Differential expression of miR-1297, miR-3191-5p, miR-4435, and miR-4465 in malignant and benign breast tumors.

OBJECTIVES: MicroRNAs (miRs) are a class of small non-coding RNAs which are associated with tumor growth and progression. In the present study, we assessed the expression of selected miRs in malignant, benign, and adjacent normal breast tissues. MATERIALS AND METHODS: The expression of miR-1297, miR-3191-5P, miR-4435, and miR-4465 were evaluated in malignant (n=50), benign (n=35), and adjacent normal breast tissues (n=20) using qRT-PCR. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were generated for evaluating the diagnostic values of miRs. To evaluate diagnostic efficacy, miRs-based score was obtained using the logistic regression model. RESULTS: Among malignant tumors, the expression of miR-1297, miR-3191-5p, and miR-4435 was significantly lower (P=0.024, P<0.001 and P=0.031), respectively. The expression of miR-4465 was higher (P=0.023) than that of normal tissue. The expression of these miRs was lower than those of benign tumors (P<0.01, P<0.001, P<0.0001, and P<0.01, respectively). We observed a positive correlation between miR-4465 expression levels and tumor stage (P=0.042) and a negative correlation with grade and Ki-67 score (P<0.05). The AUCs for miR-1297, miR-3191-5p, miR-4435, and miR-4465 in malignant tumors versus normal tissues were 0.784, 0.700, 0.976, and 0.865 and versus benign tumors they were 0.938, 0.857, 0.981, and 0.785, respectively. The optimal logit(P) value of 0.262 distinguished malignant from normal subjects with a sensitivity of 0.91, specificity of 0.85, and an overall accuracy of 0.89. CONCLUSION: The panel of these miRs are suggested as possible onco-miRs(miR-4465) or tumor suppressor-miRs (miR-3191-5P, miR-1297, miR-4435). Overall, our results indicated that these miRs could be introduced as diagnostic biomarkers in breast cancer patients.

Expression level of VLDL receptor and VLDL-c levels in the malignant and benign breast tumors: The correlation with miRNA-4465 and miRNA-1297.

Breast cancer as one of the most prevalent cancers has high morbidity and mortality. Very low-density lipoprotein receptor (VLDLR) is a multifunctional receptor which plays a principal role in the tumor development through affecting cell metastasis and proliferation. The VLDLR as a target for miRNA-4465 and miRNA-1297 was predicted using bioinformatics analysis. Tissue specimens of malignant (n = 50), benign (n = 35) and corresponding normal breast (n = 20) were considered to evaluate the expression of VLDLR using RT-qPCR and western blotting. The VLDL cholesterol (VLDL-C) levels were quantified using a colorimetric assay. The relative VLDLR expression was found in the malignant tumors, which was significantly lower than that in the normal tissues (P<0.05). The expression levels of VLDLR had no significant difference between malignant and benign tissues (P>0.05). Correlation analysis revealed that the VLDLR expression level had a direct correlation with miRNA-1297 (R=0.566, P<0.05), but a reverse one with miRNA-4465 (R = -0.663, P<0.0001). The VLDL-C level in the malignant and normal tissues was lower than that in the benign tumors, which was not significant (P>0.05). The expression levels of VLDLR in E+P-H- (ER+,PR-,HER2-) tumors were higher than those in other subtypes (P<0.05). Furthermore, a negative correlation was found between the VLDLR expression level and the Ki 67% score. These data revealed that the lower expression of VLDLR mediated by the high expression levels of miRNA-4465 may be involved in the development of breast cancer. These results might provide some evidence for the effect of VLDLR on the breast cancer.

Evaluation of LDL receptor and Scavenger Receptor, Class B, Type 1 in the malignant and benign breast tumors: The correlation with the expression of miR-199a-5p, miR-199b-5p and miR-455-5p.

AIMS: The purpose of present study was to examine the correlations of LDL (LDLR) and HDL (SR-B1) receptors with lipoproteins, miR-199a-5p, miR-199b-5p, miR-455-5p in the malignant and benign breast tumors. METHODS: Total cholesterol-rich-lipoproteins and the receptors were determined using enzymatic-homogeneous and ELISA methods. The expression levels of miRNAs were detected by qRT-PCR. RESULTS: Receptor expressions and lipoproteins concentration were significantly higher in the malignant tumors (p < 0.05). Positive correlation was found for LDLR with Ki67% and Her2+. HDL-C content of TNBC tumors was higher than those of Non-TNBC (p < 0.05). The expression level of miR-199a-5p was found to be downregulated significantly in the malignant tumors of <2 cm, TNBC, HER2- or stage3. The expression of miR-199b-5p was downregulated in the malignant tumors and was negatively associated with TNBC, stage and Her2+. The expression of miR-455-5p was significantly correlated with Her2- (p < 0.05). A positive correlation was observed for SR-B1 or LDLR with HDL-C or LDL-C and also for SR-B1 with LDLR, although a reverse association was detected for the expression of miR-199b-5p with LDLR in the malignant tumors (p < 0.05). No significant correlations were found for miR-199a-5p or miR-455-5p with LDLR or SR-B1 expressions and also for LDL-C and SR-B1 with clinicopathological features (p ≥ 0.05). CONCLUSIONS: Mechanisms potentially involved in the present findings may be due to the lipid internalization and lipoprotein consumption through LDLR and SR-B1 over expression. It is noteworthy that the expression of miR-199b-5p is negatively correlated with LDLR which may suggest it as a suppressor for LDLR expression in the breast cancer.

Soluble guanylate cyclase isoenzymes: The expression of α1, α2, ß1, and ß2 subunits in the benign and malignant breast tumors.

Soluble guanylate cyclase (sGC) encompasses α and ß subunits. This study examined the expression of α1, α2, ß1, and ß2 subunits in the malignant and benign breast tumors using the Western blot analysis. Both benign and malignant tumors showed a significantly higher expression of the α1 subunit in comparison with normal tissues (p < 0.0001). In contrast, the expression of α2 and ß2 sGC were significantly lower in these tumors than normal tissues (p < .0015 and p < .001, p < .007 and p < .0001, respectively). The expression level of α1 sGC was significantly correlated with ER + PR+ (p < .0001). A significant correlation was also detected for sGC-α1 and -α2 expression with c-erbB2-negative status (p < .01). However, the expression level of sGC was not associated with tumor stage, tumor grade, or other clinicopathological features. In conclusion, as the expression of α1 sGC is upregulated and α2 and ß2 sGC are downregulated in malignant breast tumors. Variations in the expression of sGC isoenzymes may be suggested as an indicator to confirm the enzyme antitumor activity.

Differential expression of alternative transcripts of soluble guanylyl cyclase, GYCY1a3 and GUCY1b3 genes, in the malignant and benign breast tumors.

Extensive alterations in splicing is one of the molecular indicator for human cancers. Soluble guanylyl cyclase (sGC), an obligatory heterodimer, is composed of α1 and ß1 subunits. Each subunit is encoded by a separate gene, GUCY1a3 and GUCY1b3, correspondingly. sGC activity has been regulated by an alternative splicing and it has an important effect on the breast cancer. sGC alternative splicing has been evaluated in the 55 malignant, 25 benign and 30 normal breast tissues using qRT-PCR and RT-PCR. The differences between groups were analyzed by Mann-Whitney U. The expression of six different splice forms have been detected, three for α1 and three for ß1 sGC. Expressions of Tr1, Tr2 ß1 sGC and Tr7, Tr6 α1 sGC mRNA in the malignant breast tumors were significantly lower than those of benign and normal breast tissues. However, the expression of Tr3 α1 sGC mRNA was significantly higher than that of benign and normal tissues. Present data have provided some evidences for an alteration in the expression of α1 and ß1 sGC alternative splicing forms which may contribute to the loss of sGC functions in the breast cancer. The observed information might be discussed by the cGMP status.

Hypermethylation pattern of ESR and PgR genes and lacking estrogen and progesterone receptors in human breast cancer tumors: ER/PR subtypes.

BACKGROUND: The option of endocrine therapy in breast cancer remains conventionally promising. OBJECTIVE: We aimed to investigate how accurately the pattern of hypermethylation at estrogen receptor (ESR) and progesterone receptor (PgR) genes may associate with relative expression and protein status of ER, PR and the combinative phenotype of ER/PR. METHODS: In this consecutive case-series, we enrolled 139 primary diagnosed breast cancer. Methylation specific PCR was used to assess the methylation status (individual test). Tumor mRNA expression levels were evaluated using real-time RT-PCR. Immunohistochemistry data was used to present hormonal receptor status of a tumor (as test reference). RESULTS: Methylation at ESR1 was comparably frequent in ER-breast tumors (83.0%, P< 0.001; sensitivity = 83.0%, specificity = 65.2% and diagnostic odds ratio, DOR = 12.0) and strongly correlated with ER-/PR- conditions (Cramer's V= 0.44, P< 0.001). Methylated PgRb promoter frequently was observed in tumors recognised as ER- or negative ER/PR (77.1%, P< 0.01). Assessment of DNA methylation of ESR1 harbouring methylation at PgRb was a case significantly suggested to be able to detect the lack of ER/PR expressions (55.6%, P< 0.01; sensitivity = 80.6%, specificity = 68.7% and DOR = 8.7). However, methylated PgRb was quite acceptable determinant to contribute with methylated ESR1 to rank tumors as ER-/PR- (64.4%, P< 0.01; sensitivity = 78.0%, specificity = 62.5% and DOR = 6.0). CONCLUSIONS: Despite the methylation status of ESR1 showed preponderant contribution to tumoral phenotypes of ER- and ER-/PR-, the hypermethylation of PgRb seem another epigenetic signalling variable actively associate with methylated ESR1 to show lack of ER+/PR+ tumors in breast cancer.

Mutation Screening of BRCA Genes in 10 Iranian Males with Breast Cancer.

Male breast cancer is a rare disease with an increasing trend. Due to limited information especially about the genetic basis of the disease in Iran and the lower age of its onset, the disease requires more attention. The aim of this study was to screen the male patients with breast cancer for BRCA mutations as well as tissue markers of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor (HER-2) and cytokeratin 5/6 (CK5/6). Ten Iranian males with breast cancer were selected regardless of their histologic subtypes, age and family history from patients referred to Mehrad, Day and Parsian hospitals in Tehran, Iran, during a two-year period. Paraffin blocks of the tumoral regions were tested for ER, PR, HER-2 and CK5/6 immunostaining. DNA extraction was carried out on the EDTA blood samples followed by Sanger sequencing. Immunohistochemistry results for ER, and PR were negative in 2 out of 10 patients, while the results of HER-2 and CK5/6 were negative in all the cases. A missense mutation in exon 18 of BRCA1 and a nonsense mutation in exon 25 of in BRCA2 were detected in one patient each. Both patients belonged to luminal A subtype. Despite the low number of patients in this study, it could be concluded that mutations in BRCA1 and BRCA2 occur in male breast cancer patients of luminal A subtype. The negative status of the tissue markers could not be used for the prediction of BRCA mutations.

Evaluation of RIP1K and RIP3K expressions in the malignant and benign breast tumors.

Receptor-interacting protein kinase 1 (RIP1K) and RIP3K belong to RIPK family, which regulate cell survival and cell death. In the present investigation, the expression levels of RIP1K and RIP3K were evaluated in the 30 malignant, 15 benign, and 20 normal breast tissues, and their correlation with clinicopathological characteristics was also studied. The expression levels of RIP1K and RIP3K were determined, by western blot analysis. The relative RIP1K expression was significantly higher in the malignant and benign tumors when compared to those of normal tissues (P < 0.0001 and P < 0.001, respectively). However, the expression level of RIP3K was significantly lower in the malignant tumors than those of normal and benign values (P < 0.001 and P < 0.01, respectively). Positive significant correlation was found for RIP1K expression with tumor size (P < 0.001), grades (P < 0.0001), and c-erbB2 (P < 0.001), but negative significant correlation was detected with patient's age (P < 0.001), estrogen receptor (ER) (P < 0.001), progesterone receptor (PR) (P < 0.01), and P53 (P<0.01) status. RIP3K expression was significantly lower in the pre-menopauses (P < 0.01), grade III (P < 0.05), ER-negative (P < 0.05), and c-erbB2-negative malignant tumors, but no correlation was detected with tumor size, PR, and P53 status. No significant correlation was observed for RIP1K and RIP3K expressions with Ki67 and Her2. Based on the present results, it is concluded that reduction of RIP3K expression in the malignant breast tumor might be an important evidence to support the antitumor activity of this enzyme in vivo. However, RIP1K expression was shown to be higher in the malignant breast tumors than those of normal and benign breast tissues, which probably designates as a poor prognostic factor.

Double Heterozygosity of BRCA2 and STK11 in Familial Breast Cancer Detected by Exome Sequencing.

BACKGROUND: Germ-line mutations of BRCA1 and BRCA2 genes are responsible for approximately 25-30% of dominantly inherited familial breast cancers; still a big part of genetic component is unknown. The aim of this study was to investigate genetic causes of familial breast cancer in a pedigree with recessive pattern of inheritance. METHODS: We applied exome sequencing as a useful approach in heterogeneous diseases gene identification in present study for familial breast cancer. Sanger sequencing was applied for validation and segregation analysis of mutations. RESULTS: Here, we describe a family with three affected sisters of early-onset invasive ductal carcinoma due to heterozygous frame shift mutation rs80359352 in BRCA2 gene as the first report in Iranian patients in association with a novel missense SNP of STK11 (p.S422G). These mutations are inherited from their normal father. CONCLUSION: Despite apparent recessive pattern of inheritance a dominant gene (here BRCA2) can be involved in pathogenesis of hereditary breast cancer which can be explained by incomplete penetrance of BRCA2 mutations.

Association of folate and other one-carbon related nutrients with hypermethylation status and expression of RARB, BRCA1, and RASSF1A genes in breast cancer patients.

UNLABELLED: Dietary methyl group donors could influence the hypermethylation status of certain putative genes. The present study explored the possible associations of dietary intake of one-carbon metabolism-related nutrients with promoter hypermethylation status and expression of retinoic acid receptor-beta (RARB), breast cancer-1 (BRCA1), and Ras association domain family-1, isoform A (RASSF1A) genes in Iranian women with breast cancer (BC). The hypermethylation status was investigated in 146 dissected BC tissue samples using methylation-specific PCR. The expression level was evaluated by real-time RT-PCR. Dietary nutrients were estimated using a validated 136-item food frequency questionnaire. Expression levels of the genes were associated with the unmethylated status of related promoters (p < 0.05). The crude dietary folate and adjusted cobalamin intakes were inversely associated with methylated RARB and BRCA1. Low intake of residual folate and cobalamin was correlated with the methylated status of RARB for subjects at <48 years of age, and folate alone was linked to BRCA1 at >48 years of age. High dietary intake of riboflavin and pyridoxine was the only determinant of the methylated promoter of RARB at odds ratios (ORs) of 4.15 (95 % confidence interval (CI) 1.28-13.50) and 2.53 (95 % CI 1.14-3.83) in multivariate models, respectively. One-carbon nutrients most often correlated inversely with the methylation-influenced expression of RARB. Although high folate intake increased the chance of unmethylation-dependent overexpression of BRCA1 3-fold, cobalamin and methionine were inversely linked to methylation-mediated expression. Nutritional epigenomics less actively influenced RASSF1A. These findings provide new insights into and a basic understanding of the selective contributions of individual B vitamins on hypermethylation and methylation-related expression of RARB and BRCA1 in BC. KEY MESSAGE: Hypermethylation at promoters of RARB, BRCA1, and RASSF1A is associated with reduced transcript levels of the respective gene in primary breast cancer tissue samples. Dietary folate and cobalamin intake is inversely associated with methylated RARB and BRCA1. High dietary intake of riboflavin and pyridoxine is associated with increased methylation in the RARB promoter. There is evidence for the age-dependent effects of nutrient intake on promoter methylation status. Bioavailability to the pool of nutrients might determine selectivity.

Personalized evolutionary hypothesis in genomics and auxiliary lymph node through diverse subtelomeric signal profile.

Few available data on the genomic-somatic evolution in breast cancer create limitation to provide the appropriate clinical managements. As an example, human subtelomeres (ST) are diverse-prone and variable targets. STs, as hot spots, have positive and negative impacts on the status of health and malady. We showed higher subtelomere signal copy number (SCN) of specific chromosomes in genomics than in auxiliary lymph node (ALN). Dissimilarity of signal intensity (SI) is found for all chromosomes. Significantly higher SI in genomics than in ALN cells were specified as chromosomes 5, 6, 9-12, 16-19 for weak; 1, 5-9, 19, X for medium; and 2, 5, 9, 10, 16, 18 for strong SI. For lacking, and presence of one and two SCNs; p/q ratio reflected differences for all chromosomes; but, 2, 3, 5, 7, 8, 10, 16, 18, 20, and X chromosomes were involved for three SCN. Chromosomes 1, 4, 9, 12, 17-19 lacked three SCN in ALN and lymphocytes. Weak SI ratio was higher in p- than in q-arm in majority of chromosomes. Manner of evolution and diversity in p- and q-arms is expressive of a novel definition as two diverse domains with a personalized insight. These data have been accompanied by periodic charts as ST array profiles which provide specific and individualized pattern in breast neoplasm. Such profiling at genomics level could be considered as a prediction through the patients' life. Moreover, subtelomere territory by interacting with protein expression of Ki67, cyclin D1, and cyclin E; and molecular targets including telomere length at genomics and somatic level provides package of information to bridge cancer cell biology to the cancer clinic as "puzzling paradigm."

Plasma total homocysteine level in association with folate, pyridoxine, and cobalamin status among Iranian primary breast cancer patients.

Recently the elevated plasma total homocysteine (tHcy) concentration has been concerned as the secondary feature of tumoral proliferation and enhances the likelihood of thrombogenesis in cancer patients. The objective of this study was to determine the associations between folate, cobalamin, and pyridoxine with fasting plasma tHcy concentration in breast cancer (BC) patients. The intake levels of nutrients were assessed using a validated food frequency questionnaire in 141 newly diagnosed BC patients. The plasma tHcy and pyridoxal-5-phosphate were measured using high performance liquid chromatography with fluorescence detector. Plasma tHcy levels were observed to be significantly higher among BC participants with Stage III where the plasma concentrations of folate was also comparatively less (P < 0.05) than other stages. Dietary pyridoxine was even being consumed less at this stage (P < 0.05). The plasma, dietary, and residual variables of folate were inversely correlated with plasma tHcy concentration (P < 0.05). Dietary cobalamin was also associated negatively with tHcy (P < 0.05). The odds ratio of comparing the highest tertile of plasma cobalamin (>394 pmol/l) and folate (>11.4 ng/ml) vs. the lowest categories were associated with reduced odds of high tHcy occurrence with 0.20 (95% confidence interval: 0.04-0.98) and 0.14 (95% confidence interval: 0.03-0.64), respectively. In conclusion, nutrition-related methyl-group insufficiency could lead to imbalance in tHcy metabolism, as a possible cancer marker.

Essential oil variation in the populations of Artemisia spicigera from northwest of Iran: chemical composition and antibacterial activity.

CONTEXT: Artemisia spicigera C. Koch (Asteraceae) is a perennial shrubby herb and is generally distributed in Armenia, Iran, and Middle Anatolia. This species traditionally has been used in medicines. OBJECTIVE: The aim of this research is to study the chemical composition and antibacterial activity of essential oils from Artemisia spicigera populations in northwest of Iran. MATERIALS AND METHODS: The essential oil of A. spicigera was obtained by hydrodistillation from eight populations collected from different regions of East Azerbaijan and West Azerbaijan provinces (Iran) and analyzed by gas chromatography-mass spectrometry (GC-MS). The antibacterial activity of the oils was investigated against four Gram-positive and four Gram-negative bacteria using MIC determinations and the agar-gel diffusion method. RESULTS: Fourteen compounds were identified as the main components of the essential oils and the most abundant constituents are 1,8-cineole, camphor, α-thujone, camphene, ß-thujone and p-cymene. Essential oil of population No. 1 showed the highest activity against Escherichia coli, Enterobacter aerogenes, Serratia marcescens and Staphylococcus aureus but the highest activity against St. saprophyticus, Bacillus megaterium, and B. cereus was found with population No. 6 and for Citrobacter amalonaficus with population No. 5. MIC values of essential oils ranged from 6 µg/mL against Bacillus megaterium to 12 µg/mL against Citrobacter amalonaficus. DISCUSSION: This study demonstrates the occurrence of 1,8-cineole/camphor/camphene chemotype of A. spicigera but there is also significant chemical variation between the studied populations. The findings showed the studied oils have good antibacterial activity, and thus potential to be used as natural health products.

Evaluation of PDE5 and PDE9 expression in benign and malignant breast tumors.

BACKGROUND AND AIMS: Phosphodiesterases 5 and 9 (PDE5, PDE9) are enzymes responsible for regulating second messenger signaling by hydrolyzing 3',5' cyclic guanosine monophosphate (cGMP). PDE isoforms are deregulated in some types of human cancer. The present study was carried out to evaluate the expression of phosphodiesterase isoenzymes, PDE5 and PDE9, in benign and malignant breast tumors. METHODS: The expression levels of PDE5 and PDE9 were assayed in malignant and benign breast tumors and corresponding normal breast tissues using quantitative real-time RT-PCR. Moreover, the correlation between PDE5, PDE9 relative expression and clinicopathological characteristics were analyzed. RESULTS: The relative expressions of PDE5 and PDE9 in malignant tumors were significantly higher than those of respective normal breast tissues and benign tumors (5.5-fold, p <0.001 and 6-fold, p <0.001, respectively). Furthermore, a significant positive correlation was found between PDE5 and PDE9 overexpression and tumor grade, stage, and lymph node involvement. However, a negative correlation was observed with age. CONCLUSIONS: Based on the present results, it is concluded that assessment of PDE5 and PDE9 expression may be useful in the differential diagnosis of benign and malignant breast disease and successful treatment of breast cancer. To the best of our knowledge, this is the first study to show that PDE5 and PDE9 expression levels are higher in malignant breast tumors than those of normal and benign breast tissue.

Expression of cGMP-dependent protein kinase, PKGIα, PKGIß, and PKGII in malignant and benign breast tumors.

Cyclic GMP-dependent protein kinases (PKG) constitute a small family of enzymes that are encoded by two genes. Two major forms of PKG have been identified in mammalian cells, PKG I and PKG II. In addition, there are two splice variants of PKG I, which are designated as Iα and Iß. There are increasing evidences that PKG can play an important role in the inhibition of cell proliferation and induction of apoptosis. In our previous studies, the inhibitory effects of cGMP/PKG on the cell growth were indicated using breast cancer cell lines. Accordingly, the present study was designed to compare the expression levels of three PKG isoforms in normal, benign, and malignant breast tissues. The expression level of PKG isoforms was assayed using quantitative real-time RT-PCR. The correlation between relative expression of PKG isoforms and clinicopathological characteristics were also analyzed. Downregulation of PKG isoforms was observed in the malignant and benign tumors when compared to those of respective normal tissues. No significant correlation was found between PKGIα, PKGIß, and PKGII expression and clinicopathological features. The present study is the first to evaluate the expression level of PKG isoforms PKGIα, PKGIß, and PKGII in the malignant and benign breast tumors. Reduction in the PKG expression is an important evidence to support the antitumor activity of this enzyme in vivo.

ER-negative /PR-positive breast carcinomas or technical artifacts in immunohistochemistry?

BACKGROUND: Evaluation of estrogen (ER) and progesterone (PR) receptors is important in the management and prognosis of breast cancer patients. Immunohistochemistry (IHC) is currently the worldwide accepted methodology for detection of ER/PR receptors in breast carcinomas. However, technical artifacts may alter the results. Since most authorities believe that there are no true ER-negative/PR-positive breast tumors, therefore we hypothesized that technical artifact in IHC might cause ER-negative/PR positive cases. METHODS: The clinical records of 2432 patients treated by surgery at six community hospitals for different histologic subtypes of breast carcinoma were reviewed. Among them, 43 (1.8%) patients reported as ER-negative/PR-positive were re-evaluated in a reference laboratory. Expressions of ER and PR were evaluated by IHC on the same paraffin block used for the initial testing. RESULTS: The repeat study showed that of the 43 patients with the initial results of ER-negative/PR-positive, 24 (55.8%) were ER-positive/PR-positive, 15 (34.9%) were ER-negative/PR-negative, and 4 (9.3%) were ER-positive/PR-negative. In none of the 43 cases were the initial results (ER-negative/PR-positive) confirmed. CONCLUSION: Technical artifacts in IHC may alter ER/PR results in breast carcinomas. The technical factors affecting steroid receptor IHC ought to be properly controlled to provide reliable results.

Evolutionary hypothesis of telomere length in primary breast cancer and brain tumour patients: a tracer for genomic-tumour heterogeneity and instability.

It was previously reported that tumour samples had shorter telomeres than the surrounding normal tissue. Hereby, the initial sign of correlation between malignant tissue and telomere behaviour could be noticed. Bridging knowledge between germ and somatic cells could facilitate understanding cellular evolution. The aim of our investigation was to provide evidence for the evolutionary hypothesis of TL (telomere length) in primary BC (breast cancer) and BTs (brain tumours), which might be applied as a prognostic and/or predictive marker. DNA extraction from the frozen tissues was performed using high pure PCR template preparation kit. Standard protocol of Telo TTAGGG Telomere Length Assay kit, a non-radioactive chemiluminescent assay, was used. The protein expression in extracted cells was analysed by immunofluorescence. We also detected telomerase activity. The G/T (genomic/tumour ratio) for TL in two groups of patients affected with primary BC and primary BT revealed significant differences in both BC patients (P = 0.025) and in BTs (P = 0.001). The pattern of telomere signals by Q-FISH (quantitative fluorescent in situ hybridization) show that in all samples, except one, SI (signal intensity) has been significantly decreased in tissue related to blood, either in BC patients or in patients with BTs (0.041≥P≥0.001). However, the data achieved by Q-FISH support the results of Southern blot. These data reflect a significant diversity either in BC or in BT patients, providing evidence for the evolutionary hypothesis of TL in cancer development and progression.

Importance of ATM gene as a susceptible trait: predisposition role of D1853N polymorphism in breast cancer.

The involvement of ATM gene and specifically, the important role of D1853N polymorphism, as a three-hit hypothesis has been previously reported in an Iranian proband affected with brain tumor and this polymorphism could be screened in her relatives as well. The aim of present study was to investigate the involvement of D1853N polymorphism as a predisposition factor in 129 Iranian patients affected with primary breast cancer and 248 sex- and age-matched healthy controls. Mutant allele-specific PCR amplification (MASA) assay was performed to analyze the D1853N polymorphism in the ATM gene. The frequency of D1853N polymorphism in cases, internal and external controls was 31.0% (40/129), 26.9% (28/104) and 12.5% (18/144), respectively. The frequency of D1853N in total control groups, including normal external control and pedigree internal control, was 18.6% (46/248). The odds ratio was calculated with the logistic regression test, with an estimated relative risk of 2.579 (P=0.005). The significant difference was observed between the patient-carriers of this alteration and external controls (P=0.001). The number of controls harboring D1853N polymorphism was higher in internal control compared to external controls, and the difference was statistically significant (P=0.004). The significant difference was observed between the patient-carriers and external controls and could be considered as a predisposing and diagnostic marker in the population and specifically in the cancer-prone pedigrees.

Androgen receptor gene CAG repeat polymorphism and breast cancer risk in Iranian women: a case-control study.

We investigated the association between polymorphic expansion of trinucleotide CAG repeats in androgen receptor (AR) gene and breast cancer risk among Iranian women in a matched case-control study. There was a strong overall association between per CAG repeat increments in average repeat length and the risk of the malignancy [OR=3.56; 95% CI, 2.80-5.29]. Women carrying one or two alleles with [CAG]n repeat ≥22 units were at increased risk of breast cancer [OR=2.03; 95% CI, 1.56-2.6]. The risk was significantly increased in homozygous longer repeats, versus homozygous alleles <22. We observed reduced risk of developing the tumor in positive familial breast cancer subjects carrying repeats ≥22 and 23. Homozygosity for the longer [CAG]n repeats may be linked to the increased breast cancer risk. In contrast to previous reports, longer AR [CAG]n repeat alleles may decline the risk among women with a familial breast cancer.

The effect of modifiable potentials on hypermethylation status of retinoic acid receptor-beta2 and estrogen receptor-alpha genes in primary breast cancer.

Epigenetic silencing of retinoic acid receptor-beta2 (RARbeta2) and estrogen receptor-alpha (ERalpha) expressions have been revealed to be important in the development of approaches for diagnosis and therapy of breast cancer. We aimed to explore the correlation of some potential factors with the hypermethylation status of RARbeta2 and ERalpha genes among Iranian breast cancer patients. The hypermethylation status was investigated in 137 dissected tissues from primary breast cancer patients through methylation-specific PCR. Overall, the methylation frequencies of RARbeta2 and ERalpha genes were observed in 36.5 and 51.1% of participants, respectively. The hypermethylated RARbeta2 was associated with younger age at diagnosis and negative family history of breast cancer. The hypermethylation of ERalpha was correlated positively with smoking, duration of estradiol exposure, ER-negativity in tumors and body mass index (at 5 years ago). The plasma levels of folate and vitamin B(12) were inversely related to the hypermethylation status of ERalpha, after controlling for covariates. The risk of ERalpha hypermethylation was increased with high plasma level of total homocysteine. In conclusion, our data provide new insights into the possible effect of some lifestyle-related factors on the aberrant methylation drift of ERalpha and RARbeta2 genes in breast cancer.