Prognostic significance of EZH2 and ARID1A expression in urothelial carcinoma: an immunohistochemical study.

Enhancer of zeste homolog 2 (EZH2) and AT-rich interactive domain 1A (ARID1A) expression in urothelial carcinoma (UC) has not been well studied. ARID1A is a novel tumor suppressor gene coding for a chromatin remodeling protein that is mutated in urinary bladder cancer. The enhancer of zeste homolog 2 (EZH2) is a transcriptional repressor involved in gene silencing. Amplification of EZH2 has been reported in several malignancies. This study analyzed the immunohistochemical expression of EZH2 and ARID1A in 56 cases of UC that included (n = 21) cases of radical cystectomy and (n = 35) cases of transurethral resections of bladder tumor (TURBT) with muscle fibers and immunotherapy with adjuvant intravesical bacillus Calmette-Guerin (BCG). The predicting role of both markers for tumor recurrence and recurrence-free survival (RFS) was also analyzed. High EZH2 marker expression was observed in 75% of cases while 78.6% of cases had low ARID1A marker expression. There was a significant negative correlation between the two markers where high EZH2 and low ARID1A expression was significantly associated with higher tumor grade, stage, presence of muscle invasion, lymph node metastasis, presence of concomitant carcinoma in situ (CIS) and higher incidence of recurrence with shorter RFS rate. It was concluded that EZH2 and ARID1A play a role in tumor carcinogenesis and differentiation and could be considered as independent prognostic factors in UC and for use as a potential therapeutic target.

Design and Synthesis of New Quinoxaline Derivatives as Potential Histone Deacetylase Inhibitors Targeting Hepatocellular Carcinoma: In Silico, In Vitro, and SAR Studies.

Guided by the structural optimization principle and the promising anticancer effect of the quinoxaline nucleus, a new series of novel HDAC inhibitors were designed and synthesized. The synthesized compounds were designed to bear the reported pharmacophoric features of the HDAC inhibitors in addition to an extra moiety to occupy the non-used vacant deep pocket of the HDAC receptor. The newly prepared compounds were evaluated for their in vitro anti-proliferative activities against HepG-2 and HuH-7 liver cancer cell lines. The tested compounds showed promising anti-proliferative activities against both cell lines. The most active ten candidates (6 c , 6 d , 6 f , 6 g , 6 k , 6 l , 7 b , 8, 10 h , and 12) were further evaluated for their effect on the gene expression levels of Bax as an apoptotic marker and Bcl-2 as an anti-apoptotic one. Moreover, they were evaluated for their ability to inhibit histone deacetylase (HDAC1, HDAC4, and HDAC6) activities. Compound 6 c achieved the best cytotoxic activities on both HepG-2 and HuH-7 cell lines with IC50 values of 1.53 and 3.06 µM, respectively, and also it showed the most inhibitory activities on HDAC1, HDAC4, and HDAC6 with IC50 values of 1.76, 1.39, and 3.46 µM, respectively, compared to suberoylanilide hydroxamic acid (SAHA) as a reference drug (IC50 = 0.86, 0.97, and 0.93 µM, respectively). Furthermore, it achieved a more characteristic arrest in the growth of cell population of HepG-2 at both G0/G1 and S phases with 1.23-, and 1.18-fold, respectively, compared to that of the control, as determined by cell cycle analysis. Also, compound 6 c showed a marked elevation in the AnxV-FITC apoptotic HepG-2 cells percentage in both early and late phases increasing the total apoptosis percentage by 9.98-, and 10.81-fold, respectively, compared to the control. Furthermore, docking studies were carried out to identify the proposed binding mode of the synthesized compounds towards the prospective target (HDAC4). In silico ADMET and toxicity studies revealed that most of the synthesized compounds have accepted profiles of drug-likeness with low toxicity. Finally, an interesting SAR analysis was concluded to help the future design of more potent HDACIs in the future by medicinal chemists.

Genomic alterations in the F8 gene correlating with severe hemophilia A in Egyptian patients.

BACKGROUND: Hemophilia A (HA) is an inherited X-linked recessive coagulation disorder caused by factor VIII (F8) deficiency. F8 rearrangements involving intron 22 (int22) and intron 1 (int1) account for almost half of severe HA phenotype also a hotspot exon 14 provides numerous mutational patterns. This study aims to identify F8 gene mutations among Egyptian HA patients. METHODS: DNA samples from 60 HA patients were screened for int22 and int1 rearrangements using simplified inverse shifting PCR (IS-PCR) followed by exon 14 sequencing. Also, four uncharacterized patients were studied by targeted exome sequencing. RESULTS: In 33.3% of the studied patients, we identified three int22 rearrangements, three exon 14 mutations (two frameshift; one novel (NM_000132.3:c.2734_2735delAA, p.(N912Ffs*6)), a second reported mutation (NM_000132.3:c.3091_3094delAGAA, p.(K1031Lfs*9)), and one nonsense mutation (NM_000132.3:c.2440C>T, p.(R814*)). All identified mutations were detected in patients with severe HA phenotype. Targeted exome sequencing could not detect any known pathogenic variants. CONCLUSION: Intron 22 rearrangement and exon 14 mutations correlate with most severe hemophilia A Egyptian patients.

The Relation between Serum Hepcidin, Ferritin, Hepcidin: Ferritin Ratio, Hydroxyurea and Splenectomy in Children with ß-Thalassemia.

BACKGROUND: Hepcidin, a small peptide hormone, is established as the main regulator of iron homeostasis. AIM: To estimate serum hepcidin, ferritin, and hepcidin: ferritin ratio in ß-thalassemia patients and to determine the effect of splenectomy and hydroxyurea on serum hepcidin. METHODS: A study was conducted on 30 thalassemia major (ßTM), 29 thalassemia intermedia (ßTI) and 29 healthy children's controls. Data were collected by patient interviewing where detailed history-taking and thorough clinical examinations were carried out. Serum ferritin and hepcidin were measured by ELISA assay (Bioneovan Co. Ltd Beijing, China). RESULTS: Βeta-thalassemia patients had higher serum ferritin, serum hepcidin and lower Hb and hepcidin: ferritin ratio compared to the controls (p < 0.001, 0.010, 0.001, 0.001) respectively. Β-TM patients had higher mean serum hepcidin and serum ferritin compared to ß-TI, with statistically significant difference (P = 0.042, P < 0.001, respectively). Twenty-one patients out of 29 ßTI was on hydroxyurea therapy; these patients had significantly lower levels of serum ferritin (P < 0.004) and significantly higher levels of Hb (P < 0.004). Serum ferritin was statistically significantly higher in splenectomized patients P < 0.009. Serum hepcidin level was insignificantly higher in splenectomized patients than non-splenectomized patients (21.6 ± 14.75, 17.76 ± 10.01 ng/mL). Hepcidin showed a significantly positive correlation with hepcidin: ferritin ratio in all studied groups. CONCLUSION: Serum hepcidin was elevated in ß-thalassemia children with more evident elevation in ßTM patients. Splenectomy played no major role in hepcidin regulation. Knowing that hepcidin in serum has a dynamic and multi-factorial regulation, individual evaluation of serum hepcidin and follow up, e.g. every 6 months could be valuable, and future therapeutic hepcidin agonists could be helpful in management of iron burden in such patient.

Diagnostic accuracy of immunohistochemical markers in differentiation between basal cell carcinoma and trichoepithelioma in small biopsy specimens.

BACKGROUND: The distinction of trichoepithelioma from basal cell carcinoma in small superficial biopsies is important but often challenging. This has inspired many scientists to test the validity of immunohistochemical markers in the differential diagnosis. OBJECTIVES: To develop an immunohistochemical protocol that helps in differentiation between both trichoepithelioma (TE) and basal cell carcinoma (BCC) with higher sensitivity and specificity. METHODS: Using standard immunohistochemical techniques, we examined 10 TEs and 19 BCCs for the expression of CK19, Ki-67, androgen receptors (AR), CD10, and PHLDA1. RESULTS: Immunoreactivity of AR, Ki-67, and CD10 in tumor cells was significantly higher in BCC than TE with a diagnostic accuracy in BCC of 75.5%, 75.8%, and 79.3% respectively, whereas immunoreactivity of PHLDA1 in tumor cells and stromal CD10 was significantly higher in TE than BCC with a diagnostic accuracy in TE of 100% and 82.8%, respectively. In contrast, immunoreactivity for CK19 showed no statistically significant differences between both tumors. CONCLUSION: The analysis of CD10, Ki-67, and PHLDA1 can be used as a helpful immunohistochemical panel in the distinction between TE and BCC especially in small and superficial biopsies.

Clinical significance of serum clusterin as a biomarker for evaluating diagnosis and metastasis potential of viral-related hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is an increasing problem in Egypt. Clusterin has been reported to play a significant role in tumorigenesis. AIM: The aim of this study is to evaluate clusterin as a marker for evaluating diagnosis and metastasis potential of viral-related HCC. METHODS: Eighty patients with HCC, 30 patients with liver cirrhosis, 30 patients with chronic hepatitis and 30 healthy controls were enrolled in study. Estimation of serum clusterin was done by enzyme linked immunosorbent assay. RESULTS: Serum clusterin levels were significantly increased in patients with HCC. Serum clusterin was highly increased in patients with poorly differentiated tumor, capsular infiltration, portal vein invasion and lymph node infiltration. Receiver operator characteristic curve showed that clusterin had a greater area under curve value (0.95) than that of alpha fetoprotein (0.85). At cutoff value of 128 µg/mL, serum clusterin yielded (90%) sensitivity and (87%) specificity for predicting HCC. CONCLUSION: We concluded that clusterin is a promising useful marker for diagnosis of HCC. Clusterin might be deemed as a useful marker for predicting the progression and metastasis potential of HCC.

Assessment of Her-2/neu gene amplification status in breast carcinoma with equivocal 2+ Her-2/neu immunostaining.

BACKGROUND AND PURPOSE: Amplification of Her-2/neu gene occurs in 25-30% of breast carcinomas. FDA approved trastuzumab (Herceptin) is effective only in tumors having the gene amplification. Immunohistochemistry (IHC) for Her-2/neu protein is widely used but false positive and false negative results exist. Fluorescence in-situ hybridization (FISH) has both excellent sensitivity and specificity in detecting Her-2/neu amplification. Comparative studies have shown discordant results in proportion of cases with equivocal 2+ immunostain. This study is thus conducted to ascertain the frequency of Her-2/neu gene amplification by FISH in breast carcinoma specified as score 2+ by IHC and to correlate these findings with parameters of prognosis in breast cancer. METHODS: From October 2008 till May 2010 all paraffin blocks from cases with invasive breast carcinoma which were scored as 2+ by IHC were eligible for the study, there were 50 cases. Immunohistochemical evaluation of Her-2/neu was performed using the HercepTest. All cases were immunohistochemically evaluated for ER and PR. FISH was performed using FDA approved Path-Vysion Her-2/neu/CEP 17 dual color probe. RESULTS: Nine cases (18%) out of 50 cases scored as Her-2/neu 2+ by IHC showed true gene amplification with a median value of scoring ratio 4.28 ranging from 2.37 to 13.26. Another two cases showed low level of amplification but when corrected for Her-2/neu/CEP ratio they did not show true amplification as they were associated with polysomy 17. With the exception of tumor size, neither patient's age, histologic grade nor lymph node status were correlated with Her-2/neu gene amplification. Significant inverse correlation existed between Her-2/neu gene amplification and ER (P=0.01), PR status (P<0.001). CONCLUSION: Even though FISH is a more complex and expensive procedure, it should be considered the method of choice for assessment of Her-2/neu gene status especially for equivocal cases by IHC that are not accompanied by true gene amplification in the majority of breast carcinoma cases.

Did salvage ICE chemotherapy improve the outcome in primary resistant/relapsing stage III/IV neuroblastoma?

BACKGROUND AND PURPOSE: Neuroblastoma is the most common extracranial and deadly solid tumor in children. It accounts for 15% of the deaths from cancer in the pediatric age group. Approximately half of the newly diagnosed children are at "high risk" of treatment failure. The aim of this study is to evaluate the response rate of salvage chemotherapy by the ICE (Ifosfamide, Carboplatin, and Etoposide) regimen when administered to previously treated primary refractory or progressive high risk neuroblastoma patients. PATIENTS AND METHODS: Sixty-six patients from the National Cancer Institute (NCI), Cairo University and the Children Cancer Hospital Egypt (CCHE) received salvage chemotherapy (ICE) either due to primary resistance in 51/66 (77.2%) or due to disease progression on primary chemotherapy in 15/66 (22.8%). RESULTS: They were 40 males (60.6%) and 26 females (39.4%). Patients' age ranged between 3 months and 12.5 years. The most common tumor site was suprarenal, followed by retroperitoneal mass. Two patients (3%) died from chemotherapy toxicity during ICE administration. Evaluation of tumor response in the remaining 64 patients showed the following: CR/PR in 24 patients (36.5%), SD in 11 patients (16.6%), and PD in 29 patients (43.9%). Fourteen patients (21.2%) were considered eligible for auto BMT, while 50/64 patients (78.8%) failed this second line (salvage) chemotherapy and had palliative lines of therapy. By the end of the study (May 2010), 47/66 (71.2%) of the patients were still alive, while 19/66 (28.8%) were dead. Two out of 14 patients (14.2%) who underwent HSCT died from post transplantation disease progression, while 12/14 (85.8%) were in CCR. CONCLUSION: Chemotherapy by ICE for primary resistant or progressive stage III/IV NB seems well tolerated. With a 36.6% response rate, 18% CCR, and 3% treatment mortality rate, it could be considered a good salvage therapy in the category of patients who are condemned for palliation.

Plasma visfatin and retinol binding protein-4 levels in patients with type 2 diabetes mellitus and their relationship to adiposity and fatty liver.

OBJECTIVES: We investigated whether plasma visfatin and binding protein-4 (RBP-4) levels correlate with obesity and type 2 diabetes mellitus (T2DM). DESIGN AND METHODS: Two groups were enrolled: Group 1: 40 patients with T2DM and Group 2: 40 age- and gender-matched healthy controls. Both groups were subdivided according to body mass index (BMI) into non-obese (BMI < 25 kg/m(2)) and obese subjects (BMI ≥ 30 kg/m(2)) (20 each). RESULTS: Plasma visfatin and RBP-4 levels were significantly increased in T2DM patients compared with controls with similar BMI values (for both p<0.001). Plasma visfatin and RBP-4 concentrations correlated with BMI, waist/hip ratio, insulin and homeostatic model assessment insulin resistance (HOMA(IR)). Visfatin and RBP-4 correlated with visceral fat and liver fat in diabetic patients (for both p<0.001). CONCLUSION: Visfatin level was increased in T2DM, possibly related to hyperglycemia. Plasma RBP-4 correlated positively with liver fat and HOMA(IR) which may reflect its effects on hepatic insulin sensitivity.

The role of PTPN22 gene polymorphism in childhood immune thrombocytopenic purpura.

Immune thrombocytopenia is an autoimmune disorder characterized by antibody-mediated platelet destruction. A protein tyrosine phosphatase (PTPN22) present in lymphocytes is an important negative regulator of signal transduction for the T-cell receptor-MHC complex and has been associated with autoimmune disorders that produce autoantibodies. The present study investigated the frequency of the 1858C>T single-nucleotide polymorphism (SNP) in the PTPN22 gene in idiopathic thrombocytopenic purpura (ITP) patients. This case series study included 50 children with ITP, 24 acute and 26 chronic cases, and 50 normal children as a control group. All were subjected to clinical history and laboratory investigations including complete blood count, genotyping of PTPN22 1858C/T SNP by polymerase chain reaction-restriction fragment length polymorphism and platelet antibodies using platelets suspension immunofluorescence test for the cases. Thirteen patients (26%) were positive for the PTPN22 1858C>T SNP. Three patients were homozygous for the mutation and 10 were heterozygous. Comparison of the 26% of the ITP patients who were positive for the PTPN22 1858C>T mutation with the 6% positive in the control group yielded a P value of 0.006. Antiplatelet antibodies were detected in five patients (20.8%) with acute ITP and in three patients (11.5%) with chronic ITP; no significant association between the presence of PTPN22 1858C>T mutation and the presence of antiplatelet antibodies was detected. The prevalence of PTPN22 gene mutation was higher in ITP patients, thus it may be considered as a genetic risk factor in the development of ITP in Egyptian children.

Association of fetuin-A and cardiac calcification and inflammation levels in hemodialysis patients.

BACKGROUND: Vascular calcification is commonly found in chronic kidney disease (CKD) patients and it is one of the predictors of cardiovascular death. Recently, several studies have demonstrated that low fetuin-A levels are associated with mortality in uremic patients. Objectives. To investigate the importance of non-traditional risk factors of calcification including fetuin-A, IL-6 and high sensitivity CRP (hsCRP) in hemodialysis patients and their relationship to the extent of cardiac calcification by means of multislice computed tomography (MSCT), and echocardiography. PATIENTS AND METHODS: The study was conducted on 70 hemodialysis patients as well as 20 healthy control subjects. All patients were subjected to MSCT for evaluation of calcium score in the coronary arteries as well as echocardiography for detecting valvular calcification. In addition, the patients were sampled for evaluation of inflammatory markers such as hsCRP and IL-6 and also fetuin-A. RESULTS: Mean serum fetuin-A was significantly lower in hemodialysis patients than controls subjects. By dividing the patients into tertiles of serum fetuin-A, a significant association between low levels of fetuin-A and high calcium score and valvular calcification were found. Multiple regression analysis showed that calcium scoring and IL-6 were the most independent risk factors for serum fetuin-A levels. CONCLUSION: Serum fetuin-A showed important association with coronary, valvular calcification and inflammation in hemodialysis patients. Assessment of both cardiac calcification and serum levels of fetuin-A may be of value to identify those subjects at higher risk of development and progression of vascular lesion and may be a novel therapeutic approach.