AIM: To investigate the prognostic significance of c-Kitgen emutation and DNA ploidy in gastointestinal stromal tumors (GISTs).METHODS: A total of 55 cases of GISTs were studied for the expression of c-Kit by immunohi...AIM: To investigate the prognostic significance of c-Kitgen emutation and DNA ploidy in gastointestinal stromal tumors (GISTs).METHODS: A total of 55 cases of GISTs were studied for the expression of c-Kit by immunohistochemistry, and the c-Kit gene mutations in exons 9, 11, 13, and 17 were detected by polymerase chain reaction-single strand confirmation polymarphism (PCR-SSCP) and denaturing high performance liquid chromatography (D-HPLC) techniques. DNA ploidy was determined by flow cytometry.RESULTS: Of the 55 cases of GISTs, 53 cases (96.4%) expressed c-Kit protein. The c-Kit gene mutations of exons 11 and 9 were found in 30 (54.5%) and 7 cases (12.7%),respectively. No mutations were found in exons 13 and 17.DNA aneuploidy was seen in 10 cases (18.2%). The c-Kit mutation positive GISTs were larger in size than the negative GISTs. The aneuploidy tumors were statistically associated with large size, high mitotic counts, high risk groups, high cellularity and severe nuclear atypia, and epithelioid type.There was a tendency that c-Kit mutations were more frequently found in aneuploidy GISTs.CONCLUSION: DNA aneuploidy and c-Kit mutations can be considered as prognostic factors in GISTs.展开更多
BACKGROUND Nucleic acid isolation from formalin-fixed, paraffin-embedded tissue(FFPET)samples is a daily routine in molecular pathology laboratories, but extraction from FFPET is not always easily achieved. Choosing t...BACKGROUND Nucleic acid isolation from formalin-fixed, paraffin-embedded tissue(FFPET)samples is a daily routine in molecular pathology laboratories, but extraction from FFPET is not always easily achieved. Choosing the right extraction technique is key for further examinations.AIM To compare the performance of four commercially available kits used for DNA extraction in routine practice.METHODS DNA isolation was performed on 46 randomly selected formalin-fixed, paraffinembedded(FFPE) colorectal adenocarcinoma(CRC) surgical specimens. Four commercially available extraction kits were used: two for manual DNA extraction(the Pure Link Genomic DNA Mini Kit from Invitrogen and the High Pure FFPE DNA Isolation Kit from Roche) and two for automated DNA extraction(the i Prep Genomic DNA Kit from Invitrogen and the Magna Pure LC DNA Isolation Kit from Roche). The DNA concentration and quality(odds ratio) among the four systems were compared. The results were correlated with the clinicopathological aspects of CRC cases: age, gender, localization, macro-and microscopic features,lymph node metastases, and the lymph node ratio.RESULTS The highest DNA concentration was obtained using the manual kits: 157.24 ±62.99 ng/μL for the Pure Link Genomic DNA Mini Kit and 86.64 ng/μL± 43.84 for the High Pure FFPE DNA Isolation Kit(P < 0.0001). Lower concentrations were obtained with automated systems: 20.39 ± 21.19 ng/μL for the Magna Pure LC DNA Isolation Kit and 8.722 ± 6.408 ng/μL for the i Prep Genomic DNA Kit,with differences between the systems used(P < 0.0001). The comparison between age, gender, tumor localization, pT or pN stage and the lymph node ratio indicated no statistically significant difference in DNA concentration using any of the nucleic acid isolation kits. DNA concentration was influenced by the macroscopic features and grade of differentiation. A higher DNA concentration was obtained for well-differentiated polypoid colorectal adenocarcinomas(CRCs), compared with undifferentiated ulcero-infiltrative carcinomas,irrespective of the kit used.CONCLUSION For research or diagnosis that needs high DNA concentrations, manual methods of DNA isolation should be used. A higher amount of DNA can be obtained from polypoid-type differentiated CRCs. Automated systems confer comfort and a lower amount of DNA that is, however, sufficient for classic polymerase chain reaction(PCR) and real-time quantitative PCR molecular examinations. All four commercially available kits can be successfully used in daily practice.展开更多
文摘AIM: To investigate the prognostic significance of c-Kitgen emutation and DNA ploidy in gastointestinal stromal tumors (GISTs).METHODS: A total of 55 cases of GISTs were studied for the expression of c-Kit by immunohistochemistry, and the c-Kit gene mutations in exons 9, 11, 13, and 17 were detected by polymerase chain reaction-single strand confirmation polymarphism (PCR-SSCP) and denaturing high performance liquid chromatography (D-HPLC) techniques. DNA ploidy was determined by flow cytometry.RESULTS: Of the 55 cases of GISTs, 53 cases (96.4%) expressed c-Kit protein. The c-Kit gene mutations of exons 11 and 9 were found in 30 (54.5%) and 7 cases (12.7%),respectively. No mutations were found in exons 13 and 17.DNA aneuploidy was seen in 10 cases (18.2%). The c-Kit mutation positive GISTs were larger in size than the negative GISTs. The aneuploidy tumors were statistically associated with large size, high mitotic counts, high risk groups, high cellularity and severe nuclear atypia, and epithelioid type.There was a tendency that c-Kit mutations were more frequently found in aneuploidy GISTs.CONCLUSION: DNA aneuploidy and c-Kit mutations can be considered as prognostic factors in GISTs.
基金the University of Medicine,Pharmacy,Science and Technology Research Grant,No.275/11.01.2017
文摘BACKGROUND Nucleic acid isolation from formalin-fixed, paraffin-embedded tissue(FFPET)samples is a daily routine in molecular pathology laboratories, but extraction from FFPET is not always easily achieved. Choosing the right extraction technique is key for further examinations.AIM To compare the performance of four commercially available kits used for DNA extraction in routine practice.METHODS DNA isolation was performed on 46 randomly selected formalin-fixed, paraffinembedded(FFPE) colorectal adenocarcinoma(CRC) surgical specimens. Four commercially available extraction kits were used: two for manual DNA extraction(the Pure Link Genomic DNA Mini Kit from Invitrogen and the High Pure FFPE DNA Isolation Kit from Roche) and two for automated DNA extraction(the i Prep Genomic DNA Kit from Invitrogen and the Magna Pure LC DNA Isolation Kit from Roche). The DNA concentration and quality(odds ratio) among the four systems were compared. The results were correlated with the clinicopathological aspects of CRC cases: age, gender, localization, macro-and microscopic features,lymph node metastases, and the lymph node ratio.RESULTS The highest DNA concentration was obtained using the manual kits: 157.24 ±62.99 ng/μL for the Pure Link Genomic DNA Mini Kit and 86.64 ng/μL± 43.84 for the High Pure FFPE DNA Isolation Kit(P < 0.0001). Lower concentrations were obtained with automated systems: 20.39 ± 21.19 ng/μL for the Magna Pure LC DNA Isolation Kit and 8.722 ± 6.408 ng/μL for the i Prep Genomic DNA Kit,with differences between the systems used(P < 0.0001). The comparison between age, gender, tumor localization, pT or pN stage and the lymph node ratio indicated no statistically significant difference in DNA concentration using any of the nucleic acid isolation kits. DNA concentration was influenced by the macroscopic features and grade of differentiation. A higher DNA concentration was obtained for well-differentiated polypoid colorectal adenocarcinomas(CRCs), compared with undifferentiated ulcero-infiltrative carcinomas,irrespective of the kit used.CONCLUSION For research or diagnosis that needs high DNA concentrations, manual methods of DNA isolation should be used. A higher amount of DNA can be obtained from polypoid-type differentiated CRCs. Automated systems confer comfort and a lower amount of DNA that is, however, sufficient for classic polymerase chain reaction(PCR) and real-time quantitative PCR molecular examinations. All four commercially available kits can be successfully used in daily practice.
