Feasibility of next-generation sequencing in clinical practice: results of a pilot study in the Department of Precision Medicine at the University of Campania 'Luigi Vanvitelli'.

Author(s): De Falco V, Poliero L, Vitello PP, Ciardiello D, Vitale P, Zanaletti N, Giunta EF, Terminiello M, Caputo V, Carlino F, Di Liello R, Ventriglia A, Famiglietti V, Martinelli E, Morgillo F, Orditura M, De Vita F, Fasano M, Napolitano S, Martini G, Della Corte CM, Franco R, Altucci L, Ciardiello F, Troiani T

Publication: ESMO Open, 2020, Vol. 5, Page

PubMed ID: 32234730 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to assess potential differences in the success rate of a hybrid capture-based next generation sequencing (NGS) assay among formalin-fixed, paraffin-embedded (FFPE) cytology, biopsy, and surgically resected tumor specimens; the potential impact of the duration of FFPE specimen storage (< or ≥5 y), as blocks or slide-mounted sections was also examined.

Conclusion of Paper

Out of the 122 specimens that underwent targeted NGS using the F1CDx assay, analysis was successful for 84 specimens (68.85%). Assay failure was due to insufficient tissue for analysis in 53% of failing specimens (20/38 specimens) and a laboratory failure possibly linked to poor quality in 47% of specimens (18/38). Success rates for targeted NGS with the F1CDx assay were much higher in tissue (biopsy and surgical resections) than cytology specimens (73% versus 12.5%, respectively). F1CDx success rates were modestly but significantly higher in surgically resected tissue specimens than in biopsy specimens (80% versus 60%, respectively; p=0.041). F1CDx success rates were also higher for FFPE blocks than slide-mounted FFPE tissue sections (79% versus 55%, respectively; p=0.026) due to a high lab failure rate of FFPE slide-mounted sections. While F1CDx success rate was not influenced by the storage duration of the FFPE specimen, the percentage of specimens that were considered a laboratory failure due to low quality was higher in FFPE specimens stored for ≥5 y compared to those stored for shorter durations (80% versus 44%, respectively). The most common alterations (amplification; substitutions; gene truncation, deletion, rearrangement) among the patients examined involved TP53, KRAS, and APC genes (46%, 20%, and 14%, respectively).

Studies

Study Purpose

The purpose of this study was to assess potential differences in the success rate of a hybrid capture-based next generation sequencing (NGS) assay among formalin-fixed, paraffin-embedded (FFPE) cytology, biopsy, and surgically resected tumor specimens; the potential impact of the duration of FFPE specimen storage (< or ≥5 y), as blocks or slide-mounted sections was also examined. A total of 122 tumor specimens (40 biopsy specimens, 74 surgical resection specimens, 8 cytology specimens) were collected from 114 patients diagnosed with a neoplastic carcinoma, a benign tumor, or a soft tissue sarcoma that were formalin-fixed and paraffin embedded and stored as either an FFPE block or a FFPE slide-mounted section. No additional details of formalin fixation, processing, and storage conditions were provided. FFPE specimens were analyzed using the capture-based NGS platform FoundationOne CDx (F1CDx) assay, which detects deletion and copy number alterations 0f 324 genes as well as identifies tumor mutation burden, microsatellite instability, and specific gene rearrangements. Tissue requirements of the F1CDx assay include ≥0.6 mm3 of tissue, tumor content ≥20%, and a minimum of 55 ng of genomic DNA (gDNA) for both quality control and library construction. DNA from FFPE specimens were sequenced on an Illumina HiSeq 4000 platform at a targeted depth of >500X. The F1CDx assay includes a DNA extraction, although details were not provided.

Summary of Findings:

Out of the 122 specimens that underwent targeted NGS using the F1CDx assay, analysis was successful for 84 specimens (68.85%). Assay failure was due to insufficient tissue for analysis in 53% of failed specimens (20/38 specimens) and a laboratory failure possibly linked to poor quality in 47% of specimens (18/38). Success rates for targeted NGS with the F1CDx assay were much higher in tissue (biopsy and surgical resections) than cytology specimens (73% versus 12.5%, respectively), as results were only successfully obtained from one of the eight cytology specimens analyzed although the authors did not specify the source of the high failure rate. F1CDx success rates were modestly but significantly higher in surgically resected tissue specimens than in biopsy specimens (80% versus 60%, respectively; p=0.041). F1CDx success rates were also higher for FFPE blocks compared to slide-mounted FFPE tissue sections (79% versus 55%, respectively; p=0.026) due to a high lab failure rate of slide-mounted FFPE specimens. While F1CDx success rate was not influenced by the age of the FFPE specimen, as 69% (74/107) of the FFPE specimens stored for less than 5 years and 67% (10/15) of those stored for 5 y or longer were successful, the percentage of specimens that were considered a laboratory failure due to low quality was higher in FFPE specimens stored for ≥5 y compared to those stored for shorter durations (80% versus 44%, respectively). The most common alterations (amplification; substitutions; gene truncation, deletion, rearrangement) among the patients examined were in TP53, KRAS, and APC genes (46%, 20%, and 14%, respectively).

Biospecimens

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma
Neoplastic - Benign
Neoplastic - Sarcoma

Platform:

Analyte	Technology Platform
DNA	DNA sequencing

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Biospecimen Acquisition	Method of tissue acquisition	Biopsy Surgical resection Unspecified cytology specimen
Storage	Storage conditions	FFPE blocks FFPE slide-mounted sections
Storage	Storage duration	<5 y ≥5 y

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