For first-line non-small-cell lung cancer(NSCLC) therapy,detecting mutation status of the epidermal growth factor receptor(EGFR) gene constitutes a prudent test to identify patients who are most likely to benefit ...For first-line non-small-cell lung cancer(NSCLC) therapy,detecting mutation status of the epidermal growth factor receptor(EGFR) gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor(TKI) therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded(FFPE) tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction(PCR) are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100(pH=10.0) was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs(bp).However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.展开更多
BACKGROUND Mycobacterium mucogenicum(M.mucogenicum)belongs to the group of rapidly growing Nontuberculous mycobacteria.This microorganism is associated with a wide spectrum of infectious diseases.Due to a low detectio...BACKGROUND Mycobacterium mucogenicum(M.mucogenicum)belongs to the group of rapidly growing Nontuberculous mycobacteria.This microorganism is associated with a wide spectrum of infectious diseases.Due to a low detection rate or the time required for conventional culture methodology,a rapid and broad-spectrum method is necessary to identify rare pathogens.CASE SUMMARY A 12-year-old immunocompetent girl presented with painful masses for five months.The first mass was found in the right upper quadrant of the abdomen,and was about 1 cm×1.5 cm in size,tough but pliable in texture,with an irregular margin and tenderness.An abscess gradually formed and ulcerated with suppuration of the mass.Three new masses appeared on the back one by one.Chest computed tomography showed patchy and streaky cloudy opacities in both lungs.Needle aspiration of the abscess was performed,but the smear and conventional culture were negative,and the pathological examination showed no pathogens.We then performed next-generation sequencing using a formalinfixed,paraffin-embedded specimen to identify the pathogen.A significantly high abundance of M.mucogenicum was detected.The patient’s abscesses gradually decreased in size,while inflammation in both lungs improved following 12-wk of treatment.No recurrence was observed four months after the end of the one-year treatment period.CONCLUSION Next-generation sequencing is a promising tool for the rapid and accurate diagnosis of rare pathogens,even when using a formalin-fixed,paraffin-embedded specimen.展开更多
Background: Differential diagnosis of follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA) is often difficult since presence or absence of capsular/vascular invasion can not be determined by preope...Background: Differential diagnosis of follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA) is often difficult since presence or absence of capsular/vascular invasion can not be determined by preoperative fine needle aspiration cytology, and may not be judged unanimously on permanent sections even among experienced pathologists. Determination of molecular-genetic factors such as trefoil factor 3 (TFF3) mRNA in the follicular thyroid tumors may be useful aid to improve the accuracy of diagnosis, though it is considered to be unstable and relatively low concentrated genetic substance. Purpose of our study is to investigate expression level of TFF3 mRNA of thyroid follicular tumors using formalin-fixed, paraffin-embedded (FFPE) tissue. Methods: Study population included FFPE sections from 19 FTC cases, 20 FTA cases, 11 adenomatous goiter (G) cases and 12 samples of normal thyroid tissue (N) adjacent to thyroid tumors. RNeasy FFPE kit was used for extraction of total RNA. Purification and concentration values were determined by spectrophotometer. Extracted RNA was used for cDNA synthesis in reverse transcription. Synthesized cDNA subsequently proceeded for relative quantification of TFF3 mRNA by RT-qPCR using TFF3 primers. Glyceroldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthin phosphorobosyltransferase1 (HPRT1) were used as control genes. The mean and standard deviation of TFF3 mRNA expression level were analyzed by software Multiplate RQ. Results: Extraction by the FFPE kit yielded high concentration of RNA in all cases. Purification values were 1.8 in average. Concentration values were significantly higher in FTC and FTA relative to G and N tissues, possibly due to high density of thyrocytes in the samples. Relative quantification of TFF3 mRNA expression level showed broad ranges both in FTC and FTA, while the analyses in G and N tissues indicated narrow ranges. Conclusion: FFPE tissues from thyroid follicular tumors can be used for measurement of unstable and low concentrated genetic substances such as TFF3 mRNA. Its diagnostic value yet remains to be determined.展开更多
Stains are applied to impart contrast to the tissue and identify particular features of interest. However, the use of synthetic dyes as staining reagents has been associated with significant human health challenges an...Stains are applied to impart contrast to the tissue and identify particular features of interest. However, the use of synthetic dyes as staining reagents has been associated with significant human health challenges and pollution of the ecosystem. These developments have necessitated a shift towards using natural dyes that are eco-friendlier and readily available. We investigated the staining reaction patterns of teak tree leaves (Tectona grandis) dye extracts and explored their suitability as a cytoplasmic stain in micromorphological assessments. Dye extracts were prepared using acetone, methanol, and ethanol as solvents from air-dried (under shade) teak tree young leaves. The dye extracts were applied as a counterstain and evaluated against eosin in formalin-fixed paraffin-embedded (FFPE) bovine tissue sections at varying concentrations and different staining times. Teak tree leaves (Tectona grandis) dye extracts produced relatively varying staining intensities of reddish-brown cytoplasmic coloration when used on bovine tissue at different concentrations and staining times comparable to eosin and with blue-purple hematoxylin nuclear stain. The present study showed that Tectona grandis leaf dye extracts provide an excellent cytoplasmic staining pattern and can be used as an alternative counterstain in routine H&E staining techniques.展开更多
Background: MicroRNA (miRNA) regulates post-transcriptional gene expression through binding to complementary sites of target messenger RNA, including that from oncogenes or tumor suppressor genes. This study planned t...Background: MicroRNA (miRNA) regulates post-transcriptional gene expression through binding to complementary sites of target messenger RNA, including that from oncogenes or tumor suppressor genes. This study planned to pursue the possibility that circulating miRNA could be used for the early diagnosis of diffuse large B-cell lymphoma (DLBCL). Materials and Methods: Expression levels of miRNA obtained from serum, exosome-enriched serum, and formalin-fixed paraffin-embedded (FFPE) tissue were evaluated. Samples were collected from patients with newly diagnosed DLBCL (n = 33) or healthy volunteers (n = 22). Based on the results of previous reports, ten miRNAs were selected and expression levels were analyzed by the quantitative real-time PCR. Results: The expression levels of hsa-miR-15a-3p, hsa-miR-21-5p, hsa-miR-181a-5p, and hsa-miR-210-5p differed significantly between DLBCL patients and controls in serum and/or exosome-enriched serum, but not in FFPE tissue. In contrast, expression levels of hsa-miR-155-5p in FFPE tissue were significantly higher in DLBCL patients, as previously reported. Conclusion: We confirmed that some miRNAs were differentially expressed in serum from DLBCL patients as previously reported. Measurement of these miRNA in exosome-enriched serum did not improve the accuracy in the differential diagnosis of DLBCL. In addition, these miRNAs seem to be produced outside of lymphoma tissue.展开更多
The sensitivity of PCR was determined for detection of HBV DNA in paraffin-embed-ded liver tissue with different methods for sample preparation.Of 8 cases of HBeAg-positiveHBV infection,HBV DNA was detected in 6 by ex...The sensitivity of PCR was determined for detection of HBV DNA in paraffin-embed-ded liver tissue with different methods for sample preparation.Of 8 cases of HBeAg-positiveHBV infection,HBV DNA was detected in 6 by extracting DNA from both frozen and dewaxedsamples,but in none by direct reaction.Of 12 cases subjected to Southern blot hybridization,HBV DNA was detected in 7 by this technique,in 10 by PCR with both methods of DNA extrac-tion and in 3 by direct PCR.The results showed that PCR was sensitive and was comparablewith blot hybridization in detecting intrahepatic HBV DNA.In comparison between differentmethods of sample preparation,the viral detection rate from the dewaxed samples was near thatfrom the frozen ones,while by the direct reaction HBV DNA could be detected only in a fewsamples with high level of infection.展开更多
BACKGROUND Studies correlating specific genetic mutations and treatment response are ongoing to establish an effective treatment strategy for gastric cancer(GC).To facilitate this research,a cost-and time-effective me...BACKGROUND Studies correlating specific genetic mutations and treatment response are ongoing to establish an effective treatment strategy for gastric cancer(GC).To facilitate this research,a cost-and time-effective method to analyze the mutational status is necessary.Deep learning(DL)has been successfully applied to analyze hematoxylin and eosin(H and E)-stained tissue slide images.AIM To test the feasibility of DL-based classifiers for the frequently occurring mutations from the H and E-stained GC tissue whole slide images(WSIs).METHODS From the GC dataset of The Cancer Genome Atlas(TCGA-STAD),wildtype/mutation classifiers for CDH1,ERBB2,KRAS,PIK3CA,and TP53 genes were trained on 360×360-pixel patches of tissue images.RESULTS The area under the curve(AUC)for the receiver operating characteristic(ROC)curves ranged from 0.727 to 0.862 for the TCGA frozen WSIs and 0.661 to 0.858 for the TCGA formalin-fixed paraffin-embedded(FFPE)WSIs.The performance of the classifier can be improved by adding new FFPE WSI training dataset from our institute.The classifiers trained for mutation prediction in colorectal cancer completely failed to predict the mutational status in GC,indicating that DL-based mutation classifiers are incompatible between different cancers.CONCLUSION This study concluded that DL could predict genetic mutations in H and E-stained tissue slides when they are trained with appropriate tissue data.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
AIM:to study the prevalence of human papillomavirus(HPV) in esophageal carcinoma in tangshan,China,a high-incidence area.METHODS:Formalin-fixed,paraffin-embedded tissue specimens from 198 patients who were pathologica...AIM:to study the prevalence of human papillomavirus(HPV) in esophageal carcinoma in tangshan,China,a high-incidence area.METHODS:Formalin-fixed,paraffin-embedded tissue specimens from 198 patients who were pathologically diagnosed with esophageal squamous cell carcinoma from 2011 to 2013 were obtained from a pathology department in Tangshan.DNA was extracted from all198 specimens to detect HPV by polymerase chain reaction(PCR).β-globin PCR was performed to check the quality of the DNA extraction procedure.PCR was performed to detect a wide range of HPV types,and type-specific PCR was performed to detect HPV types16 and 18.Negative and positive controls were used for HPV 16 and 18 detection.RESULTS:the DNA extraction method in this study appeared to be more effective than other previously reported methods.After DNA extraction,more than98%of the tissue specimens had an acceptable result in the DNA qualification test(β-globin PCR).the overall prevalence of HPV in tumor tissues by GP6+/GP5+PCR was 79.79%,and the prevalence of HPV types16 and 18 was 40.40%and 47.47%,respectively.PCR demonstrated the presence of HPV,and direct sequencing confirmed the HPV genotypes.All HPVpositive PCR products were checked by DNA sequence analysis using DNAman and compared with the known HPV sequences listed in the basic Local Alignment Search tool database to evaluate the HPV types.this analysis confirmed the presence of HPV types 16 and18.CONCLUSION:DNA of high-risk HPV types 16 and 18is present in esophageal tumors,implicating HPV as a possible etiologic factor for esophageal squamous cell carcinoma.展开更多
Formalin-fixed and paraffin-embedded(FFPE)tissues provide a wealth of pathological information crucial for clinical and forensic examinations.Formalin induces robust complexes between DNA and proteins,impacting DNA ex...Formalin-fixed and paraffin-embedded(FFPE)tissues provide a wealth of pathological information crucial for clinical and forensic examinations.Formalin induces robust complexes between DNA and proteins,impacting DNA extraction and complicating short tandem repeat(STR)typing for personal identification and paternity testing.Here,we present a case of paternity testing involving one FFPE tissue and one blood specimen.We compared four DNA extraction methods and analyzed the obtained products from the most successful approach.To ensure robust statistical support,we used a combination of three STR kits for the analysis.This case demonstrates the viability of using multiple kits in tandem for STR profiling of FFPE tissues.展开更多
Both fresh-frozen and formalin-fixed,paraffinembedded(FFPE)human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases,especially neurodegenerative disorders.To iden...Both fresh-frozen and formalin-fixed,paraffinembedded(FFPE)human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases,especially neurodegenerative disorders.To identify the optimal method for DNA extraction from human brain tissue,we compared methods on differently-processed tissues.Fragments of LRRK2 and MAPT(257 bp and 483 bp/245 bp)were amplified for evaluation.We found that for FFPE samples,the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method(successful DNA extraction from 76%versus 33%of samples).PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples.In the fresh-frozen samples,the DNA extraction success rate was 100%using either a commercial kit(QIAamp DNA Micro)or a laboratorybased method(sample boiling in 0.1 mol/L NaOH,followed by proteinase K digestion,and then DNA extraction using Chelex-100)regardless of PCR amplicon length or tissue storage time.Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples,fresh brain tissue is recommended for DNA extraction in future neuropathological studies.展开更多
基金Supported by the Jilin Science & Technology Development Plan,China(No.201201060)the Scientific Research Foundation of Jilin Province,China(No.20100942)+1 种基金the Fund of Developing and Reforming Community of Jilin Province,China(No.2010-1928)the Health Scientific Research Foundation of Jilin Province,China(Nos.2009z081,2010Z083)
文摘For first-line non-small-cell lung cancer(NSCLC) therapy,detecting mutation status of the epidermal growth factor receptor(EGFR) gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor(TKI) therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded(FFPE) tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction(PCR) are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100(pH=10.0) was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs(bp).However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.
基金Supported by the Clinical Research Foundation of the Third Affiliated Hospital of Sun Yat-Sen University,No.YHJH201904National Science and Technology Major Project,No.2018ZX10302204.
文摘BACKGROUND Mycobacterium mucogenicum(M.mucogenicum)belongs to the group of rapidly growing Nontuberculous mycobacteria.This microorganism is associated with a wide spectrum of infectious diseases.Due to a low detection rate or the time required for conventional culture methodology,a rapid and broad-spectrum method is necessary to identify rare pathogens.CASE SUMMARY A 12-year-old immunocompetent girl presented with painful masses for five months.The first mass was found in the right upper quadrant of the abdomen,and was about 1 cm×1.5 cm in size,tough but pliable in texture,with an irregular margin and tenderness.An abscess gradually formed and ulcerated with suppuration of the mass.Three new masses appeared on the back one by one.Chest computed tomography showed patchy and streaky cloudy opacities in both lungs.Needle aspiration of the abscess was performed,but the smear and conventional culture were negative,and the pathological examination showed no pathogens.We then performed next-generation sequencing using a formalinfixed,paraffin-embedded specimen to identify the pathogen.A significantly high abundance of M.mucogenicum was detected.The patient’s abscesses gradually decreased in size,while inflammation in both lungs improved following 12-wk of treatment.No recurrence was observed four months after the end of the one-year treatment period.CONCLUSION Next-generation sequencing is a promising tool for the rapid and accurate diagnosis of rare pathogens,even when using a formalin-fixed,paraffin-embedded specimen.
文摘Background: Differential diagnosis of follicular thyroid carcinoma (FTC) from follicular thyroid adenoma (FTA) is often difficult since presence or absence of capsular/vascular invasion can not be determined by preoperative fine needle aspiration cytology, and may not be judged unanimously on permanent sections even among experienced pathologists. Determination of molecular-genetic factors such as trefoil factor 3 (TFF3) mRNA in the follicular thyroid tumors may be useful aid to improve the accuracy of diagnosis, though it is considered to be unstable and relatively low concentrated genetic substance. Purpose of our study is to investigate expression level of TFF3 mRNA of thyroid follicular tumors using formalin-fixed, paraffin-embedded (FFPE) tissue. Methods: Study population included FFPE sections from 19 FTC cases, 20 FTA cases, 11 adenomatous goiter (G) cases and 12 samples of normal thyroid tissue (N) adjacent to thyroid tumors. RNeasy FFPE kit was used for extraction of total RNA. Purification and concentration values were determined by spectrophotometer. Extracted RNA was used for cDNA synthesis in reverse transcription. Synthesized cDNA subsequently proceeded for relative quantification of TFF3 mRNA by RT-qPCR using TFF3 primers. Glyceroldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthin phosphorobosyltransferase1 (HPRT1) were used as control genes. The mean and standard deviation of TFF3 mRNA expression level were analyzed by software Multiplate RQ. Results: Extraction by the FFPE kit yielded high concentration of RNA in all cases. Purification values were 1.8 in average. Concentration values were significantly higher in FTC and FTA relative to G and N tissues, possibly due to high density of thyrocytes in the samples. Relative quantification of TFF3 mRNA expression level showed broad ranges both in FTC and FTA, while the analyses in G and N tissues indicated narrow ranges. Conclusion: FFPE tissues from thyroid follicular tumors can be used for measurement of unstable and low concentrated genetic substances such as TFF3 mRNA. Its diagnostic value yet remains to be determined.
文摘Stains are applied to impart contrast to the tissue and identify particular features of interest. However, the use of synthetic dyes as staining reagents has been associated with significant human health challenges and pollution of the ecosystem. These developments have necessitated a shift towards using natural dyes that are eco-friendlier and readily available. We investigated the staining reaction patterns of teak tree leaves (Tectona grandis) dye extracts and explored their suitability as a cytoplasmic stain in micromorphological assessments. Dye extracts were prepared using acetone, methanol, and ethanol as solvents from air-dried (under shade) teak tree young leaves. The dye extracts were applied as a counterstain and evaluated against eosin in formalin-fixed paraffin-embedded (FFPE) bovine tissue sections at varying concentrations and different staining times. Teak tree leaves (Tectona grandis) dye extracts produced relatively varying staining intensities of reddish-brown cytoplasmic coloration when used on bovine tissue at different concentrations and staining times comparable to eosin and with blue-purple hematoxylin nuclear stain. The present study showed that Tectona grandis leaf dye extracts provide an excellent cytoplasmic staining pattern and can be used as an alternative counterstain in routine H&E staining techniques.
文摘Background: MicroRNA (miRNA) regulates post-transcriptional gene expression through binding to complementary sites of target messenger RNA, including that from oncogenes or tumor suppressor genes. This study planned to pursue the possibility that circulating miRNA could be used for the early diagnosis of diffuse large B-cell lymphoma (DLBCL). Materials and Methods: Expression levels of miRNA obtained from serum, exosome-enriched serum, and formalin-fixed paraffin-embedded (FFPE) tissue were evaluated. Samples were collected from patients with newly diagnosed DLBCL (n = 33) or healthy volunteers (n = 22). Based on the results of previous reports, ten miRNAs were selected and expression levels were analyzed by the quantitative real-time PCR. Results: The expression levels of hsa-miR-15a-3p, hsa-miR-21-5p, hsa-miR-181a-5p, and hsa-miR-210-5p differed significantly between DLBCL patients and controls in serum and/or exosome-enriched serum, but not in FFPE tissue. In contrast, expression levels of hsa-miR-155-5p in FFPE tissue were significantly higher in DLBCL patients, as previously reported. Conclusion: We confirmed that some miRNAs were differentially expressed in serum from DLBCL patients as previously reported. Measurement of these miRNA in exosome-enriched serum did not improve the accuracy in the differential diagnosis of DLBCL. In addition, these miRNAs seem to be produced outside of lymphoma tissue.
文摘The sensitivity of PCR was determined for detection of HBV DNA in paraffin-embed-ded liver tissue with different methods for sample preparation.Of 8 cases of HBeAg-positiveHBV infection,HBV DNA was detected in 6 by extracting DNA from both frozen and dewaxedsamples,but in none by direct reaction.Of 12 cases subjected to Southern blot hybridization,HBV DNA was detected in 7 by this technique,in 10 by PCR with both methods of DNA extrac-tion and in 3 by direct PCR.The results showed that PCR was sensitive and was comparablewith blot hybridization in detecting intrahepatic HBV DNA.In comparison between differentmethods of sample preparation,the viral detection rate from the dewaxed samples was near thatfrom the frozen ones,while by the direct reaction HBV DNA could be detected only in a fewsamples with high level of infection.
文摘BACKGROUND Studies correlating specific genetic mutations and treatment response are ongoing to establish an effective treatment strategy for gastric cancer(GC).To facilitate this research,a cost-and time-effective method to analyze the mutational status is necessary.Deep learning(DL)has been successfully applied to analyze hematoxylin and eosin(H and E)-stained tissue slide images.AIM To test the feasibility of DL-based classifiers for the frequently occurring mutations from the H and E-stained GC tissue whole slide images(WSIs).METHODS From the GC dataset of The Cancer Genome Atlas(TCGA-STAD),wildtype/mutation classifiers for CDH1,ERBB2,KRAS,PIK3CA,and TP53 genes were trained on 360×360-pixel patches of tissue images.RESULTS The area under the curve(AUC)for the receiver operating characteristic(ROC)curves ranged from 0.727 to 0.862 for the TCGA frozen WSIs and 0.661 to 0.858 for the TCGA formalin-fixed paraffin-embedded(FFPE)WSIs.The performance of the classifier can be improved by adding new FFPE WSI training dataset from our institute.The classifiers trained for mutation prediction in colorectal cancer completely failed to predict the mutational status in GC,indicating that DL-based mutation classifiers are incompatible between different cancers.CONCLUSION This study concluded that DL could predict genetic mutations in H and E-stained tissue slides when they are trained with appropriate tissue data.
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.
基金Supported by Chinese Center for Disease Control and Prevention and the State Key Laboratory for Infectious Disease Prevention and Control,No.2011SKLID103Beijing Key Laboratory of Environmental and Viral Oncology,College of Life Science and Bio-Engineering,Beijing University of Technology(Grant sponsor:State 863 projects,No.2012AA02A404,No2014ZX10005002 and No.PXM2014_014204_07_000046)
文摘AIM:to study the prevalence of human papillomavirus(HPV) in esophageal carcinoma in tangshan,China,a high-incidence area.METHODS:Formalin-fixed,paraffin-embedded tissue specimens from 198 patients who were pathologically diagnosed with esophageal squamous cell carcinoma from 2011 to 2013 were obtained from a pathology department in Tangshan.DNA was extracted from all198 specimens to detect HPV by polymerase chain reaction(PCR).β-globin PCR was performed to check the quality of the DNA extraction procedure.PCR was performed to detect a wide range of HPV types,and type-specific PCR was performed to detect HPV types16 and 18.Negative and positive controls were used for HPV 16 and 18 detection.RESULTS:the DNA extraction method in this study appeared to be more effective than other previously reported methods.After DNA extraction,more than98%of the tissue specimens had an acceptable result in the DNA qualification test(β-globin PCR).the overall prevalence of HPV in tumor tissues by GP6+/GP5+PCR was 79.79%,and the prevalence of HPV types16 and 18 was 40.40%and 47.47%,respectively.PCR demonstrated the presence of HPV,and direct sequencing confirmed the HPV genotypes.All HPVpositive PCR products were checked by DNA sequence analysis using DNAman and compared with the known HPV sequences listed in the basic Local Alignment Search tool database to evaluate the HPV types.this analysis confirmed the presence of HPV types 16 and18.CONCLUSION:DNA of high-risk HPV types 16 and 18is present in esophageal tumors,implicating HPV as a possible etiologic factor for esophageal squamous cell carcinoma.
基金supported by the Chengdu Science and Technology Program(No.2022-YF05-02026-SN).
文摘Formalin-fixed and paraffin-embedded(FFPE)tissues provide a wealth of pathological information crucial for clinical and forensic examinations.Formalin induces robust complexes between DNA and proteins,impacting DNA extraction and complicating short tandem repeat(STR)typing for personal identification and paternity testing.Here,we present a case of paternity testing involving one FFPE tissue and one blood specimen.We compared four DNA extraction methods and analyzed the obtained products from the most successful approach.To ensure robust statistical support,we used a combination of three STR kits for the analysis.This case demonstrates the viability of using multiple kits in tandem for STR profiling of FFPE tissues.
基金supported by a Personnel Training Award from the Department of Health, Hebei Province, a Goldstar Award from the University of New South Wales, and an NHMRC Senior Principal Research Fellowship (630434)
文摘Both fresh-frozen and formalin-fixed,paraffinembedded(FFPE)human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases,especially neurodegenerative disorders.To identify the optimal method for DNA extraction from human brain tissue,we compared methods on differently-processed tissues.Fragments of LRRK2 and MAPT(257 bp and 483 bp/245 bp)were amplified for evaluation.We found that for FFPE samples,the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method(successful DNA extraction from 76%versus 33%of samples).PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples.In the fresh-frozen samples,the DNA extraction success rate was 100%using either a commercial kit(QIAamp DNA Micro)or a laboratorybased method(sample boiling in 0.1 mol/L NaOH,followed by proteinase K digestion,and then DNA extraction using Chelex-100)regardless of PCR amplicon length or tissue storage time.Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples,fresh brain tissue is recommended for DNA extraction in future neuropathological studies.