Neuroprotective effects of vitamin D in an Alzheimer's disease rat model: Improvement of mitochondrial dysfunction via calcium/calmodulin-dependent protein kinase kinase 2 activation of Sirtuin1 phosphorylation.

Mitochondrial dysfunction is an early event in Alzheimer's disease (AD) pathogenesis. To assess the impact of vitamin D3 (Vit.D) on neurogenesis, we investigated its role in mitigating cognitive impairment and mitochondrial dysfunction through calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2)-mediated phosphorylation of Sirtuin1 (SIRT1) in an aluminum-chloride-D-galactose (AlCl3-D-gal)-induced AD rat model. Rats were distributed into four groups: control, AlCl3 + D-gal (10 + 60 mg/kg, ip), Vit.D (500 IU/kg, po), and AlCl3 + D-gal+Vit.D. Novel object recognition (NOR), Morris Water Maze, and passive avoidance (PA) tests were used to measure memory abilities. The hippocampal tissue was used to assess vitamin D3 receptor (VDR) and peroxisome-proliferator-activated-receptor-γ-coactivator-1α (PGC-1α) expression by quantitative real-time polymerase chain reaction (qRT-PCR), CAMKK2, p-SIRT1, phosphorylated-AMP-activated protein kinase (p-AMPK), dynamin-related-protein-1 (Drp1), and mitofusin-1 (Mnf1) proteins by western blot and Ca2+ levels, endothelial nitic oxide synthase (eNOS), superoxide dismutase (SOD), amyloid beta (Aß), and phospho tau (p-Tau) via enzyme-linked immunosorbent assay(ELISA) in addition to histological and ultrastructural examination of rat's brain tissue. Vit.D-attenuated hippocampal injury reversed the cognitive decline and Aß aggregation, and elevated p-Tau levels in the AlCl3 + D-gal-induced AD rat model. In AlCl3 + D-gal-exposed rats, Vit.D induced VDR expression, normalized Ca2+ levels, elevated CAMKK2, p-AMPK, p-SIRT1, and PGC-1α expression. Vit.D reduced Drp1, induced Mnf1, increased mitochondrial membrane potential, preserved mitochondrial structure, restored normal mitochondrial function, and retained normal eNOS level and SOD activity in AlCl3 + D-gal rats. In conclusion, our findings proved that Vit.D may ameliorate cognitive deficits in AlCl3 + D-gal-induced AD by restoring normal mitochondrial function and reducing inflammatory and oxidative stress via CAMKK2-AMPK/SIRT1 pathway upregulation.

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.

Long non-coding RNAs as novel prognostic biomarkers for breast cancer in Egyptian women.

Breast cancer (BC), the most common type of malignant tumor, is the leading cause of death, having the highest incidence rate among women. The lack of early diagnostic tools is one of the clinical obstacles for BC treatment. The current study was designed to evaluate a panel of long non-coding RNAs (lncRNAs) BC040587, HOTAIR, MALAT1, CCAT1, CCAT2, PVT1, UCA1, SPRY4-IT1, PANDAR, and AK058003-and two mRNAs (SNCG, BDNF) as novel prognostic biomarkers for BC. This study was ethically approved by the Institutional Review Board of the National Cancer Institute, Cairo University. Our study included 75 women recently diagnosed with BC and 25 healthy women as normal controls. Patients were divided into three groups: 24 with benign breast diseases, 28 with metastatic breast cancer (MBC, stage IV), and 23 with non-metastatic breast cancer (NMBC, stage III). LncRNA and mRNA expression levels were measured in patient plasma using quantitative real-time PCR. We found that 10 lncRNAs (BCO40587, HOTAIR, PVT1, CCAT2, PANDAR, CCAT1, UCA1, SPRY4-IT1, AK058003, and MALAT1) and both mRNAs demonstrated at least a 2-fold change in expression with a more than 95% probability of significance. BCO40587 and SNCG were significantly up-regulated in MBC and NMBC patients (3.2- and 4-fold, respectively) compared with normal controls. The expression of UCA1 was repressed by 1.78-fold in MBC and NMBC patients compared with those with benign diseases. SPRY4-IT1 was down-regulated by 1.45-fold in MBC patients compared with NMBC and benign disease patients. Up-regulation of lncRNAs plays an important role in BC development. SNCG and BCO40587 may be potential prognostic markers for BC.The organization number is IORG0003381 (IRB No: IRB00004025).

Epigenetic inactivation of DNA repair genes as promising prognostic and predictive biomarkers in urothelial bladder carcinoma patients.

We sought to examine epigenetic inactivation of DNA damage repair (DDR) genes as prognostic and predictive biomarkers for urothelial bladder cancer (UBC) as there are currently no reliable prognostic biomarkers that identify UBC patients who would benefit from chemotherapy. Genome-wide DNA methylome using the cancer genome atlas-bladder cancer (TCGA-BLCA) datasets (primary tumors = 374 and normal tissues = 37) was performed for 154 DDR genes. The most two significant differentially methylated genes, Retinoblastoma binding protein 8 (RBBP8) and MutS homologue 4 (MSH4), between primary tumors and normal tissues of TCGA-BLCA were validated by methylation-specific PCR (MSP) in UBC (n = 70) compared to normal tissues (n = 30). RBBP8 and MSH4 expression was measured using qRT-PCR. We developed a predictive model for therapeutic response based on the RBBP8- and MSH4-methylation along with patients' clinical features. Then, we assessed the prognostic significance of RBBP8 and MSH4. RBBP8- and MSH4 methylation and corresponding gene downregulation significantly associated with muscle-invasive phenotype, prolonged progression-free survival (PFS) and increased susceptibility to cisplatin chemotherapy in UBC. Promoter methylation of RBBP8 and MSH4 was positively correlated with each other and with their corresponding gene repression. The best machine-learning classification model predicted UBC patients' response to cisplatin-based chemotherapy with an accuracy of 90.05 ± 4.5%. Epigenetic inactivation of RBBP8 and MSH4 in UBC could sensitize patients to DNA-damaging agents. A predictive machine-learning modeling approach based on the clinical features along with RBBP8- and MSH4-methylation might be a promising tool for stratification of UBC responders from nonresponders to chemotherapy.

Impact of HOXB4 and PRDM16 Gene Expressions on Prognosis and Treatment Response in Acute Myeloid Leukemia Patients.

Introduction: Acute myeloid leukemia (AML) is the most common type of leukemia among adults and is characterized by various genetic abnormalities. HOXB4 and PRDM16 are promising markers of AML. Our objective is to assess the potential roles of HOXB4 and PRDM16 as prognostic and predictive markers in newly diagnosed AML patients and determine the correlation between their expressions and other prognostic markers as FLT3-ITD, NPM1 exon 12 mutations, response to treatment, and patient's survival. Methods: This study included 83 de novo AML adult patients. All patients were subjected to clinical, morphological, cytochemical, and molecular analysis to detect HOXB4 and PRDM16 gene expressions and FLT3-ITD, NPM1 exon 12 mutations. Results: The results showed that a low expression of HOXB4 was found in 31.3% of AML patients, whereas a high expression of PRDM16 was evident in 33.8% of AML patients. FLT3-ITD mutations were detected in 6 patients (7.2%), while NPM1 exon 12 mutations were detected in 7 patients (19.4%) out of 36 patients with intermediate genetic risk. Out of the 50 patients who achieved complete remission (CR), relapse occurred in 16% of the cases. Low expression of HOXB4 and high expression of PRDM16 were associated with CR of 32% and 28%, respectively, and a short overall survival (OS) and disease-free survival (DFS). Conclusion: Further larger study should be conducted to verify that high PRDM16 and low HOXB4 gene expressions could be used as a poor prognostic predictor for AML. The correlation between PRDM16 and HOXB4 gene expressions and FLT3-ITD and NPM1 exon 12 mutations might have a role on CR, relapse, OS, and, however, this should be clarified in analysis with a larger number of samples.

The prognostic role of PD-1, PD-L1, ALK, and ROS1 proteins expression in non-small cell lung carcinoma patients from Egypt.

BACKGROUND: Programmed death ligand-1 (PD-L1), anaplastic lymphoma kinase (ALK), and c-ros oncogene1 (ROS1) expression may influence the prognosis of non-small cell lung carcinoma (NSCLC). We aimed to investigate the prognostic and predictive significance of PD-1/PD-L1 along with c-ros ROS1 and ALK in NSCLC patients. METHODS: Immunohistochemistry used to identify ALK, ROS1, PD-1, and PD-L1 proteins expression as well as ROS1 rearrangement via fluorescence in situ hybridization, in 70 NSCLC patients. Results were related to clinicopathological feature, survival, and treatment response. RESULTS: Expression of ROS1, ALK, PD-1, and PD-L1 and ROS1-rearrangement were detected in 18.57%, 54.29%, 84.29%, 87.14%, and 15.71% of the cases, respectively. No association was found between ROS1, PD-1, and PD-L1 and any clinicopathological features, survival, or treatment outcome. ALK expression significantly associated with stage-IV and left-sided tumors. Epidermal growth factor receptor (EGFR) mutation and ALK-positive patients had significantly reduced progression-free survival than patients with wild type EGFR [HR: 1.99, 95% CI: 1.37-2.93, p < 0.001] and negative-ALK expression [HR: 1.46, 95% CI: 1.03-2.07, p = 0.03]. In multivariate analysis, lymph node metastasis, EGFR-mutations, and ALK were independent predictors of NSCLC. PD-L1 expression was significantly correlated with PD-1 but not with ROS1, ALK, or EGFR-mutation. CONCLUSION: Positive ALK expression and EGFR-mutations are independent adverse predictors of NSCLC. Overexpression of PD-1/PD-L1 is not a significant prognostic marker in NSCLC patients receiving chemotherapy, making them susceptible to immunotherapy. Since PD-1/PD-L1 expression is independent to oncogenic driver mutations, future studies into specific immune checkpoint inhibitors combined with targeted therapies for individualized treatment of NSCLC is warranted. Positive ALK expression and EGFR mutations are independent risk factors for NSCLC. Overexpression of PD-1/PD-L1 is not a significant prognostic factor in patients with NSCLC who are receiving chemotherapy, making them immunotherapy susceptible. Given that PD-1/PD-L1 expression is not dependent on oncogenic driver mutations, additional research into specific immune checkpoint inhibitors in combination with targeted therapies for the treatment of NSCLC on an individual basis is warranted.

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.

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.

Genome sequencing of SARS-CoV-2 in a cohort of Egyptian patients revealed mutation hotspots that are related to clinical outcomes.

BACKGROUND: Severe acute respiratory syndrome-2 (SARS-CoV-2) exhibits a broad spectrum of clinical manifestations. Despite the fact that SARS-CoV-2 has slower evolutionary rate than other coronaviruses, different mutational hotspots have been identified along the SARS-CoV-2 genome. METHODS: We performed whole-genome high throughput sequencing on isolates from 50 Egyptian patients to see if the variation in clinical symptoms was related to mutations in the SARS-CoV-2 genome. Then, we investigated the relationship between the observed mutations and the clinical characteristics of the patients. RESULTS: Among the 36 most common mutations, we found two frameshift deletions linked to an increased risk of shortness of breath, a V6 deletion in the spike glycoprotein's signal peptide region linked to an increased risk of fever, longer fever duration and nasal congestion, and L3606-nsp6 deletion linked to a higher prevalence of cough and conjunctival congestion. S5398L nsp13-helicase was linked to an increased risk of fever duration and progression. The most common mutations (241, 3037, 14,408, and 23,403) were not linked to clinical variability. However, the E3909G-nsp7 variant was more common in children (2-13 years old) and was associated with a shorter duration of symptoms. The duration of fever was significantly reduced with E1363D-nsp3 and E3073A-nsp4. CONCLUSIONS: The most common mutations, D614G/spike-glycoprotein and P4715L/RNA-dependent-RNA-polymerase, were linked to transmissibility regardless of symptom variability. E3909G-nsp7 could explain why children recover so quickly. Nsp6-L3606fs, spike-glycoprotein-V6fs, and nsp13-S5398L variants may be linked to clinical symptom worsening. These variations related to host-virus interactions might open new therapeutic avenues for symptom relief and disease containment.

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.

Mechanistic insights into the protective effects of chlorogenic acid against indomethacin-induced gastric ulcer in rats: Modulation of the cross talk between autophagy and apoptosis signaling.

BACKGROUND: This study aimed to investigate the gastroprotective effect of chlorogenic acid (CGA) against Indomethacin (IND)-induced gastric ulcer (GU) in rats and its underlying mechanism, especially through autophagic and apoptotic pathways. METHODS: Seventy-five rats were divided into five groups; control, IND (50 mg/kg, p.o.), CGA (100 mg/kg, p.o., 14 days), IND pretreated with CGA (50 mg/kg or 100 mg/kg, p.o., 14 days). The stomach tissues were examined to calculate the ulcer index and analyze markers of autophagy (beclin-1, LC3-II/LC3-I and p62), lysosomal function (cathepsin-D) and apoptosis (Bcl-2, Bax and caspase-3), along with expression of Akt/mTOR pathway using western blot or ELISA techniques. In addition, viability of gastric mucosal cells was detected by flowcytometry. Structural changes were assessed histologically, while autophagic and apoptotic changes of gastric mucosa were observed by transmission electron microscopy. RESULTS: CGA exhibited a dose-dependent gastroprotective effect by reversing IND-induced accumulation of autophagic vacuoles, significant reduction in beclin-1, LC3-II/LC3-I, and p62 levels, and down-regulation of p-Akt/p-mTOR expression. CGA100 also restored normal autolysosomal function by modulation of cathepsin-D levels. Furthermore, pretreatment with CGA100 was significantly associated with an increase in antiapoptotic protein Bcl-2 along with a decrease in proapoptotic Bax and caspase-3 proteins in such a way that impairs IND-induced apoptosis. This was confirmed by CGA-induced significant decrease in annexin V+ cells. CONCLUSIONS: The natural compound CGA offers a novel gastroprotective intervention against IND-induced GU through restoration of normal autophagic flux, impairment of apoptosis in a crosstalk mechanism mediated by Akt/mTOR pathway reactivation, and alleviation of IND-induced lysosomal dysfunction.

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.

The possible role of Dickkopf-1, Golgi protein- 73 and Midkine as predictors of hepatocarcinogenesis: a review and an Egyptian study.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death worldwide. The use of alpha fetoprotein (AFP) alone was not an accurate biomarker for HCC despite its high specificity. Therefore, we assessed the possible role of serum biomarkers that have been mentioned briefly in previous studies on Egyptian patients ion top of HCV. However these studies included small number of patients and did not assess the different stages of hepatocarcinogenesis. In the current study we assessed 1) the expression levels of Golgi protein 37(GP73),Midkine (MDK) and Dickkopf-1(DKK-1) proteins separately and in combination at different stages of hepatocarcinogenesis. GP73, MDK and DKK-1 proteins were assessed in 238 individuals divided into 4 groups (HCC, chronic HCV, and chronic HCV with cirrhosis and healthy subjects as a control) Serum levels of GP73, MDK, and DKK-1 were assessed in all subjects by ELISA. Serum levels of the studied markers were significantly higher in HCC compared to other groups (p < 0.001). The ROC curve analysis for the studied markers showed 1) 88.5% sensitivity, 80.6% specificity, 69% PPV, 93.5% NPV and (AUC 0.91)for MDK; 2) 93.6%, 86.9%, 77.7%, 96.5% for DKK-1. 3) 91%, 85%, 74.7%, 95% (AUC 0.96) for GP73 and 4) 74.4%, 84.4%, 69.9%, 87.1% (AUC 0.81) for AFP. Serum levels of GP73, MDK, and DKK-1 are comparable to AFP as promising predictor biomarkers for HCC patients from Egypt. A two markers panel including Gp73 and DKK-1 showed the highest specificity and sensitivity among different markers combinations.

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 impact of hypermethylation for a panel of tumor suppressor genes and cell of origin subtype on diffuse large B-cell lymphoma.

Diffuse Large B-cell lymphoma (DLBCL) is an aggressive disease with heterogeneous outcome and marked variable response to chemotherapy. We assessed promoter hypermethylation (PM) for a panel of tumor suppressor genes in 75 DLBCLs compared to 20 lymphoid hyperplasia (LH) and 30 normal control, using methylation specific PCR. Results were correlated to patients' clinic-pathological characteristics, immunophenotyping, and patients' outcome. DAPK1, RUNX3, MT1G, MGMT, CDH1 and p16 PM were detected in 38.7% (29/75), 49.3% (37/75), 46.7% (35/75), 44% (33/75), 49.3% (37/75) and 42.7% (32/75);respectively, of DLBCL patients compared to LH group (P < 0.05). Aberrant PM of RUNX3, MGMT, CDH1 and p16 was significantly higher in non-germinal central B-cell like (non-GCB) compared to GCB (58.3% vs. 33.3%, 56.2% vs. 22.2, 62.5% vs. 25.9, and 56.2% vs. 18.5%, respectively). PM of studies genes in DLBCL associated significantly with worse survival outcome and resistance to chemotherapy (P ≤ 0.01). In non-GCB group, DAPK1, MT1G, RUNX3, CDH1 and p16 PM associated significantly with reduced DFS (P ≤ 0.004) and OS (P ≤ 0.015). Multivariate analysis indicated that RUNX3 and CDH1 PM were independent prognostic factors for OS (P = 0.03 and 0.04; respectively), while DAPK1, RUNX3 and MT1G PM were independent prognostic factors for DFS (P = 0.002, 0.037& 0.007; respectively). DAPK1, RUNX3, MT1G, CDH1 and p16 PM are promising prognostic and/or predictive markers for non-GCB independent of IPI. Upregulation of those genes using new demethylating agents is a promising approach that sensitize chemoresistant DLBCL patients, especially the non-GCB subtype.

Microsatellite instability profiling in Egyptian bladder cancer patients: A pilot study.

Microsatellite alterations have been implicated in the pathogenesis of many cancers; however, they are still not well addressed in the bladder cancer (BC) of Egyptian population. We assessed microsatellite instability (MSI) profile and loss of heterozygosity (LOH) using 13 microsatellite markers in tumor tissue samples and urine sediments obtained from 30 Egyptian patients with BC. The concordance between MSI in tumor tissue and urine samples was determined, and correlated to relevant clinicopathologic features. We found that MSI was more frequent than LOH (100% and 46.7%, respectively). D16S310, MBP, and IFN-α showed the highest MSI frequency in urine samples (70%, 70%, and 66.67%, respectively), while MBP, ACTBP2, and D9S171 (66.67%, 63.33%, and 60%, respectively) were the most frequently detected in tumor tissues. All assessed MSI markers correlated significantly with pathologic subtype (being more frequent in TCC) and with hematuria. The concordance between tissue and urine samples was statistically significant for D16S476, D9S171, FGA, and ACTBP2 (P = 0.04, 0.015, 0.02, and 0.007, respectively). When we combined D16S476 and D9S171, the sensitivity, specificity, positive predictive value, and negative predictive value for the diagnosis of BC were 80.0%, 75.0%, 82.8%, and 71.4%, respectively. Accordingly, we concluded that MSI in urine sediments could be a potential tool for the diagnosis of BC.

Prognostic significance of circulating tumor cells (CTCs) in Egyptian non-metastatic colorectal cancer patients: A comparative study for four different techniques of detection (Flowcytometry, CellSearch, Quantitative Real-time PCR and Cytomorphology).

PURPOSE: We assessed CTCs counts in NMCRC patients using four different techniques. METHODS: CTCs were detected in 63 NMCRC patients, 40 benign bowel diseases (BBD) and 40 normal controls (NC) using, flow-cytometry (FCM), CellSearch (CS), cytomorphology and quantitative real time (qPCR) for CK19, MUC1, CD44, CD133, ALDH1 expression. Results were correlated to progression free (PFS) and overall (OS). RESULTS: Positive CTCs (≥4 cells /7.5 mL blood) were detected in 50.8% (32/63) NMCRC by FCM and 7.5% (3/40) BBD (p < .001). CTCs were detected in 34/63 (54%) NMCRC, 4/40 (10%) BBD (p < .001) by CS. CK19, MUC1, CD44, CD133 and ALDH1 were expressed in 35 (55.6%), 29 (46.0%), 28 (44.4%), 26 (41.3%) and 25 (41.3%) cases of NMCRC. In BBD 4/40 (10%) cases expressed CK19, MUC1 and CD44, while 2/40 (5%) expressed CD133. Cytomorphology showed the lowest sensitivity (47.6%) and specificity (90%) for CTCs detection. The combined use of FCM or CS with CTCs-mRNA markers improved the sensitivity and specificity to 68.3%, and 95.0%; respectively. Positive CTCs and mRNA markers expression were significantly associated with shorter 5-yr PFS and OS. In multivariate analysis, CTCs mRNA markers were independent prognostic factors for PFS and OS. CONCLUSIONS: Enumeration of CTCs by FCM and RNA expression for specific colon cancer markers are comparable to CS regarding sensitivity and specificity. CTCs also represent novel therapeutic targets for NMCRC cases.

MiRNAs as molecular biomarkers in stage II egyptian colorectal cancer patients.

AIM: To assess the role of aberrant miRNAs expressions in stage II CRC patients from Egypt. METHODS: Tumor tissue samples were obtained from 124 CRC stage II patients compared to 100 healthy controls for assessing miRNAs expression using; 1) a cataloged 84-miRNAs PCR array panel, and 2) another five miRNAs (miR-21, miR-137, miR-145, miR-320 and miR-498) that have been reported in previous studies to have a role in CRC, by quantitative real time PCR (qPCR). The results were correlated to patients' characteristics, response to treatment and survival. RESULTS: There were 17 out of 84 miRNAs differentially expressed in the CRC patients. Twenty six miRNAs were significantly differentially expressed in the female CRC patients, while 16 miRNAs were significantly differentially expressed in the male CRC patients. Only, five miRNAs (miR-21, Let-7a-5p, miR-100-5p, miR-200c-3p and miR-23b-3p) were significantly common deregulated in CRC patients regardless gender. miR-21 was overexpressed in 48.4% of the patients and it was significantly downregulated in females and over expressed in males. In univariate analysis; performance status, over-expression of miR-21 and miR-498 and reduced miR-137, miR-145, and miR-320 associated significantly with reduced DFS and OS whereas in multivariate analysis; miR-498 and miR-320 were independent prognostic factors for DFS and miR-21 was independent prognostic factors for OS. CONCLUSION: miRNAs expression differs significantly between male and female stage II CRC patients, miR-21, Let-7a-5p, miR-100-5p, miR-200c-3p and miR-23b-3p could be used as common diagnostic biomarkers for CRC. On the other hand, a three miRNAs panel (miR-21, miR-498 and miR-320) can predict recurrence and survival in those patients.

Role of relevant immune-modulators and cytokines in hepatocellular carcinoma and premalignant hepatic lesions.

AIM: To assess the levels of different immune modulators in patients with hepatocellular carcinoma (HCC), in relation to other hepatic diseases. METHODS: Eighty-eight patients were included in the current study and represented patients with HCC (20), liver cirrhosis (28) and chronic hepatitis (CH; 25), and normal controls (NC; 15). Peripheral blood was isolated for immunophenotyping of active myeloid dendritic cells (mDCs; CD1c and CD40), mature inactive myeloid cells (CD1c and HLA), active plasmacytoid cells (pDCs; CD303 and CD40), mature inactive pDCs (CD30 and HLA), active natural killer (NK) cells (CD56 and CD161), active NK cells (CD56 and CD314) and inactive NK cells (CD56 and CD158) was done by flow cytometry. Serum levels of interleukin (IL)-2, IL-10, IL-12, IL-1ß, interferon (IFN)-α, IFN-γ and tumor necrosis factor (TNF)-αR2 were assessed by ELISA. RESULTS: Active mDCs (CD1C+/CD40+) and inactive mDCs (CD1c+/HLA+) were significantly decreased in HCC patients in relation to NC (P < 0.001). CD40+ expression on active pDCs was decreased in HCC patients (P < 0.001), and its level was not significantly changed among other groups. Inactive pDCs (CD303+/HLA+), inactive NKs (CD56+/CD158+) and active NKs (CD56+/CD161+) were not statistically changed among the four groups studied; however, the latter was increased in CH (P < 0.05). NKG2D was statistically decreased in HCC, CH and cirrhosis (P < 0.001), and it was not expressed in 63% (12/20) of HCC patients. There was significant decrease of IL-2, IFN-α and IFN-γ (P < 0.001), and a significant increase in IL-10, IL-1ß, and TNF-αR2 (P <0.01, P < 0.001 and P < 0.001; respectively) in HCC patients. There was inverted correlation between IL-12 and IL-1ß in HCC (r = -0.565, P < 0.01), with a strong correlation between pDCs (CD303+/CD40+) and NKs (CD56+/CD161+; r = 0.512, P < 0.05) as well as inactive mDCs (CD1c+/HLA+) and inactive NK cells (CD56+/CD158+; r = 0.945, P < 0.001). CONCLUSION: NKG2D, CD40, IL-2 and IL-10 are important modulators in the development and progression of HCC.

Aberrant expression of miRNAs predicts recurrence and survival in stage-II colorectal cancer patients from Egypt

Background: Patients with stage II CRC have a varying survival outcome. Therefore, it is critical to identify prognostic biomarkers that can define more aggressive forms of the disease. We assessed the expression levels of five miRNAs that have been previously addressed in relation to the development and progression of solid and hematological tumors. Methods: We measured the expression levels of miR-21, miR-137, miR-145, miR-320 and miR-498in stage II CRC patients from Egypt (124 tissues and 41 blood samples) by quantitative real time PCR (qPCR). The results were correlated with relevant clinicopathological factors, response to treatment and survival rates of the patients. Results: miR-137, miR-145 and miR-320 were significantly reduced in 39.5%, 48.4% and 52.4%; respectively whereas miR-21 and miR-498 were significantly overexpressed in 48.4% and 40.3% of the CRC tissues compared to the control group. In patients' blood, miR-137, miR-145 and miR-320 were significantly reduced in 46.3%, 46.3% and 51. 2%; respectively whereas mir-21 and miR-498 were significantly overexpressed in 46.3% and 43.9% of the cases, respectively. The concordance between tissue and blood was weak for miR-320 and miR-145 (kappa 40-65%), intermediate for miR-498 and miR-137 (kappa 65-75%) and strong for miR-21 (kappa 75-85%). In univariate analysis performance status, over-expression of miR-21 and miR-498 and reduced miR-137, miR-145, and miR-320 associated significantly with reduced DFS and OS. However, in multivariate analysis, miR-498 and miR-320 were independent prognostic factors for DFS whereas miR-21 was independent prognostic factors for OS. Conclusions: miRNAs play an important role in the development and progression of stage II CRC. A five markers panel (miR-21, miR-498, miR-137, miR-145 and miR-320) can predict recurrence and survival in stage II CRC patients from Egypt (AU)