Mahonia aquifolium Extracts Promote Doxorubicin Effects against Lung Adenocarcinoma Cells In Vitro.

Mahonia aquifolium and its secondary metabolites have been shown to have anticancer potential. We performed MTT, scratch, and colony formation assays; analyzed cell cycle phase distribution and doxorubicin uptake and retention with flow cytometry; and detected alterations in the expression of genes involved in the formation of cell-cell interactions and migration using quantitative real-time PCR following treatment of lung adenocarcinoma cells with doxorubicin, M. aquifolium extracts, or their combination. MTT assay results suggested strong synergistic effects of the combined treatments, and their application led to an increase in cell numbers in the subG1 phase of the cell cycle. Both extracts were shown to prolong doxorubicin retention time in cancer cells, while the application of doxorubicin/extract combination led to a decrease in MMP9 expression. Furthermore, cells treated with doxorubicin/extract combinations were shown to have lower migratory and colony formation potentials than untreated cells or cells treated with doxorubicin alone. The obtained results suggest that nontoxic M. aquifolium extracts can enhance the activity of doxorubicin, thus potentially allowing the application of lower doxorubicin doses in vivo, which may decrease its toxic effects in normal tissues.

Germline mutations in cancer susceptibility genes in high grade serous ovarian cancer in Serbia.

Clinical criteria for genetic testing of genes other than BRCA1/2 in epithelial ovarian cancer (EOC) still do not exist. We assessed the frequency and predictors of deleterious mutations in 19 cancer predisposition genes in high-grade serous ovarian cancer (HGSOC) in Serbia. Next-generation sequencing was used to identify germline mutations in the whole coding regions of a gene panel. Patients' characteristics and sequencing data were summarized with descriptive statistics and compared using chi-square test. Among 131 HGSOC patients, 23 had BRCA1 (17.6%) while 5 had BRCA2 (3.8%) mutation. In addition, 9 (6.9%) pathogenic mutations were detected in other genes including BRIP1 (n = 2;1.5%), CHEK2 (n = 2;1.5%), NBN (n = 3;2.3%) and RAD51C (n = 2;1.5%). Factors that predicted for BRCA1/2 mutations were: breast and ovarian cancers in the same patient (p = 0.031), young age of EOC (p = 0.029), menstrual status (p = 0.004) and family history of cancer (p < 0.0001). However, these factors did not predict for mutations in other cancer susceptibility genes. Applying established referral criteria for genetic testing in Serbia will help identify BRCA1/2 mutation carriers but will not help identify mutations in other cancer susceptibility genes. Until better predictors emerge we should be performing wider genetic testing of EOC in order to identify all mutation carriers.

Association of uPA and PAI-1 tumor levels and 4G/5G variants of PAI-1 gene with disease outcome in luminal HER2-negative node-negative breast cancer patients treated with adjuvant endocrine therapy.

BACKGROUND: The aim of this study was to evaluate the prognostic potential of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) tumor tissue levels and examine the association between these biomarkers and classical prognostic factors in early node-negative luminal breast cancer patients. The clinical value of 4G/5G variants of PAI-1 gene was evaluated. PATIENTS AND METHODS: This study involved 81 node-negative, estrogen receptor-positive and/or progesterone receptor-positive and human epidermal growth factor receptor 2-negative operable breast cancer patients who underwent radical surgical resection and received adjuvant endocrine therapy. Determination of uPA and PAI-1 concentrations in the breast cancer tissue extracts was performed using FEMTELLE® uPA/PAI-1 ELISA. An insertion (5G)/deletion (4G) polymorphism at position - 675 of the PAI-1 gene was detected by PCR-RFLP analysis. RESULTS: Our research showed that patients with uPA tumor tissue levels higher than 3 ng/mg of protein had significantly reduced disease-free survival (DFS) and overall survival (OS) when compared to patients with uPA tumor tissue levels lower or equal to 3 ng/mg of protein. Patients with PAI-1 tumor tissue levels higher than 14 ng/mg of protein had significantly decreased OS in comparison with patients with PAI-1 tumor tissue levels lower or equal to 14 ng/mg of protein. ROC analysis confirmed the uPA and PAI-1 discriminative potential for the presence/absence of relevant events in these patients and resulted in higher cut-off values (5.65 ng/mg of protein for uPA and 27.10 ng/mg of protein for PAI-1) than standard reference cut-off values for both biomarkers. The prognostic importance of uPA and PAI-1 ROC cut-off values was confirmed by the impact of uPA higher than 5.65 ng/mg of protein and PAI-1 higher than 27.10 ng/mg of protein on poorer DFS, OS and event-free survival (EFS). We observed that patients with dominant allele in PAI-1 genotype (heterozygote and dominant homozygote, - 675 4G/5G and - 675 5G/5G) had significantly increased DFS, OS and EFS when compared with patients with recessive homozygote genotype (- 675 4G/4G). CONCLUSION: Our study indicates that uPA and PAI-1 tumor tissue levels and 4G/5G variants of PAI-1 gene might be of prognostic significance in early node-negative luminal HER2-negative breast cancer patients treated with adjuvant endocrine therapy.

Highly selective anthraquinone-chalcone hybrids as potential antileukemia agents.

A series of 23 novel anthraquinone-chalcone hybrids containing amide function was synthesized and structurally characterized. Sixteen compounds exerted strong cytotoxic activities against K562, Jurkat and HL-60 leukemia cell lines and significantly lower cytotoxic effects against normal MRC-5 cells, indicating very high selectivity in their anticancer action. The compounds 6g, 6u and 6v activate apoptosis in K562 cells through the extrinsic and intrinsic apoptotic pathway. The compound 6e triggered apoptosis in K562 cells only through the extrinsic apoptotic pathway. Treatment of K562 cells with each of these four compounds caused decrease in the expression levels of MMP2, MMP9, and VEGF, suggesting their anti-invasive, antimetastatic and antiangiogenic properties. The compounds 6g and 6v downregulated expression levels of miR-155 in K562 cells, while compounds 6e and 6u upregulated miR-155 levels in treated cells, in comparison with control cells. The structure-based 3-D QSAR models for 6f, 6e, 6i and 6l describe pro-apoptotic activity against caspase-3.

Promoter hypermethylation of p16, BRCA1 and RASSF1A genes in triple-negative breast cancer patients from Serbia.

PURPOSE: In order to investigate if aberrant promoter methylation of p16, BRCA1 and RASSF1A genes contributes to biological behavior of triple-negative breast cancer (TNBC), marked as the most aggressive phenotype of breast cancer, we compared the hypermethylation pattern between TNBC and ER+PR+Her2- breast cancer. METHODS: 131 patients with histologically confirmed breast cancers were included - 61 TNBC and 70 ER+PR+Her2- cases. The patients were followed up for 1-87 months (median 78). DNA from tumor tissues was isolated by the salting out procedure. The methylation status was assessed by nested methylation-specific PCR after bisulfite modification of DNA. RESULTS: The frequency of p16 hypermethylated breast cancer cases was significantly higher in TNBC than in ER+PR+Her2- group (33; 54.1% vs. 20; 28.6%, p=0.00298). Co-methylated p16 and RASSF1A genes were more frequent in the TNBC than in ER+PR+Her2- group (20; 32.8% vs. 10; 14.3%, p=0.0225). The same result was observed when hypermethylated BRCA1 gene was added in the analysis: 12; 19.7% vs. 3; 4.3%, p=0.00791. Although there was significant difference in disease-free survival (DFS) and overall survival (OS) between TNBC and ER+PR+Her2- group, further analysis of co-methylation of p16 and RASSF1A (p16+RASSF1A+) showed that DFS was significantly shorter in the patients with both genes co-methylated in TNBC than in ER+PR+Her-2- group (8/20; 40% vs. 2/10; 20%, p=0.03272). CONCLUSIONS: The obtained data indicate that hypermethylated p16 and RASSF1A cell-cycle inhibitor genes might be considered as biomarkers for bad prognosis in breast cancer. Hypermethylation of these genes may influence the clinical disease course, distinguishing a particular group of TNBC patients with even more aggressive phenotype.

Evaluation of the Functional Capacity of NK Cells of Melanoma Patients in an In Vitro Model of NK Cell Contact with K562 and FemX Tumor Cell Lines.

NK cells of metastatic melanoma (MM) patients display impaired function, making them incapable to mount an effective antitumor response. In this study, we evaluated immunophenotypic characteristics and functional capacity of CD3-CD16+ NK cells of MM patients in an in vitro model based on NK cell contact with an NK sensitive, K562, and a tumor-specific, melanoma FemX tumor cell line. Although our results indicate similar NK cell antitumor cytotoxic potential of MM patients in contact with both cell lines based on the expression of CD107a degranulation marker, there is a discrepancy in NK cell IFNγ production, as it is not significantly induced by FemX tumor cells, found to be, contrary to K562, HLA class I positive. Furthermore, we show NKG2D receptor downregulation by K562 tumor cell line, only. This may result from the obtained higher gene expression of TGFß and VEGFA growth factors in K562 tumor cells that can negatively regulate NKG2D expression. Additionally, aside from postcontact downmodulation of activating CD16 receptor, there are no significant changes in the expression of CD161, CD158a, and CD158b NK cell receptors. Therefore, the applied in vitro model shows that, compared to the full NK cell functional capacity of MM patients displayed in a tumor-sensitive setting represented by contact with K562 cells, tumor-specific melanoma setting provided by FemX tumor cells leads to reduced NK functional potential. The obtained insight into NK cell capacity may be of use for evaluation of the state of disease and can help in selecting effective immunotherapeutic agents for MM patients.

Synthesis, antioxidant and antiproliferative activities of 1,3,4-thiadiazoles derived from phenolic acids.

Two 2-amino-1,3,4-thiadiazoles containing phenolic hydroxyl groups were combined with different carboxylic acid chlorides giving sixteen amide derivatives with good antioxidant and antiproliferative potential. The compound 3'c with an adamantane ring displayed excellent DPPH radical scavenging activity and good cytotoxic activity against human acute promyelocytic leukemia HL-60 cells, while 1,3,4-thiadiazole 3'h with 4-chlorophenyl moiety was found to be the most effective in inhibition of survival of lung carcinoma A549 cells. All examined thiadiazoles except 3a and 3'a exerted higher cytotoxic activities on A549 and HL-60 cancer cells when compared with normal fibroblasts MRC-5, pointing to selectivity in their antiproliferative action. Some of the most active novel compounds 3c, 3'c, 3'g and 3'h induced significant increase in the percentage of HL-60 cells in the subG1 cell cycle phase in comparison with the control cells. The induction of cell death in HL-60 cells by these compounds was at least partially dependent on activation of caspase-3 and caspase-8. The compounds 3c and 3'c exerted strong antiangiogenic activity. Furthermore, compounds 3c, 3'c, 3'g and 3'h showed the ability to down-regulate the MMP2 and VEGFA expression levels in the treated HL-60 cells when compared with the control cell samples.

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.

Li-Fraumeni Syndrome With Six Primary Tumors-Case Report.

Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome associated with a high, lifetime risk of a broad spectrum of cancers caused by pathogenic germline TP53 mutations. Numerous different germline TP53 mutations have been associated with LFS, which has an exceptionally diverse clinical spectrum in terms of tumor type and age of onset. Our patient has developed six asynchronous tumors to date: a phyllode tumor of the breast, a pheochromocytoma, a rosette-forming glioneuronal tumor (RGNT), an adrenocortical carcinoma (ACC), a ductal carcinoma of the breast, and a thymoma. The occurrence of such a number of rare tumors is sporadic even among in the population of patients living with cancer predisposition syndromes. In this instance, the omission of pretest genetic counseling and thorough family tree analysis prior to selecting the test led to the oversight of an underlying TP53 likely pathogenic mutation (classified as Class 4). This emphasizes the necessity for such counseling to prevent overlooking crucial genetic information. Neglecting this step could have had profound implications on the patient's treatment, particularly considering the early onset and occurrence of multiple tumors, which typically raise suspicion of a hereditary component. The implications for family members must be considered.

Serbian high-risk families: extensive results on BRCA mutation spectra and frequency.

Mutations in BRCA genes elevate risk for breast and ovarian cancer. These mutations are population specific. As there are no data on BRCA mutation screening on larger number of probands in Serbia to date, aim of this study was to determine types and frequencies of BRCA mutations in individuals from high-risk families from Serbia, as well as to determine which BRCA mutations may be considered as founder for Serbian population. We analyzed 94 probands and detected 9 frameshift mutations in 12 individuals, 1 benign BRCA2 nonsense mutation and numerous missense and synonymous mutations in both genes. Frequency of frameshift mutations is 12.77%. In addition to two novel mutations detected in our population we reported previously, we detected another novel mutation--c.7283delT in BRCA2 exon 14. None of the detected deleterious mutations may be considered as founder mutations for Serbian population, as each of them was found in no more than two high-risk families. This mutation diversity is most probably due to high migration rate in history of this part of Europe. Interpretation of genetic testing results with missense mutations of unknown clinical importance is very challenging and should be approached with caution, using all available data sources for results' interpretation.

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.

Biological evaluation of transdichloridoplatinum(II) complexes with 3- and 4-acetylpyridine in comparison to cisplatin.

BACKGROUND: In our previous study we reported the synthesis and cytotoxicity of two trans-platinum(II) complexes: trans-[PtCl2(3-acetylpyridine)2] (1) and trans-[PtCl2(4-acetylpyridine)2] (2), revealing significant cytotoxic potential of 2. In order to evaluate the mechanism underlying biological activity of both trans-Pt(II) isomers, comparative studies versus cisplatin were performed in HeLa, MRC-5 and MS1 cells. MATERIALS AND METHODS: The cytotoxic activity of the investigated complexes was determined using SRB assay. The colagenolytic activity was determined using gelatin zymography, while the effect of platinum complexes on matrix metalloproteinases 2 and 9 mRNA expression was evaluated by quantitative real-time PCR. Apoptotic potential and cell cycle alterations were determined by FACS analyses. Western blot analysis was used to evaluate the effect on expression of DNA-repair enzyme ERCC1, and quantitative real-time PCR was used for the ERCC1 mRNA expression analysis. In vitro antiangiogenic potential was determined by tube formation assay. Platinum content in intracellular DNA and proteins was determined by inductively coupled plasma-optical emission spectrometry. RESULTS: Compound 2 displayed an apparent cytoselective profile, and flow cytometry analysis in HeLa cells indicated that 2 exerted antiproliferative effect through apoptosis induction, while 1 induced both apoptosis and necrosis. Action of 1 and 2, as analyzed by quantitative real-time PCR and Western blot, was associated with down-regulation of ERCC1. Both trans-complexes inhibited MMP-9 mRNA expression in HeLa, while 2 significantly abrogated in vitro tubulogenesis in MS1 cells. CONCLUSIONS: The ability of 2 to induce multiple and selective in vitro cytotoxic effects encourages further investigations of trans-platinum(II) complexes with substituted pyridines.

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.

Mutational profile of hereditary breast and ovarian cancer - Establishing genetic testing guidelines in a developing country.

PURPOSE: Because many countries lack the capacity to follow the international guidelines for genetic testing, we suggest the specific approach for establishing local genetic testing guidelines that could be applied in developing countries. We focus on hereditary breast (BC) and ovarian cancer (OC) in Serbia. METHODS: From the cohort of 550 persons who were referred for genetic counseling at the Institute for Oncology and Radiology of Serbia, 392 were selected. Personal and family histories were collected and germline DNA was sequenced with NGS in a panel of 20 genes. RESULTS: Pathogenic (PV) and likely-pathogenic variants (LPV) were detected in 8 genes with the frequency of 23.7%. The most frequent were in BRCA1(7.6%), BRCA2(4.8%), PALB2(4.1%) and CHEK2(3.8%). They were also detected in ATM(1.8%), NBN(0.8%), TP53(0.5%) and RAD51C(0.3%). Whereas high carrier probability (CP), bilateral BC, BC and OC in the same patient and family history (FH) of BC/OC, were the strongest predictors for BRCA1/2 PV/LPV, lower CP values and early age of BC onset without FH were associated with higher frequency of PALB2 and CHEK2 PV/LPV. CONCLUSIONS: Population specific studies to identify specific mutational patterns and predictors of PV/LPV should be conducted in order to make scientifically sound and cost-effective guidelines for genetic testing in developing countries.

Pathological Diagnosis, Work-Up and Reporting of Breast Cancer 1st Central-Eastern European Professional Consensus Statement on Breast Cancer.

This text is based on the recommendations accepted by the 4th Hungarian Consensus Conference on Breast Cancer, modified on the basis of the international consultation and conference within the frames of the Central-Eastern European Academy of Oncology. The recommendations cover non-operative, intraoperative and postoperative diagnostics, determination of prognostic and predictive markers and the content of cytology and histology reports. Furthermore, they address some specific issues such as the current status of multigene molecular markers, the role of pathologists in clinical trials and prerequisites for their involvement, and some remarks about the future.

Histone Deacetylase 7 Gene Overexpression Is Associated with Poor Prognosis of Triple-Negative Breast Cancer Patients.

Background: Differential expressions of cancer-associated genes, including histone deacetylases (HDACs), were identified in distinctive molecular subtypes of breast cancer. Compared with hormone receptor-positive breast cancer, triple-negative (TNBC, ER-PR-HER2-) is the most aggressive form of breast cancer. Aims: To determine the association of HDAC7 mRNA expression levels with clinicopathological features and patients' survival with TNBC or ER+PR+HER2- breast cancers. Methods: Total RNA was extracted from 61 TNBC and 74 ER+PR+Her2- tumors. Relative gene expression was evaluated by SYBR Green RT-PCR, normalized to glyceraldehyde-3-phosphate dehydrogenase. The HDAC7 mRNA expression was defined as high or low, according to receiver operating characteristic analysis. Kaplan-Meier and Cox regression analyses for overall survival were assessed to evaluate the prognostic relevance of HDAC7 overexpression. Results: The HDAC7 overexpression was predominantly found in invasive ductal carcinomas (p = 0.023), high histologic grade (p = 0.007), and high nuclear grade tumors (p = 0.030). TNBC subtypes had a significantly lower mean HDAC7 gene expression compared with ER+PR+HER2- tumors (p = 0.005). However, HDAC7 overexpression predicted unfavorable survival of TNBC patients (p = 0.003). Multivariate Cox regression analysis indicated that recurrences (hazard ratio [HR] = 5.432, p = 0.003), and HDAC7 overexpression (HR = 9.287, p = 0.033) persisted as independent prognostic factors for poor survival of TNBC patients. Conclusions: HDAC7 mRNA overexpression is associated with poor survival in patients with TNBC tumors.

3D HeLa spheroids as a model for investigating the anticancer activity of Biginelli-hybrids.

In previous study, we examined the anticancer effects of novel Biginelli-hybrids against HeLa cell line on 2D monolayer culture. The five most effective compounds were chosen for further analysis of their anticancer activity against HeLa spheroids. Using the 3D models implies the possible differences in anticancer effects and mechanisms of activity of tested compounds. The compounds 4c and 4d exerted the strongest activity against 3D HeLa spheroids and induced to some extent loosened cell-cell contacts in spheroids, leading to the largest reduction in the diameter of the spheroids. Additionally, the highest accumulation of the cells in the subG1 phase of the cell cycle was observed after the treatment with compounds 4d and 4c, while the compound 4f led to the G2/M arrest. The invasion potential of treated HeLa cells in spheroids was monitored by imaging of spheroids embedded in a matrix made of matrigel and collagen and by determination of MMP2, MMP9, and VEGF gene expression levels. The compound 4l did not show invasion-suppressive activity, while the compounds 4c and 4d exerted the strongest anti-invasive activity.

Exploring the real-world effect of the SARS-CoV-2 pandemic on the molecular diagnostics for cancer patients and high-risk individuals.

Background: The SARS-CoV-2 pandemic introduced a global distraction effect in cancer patients' care. The aim of this study was to explore the effect of the pandemic on the largest molecular diagnostics center for cancer patients and high-risk individuals in Serbia.Research design and methods: EGFR, KRAS/NRAS, BRAF, and BRCA1/2 mutation testing were performed by qPCR and NGS. NGS was used for panel testing of hereditary breast/ovarian cancer and cancers associated with Lynch syndrome. The analytical output during the state of emergency (SoE) was compared to the period before and after the outbreak using one-way ANOVA. Statistical significance was set at p < 0.05.Results: A 38% reduction in the number of analysis was detected during the SoE. After the SoE, a 19% reduction was noted compared to SoE and 50% compared to the period before the SoE (p = 0.038). Three of the 48 scheduled appointments for pretest genetic counseling were carried out during the SoE, but the number of NGS tests increased by 50%.Conclusions: The SARS-CoV-2 pandemic had a profound negative effect on the diagnostic output of our centralized molecular diagnostics center. The only positive effect was shortening of waiting lists for hereditary cancer patients and high-risk individuals.