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Objective:To investigate the radiosensitizing effect of silencing breast cancer susceptibility gene 1 (shBRCA1) expression on MDA-MB-231 breast cancer bearing nude mice by 3′-deoxy-3′- 18F-fluorothymidine ( 18F-FLT) microPET/CT imaging. Methods:Twenty-four BALB/c nude mice were divided into 4 groups ( n=6 in each group) according to the random number table method, namely negative control (NC) group, NC+ radiotherapy group, shBRCA1 group and shBRCA1+ radiotherapy group. 18F-FLT microPET/CT imaging was performed before and 24 h after radiotherapy. The changes of SUV max before and after radiotherapy were compared among 4 groups, and the total proliferative volume (TPV) of tumors in each group after treatment was also analyzed. The expression of Ki-67 in tumor tissues was analyzed by immunohistochemistry. Data were analyzed by paired t test, one-way analysis of variance, least significant difference t test and Pearson correlation analysis. Results:Breast cancer cells targeting the BRCA1 were constructed. Before radiotherapy, the differences of SUV max among the NC group, NC+ radiotherapy group, shBRCA1 group and shBRCA1+ radiotherapy group were not statistically significant (1.034±0.137, 1.031±0.152, 1.028±0.169 and 1.026±0.156; F=0.00, P=0.999). Twenty-four hours after the end of the four times of radiotherapy, the differences of SUV max among the 4 groups were statistically significant (1.367±0.100, 0.781±0.079, 1.306±0.213 and 0.597±0.129; F=44.77, P<0.001), with lower SUV max in the shBRCA1+ radiotherapy group compared with the NC+ radiotherapy group ( t=2.98, P=0.014). The SUV max of the NC+ radiotherapy group and shBRCA1+ radiotherapy group were reduced compared with those before radiotherapy ( t values: 5.82, 5.44, P values: 0.002, 0.003), while SUV max of the NC group and shBRCA1 group increased compared with those before radiotherapy ( t values: -4.47, -3.98, P values: 0.007, 0.011). TPV was smaller in the shBRCA1+ radiotherapy group compared with that in the NC+ radiotherapy group (0.48±0.03 vs 0.61±0.07; F=25.36, t=3.82, P=0.003). Immunohistochemical assays showed that Ki-67 was less expressed in the shBRCA1+ radiotherapy group than that in the NC+ radiotherapy group (0.286±0.072 vs 0.476±0.093; F=15.73, t=3.61, P=0.007). Correlation analysis showed a positive correlation between Ki-67 expression and SUV max ( r=0.83, P<0.001). Conclusion:18F-FLT microPET/CT imaging can evaluate the radiosensitizing effect of shBRCA1 expression on MDA-MB-231 breast cancer bearing nude mice.
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To analyze the occurrence of genetic mutations in a sample of patients with high risk of breast cancer in Florianopolis/ SC from December 1st, 2021, to January 31, 2022. Methods: An observational, descriptive and retrospective study carried out through data collection of a preexisting database. A total of 194 tests were analyzed. Of these, 192 met the inclusion criteria and composed the final sample of 205 genes. Data were classified and reported the frequency and percentage of the variables: gene and presence or absence of mutation. Results: Mean age of the analyzed patients was 52.3 years, and most underwent the test due to personal history of breast cancer (80%). Clinical significance classification showed that, of the 192 gene panels, 62% were variants of uncertain significance; 14% were pathogenic; and 24%, negative. Of the 205 mutations, the most prevalent genes were: ATM 8.7%, MUTYH 5.8%, POLE 5.8%, BRCA2 4.8%, MSH6 4.8% and RECQL4 4.8%. Of the pathogenic tests regarding genetic predisposition to cancer (n=38/14.1%), the most common mutations were MUTYH (23%) and BRCA1 (15%), with mean age of 52 years (±14.3). In variants of uncertain significance panels (n=168/62%) the frequency rates were ATM (7.7%), POLE (7.1%) and MSH6 (5.9%) genes. The high penetrance genes were present in 18% of the genetic predisposition to cancer panels. Of those with positive family history (n=40), 19% of the genes were pathogenic, 53% were variants of uncertain significance; and 26% were negative. Furthermore, in patients with pathogenic mutations and positive family history (n=11), the most common mutations were in BRCA1 (27%) and BRCA2 (27%). Of the patients who tested due to personal history (n=152), 64% of the genes presented variants of uncertain significance, 13% were pathogenic and 22% were negative.
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Objective:To investigate the expressions and clinical significances of breast cancer susceptibility gene 1 (BRCA1) and tubulin β3 (TUBB3) in patients with gastric cancer, so as to provide a basis for accurate diagnosis and treatment of gastric cancer.Methods:The data of 46 hospitalized patients with gastric cancer who underwent gastroscopebiopsy or operation in Maanshan People's Hospital in Anhui Province from December 2018 to May 2020 were collected. The expressions of BRCA1 and TUBB3 in tumor tissues and peritumoral tissues were determined by immunohistochemistry. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect BRCA1 and TUBB3 mRNA expressions in tumor tissues and peritumoral tissues. The correlations between expressions of BRCA1 and TUBB3 in gastric cancer tissues and clinicopathologic features were analyzed.Results:The positive rates of BRCA1 and TUBB3 proteins in gastric cancer tissues were higher than those in peritumoral tissues [43.5% (20/46) vs. 16.7% (5/30), 65.2% (30/46) vs. 6.7% (2/30), both P < 0.05]. qRT-PCR showed that the relative expression of BRCA1 mRNA in gastric cancer tissues was higher than that in peritumoral tissues (15.5±6.8 vs. 5.0±1.6, t = 9.41, P < 0.01); the relative expression of TUBB3 mRNA in gastric cancer tissues was higher than that in peritumoral tissues (22.1±6.3 vs. 5.7±1.9, t = 3.51, P < 0.01). The positive rate of TUBB3 protein in female patients was lower than that in male patients [15.4% (2/13) vs. 84.8% (28/33)], the positive rate of BRCA1 protein in patients with positive human epidermal growth factor receptor 2 (HER2) was higher than that in patients with negative HER2 [87.5% (7/8) vs. 47.4% (18/38)], the positive rate of BRCA1 protein in patients with family history was higher than that in patients without family history [85.7% (6/7) vs. 35.9% (14/39)], and the differences were statistically significant (all P < 0.05). The positive expressions of BRCA1 and TUBB3 proteins in gastric cancer tissues were both correlated with tumor stage and differentiation (all P < 0.05), and the expressions of BRCA1 and TUBB3 proteins were correlated ( χ2 = 33.52, P < 0.01). Conclusions:BRCA1 and TUBB3 may be related to the occurrence and development of gastric cancer, and there may be a certain relationship between BRCA1 and TUBB3, BRCA1 and HER2. BRCA1 and TUBB3 may have significances in the diagnosis and treatment of gastric cancer.
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Breast cancer is the second most common cancer in Korean women. Germline mutations in the BRCA1 and BRCA2 genes cause hereditary breast cancer and are detected in 15–20% of hereditary breast cancer. We investigated the BRCA1 and BRCA2 mutations in 114 familial breast cancer patients using next-generation sequencing. We confirmed 20 different mutations of BRCA1 and BRCA2 in 25 subjects (21.9%). Two such mutations in eight patients were novel (not reported in any variant database or previous study). Six mutations have been reported as disease-causing mutations in public databases. Seven mutations were found only in a single nucleotide polymorphism database and one mutation has been reported in Korea. The BRCA1/2 mutation frequency was similar to that of other studies on familial breast cancer patients in the Korean population. Further studies should examine more cases and mutations of whole exons.
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Female , Humans , BRCA1 Protein , BRCA2 Protein , Breast Neoplasms , Breast , Exons , Genes, BRCA2 , Germ-Line Mutation , Korea , Mutation Rate , Polymorphism, Single NucleotideABSTRACT
Objective@#To investigate the expression of BRCA-associated protein 1 (BAP1) in malignant mesothelioma, non-small cell lung cancer and carcinosarcoma, and its application in the differential diagnosis.@*Methods@#Twenty-two cases of malignant mesothelioma including 17 epithelioid type, 2 sarcomatoid type and 3 biphasic type were collected.As the study control, 80 non-small cell lung cancers infringement pleural membrane(including 40 lung adenocarcinomas and 40 lung squamous cell carcinomas) and 15 carcinosarcomas were included. BAP1 expression was detected using immunohistochemical method. A differential diagnosis antibody panel, including calretinin, WT1, CK5/6, D2-40, CAM5.2, CEA, TTF1, Napsin A, p63 and p40 was tested in all cases.@*Results@#All 80 cases of non-small cell lung cancer and 15 cases of carcinosarcoma were BAP1 positive. In contrast, 64% (14/22) of malignant mesotheliomas lost BAP1 expression (P<0.01). Addition of BAP1 to the mesothelioma marker panel, the diagnostic accuracy of malignant mesothelioma was enhanced to 93%. Focal expression of BAP1 in tumors suggested multiclonal evolution of mesothelioma.@*Conclusions@#Loss of BAP1 expression helps to confirm the diagnosis of malignant mesothelioma whereas all non-small cell lung cancer expresses BAP1. It is therefore recommended that BAP1 can be used in conjunction with other immunohistochemical markers to improve the diagnostic accuracy of malignant mesothelioma.
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Germline mutations in the BRCA1 and BRCA2 genes are strong genetic factors for predispositions to breast, ovarian, and other related cancers. This report describes a family with a history of breast and ovarian cancers that harbored a novel BRCA1 germline mutation. A single nucleotide deletion in intron 20, namely c.5332+4delA, was detected in a 43-year-old patient with breast cancer. This mutation led to the skipping of exon 20, which in turn resulted in the production of a truncated BRCA1 protein that was 1773 amino acids in length. The mother of the proband had died due to ovarian cancer and had harbored the same germline mutation. Ectopically expressed mutant BRCA1 protein interacted with the BARD1 protein, but showed a reduced transcriptional function, as demonstrated by the expression of cyclin B1. This novel germline mutation in the BRCA1 gene caused familial breast and ovarian cancers.
Subject(s)
Adult , Humans , Amino Acids , BRCA1 Protein , Breast Neoplasms , Breast , Cyclin B1 , Exons , Genes, BRCA1 , Genes, BRCA2 , Germ-Line Mutation , Introns , Mothers , Ovarian NeoplasmsABSTRACT
Objective To investigate the role of the predictive prognostic value of BRCA1 screened by whole genome expression profiling in ductal carcinoma in situ.Methods Collected 4 cases of breast ductal carcinoma and 4 cases of breast invasive ductal carcinoma fresh samples from January 2014 to June 2014,and the difference of BRCA1 expression on whole genone expression profiling was analyzed by microarray comparative genomic hybridization.The expression of BRCA1 was detected by immunohistochemistry in 70 cases of ductal carcinoma in situ of the breast,and the prognosis of intraductal carcinoma was evaluated.Results BRCA1 gene differentially expressed in invasive ductal carcinoma and ductal carcinoma in situ by screening.The positive rate of BRCA1 protein in breast ductal carcinoma in 14.3% (10/70),its expression had no significant relationship with age (P =0.959),menopause (P =0.959),tumor size (P =0.627),axillary lymph node status (P =1.000),HR status (P =0.958),HER-2 status (P =1.000),P53 expression (P =0.460).ductal carcinoma with micro-infiltration ratio in BRCA1 negative group was higher than BRCA1-positive group (P =0.043).The median follow-up of 47 months,Disease-free survival rate of all was 97.1%.Disease-free survival of BRCA1 negative group and BRCA1-positive group had no significant difference (96.7% vs 100%,P =0.569),over all survival of BRCA1 negative and positive groups was 100%.Conclusions BRCA1 expression may not predict the prognosis of intraductal carcinoma,but ductal carcinoma in situ with microinvasion group ratio in BRCA1 negative was higher than ductal carcinoma in situ group,BRCA1 may take affect within ductal carcinoma infiltration process work.
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PURPOSE: Trabectedin induces synthetic lethality in tumor cells carrying defects in homologous recombinant DNA repair. We evaluated the effect of concomitant inhibition of nucleotide-excision repair and poly (ADP-ribose) polymerase (PARP) activity with trabectedin and PARP inhibitors, respectively, and whether the synthetic lethality effect had the potential for a synergistic effect in breast cancer cell lines. Additionally, we investigated if this approach remained effective in BRCA1-positive breast tumor cells. METHODS: We have evaluated the in vitro synergistic effect of combinations of trabectedin and three different PARP inhibitors (veliparib, olaparib, and iniparib) in four breast cancer cell lines, each presenting a different BRCA1 genetic background. Antiproliferative activity, DNA damage, cell cycle perturbations and poly(ADP-ribosyl)ation were assessed by MTT assay, comet assay, flow cytometry and western blot, respectively. RESULTS: The combination of trabectedin and olaparib was synergistic in all the breast cancer cell lines tested. Our data indicated that the synergy persisted regardless of the BRCA1 status of the tumor cells. Combination treatment was associated with a strong accumulation of double-stranded DNA breaks, G2/M arrest, and apoptotic cell death. Synergistic effects were not observed when trabectedin was combined with veliparib or iniparib. CONCLUSION: Collectively, our results indicate that the combination of trabectedin and olaparib induces an artificial synthetic lethality effect that can be used to kill breast cancer cells, independent of BRCA1 status.
Subject(s)
Blotting, Western , BRCA1 Protein , Breast Neoplasms , Breast , Cell Cycle , Cell Death , Cell Line , Comet Assay , DNA Breaks, Double-Stranded , DNA Damage , DNA, Recombinant , Drug Combinations , Flow CytometryABSTRACT
Objective: To investigate the effects of doxorubicin on the expressions of DNA-damage/repair-related proteins BRCA1 (breast cancer-associated protein 1) and PARP-1 [poly(ADP-ribose) polymerase-1] in BRCA1 wild-type breast cancer MCF-7 cells. Methods: The MCF-7 cells were treated with doxorubicin, then the expressions of BRCA1 and PARP-1 and the activity of PARP-1 in the cells recovering after different time peroids were detected by Western blotting. The apoptotic rates of MCF-7 cells and SKBR3.0 cells (BRCA1 mutant-type breast cancer cells) after intervention with doxorubicin and PARP-1 inhibitor 3-ABA (3-aminobenzamide) were detected by FCM (flow cytometry). Results: After MCF-7 cells were treated with different concentrations of doxorubicin for 24 h and then recovered for 12 h, the expression of PAR [poly(ADP-ribose)], an active product of PARP-1, was increased in a dose-dependent manner (P 0.05). Both doxorubicin and 3-ABA alone could significantly induce the apoptosis of MCF-7 cells (P < 0.05), and the combination of the two could further increase the apoptosis of BRCA1 wild-type breast MCF-7 cells (P < 0.05). Doxorubicin in combination with 3-ABA could also induce the apoptosis of BRCA1 mutant-type SKBR3.0 cells, and this effect was stronger than that in MCF-7 cells (P < 0.05). Conclusion: Doxorubicin intervention can affect the activity of DNA-damage/repair-related protein PARP-1 and the expression of BRCA1. Both doxorubicin and PARP-1 inhibitor 3-ABA can induce the apoptosis of MCF-7 cells, and the combination of the two can increase this apoptosis-inducing effect. Copyright © 2013 by TUMOR.
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PURPOSE: To study the subcellular localization with flow-cytometry and to evaluate their prognostic values. MATERIALS AND METHODS: The breast tissues were obtained from 28 patients with breast cancer and 6 patients with benign mass. The expression of BRCA1 protein was analyzed with the flow cytometry(Coulter Epics-XL, Coulter Corps, FL, USA) using the monoclonal antibody(BRCA1(Ab-1), Calbiochem, MA, USA) before and after nuclear and cytoplasmic permeabilization in association with DNA ploidy analysis. Several BRCA1 protein indices were derived including 95 percentile channel fluorescence(95% CF) and mean channel fluorescence(MCF) and percentage of BRCA 1 positive cell population arbitarily defined as those above 0.12 channel fluorescence. RESULTS: Cytoplasmic 95% CF were higher in breast cancer(n=28, 0.65+/-0.26) than in benign mass(n=6, 0.40+/-0.13, p=0.0211). Cytoplasmic BRCAl positive cell percentages were significantly higher in malignant tissues(24.0+/-10.3) than in benign mass(43.4+/-15.2, p=0.0059). Cytoplasmic BRCA1 positive cell percentages were significantly different according to the stages(stage I vs II, 32.6+/-9.8 vs 48.3+/-18.8, p=0.048, stage I vs stage III, 32.6+/-9.8 vs 47.0+/-10.9, p=0.010). The BRCA1 protein indices were not significantly correlated with histologic grades and DNA indices(aneuploidy, S phase and proliferation fractions). CONCLUSIONS: Flowcytometric assay offers an alternative approach to evaluating BRCA1 protein status of breast cancer tissue and detection of cytoplasmic BRCA1 protein by this method may help to understand the role of BRCA1 in breast cancer cell biology. The further study on cytoplasmic or nuclear BRCA1 protein in association with clinical therapeutic response or prognosis seems to be warranted.
Subject(s)
Humans , BRCA1 Protein , Breast Neoplasms , Breast , Cytoplasm , DNA , Flow Cytometry , Fluorescence , Ploidies , Prognosis , S PhaseABSTRACT
Purpose:To study the relationship between the expression of the BRCA 1 protein and clinico pathological characteristics of breast cancer,and to clarify the correlation of the expression of the BRCA 1 protein with expression of p53 and c erbB 2 . Methods:The expressions of BRCA 1 , p53 and c erbB 2 of breast cancer tissues taken from 60 breast cancer patients as well as 10 benign breast disease patients were examined using immunohistochemical LDP method. All tissues were taken from formalin fixed and paraffin embedded breast tumor specimens. We analyzed the correlation of the results with other parameters which included age of onset, family history, histological grade, status of estrogen receptor and progesterone receptor, axillary nodal status and so on. Results:The expression of BRCA 1 was 61.66% (37/60) in breast cancer patients and 0 (0/10) in benign diseases patients. The protein expressed was mainly located in the cytoplasm. The correlation between age of onset and the expression of BRCA 1 was significant (r=-0.295, P