Amira Salah El-Din Youssef

Breast cancer (BC) is a multifactorial disease influenced by both genetic and environmental factors. Pathogenic variants in BRCA1 and BRCA2 significantly increase hereditary BC risk, while oncogenic viruses such as human Papillomavirus (HPV) and human mammary tumor virus ( HMTV ) may modulate disease development. Population-specific studies integrating genetic, clinicopathological, and viral data remain limited. Genomic DNA was extracted from 48 BC patients (19 fresh tissues, 29 FFPE samples). BRCA1/2 libraries were prepared using the Devyser BRCA NGS kit and sequenced on the Illumina MiSeq platform. Variants were called using Freebayes and annotated with ANNOVAR. The DNA of HMTV and HPV were detected by PCR. Associations between BRCA variants, clinicopathological features, and viral status were investigated using statistical and rule-based analyses. Exonic variants constituted 72% of all detected variants in BRCA1 and 97% in BRCA2 , whereas intronic variants accounted for 23% and 1%, respectively. Variants were most frequently identified within BRCA1 exons 9–10 and BRCA2 exon 11. This distribution may reflect the large size and structural complexity of these regions, as well as cohort-specific genetic characteristics. Among the 48 patients, 46 (95.8%) were diagnosed with invasive breast carcinoma, predominantly grade II tumors, and more than 30% reported a family history of breast cancer. Pathogenic BRCA1/2 variants were detected in 39.6% of cases, with BRCA1 alterations occurring more frequently than BRCA2 alterations. Several BRCA1/2 variants showed significant associations with HPV or HMTV DNA detection, while the BRCA2 p.Pro1639Ser variant appeared to be associated with a reduced likelihood of viral DNA positivity. Furthermore, specific pathogenic BRCA variants co-occurred with HPV or HMTV DNA, suggesting potential interactions between host genetic susceptibility and viral presence. Carriers of BRCA variants tended to present with larger tumors, higher histological grade, left-sided disease, microcalcifications, and a greater prevalence of hormone receptor–positive breast cancer. This study presents the first comprehensive investigation of BRCA1/2 variants in conjunction with HPV and HMTV DNA detection in a cohort of Egyptian women with breast cancer. The findings reveal a distinct BRCA1/2 variant profile, identify novel variants, and provide preliminary evidence of potential associations between host genetic variation and viral DNA detection. These results broaden the current understanding of the genetic and viral landscape of breast cancer in the Egyptian population and highlight possible interactions that merit further investigation. Given the exploratory nature of the observed associations, validation in larger, independent cohorts, supported by functional and clinical correlation studies, is required. Such efforts may contribute to improving risk assessment, patient stratification, and the development of tailored preventive and management strategies for this population.

Background: Rosmarinus officinalis L. is a medicinally important aromatic plant rich in bioactive secondary metabolites with diverse therapeutic properties. This study aimed to characterize the phytochemical profile of R. officinalis leaf extracts, isolate carnosic acid as a major bioactive diterpene, and evaluate its biological activities. Methods: Leaf extracts were prepared using solvents of increasing polarity and analyzed by phytochemical screening and UHPLC/QTOF-MS. Carnosic acid was isolated by thin-layer chromatography and assessed for antibacterial, antibiofilm, antioxidant, anti-inflammatory, antidiabetic, and antiproliferative activities using in vitro assays. Expression of apoptosis-related genes was also investigated. Results: Methanolic and ethanolic extracts exhibited the highest abundance of phenolic compounds and secondary metabolites, whereas the hexane extract showed lower phytochemical content. UHPLC/QTOF-MS identified seven major metabolites, including phenolic acids, flavonoids, and abietane-type diterpenes. Purified carnosic acid demonstrated potent antibacterial activity (MIC: 10–23 μg/mL) and inhibited biofilm formation by up to 90%. Strong antioxidant activity was observed, with DPPH and ABTS radical-scavenging IC₅₀ values of 125 and 130 μg/mL, respectively. The compound also exhibited notable anti-inflammatory activity and markedly inhibited α-amylase and α-glucosidase activities. Furthermore, carnosic acid exhibited dose-dependent antiproliferative activity against MCF-7, HepG2, and MCF-10A cells, reducing cell viability to 10.8%, 16.9%, and 70.4 ± 1.8%, respectively, at 250 μg/mL, with corresponding IC₅₀ values of 28.3, 37.8, and >250 μg/mL, respectively. Gene expression analysis revealed upregulation of BAX and downregulation of BCL2, indicating activation of mitochondrial-mediated apoptosis. Conclusions:R. officinalis leaves represent a valuable source of multifunctional phytochemicals, particularly carnosic acid. Its broad-spectrum biological activities and apoptosis-inducing potential support its promising application in pharmaceutical, nutraceutical, and biomedical fields.

Background: Nigella sativa (black cumin) seeds are renowned for their ethnomedicinal significance and are rich in bioactive phytochemicals, which contribute to food preservation and the prevention of various diseases through their antimicrobial and antioxidant properties. Accordingly, this study aimed to characterize the phytochemical composition of N. sativa seed extracts, isolate thymoquinone, and assess their antibacterial, antibiofilm, antioxidant, anti-inflammatory and antidiabetic activities. Methods: Nigella sativa seed extracts were prepared using solvents of increasing polarity and analyzed for phytochemical content. Metabolite profiling was performed using UHPLC/QTOF-MS. Thymoquinone, the major constituent, was isolated via thin-layer chromatography (TLC), further purified using semi-preparative reverse-phase high-performance liquid chromatography (RP-HPLC), and evaluated in vitro for antibacterial, antibiofilm, antioxidant, anti-inflammatory, and antidiabetic activities. Results: Extraction yields ranged from 5.5% to 8.4% (w/w), with methanol yielding the highest phenol (6.34 ± 0.31 mg GAE/mL) and flavonoid (5.12 ± 0.26 mg QE/mL) contents. UHPLC/QTOF-MS revealed a chemically diverse profile dominated by thymoquinone (58% relative abundance), alongside p-cymene, carvacrol, longifolene, and nigellidine. Thymoquinone (Rf = 0.56) was initially isolated from the methanolic extract with a yield of 270 mg/g and further purified from preparative TLC fractions using semi-preparative RP-HPLC, affording 82 mg of >95% pure compound with a 68.3% recovery, suitable for subsequent biological assays. It inhibited Gram-positive and Gram-negative bacteria, with MICs of 62.5 µg/mL against Staphylococcus aureus, Bacillus subtilis, and Listeria monocytogenes; 125–250 µg/mL against Escherichia coli and Salmonella typhimurium; and 500 µg/mL against Pseudomonas aeruginosa. Thymoquinone reduced biofilm formation (>80% at 25–50 µg/mL; MBIC₅₀ ≈ 5.4–11.6 µg/mL), exhibited antioxidant activity (DPPH IC₅₀ = 52.3 ± 2. 1 µg/mL; ABTS IC₅₀ = 41.6 ± 1.9 µg/mL), stabilized erythrocyte membranes (IC₅₀ ≈ 14.8 µg/mL), and inhibited carbohydrate-hydrolyzing enzymes, with stronger inhibition of α-glucosidase (~92%) than α-amylase (~84%) at 128 µg/mL. Conclusions: Thymoquinone is a major bioactive constituent of N. sativa seeds, exhibiting consistent multi-target in vitro activity. These findings highlight its functional relevance and in vivo investigations to establish therapeutic potential.

Background: Colorectal cancer (CRC) is among the five leading causes of cancer incidence and mortality. During the past decade, the role of the gut microbiota and its dysbiosis in colorectal tumorigenesis has been emphasized. Metagenomics and amplicon-based microbiome profiling provided insights into the potential role of microbial dysbiosis in the development of CRC. Aim: To address the scarcity of information on differential microbiome composition of tumor tissue in comparison to adenomas and the lack of such data from Egyptian patients with CRC. Materials and methods: Long-read nanopore sequencing of 16S rRNA amplicons was used to profile the colonic microbiota from fresh colonoscopic biopsy samples of Egyptian patients with CRC and patients with colonic polyps. Results: Species richness of CRC lesions was significantly higher than that in colonic polyps (p-value = 0.0078), while evenness of the CRC group was significantly lower than the colonic polyps group (p-value = 0.0055). Both species richness and Shannon diversity index of the late onset CRC samples were significantly higher than those of the early onset ones. The Firmicutes-to-Bacteroidetes (F/B) ratio was significantly higher in the CRC group than in the colonic polyps group (p-value = 0.0054), and significantly higher in samples from early-onset CRC. The Enterococcus spp. were significantly overabundant in patients with rectal cancer and early-onset CRC, while Staphylococcus spp. were significantly higher in patients with sigmoid cancer and late-onset CRC. In addition, the relative abundance of Fusobacterium nucleatum was significantly higher in CRC patients. Conclusion: Differentiating trends were identified at phylum, genus, and species levels, despite the inter-individual differences. In summary, this study addressed the microbial dysbiosis associated with CRC and colonic polyps groups, paving the way for a better understanding of the pathogenesis of early and late-onset CRC in Egyptian patients.

This study aimed to identify BRCA1/2 mutational patterns in the tissue and blood of Egyptian colorectal cancer (CRC) patients and to study the possible correlation of this mutational pattern with Human papillomavirus (HPV) infection. Eighty-two colonoscopic biopsies and forty-six blood samples were collected from Egyptian CRC patients, as well as blood samples of age and sex-matched healthy controls (n = 43) were enrolled. The libraries were performed using Qiaseq Human BRCA1 and BRCA2 targeted DNA panel and sequenced via Ion proton sequencer. Also, the CRC tissues were subjected to conventional PCR targeting the HPV Late 1 (L1) region. Our analysis revealed that the BRCA-DNA damage pathway had been altered in more than 65% of the CRC patients. Comparing tissue and blood samples from CRC patients, 25 somatic mutations were found exclusively in tissue, while 41 germline mutations were found exclusively in blood. Additionally, we identified 23 shared BRCA1/2 pathogenic (PVs) mutations in both blood and tissue samples, with a significantly higher frequency in blood samples compared to tissue samples. The most affected exon in BRCA1 was exon 10, while the most affected exons in BRCA2 were 11, 14, 18, 24, and 27 exons. Notably, we revealed an ethnic-related cluster of polymorphism variants in our population closely related to South Asian and African ethnicities. Novel PVs were identified and submitted to the ClinVar database. HPV was found in 23.8% of the CRC tissues, and 54% of HPV-positive cases had somatic BRCA1/2 PVs. The results of this research point to a possible connection between infection with HPV and BRCA1/2 mutations in the occurrence of colorectal cancer in the Egyptian population, which has a mixed ethnic background. Our data also indicate that liquid biopsy (blood samples) may be more representative than tissue samples for detecting BRCA1/2 mutations. These findings may have implications for cancer screening and the development of personalized, targeted therapies, such as PARP inhibitors, which can effectively target BRCA1/2 mutations.

Background: Precision oncology has been increasingly used in clinical practice and rapidly evolving in the oncology field. Thus, this study was performed to assess the frequency of germline mutations in early and late onset familial breast cancer (BC) Egyptian patients using multi-gene panel sequencing to better understand the contribution of the inherited germline mutations in BC predisposition. Moreover, to determine the actionable deleterious mutations associated with familial BC that might be used as biomarker for early cancer detection. Methods: Whole blood samples were collected from 101 Egyptian patients selected for BC family history, in addition to 50 age-matched healthy controls. A QIAseq targeted DNA panel (human BC panel) was used to assess the frequency of germline mutations. Results: A total of 58 patients (57.4%) out of 101 were found to have 27 deleterious germline mutations in 11 cancer susceptibility genes. Of them, 32 (31.6%) patients carried more than one pathogenic mutation and each one carried at least one pathogenic mutation. The major genes harboring the pathogenic mutations were: ATM, BRCA2, BRCA1, VHL, MSH6, APC, CHEK2, MSH2, MEN1, PALB2, and MUTYH. Thirty-one patients (30.6%) had BRCA2 mutations and twenty (19.8%) had BRCA1 mutations. Our results showed that exon 10 and exon 11 harbored 3 and 5 mutations, respectively, in BRCA1 and BRCA2 genes. Our analysis also revealed that the VHL gene significantly co-occurred with each of the BRCA2 gene (p = 0.003, event ratio 11/21), the MSH2 gene (p = 0.01, 4/10), the CHEK2 gene (p = 0.02, 4/11), and the MSH6 gene (p = 0.04, 4/12). In addition, the APC gene significantly co-occurred with the MSH2 gene (p = 0.01, 3/7). Furthermore, there was a significant mutually exclusive event between the APC gene and the ATM gene (p = 0.04, 1/36). Interestingly, we identified population specific germline mutations in genes showing potentials for targeted therapy to meet the need for incorporating precision oncology into clinical practice. For example, the mutations identified in the ATM, APC, and MSH2 genes. Conclusions: Multi-gene panel sequencing was used to detect the deleterious mutations associated with familial BC, which in turns mitigate the essential need for implementing next generation sequencing technologies in precision oncology to identify cancer predisposing genes. Moreover, identifying DNA repair gene mutations, with focus on non-BRCA genes, might serve as candidates for targeted therapy and will be increasingly used in precision oncology.

This study aimed to investigate the impact of somatic mutations on various interleukin signaling pathways associated with grade II invasive breast cancer (BC) in Egyptian patients to broaden our understanding of their role in promoting carcinogenesis. Fifty-five grade II invasive BC patients were included in this study. Data for somatic mutations in 45 BC patients were already available from a previous study. Data for somatic mutations of 10 new BC patients were included in the current study. Somatic mutations were identified using targeted next-generation sequencing (NGS) to study their involvement in interleukin signaling pathways. For pathway analysis, we used ingenuity variant analysis (IVA) to identify the most significantly altered pathways. We identified somatic mutations in components of the interleukin-2, interleukin-6, and inter-leukin-7 signaling pathways, including mutations in JAK1, JAK2, JAK3, SOCS1, IL7R, MCL1, BCL2, MTOR, and IL6ST genes. Interestingly, six mutations which were likely to be novel deleterious were identified: two in the SCH1 gene, two in the IL2 gene, and one in each of the IL7R and JUN genes. According to IVA analysis, interleukin 2, interleukin 6, and interleukin 7 signaling pathways were the most altered in 34.5%, 29%, and 23.6% of our BC group, respectively. Our multigene panel sequencing analysis reveals that our BC patients have altered interleukin signaling pathways. So, these results highlight the prominent role of interleukins in the carcinogenesis process and suggest its potential role as promising candidates for personalized therapy in Egyptian patients.

This study aims at identifying common pathogenic somatic mutations at different stages of colorectal carcinogenesis in Egyptian patients. Our cohort included colonoscopic biopsies collected from 120 patients: 20 biopsies from patients with inflammatory bowel disease, 38 from colonic polyp patients, and 62 from patients with colorectal cancer. On top of this, the cohort included 20 biopsies from patients with non-specific mild to moderated colitis. Targeted DNA sequencing using a customized gene panel of 96 colorectal related genes running on the Ion Torrent NGS technology was used to process the samples. Our results revealed that 69% of all cases harbored at least one somatic mutation. Fifty-seven genes were found to carry 232 somatic non-synonymous variants. The most frequently pathogenic somatic mutations were localized in TP53, APC, KRAS, and PIK3CA. In total, 16 somatic mutations were detected in the CRC group and in either the IBD or CP group. In addition, our data showed that 51% of total somatic variants were CRC-specific variants. The average number of CRC-specific variants per sample is 2.4. The top genes carrying CRC-specific mutations are APC, TP53, PIK3CA, FBXW7, ATM, and SMAD4. It seems obvious that TP53 and APC genes were the most affected genes with somatic mutations in all groups. Of interest, 85% and 28% of the APC and TP53 deleterious somatic mutations were located in Exon 14 and Exon 3, respectively. Besides, 37% and 28% of the total somatic mutations identified in APC and TP53 were CRC-specific variants, respectively. Moreover, we identified that, in 29 somatic mutations in 21 genes, their association with CRC patients was unprecedented. Ten detected variants were likely to be novel: six in PIK3CA and four variants in FBXW7. The detected P53, Wnt/βcatenin, Angiogenesis, EGFR, TGF-β and Interleukin signaling pathways were the most altered pathways in 22%, 16%, 12%, 10%, 9% and 9% of the CRC patients, respectively. These results would contribute to a better understanding of the colorectal cancer and in introducing personalized therapies for Egyptian CRC patients.

Background: Colorectal cancer (CRC) incidence is progressively increasing in Egypt. Unfortunately, there is inadequate knowledge of the acquired somatic mutations in Egyptian CRC patients which limit our understanding of its progression. To the best of our knowledge, our study is the first to sequence multiple-gene panel to identify the somatic mutation pattern associated with CRC disease progression in a cohort of Egyptian patients. Custom 72 genes, which are frequently associated with CRC, were sequenced using Qiaseq UMI-based targeted DNA panel in 120 fresh tissues classified into; inflammatory bowel disease (IBD; n=20), colonic polyp (CP; n=38) and CRC (n=62) as well as 20 biopsies with non-specific colitis served as a control group (n=20). Results: Using Ingenuity Variant Analysis (IVA), we revealed that APC, TP53 & ATM genes harbored the highly frequent CRC-specific somatic mutations (15, 11 & 6, respectively). We also identified common somatic mutations (predictors) that were associated with disease progression from colitis to CRC; APC (c.1742delA (65%)), TP53 (c.121delG (58%), c.215C>G (52%)), ATM (c.640delT (16%)), IGF2 (c.677delG (56%)), RET (c.2071G>A (37%)), ACVR2A (c.1310delA (26%)), PIK3CA (c.1173A>G (16%)) & KIT (c.1621A>C (13%)). Furthermore, pathway analysis using Ingenuity Pathway Analysis (IPA) showed that Wnt/βcatenin, ATM signaling, RTK-RAS and TGF-β were the most altered pathways in the CRC group (73%. 72%, 40% & 36%, respectively).Conclusion: In this data set, we shed the light on the most frequent somatic mutations and the most altered pathways that are crucial for understanding colorectal cancer predisposition and developing personalized therapies for the Egyptian CRC patients.

The identification of new high-sensitivity and high-specificity markers for hepatocellular carcinoma (HCC) is essential. We aimed at identifying serum microRNAs (miRNAs) as potential biomarkers for early detection of HCC on top hepatitis C virus (HCV) infection. We investigated serum expression of 13 miRNAs in 384 patients with HCV-related chronic liver disease (192 with HCC, 96 with liver cirrhosis (LC), and 96 with chronic hepatitis C (CHC)) in addition to 96 healthy participants enrolled as a control group. The miRNA expression was performed using real-time quantitative PCR-based SYBR Green custom miRNA arrays. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic performance of miRNA panels for early detection of HCC. Using miRNA panel of miR-122, miR-885-5p, and miR-29b with alpha fetoprotein (AFP) provided high diagnostic accuracy (AUC = 1) for early detection of HCC in normal population while using miRNA panel of miR-122, miR-885-5p, miR-221, and miR-22 with AFP provided high diagnostic accuracy (AUC = 0.982) for early detection of HCC in LC patients. However, using miRNA panel of miR-22 and miR-199a-3p with AFP provided high diagnostic accuracy (AUC = 0.988) for early detection of HCC in CHC patients. We identified serum miRNA panels that could have a considerable clinical use in early detection of HCC in both normal population and high-risk patients.