Pub Date : 2024-09-30DOI: 10.1016/j.jmoldx.2024.08.008
Mohamed A. Jama , N. Scott Reading , Eric Fredrickson , Sherin Shaaban , Yuan Ji
Mutation analysis provides confirmation of a clinical and radiological diagnosis of thanatophoric dysplasia types I and II (TD I and II). We developed a single multiplexed PCR and a single-nucleotide extension (SNE) assay to identify 14 common mutations causing 99% of TD I and TD II, including the challenging three adjacent mutations in the stop codon of exon 18 of the FGFR3 gene. The assay design also provides a solution for resolving SNE PCR product sizing using performance optimized polymer-7. The assay was validated using 37 previously characterized, de-identified patient samples representing the nine wild-types and 10 of 14 mutant genotypes. Four artificial templates were synthesized to mimic four TD I mutations not represented in the available patient samples. Fragment size and fluorophore channel for each SNE product from 10 samples and the four artificial templates were used to define bins and panels for analysis with GeneMarker version 3.0 and GeneMapper version 6.0 software. Allele calls (bin placement within the panels) were verified using the remaining 27 previously characterized samples. This TD I and II PCR and SNE assay is a robust multiplexed assay, streamlined, to identify 14 mutations in one single reaction. This assay has a shorter turnaround time in comparison to traditional Sanger or next-generation sequencing.
{"title":"A Single Multiplex PCR and Single-Nucleotide Extension Assay for the Detection of Common Thanatophoric Dysplasia I and II Mutations","authors":"Mohamed A. Jama , N. Scott Reading , Eric Fredrickson , Sherin Shaaban , Yuan Ji","doi":"10.1016/j.jmoldx.2024.08.008","DOIUrl":"10.1016/j.jmoldx.2024.08.008","url":null,"abstract":"<div><div>Mutation analysis provides confirmation of a clinical and radiological diagnosis of thanatophoric dysplasia types I and II (TD I and II). We developed a single multiplexed PCR and a single-nucleotide extension (SNE) assay to identify 14 common mutations causing 99% of TD I and TD II, including the challenging three adjacent mutations in the stop codon of exon 18 of the <em>FGFR3</em> gene. The assay design also provides a solution for resolving SNE PCR product sizing using performance optimized polymer-7. The assay was validated using 37 previously characterized, de-identified patient samples representing the nine wild-types and 10 of 14 mutant genotypes. Four artificial templates were synthesized to mimic four TD I mutations not represented in the available patient samples. Fragment size and fluorophore channel for each SNE product from 10 samples and the four artificial templates were used to define bins and panels for analysis with GeneMarker version 3.0 and GeneMapper version 6.0 software. Allele calls (bin placement within the panels) were verified using the remaining 27 previously characterized samples. This TD I and II PCR and SNE assay is a robust multiplexed assay, streamlined, to identify 14 mutations in one single reaction. This assay has a shorter turnaround time in comparison to traditional Sanger or next-generation sequencing.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1102-1108"},"PeriodicalIF":3.4,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142367258","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-30DOI: 10.1016/j.jmoldx.2024.08.007
Silvia A. Longhi , Lady J. García-Casares , Arturo Muñoz-Calderón , Lucía Irazu , Marcelo A. Rodríguez , Gustavo Landfried , Julio Alonso-Padilla , Alejandro G. Schijman , Chagas-Group
Timely diagnosis of vertical Trypanosoma cruzi infections involves microscopy-based detection of circulating parasites from peripheral blood, which lacks sensitivity and is operator dependent. Consequently, most children born to T. cruzi–infected mothers are required to undergo serological testing after 9 months, which risks loss to follow-up. Alternatively, the loop-mediated isothermal amplification (LAMP) test for T. cruzi DNA offers high analytical and clinical performance and is easy to use in low-complexity laboratories. Recently, we optimized this technique using an ultrarapid DNA extraction method combined with the LAMP in dried blood spots (DBSs) on Flinders Technology Associates cards. The procedure has been implemented in 10 public maternities across Paraguay, Bolivia, and Argentina, involving the training of 14 technicians. Operators' performance was evaluated using a standardized DBS testing panel for harmonization, including negative controls and DBS samples artificially contaminated with T. cruzi at 50 and 20 cells/mL. There was strong agreement (ĸ = 0.924) for controls and 50 cells/mL samples, and good agreement (ĸ = 0.718) across all testing panels, even at the detection limit of the test. A prospective study collected 306 DBSs from 222 newborns at birth and/or 2 months, detecting T. cruzi microscopically in four cases. LAMP identified eight positive cases and perfectly aligned with real-time PCR (ĸ = 1), demonstrating higher sensitivity than microscopic observation for early detection of infection in infants.
{"title":"Interlaboratory Harmonization Study and Prospective Evaluation of the PURE–Trypanosoma cruzi–Loop-Mediated Isothermal Amplification Assay for Detecting Parasite DNA in Newborn's Dried Blood Spots","authors":"Silvia A. Longhi , Lady J. García-Casares , Arturo Muñoz-Calderón , Lucía Irazu , Marcelo A. Rodríguez , Gustavo Landfried , Julio Alonso-Padilla , Alejandro G. Schijman , Chagas-Group","doi":"10.1016/j.jmoldx.2024.08.007","DOIUrl":"10.1016/j.jmoldx.2024.08.007","url":null,"abstract":"<div><div>Timely diagnosis of vertical <em>Trypanosoma cruzi</em> infections involves microscopy-based detection of circulating parasites from peripheral blood, which lacks sensitivity and is operator dependent. Consequently, most children born to <em>T. cruzi</em>–infected mothers are required to undergo serological testing after 9 months, which risks loss to follow-up. Alternatively, the loop-mediated isothermal amplification (LAMP) test for <em>T. cruzi</em> DNA offers high analytical and clinical performance and is easy to use in low-complexity laboratories. Recently, we optimized this technique using an ultrarapid DNA extraction method combined with the LAMP in dried blood spots (DBSs) on Flinders Technology Associates cards. The procedure has been implemented in 10 public maternities across Paraguay, Bolivia, and Argentina, involving the training of 14 technicians. Operators' performance was evaluated using a standardized DBS testing panel for harmonization, including negative controls and DBS samples artificially contaminated with <em>T. cruzi</em> at 50 and 20 cells/mL. There was strong agreement (ĸ = 0.924) for controls and 50 cells/mL samples, and good agreement (ĸ = 0.718) across all testing panels, even at the detection limit of the test. A prospective study collected 306 DBSs from 222 newborns at birth and/or 2 months, detecting <em>T. cruzi</em> microscopically in four cases. LAMP identified eight positive cases and perfectly aligned with real-time PCR (ĸ = 1), demonstrating higher sensitivity than microscopic observation for early detection of infection in infants.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1055-1064"},"PeriodicalIF":3.4,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142367259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-26DOI: 10.1016/j.jmoldx.2024.08.004
Carmen Rubio-Alarcón , Ellen Stelloo , Daan C.L. Vessies , Iris van 't Erve , Nienke J. Mekkes , Joost Swennenhuis , Soufyan Lakbir , Elisabeth J. van Bree , Marianne Tijssen , Pien Delis-van Diemen , Mirthe Lanfermeijer , Theodora Linders , Daan van den Broek , Cornelis J.A. Punt , Jaap Heringa , Gerrit A. Meijer , Sanne Abeln , Harma Feitsma , Remond J.A. Fijneman
Structural variants (SVs) caused by chromosomal rearrangements in common fragile sites or long interspersed nuclear element (LINE) retrotranspositions are highly prevalent in colorectal cancer. However, methodology for the targeted detection of these SVs is lacking. This article reports the use of formalin-fixed, paraffin-embedded targeted-locus capture (FFPE-TLC) sequencing as a novel technology for the targeted detection of tumor-specific SVs. Analysis of 29 FFPE colorectal tumor samples and 8 matched normal samples revealed tumor-specific SVs in 24 patients (83%), with a median of 2 SVs per patient (range, 1 to 21). A total of 104 SVs were found in the common fragile site–associated genes MACROD2, PRKN, FHIT, and WWOX in 18 patients (62%), and 39 SVs caused by three LINE transposable elements were found in 15 patients (52%). Tumor specificity of SVs was independently verified by droplet digital PCR of tumor tissue DNA, and their applicability as plasma circulating tumor DNA biomarkers was demonstrated. FFPE-TLC sequencing enabled the detection of tumor-specific SVs caused by chromosomal rearrangements and LINE retrotranspositions in FFPE tissue. Therefore, FFPE-TLC sequencing facilitates the investigation of the biological and clinical effects of SVs using FFPE material from (retrospective) cohorts of cancer patients and has potential clinical applicability in the detection of SV biomarkers in the routine molecular diagnostics setting.
{"title":"High Prevalence of Chromosomal Rearrangements and LINE Retrotranspositions Detected in Formalin-Fixed, Paraffin-Embedded Colorectal Cancer Tissue","authors":"Carmen Rubio-Alarcón , Ellen Stelloo , Daan C.L. Vessies , Iris van 't Erve , Nienke J. Mekkes , Joost Swennenhuis , Soufyan Lakbir , Elisabeth J. van Bree , Marianne Tijssen , Pien Delis-van Diemen , Mirthe Lanfermeijer , Theodora Linders , Daan van den Broek , Cornelis J.A. Punt , Jaap Heringa , Gerrit A. Meijer , Sanne Abeln , Harma Feitsma , Remond J.A. Fijneman","doi":"10.1016/j.jmoldx.2024.08.004","DOIUrl":"10.1016/j.jmoldx.2024.08.004","url":null,"abstract":"<div><div>Structural variants (SVs) caused by chromosomal rearrangements in common fragile sites or long interspersed nuclear element (LINE) retrotranspositions are highly prevalent in colorectal cancer. However, methodology for the targeted detection of these SVs is lacking. This article reports the use of formalin-fixed, paraffin-embedded targeted-locus capture (FFPE-TLC) sequencing as a novel technology for the targeted detection of tumor-specific SVs. Analysis of 29 FFPE colorectal tumor samples and 8 matched normal samples revealed tumor-specific SVs in 24 patients (83%), with a median of 2 SVs per patient (range, 1 to 21). A total of 104 SVs were found in the common fragile site–associated genes <em>MACROD2</em>, <em>PRKN</em>, <em>FHIT</em>, and <em>WWOX</em> in 18 patients (62%), and 39 SVs caused by three LINE transposable elements were found in 15 patients (52%). Tumor specificity of SVs was independently verified by droplet digital PCR of tumor tissue DNA, and their applicability as plasma circulating tumor DNA biomarkers was demonstrated. FFPE-TLC sequencing enabled the detection of tumor-specific SVs caused by chromosomal rearrangements and LINE retrotranspositions in FFPE tissue. Therefore, FFPE-TLC sequencing facilitates the investigation of the biological and clinical effects of SVs using FFPE material from (retrospective) cohorts of cancer patients and has potential clinical applicability in the detection of SV biomarkers in the routine molecular diagnostics setting.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1065-1080"},"PeriodicalIF":3.4,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331459","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-26DOI: 10.1016/j.jmoldx.2024.09.002
Alba Abras
{"title":"Isothermal Nucleic Acid Amplification as a Promising and Versatile Diagnostic Approach for Point-of-Care Testing of Congenital Chagas Disease","authors":"Alba Abras","doi":"10.1016/j.jmoldx.2024.09.002","DOIUrl":"10.1016/j.jmoldx.2024.09.002","url":null,"abstract":"","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1042-1044"},"PeriodicalIF":3.4,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-24DOI: 10.1016/j.jmoldx.2024.09.001
Emily S. Winn-Deen , Laura T. Bortolin , Daniel Gusenleitner , Kelly M. Biette , Karen Copeland , Aleksandra Gentry-Maharaj , Sophia Apostolidou , Anthony D. Couvillon , Daniel P. Salem , Sanchari Banerjee , Jonian Grosha , Ibukunoluwapo O. Zabroski , Christopher R. Sedlak , Delaney M. Byrne , Bilal F. Hamzeh , MacKenzie S. King , Lauren T. Cuoco , Peter A. Duff , Brendan J. Manning , Troy B. Hawkins , Usha Menon
The low incidence of ovarian cancer (OC) dictates that any screening strategy needs to be both highly sensitive and highly specific. This study explored the utility of detecting multiple colocalized proteins or glycosylation epitopes on single tumor-associated extracellular vesicles from blood. The novel Mercy Halo Ovarian Cancer Test (OC Test) uses immunoaffinity capture of tumor-associated extracellular vesicles, followed by proximity-ligation real-time quantitative PCR to detect combinations of up to three biomarkers to maximize specificity, and measures multiple combinations to maximize sensitivity. A high-grade serous carcinoma (HGSC) case-control training set of EDTA plasma samples from 397 women was used to lock down the test design, the data interpretation algorithm, and the cutoff between cancer and noncancer. Performance was verified and compared with cancer antigen 125 in an independent blinded case-control set of serum samples from 390 women (132 controls, 66 HGSC, 83 non-HGSC OC, and 109 benign). In the verification study, the OC Test showed a specificity of 97.0% (128/132; 95% CI, 92.4%–99.6%), a HGSC sensitivity of 97.0% (64/66; 95% CI, 87.8%–99.2%), and an area under the curve of 0.97 (95% CI, 0.93–0.99) and detected 73.5% (61/83; 95% CI, 62.7%–82.6%) of the non-HGSC OC cases. This test exhibited fewer false positives in subjects with benign ovarian tumors, nonovarian cancers, and inflammatory conditions when compared with cancer antigen 125. The combined sensitivity and specificity of this new test suggests that it may have potential in OC screening.
{"title":"Improving Specificity for Ovarian Cancer Screening Using a Novel Extracellular Vesicle–Based Blood Test","authors":"Emily S. Winn-Deen , Laura T. Bortolin , Daniel Gusenleitner , Kelly M. Biette , Karen Copeland , Aleksandra Gentry-Maharaj , Sophia Apostolidou , Anthony D. Couvillon , Daniel P. Salem , Sanchari Banerjee , Jonian Grosha , Ibukunoluwapo O. Zabroski , Christopher R. Sedlak , Delaney M. Byrne , Bilal F. Hamzeh , MacKenzie S. King , Lauren T. Cuoco , Peter A. Duff , Brendan J. Manning , Troy B. Hawkins , Usha Menon","doi":"10.1016/j.jmoldx.2024.09.001","DOIUrl":"10.1016/j.jmoldx.2024.09.001","url":null,"abstract":"<div><div>The low incidence of ovarian cancer (OC) dictates that any screening strategy needs to be both highly sensitive and highly specific. This study explored the utility of detecting multiple colocalized proteins or glycosylation epitopes on single tumor-associated extracellular vesicles from blood. The novel Mercy Halo Ovarian Cancer Test (OC Test) uses immunoaffinity capture of tumor-associated extracellular vesicles, followed by proximity-ligation real-time quantitative PCR to detect combinations of up to three biomarkers to maximize specificity, and measures multiple combinations to maximize sensitivity. A high-grade serous carcinoma (HGSC) case-control training set of EDTA plasma samples from 397 women was used to lock down the test design, the data interpretation algorithm, and the cutoff between cancer and noncancer. Performance was verified and compared with cancer antigen 125 in an independent blinded case-control set of serum samples from 390 women (132 controls, 66 HGSC, 83 non-HGSC OC, and 109 benign). In the verification study, the OC Test showed a specificity of 97.0% (128/132; 95% CI, 92.4%–99.6%), a HGSC sensitivity of 97.0% (64/66; 95% CI, 87.8%–99.2%), and an area under the curve of 0.97 (95% CI, 0.93–0.99) and detected 73.5% (61/83; 95% CI, 62.7%–82.6%) of the non-HGSC OC cases. This test exhibited fewer false positives in subjects with benign ovarian tumors, nonovarian cancers, and inflammatory conditions when compared with cancer antigen 125. The combined sensitivity and specificity of this new test suggests that it may have potential in OC screening.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1129-1148"},"PeriodicalIF":3.4,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-24DOI: 10.1016/j.jmoldx.2024.08.006
Daniel P. Salem , Laura T. Bortolin , Dan Gusenleitner , Jonian Grosha , Ibukunoluwapo O. Zabroski , Kelly M. Biette , Sanchari Banerjee , Christopher R. Sedlak , Delaney M. Byrne , Bilal F. Hamzeh , MacKenzie S. King , Lauren T. Cuoco , Timothy Santos-Heiman , Gabrielle N. Barcaskey , Katherine S. Yang , Peter A. Duff , Emily S. Winn-Deen , Toumy Guettouche , Dawn R. Mattoon , Eric K. Huang , Joseph C. Sedlak
Detection of cancer early, when it is most treatable, remains a significant challenge because of the lack of diagnostic methods sufficiently sensitive to detect nascent tumors. Early-stage tumors are small relative to their tissue of origin, heterogeneous, and infrequently manifest in clinical symptoms. The detection of early-stage tumors is challenging given the lack of tumor-specific indicators (ie, protein biomarkers, circulating tumor DNA) to enable detection using a noninvasive diagnostic assay. To overcome these obstacles, we have developed a liquid biopsy assay that interrogates circulating extracellular vesicles (EVs) to detect tumor-specific biomarkers colocalized on the surface of individual EVs. We demonstrate the technical feasibility of this approach in human cancer cell line–derived EVs, where we show strong correlations between assay signal and cell line gene/protein expression for the ovarian cancer–associated biomarkers bone marrow stromal antigen-2, folate receptor-α, and mucin-1. Furthermore, we demonstrate that detecting distinct colocalized biomarkers on the surface of EVs significantly improves discrimination performance relative to single biomarker measurements. Using this approach, we observe promising discrimination of high-grade serous ovarian cancer versus benign ovarian masses and healthy women in a proof-of-concept clinical study.
由于缺乏足够灵敏的诊断方法来检测新生肿瘤，因此在癌症最容易治疗的早期发现癌症仍然是一项重大挑战。早期肿瘤相对于其原发组织而言较小，具有异质性，并且很少表现出临床症状。由于缺乏丰富的肿瘤特异性指标（如蛋白质生物标志物、循环肿瘤 DNA 等），因此使用非侵入性诊断方法检测肿瘤的存在变得更加困难。为了克服这些障碍，我们开发了一种液体活检检测方法，该方法可检测循环细胞外囊泡 (EV)，从而检测单个 EV 表面共定位的肿瘤特异性生物标记物。我们在人类癌细胞系衍生的EV中证明了这种方法的技术可行性，我们发现检测信号与卵巢癌相关生物标记物BST2、FOLR1和MUC1的细胞系基因/蛋白表达之间存在很强的相关性。此外，我们还证明，与单一生物标记物测量相比，检测 EV 表面不同的共定位生物标记物能显著提高判别性能。利用这种方法，我们在一项概念验证临床研究中观察到了区分高级别浆液性卵巢癌与良性卵巢肿块和健康妇女的良好效果。
{"title":"Colocalization of Cancer-Associated Biomarkers on Single Extracellular Vesicles for Early Detection of Cancer","authors":"Daniel P. Salem , Laura T. Bortolin , Dan Gusenleitner , Jonian Grosha , Ibukunoluwapo O. Zabroski , Kelly M. Biette , Sanchari Banerjee , Christopher R. Sedlak , Delaney M. Byrne , Bilal F. Hamzeh , MacKenzie S. King , Lauren T. Cuoco , Timothy Santos-Heiman , Gabrielle N. Barcaskey , Katherine S. Yang , Peter A. Duff , Emily S. Winn-Deen , Toumy Guettouche , Dawn R. Mattoon , Eric K. Huang , Joseph C. Sedlak","doi":"10.1016/j.jmoldx.2024.08.006","DOIUrl":"10.1016/j.jmoldx.2024.08.006","url":null,"abstract":"<div><div>Detection of cancer early, when it is most treatable, remains a significant challenge because of the lack of diagnostic methods sufficiently sensitive to detect nascent tumors. Early-stage tumors are small relative to their tissue of origin, heterogeneous, and infrequently manifest in clinical symptoms. The detection of early-stage tumors is challenging given the lack of tumor-specific indicators (ie, protein biomarkers, circulating tumor DNA) to enable detection using a noninvasive diagnostic assay. To overcome these obstacles, we have developed a liquid biopsy assay that interrogates circulating extracellular vesicles (EVs) to detect tumor-specific biomarkers colocalized on the surface of individual EVs. We demonstrate the technical feasibility of this approach in human cancer cell line–derived EVs, where we show strong correlations between assay signal and cell line gene/protein expression for the ovarian cancer–associated biomarkers bone marrow stromal antigen-2, folate receptor-α, and mucin-1. Furthermore, we demonstrate that detecting distinct colocalized biomarkers on the surface of EVs significantly improves discrimination performance relative to single biomarker measurements. Using this approach, we observe promising discrimination of high-grade serous ovarian cancer versus benign ovarian masses and healthy women in a proof-of-concept clinical study.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1109-1128"},"PeriodicalIF":3.4,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-24DOI: 10.1016/j.jmoldx.2024.08.005
Elena Aramendía Cotillas , Carina Bernardo , Srinivas Veerla , Fredrik Liedberg , Gottfrid Sjödahl , Pontus Eriksson
Stratification of cancer into biologically and molecularly similar subgroups is a cornerstone of precision medicine. The Lund Taxonomy classification system for urothelial carcinoma aims to be applicable across the whole disease spectrum including both non–muscle-invasive and invasive bladder cancer. A successful classification system is one that can be robustly and reproducibly applied to new samples. However, transcriptomic methods used for subtype classification are affected by analytic platform, data preprocessing, cohort composition, and tumor purity. Furthermore, only limited data have been published evaluating the transferability of existing classification algorithms to external data sets. In this study, a single sample classifier was developed based on in-house microarray and RNA-sequencing data, intended to be broadly applicable across studies and platforms. The new classification algorithm was applied to 10 published external bladder cancer cohorts (n = 2560 cases) to evaluate its ability to capture characteristic subtype-associated gene expression signatures and complementary data such as mutations, clinical outcomes, treatment response, or histologic subtypes. The effect of sample purity on the classification results was evaluated by generating low-purity versions of samples in silico. The classifier was robustly applicable across different gene expression profiling platforms and preprocessing methods and was less sensitive to variations in sample purity.
{"title":"A Versatile and Upgraded Version of the LundTax Classification Algorithm Applied to Independent Cohorts","authors":"Elena Aramendía Cotillas , Carina Bernardo , Srinivas Veerla , Fredrik Liedberg , Gottfrid Sjödahl , Pontus Eriksson","doi":"10.1016/j.jmoldx.2024.08.005","DOIUrl":"10.1016/j.jmoldx.2024.08.005","url":null,"abstract":"<div><div>Stratification of cancer into biologically and molecularly similar subgroups is a cornerstone of precision medicine. The Lund Taxonomy classification system for urothelial carcinoma aims to be applicable across the whole disease spectrum including both non–muscle-invasive and invasive bladder cancer. A successful classification system is one that can be robustly and reproducibly applied to new samples. However, transcriptomic methods used for subtype classification are affected by analytic platform, data preprocessing, cohort composition, and tumor purity. Furthermore, only limited data have been published evaluating the transferability of existing classification algorithms to external data sets. In this study, a single sample classifier was developed based on in-house microarray and RNA-sequencing data, intended to be broadly applicable across studies and platforms. The new classification algorithm was applied to 10 published external bladder cancer cohorts (<em>n</em> = 2560 cases) to evaluate its ability to capture characteristic subtype-associated gene expression signatures and complementary data such as mutations, clinical outcomes, treatment response, or histologic subtypes. The effect of sample purity on the classification results was evaluated by generating low-purity versions of samples <em>in silico</em>. The classifier was robustly applicable across different gene expression profiling platforms and preprocessing methods and was less sensitive to variations in sample purity.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1081-1101"},"PeriodicalIF":3.4,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142331457","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-12DOI: 10.1016/j.jmoldx.2024.08.002
Mario A. González-Carracedo , Esther Herrera-Luis , María Marco-Simancas , Ainhoa Escuela-Escobar , Elena Martín-González , Olaia Sardón-Prado , Paula Corcuera , Jose M. Hernández-Pérez , Fabián Lorenzo-Díaz , José A. Pérez-Pérez
α-1 Antitrypsin (AAT) is an acute-phase reactant with immunomodulatory properties that mainly inhibits neutrophil elastase. Low serum levels cause AAT deficiency (AATD), an underdiagnosed condition that predisposes to pulmonary and hepatic diseases. The SERPINA1 gene, which encodes AAT, contains >500 variants. PI∗Z and PI∗S alleles are the most diagnosed causes of AATD, but the role of the SERPINA1 haplotypes in AAT function remains unknown. SERPINA1 gene was PCR amplified from 94 patients with asthma, using primers with tails for indexing. Sequencing libraries were loaded into a MinION-Mk1C, and MinKNOW was used for basecalling and demultiplexing. Nanofilt and Minimap2 were used for filtering and mapping/alignment. Variant calling/phasing were performed with PEPPER-Margin-DeepVariant. SERPINA1 gene was 100% covered for all samples, with a minimum sequencing depth of 500×. A total of 75 single-nucleotide variants (SNVs) and 4 insertions/deletions were detected, with 45 and 2 of them highly polymorphic (minor allele frequency >0.1), respectively. Nine of the SNVs showed differences in allele frequencies when compared with the overall Spanish population. More than 90% of heterozygous SNVs were phased, yielding 91 and 58 different haplotypes for each SERPINA1 amplified region. Haplotype-based linkage disequilibrium analysis suggests that a recombination hotspot could generate variation in the SERPINA1 gene. The proposed workflow enables haplotype-aware genotyping of the SERPINA1 gene by nanopore sequencing, which will allow the development of novel AATD diagnostic strategies.
{"title":"Haplotype-Aware Detection of SERPINA1 Variants by Nanopore Sequencing","authors":"Mario A. González-Carracedo , Esther Herrera-Luis , María Marco-Simancas , Ainhoa Escuela-Escobar , Elena Martín-González , Olaia Sardón-Prado , Paula Corcuera , Jose M. Hernández-Pérez , Fabián Lorenzo-Díaz , José A. Pérez-Pérez","doi":"10.1016/j.jmoldx.2024.08.002","DOIUrl":"10.1016/j.jmoldx.2024.08.002","url":null,"abstract":"<div><div>α-1 Antitrypsin (AAT) is an acute-phase reactant with immunomodulatory properties that mainly inhibits neutrophil elastase. Low serum levels cause AAT deficiency (AATD), an underdiagnosed condition that predisposes to pulmonary and hepatic diseases. The <em>SERPINA1</em> gene, which encodes AAT, contains >500 variants. <em>PI∗Z</em> and <em>PI∗S</em> alleles are the most diagnosed causes of AATD, but the role of the <em>SERPINA1</em> haplotypes in AAT function remains unknown. <em>SERPINA1</em> gene was PCR amplified from 94 patients with asthma, using primers with tails for indexing. Sequencing libraries were loaded into a MinION-Mk1C, and MinKNOW was used for basecalling and demultiplexing. Nanofilt and Minimap2 were used for filtering and mapping/alignment. Variant calling/phasing were performed with PEPPER-Margin-DeepVariant. <em>SERPINA1</em> gene was 100% covered for all samples, with a minimum sequencing depth of 500×. A total of 75 single-nucleotide variants (SNVs) and 4 insertions/deletions were detected, with 45 and 2 of them highly polymorphic (minor allele frequency >0.1), respectively. Nine of the SNVs showed differences in allele frequencies when compared with the overall Spanish population. More than 90% of heterozygous SNVs were phased, yielding 91 and 58 different haplotypes for each <em>SERPINA1</em> amplified region. Haplotype-based linkage disequilibrium analysis suggests that a recombination hotspot could generate variation in the <em>SERPINA1</em> gene. The proposed workflow enables haplotype-aware genotyping of the <em>SERPINA1</em> gene by nanopore sequencing, which will allow the development of novel AATD diagnostic strategies.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 11","pages":"Pages 971-987"},"PeriodicalIF":3.4,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142299819","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-11DOI: 10.1016/j.jmoldx.2024.08.001
Hans-Peter Adams , Matthew C. Hiemenz , Kay Hertel , Frederike Fuhlbrück , Mara Thomas , James Oughton , Helle Sorensen , Ulrich Schlecht , Justin M. Allen , Martina Cantone , Sophie Osswald , David Gonzalez , Eli Pikarsky , Muriel De Vos , Ed Schuuring , Thomas Wieland
Increased adoption of personalized medicine has brought comprehensive genomic profiling (CGP) to the forefront. However, differences in assay, bioinformatics, and reporting systems and lack of understanding of their complex interplay are a challenge for implementation and achieving uniformity in CGP testing. Two commercially available, tissue-based, in-house CGP assays were compared, in combination with a tertiary analysis solution in a research use only (RUO) context: the AVENIO Tumor Tissue CGP RUO Kit paired with navify Mutation Profiler (RUO) software and the TruSight Oncology 500 RUO assay paired with PierianDx Clinical Genomics Workspace software. Agreements and differences between the assays were assessed for short variants, copy number alterations, rearrangements, tumor mutational burden, and microsatellite instability, including variant categorization and clinical trial-matching (CTM) recommendations. Results showed good overall agreement for short variant, known gene fusion, and microsatellite instability detection. Important differences were obtained in tumor mutational burden scoring, copy number alteration detection, and CTM. Differences in variant and biomarker detection could be explained by bioinformatic approaches to variant calling, filtering, tiering, and normalization; differences in CTM, by underlying reported variants and conceptual differences in system parameters. Thus, distinctions between different approaches may lead to inconsistent results. Complexities in calling, filtering, and interpreting variants illustrate key considerations for implementation of any high-quality CGP in the laboratory and bringing uniformity to genomic insight results.
{"title":"Comparison of Results from Two Commercially Available In-House Tissue-Based Comprehensive Genomic Profiling Solutions","authors":"Hans-Peter Adams , Matthew C. Hiemenz , Kay Hertel , Frederike Fuhlbrück , Mara Thomas , James Oughton , Helle Sorensen , Ulrich Schlecht , Justin M. Allen , Martina Cantone , Sophie Osswald , David Gonzalez , Eli Pikarsky , Muriel De Vos , Ed Schuuring , Thomas Wieland","doi":"10.1016/j.jmoldx.2024.08.001","DOIUrl":"10.1016/j.jmoldx.2024.08.001","url":null,"abstract":"<div><div>Increased adoption of personalized medicine has brought comprehensive genomic profiling (CGP) to the forefront. However, differences in assay, bioinformatics, and reporting systems and lack of understanding of their complex interplay are a challenge for implementation and achieving uniformity in CGP testing. Two commercially available, tissue-based, in-house CGP assays were compared, in combination with a tertiary analysis solution in a research use only (RUO) context: the AVENIO Tumor Tissue CGP RUO Kit paired with navify Mutation Profiler (RUO) software and the TruSight Oncology 500 RUO assay paired with PierianDx Clinical Genomics Workspace software. Agreements and differences between the assays were assessed for short variants, copy number alterations, rearrangements, tumor mutational burden, and microsatellite instability, including variant categorization and clinical trial-matching (CTM) recommendations. Results showed good overall agreement for short variant, known gene fusion, and microsatellite instability detection. Important differences were obtained in tumor mutational burden scoring, copy number alteration detection, and CTM. Differences in variant and biomarker detection could be explained by bioinformatic approaches to variant calling, filtering, tiering, and normalization; differences in CTM, by underlying reported variants and conceptual differences in system parameters. Thus, distinctions between different approaches may lead to inconsistent results. Complexities in calling, filtering, and interpreting variants illustrate key considerations for implementation of any high-quality CGP in the laboratory and bringing uniformity to genomic insight results.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 11","pages":"Pages 1018-1033"},"PeriodicalIF":3.4,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142264102","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-03DOI: 10.1016/j.jmoldx.2024.06.007
Shimul Chowdhury , Michael Kesling , Micah Collins , Vanessa Lopez , Yuan Xue , Glenn Oliveira , Verena Friedl , Anna Bergamaschi , David Haan , Wayne Volkmuth , Samuel Levy
Early detection of pancreatic cancer has been shown to improve patient survival rates. However, effective early detection tools to detect pancreatic cancer do not currently exist. The Avantect Pancreatic Cancer Test, leveraging the 5-hydroxymethylation [5-hydroxymethylcytosine (5hmC)] signatures in cell-free DNA, was developed and analytically validated to address this unmet need. We report a comprehensive analytical validation study encompassing precision, sample stability, limit of detection, interfering substance studies, and a comparison with an alternative method. The assay performance on an independent case-control patient cohort was previously reported with a sensitivity for early-stage (stage I/II) pancreatic cancer of 68.3% (95% CI, 51.9%–81.9%) and an overall specificity of 96.9% (95% CI, 96.1%–97.7%). Precision studies showed a cancer classification of 100% concordance in biological replicates. The sample stability studies revealed stable assay performance for up to 7 days after blood collection. The limit of detection studies revealed equal results between early- and late-stage cancer samples, emphasizing strong early-stage performance characteristics. Comparisons of concordance of the Avantect assay with the enzymatic methyl sequencing (EM-Seq) method, which measures both methylation (5-methylcytosine) and 5hmC, were >95% for all samples tested. The Avantect Pancreatic Cancer Test showed strong analytical validation in multiple validation studies required for laboratory-developed test accreditation. The comparison of 5hmC versus EM-Seq further validated the 5hmC approach as a robust and reproducible assay.
{"title":"Analytical Validation of an Early Detection Pancreatic Cancer Test Using 5-Hydroxymethylation Signatures","authors":"Shimul Chowdhury , Michael Kesling , Micah Collins , Vanessa Lopez , Yuan Xue , Glenn Oliveira , Verena Friedl , Anna Bergamaschi , David Haan , Wayne Volkmuth , Samuel Levy","doi":"10.1016/j.jmoldx.2024.06.007","DOIUrl":"10.1016/j.jmoldx.2024.06.007","url":null,"abstract":"<div><p>Early detection of pancreatic cancer has been shown to improve patient survival rates. However, effective early detection tools to detect pancreatic cancer do not currently exist. The Avantect Pancreatic Cancer Test, leveraging the 5-hydroxymethylation [5-hydroxymethylcytosine (5hmC)] signatures in cell-free DNA, was developed and analytically validated to address this unmet need. We report a comprehensive analytical validation study encompassing precision, sample stability, limit of detection, interfering substance studies, and a comparison with an alternative method. The assay performance on an independent case-control patient cohort was previously reported with a sensitivity for early-stage (stage I/II) pancreatic cancer of 68.3% (95% CI, 51.9%–81.9%) and an overall specificity of 96.9% (95% CI, 96.1%–97.7%). Precision studies showed a cancer classification of 100% concordance in biological replicates. The sample stability studies revealed stable assay performance for up to 7 days after blood collection. The limit of detection studies revealed equal results between early- and late-stage cancer samples, emphasizing strong early-stage performance characteristics. Comparisons of concordance of the Avantect assay with the enzymatic methyl sequencing (EM-Seq) method, which measures both methylation (5-methylcytosine) and 5hmC, were >95% for all samples tested. The Avantect Pancreatic Cancer Test showed strong analytical validation in multiple validation studies required for laboratory-developed test accreditation. The comparison of 5hmC versus EM-Seq further validated the 5hmC approach as a robust and reproducible assay.</p></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 10","pages":"Pages 888-896"},"PeriodicalIF":3.4,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1525157824001582/pdfft?md5=adc6e94f71f3c1506c11dec2c7acce89&pid=1-s2.0-S1525157824001582-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142127210","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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