The High Frequency of PIK3CA Mutations in Iranian Breast Cancer Patients.

In breast cancer, somatic mutations of PIK3CA oncogene are common. We investigated the mutational status of exons 9 and 20 of the PIK3CA gene by polymerase chain reaction and direct sequencing in 80 breast tumors, and observed that 45% of these contained PIK3CA mutations in the mentioned exons. These mutations were found more in progesterone receptor positive and Her2- tumors, but this association did not reach a statistically significant level. Also, we observed a significant association between PIK3CA mutations and low-grade tumors. In our study, PIK3CA mutations were related to good and moderate prognosis in breast cancer patients.

Epigenetic marks in estrogen receptor alpha CpG island correlate with some reproductive risk factors in breast cancer.

Reproductive backgrounds, such as age at menarche and menopause, age of first full-term pregnancy (FFTP), number of full-term deliveries and oral contraceptive use are main hormone-related risk factors of breast cancer. It seems that the mentioned factors may affect the risk of breast cancer by enhancing the duration of exposure to estrogen as a potent carcinogen for breast tissue, but the molecular mechanism which links each risk factor to breast cancer is unclear. Estrogen mainly works via its nuclear receptor (ERα). As epigenetic alterations such as CpG methylation are potential links between endogenous or exogenous exposures and genome, we hypothesized that hormone-related risk factors may correlate with the epigenetic marks of the ERα promoter in breast tumors. In the present study, the CpG methylation status of the ERα gene in 99 samples of breast tumors belonged to women with different reproductive histories was evaluated. The reproductive history data were collected from patients. ERα CpG methylation was investigated by methylation specific PCR in DNA samples were obtained from the breast tumors. We could show that some of the hormone-related risk factors (early FFTP and increased number of pregnancies) were inversely correlated with epigenetic marks in ERα gene in breast tumors. Other hormone-related risk factors such as age of menarche and menopause and oral contraceptive use did not show any association with ERα methylation. It seems that pregnancy-related risk factors in comparison with other hormone-related factors work via different mechanism. As ERα methylation is a poor prognosis marker in breast tumors, its association with some modifiable reproductive risk factors (FFTP age and numbers of pregnancies) reiterates the importance of programming reproductive life style not only for prevention of breast cancer but also in favoring the prognosis of the affected women. The exact molecular mechanisms of the observed correlation need more investigation in the future.

The mitochondrial ATPase6 gene is more susceptible to mutation than the ATPase8 gene in breast cancer patients.

BACKGROUND: Breast cancer is the most common malignancy in women throughout the world. Mitochondria play important roles in cellular energy production, free radical generation and apoptosis. Identification of mitochondrial DNA mutations and/or polymorphisms as cancer biomarkers is rapidly developing in molecular oncology research. METHODS: In this study, the DNA alterations of the mitochondrial ATPase 6 and 8 genes were investigated in 49 breast cancer patients using PCR amplification and direct DNA sequencing on mtDNA. A possible association between these variants and tumorigenesis was assessed. Furthermore, the impact of non-synonymous substitutions on the amino acid sequence was evaluated using the PolyPhen-2 software. RESULTS: Twenty eight distinct somatic mitochondrial DNA variants were detected in tumor tissues but not in the corresponding adjacent non-tumor tissues. Among these variants, 9 were observed for the first time in breast cancer patients. The mtDNA variants of A8384 (T7A), T8567C (I14T), G8572A (G16S), A9041G (H172R) and G9055A (A177T) showed the most significant effects probably due to damaging changes to the resulting protein. Furthermore, non-synonymous amino acid changing variants were more frequent in the ATPase6 gene compared to the ATPase8 gene. CONCLUSION: Our results showed that the ATPase6 gene is more susceptible to variations in breast cancer and may play an important role in tumorigenesis by changing the energy metabolism level in cancer cells.

Accuracy of estrogen and progesterone receptor assessment in core needle biopsy specimens of breast cancer.

BACKGROUND: Diagnosis of breast cancer is completed through core needle biopsy (CNB) of the tumors but there is controversy on the accuracy of hormone receptor results on CNB specimens. OBJECTIVES: We undertook this study to compare the results of hormone receptor assessment in CNB and surgical samples on our patients. PATIENTS AND METHODS: Hormone receptor status was determined in CNB and surgical samples in breast cancer patients whose CNB and operation had been performed in this institute from 2009 to 2011 and had not undergone neoadjuvant chemotherapy. RESULTS: About 350 patients, 60 cases met all the criteria for entering the study. The mean age was 49.8 years. Considering a confidence interval (CI) of 95%, the sensitivity of ER and PR assessment in CNB was 92.9% and 81%, respectively and the specificity of both was 100%. The Accuracy of CNB was 98% for ER and 93% for PR. CONCLUSIONS: Our results confirm the acceptable accuracy of ER assessment on CNB. The subject needs further investigation in developing countries where omission of the test in surgical samples can be cost and time-saving.

Lack of detection of the mouse mammary tumor-like virus (MMTV) Env gene in Iranian women breast cancer using real time PCR.

BACKGROUND: Mouse mammary tumor virus (MMTV) is the major cause of mammary tumors in mice. There is limited controversial evidence about the probable etiologic role of MMTV- like virus in human breast cancer. MATERIALS AND METHODS: A total of 40 Formalin fixed paraffin embedded samples with diagnosis of breast cancer were collected in a period of 3 years from cancer institute of Iran. We selected both pre-menopausal and post-menopausal patients with different histologic grades and different ethnic groups. We evaluated presence of MMTV-like virus env gene through real time PCR method. RESULTS: Forty patients (20 pre and 20 post- menopausal women) were evaluated with the mean age of 49.67. The average tumor size was 39 mm. None of the studied samples were positive for MMTV-like virus env gene target sequences. CONCLUSIONS: We found no evidence on the potential role of MMTV-like virus in the carcinogenicity of breast cancer among Iranian women.

The immunohistochemical characterization of MMP-2, MMP-10, TIMP-1, TIMP-2, and podoplanin in oral squamous cell carcinoma.

OBJECTIVES: The aim of this study was to immunohistochemically evaluate the expression of matrix metalloproteinase (MMP)-1, MMP- 2, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and podoplanin in oral squamous cell carcinoma (OSCC). Immunohistochemical staining of podoplanin-positive lymphatic vessel density (LVD) was also assessed. STUDY DESIGN: Forty cases of OSCC were analyzed by immunohistochemistry. RESULTS: MMP-2, MMP-10, TIMP-1, TIMP-2, and podoplanin were detected in each of the 40 OSCC cases. The expression of MMP-2 was significantly correlated with histologic grade. The expression of podoplanin was positively correlated with gender and negatively correlated with tumor size. A significant positive correlation was also detected between LVD and the presence of lymph node metastases, gender, age, and diameter of the lymph node (if involved), as well as histologic grade. CONCLUSIONS: The results are suggestive of important roles that MMP-2, MMP-10, TIMP-2, and podoplanin play in pathologic processes of OSCC, including invasion. Our findings also suggest that LVD may play a role in lymphatic metastasis and tumor progression.

Expression analysis of MiR-21, MiR-205, and MiR-342 in breast cancer in Iran.

MicroRNAs (miRNAs) are short non-coding RNA molecules characterized by their regulatory roles in cancer and gene expression. We analyzed the expression of miR-21, miR-205, and miR-342 in 59 patients with breast cancer. Samples were divided into three different groups according to their immunohistochemistry (IHC) classification: ER-positive and/or PR-positive group (ER+ and/or PR+; group I); HER2-positive group (HER2+; group II); and ER/PR/HER2- negative (ER-/PR-/HER2-; group III) as the triple negative group. The expression levels of the 3 miRNAs were analyzed in the tumor samples and the compared with the normal neighboring dissected tumor (NNDT) samples in all three groups. The expression of miR-21 was similar in all three groups. In patients positive for P53 by IHC, positive for axillary lymph node metastasis and higher tumor stages, it appeared to have significantly elevated. However, significant increase was not found among the 18 fibroadenoma samples. Both miR-205 and miR-342 expressions were significantly down regulated in group III. We conclude that miR-21 does not discriminate between different breast cancer groups. In contrast, miR-205 and miR-342 may be used as potential biomarkers for diagnosis of triple negative breast cancer.

Helicobacter pylori Omp18 and its application in serologic screening of infection.

Helicobacter pylori (Hp) is a major risk factor for gastrointestinal disorders including gastric cancer. We evaluated host serum antibody responses toward outer membrane protein18 in comparison with Urease A and B subunits. omp18 and ureA-ureB gene fragments were PCR amplified, cloned, and expressed in E. coli expression system. The expressed proteins were visualized on SDS-PAGE and confirmed by immuno-blotting. Purified proteins were applied in western blotting assays in comparison with local and foreign ELISA kits. ROC curve analysis identified the optimum cut-off points for each protein. rOmp18 represented the highest rates of sensitivity (94%), specificity (89%), PPV (97.4%), NPV (77.4%), and accuracy (93.2%) in comparison with urease A and B subunits. These immunologic indices were in "substantial" agreement (Κ = 0.7) with the gold standard tests for Hp detection. This study recommends Hp conserved Omp18 as a reliable serologic marker for accurate detection of Hp infection particularly for application in population screening approaches.

Heterologous immunization, a way out of the problem of monoclonal antibody production against carcinoma antigen 125.

BACKGROUND: Monoclonal antibodies (mAbs) are essential tools for many molecular immunology investigations, epitope mapping and molecular modelling, clinical laboratory diagnostic tests and immunotherapy. Humoral immune response of immunized animals largely depends on the nature of antigen and the immunization technique. Polysaccharides and heavily-glycosylated proteins are very elusive targets incapable of mounting long-lasting, high affinity antibody responses. Carcinoma antigen 125 (CA 125), a well known tumor marker of ovarian cancer, is a mucin type antigen consisting of repetitive units of heavily glycosylated moieties which render production of mAbs very difficult. OBJECTIVE: To evaluate the efficacy of heterologous antigen preparations as a way of mouse immunization in the production of anti-CA 125 mAb. METHODS: Two different protocols of immunization were used for priming of NMRI mice. In the first method, mice conventionally immunized by three intraperitoneal injections of purified CA 125 and boosted by the antigen three days before fusion. In the second approach, mice were primed by three intraperitoneal injections of living CA 125 positive cells of OVCAR-3 cell line, and boosted by intravenous injection of the purified extracellular domain of CA 125. Production of mAb was performed by standard hybridoma technology and mAbs were characterized by different immunoassays. RESULTS: The first method failed to produce stable clones despite six time fusion. A total of ten stable clones, however, were produced in the second approach. Some of the clones were characterized and found to have excellent immunoreactivity when tested by ELISA assay, western blotting, intracellular and surface immunofluorescent staining of OVCAR-3 cell line and immunohistochemical staining of ovarian cancer tissues. CONCLUSION: Altogether the results of the present study clearly showed that heterologous antigen preparation is the method of choice for immunization when production of monoclonal antibody against highly glycosylated poorly immunogenic antigens is concerned.

Corticotroph carcinoma of the pituitary: a clinicopathological study. Report of four cases.

To understand the relationship between pituitary adenoma and carcinoma, four adrenocorticotropic hormone-producing pituitary adenomas and corresponding metastatic carcinomas were studied. All were functional macroadenomas (three cases of Nelson syndrome and one of Cushing disease) that initially invaded the sella turcica and occurred in women ranging in age from 17 to 66 years (mean 45 years). Metastases (two craniospinal and two systemic) occurred after latency periods of 6 to 13 years. Histological specimens were immunostained for pituitary hormones, Ki-67 antigen (MIB-1), p53 and p27 proteins, D-type cyclins, and glucocorticoid receptor messenger (m)RNA. The DNA content of the specimens was assessed using Feulgen stain. Reactivities were quantified by digital image analysis. Primary/recurrent lesions and metastatic tumors differed according to their respective mean mitotic indices (1.2/10 hpf compared with 4.3/10 hpf), MIB-1 labeling (1.7% compared with 8%), p53 staining (37.3% compared with 49.9%), and p27 labeling (48% compared with 25%). Cyclin D, immunoreactivity provided no prognostically significant information. Glucocorticoid receptor mRNA was detected in all cases. Results of a ploidy analysis were variable and nonprognostic. In keeping with the 2000 World Health Organization classification of endocrine neoplasms, our findings support the concept that primary tumors that exhibit mitotic activity, an increased (> 3%) MIB-1 labeling index, and/or p53 immunoreactivity should be termed "atypical adenomas" to denote their aggressive potential and the possibility of future malignant transformation.