Nucleic Acid Quality Assessment is Critical to the Success of the Oncomine Dx Target Test for Lung Cancer

Nagakubo, Yuki; Hirotsu, Yosuke; Amemiya, Kenji; Mochizuki, Hitoshi; Tsutsui, Toshiharu; Kakizaki, Yumiko; Miyashita, Yoshihiro; Higuchi, Rumi; Nakagomi, Takahiro; Goto, Taichiro; Oyama, Toshio; Omata, Masao

doi:10.1007/s40291-023-00653-2

Cited by 6 publications

(4 citation statements)

References 26 publications

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“…The DV200 values, assessing the proportion of fragments exceeding 200 nucleotides and recognized as a crucial indicator of RNA quality, consistently surpassed the 66.1% threshold established by Nagakubo et al [21] across all our samples (Table 1). Moreover, the nSolver software (version 4, nanoString Technologies Inc., Seattle, WA, USA) did not flag any samples for suboptimal quality.…”

Section: Rna Qualitysupporting

confidence: 62%

Molecular Behavior of α-Synuclein Is Associated with Membrane Transport, Lipid Metabolism, and Ubiquitin–Proteasome Pathways in Lewy Body Disease

Kon,

Lee,

Martinez-Valbuena

et al. 2024

IJMS

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Lewy body diseases (LBDs) feature α-synuclein (α-syn)-containing Lewy bodies, with misfolded α-syn potentially propagating as seeds. Using a seeding amplification assay, we previously reported distinct α-syn seeding in LBD cases based on the area under seeding curves. This study revealed that LBD cases showing different α-syn seeding kinetics have distinct proteomics profiles, emphasizing disruptions in mitochondria and lipid metabolism in high-seeder cases. Though the mechanisms underlying LBD development are intricate, the factors influencing α-syn seeding activity remain elusive. To address this and complement our previous findings, we conducted targeted transcriptome analyses in the substantia nigra using the nanoString nCounter assay together with histopathological evaluations in high (n = 4) and low (n = 3) nigral α-syn seeders. Neuropathological findings (particularly the substantia nigra) were consistent between these groups and were characterized by neocortical LBD associated with Alzheimer’s disease neuropathologic change. Among the 1811 genes assessed, we identified the top 20 upregulated and downregulated genes and pathways in α-syn high seeders compared with low seeders. Notably, alterations were observed in genes and pathways related to transmembrane transporters, lipid metabolism, and the ubiquitin–proteasome system in the high α-syn seeders. In conclusion, our findings suggest that the molecular behavior of α-syn is the driving force in the neurodegenerative process affecting the substantia nigra through these identified pathways. These insights highlight their potential as therapeutic targets for attenuating LBD progression.

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Section: Rna Qualitysupporting

confidence: 62%

Molecular Behavior of α-Synuclein Is Associated with Membrane Transport, Lipid Metabolism, and Ubiquitin–Proteasome Pathways in Lewy Body Disease

Kon,

Lee,

Martinez-Valbuena

et al. 2024

IJMS

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“…These samples were then subjected to various fixation times of 1, 2, 4, 6, 12, 24, 48, 72, 120, 168, and 240 h. FFPE tissues were processed on the Tissue-Tek VIP ® 6 AI Tissue Processor (Sakura Finetek, Tokyo, Japan) with the following steps: 70% alcohol (one cycle) for 1 h; 95% alcohol (one cycle) for 1 h;100% alcohol (five cycles) for 1 h each (total of 5 h); xylene (three cycles) for 1 h each (total of 3 h); and paraffin infiltration for two cycles for 45 min and two cycles for 1 h each (a total of 3.5 h). 25…”

Section: Preparation Of Ffpe Tissuesmentioning

confidence: 99%

“…Therefore, these two methods are important in quantifying RNA for NGS. 25,31 The recommended fixation time in the CDx guidance ranges from 6 to 72 h. 8,[32][33][34][35][36] Insufficient fixation leads to the loss of antigenicity, adversely affecting immunohistochemistry diagnosis and patient outcomes. 32,33 Therefore, it is crucial to fix the specimens within the recommended guidelines.…”

Section: Number Of Reads Of Internal Control Genes Fixation Time (H)mentioning

confidence: 99%

Influence of formalin fixation duration on RNA quality and quantity from formalin‐fixed paraffin‐embedded hepatocellular carcinoma tissues

Amemiya,

Hirotsu,

Nagakubo

et al. 2023

Pathology International

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Analyzing RNA samples from formalin‐fixed paraffin‐embedded (FFPE) tissues is essential for precision medicine. We investigated RNA quantity and quality from FFPE tumor tissues fixed in formalin for various times and compared sequencing metrics from next‐generation sequencing (NGS). Hepatocellular carcinoma (HCC) tissues were fixed in 10% neutral buffered formalin (1–240 h) and FFPE blocks were prepared. Total RNA was extracted, and the quantity and quality were assessed using the NanoDrop, Qubit and Bioanalyzer. After preparing sequencing libraries, NGS was performed on the Oncomine Dx Multi‐CDx system. Total RNA yields of all samples met the threshold required for NGS, but longer fixation times resulted in decreased total RNA and long RNA fragment (>200 nt) yields. NGS analysis showed fewer sequencing reads of internal control genes from RNA with longer fixation times. RNA extracted from FFPE blocks stored for 500 days had reduced RNA yield and quality compared with RNA obtained from FFPE blocks prepared immediately. In conclusion, short and over‐fixation should be avoided because of their negative impact on sequencing quality. Fixation process should be finished promptly within recommended guidelines (6–72 h) for cancer patients.

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“…In Japan, the Oncomine Dx Target Test (ODxTT) has been used as a companion diagnostic test for patients with lung cancer. 11 ODxTT comprises DNA-based and RNA-based assays that detect genetic abnormalities such as single-nucleotide variants, insertions and deletions, and fusion genes. Currently, ODxTT is used as a companion diagnostic test for mutations including EGFR mutations, BRAF V600E, ALK fusion, ROS1 fusion, and RET fusion.…”

Section: Introductionmentioning

confidence: 99%

Brief Report: Tepotinib as a Treatment Option in MET Exon 14 Skipping-Positive Lung Cancers—Investigating Discordance Between ArcherMET and the Oncomine Dx Target Test

Nucleic Acid Quality Assessment is Critical to the Success of the Oncomine Dx Target Test for Lung Cancer

Cited by 6 publications

References 26 publications

Molecular Behavior of α-Synuclein Is Associated with Membrane Transport, Lipid Metabolism, and Ubiquitin–Proteasome Pathways in Lewy Body Disease

Molecular Behavior of α-Synuclein Is Associated with Membrane Transport, Lipid Metabolism, and Ubiquitin–Proteasome Pathways in Lewy Body Disease

Influence of formalin fixation duration on RNA quality and quantity from formalin‐fixed paraffin‐embedded hepatocellular carcinoma tissues

Brief Report: Tepotinib as a Treatment Option in MET Exon 14 Skipping-Positive Lung Cancers—Investigating Discordance Between ArcherMET and the Oncomine Dx Target Test

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