Tumor Mutation Burden Prediction Model in Egyptian Breast Cancer patients based on Next Generation Sequencing

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

Somatic Mutations Alter Interleukin Signaling Pathways in Grade II Invasive Breast Cancer Patients: An Egyptian Experience

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

Frequency of Pathogenic Germline Mutations in Early and Late Onset Familial Breast Cancer Patients Using Multi-Gene Panel Sequencing: An Egyptian Study

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

Promotor methylation: Does it affect response to therapy in chronic hepatitis C (G4) or fibrosis?

Background and aim. DNA methylation plays a critical role in the control of important cellular processes. The present study assessed the impact of promoter methylation (PM) of some genes on the antiviral response to antiviral therapy and it’s relation to the presence of fibrosis in HCV-4 infected patients from Egypt.Material and methods. Clinical, laboratory and histopathological data of 53 HCV-4 infected patients who were subjected to combined antiviral therapy were collected; patients were classified according to their response to treatment and the fibrosis status. The methylation profiles of the studied groups were determined using the following genes: APC, P14ARF, P73, DAPK, RASSF1A, and O6MGMT in patients’ plasma.Results. O6MGMT and P73 showed the highest methylation frequencies (64.2 and 50.9%) while P14 showed the lowest frequency (34%). Sustained virological response (SVR) was 54.7%with no significant difference in clinico-pathological or laboratory features between the studied groups. PM of O6MGM was significantly higher in non-responders (p value 0.045) while DAPK showed high methylation levels in responders with no significance (p value: 0.09) andPM of RASSF1A was significantly related to mild fibrosis (p value: 0.019). No significant relations were reported between PM of any of the studied genes and patients’ features.Conclusion. PM of some Tumor Suppressor genes increases in chronic active HCV-4. However, only 06MGMT can be used as a predictor of antiviral response and RASSF1A as a marker of marked fibrosis in this small set of patients. An extended study, including more patients is required to validate the results of this preliminary study

Multigene Panel Sequencing Reveals Cancer-Specific and Common Somatic Mutations in Colorectal Cancer Patients: An Egyptian Experience

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

MicroRNA Signatures for circulating CD133-positive cells in hepatocellular carcinoma with HCV infection

<div><p>Aim</p><p>Molecular characterization of the CD133+ stem cells associated with hepatocarinogensis through identifying the expression patterns of specific microRNAs (miRNAs).</p><p>Methods</p><p>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.</p><p>Results</p><p>The differential expression of the 13 studied miRNAs in CD133+ cells separated from the HCC patients' peripheral blood compared to the controls revealed that <i>miR-602</i>, <i>miR-181b</i>, <i>miR-101</i>, <i>miR-122</i>, <i>miR-192</i>, <i>miR-125a-5p</i>, and <i>miR-221</i> were significantly up regulated (fold change = 1.8, 1.7, 2, 5.4, 1.6, 2.9 & 1.5 <i>P</i> 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; <i>miR-602</i>, miR-122, <i>miR-181b</i>, <i>miR-125a-5p</i>, and <i>miR-192</i> were significantly up regulated (fold change = 13, 3.1, 2.8, 1.6 & 1.56, <i>P</i> value = 0.01, 0.001, 0.000004, 0.002 & 0.007 respectively). Upon comparing the HCC group to the LC group; <i>miR-199a-3p</i>, <i>miR-192</i>, <i>miR-122</i>, <i>miR-181b</i>, <i>miR-224</i>, <i>miR-125a-5p</i>, and <i>miR-885-5p</i> were significantly up regulated (fold change = 5, 6.7, 2.3, 3, 2.5, 4.2 & 39.5 <i>P</i> value = 0.001025, 0.000024, 0.000472, 0.000278, 0.000004, 0.000075 & 0.0000001 respectively) whereas <i>miR-22</i> was significantly down regulated (fold change = 0.57 <i>P</i> value = 0.00002). Only, <i>miR-192</i>, <i>miR-122</i>, <i>miR-181b</i> and <i>miR-125a-5p</i> were significant common miRNAs in CD133+ cells of the HCC group compared to the other non-malignant groups.</p><p>Conclusion</p><p>We identified a miRNA panel comprised of four miRNAs (<i>miR-192</i>, <i>miR-122</i>, <i>miR-181b</i> and <i>miR-125a-5p</i>) 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.</p></div

MicroRNA Signatures for circulating CD133-positive cells in hepatocellular carcinoma with HCV infection - Fig 8

<p>A) Histogram showing the differential expression of the 13 miRNAs in CD133+ cells of the control group (BM) versus the control group (PB). B) Histogram showing the differential expression of the 13 studied miRNAs in CD133+cells of the LC group (BM) versus the LC group (PB). C) Histogram showing the differential expression of the 13 miRNAs in CD133+ cells of the LC group (BM) versus the control group (BM). "*" miRNA is significant at 0.05 level while "**" miRNA is significant at 0.01 level.</p

Functional annotation of miRNAs and their target genes in CD133+ cells of HCC.

<p>Functional annotation of miRNAs and their target genes in CD133+ cells of HCC.</p

MicroRNA Signatures for circulating CD133-positive cells in hepatocellular carcinoma with HCV infection - Fig 3

<p>A) The multi group plot provided both a bar chart and line graph representation (with optional error bars) for differential expression of the13 miRNAs in CD133+ cells of the three groups (PB) compared to control group (PB) where group 1 represented CHC group, group 2 represented LC group and group 3 represented HCC group. B) The heat map with dendrograms representing co-regulated miRNAs across all groups.</p

MicroRNA Signatures for circulating CD133-positive cells in hepatocellular carcinoma with HCV infection - Fig 5

<p>A) Histogram showing the differential expression of the 13 miRNAs in CD133+ cells the CHC group (PB) versus the control group (PB). B) Histogram showing the differential expression of the 13 miRNAs in CD133+cells the LC group (PB) versus the control group (PB). C) Histogram showing the differential expression of the 13 miRNAs in CD133+ cells of the HCC group (PB) versus the control group (PB). D) Histogram showing the differential expression of the 13 miRNAs in CD133+ of the HCC group (PB) versus the CHC group (PB). E) Histogram showing the differential expression of the 13 miRNAs in CD133+ of the HCC group (PB) versus the LC group (PB). "*" miRNA is significant at 0.05 level while "**" miRNA is significant at 0.01 level.</p