Formalin-fixed paraffin-embedded sample conditions for deep next generation sequencing

Abstract

Introduction Precision medicine is only possible in oncology practice if targetable genes in fragmented DNA, such as DNA from formalin-fixed paraffin-embedded (FFPE) samples, can be sequenced using next generation sequencing (NGS). The aim of this study is to examine the quality and quantity of DNA from FFPE cancerous tissue samples from surgically resected and biopsy specimens. Methods DNA was extracted from unstained FFPE tissue sections prepared from surgically resected specimens of breast, colorectal and gastric cancer, and biopsy specimens of breast cancer. A total quantity of DNA ≥60 ng from a sample was considered adequate for NGS. The DNA quality was assessed by Q-ratios, with a Q-ratio >0.1 considered sufficient for NGS. Results The Q-ratio for DNA from FFPE tissue processed with neutral-buffered formalin was significantly better than that processed with unbuffered formalin. All Q-ratios for DNA from breast, colorectal and gastric cancer samples indicated DNA levels sufficient for NGS. DNA extracted from gastric cancer FFPE samples prepared within the last seven years is suitable for NGS analysis, whereas those older than seven years may not be suitable. Our data suggested that adequate amounts of DNA can be extracted from FFPE samples, not only of surgically resected tissue but also of biopsy specimens. Conclusion The type of formalin used for fixation and the time since FFPE sample preparation affect DNA quality. Sufficient amounts of DNA can be extracted from FFPE samples of both surgically resected and biopsy tissue, thus expanding the potential diagnostic uses of NGS in a clinical setting.

Common driver mutations and smoking history affect tumor mutation burden in lung adenocarcinoma

“…We analyzed archival tissues of primary cancer from surgical specimens that were confirmed to be of high quality in the form of formalin-fixed, paraffin-embedded (FFPE) slides. 18 An independent pathologist evaluated the tumor content on hematoxylin and eosin stained slides for each study sample to ensure that > 20% tumor content was present. Where applicable, unstained slides were macrodissected to enrich for tumor content, and genomic DNA was extracted using the BiOstic FFPE Tissue DNA Isolation Kit (Mo Bio Laboratories, Inc; Carlsbad, CA).…”

“…DNA quality of the samples was confirmed by QC ratio as previously described. 18 In brief, the quality of an adequate quantity of DNA was assessed by determining the Qratio, in which 41 bp and 129 bp targets were amplified by quantitative polymerase chain reaction and compared using a KAPA Human Genomic DNA Quantification and QC Kit (KAPA Biosystems). The 41 bp assay was used for absolute quantification of DNA samples.…”

“…DNA with a Q-ratio (129 bp/41 bp) > 0.1 was designated as being of high enough quality for NGS analysis based on our previous results. 18 All sample preparation, NGS, and analytics were performed in a Clinical Laboratory Imprevement Amendments/College of American Pathologists-accredited laboratory (KEW Inc; Cambridge, MA).…”

Suitability of melanoma FFPE samples for NGS libraries: time and quality thresholds for downstream molecular tests

“…Despite the tested use of FFPE tissue in NGS [6], further studies are required to check whether archival FFPE samples are suitable for NGS [7,8]. A recent study found that FFPE samples stored for more than 7 years were not worth being included in NGS studies [9]. …”

Next generation sequencing‐based gene panel tests for the management of solid tumors

“…It is therefore important to consider geographic and ethnic differences when looking to understand the profile of genomic alterations, and apply NGS analysis to the development of therapies for Asian patients. Recently, we studied several types of solid cancers, including colorectal, gastric, lung, and breast cancer, in Japan utilizing NGS-based comprehensive genomic panel testing, 8,[11][12][13][14][15][16][17][18] and revealed some differences in genomic alterations between Asian and non-Asian populations.…”