Association between hepatitis C virus genotype 4 and renal cell carcinoma: Molecular and virological studies.

Hepatitis C virus (HCV) is the most common infection worldwide. The correlation between HCV and renal cell carcinoma (RCC) is still mysterious. Therefore, the relationship between HCV and RCC was investigated. The study included 100 patients with RCC; 32 with HCV infection, and 68 without HCV infection. Expressions of viral proteins (NS3 and NS5A) were tested using an immune electron-microscope (IEM) and immunohistochemistry (IHC). IHC and quantitative real time-PCR investigated the presentation of human proteins TP53 and p21 genes. Transmission electron (TEM) detected viral-like particles in infected RCC tissues. The gene and protein expression of P53 was higher in HCV positive versus HCV negative patients and p21 was lower in HCV positive versus HCV negative in both tumor and normal tissue samples. Viral like particles were observed by TEM in the infected tumor and normal portion of the RCC tissues and the plasma samples. The IEM showed the depositions of NS3 and NS5A in infected renal tissues, while in noninfected samples, were not observed. The study hypothesizes that a correlation between HCV and RCC could exist through successfully detecting HCV-like particles, HCV proteins, and (p53 and p21) in RCC-infected patients.

Stem cell therapy for hepatocellular carcinoma and end-stage liver disease.

Hepatocellular carcinoma (HCC) is a major health problem worldwide, especially for patients who are suffering from end-stage liver disease (ESLD). The ESLD is considered a great challenge for clinicians due to the limited chance for liver transplantation, which is the only curative treatment for those patients. Stem cell-based therapy as a part of regenerative medicine represents a promising application for ESLD patients. Many clinical trials were performed to assess the utility of bone marrow-derived stem cells as a potential therapy for patients with liver diseases. The aim of the present study is to present and review the various types of stem cell-based therapy, including the mesenchymal stem cells (MSCs), BM-derived mononuclear cells (BM-MNCs), CD34 + hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), and cancer stem cells.Though this type of therapy achieved promising results for the treatment of ESLD, however still there is a confounding data regarding its clinical application. A large body of evidence is highly required to evaluate the stem cell-based therapy after long-term follow-up, with respect to the incidence of toxicity, immunogenicity, and tumorigenesis that developed in many patients.

Deleterious and ethnic-related BRCA1/2 mutations in tissue and blood of Egyptian colorectal cancer patients and its correlation with human papillomavirus.

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.

Genetic profiling of different phenotypic subsets of breast cancer stem cells (BCSCs) in breast cancer patients.

BACKGROUND: Breast cancer stem cells (BCSCs) have a crucial role in breast carcinogenesis, development, and progression. The aim of the current study is to characterize the BCSCs through the genetic profiling of different BCSCs phenotypic subsets to determine their related genetic pathways. METHODS: Fresh tumor tissue samples were obtained from 31 breast cancer (BC) patients for (1) Mammosphere culture. (2) Magnetic separation of the BCSCs subsets using CD24, CD44, and CD326 Microbeads. (3) Flow cytometry (FCM) assay using CD44, CD24, and EpCAM. (4) RT-PCR profiler Arrays using stem cell (SC) panel of 84 genes for four group of cells (1) CD44+/CD24-/EpCAM- BCSCs, (2) CD44+/CD24- /EpCAM+ BCSCs, (3) mammospheres, and (4) normal breast tissues. RESULTS: The BCSCs (CD44+/CD24-/EpCAM-) showed significant downregulation in 13 genes and upregulation in 15, where the CD44, GJB1 and GDF3 showed the maximal expression (P = 0.001, P = 0.003 and P = 0.007); respectively). The CD44+/CD24-/EpCAM+ BCSCs showed significant upregulation in 28 genes, where the CD44, GDF3, and GJB1 showed maximal expression (P < 0.001, P = 0.001 and P = 0.003; respectively). The mammospheres showed significant downregulation in 9 genes and a significant upregulation in 35 genes. The maximal overexpression was observed in GJB1 and FGF2 (P = 0.001, P = 0.001; respectively). The genes which achieved significant overexpression in all SC subsets were CD44, COL9A1, FGF1, FGF2, GDF3, GJA1, GJB1, GJB2, HSPA9, and KRT15. While significant downregulation in BMP2, BMP3, EP300, and KAT8. The genes which were differentially expressed by the mammospheres compared to the other BCSC subsets were CCND2, FGF3, CD4, WNT1, KAT2A, NUMB, ACAN, COL2A1, TUBB3, ASCL2, FOXA2, ISL1, DTX1, and DVL1. CONCLUSION: BCSCs have specific molecular profiles that differ according to their phenotypes which could affect patients' prognosis and outcome.

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.

Her2/EGFR-PDGFR pathway aberrations associated with tamoxifen response in metastatic breast cancer patients.

BACKGROUND: Metastatic breast cancer (MBC) is a major health problem worldwide. Some patients improve on tamoxifen and others do not respond to treatment. Therefore, the aim of the current study is to assess genetic aberrations in the Her2/EGFR-PDGFR pathway associated with tamoxifen response in MBC patients. METHODS: This is a retrospective cohort study, including 157 hormone receptors positive, locally recurrent inoperable and/or MBC patients on tamoxifen treatment. Patients were categorized into 78 (49.7%) tamoxifen responders and 79 (50.3%) tamoxifen non-responder patients. Genetic aberrations of 84 genes involved in the Her2/EGFR-PDGFR pathway were assessed in the tumor tissue samples obtained from the patients using SA-Bioscience assay. The identified panel was correlated to patients' response to treatment, to detect the differentially expressed genes in tamoxifen responders and non-responders. RESULTS: One hundred twenty-three (78.3%) patients were estrogen receptor (ER) and progesterone receptor (PR) positive, 108 (68.8%) were ER only positive, and 78 (49.7%) were PR only positive. There were 56 genes overexpressed in the refractory group compared to responders. However, only five out of these 56 genes, Janus kinase 1 (JAK1), collagen type I alpha 1 (COL1A1), GRB2-associated binding protein 1 (GAB1), fibronectin-1 (FN1), and MAP kinase-interacting serine/threonine-protein kinase (MKNK1), showed statistical significance between the two groups. Patients with bone metastasis showed a better response to treatment compared to those with metastatic deposits in other sites such as visceral metastasis (P < 0.005). CONCLUSIONS: Genetic profiling using simple quantitative real-time polymerase chain reaction (qRT-PCR) protocols could be used to assess response to tamoxifen treatment in MBC patients. According to our data, a five-gene panel in the EGFR pathway (JAK1, COL1A1, GAB1, FN1 and MKNK1) could be used to categorize MBC patients into groups according to treatment response.

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/ßcatenin, Angiogenesis, EGFR, TGF-ß 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.

Estrogen Receptor Gene Polymorphism as a Possible Genetic Risk Factor for Treatment Response in ER-Positive Breast Cancer Patients.

Estrogen receptor-α (ESR1) single nucleotide polymorphisms (SNPs) have been related to breast cancer (BC) susceptibility. In this retrospective study we investigated ESR1 SNPs in association with survival and treatment response in BC patients. Seven ESR1 SNPs were genotyped using TaqMan probe assay in 100 formalin-fixed paraffin embedded blocks of Egyptian ER+BC patients. Log-binomial regression was used to assess the association of 5 ESR1 SNPs with relative risk of non-response to adjuvant-hormonal treatment. We compared the performance of five machine learning classification models for prediction of treatment response. Predictive models were developed using rs1801132, rs2228480, and rs9322354 that were significantly associated with increased risk for non-response along with the relevant clinical features. Survival analysis was performed to detect prognostic significance of ESR1 SNPs in ESR+BC patients. rs1801132 (C), rs2228480 (A), and rs9322354 (G) minor alleles significantly increased the risk of non-response to tamoxifen by more than 81, 84, and 117%, respectively, in ER+BC patients on anthracycline/anthracycline-taxanes-based chemotherapy. Multivariate Cox regression survival analysis revealed that rs1801132 (C) and large tumor size were independent predictors for poor survival outcome in ER+BC. The best response predictive model was a combination random forest, K-nearest neighbor, and decision tree having an area under the curve of 0.94 and an accuracy of 90.8%. Our proposed predictive model based on ESR1 rs1801132, rs2228480, and rs9322354 SNPs represents a promising genetic risk stratification for selection patients who could benefit from tamoxifen therapy in such a way that might facilitate personalized medicine required to improve ER+BC patients' outcome.

Identification of Different miRNAs and Their Relevant miRNA Targeted Genes Involved in Sister Chromatid Cohesion and Segregation (SCCS)/chromatin Remodeling Pathway on T1G3 Urothelial Carcinoma (UC) Response to BCG Immunotherapy.

BACKGROUND: Till now, no definite clinical or laboratory marker can predict the recurrence or progression of T1 G3 urothelial carcinoma (UC). Genetic aberrations of the chromatin remodeling genes and sister chromatid cohesion and segregation (SCCS) were identified in UC. Here we investigated the impact of novel miRNAs and their targeted expressed SCCS and chromatin remodeling genes on T1G3 UC response to Bacillus Calmette-Guérin (BCG) therapy. METHODS: One hundred tissue samples were obtained from NMIBC patients. Gene expression and immunohistochemical assay of STAG2, ARID1A, NCOR1and UTX were assessed. MiRNA analysis for their targeting miRNAs (miR-21, miR-31, Let7a and miR-199a) was carried out. Assessed genes were compared between responders and no responders to BCG. Univariate and multivariate analysis of predictors of disease recurrence and progression were performed using cox regression analysis. RESULTS: Thirty-two and 22 patients developed recurrence and progression to MIBC (BCG non-responders). BCG non-responders showed statistically significant higher expression of miR-21 and their targeted STAG2, miR-199a and NCOR1 gene (P < .001), and lower expression of miR-31, Let7a, ARID1A and UTX genes (P < .001). Higher miR-199a (P = .006) and lower miR-31 (P = .01), ARID1A (P = .008) and UTX (P = .03) were independent predictor of higher tumor recurrence. Recurrent disease (P = .003), higher expression of STAG2 (P = .01), NCOR1 (P = .01) and miR-21 (P = .03) genes and lower expression of miR-31 (P = .02), Let7a (P = .04) and ARID1A (P = .04) genes were the independent predictor of disease progression. CONCLUSION: Upregulation of STAG2 and NCOR1 and down regulation of ARID1A and UTX genes and their targeting miRNAs were associated with UC non-response to BCG.

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.

Prognostic influence of microsatellite alterations of muscle-invasive bladder cancer treated with radical cystectomy.

OBJECTIVE: To examine the prognostic effect of microsatellite instability (MSI) and loss of heterozygosity (LOH) on cancer-specific survival (CSS) in patients with muscle-invasive bladder cancer (MIBC). PATIENTS AND METHODS: The liquid nitrogen-preserved specimens of 220 patients between March 2009 and December 2012 were analyzed for the presence of MSI and LOH in 12 loci (ACTBP2, D16S310, D16S476, D18S51, D4S243, D9S162, D9S171, D9S747, FGA, INF-α, MBP, MJD) using polymerase chain reaction. MSI was defined as MSI-stable, MSI-Low, or MSI-High if instability was detected in 0, 1, or 2 or more of the examined markers, respectively. The association between MSI-High and LOH and CSS was analyzed using uni- and multivariate analyses and the degree of agreement between tumor and urine samples were determined. RESULTS: MSI were found in 1030 (39%) and 1148 (43.5%) in tumor and urine specimens, respectively (Kappa = 0.77). On the other hand, LOH was found in 163 (6.2%) of tumor tissues and 44 (1.7%) in urine specimens (Kappa = 0.34). Microsatellite alterations were significantly associated with worse CSS at 1- and 5-year in tumor tissue (95% and 83.7% vs. 65.8% and 3.5%, respectively; P < 0.001) and in urine sample (90% and 64% vs. 46.5% and 9.3%, respectively; P < 0.001). MSI and/or LOH was an independent predictor of CSS (HR: 9.8; 95%CI: 5.1-18.9; P < 0.001). CONCLUSIONS: Microsatellite alterations were potentially an independent predictor of CSS in patients with MIBC. The agreement was good between tumor and urine MSI but weak for LOH.

Prevalence of MMTV-Like env Sequences and Its Association with BRCA1/2 Genes Mutations Among Egyptian Breast Cancer Patients.

BACKGROUND: Mouse mammary tumor virus (MMTV) is thought to have a role in human breast cancer (BC) pathogenesis. BRCA1 and 2 genes mutations are well-established risk factors for BC. The purpose of this study was to evaluate the presence of MMTV in familial and non-familial Egyptian breast cancer patients. We also aimed to establish a correlation between BRCAs genes mutations and MMTV infection in those patients. PATIENTS AND METHODS: The study was included 80 BC patients and 10 healthy women were included as a control group. We used PCR to amplify a 250-bp MMTV-like env sequence. We also used PCR followed by direct sequencing to identify the genetic variation of exons 2, 13, 19 of BRCA1 gene and exon 9 and region f of exon 11 of BRCA2 gene. High resolution melting (HRM) analysis was used to screen the selected exons of BRCA1/2 genes in order to detect different variants. RESULTS: MMTV DNA-like env sequences were detected in 70%, 76% of familial and non-familial BC patients, respectively, and it was not detected in any of the control subjects. The presence of viral sequences was associated with larger tumor size in the sporadic patients. Seventy BC patients showed variations in BRCA1/2 genes according to HRM analysis and sequencing analysis showed two different sequences of polymorphism among 22 familial and non-familial BC patients. CONCLUSION: MMTV DNA was present among BC patients and it was associated with increased tumor growth. This indicates a potential role for MMTV in BC patients with and without deleterious mutation in BRCA1/2 genes.

Assessment of the Circulating Tumor Cells and Microsatellite Instability in Colorectal Cancer Patients: Prognostic and Diagnostic Value.

BACKGROUND: Microsatellite instability (MSI) and circulating tumor cells (CTCs) play important roles in the diagnosis, prognosis and management of colorectal cancer (CRC) patients. METHODS: CTCs and MSI were assessed in the blood and representative tumor tissues of 100 CRC patients by flow cytometry (FCM) and PCR amplification. The data were correlated to relevant clinicopathological features of the patients, progression-free survival (PFS) and overall survival (OS) rates. RESULTS: MSI-high was detected in 44 (44.0%) patients, MSI-low in 37 (37%), and microsatellite stable (MSS) in 19 (19.0%) patients (P=0.007). The baseline CTCs count (<4 cells/7mL blood) was reported in 39% of the patients, and CTCs ≥4 cells/7mL blood in 61% of the patients (P=0.028). Improved PFS and OS rates were associated significantly with MSI-high (P<0.001), decreased CTC levels during the course of treatment (P<0.001) and post-treatment CTCs (P=0.008). There was no significant association between MSI-high and PFS or OS in early-stage patients (P=0.187 and P=0.187; respectively); however, it was associated significantly with better PFS and OS in late-stage patients (P<0.001). Multivariate analysis showed that only a change in serial CTC levels is considered an independent prognostic factor for OS (P<0.012). Post-treatment CTCs level, serial CTCs level changes during the course of treatment, lymph nodes and distant metastasis were independent prognostic factors for PFS (P<0.001, P= 0.047, P=0.001 and P<0.001; respectively). CONCLUSION: MSI and CTCs could be used as accurate, reliable and sensitive diagnostic and prognostic biomarkers for CRC patients' survival rates and outcomes.

Genomic characterization of SARS-CoV-2 in Egypt.

Introduction: The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the globe, causing a pandemic. In Egypt over 115,000 individuals were infected so far. Objective: In the present study, the objective is to perform a complete genome sequence of SAR-CoV2 isolated from Egyptian coronavirus disease (COVID-19) patients. Methods: Nasopharyngeal swabs were collected from 61 COVID-19 patients who attended at National Cancer Institute, Kasr Al-Aini Hospital and the army hospital. Viral RNA was extracted and whole genomic sequencing was conducted using Next Generation Sequencing. Results: In all cases, the sequenced virus has at least 99% identity to the reference Wuhan 1. The sequence analysis showed 204 distinct genome variations including 114 missense mutations, 72 synonymous mutations, 1 disruptive in-frame deletion, 7 downstream gene mutations, 6 upstream gene mutations, 3 frame-shift deletions, and 1 in-frame deletion. The most dominant clades were G/GH/GR/O and the dominant type is B. Conclusion: The whole genomic sequence of SARS-CoV2 showed 204 variations in the genomes of the Egyptian isolates, where the Asp614Gly (D614G) substitution is the most common among the samples (60/61). So far, there were no strikingly variations specific to the Egyptian population, at least for this set of samples.

BRCA1 and BRCA2 truncating mutations and variants of unknown significance in Egyptian female breast cancer patients.

BACKGROUND: Breast cancer is the most common malignancy among women worldwide and the leading cause of cancer death in economically developing countries. We sought to study the contribution of BRCA1/2 mutations to the burden of breast cancer in Egypt. PATIENTS AND METHODS: 103 Egyptian female breast cancer patients, unselected for age of onset or family history, were included in the study. Mutational screening of some exons of BRCA1/2 genes was performed using High Resolution Melting analysis followed by direct sequencing of detected variants. RESULTS: Twenty sequence variants were identified. According to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, 8 variants were classified as pathogenic (Class 5), 1 as likely pathogenic and 11 as variants of unknown significance (Class 3). The pathogenic variants comprised 5 novel frameshift mutations; BRCA1 c.5205delA and BRCA2 (c.3641delT, c.3291dupT, c.3292delA, and c.787dupA) mutations; 1 novel nonsense mutation (BRCA2 c.3280A>T) and 2 previously described missense mutations (BRCA1 c.117T>G, c.110C>A). CONCLUSION: This study provides the results of our attempt to delineate the genetic aspect of breast cancer among the Egyptian population and emphasizes the necessity of implementing screening strategies for early diagnosis and counseling for breast cancer in Egypt.

The role of circulating tumor cells and K-ras mutations in patients with locally advanced rectal cancer: a prospective study.

Rectal cancer is a common malignancy with a relatively poor prognosis. We assessed the possible prognostic and predictive role(s) of circulating tumor cells (CTCs) and K-ras mutations in locally advanced rectal carcinoma (LARC) patients. CTCs number and K-ras mutation status were assessed in the Peripheral blood and tumor tissue samples of 60 patients with LARC compared to control group (normal rectal mucosa). Data were correlated to relevant clinico-pathological features, response to treatment, disease free (DFS) and overall survival (OS) rates. K-ras mutations were present in 24/60 (40%) patients. Baseline CTCs (< 5 cells/7 ml blood) were detected in 23/60 (38.3%) patients, and 37 (61.7%) had baseline CTCs (≥ 5 cells/7 ml) blood (P = 0.071). Serial sampling showed a decrease in CTCs levels in 40 (66.7%) patients and increase in 20 (33.3%) patients (P = 0.01). Patients with K-ras mutations had a significantly poor response to treatment, with reduced DFS and OS rates (P = 0.001, 0.004, and 0.001; respectively). Similarly, decreased CTCs levels during treatment associated significantly with better pathological responses (P = 0.003). Multivariate analysis demonstrated that K-ras mutation and baseline CTCs are independent prognostic factors for DFS (P = 0.014 and 0.045; respectively) and OS (P = 0.002 and 0.045; respectively). The presence of mutant K-ras and baseline CTCs ≥ 5 cells associated significantly with poor pathological response, shorter DFS and OS rates compared to those with either K-ras mutation or CTCs ≥ 5 cells only (P = 0.014, 0.005 and 0.001, respectively). K-ras mutations, baseline and serial CTCs changes represent good prognostic and predictive factors for LARC patients.

Targeted next generation sequencing identifies somatic mutations in a cohort of Egyptian breast cancer patients.

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. TP53 and PIK3CA were the most top two frequently mutated genes. We detected 15 different somatic mutations in TP53 and 8 different ones in PIK3CA, each in 27 samples (58.7%). According to Clinvar database; we found 19 pathogenic somatic mutations: 7 in Tp53, 5 in PIK3CA, and single variants of VHL, STK11, AKT1, KRAS, IDH2, PTEN and ERBB2. We also identified 5 variants with uncertain significance (4 in TP53 and 1 in CEBPA) and 4 variants with conflicting interpretations of pathogenicity (2 in TP53 and 1 in each of APC and JAK3). Moreover, one drug response variant (p.P72R) in TP53 was detected in 8 samples. Furthermore, four novel variants were identified in JAK2, MTOR, KIT and EPHB. 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.

The Role of Breast Cancer Stem Cells and Some Related Molecular Biomarkers in Metastatic and Nonmetastatic Breast Cancer.

BACKGROUND: Breast cancer stem cells (BCSCs) play important role(s) in the development and progression of invasive duct carcinoma (IDC). We assessed the role of BCSC marker expression and the number of mammospheres in cultures of breast cancer (BC) tissues and correlated these data to relevant clinicopathologic features of the patients and overall survival (OS). METHODS: Fresh tumor tissue samples were collected from 44 Egyptian female patients with IDC of the breast and 25 healthy women undergoing reduction mammoplasty as a control. The mammosphere number and the RNA expression levels of some cancer stem cell-related genes (PTEN, PI3K, AKT, Wnt, and ß-catenin) were assessed by reverse-transcriptase polymerase chain reaction at different stages of BCSC differentiation compared with control samples. RESULTS: The number of CD44+CD24-/low cells associated significantly at the end of culture with the expression level of Wnt, ß-catenin, and distant metastasis (P < .001, P = .015 and P = .003, respectively). There was significant association between the mammosphere number and CD44+CD24-/low cells as well as AKT expression (P = .040 and .021, respectively). PTEN messenger RNA expressed significantly in BC (P < .05). Wnt-RNA expression associated significantly with high tumor stage, positive lymph node status, Her2-neu overexpression, and metastasis (P = .009, .012, .026, and .001, respectively), whereas OS associated significantly with distant metastasis, Wnt, and PTEN expressions (P < .001, P = .001, P = .014, respectively). CONCLUSION: BCSCs and their related genes (PTEN, PI3K, AKT, Wnt, and ß-catenin) play important roles in the development and progression of BC and they can be used as potential prognostic and predictive biomarkers for patients with BC or as target therapy.

The role of CD68+ macrophage in classical Hodgkin lymphoma patients from Egypt.

BACKGROUND: CD68+ tumor-associated macrophages (TAM) play an important role in the progression of classical Hodgkin lymphoma (cHL). We assessed the role of CD20 and CD68 + TAM in a cohort of cHL patients from Egypt and correlated the number of CD68 + cells with patients' characteristics, response to treatment, overall and progression free survival rates (OS & PFS). METHODS: CD20 expression and CD68 + TAM numbers were assessed in representative tumor tissues obtained from 81 cHL patients using flowcytometry (FCM), immunohistochemistry (IHC), and Rt-PCR techniques. RESULTS: The expression levels of CD68 protein by IHC was high in 27 (33.3%), moderate in 15 (18.5%), low in 15 (18.5%), and negative in 24 (29.6%) patients (p = 0.13). CD68-mRNA expression was high in 43/81(53.1%), and low in 38(46.9%) patients (p = 0.6). The number of CD68 + TAM (by FCM) was low (< 20 cells) in 42/81 (51.9%), and high (≥20 cells) in 39/81 (48.1%) patients (p = 0.74). CD68 expression (by FCM, IHC& Rt-PCR) associated significantly with poor response to treatment, decreased CD20 expression, reduced OS and PFS rates (p < 0.001 for all). CD68 expression (by Rt-PCR only) associated significantly with advanced disease stage (p = 0.04). The age of the patients, high CD20 expression & high CD68+ macrophage number were independent prognostic factors for OS (p= 0.02, p = 0.008 & p = 0.009; respectively). However, the age of the patient, high CD20, and high CD68+ macrophage expression (by FCM&IHC) were independent prognostic factors for DFS (p. = 0.004, p. = 0.01, p. = 0.007 and p. = 0.01; respectively). CONCLUSION: CD68 + TAM expression (by Rt-PCR, FCM and/or IHC) can identify patients with poor response to treatment and reduced survival rates (OS& PFS). Assessment of CD68 + positive macrophages by FCM is superior to other methods (Rt-PCR and IHC) as a prognostic factor for DFS and OS rates.

The Prognostic Significance of 5-Fluorouracil Induced Inflammation and Immuno-Modulation in Colorectal Cancer Patients.

AIM: The change in the levels of peripheral inflammatory markers together with EGFR in relation to 5- fluorouracil (5-FU) therapy was evaluated for their prognostic significance in colorectal cancer (CRC) patients. PATIENTS AND METHODS: Expression levels of COX2, IL6, IL1ß, EGFR, IL10, and TNFα were determined with quantitative real-time PCR (qPCR) in the peripheral blood of 90 CRC patients. The inflammatory response was correlated with patients' clinical features, disease-free survival (DFS), and overall survival (OS). RESULTS: After 6 months of 5-FU therapy, increased inflammatory response was found to be associated with smoking, T3 or T4 tumors, performance status (PS) III, positive lymph nodes, distant metastasis, and gastrointestinal (GIT) toxicity. The combination of COX2 with interleukins in a predictive equation for DFS was significant in patients with over-expression of EGFR. DFS and OS rates were reduced in patients with increased COX2, IL6, IL10, and TNFα expression with 5-FU therapy. Significant hazard of disease progression was associated with smoking (HR=1.27, P=0.004), 5-FU induction of COX2, and IL6 expression (HR=1.35, P=0.001 and HR=1.27, P=0.004, respectively). Moreover, smoking, 5-FU induction of IL6, TNFα, and IL10 expression are found to be independent prognostic factors for OS (P=0.003, 0.003, 0.002, and 0.002, respectively). CONCLUSION: The peripheral effects of 5-FU therapy have shown a significant impact on the treatment outcome of CRC patients.