Tissue recommendations for precision cancer therapy using next generation sequencing: a comprehensive single cancer center's experiences.

Minho Cho,Seunggwan Lee,Jeeyun Lee,Soomin Ahn,Se Hoon Park,Duk Hwan Kim,Ho Yeong Lim,Joon Oh Park,Heejin Bang,Michael Van Vrancken,Won Ki Kang,Mineui Hong,Young Suk Park,Seungtae Kim,Keunchil Park,Jong Mu Sun ,Woong-Yang Park ,Myung Ju Ahn ,Se‐Hoon Lee ,Kyoung Mee Kim

doi:10.18632/oncotarget.17199

Minho Cho, Seunggwan Lee + Show 18 more

Open Access

https://doi.org/10.18632/oncotarget.17199

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Abstract

To generate accurate next-generation sequencing (NGS) data, the amount and quality of DNA extracted is critical. We analyzed 1564 tissue samples from patients with metastatic or recurrent solid tumor submitted for NGS according to their sample size, acquisition method, organ, and fixation to propose appropriate tissue requirements.Of the 1564 tissue samples, 481 (30.8%) consisted of fresh-frozen (FF) tissue, and 1,083 (69.2%) consisted of formalin-fixed paraffin-embedded (FFPE) tissue. We obtained successful NGS results in 95.9% of cases. Out of 481 FF biopsies, 262 tissue samples were from lung, and the mean fragment size was 2.4 mm. Compared to lung, GI tract tumor fragments showed a significantly lower DNA extraction failure rate (2.1 % versus 6.1%, p = 0.04). For FFPE biopsy samples, the size of biopsy tissue was similar regardless of tumor type with a mean of 0.8 × 0.3 cm, and the mean DNA yield per one unstained slide was 114 ng. We obtained highest amount of DNA from the colorectum (2353 ng) and the lowest amount from the hepatobiliary tract (760.3 ng) likely due to a relatively smaller biopsy size, extensive hemorrhage and necrosis, and lower tumor volume. On one unstained slide from FFPE operation specimens, the mean size of the specimen was 2.0 × 1.0 cm, and the mean DNA yield per one unstained slide was 1800 ng.In conclusions, we present our experiences on tissue requirements for appropriate NGS workflow: > 1 mm2 for FF biopsy, > 5 unstained slides for FFPE biopsy, and > 1 unstained slide for FFPE operation specimens for successful test results in 95.9% of cases.

Highlights

We present our experiences on tissue requirements for appropriate next-generation sequencing (NGS) workflow: > 1 mm2 for FF biopsy, > 5 unstained slides for formalin-fixed paraffin-embedded (FFPE) biopsy, and > 1 unstained slide for FFPE operation specimens for successful test results in 95.9% of cases
The molecular assessment of malignant tumors has become increasingly important in recent years in this era of precision cancer therapy and personalized medicine
Fresh frozen (FF) tissue is preferred over formalin-fixed paraffin-embedded (FFPE) tissue, FFPE is typically used more due to practical concerns

Summary

Introduction

The molecular assessment of malignant tumors has become increasingly important in recent years in this era of precision cancer therapy and personalized medicine. Such studies typically give information regarding possible therapeutic targets as well as important prognostic information. Recent advances in highthroughput next-generation sequencing (NGS) technology have substantially reduced the cost and increased the workflow speed in helping to detect important genetic alterations [1, 2]. The importance of the quality of the DNA extracted cannot be overemphasized as it affects the subsequent sequencing quality and final results. The amount and quality of starting DNA used for NGS depends mainly on the desired application or workflow to create the appropriate library. The Illumina platform usually requires > 100 ng of DNA input, and the initial fragment size range is recommended to be between 150–200 base pairs in length

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