Zekri, A. - R. N., E. R. El-Sisi, A. S. E. - D. Youssef, M. M. Kamel, A. Nassar, O. S. Ahmed, M. El Kassas, A. B. Barakat, A. I. Abd El-Motaleb, and A. A. Bahnassy, "MicroRNA Signatures for circulating CD133-positive cells in hepatocellular carcinoma with HCV infection.", PloS one, vol. 13, issue 3, pp. e0193709, 2018. Abstract

AIM: Molecular characterization of the CD133+ stem cells associated with hepatocarinogensis through identifying the expression patterns of specific microRNAs (miRNAs).

METHODS: We investigated the expression pattern of 13 miRNAs in purified CD133+ cells separated from the peripheral blood of healthy volunteers, chronic hepatitis C (CHC), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) patients a long with bone marrow samples from the healthy volunteers and the LC patients using custom miScript miRNA PCR array.

RESULTS: The differential expression of the 13 studied miRNAs in CD133+ cells separated from the HCC patients' peripheral blood compared to the controls revealed that miR-602, miR-181b, miR-101, miR-122, miR-192, miR-125a-5p, and miR-221 were significantly up regulated (fold change = 1.8, 1.7, 2, 5.4, 1.6, 2.9 & 1.5 P value = 0.039, 0.0019, 0.0013, 0.0370, 00024, 0.000044 &0.000007 respectively). As for the HCC group compared to the CHC group; miR-602, miR-122, miR-181b, miR-125a-5p, and miR-192 were significantly up regulated (fold change = 13, 3.1, 2.8, 1.6 & 1.56, P value = 0.01, 0.001, 0.000004, 0.002 & 0.007 respectively). Upon comparing the HCC group to the LC group; miR-199a-3p, miR-192, miR-122, miR-181b, miR-224, miR-125a-5p, and miR-885-5p were significantly up regulated (fold change = 5, 6.7, 2.3, 3, 2.5, 4.2 & 39.5 P value = 0.001025, 0.000024, 0.000472, 0.000278, 0.000004, 0.000075 & 0.0000001 respectively) whereas miR-22 was significantly down regulated (fold change = 0.57 P value = 0.00002). Only, miR-192, miR-122, miR-181b and miR-125a-5p were significant common miRNAs in CD133+ cells of the HCC group compared to the other non-malignant groups.

CONCLUSION: We identified a miRNA panel comprised of four miRNAs (miR-192, miR-122, miR-181b and miR-125a-5p) that may serve as a molecular tool for characterization of the CD133+ cells associated with different stages of hepatocarinogensis. This panel may aid in developing a new target therapy specific for those CD133+ cells.

Zekri, A. - R. N., A. S. E. - D. Youssef, M. M. Lotfy, R. Gabr, O. S. Ahmed, A. Nassar, N. Hussein, D. Omran, E. medhat, S. Eid, et al., "Circulating Serum miRNAs as Diagnostic Markers for Colorectal Cancer.", PloS one, vol. 11, issue 5, pp. e0154130, 2016. Abstract

AIM: The study was designed to assess the possibility of using circulating miRNAs (serum miRNAs) as diagnostic biomarkers in colorectal cancer (CRC) and to identify their possibility as candidates for targeted therapy.

METHODS: The study involved two sample sets: 1- a training set which included 90 patients with colorectal related disease (30 with CRC, 18 with inflammatory bowel disease (IBD), 18 with colonic polyps (CP) and 24 with different colonic symptoms but without any colonoscopic abnormality who were enrolled as control group) and 2- a validation set which included 100 CRC patients. Serum miRNAs were extracted from all subjects to assess the expression profiles for the following miRNAs (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-146a, miR-223, miR-24, miR-454, miR-183, miR-135a, miR- 135b and miR- 92a) using the custom miScript miRNA PCR-based sybergreen array. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic performance of the studied miRNAs for colorectal cancer diagnosis.

RESULTS: Data analysis of miRNA from the training set showed that; compared to control group, only miR-19b was significantly up-regulated in patients with IBD group (fold change = 5.24, p = 0.016), whereas in patients with colonic polyps, miR-18a was significantly up-regulated (fold change = 3.49, p-value = 0.018). On the other hand, miR-17, miR-19a, miR-20a and miR-223 were significantly up-regulated (fold change = 2.35, 3.07, 2.38 and 10.35; respectively and p-value = 0.02, 0.015, 0.017 and 0.016; respectively in CRC patients. However, the validation set showed that only miR-223 was significantly up-regulated in CRC patients (fold change = 4.06, p-value = 0.04).

CONCLUSION: Aberrant miRNA expressions are highly involved in the cascade of colorectal carcinogenesis. We have found that (miR-17, miR-19a, miR-20a and miR-223) could be used as diagnostic biomarkers for CRC. On the other hand, miR-19b and miR-18a could be used as diagnostic biomarkers for CP and IBD respectively.

Zekri, A. - R. N., A. S. E. - D. Youssef, E. D. El-Desouky, O. S. Ahmed, M. M. Lotfy, A. A. - M. Nassar, and A. A. Bahnassey, "Serum microRNA panels as potential biomarkers for early detection of hepatocellular carcinoma on top of HCV infection.", Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, vol. 37, issue 9, pp. 12273-12286, 2016. Abstract

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.

Youssef, A. S. E. - D., A. R. N. Zekri, M. Mohanad, S. A. Loutfy, N. F. Abdel Fattah, M. H. Elberry, A. A. El Leithy, A. El-Touny, A. S. Rabie, M. Shalaby, et al., "Deleterious and ethnic-related BRCA1/2 mutations in tissue and blood of Egyptian colorectal cancer patients and its correlation with human papillomavirus.", Clinical and experimental medicine, vol. 23, issue 8, pp. 5063-5088, 2023. Abstract

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.

Nassar, A., A. - R. N. Zekri, M. M. Kamel, M. H. Elberry, M. M. Lotfy, M. G. Seadawy, Z. K. Hassan, H. K. Soliman, A. M. Lymona, and A. S. E. - D. Youssef, "Frequency of Pathogenic Germline Mutations in Early and Late Onset Familial Breast Cancer Patients Using Multi-Gene Panel Sequencing: An Egyptian Study.", Genes, vol. 14, issue 1, 2022. Abstract

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: , , , , , , , , , , and . Thirty-one patients (30.6%) had mutations and twenty (19.8%) had mutations. Our results showed that exon 10 and exon 11 harbored 3 and 5 mutations, respectively, in and genes. Our analysis also revealed that the gene significantly co-occurred with each of the gene ( = 0.003, event ratio 11/21), the gene ( = 0.01, 4/10), the gene ( = 0.02, 4/11), and the gene ( = 0.04, 4/12). In addition, the gene significantly co-occurred with the gene ( = 0.01, 3/7). Furthermore, there was a significant mutually exclusive event between the gene and the gene ( = 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 , , and 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.

Nassar, A., A. M. Lymona, M. M. Lotfy, A. S. E. - D. Youssef, M. Mohanad, T. M. Manie, M. M. G. Youssef, I. G. Farahat, and A. - R. N. Zekri, "Tumor Mutation Burden Prediction Model in Egyptian Breast Cancer patients based on Next Generation Sequencing.", Asian Pacific journal of cancer prevention : APJCP, vol. 22, issue 7, pp. 2053-2059, 2021. Abstract

OBJECTIVES: This study aimed to identify the tumor mutation burden (TMB) value in Egyptian breast cancer (BC) patients. Moreover, to find the best TMB prediction model based on the expression of estrogen (ER), progesterone (PR), human epidermal growth factor receptor 2 (HER-2), and proliferation index Ki-67.

METHODS: The Ion AmpliSeq Comprehensive Cancer Panel was used to determine TMB value of 58 Egyptian BC tumor tissues. Different machine learning models were used to select the optimal classification model for prediction of TMB level according to patient's receptor status.

RESULTS: The measured TMB value was between 0 and 8.12/Mb. Positive expression of ER and PR was significantly associated with TMB ≤ 1.25 [(OR =0.35, 95% CI: 0.04-2.98), (OR = 0.17, 95% CI= 0.02-0.44)] respectively. Ki-67 expression positive was significantly associated with TMB >1.25 than those who were Ki-67 expression negative (OR = 9.33, 95% CI= 2.07-42.18). However, no significant differences were observed between HER2 positive and HER2 negative groups. The optimized logistic regression model was TMB = -27.5 -1.82 ER - 0.73 PR + 0.826 HER2 + 2.08 Ki-67.

CONCLUSION: Our findings revealed that TMB value can be predicted based on the expression level of ER, PR, HER-2, and Ki-67.

Nassar, A., M. Abouelhoda, O. Mansour, S. A. Loutfy, M. M. Hafez, M. Gomaa, A. Bahnassy, A. S. E. - D. Youssef, M. M. Lotfy, Hoda Ismail, et al., "Targeted next generation sequencing identifies somatic mutations in a cohort of Egyptian breast cancer patients.", Journal of advanced research, vol. 24, pp. 149-157, 2020. Abstract

Breast cancer (BC) incidence is progressively increasing in Egypt. However, there is insufficient knowledge of the acquired somatic mutations in Egyptian BC patients which limit our understanding of its progression. To the best of our knowledge, this is the first Egyptian cohort to sequence a multiple-gene panel of cancer related genes on BC patients. Four hundred and nine cancer related genes were sequenced in 46 fresh breast tumors of Egyptian BC patients to identify somatic mutations and their frequencies. and were the most top two frequently mutated genes. We detected 15 different somatic mutations in and 8 different ones in , each in 27 samples (58.7%). According to Clinvar database; we found 19 pathogenic somatic mutations: 7 in , 5 in , and single variants of , , , , and We also identified 5 variants with uncertain significance (4 in and 1 in ) and 4 variants with conflicting interpretations of pathogenicity (2 in and 1 in each of and ). Moreover, one drug response variant (p.P72R) in was detected in 8 samples. Furthermore, four novel variants were identified in , , and . Further analysis, by Ingenuity Variant Analysis software (IVA), showed that PI3K/AKT signaling is altered in greater than 50% of Egyptian BC patients which implicates PI3K/AKT signaling as a therapeutic target. In this cohort, we shed the light on the most frequently detected somatic mutations and the most altered pathway in Egyptian BC patients.

Youssef, A. S. E. - D., M. A. Abdel-Fattah, M. M. Lotfy, A. Nassar, M. Abouelhoda, A. O. Touny, Z. K. Hassan, M. M. Eldin, A. A. Bahnassy, H. Khaled, et al., "Multigene Panel Sequencing Reveals Cancer-Specific and Common Somatic Mutations in Colorectal Cancer Patients: An Egyptian Experience.", Current issues in molecular biology, vol. 44, issue 3, pp. 1332-1352, 2022. Abstract

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 , , , and 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 , , , , , and It seems obvious that and genes were the most affected genes with somatic mutations in all groups. Of interest, 85% and 28% of the and deleterious somatic mutations were located in Exon 14 and Exon 3, respectively. Besides, 37% and 28% of the total somatic mutations identified in and 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 and four variants in . 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.

Nassar, A., A. R. N. Zekri, M. H. Elberry, A. M. Lymona, M. M. Lotfy, M. Abouelhoda, and A. S. E. - D. Youssef, "Somatic Mutations Alter Interleukin Signaling Pathways in Grade II Invasive Breast Cancer Patients: An Egyptian Experience.", Current issues in molecular biology, vol. 44, issue 12, pp. 5890-5901, 2022. Abstract

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 , and genes. Interestingly, six mutations which were likely to be novel deleterious were identified: two in the gene, two in the gene, and one in each of the and 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.

Zekri, A. R., A. A. Nassar, M. N. Elrouby, H. I. Shousha, and A. B. Barakat, "Disease progression from chronic hepatitis C to cirrhosis and hepatocellular carcinoma is associated with increasing DNA promoter methylation", Asian Pac J Cancer Prev., vol. 14, issue 11, pp. 6721-6, 2014.