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

Author(s): Nagakubo Y, Hirotsu Y, Amemiya K, Mochizuki H, Tsutsui T, Kakizaki Y, Miyashita Y, Higuchi R, Nakagomi T, Goto T, Oyama T, Omata M

Publication: Mol Diagn Ther, 2023, Vol. 27, Page 513-523

PubMed ID: 37198423 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper sought to identify factors that influence the success and failure rates of tissue, cell block, and cytology specimens analyzed by the Oncomine Dx Target Test (ODxTT) using specimens collected from patients diagnosed with lung cancer.  The following factors were investigated for their influence on the NGS-based ODxTT: tissue type (146 lung, 47 lymph node, 10 pleura, 9 pleural effusion, 5 bone, 3 bronchus, 2 soft tissue, and 1 liver specimens), collection method (surgical resection, n=86; endobronchial ultrasonography with a guide sheath, n =45; endobronchial ultrasound-guided transbronchial needle aspiration, n = 41; transbronchial biopsy, n = 23; aspiration, n = 9; thoracoscopy, n = 7; ultrasound-guided biopsy, n = 6; computed tomography-guided biopsy, n = 5; endoscopic ultrasound-guided fine-needle aspiration, n = 1), sample type (206 tissues, 7 cell blocks, 10 cytology specimens), whether or not microdissection was performed (67 were microdissected, 156 were not), RNA integrity metrics, and DNA and RNA concentration. In total 223 samples from 218 patients (77 female, 141 male) diagnosed with lung cancer (163 adenocarcinomas, 39 squamous cell carcinomas, 14 non-small cell lung cancer, 2 large-cell carcinomas, 1 large-cell neuroendocrine carcinoma, 1 neuroendocrine carcinoma,2 pleomorphic cancer, 1 adenosquamous carcinoma) were used in the study. DNA and RNA were extracted from either two (surgical resection specimens) or 12 (all other collection methods) 5 µm-thick FFPE sections using the Ion Torrent Dx Total Nucleic Acid Isolation Kit; RNA samples were treated with DNase prior to extraction. DNA and RNA samples were stored at -80°C until analysis.  DNA and RNA concentration was quantified using a Qubit 3.0 machine and the Ion Torrent Dx RNA Quantification Kit.  RNA degradation was assessed by DV200 (the percentage of RNA fragments >200 bp) using the Agilent RNA 6000 Pico Kit and a bioanalyzer.  Libraries were constructed using 10 ng of DNA or RNA (or less if required).  cDNA was synthesized from RNA using the IonTorrent Dx cDNA Synthesis Kit, and the Ion PGM Dx Library Kit was used to amplify targeted regions. Libraries were purified and equalized with the Ion PGM Dx Library Equalizer Kit, enriched with the Ion OneTouch Dx Template Kit and an Ion One Touch ES Dx instrument, and sequenced with the Ion PGM Dx Sequencing Kit and a Ion PGM 318 Dx Chip on a Ion PGM Dx sequencer; data was analyzed with Torrent Suite Dx software.  Any of the following resulted in a DNA sequencing failure: “mean alignment quality 20 read length ≥90 bp, percent reads ≥0.7, samples that resulted in “No Call” after variant call. Any of the following resulted in a RNA sequencing failure: “mappable fusion reads ≥5000”, “’No Call’ for mutations related to companion diagnostics”.  RNA quality was also assessed using the following internal control genes: hydroxymethylbilane synthase (HMBS), integrin beta 7 (ITGB7), low density lipoprotein receptor-related protein 1 (LRP1), myelocytomatosis oncogene (MYC), and TATA box binding protein (TBP).

   Summary of Findings:

   Success rates for the NGS-based ODxTT assay did not differ based on collection method, tissue type, or whether microdissection was, or was not, conducted.  ODxTT success rates did differ significantly among the sample types examined (cell block= 100%, surgical resection=99%, cytology=80%; p=0.015).  More RNA than DNA was isolated from the 223 specimens destined for ODxTT analysis (36.8 ng/µl versus 21.6 ng/µl, respectively) and the yields of the two were weakly albeit significantly correlated (r²=0.38, p<0.001).  Of the 223 samples analyzed, 97.8% (218) had nucleic acid concentrations that were at or above the concentrations recommended for the ODxTT assay (DNA ≥0.83 ng/µl, RNA ≥1.42 ng/µl).  One sample (cytology specimen) failed to meet the DNA concentration threshold, three samples (all surgically resected specimens) failed to meet the RNA concentration threshold, and one sample (cytology specimen) failed to meet both thresholds.  ODxTT analysis was successful for 98.2% (219/223) of samples, which included four of the five samples that did not meet nucleic acid concentration thresholds (the remaining sample generated results for RNA but not DNA).  The four samples that failed ODxTT analysis included two cytology specimens (collected by endobronchial ultrasound-guided transbronchial needle aspiration and endobronchial ultrasonography with a guide sheath) that failed DNA analysis (DNA analysis failure) and two surgical resections specimens that failed RNA analysis.  Samples that failed DNA ODxTT analysis had significantly lower DNA concentrations than those that were successful (p=0.017). Samples that failed RNA ODxTT analysis had a DV200 value <30.  Samples with a DV200 <30 also had a significantly lower total number of reads compared to those with a DV200 ≥30 (p=0.013) and a lower number of reads for the five internal control transcripts.  DV200 was not correlated to RNA amount and RNA concentration did not differ among samples that failed ODxTT analysis and those that were successfully analyzed.

   Biospecimens

   • Tissue - Lung
   • Tissue - Lymph Node
   • Cell - Pleural Fluid
   • Tissue - Liver
   • Tissue - Bone
   • Tissue - Bronchus

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	Next generation sequencing
   RNA	Next generation sequencing
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Method of tissue acquisition	Surgical resection Ultrasound-guided fine needle aspiration Ultrasound-guided biopsy Fine needle aspiration Endoscopic biopsy
   Biospecimen Acquisition	Biospecimen location	Bone Bronchus Liver Lymph node Lung Pleural effusion Soft tissue
   Biospecimen Aliquots and Components	Cell capture method	Microdissection Performed Microdissection Not Performed

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