Laboratory validation of formal concept analysis of the methylation status of microarray-detected genes in primary breast cancer.

Breast cancer is the leading cause of cancer-related mortality. DNA methylations play important roles in cancer development and progression. Formal concept analysis was previously utilized for data mining hypermethylated and hypomethylated genes in breast cancer molecular subtypes in illumina methylation-based microarray database, to laboratory validate their outputs; HS3ST2 (heparan sulfate d-glucosaminyl 3-O-sulfonyl transferase-2) and MUC1 (mucin-1) were retrieved. Both play important roles in progression and invasion of breast cancer. The methylation status of both genes was laboratory validated using methylation-based polymerase chain reaction in breast cancer subtypes luminal A (early stages) and luminal B (late stages) in comparison with benign conditions and normal breast to conclude their roles in tumor invasion and to validate the newly developed algorithm (formal concept analysis). Significant cancer-specific hypermethylation of HS3ST2 was detected in luminal B (chi square = 30.6, p = 0.000), while significant cancer-specific hypomethylation of MUC1 was detected in luminal B (chi square = 30.5, p = 0.001) breast cancer. The median levels of the percentage of methylated allele of both genes were significantly discriminative between luminal A and luminal B subtypes and benign and healthy control groups. Detection of MUC1 and HS3ST2 promoter methylation status appears to be useful molecular markers for assessing the progressive state of the disease and could be helpful in discriminating breast cancer molecular subtypes. These results validate the methylation-based microarray analysis, thus trust their output in the future.

Diagnostic value of serum kallikrein-related peptidases 6 and 10 versus CA125 in ovarian cancer.

OBJECTIVES: To assess the diagnostic value of serum KLK6 and KLK10 in patients with ovarian tumor in comparison to serum CA125. METHODS: Based on clinical and sonographic findings, 90 patients were consecutively recruited at the Gynecological Oncology Unit, Ain Shams University Maternity Hospital. Preoperative serum KLK6 and/or KLK10 were determined by enzyme-linked immunosorbent assay technique. The patients' final diagnoses were those of the histopathological reports. RESULTS: There were 27 malignant versus 63 benign cases. Serum markers' diagnostic specificity and sensitivity were 80.3/72.7, 56.8/64.0, and 39.53/58.3 for CA125, KLK6, and KLK10, respectively. Combination of CA125 with either of the other 2 markers revealed diagnostic enhancement with KLK10 (85.37/73.00) but not with KLK6 (42.86/86.36). CONCLUSIONS: In ovarian cancer, serum KLK6 and KLK10 may have much lower overall sensitivities than serum CA125. However, whereas serum KLK6 may improve the sensitivity of CA125, serum KLK10 may have the highest specificity among the 3 markers.

KLK10 exon 3 unmethylated PCR product concentration: a new potential early diagnostic marker in ovarian cancer? - A pilot study.

BACKGROUND: KLK10 exon 3 hypermethylation correlated to tumor-specific lack of KLK10 expression in cancer cell lines and primary tumors. In the present study we investigate the possible role of KLK10 exon 3 methylation in ovarian tumor diagnosis and prognosis. RESULTS: Qualitative methylation-specific PCR (MSP) results did not show statistically significant differences in patient group samples (normal and tumor) where all samples were positive only for the unmethylated-specific PCR except for two malignant samples that were either doubly positive (serous carcinoma) or doubly negative (Sertoli-Leydig cell tumor) for the two MSP tests. However, KLK10 exon 3 unmethylated PCR product concentration (ng/µl) showed statistically significant differences in benign and malignant patient group samples; mean ± SD (n): tumor: 0.077 ± 0.035 (14) and 0.047 ± 0.021 (15), respectively, p-value = 0.011; and normal: 0.094 ± 0.039 (7) and 0.046 ± 0.027 (6), respectively, p-value = 0.031. Moreover, ROC curve analysis of KLK10 exon 3 unmethylated PCR product concentration in overall patient group samples showed good diagnostic ability (AUC = 0.778; p-value = 0.002). Patient survival (living and died) showed statistically significant difference according to preoperative serum CA125 concentration (U/ml); median (n): 101.25 (10) and 1252 (5), respectively, p-value = 0.037, but not KLK10 exon 3 unmethylated PCR product concentration (ng/µl) in overall malignant patient samples; mean ± SD (n): 0.042 ± 0.015 (14) and 0.055 ± 0.032 (7), p-value = 0.228. CONCLUSION: To the best of our knowledge, this is the first report on KLK10 exon 3 unmethylated PCR product concentration as potential early epigenetic diagnostic marker in primary ovarian tumors. Taken into account the limitations in our study (small sample size and semi-quantitative PCR product analysis) further studies are strongly recommended.

Investigation of long noncoding RNAs expression profile as potential serum biomarkers in patients with hepatocellular carcinoma.

There is an increasing interest in using long noncoding RNAs (lncRNAs) as biomarkers in cancer. Predictive biomarkers in hepatocellular carcinoma (HCC) have great benefit in the choice of therapeutic modality for HCC. The aim of this study is to assess lncRNA-urothelial carcinoma associated-1 (lncRNA-UCA1) and WD repeat containing, antisense to TP53 (WRAP53) expression as novel noninvasive biomarkers for diagnosis of HCC in sera of HCC patients compared with chronic hepatitis C virus (HCV) patients and healthy volunteers and to analyze their relationship with respect to the clinicopathologic features. We retrieved HCC characteristic lncRNAs, lncRNA-UCA1 and lncRNA-WRAP53, based on the microarray signature profiling (released by LncRNADisease database). Quantitative reverse-transcriptase polymerase chain reaction assay (RT-qPCR) was then used to evaluate the expression of selected lncRNAs in the serum of 160 participants. Furthermore, in 20 of 82 HCC cases involved in the study, we examined the expression of lncRNA-UCA1 and lncRNA-WRAP53 in 20 HCC tissues and adjacent nontumor tissues and analyzed its correlation with the serum level of these lncRNAs. The prognostic significance of the investigated parameters in HCC patients was explored. We found that lncRNA-UCA1 and lncRNA-WRAP53 were significantly higher in sera of HCC than those with chronic HCV infection or healthy volunteers. Our data suggested that the increased expression of UCA1 and WRAP53 was associated with advanced clinical parameters in HCC. Of note, tissue levels of the chosen lncRNAs strongly correlate with their sera level. The combination of both lncRNAs with serum alpha fetoprotein resulted in improved sensitivity to 100%. The median follow-up period was 21.5 months. LncRNA-WRAP53 was significant independent prognostic markers in relapse-free survival. LncRNA-UCA1 and lncRNA-WRAP53 upregulation may serve as novel serum biomarkers for HCC diagnosis and prognosis.

Chronic myelogenous leukemia in sickle cell/beta 0-thalassemia.

Sickle cell/beta (0)-thalassemia (S/ß(0)-thal) is a compound heterozygous state for ßS and ß(0) thalassemia. There are rare reported cases of patients with sickle cell disease who developed hematological neoplasms including myeloid and lymphoid conditions; however, to the best of our knowledge, chronic myelogenous leukemia (CML) occurring in S/ß(0) -thal has been reported in one case and this is the second such report.

Hyperthyrotropinemia in relation to endothelial dysfunction in obese adolescents.

BACKGROUND: It has been shown that isolated elevation of thyroid stimulating hormone (hyperthyrotropinemia) is quite frequent in obese adolescents, but it is not clear whether this condition entails an increased risk related to body mass index (BMI). AIM: The aim of the current study was to investigate the relationship between hyperthyrotropinemia and endothelial dysfunction and other cardiovascular risk factors in obese adolescents. METHODS: Seventeen obese adolescents with hyperthyrotropinemia (TSH ≥ 4.2 µUI/ml) (group 1), 21 obese adolescents with normal thyroid stimulating hormone (TSH) level (group 2) and 10 age- and sex-matched healthy lean controls were included in the study. Fasting blood glucose (FBG), lipid profile, insulin resistance (HOMA-IR) and thyroid hormones were determined. All participants were subjected to ultrasound recording of brachial artery diameter at rest and after reactive hyperemia (FMD) for assessment of endothelial function. RESULTS: BMI and free triiodothyronine (fT3) levels were higher in obese adolescents with hyperthyrotropinemia (group 1) than in those with normal TSH (group 2) (p < 0.05), but there were no significant differences between the two groups in other metabolic parameters (lipid profile and HOMA-IR). The FMD was significantly low in obese adolescents (groups 1 and 2) relative to controls (3.4 ± 18% in group 1 and 3.6 ± 2.2% in group 2 vs. 10.4 ± 3.1% in control subjects), but there was no statistical difference between obese adolescents with hyperthyrotropinemia (group 1) and those with normal TSH (group 2). There were positive correlations between TSH and BMI (p = 0.03) and between fT3 and BMI (p = 0.04), but no correlation between TSH and lipid profile, HOMA-IR or FMD. CONCLUSIONS: Hyperthyrotropinemia is not related to endothelial dysfunction or metabolic risk factors in obese adolescents.

Expression of indoleamine 2,3-dioxygenase in acute myeloid leukemia and the effect of its inhibition on cultured leukemia blast cells.

Indoleamine 2,3-dioxygenase (IDO), a catabolizing enzyme of tryptophan, is a novel immunosuppressive agent blocking T-cell activation in neoplastic cells, including acute myeloid leukemia (AML) cells. IDO inhibitors as 1-methyl tryptophan (1MT) can abrogate IDO enzymatic activity and may result in an effective immune response. Mononuclear cells (MNCs) were separated from peripheral blood of 25 AML patients and 25 normal adults. IDO expression was detected by RT-PCR and its enzymatic activity by a colorimetric method. MNCs were cultured and the effects of Adriamycin, 1MT and a mixture of both on blast and lymphocyte cell counts after 24 and 72 h were detected. IDO mRNA and activity were detected in 52% of patients and absent in normal subjects. There was a significant correlation between IDO mRNA expression and its enzymatic activity in AML. IDO activity was correlated positively with patient's ages and negatively with hemoglobin levels. There was a significant inhibition of blast cells proliferation with Adriamycin and more inhibition when combined with 1MT. The inhibition was more after 72 h more than 24 h of culture. However, using 1MT alone showed no significant inhibitory effect on blast cells, with a significant increase in lymphocyte counts. Our study confirms the role of indoleamine 2,3-dioxygenase in tumor-induced immune tolerance and points to the possible benefit of 1-methyl tryptophan as immunotherapeutic enhancing the anticancer effects of traditional chemotherapeutics.

Detection of Epstein-Barr virus in breast carcinoma in Egyptian women.

BACKGROUND: The association of oncogenic EBV with breast carcinoma (BC) is still in controversy. AIM OF WORK: Assess the association of EBV with BC in Egyptian women and find possible relationship between prognostic factors of BC and EBV detection. SUBJECTS AND METHODS: Paraffin-embedded sections from 40 female patients with primary invasive BC; ductal (n=32) and lobular (n=8) and breast tissues from patients with fibrocystic disease (n=20) as control were screened for presence of EBV by EBV nuclear antigen-1 (EBNA-1) immunostaining and by PCR for EBV-DNA. RESULTS: 10/40 (25%) of the BC specimens stained positively for EBNA-1; EBNA-1 expression was restricted to a fraction 5%-60% of tumor epithelial cells. EBV-DNA was detected in 8/10 of BC specimens positive for EBNA-1. Control specimens were negative by both techniques. 7/8 (87.5%) of EBV-DNA positive tumors were associated with >3 lymph nodes involvement. CONCLUSION: EBV is associated with some invasive BC in Egyptian females and may play a role in their etiology.

Local expression of IL-4 and IL-5 in perennial allergic rhinitis and their modulation by topical corticosteroid therapy.

Perennial allergic rhinitis (PAR) is an increasing problem for which new and exciting therapies are being developed. A T(H)2-polarized cytokine pattern is thought to predominate regulating local IgE synthesis and cell recruitment in PAR and the development of intranasal steroids has resulted in several agents with quick actions, localized effects and great efficacy in its management. The aim of work was to determine the differences in the local expression of IL-4 and IL-5 in patients with PAR compared to non-atopic healthy controls and investigate the relationship between the expression of these cytokines and the clinical aspects of the disease. Also to evaluate local expression of these cytokines in some of these patients before and after treatment with intranasal steroids (fluticasone proprionate). Nasal biopsies from 37 patients with PAR before therapy and from 8 of them after receiving corticosteroids as local nasal spray were taken. PAR was confirmed by a history of perennial nasal blockage, discharge, and/or sneeze for at least 2 years before the study and by positive skin prick test. Also nasal biopsies were taken from 20 age and gender matched non-atopic controls. Biopsies were analyzed using a reverse transcription-polymerase chain reaction (RT-PCR) to investigate local expression of IL-4 mRNA. Enzyme immunoassay was used for estimation of IL-5 levels in the nasal mucosa. By using the ROC curve; (11 pg/ml) was estimated as a cut-off value for IL-5 where levels below this cut off were considered negative. This study showed that the most common causative allergens in PAR were mite dust, followed by wool & pigeon then mixed moulds. There was a significant relation between expression of IL-4 and IL-5 and the occurrence of allergic rhinitis where mRNA of IL-4 was detected in 17/37 [46%] of patient group and in 3/20 (15%) of the control group (P < 0.05). IL-5 levels were more than the calculated cut off value in 22/37 (59.5%) of patient group as compared to 4/20 (20%) in the control group (P < 0.01). Also a high significant association was found between IL-4 & IL-5 (P < 0.01) in patient group. However, no significant relation was found between signs & symptoms of AR or patients' age or gender and cytokines expression. Corticosteroid nasal spray treatment showed a significant reduction in IL-4 gene expression and IL-5 positivity (P < 0.05). It is concluded that IL-4 & IL-5 have an important role in the pathogenesis of PAR and corticosteroid nasal spray is effective in exerting an immunomodulatory activity by reducing IL-4 & IL-5 expression.

Implication of intercellular adhesion molecule-1 (ICAM-1) and serum N(G)-hydroxy-L-arginine (L-NHA) in the pathogenesis of systemic sclerosis.

In a trial to throw light on the implication of intercellular adhesion molecule-1 (ICAM-1) and N(G)-hydroxy-L-arginine (L-NHA) in the pathogenesis of systemic sclerosis or Scleroderma, (SSc), their serum levels were estimated in twenty SSc patients using ELISA and high performance liquid chromatography respectively. In situ "local" expression of ICAM-1 in lesional skin of these patients was also assessed using biotinstreptavidin amplified detection system. Patients were divided into 3 groups according to the cutaneous extension of sclerosis (Grades I; II & III). A significant (P < 0.001) difference was found between patients (n = 20) and controls (n = 10) regarding soluble ICAM-1 (s ICAM-1) and L-NHA levels. Among patients, a significant difference (P < 0.001, 0.05 respectively) in sICAM-1 & L-NHA serum levels was found between patients who had musculoskeletal manifestations and those who had not. A significant (P < 0.001) difference in L-NHA level was found between patients with grade I, II, III. Among patients, there was a negative correlation (r = -0.413) between serum sICAM-1 and the duration of the disease, and a positive correlation (r = +0.514) between sICAM-1 and L-NHA serum levels. 4 patients (23.6%) showed mild immunostaining, 8 patients (47%) showed moderate staining, and 5 patients (29.4%) showed intense staining, while control specimens showed negative immunostaining. In conclusion, ICAM-1 and serum L-NHA are probably implicated in the pathogenesis of SSc. Elevated sICAM-1 and L-NHA serum could be used as a quantitative marker of tissue sclerosis, allowing better follow up of patients.

Preoperative serum inhibin levels in patients with ovarian tumors.

AIM: To assess the value of preoperative determination of serum inhibin levels in the prediction of malignancy in women with ovarian tumors. The prediction of malignancy not only helps patient counseling regarding prognosis and extent of surgery but also allows for proper specialist referral. METHODS: Fifty women with clinically diagnosed ovarian tumors before surgery (patients group) and 32 healthy non-pregnant women in the early follicular phase of their cycle (controls) were studied. Serum inhibin (total) levels and CA125 were determined using immunoenzymometric assay and enzyme immunoassay, respectively. RESULTS: In the patients group, 31 women had malignant ovarian tumors and 19 had benign tumors. Mean (SEM) serum inhibin levels were 0.94 (0.13) U/mL in the control group, 0.91 (0.7) U/mL in women with benign tumors and 1.9 (0.12) U/mL in women with malignant tumors; the differences are statistically significant (P < 0.01). Inhibin levels were significantly higher in women with late-stage malignant ovarian tumors (III and IV) than in early stages (I and II) and showed no significant difference in relation to menopausal status or the presence of ascites. Taking the 95th centile inhibin level in the control group (1.155 U/mL) as a discriminator level, all women with benign tumors were negative while 67.7% of women with malignant tumors were positive. The inhibin level testing predicted malignancy with a sensitivity of 67.7%, 100% specificity, 100% positive predictive value and 65.52% negative predictive value. Combining CA125 levels (>35 U/mL) with serum inhibin levels (> 1.155 U/mL) improves sensitivity of predicting malignancy to 83.87% and negative predictive value to 75% while maintaining a specificity of 100% and a positive predictive value of 100%. Combining serum inhibin with sonography achieves 96.77% sensitivity and 73.68% specificity for detecting malignancy. CONCLUSION: Preoperative serum inhibin levels in women with ovarian tumors are useful in the prediction of malignancy especially when combined with sonography.