Association of genetic polymorphisms CYP2A6*2 rs1801272 and CYP2A6*9 rs28399433 with tobacco-induced lung Cancer: case-control study in an Egyptian population.

BACKGROUND: Several studies have reported the role of CYP2A6 genetic polymorphisms in smoking and lung cancer risk with some contradictory results in different populations. The purpose of the current study is to assess the contribution of the CYP2A6*2 rs1801272 and CYP2A6*9 rs28399433 gene polymorphisms and tobacco smoking in the risk of lung cancer in an Egyptian population. METHODS: A case-control study was conducted on 150 lung cancer cases and 150 controls. All subjects were subjected to blood sampling for Extraction of genomic DNA and Genotyping of the CYP2A6 gene SNPs (CYP2A6*2 (1799 T > A) rs1801272 and CYP2A6*9 (- 48 T > G) rs28399433 by Real time PCR. RESULTS: AC and CC genotypes were detected in CYP2A6*9; and AT genotype in CYP2A6*2. The frequency of CYP2A6*2 and CYP2A6*9 were 0.7% and 3.7% respectively in the studied Egyptian population. All cancer cases with slow metabolizer variants were NSCLC. Non-smokers represented 71.4% of the CYP2A6 variants. There was no statistical significant association between risk of lung cancer, smoking habits, heaviness of smoking and the different polymorphisms of CYP2A6 genotypes. CONCLUSION: The frequency of slow metabolizers CYP2A6*2 and CYP2A6*9 are poor in the studied Egyptian population. Our findings did not suggest any association between CYP2A6 genotypes and risk of lung cancer.

The role of BRCA1-IRIS in the development and progression of triple negative breast cancers in Egypt: possible link to disease early lesion.

BACKGROUND: Breast cancer is the most globally diagnosed female cancer, with the triple negative breast cancer (TNBC) being the most aggressive subtype of the disease. In this study we aimed at comparing the effect of BRCA1-IRIS overexpression on the clinico-pathological characteristics in breast cancer patients with TNBC or non-TNBC in the largest comprehensive cancer center in Egypt. METHODS: To reach this goal, we conducted an observational study at the National Cancer Institute (NCI), Cairo University (Cairo, Egypt). The data on all diagnosed breast cancer patients, between 2009 and 2012, were reviewed. BRCA1-IRIS expression measured using real time RT/PCR in these patients' tumor samples was correlated to tumor characteristics, such as to clinico-pathological features, therapeutic responses, and survival outcomes. RESULTS: 96 patients were enrolled and of these 45% were TNBC, and 55% were of other subtypes (hereafter, non-TNBC). All patients presented with invasive ductal carcinomas. No significant difference was observed for risk factors, such as age and menopausal status between the TNBC and the non-TNBC groups except after BRCA1-IRIS expression was factored in. The majority of the tumors in both groups were ≤5 cm at surgery (p = 0.013). However, in the TNBC group, ≤5 cm tumors were BRCA1-IRIS-overexpressing, whereas in the non-TNBC group they were BRCA1-IRIS-negative (p = 0.00007). Most of the TNBC patients diagnosed with grade 1 or 2 were BRCA1-IRIS-overexpressing, whereas non-TNBCs were IRIS-negative (p = 0.00035). No statistical significance was measured in patients diagnosed with grade 3 tumors. Statistically significant difference between TNBCs and non-TNBCs and tumor stage with regard to BRCA1-IRIS-overexpression was observed. Presence of axillary lymph node metastases was positively associated with BRCA1-IRIS overexpression in TNBC group, and with BRCA1-IRIS-negative status in the non-TNBC group (p = 0.00009). Relapse after chemotherapy (p < 0.00001), and local recurrence/distant metastasis after surgery (p = 0.0028) were more pronounced in TNBC patients with BRCA1-IRIS-overexpressing tumors compared to non-TNBC patients. Finally, decreased disease-free survival in TNBC/BRCA1-IRIS-overexpressing patients compared to TNBC/BRCA1-IRIS-negative patients, and decreased overall survival in TNBC as well as non-TNBC patients was driven by BRCA1-IRIS overexpression. CONCLUSIONS: TNBC/BRCA1-IRIS-overexpressing tumors are more aggressive than TNBC/BRCA1-IRIS-negative or non-TNBC/BRCA1-IRIS-overexpressing or both negative tumors. Further studies are warranted to define whether BRCA1-IRIS drives the early TNBC lesions growth and dissemination and whether it could be used as a diagnostic biomarker and/or therapeutic target for these lesions at an early stage setting.

Molecular biomarkers for prediction of response to treatment and survival in triple negative breast cancer patients from Egypt.

BACKGROUND: Triple negative breast cancer (TNBC) is an aggressive phenotype of breast cancer with reduced survival and poor prognosis. Increased VEGF-A, IGF-I, IGF-IR and TGF-ß1 expressions were detected in breast cancer. However, little is known about their prognostic and predictive roles in TNBC. AIM: We assessed the possible prognostic and predictive values of VEGF-A, IGF-I/IGF-IR and TGF-ß1 in TNBC cases by measuring their protein and mRNA expression in TNBC and non-TNBC cases. METHODS: VEGF-A, IGF-I, IGF-IR and TGF-ß1 RNA and their corresponding proteins were assessed in 43 TNBCs, 53 non-TNBCs and 30 normal breast tissues (NBT) by real time PCR (qPCR) and immunohistochemistry (IHC); respectively. Results were related to clinico-pathological factors, response to treatment and survival rates. RESULTS: Increased mRNA expression of VEGF-A, IGF-I, and IGF-IR was significantly higher in TNBC (65.1%, 65.1%, and 72.1%) than non-TNBC (28.1%, 33.96% and 28.3%) and NBT (0.00%) (P<0.001). Similarly, TNBC patients were significantly associated with high expression of VEGF-A, IGF-I, and IGF-IR proteins (67.44%, 62.79% and 83.72%) than non-TNBC (20.75%, 35.86% and 20.75%) and NBT (0.00%) (P<0.001). Protein and RNA expression levels of all studied markers showed high concordance in all investigated patients (correlation coefficient exceeding 0.5 and 0.4, respectively). In the TNBC group, metastasis and poor response to treatment were significantly associated with VEGF-A (P<0.001, P=0.007, respectively), IGF-I (P<0.001, P<0.001, respectively), IGF-IR (P=0.001, P=0.015, respectively) and TGF-ß1 (P<0.001, P=0.007, respectively) protein levels. Multivariate logistic regression showed that IGF-I was the only independent prognostic factor for reduced OS (P=0.034) and DFS (P=0.026) in the TNBC patients. CONCLUSIONS: VEGF-A, IGF-I and IGF-IR play an important role in the development and progression of TNBC compared to non-TNBC. Therefore, they could be used as prognostic and predictive biomarkers as well as candidates for targeted therapy. However, only IGF-I can predict survival in those patients.

Final Results of RIGHT Choice: Ribociclib Plus Endocrine Therapy Versus Combination Chemotherapy in Premenopausal Women With Clinically Aggressive Hormone Receptor-Positive/Human Epidermal Growth Factor Receptor 2-Negative Advanced Breast Cancer.

PURPOSE: A head-to-head comparison of efficacy between a cyclin-dependent kinase 4/6 inhibitor plus endocrine therapy (ET) versus combination chemotherapy (CT) has never been reported in patients with clinically aggressive hormone receptor-positive, human epidermal growth factor receptor 2-negative (HR+/HER2-) advanced breast cancer (ABC). METHODS: In this open-label, multicenter, randomized phase II trial, pre/perimenopausal women with clinically aggressive HR+/HER2- ABC were randomly assigned 1:1 to first-line ribociclib (600 mg once daily; 3 weeks on, 1 week off) plus letrozole/anastrozole and goserelin or investigator's choice of combination CT (docetaxel plus capecitabine, paclitaxel plus gemcitabine, or capecitabine plus vinorelbine). The primary end point was progression-free survival (PFS). RESULTS: Among 222 patients randomly assigned to ribociclib plus ET (n = 112) or combination CT (n = 110), 150 (67.6%) had symptomatic visceral metastases, 41 (18.5%) had rapid disease progression per investigator's judgment, and 31 (14.0%) had symptomatic nonvisceral disease. Overall, 106 (47.7%) patients had investigator-assessed visceral crisis. The median follow-up time was 37.0 months. At data cutoff, 31.3% (ribociclib arm) and 15.5% (CT arm) of patients had completed study treatment and transitioned to post-trial access. The median PFS was 21.8 months (ribociclib plus ET; [95% CI, 17.4 to 26.7]) and 12.8 months (combination CT; [95% CI, 10.1 to 18.4); hazard ratio, 0.61 [95% CI, 0.43 to 0.87]; P = .003. The overall response rates and the median time to response in the ribociclib versus CT arms, respectively, were 66.1% and 61.8% and 4.9 months and 3.2 months (hazard ratio, 0.76 [95% CI, 0.55 to 1.06]). Lower rates of symptomatic adverse events were observed in the ribociclib versus CT arm. CONCLUSION: First-line ribociclib plus ET showed a significant PFS benefit, similar response rates, and better tolerability over combination CT in patients with clinically aggressive HR+/HER2- ABC.

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.

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.

Genetic polymorphisms of human cytochrome P450 CYP1A1 in an Egyptian population and tobacco-induced lung cancer.

BACKGROUND: Cytochrome P450 CYP1A1 helps detoxify the potential carcinogens in tobacco smoke, it was reported that polymorphisms in the coding gene result in variation in the expression and activity levels which alter metabolism and clearance of carcinogens and therefore modify cancer risk. In this work, we aimed to identify CYP1A1 gene polymorphisms associated with lung cancer in Egyptian population and to examine the interaction effect with Tobacco smoking in modulating disease risk. METHODS: A case-control study was conducted on 150 unrelated lung cancer patients and 150 unrelated control subjects. Genomic DNA was extracted and sequencing analysis of CYP1A1 gene was performed on ABI PRISM 3100 genetic analyzer. RESULTS: Three variants in CYP1A1 gene were identified in heterozygous forms in lung cancer patients I462V, T461N and I286T. A combined variant T461N/ I462V associated with lung cancer and those who carried this variant were 2-times more likely to develop lung cancer (OR = 2.03, 95% CI = 1.81-2.29, P = 0.04), specially the non-small cell type (NSCLC) (OR = 2.20, 95% CI = 1.93-2.50, P = 0.02). Wild type was more frequent among smoker controls (83.3%) compared to smoker lung cancer patients (54.8%), P = 0.03. Association studies to examine the interaction effect of identified variants with Tobacco smoking in modulating disease risk showed no significant associations. Identified polymorphisms showed no significant implication on the stage or the prognosis of the disease. CONCLUSION: Our findings support that CYP1A1 polymorphisms play a role in the pathogenesis of lung cancer. In Egyptian population, CYP1A1 I462V, T461N and I286T variants were identified among lung cancer patients and combined T461N/ I462V was a risk variant for NSCLC in non smokers.

BRCA1-IRIS overexpression promotes and maintains the tumor initiating phenotype: implications for triple negative breast cancer early lesions.

Tumor-initiating cells (TICs) are cancer cells endowed with self-renewal, multi-lineage differentiation, increased chemo-resistance, and in breast cancers the CD44+/CD24-/ALDH1+ phenotype. Triple negative breast cancers show lack of BRCA1 expression in addition to enhanced basal, epithelial-to-mesenchymal transition (EMT), and TIC phenotypes. BRCA1-IRIS (hereafter IRIS) is an oncogene produced by the alternative usage of the BRCA1 locus. IRIS is involved in induction of replication, transcription of selected oncogenes, and promoting breast cancer cells aggressiveness. Here, we demonstrate that IRIS overexpression (IRISOE) promotes TNBCs through suppressing BRCA1 expression, enhancing basal-biomarkers, EMT-inducers, and stemness-enforcers expression. IRISOE also activates the TIC phenotype in TNBC cells through elevating CD44 and ALDH1 expression/activity and preventing CD24 surface presentation by activating the internalization pathway EGFR→c-Src→cortactin. We show that the intrinsic sensitivity to an anti-CD24 cross-linking antibody-induced cell death in membranous CD24 expressing/luminal A cells could be acquired in cytoplasmic CD24 expressing IRISOE TNBC/TIC cells through IRIS silencing or inactivation. We show that fewer IRISOE TNBC/TICs cells form large tumors composed of TICs, resembling TNBCs early lesions in patients that contain metastatic precursors capable of disseminating and metastasizing at an early stage of the disease. IRIS-inhibitory peptide killed these IRISOE TNBC/TICs, in vivo and prevented their dissemination and metastasis. We propose IRIS inactivation could be pursued to prevent dissemination and metastasis from early TNBC tumor lesions in patients.

Transforming growth factor-ß, insulin-like growth factor I/insulin-like growth factor I receptor and vascular endothelial growth factor-A: prognostic and predictive markers in triple-negative and non-triple-negative breast cancer.

In the current study, the prognostic and predictive values of serum transforming growth factor-ß1 (TGF-ß1), insulin-like growth factor I (IGF-I)/IGF-I receptor (IGF-IR) and vascular endothelial growth factor-A (VEGF-A) were evaluated in triple-negative and non-triple-negative breast cancer (TNBC and non-TNBC). The aim was to identify a group of serological biomarkers and to identify possible candidates for targeted therapy in patients with TNBC and non-TNBC. Protein levels of TGF-ß1, IGF-I/IGF-IR and VEGF-A in the serum were measured in 43 TNBC, 53 non­TNBC and 20 normal control participants using quantitative ELISA assays. Results were correlated against standard prognostic factors, response to treatment and survival. TNBC was identified to be associated with poor prognosis and serum levels of VEGF-A and IGF/IGF-IR were significantly higher in the TNBC group compared with the non-TNBC group. IGF-IR and VEGF-A overexpression was observed to be correlated with TGF-ß1 expression and all of the markers investigated were associated with metastasis and disease progression. In the multivariate analysis, VEGF-A, IGF-I and IGF-IR were observed to be independent predictors for overall survival, whereas TGF-ß1 and lymph node status were identified as independent predictors for disease-free survival. The overall response rate was significantly lower in patients with TNBC and those with high levels of TGF-ß1, IGF-I/IGF-IR and VEGF-A. In view of the present results, it was concluded that TGF-ß1, IGF-I/IGF-IR and VEGF-A overexpression is associated with the presence of aggressive tumors, which exhibit an increased probability of metastasis, a poor response to treatment and reduced survival rate. This indicates that VEGF-A, IGF-IR and IGF-I have the potential to be used as surrogate biomarkers and are promising candidates for targeted therapy, particularly in patients with TNBC.

Circulating tumor and cancer stem cells in hepatitis C virus-associated liver disease.

AIM: To assess the role of circulating tumor cells (CTCs) and cancer stem cells (CSCs) in hepatitis C virus (HCV)-associated liver disease. METHODS: Blood and/or tissue samples were obtained from HCV (genotype 4)-associated hepatocellular carcinoma patients (HCC; n = 120), chronic hepatitis C patients (CH; n = 30) and 33 normal control subjects (n = 33). Serum levels of alpha-fetoprotein (AFP), alkaline phosphatase, and alanine and aspartate aminotransferases were measured. Cytokeratin 19 (CK19) monoclonal antibody was used to enumerate CTCs, and CD133 and CD90 were used to enumerate CSCs by flow cytometry. The expression levels of the CSCs markers (CD133 and CD90) as well as telomerase, melanoma antigen encoding gene 1 (MAGE1) and MAGE3 were assessed by RT-PCR and quantitative real-time polymerase chain reactions. The number of CTCs and/or the expression levels of CK19, CD133, telomerase, MAGE1 and MAGE3 were correlated to the standard clinicopathologic prognostic factors and disease progression. RESULTS: Levels of AFP, alkaline phosphatase and aspartate aminotransferase were significantly different among the HCC, CH and control groups (P < 0.001), whereas alanine aminotransferase differed significantly between patient (HCC and CH) and control groups (P < 0.001). At the specified cutoff values determine by flow cytometry, CK19 (49.8), CD90 (400) and CD133 (73) were significantly higher in the blood of HCC patients compared to those in the CH and control groups (P < 0.001). On the other hand, CD133 at a 69.5 cutoff was significantly higher in the CH compared to the control group (P ≤ 0.001). Telomerase, MAGE1 and MAGE3 RNA were expressed in 55.71%, 60.00% and 62.86% of the HCC patients, respectively, but were not detected in patients in the CH or control groups, which were statistically significant (Ps < 0.001). The expression levels of telomerase, CD90, MAGE3, CD133 and CK19 were all significantly associated with high tumor grade and advanced stage in HCC patients (all Ps < 0.05). CONCLUSION: CTC counts and AFP, CK19, telomerase, and MAGE1/MAGE3 expression predict disease progression in patients with HCV, whereas telomerase, MAGE3, CD90, CD133 and CK19 are prognostic markers in HCC.

Plasma vascular endothelial growth factor 165 in advanced non-small cell lung cancer.

Currently, there is no serum marker that is routinely recommended for lung cancer. Therefore, the aim of the present study was to demonstrate that plasma vascular endothelial growth factor 165 (VEGF 165) may be a potential marker for advanced lung cancer. Lung cancer is the leading cause of cancer-related mortality worldwide, therefore, it is important to develop novel diagnostic techniques. The present prospective case control study included two groups of patients; a control group of healthy volunteers and a second group of patients with advanced non-small cell lung cancer (NSCLC). The plasma VEGF 165 levels were measured at baseline by ELISA prior to the first-line gemcitabine-cisplatin regimen. The high VEGF 165 expression level cut-off was >703 pg/ml, and the primary endpoint was used to compare the plasma VEGF 165 levels between the NSCLC patients and the control group subjects. The secondary endpoint was used to identify the correlations between high VEGF 165 levels and; clinical response (CR), progression-free survival (PFS) and overall survival (OS) in the advanced NSCLC patients. In total, patients with advanced NSCLC (n=35) were compared with a control group of age- and gender-matched healthy subjects (n=34). The follow-up period was between Oct 2009 and Oct 2012, with a median follow-up time of 10.5 months. The median plasma VEGF 165 level was 707 pg/ml in the NSCLC patients versus 48 pg/ml in the healthy control subjects (P<0.001). However, no significant correlation was found between the plasma VEGF 165 levels and CR (P<0.5), median PFS (P=1.00) or OS (P=0.70). Therefore, it was concluded that plasma VEGF 165 may serve as a potential diagnostic marker for advanced NSCLC.

Mistletoe preparation (Viscum Fraxini-2) as palliative treatment for malignant pleural effusion: a feasibility study with comparison to bleomycin.

BACKGROUND: Malignant pleural effusion is a common problem in patients with solid tumours. It has a significant impact on quality of life, and, hence, there is a substantial need to investigate new agents to treat it. PATIENTS AND METHODS: This is a prospective randomised controlled study, including patients with symptomatic recurrent malignant pleural effusion of different primaries. Patients were randomised into two groups: the first group received five ampoules of mistletoe preparation with defined lectin content (Viscum Fraxini-2, ATOS Pharma) diluted in 10 cc glucose 5% solution. Re-instillation was repeated every week until complete dryness of the pleural fluid was achieved (the maximum duration of the therapy was eight weeks). The second group received 60 units of bleomycin once intrapleurally. AIMS: The primary aim of this paper was to evaluate the efficacy of mistletoe preparation as a palliative treatment for malignant pleural effusions in comparison with bleomycin. The secondary aim was to evaluate the tolerability of the mistletoe preparation. RESULTS: A total of 23 patients were included and followed up during the study from December 2007 to January 2012: 13 patients received mistletoe preparation, and ten patients received bleomycin. Overall clinical response was reported in 61.5% of the mistletoe preparation arm versus 30% in bleomycin arm (p = 0.2138), 95% CI = (-0.1203, 0.6325). The toxicity of both arms was mild and manageable; the mistletoe preparation arm included fever, chills, headache, malaise, and, in two cases, allergic reaction, which was controlled by discontinuation of the drug and steroid injection. CONCLUSION: Mistletoe preparation is an efficient and well tolerated sclerosant agent which needs further investigation.