MP57-08 MOLECULAR TESTING WITH NEXT-GENERATION SEQUENCING APPEARS TO IDENTIFY BIOFILM ON PENILE PROSTHESES BETTER THAN TRADITIONAL CULTURES: THE NEW GOLD STANDARD?

Context.— Next-generation sequencing is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for patients with cancer. Oncogenic fusion transcripts are among the various classifications of genetic abnormalities present in tumors and are typically detected clinically with fluorescence in situ hybridization (FISH). However, FISH probes only exist for a limited number of targets, do not provide any information about fusion partners, cannot be multiplex, and have been shown to be limited in specificity for common targets such as ALK. Objective.— To validate an anchored multiplex polymerase chain reaction–based panel for the detection of fusion transcripts in a university hospital–based clinical molecular diagnostics laboratory. Design.— We used 109 unique clinical specimens to validate a custom panel targeting 104 exon boundaries from 17 genes involved in fusions in solid tumors. The panel can accept as little as 100 ng of total nucleic acid from PreservCyt-fixed tissue, and formalin-fixed, paraffin-embedded specimens with as little as 10% tumor nuclei. Results.— Using FISH as the gold standard, this assay has a sensitivity of 88.46% and a specificity of 95.83% for the detection of fusion transcripts involving ALK, RET, and ROS1 in lung adenocarcinomas. Using a validated next-generation sequencing assay as the orthogonal gold standard for the detection of EGFR variant III (EGFRvIII) in glioblastomas, the assay is 92.31% sensitive and 100% specific. Conclusions.— This multiplexed assay is tumor and fusion partner agnostic and will provide clinical utility in therapy selection for patients with solid tumors.

Download Full-text

PLAG1 Immunohistochemical Staining Is a Surrogate Marker for PLAG1 Fusions in Lipoblastomas

Pediatric and Developmental Pathology ◽

10.1177/10935266211043366 ◽

2021 ◽

pp. 109352662110433

Author(s):

Mikako Warren ◽

Nishant Tiwari ◽

Sabrina Sy ◽

Gordana Raca ◽

Ryan J Schmidt ◽

...

Keyword(s):

Next Generation Sequencing ◽

Gold Standard ◽

Immunohistochemical Staining ◽

Surrogate Marker ◽

Molecular Testing ◽

Protein Overexpression ◽

Conventional Cytogenetics ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing ◽

Lipomatous Tumors

Background The hallmark of lipoblastoma is a PLAG1 fusion. PLAG1 protein overexpression has been reported in sporadic PLAG1-rearranged lipoblastomas. Methods We evaluated the utility of PLAG1 immunohistochemical staining (IHC) in 34 pediatric lipomatous tumors, correlating the results with histology and conventional cytogenetics, FISH and/or next generation sequencing (NGS) results. Results The study included 24 lipoblastomas, divided into 2 groups designated as “Lipoblastoma 1” with both lipoblastoma histology and PLAG1 rearrangement (n = 16) and “Lipoblastoma 2” with lipoblastoma histology but without PLAG1 cytogenetic rearrangement (n = 8), and 10 lipomas with neither lipoblastoma histology nor a PLAG1 rearrangement. Using the presence of a fusion as the “gold standard” for diagnosing lipoblastoma (Lipoblastoma 1), the sensitivity of PLAG1 IHC was 94%. Using histologic features alone (Lipoblastoma 1 + 2), the sensitivity was 96%. Specificity, as defined by the ability to distinguish lipoma from lipoblastoma, was 100%, as there were no false positives in the lipoma group. Conclusions Cytogenetics/molecular testing is expensive and may not be ideal for detecting PLAG1 fusions because PLAG1 fusions are often cytogenetically cryptic and NGS panels may not include all partner genes. PLAG1 IHC is an inexpensive surrogate marker of PLAG1 fusions and may be useful in distinguishing lipoblastomas from lipomas.

Download Full-text

MSIplus for Integrated Colorectal Cancer Molecular Testing by Next-Generation Sequencing

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2015.05.008 ◽

2015 ◽

Vol 17 (6) ◽

pp. 705-714 ◽

Cited By ~ 22

Author(s):

Jennifer A. Hempelmann ◽

Sheena M. Scroggins ◽

Colin C. Pritchard ◽

Stephen J. Salipante

Keyword(s):

Colorectal Cancer ◽

Next Generation Sequencing ◽

Molecular Testing ◽

Next Generation ◽

Generation Sequencing

Download Full-text

Next-Generation Sequencing Technologies in Blood Group Typing

Transfusion Medicine and Hemotherapy ◽

10.1159/000504765 ◽

2019 ◽

Vol 47 (1) ◽

pp. 4-13 ◽

Cited By ~ 1

Author(s):

Daniel Fürst ◽

Chrysanthi Tsamadou ◽

Christine Neuchel ◽

Hubert Schrezenmeier ◽

Joannis Mytilineos ◽

...

Keyword(s):

Next Generation Sequencing ◽

Blood Group ◽

Large Scale ◽

Cost Effective ◽

Molecular Testing ◽

Blood Group Antigens ◽

Next Generation ◽

Sequencing Technologies ◽

Blood Group Typing ◽

Generation Sequencing

Sequencing of the human genome has led to the definition of the genes for most of the relevant blood group systems, and the polymorphisms responsible for most of the clinically relevant blood group antigens are characterized. Molecular blood group typing is used in situations where erythrocytes are not available or where serological testing was inconclusive or not possible due to the lack of antisera. Also, molecular testing may be more cost-effective in certain situations. Molecular typing approaches are mostly based on either PCR with specific primers, DNA hybridization, or DNA sequencing. Particularly the transition of sequencing techniques from Sanger-based sequencing to next-generation sequencing (NGS) technologies has led to exciting new possibilities in blood group genotyping. We describe briefly the currently available NGS platforms and their specifications, depict the genetic background of blood group polymorphisms, and discuss applications for NGS approaches in immunohematology. As an example, we delineate a protocol for large-scale donor blood group screening established and in use at our institution. Furthermore, we discuss technical challenges and limitations as well as the prospect for future developments, including long-read sequencing technologies.

Download Full-text

Next generation sequencing as the new gold standard for minimal residual disease detection in B-ALL

Journal of Laboratory and Precision Medicine ◽

10.21037/jlpm.2018.11.04 ◽

2018 ◽

Vol 3 ◽

pp. 97-97

Author(s):

Rosa Ayala ◽

Esther Onecha

Keyword(s):

Next Generation Sequencing ◽

Minimal Residual Disease ◽

Gold Standard ◽

Residual Disease ◽

Disease Detection ◽

Next Generation ◽

Minimal Residual Disease Detection ◽

Generation Sequencing ◽

Minimal Residual

Download Full-text

Routine molecular testing of resected early-stage lung adenocarcinoma with targeted next-generation sequencing demonstrates a high rate of actionable mutations

Journal of Thoracic Oncology ◽

10.1016/j.jtho.2015.12.075 ◽

2016 ◽

Vol 11 (2) ◽

pp. S44-S45 ◽

Cited By ~ 1

Author(s):

Brendon M. Stiles ◽

Abu Nasar ◽

Mohamed Kamel Hussein ◽

Galal Rafik Ghaly ◽

Mohamed Rahouma Ahmed ◽

...

Keyword(s):

Next Generation Sequencing ◽

Lung Adenocarcinoma ◽

Early Stage ◽

High Rate ◽

Molecular Testing ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Generation Sequencing

Download Full-text

Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms

PLoS ONE ◽

10.1371/journal.pone.0243683 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0243683

Author(s):

Sun Hee Rosenthal ◽

Anna Gerasimova ◽

Charles Ma ◽

Hai-Rong Li ◽

Andrew Grupe ◽

...

Keyword(s):

Next Generation Sequencing ◽

Myeloproliferative Neoplasms ◽

Simultaneous Detection ◽

Molecular Testing ◽

Next Generation ◽

Analytical Validation ◽

Bioinformatics Pipeline ◽

Myeloid Neoplasms ◽

Clinically Significant ◽

Generation Sequencing

Identification of genomic mutations by molecular testing plays an important role in diagnosis, prognosis, and treatment of myeloid neoplasms. Next-generation sequencing (NGS) is an efficient method for simultaneous detection of clinically significant genomic mutations with high sensitivity. Various NGS based in-house developed and commercial myeloid neoplasm panels have been integrated into routine clinical practice. However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS panels (e.g., CEBPA, CARL, and FLT3). We report development and validation of a 48-gene NGS panel that includes genes that are technically challenging for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. A bioinformatics pipeline that was developed in-house was used to detect single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). An analytical validation study was performed on 184 unique specimens for variants with allele frequencies ≥5%. Variants identified by the 48-gene panel were compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 unique specimens. The developed assay was applied to a large cohort (n = 2,053) of patients with suspected myeloid neoplasms. Analytical validation yielded 99.6% sensitivity (95% CI: 98.9–99.9%) and 100% specificity (95% CI: 100%). Concordance of variants detected by the 2 tested panels was 100%. Among patients with suspected myeloid neoplasms (n = 2,053), 54.5% patients harbored at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN. Together, these findings demonstrate that the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms even in technically challenging genes.

Download Full-text