| AIM: Breast cancer is one of high incidence tumor in women, and its incidence increases all over the world to threat women health annually. It is significant to early diagnose and monitor postoperative recurrence for breast cancer since early diagnose and treatment may improve survival rate and period. Not an invasion method is high specific and sensitivity. The research shows that circling nucleic acid come from tumor tissue and the close relation between tumor and mtDNA mutations. The research of circulating plasma DNA and matched paraffin tumor tissue from breast cancer patientsin our experiment confirmed that same characteristic between plasma DNA and tumor tissue. The initial experiment established a way to check mtDNA mutation in circling nucleic acid from breast cancer patients. We disscued the feasibility and significance of mtDNA mutation screening in plasma for breast cancer patients diagnosis and prognosis and made efforts to seek new gene muations.METHODS: 35 breast cancer patients (preoperation 21 patients, postoperation 14 patients)aged 27 to 82(average 52.6)were diagnosed in Xijing Hospital, Fourth Military Medical University between July 2002 to October 2002. These patients were confirmed by pathology. Among them I phrase 0 patient, IIphrase 15 patients, III phrase 6 patients, IV phrase 14 patients. The benign group is mammary gland fibrous adenomatosis (10 patients) and 10 healthy blood donors were enrolled into the research as noprmal control. Breast cancer patients pathology information were collected. DNA was extracted from plasma by using microccntrifugation column (QIAgen)and the target DNA fragments(HVRland HVR2)were amplified by PCR,each HVR amplified by two pairs of primers. 1F1R and 2F2R were responsible for HVR1; 3F3R and 4F4R were responsible for HVR2. The production was screened by DHPLC (denaturing high performance liquid chromatography), and the mutation status and sorts of mtDNA gene in circulating plasma DNA was finally confirmed by direct DNA secquencing. Mutation results were analysed with clinical characteristics.Matched paraffin tumor tissue of two patients having mutations were amplified by PCR and sequenced. Data analysis were performed with SPSS 10.0 for windows or analysis of variance.RESULTS: 6 fragments of 1F1R , 14 fragments of 2F2R, 25 fragments of 3F3R and 1 fragments of 4F4R were obtained from 35 breast cancer patients .Only 3 fragments of 2F2R and 2 fragments of 3F3R were obtained form this begnign breast disease group, but only one fragment of 3F3R was obtainted from 10 health donors. Five mutations from 35 breast cancer patients by DHPLC were sequenced. The results are 1 mutation of 1F1R (patient 22,mutation point: 16183 A-AC), 1 mutation of 2F2R(patient 11,mutation point: 16347 T-C, 16362 T-G, 16457 G-T, 16496 G-A) and 3 mutation of 3F3R(patient 4, mutation point:73A-G; patient 16, mutation point: 73 A-G; patient 26, mutation point: 124 G-GG, 129 T-TT, 131 T-TT). 1 mutation of 3F3R from the benign group by DHPLC wasnot sequenced. No mutations came from healthy group. Same mutation (mutation point: 73 ATG-GTG )were found in circling nucleic acid and matched paraffin tumor tissue from 2 cancer patients.CONCLUSION:We first amplified mtDNA in circulating plasma from breast cancer patients by direct PCR, checked mutation by DHPLC and confirmed results by sequencing. Initial result of our experiment shows that it is practical to amplify tumor related little gene fragment in circulating plasma from tumor patients , plasma DNA come from tumor tissue and found somenew mutation points. Positive amplification rate in circulating plasma from cancer group is abnormally above than that from healthy group (P<0.05) .The difference is helpful for cancer diagnosis. We found main mutation type is base replace coincidence with previous conclusion from tumor tissue. There isnot clear relation between mtDNA mutations and tuimor size and clinical phrase, but closely related with pathology differentiation. That is to say , certain mutations may me... |
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