Ola Sayed Ahmed

Disease progression from chronic hepatitis C to cirrhosis and hepatocellular carcinoma is associated with increasing DNA promoter methylation

BACKGROUND Changes in DNA methylation patterns are believed to be early events in hepatocarcinogenesis. A better understanding of methylation states and how they correlate with disease progression will aid in finding potential strategies for early detection of HCC. The aim of our study was to analyze the methylation frequency of tumor suppressor genes, P14, P15, and P73, and a mismatch repair gene (O6MGMT) in HCV related chronic liver disease and HCC to identify candidate epigenetic biomarkers for HCC prediction. MATERIALS AND METHODS 516 Egyptian patients with HCV-related liver disease were recruited from Kasr Alaini multidisciplinary HCC clinic from April 2010 to January 2012. Subjects were divided into 4 different clinically defined groups - HCC group (n=208), liver cirrhosis group (n=108), chronic hepatitis C group (n=100), and control group (n=100) - to analyze the methylation status of the target genes in patient plasma using EpiTect Methyl qPCR Array technology. Methylation was considered to be hypermethylated if >10% and/or intermediately methylated if >60%. RESULTS In our series, a significant difference in the hypermethylation status of all studied genes was noted within the different stages of chronic liver disease and ultimately HCC. Hypermethylation of the P14 gene was detected in 100/208 (48.1%), 52/108 (48.1%), 16/100 (16%) and 8/100 (8%) among HCC, liver cirrhosis, chronic hepatitis and control groups, respectively, with a statistically significant difference between the studied groups (p-value 0.008). We also detected P15 hypermethylation in 92/208 (44.2%), 36/108 (33.3%), 20/100 (20%) and 4/100 (4%) , respectively (p-value 0.006). In addition, hypermethylation of P73 was detected in 136/208 (65.4%), 72/108 (66.7%), 32/100 (32%) and 4/100 (4%) (p-value <0.001). Also, we detected O6MGMT hypermethylation in 84/208 (40.4%), 60/108 (55.3%), 20/100 (20%) and 4/100 (4%), respectively (p value <0.001. CONCLUSIONS The epigenetic changes observed in this study indicate that HCC tumors exhibit specific DNA methylation signatures with potential clinical applications in diagnosis and prognosis. In addition, methylation frequency could be used to monitor whether a patient with chronic hepatitis C is likely to progress to liver cirrhosis or even HCC. We can conclude that methylation processes are not just early events in hepatocarcinogenesis but accumulate with progression to cancer.

Serum microRNA panels as potential biomarkers for early detection of hepatocellular carcinoma on top of HCV infection

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.

Circulating Serum miRNAs as Diagnostic Markers for Colorectal Cancer

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.

Assessment of health-related quality of life in patients receiving stem cell therapy for end-stage liver disease: an Egyptian study

IntroductionThis prospective cohort study aimed to assess the influence of stem cell therapy (SCT) on health-related quality of life (HRQOL) by using the SF-36 v2 and to elucidate the influence of objective clinical variables on subjective HRQOL.MethodsThe study included 100 chronic liver disease patients (50 received SCT, and 50 received supportive medical treatment (SMT)). Both groups completed a modified SF-36 v2 form before therapy and at 1-, 3-, 6-, and 12-month intervals. Fifty healthy Egyptian volunteers were enrolled in the study and completed the SF-36 v2 form once.ResultsBoth SCT and SMT groups showed significantly lower pretherapy SF 36 v2 scores compared with healthy volunteers. In SCT-treated patients, limited complications were encountered (SF-36 v2 scores showed significant improvement in all domains throughout the follow-up period) compared with the deterioration shown by SMT patients after therapy. A significant association was detected between SF-36 v2 scores and laboratory data in SCT patients during the first month after therapy. The grade of ascites improved during the follow-up in SCT compared with SMT patients. The mean survival time was 277.56 days (95% CI, 246.217 to 308.903) for SMT and 359.300 days (95% CI, 353.022 to 365.578) for SCT patients (log rank, 0.00). Stem cell-treated patients showed no malignancies.ConclusionsSCT positively affects health-related quality of life in cirrhosis patients. The survival rate was significantly improved after SCT.

Unique Features of Germline Variation in Five Egyptian Familial Breast Cancer Families Revealed by Exome Sequencing

Genetic predisposition increases the risk of familial breast cancer. Recent studies indicate that genetic predisposition for familial breast cancer can be ethnic-specific. However, current knowledge of genetic predisposition for the disease is predominantly derived from Western populations. Using this existing information as the sole reference to judge the predisposition in non-Western populations is not adequate and can potentially lead to misdiagnosis. Efforts are required to collect genetic predisposition from non-Western populations. The Egyptian population has high genetic variations in reflecting its divergent ethnic origins, and incident rate of familial breast cancer in Egypt is also higher than the rate in many other populations. Using whole exome sequencing, we investigated genetic predisposition in five Egyptian familial breast cancer families. No pathogenic variants in BRCA1, BRCA2 and other classical breast cancer-predisposition genes were present in these five families. Comparison of the genetic variants with those in Caucasian familial breast cancer showed that variants in the Egyptian families were more variable and heterogeneous than the variants in Caucasian families. Multiple damaging variants in genes of different functional categories were identified either in a single family or shared between families. Our study demonstrates that genetic predisposition in Egyptian breast cancer families may differ from those in other disease populations, and supports a comprehensive screening of local disease families to determine the genetic predisposition in Egyptian familial breast cancer.

Early detection of hepatocellular carcinoma co-occurring with hepatitis C virus infection: A mathematical model

AIM To develop a mathematical model for the early detection of hepatocellular carcinoma (HCC) with a panel of serum proteins in combination with α-fetoprotein (AFP). METHODS Serum levels of interleukin (IL)-8, soluble intercellular adhesion molecule-1 (sICAM-1), soluble tumor necrosis factor receptor II (sTNF-RII), proteasome, and β-catenin were measured in 479 subjects categorized into four groups: (1) HCC concurrent with hepatitis C virus (HCV) infection (n = 192); (2) HCV related liver cirrhosis (LC) (n = 96); (3) Chronic hepatitis C (CHC) (n = 96); and (4) Healthy controls (n = 95). The R package and different modules for binary and multi-class classifiers based on generalized linear models were used to model the data. Predictive power was used to evaluate the performance of the model. Receiver operating characteristic curve analysis over pairs of groups was used to identify the best cutoffs differentiating the different groups. RESULTS We revealed mathematical models, based on a binary classifier, made up of a unique panel of serum proteins that improved the individual performance of AFP in discriminating HCC patients from patients with chronic liver disease either with or without cirrhosis. We discriminated the HCC group from the cirrhotic liver group using a mathematical model (-11.3 + 7.38 × Prot + 0.00108 × sICAM + 0.2574 × β-catenin + 0.01597 × AFP) with a cutoff of 0.6552, which achieved 98.8% specificity and 89.1% sensitivity. For the discrimination of the HCC group from the CHC group, we used a mathematical model [-10.40 + 1.416 × proteasome + 0.002024 × IL + 0.004096 × sICAM-1 + (4.251 × 10(-4)) × sTNF + 0.02567 × β-catenin + 0.02442 × AFP] with a cutoff 0.744 and achieved 96.8% specificity and 89.7% sensitivity. Additionally, we derived an algorithm, based on a binary classifier, for resolving the multi-class classification problem by using three successive mathematical model predictions of liver disease status. CONCLUSION Our proposed mathematical model may be a useful method for the early detection of different statuses of liver disease co-occurring with HCV infection.

Abstract 2914: Gold magneto nanoparticles induced apoptosis in liver cancer cells through changes of Ca channel pump detected by LIBS Technique and RT-PCR array

Laser-induced breakdown spectroscopy technique (LIBS) was used in thesis study to address the effect of gold magneto nanoparticles on the liver cancer cells (HepG2) by examining the variation in genotoxicity and cytotoxicity. HepG2 cell line was cultured at 105 cells/ml and expanded, after which, they were incubated with gold magneto nanoparticles at concentrations 10, 25, 50 μg/ml respectively for 12 hours. The effect of these nanoparticles in cell apoptosis as well as in autophagy was evaluated by acridine orange staining and Annexin V/propidium iodide double staining as well as apoptosis specific PCR array assay for evaluation of apoptosis in the treated cells. At a concentration of nanocomposite 10, 25, 50 μg/ml, cells showed increase in the apoptosis figure from 11.25% ± 3.1% to 28% ± 2.04%, 39% ± 3.6%, 60% ± 4.3% respectively. Apoptosis specific PCR array also, showed very high expression of TNF, BIRC3 and NFKB1 with increasing in the fold regulation ratio of 155.03,158.03 and 149.2- fold respectively. On the other hand, extra investigation of these treated cells by LIBS shows an increasing in the iron oxide nanoparticles concentration as well as calcium concentration. This increase in the calcium content in the treated cells may play an important role in the program cell apoptosis which was prove in our study by both Flow cytometry as well as by profile apoptosis PCR array. Finally, our data showed the possibility of the using LIBS as inductor of cells apoptosis by measurement Ca concentration changes in liver cancer cells (HepG2 cells). Keywords: Apaptosis, LIBS; HepG2 cell; Genotoxicity Citation Format: Ola Ahmed, Hisham Imam, Abdel-Rahman N. Zekri. Gold magneto nanoparticles induced apoptosis in liver cancer cells through changes of Ca channel pump detected by LIBS Technique and RT-PCR array. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2914. doi:10.1158/1538-7445.AM2015-2914

Abstract 5353: Characterization of breast cancer stem cells (BCSCs) in Egyptian breast cancer patients through genetic profiling using the RT-PCR and quantitative real time microarray

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Stem cells (SC) are cells that can differentiate along distinct lineages through systemic differentiation steps. The concept of self renewal is crucial to understand cancer stem cells (CSC) and the mechanisms by which current therapies might evade the available treatment modalities. The aim of the present study was to characterize breast cancer stem cells (BCSC) by genetic profiling of properly isolated BCSCs at different stages using the recently developed quantitative real time microarray technology to understand the origin and contribution BCSCs to breast cancer development and progression. Material and Methods: 60 cases of BC were subjected to enzymatic digestion to obtain single cell suspension for: 1) flowcytometric characterization of cells, 2) mammosphere culture 3) magnetic separation of different subsets of BCSCs (CD44 and CD24), and 4) RNA extraction for RT-PCR and microarray using the SABiosciencess QC PCR profiler Arrays with SC panel of 84 genes (SC specific markers, SC differentiation markers, and signaling pathway markers for SC). Samples for the QC PCR profiler Arrays were categorized into groups: 1) normal breast tissue, 2) BC cells obtained from tumor samples, 3) cells from mammosphere cultures, 4)CD44+/CD24low/EpCAM- cells, 5) CD44+/ CD24low cells. ER, PR and Her-2 assessment was also performed. Results: Discrete mammospheres of different sizes were obtained from 10 cases and IHC of these samples confirmed the SC lineage. By FCM, cells expressing CD44+/CD24low ranged from 0.1% - 79%. MYC, MYST2, DHH, BTRC AND CXCL12 genes showed over-expression in the studied groups in comparison to normal breast tissue. Comparing all studied groups, we identified 5 candidate genes DVL1 and EP300 (Notch pathway), BTRC (Wnt Pathway), DHH (Chro-Chro Modulation), MYST2 (self Renewal), NCAM1 (Cell Adhesion Molecules) which could be used for characterization and molecular target therapy for breast cancerl. Conclusions: Our QC PCR profiler Arrays showed significant differences between the studied groups shedding light on the nature and helping in proper characterization of BCSCs. Correlation between prognostic factors of BC and the number of BCSCs in tumor biopsies together with the probability of mammoshpere formation in culture denotes the contribution of CSCS to the progression of BC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5353. doi:1538-7445.AM2012-5353