Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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External Quality Assessment on Molecular Tumor Profiling with Circulating Tumor DNA-Based Methodologies Routinely Used in Clinical Pathology within the COIN Consortium.

Author(s): van der Leest P, Rozendal P, Hinrichs J, van Noesel CJM, Zwaenepoel K, Deiman B, Huijsmans CJJ, van Eijk R, Speel EJM, van Haastert RJ, Ligtenberg MJL, van Schaik RHN, Jansen MPHM, Dubbink HJ, de Leng WW, Leers MPG, Tamminga M, van den Broek D, van Kempen LC, Schuuring E

Publication: Clin Chem, 2024, Vol. , Page

PubMed ID: 38484302 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the detection of mutations in two spiked-in plasma specimens and three non-small cell lung cancer patient plasma specimens by sixteen laboratories, each of which used their own standard operating procedure for cell-free DNA (cfDNA) extraction and genotyping. Aliquots of two plasma specimens (diagnosis not specified) were spiked with 25 ng/mL of artificial cfDNA containing known mutations. Three diagnostic leukapheresis (DLA) citrate plasma specimens from patients with metastatic non-small cell lung cancer with a known tumor mutation in BRAF exon 15, EGFR exon 18–21, or KRAS exon 2-3 were obtained from a biobank. The DLA plasma was frozen at -80°C within 30 min of collection, thawed, centrifuged at 16000 g for 10 min and refrozen.  Further details of specimen collection and handling were not provided. Matched aliquots of plasma were distributed to sixteen different laboratories for analysis of BRAF exon 15, EGFR exon 18–21, and KRAS exon 2–3 mutations using the individual laboratories’ workflows. Laboratories extracted cfDNA from one 2 mL aliquot using the QIAamp Circulating Nucleic Acid Kit (one laboratory); two 2 mL aliquots using the QIAsymphony DSP Circulating DNA Kit (one laboratory) or Cobas ccfDNA Sample Preparation Kit (3 laboratories);  a 3 mL aliquot using QIAamp Circulating Nucleic Acid Kit (two laboratories) or the QIAamp MinElute ccfDNA Mini Kit (one laboratory); or a 4 mL aliquot using QIAamp Circulating Nucleic Acid Kit (3 laboratories), the Maxwell RSC LV ccfDNA Kit (two laboratories), AVENIO cfDNA Isolation Kit (one laboratory), the MagMAX Cell-Free Total Nucleic Acid Kit (one laboratory), or a custom protocol (one laboratory). cfDNA was quantified using Qubit (eight laboratories), Nanodrop (one laboratory), Tapestation (one laboratory), ddPCR (one laboratory) or not quantified (five laboratories).  Mutations were detected using ddPCR assays from Bio-Rad (six laboratories) or Naica (one laboratory), small panel real-time PCR assays from COBAS (3 laboratories), or NGS-based assays including Oncomine lung (three laboratories), Oncomine Pan-Cancer (one laboratory), Ion AmpliSeq Colon and Lung research (one laboratory), Avenio targeted (one laboratory), Avenio Expanded (one laboratory), and an in-house assay (one laboratory). Genotyping results were scored on a scale of -1 (false positive) to 1 (correct genotype assigned) with variant specific intermediate scores. The performance score was the percentage of points awarded out of total points available (mutations genotyped).

   Summary of Findings:

   Not all laboratories could report data for the 21 mutations and the accuracy of the results was variable among the mutations and the laboratories. Two of the EGFR mutations (S752_I759del and N771_H773dup) were only correctly genotyped by 31% (5 of 16) and 50% (8 of 16) of the participating laboratories, respectively, using each spike-in. In contrast, the EGFR T790M mutation was correctly genotyped by 94% (15 of 16) of laboratories using the first spiked-in specimen and 100% of the laboratories using the second spiked-in specimen. KRAS G12C was only correctly genotyped by 44-50% (7-8 of 16) of the sixteen participating laboratories using the two spiked-in specimens and 56% (9 of 16) of laboratories when the plasma of each patient with that mutation was analyzed. However, KRASG12D and KRASQ61H were correctly genotyped by 69-88% (11-14 of 16) of the participating laboratories using the two spike-in specimens. Of the eight laboratories using next generation sequencing-based assays for mutation detection, five had performance scores >0.9, with the remaining laboratories having scores of 0.26 using the Ion AmpliSeq Colon and Lung research panel and 0.66 and 0.76 for two of the three laboratories that used the Oncomine lung assay. In contrast, only one of the eight participating laboratories using a PCR-based approach had a performance score >0.90 with the remaining ranging from 0.56-0.80.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Not specified
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR
   DNA	Next generation sequencing
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAamp Circulating Nucleic Acid Kit QIAamp MinElute ccfDNA Mini Kit QIAamp DSP Circulating Nucleic Acid Kit Maxwell RSC LV ccfDNA AVENIO cfDNA Isolation Kit MagMAX Cell-Free Total Nucleic Acid Kit In house extraction protocol Cobas ccfDNA Sample Preparation Kit
   Next generation sequencing Specific	Technology platform	ddPCR assays from Bio-Rad ddPCR assay from Naica Small panel real-time PCR assay from COBAS Oncomine Lung NGS assay Oncomine Pan-Cancer NGS assay Ion AmpliSeq Colon and Lung research NGS assay Avenio targeted NGS assay Avenio Expanded NGS assay In-house NGS assay

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