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.

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.

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.

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.

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.