in the nuclei in melanoma patients' tissues. NISCH expression in primary melanoma had favorable prognostic value for female patients, but, unexpectedly, high NISCH expression predicted worse prognosis for males. Gene set enrichment analysis suggested significant sex-related disparities in predicted association of NISCH with several signaling pathways, as well as with different tumor immune infiltrate composition in male and female patients. Taken together, our results imply that nischarin may have a role in melanoma progression, but that fine-tuning of the pathways it regulates is sex-dependent. KEY MESSAGES: Nischarin is a tumor suppressor whose role has not been investigated in melanoma. Nischarin expression was downregulated in melanoma tissue compared to the normal skin. Nischarin had the opposite prognostic value in male and female melanoma patients. Nischarin association with signaling pathways differed in females and males. Our findings challenge the current view of nischarin as a universal tumor suppressor.

Detection of humoral and cellular immune response to anti-SARS-CoV-2 BNT162b2 vaccine in breastfeeding women and naïve and previously infected individuals.

This study explored humoral and cellular responses to anti-SARS-CoV-2 BNT162b2 mRNA vaccine in breastfeeding women and naïve and seropositive individuals in the first six months after vaccination.Sixty-one volunteers vaccinated with two doses of the BNT162b2 mRNA vaccine were enrolled in the study. In-house developed ELISA was used for the quantification of SARS-CoV-2 RBD-specific antibodies. Cell surface marker expression and intracellular IFN-γ analysis were carried out by flow cytometry. The concentrations of IFN-γ, IL-6 and TNF were determined by ELISA. A significant rise in anti-RBD IgG antibody levels was observed 14 days after the first vaccine dose (p < 0.0001) in serum and milk. The expression of CD28 on CD4+ T cells was significantly higher compared to baseline (p < 0.05). There was a significant increase (p ≤ 0.05) in B cell lymphocyte subset after revaccination, and increased percentage of CD80+ B cells. The expression of IFN-γ in peripheral blood lymphocytes, CD3+ T cells and serum was significantly increased (p < 0.05). No significant difference in immune response was observed between breastfeeding women and other study participants. The anti-SARS-CoV-2 BNT162b2 mRNA vaccine-induced measurable and durable immune response in breastfeeding women and in naïve and previously infected individuals.

Methylenetetrahydrofolate reductase polymorphic variants C677T and A1298C in rectal cancer in Slavic population: significance for cancer risk and response to chemoradiotherapy.

Background: Methylenetetrahydrofolate reductase (MTHFR) single nucleotide polymorphisms (SNPs) have been suggested as risk, prognostic, and predictive factors for colorectal cancer in various populations, but have not been validated so far. The aim of this study was to examine the association of MTHFR C677T (rs1801133) and A1298C (rs1801131) single nucleotide polymorphisms with the risk of rectal cancer as well as the response to neoadjuvant chemoradiotherapy (nCRT) based on 5-Fluorouracil (5-FU)/leucovorin (LV) in the locally advanced setting. Patients and methods: This case-control study included 119 healthy controls and 97 patients with locally advanced rectal cancer (LARC). For MTHFR genotyping, restriction fragment length polymorphism analysis (PCR-RFLP) was employed. Results: In silico analysis highlighted that SNPs C677T and A1298T correlate with MTHFR gene expression, and that gene expression profile correlates with cancer risk and stage. Using dominant and recessive models, it was found that the MTHFR 677CC vs. 677CT+677TT have increased risk of cancer development (odds ratio (OR): 2.27; 95% confidence interval (CI): 1.30-3.95, p = 0.002) as well as 677CC+677CT compared to 677TT (OR: 4.18, 95% CI: 1.16-14.99, p = 0.014). MTHFR 1298AA also shown increased risk for cancer development compared to 1298AC+1298CC (OR:2.0, 95% CI: 1.20-3.59, p = 0.035) Statistical analysis of combined genotypes highlighted the protective role of CT/AC combined genotype (OR: 3.15 95% CI: 1.576-6.279, p = 0.002) while the CC/AA genotype showed an increased risk for rectal cancer development (OR: 2.499, 95% CI: 1.246-5.081, p = 0.016) The carriers of the 677C/1298A haplotype had the highest risk for developing rectal cancer (OR: 1.74; 95% CI: 1.198-2.530, p = 0.002) while the 677T/1298C haplotype seems to provide a protective effect. (OR: 0.44; 95%CI 0.248-0.795, p = 0.003). No significant association with response to chemoradiotherapy was found. Conclusion: Our data point to MTHFR 667C allele and 1298A alleles as low-penetrance risk factors for rectal cancer in our population. To the best of our knowledge, this is the first study of this type performed on the Slavic population in the Western Balkan, as various population-based factors might also be significant our findings can be used for future meta-analyses and the construction of genetic cancer risk prediction panels.

Exploring novel genetic and hematological predictors of response to neoadjuvant chemoradiotherapy in locally advanced rectal cancer.

Introduction: The standard treatment for locally advanced rectal cancer (LARC) is neoadjuvant chemoradiotherapy (nCRT). To select patients who would benefit the most from nCRT, there is a need for predictive biomarkers. The aim of this study was to evaluate the role of clinical, pathological, radiological, inflammation-related genetic, and hematological parameters in the prediction of post-nCRT response. Materials and methods: In silico analysis of published transcriptomics datasets was conducted to identify candidate genes, whose expression will be measured using quantitative Real Time PCR (qRT-PCR) in pretreatment formaline-fixed paraffin-embedded (FFPE) samples. In this study, 75 patients with LARC were prospectively included between June 2020-January 2022. Patients were assessed for tumor response in week 8 post-nCRT with pelvic MRI scan and rigid proctoscopy. For patients with a clinical complete response (cCR) and initially distant located tumor no immediate surgery was suggested ("watch and wait" approach). The response after surgery was assessed using histopathological tumor regression grading (TRG) categories from postoperative specimens by Mandard. Responders (R) were defined as patients with cCR without operative treatment, and those with TRG 1 and TRG 2 postoperative categories. Non-responders (NR) were patients classified as TRG 3-5. Results: Responders group comprised 35 patients (46.6%) and NR group 53.4% of patients. Analysis of published transcriptomics data identified genes that could predict response to treatment and their significance was assessed in our cohort by qRT-PCR. When comparison was made in the subgroup of patients who were operated (TRG1 vs. TRG4), the expression of IDO1 was significantly deregulated (p < 0.05). Among hematological parameters between R and NR a significant difference in the response was detected for neutrophil-to-monocyte ratio (NMR), initial basophil, eosinophil and monocyte counts (p < 0.01). According to MRI findings, non-responders more often presented with extramural vascular invasion (p < 0.05). Conclusion: Based on logistic regression model, factors associated with favorable response to nCRT were tumor morphology and hematological parameters which can be easily and routinely derived from initial laboratory results (NMR, eosinophil, basophil and monocyte counts) in a minimally invasive manner. Using various metrics, an aggregated score of the initial eosinophil, basophil, and monocyte counts demonstrated the best predictive performance.

Exploring heterometallic bridged Pt(II)-Zn(II) complexes as potential antitumor agents.

The four novel complexes [{cis-PtCl(NH3)2(µ-4,4'-bipyridyl)ZnCl(terpy)}](ClO4)2 (C1), [{trans-PtCl(NH3)2(µ-4,4'-bipyridyl)ZnCl(terpy)}](ClO4)2 (C2), [{cis-PtCl(NH3)2(µ-pyrazine)ZnCl(terpy)}](ClO4)2 (C3) and [{trans-PtCl(NH3)2(µ-pyrazine)ZnCl(terpy)}](ClO4)2 (C4) (where terpy = 2,2':6',2''-terpyridine) were synthesized and characterized. Acid-base titrations and concentration dependent kinetic measurements for the reactions with biologically relevant ligands such as guanosine-5'-monophosphate (5'-GMP), inosine-5'-monophosphate (5'-IMP) and glutathione (GSH), were studied at pH 7.4 and 37 °C. The binding of the heterometallic bridged cis- or trans-Pt(II)-Zn(II) complexes to calf thymus DNA (CT-DNA) was studied by UV absorption and fluorescence emission spectroscopy and molecular docking. The results indicated that the complexes bind strongly to DNA, through groove binding, hydrogen bonds, and hydrophobic or electrostatic interaction. The possible in vitro DNA protective effect of cis- and trans-Pt-L-Zn complexes has shown that C3 had significant dose-dependent DNA-protective effect and the same ability to inhibit peroxyl as well as hydroxyl radicals. Antiproliferative effect of the complexes, mRNA expression of apoptosis and repair-related genes after treatment in cancer cells indicated that newly synthesized C2 exhibited highly selective cytotoxicity toward colon carcinoma HCT116 cells. Only treatment with trans analog C2 induced effect similar to the typical DNA damaging agent such as cisplatin, characterized by p53 mediated cell response, cell cycle arrest and certain induction of apoptotic related genes. Both cis- and trans-isomers C1 and C2 showed potency to elicit expression of PARP1 mRNA and in vitro DNA binding.