Pub Date : 2024-11-25DOI: 10.1016/j.jmoldx.2024.06.013
Victoria M. Pratt , Sara Akhavanfard , Jane Houldsworth , Jennifer J. Laffin , Ann M. Moyer , Honey V. Reddi , Stuart A. Scott , Matthew S. Lebo
{"title":"Twenty-Five Years of Germline Genetic Testing and What May Lie Ahead","authors":"Victoria M. Pratt , Sara Akhavanfard , Jane Houldsworth , Jennifer J. Laffin , Ann M. Moyer , Honey V. Reddi , Stuart A. Scott , Matthew S. Lebo","doi":"10.1016/j.jmoldx.2024.06.013","DOIUrl":"10.1016/j.jmoldx.2024.06.013","url":null,"abstract":"","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1038-1041"},"PeriodicalIF":3.4,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142720210","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}
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) application for targeted sequencing has made a breakthrough in the genomic research era. High diversity in the capsular polysaccharide (cps) locus of Streptococcus pneumoniae has hampered identification of the serotype. This study developed a new serotyping method for S. pneumoniae using CRISPR/Cas9–targeted sequencing with the Oxford Nanopore Technologies platform. A probe was designed at the position of the cps locus using an excision approach on two sides flanking genes between the dexB and aliA genes with approximately 20 kb. A native barcoding method was used for multiplexing. The probe will attach to a specific side followed by attachment of CRISPR/Cas9 to cut the recognition area. The study used de novo assembly to reconstruct sequence reads, which were analyzed using PneumoCRISPR, a new serotyping pipeline for Oxford Nanopore Technologies sequencing data output. Four CRISPR/Cas9 probes have been designed and recognize the cps locus of S. pneumoniae. Serotyping results align precisely with serotyping data from whole-genome sequencing. This serotyping method also allows researchers to use multiple samples in a single run. The new serotyping method based on CRISPR/Cas9–targeted sequencing holds immense promise for serotype identification of S. pneumoniae.
{"title":"A New Serotyping Method of Streptococcus pneumoniae Based on CRISPR/Cas9–Targeted Sequencing","authors":"Yustinus Maladan , Endah Retnaningrum , Budi Setiadi Daryono , Rosantia Sarassari , Ratna Fathma Sari , Sarah Azhari Balqis , Ghina Athyah Wahid , Dodi Safari","doi":"10.1016/j.jmoldx.2024.08.009","DOIUrl":"10.1016/j.jmoldx.2024.08.009","url":null,"abstract":"<div><div>Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) application for targeted sequencing has made a breakthrough in the genomic research era. High diversity in the capsular polysaccharide (<em>cps</em>) locus of <em>Streptococcus pneumoniae</em> has hampered identification of the serotype. This study developed a new serotyping method for <em>S. pneumoniae</em> using CRISPR/Cas9–targeted sequencing with the Oxford Nanopore Technologies platform. A probe was designed at the position of the <em>cps</em> locus using an excision approach on two sides flanking genes between the <em>dexB</em> and <em>aliA</em> genes with approximately 20 kb. A native barcoding method was used for multiplexing. The probe will attach to a specific side followed by attachment of CRISPR/Cas9 to cut the recognition area. The study used <em>de novo</em> assembly to reconstruct sequence reads, which were analyzed using PneumoCRISPR, a new serotyping pipeline for Oxford Nanopore Technologies sequencing data output. Four CRISPR/Cas9 probes have been designed and recognize the <em>cps</em> locus of <em>S. pneumoniae</em>. Serotyping results align precisely with serotyping data from whole-genome sequencing. This serotyping method also allows researchers to use multiple samples in a single run. The new serotyping method based on CRISPR/Cas9–targeted sequencing holds immense promise for serotype identification of <em>S. pneumoniae</em>.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1045-1054"},"PeriodicalIF":3.4,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142721844","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 : 2024-11-25DOI: 10.1016/j.jmoldx.2024.06.012
Karen L. Kaul , Timothy J. O'Leary , Barbara Zehnbauer
{"title":"Changes and Challenges in Molecular Diagnostics","authors":"Karen L. Kaul , Timothy J. O'Leary , Barbara Zehnbauer","doi":"10.1016/j.jmoldx.2024.06.012","DOIUrl":"10.1016/j.jmoldx.2024.06.012","url":null,"abstract":"","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1035-1037"},"PeriodicalIF":3.4,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142720211","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 : 2024-11-25DOI: 10.1016/j.jmoldx.2024.09.003
Ruoheng Zhang , Yimeng Shang , Joseph Cioccio , Kevin Rakszawski , Myles Nickolich , Christopher Ehmann , Yoshitaka Inoue , Seema Naik , Witold Rybka , Hong Zheng , Joseph Mierski , Brooke Silar , Jason Liao , Robert Greiner , Valerie Brown , David Claxton , Jing Ning , Shouhao Zhou , Shin Mineishi , Kentaro Minagawa , Hiroko Shike
Chimerism test was evaluated to predict leukemia relapse. Increasing mixed chimerism (IMC), defined as recipient increase ≥0.1% in peripheral blood total cell chimerism, was used as a surrogate of disease activity. Combination of quantitative PCR and short-tandem repeat method was applied to achieve high assay sensitivity. Total of 184 patients received stem cell transplant for acute myeloid leukemia (N = 110), acute lymphocytic leukemia (N = 41), myelodysplastic syndrome (N = 30), and 2389 chimerism tests (median follow-up, 1054 days). Sixty-six patients relapsed, and 118 patients did not. Cumulative incidence of relapse increased after 1 IMC or ≥2 consecutive IMCs (hazard ratios, 9.9 and 44.4, respectively). Predicted percentage relapse by day 30 after IMC was 0% (0 IMC), 10% (1 IMC), and 40% (≥2 IMCs). The last chimerism results before relapse detected IMC in 57 of 66 relapsed patients (sensitivity, 86.4%). Nine patients had no IMC before relapse (false negative) because of rapidly evolving relapse (N = 4) or extramural relapse (N = 5). In 118 patients without relapse, 158 of 1873 tests detected IMC (false positive, 8.4%; specificity, 91.6%). The false-positive rates increased with higher percentage recipient T-cell chimerism levels, indicating T-cell contamination as a cause. Chimerism monitoring predicts relapse. However, caution must be taken for false-positive or false-negative IMCs. T-cell removal can improve chimerism test specificity in patients with mixed T-cell chimerism.
对嵌合体检测进行评估，以预测白血病复发。混合嵌合体增加（IMC）被定义为受体外周血总细胞嵌合体增加≥0.1%，被用作疾病活动性的替代指标。该方法结合了定量 PCR 和短串联重复方法，以达到较高的检测灵敏度。共有184名急性髓性白血病（110人）、急性淋巴细胞白血病（41人）和骨髓增生异常综合征（30人）患者接受了干细胞移植，共进行了2389次嵌合体检测（中位随访时间为1054天）。66名患者复发，118名患者未复发。复发的累积发生率在1次IMC或连续≥2次IMC后增加（危险比分别为9.9和44.4）。IMC后第30天的预测复发率分别为0%（0次IMC）、10%（1次IMC）和40%（≥2次IMC）。66 位复发患者中有 57 位在复发前的最后一次嵌合结果中检测到了 IMC（灵敏度为 86.4%）。9名患者在复发前没有IMC（假阴性），原因是快速发展的复发（4例）或室外复发（5例）。在 118 名没有复发的患者中，1873 次检测中有 158 次检测到 IMC（假阳性，8.4%；特异性，91.6%）。受体T细胞嵌合率越高，假阳性率越高，这表明T细胞污染是一个原因。嵌合度监测可预测复发。不过，必须注意 IMC 的假阳性或假阴性。去除 T 细胞可提高混合 T 细胞嵌合患者嵌合体检测的特异性。
{"title":"Sensitivity and Specificity of Chimerism Tests in Predicting Leukemia Relapse Using Increasing Mixed Chimerism","authors":"Ruoheng Zhang , Yimeng Shang , Joseph Cioccio , Kevin Rakszawski , Myles Nickolich , Christopher Ehmann , Yoshitaka Inoue , Seema Naik , Witold Rybka , Hong Zheng , Joseph Mierski , Brooke Silar , Jason Liao , Robert Greiner , Valerie Brown , David Claxton , Jing Ning , Shouhao Zhou , Shin Mineishi , Kentaro Minagawa , Hiroko Shike","doi":"10.1016/j.jmoldx.2024.09.003","DOIUrl":"10.1016/j.jmoldx.2024.09.003","url":null,"abstract":"<div><div>Chimerism test was evaluated to predict leukemia relapse. Increasing mixed chimerism (IMC), defined as recipient increase ≥0.1% in peripheral blood total cell chimerism, was used as a surrogate of disease activity. Combination of quantitative PCR and short-tandem repeat method was applied to achieve high assay sensitivity. Total of 184 patients received stem cell transplant for acute myeloid leukemia (N = 110), acute lymphocytic leukemia (N = 41), myelodysplastic syndrome (N = 30), and 2389 chimerism tests (median follow-up, 1054 days). Sixty-six patients relapsed, and 118 patients did not. Cumulative incidence of relapse increased after 1 IMC or ≥2 consecutive IMCs (hazard ratios, 9.9 and 44.4, respectively). Predicted percentage relapse by day 30 after IMC was 0% (0 IMC), 10% (1 IMC), and 40% (≥2 IMCs). The last chimerism results before relapse detected IMC in 57 of 66 relapsed patients (sensitivity, 86.4%). Nine patients had no IMC before relapse (false negative) because of rapidly evolving relapse (N = 4) or extramural relapse (N = 5). In 118 patients without relapse, 158 of 1873 tests detected IMC (false positive, 8.4%; specificity, 91.6%). The false-positive rates increased with higher percentage recipient T-cell chimerism levels, indicating T-cell contamination as a cause. Chimerism monitoring predicts relapse. However, caution must be taken for false-positive or false-negative IMCs. T-cell removal can improve chimerism test specificity in patients with mixed T-cell chimerism.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1159-1170"},"PeriodicalIF":3.4,"publicationDate":"2024-11-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142721941","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 : 2024-10-24DOI: 10.1016/j.jmoldx.2024.03.011
Yi-Wei Tang , Barbara A. Zehnbauer
{"title":"Navigating the Flood","authors":"Yi-Wei Tang , Barbara A. Zehnbauer","doi":"10.1016/j.jmoldx.2024.03.011","DOIUrl":"10.1016/j.jmoldx.2024.03.011","url":null,"abstract":"","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 11","pages":"Pages 950-951"},"PeriodicalIF":3.4,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142512119","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 : 2024-10-24DOI: 10.1016/j.jmoldx.2024.08.003
Emily H. Essex
{"title":"25 Years of Publishing The Journal of Molecular Diagnostics","authors":"Emily H. Essex","doi":"10.1016/j.jmoldx.2024.08.003","DOIUrl":"10.1016/j.jmoldx.2024.08.003","url":null,"abstract":"","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 11","pages":"Pages 943-944"},"PeriodicalIF":3.4,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142512118","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 : 2024-10-24DOI: 10.1016/j.jmoldx.2024.06.011
Pawel Mroz , Mark D. Ewalt , Susan E. Harley , Patricia C. Tsang , Rena R. Xian , Craig R. Soderquist
{"title":"The Era of Molecular Hematopathology","authors":"Pawel Mroz , Mark D. Ewalt , Susan E. Harley , Patricia C. Tsang , Rena R. Xian , Craig R. Soderquist","doi":"10.1016/j.jmoldx.2024.06.011","DOIUrl":"10.1016/j.jmoldx.2024.06.011","url":null,"abstract":"","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 11","pages":"Pages 945-949"},"PeriodicalIF":3.4,"publicationDate":"2024-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142512120","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 : 2024-10-02DOI: 10.1016/j.jmoldx.2024.09.004
Adil Menon, Madina Sukhanova, Kevin L. Nocito, Juehua Gao, Lawrence J. Jennings, Erica R. Vormittag-Nocito
Clonal hematopoiesis (CH) and clonal cytopenia of undetermined significance (CCUS) are recently recognized diagnostic entities that serve as independent risk factors for cardiovascular disease and myeloid malignancy. CH is an incidental finding, and evaluation of the incidence of CH/CCUS-associated mutations in solid tumor next-generation sequencing samples was undertaken to better understand the prevalence of mutations in this population. A retrospective review of clinical sequencing data for solid tumor malignancies diagnosed between February 2022 and April 2023 on next-generation sequencing data was performed. Cases were reviewed for variants in genes associated with CH/CCUS. Variant allele frequencies and other factors of the sequencing data were assessed for determining risk of CH/CCUS. A total of 2479 cases were evaluated during the study period. Of these, 29 cases demonstrated potential CH/CCUS-associated mutations, with a total of 33 variants identified. These were identified in a variety of tumor types, with gliomas being the most common. Significant cardiac histories were found in over half of cases identified, and few cases had abnormal blood counts. Detailed criteria for flagging variants as suspicious for CH and recommendations for these criteria as future guidelines for reporting are described. These variants are incidental findings that require more extensive follow-up or change in therapy management using a single institutional cohort.
{"title":"Detection and Interpretation of Clonal Hematopoiesis Variants during Routine Solid Tumor Next-Generation Sequencing","authors":"Adil Menon, Madina Sukhanova, Kevin L. Nocito, Juehua Gao, Lawrence J. Jennings, Erica R. Vormittag-Nocito","doi":"10.1016/j.jmoldx.2024.09.004","DOIUrl":"10.1016/j.jmoldx.2024.09.004","url":null,"abstract":"<div><div>Clonal hematopoiesis (CH) and clonal cytopenia of undetermined significance (CCUS) are recently recognized diagnostic entities that serve as independent risk factors for cardiovascular disease and myeloid malignancy. CH is an incidental finding, and evaluation of the incidence of CH/CCUS-associated mutations in solid tumor next-generation sequencing samples was undertaken to better understand the prevalence of mutations in this population. A retrospective review of clinical sequencing data for solid tumor malignancies diagnosed between February 2022 and April 2023 on next-generation sequencing data was performed. Cases were reviewed for variants in genes associated with CH/CCUS. Variant allele frequencies and other factors of the sequencing data were assessed for determining risk of CH/CCUS. A total of 2479 cases were evaluated during the study period. Of these, 29 cases demonstrated potential CH/CCUS-associated mutations, with a total of 33 variants identified. These were identified in a variety of tumor types, with gliomas being the most common. Significant cardiac histories were found in over half of cases identified, and few cases had abnormal blood counts. Detailed criteria for flagging variants as suspicious for CH and recommendations for these criteria as future guidelines for reporting are described. These variants are incidental findings that require more extensive follow-up or change in therapy management using a single institutional cohort.</div></div>","PeriodicalId":50128,"journal":{"name":"Journal of Molecular Diagnostics","volume":"26 12","pages":"Pages 1149-1158"},"PeriodicalIF":3.4,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142373414","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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