Correlation between Mutations Found in FFPE Tumor Tissue and Paired cfDNA Samples

Content Type: Poster
Authors: Lauren Saunders and Asmita Patel

Introduction

Liquid biopsies represent a promising area of facilitating cancer research as taking blood is less invasive than tumor biopsies. The cell free DNA (cfDNA) present in the blood includes DNA derived from cancer cells and cancer biomarkers can be detected in the extracted cfDNA. However, cfDNA is a less direct view of what is happening in the tumor, and can have a different genetic profile than the tumor tissue itself.

Tumor tissue is typically removed and stored as formalin-fixed, paraffin-embedded tissue, a process that preserves the morphological structures well but chemically modifies and degrades the nucleic acids. This tissue is often used to look for cancer-associated mutations despite these difficulties; however, it does not always correlate with the mutations seen in cfDNA.

In this poster we present a comparison of matched FFPE and plasma samples to determine how many mutations are seen in both tissues. We also look at where the mutational mismatches appear in the chromosome. Different chromosomal regions can have different mismatch rates, and we use this to draw conclusions about the best chromosomal locations for biomarkers.

Extraction of DNA and RNA from FFPE with FormaPure XL

FormaPure XL is a kit from Beckman Coulter Life Sciences that improves the lysis capabilities for larger sample volumes or very difficult to digest tissues. FormaPure XL can extract both RNA and DNA or the entire lysate can be used to extract a single nucleic acid type.

Genomics FormaPure Total XL Workflow

DNA and RNA Yield: Seven 10 µm FFPE curls were extracted in triplicate with both FormaPure XL and a competitor’s kit. Yields for both RNA and DNA are shown below. Yields were measured using Quant-It Ribogreen and Picogreen, respectively. Yields varied with tissue block, but high yields were seen with both kits.

GenFind V3 Fresh Whole Blood DNA Isolation GenFind V3 Frozen Whole Blood DNA Isolation

Quality of RNA from FFPE. Comparison of FormaPure XL RNA (red) with Kit 1 RNA (blue). FormaPure XL RNA is significantly larger than Kit 1 RNA, as seen by both the traces and the DV200 values.

Genomics Poster Quality RNA Figure 3

Quality of DNA from FFPE. Comparison of FormaPure XL DNA (red) with Kit 1 DNA (blue).

Genomics Poster Quality DNA Figure 4

Extraction of cfDNA from Plasma

DNA was extracted from 1.5 mL plasma using the Apostle MiniMax kit. This kit uses a bead-based chemistry and is automatable. The workflow is shown below.

Genomics cfDNA Apostle MiniMax Workflow

DNA Yield: Yield was calculated by both Quant-it Picogreen and KAPA hgQuant using 41 bp primers. The quantification agreed well with both methods, which is expected for high quality DNA fragments.

Genomics Poster DNA Yield Figure 5

Library Preparation

Libraries were created from DNA (25 ng) extracted with FormaPure and Apostle MiniMax using the Accel-NGS 2S Hyb DNA Library Kit. Those libraries (500 ng) were hybridized to the IDT xGen Pan-cancer Panel and further amplified as described in the protocol.

Genomics Poster Library Figure 6

Library size: Representative traces of Accel-NGS 2S Hyb Libraries (left) and trace of pooled samples after IDT hybridization and amplification. Libraries are in the 200 – 400 bp range as expected.

Sequence Quality

Sequencing was done on a NextSeq and Illumina BWA Amplification was used to create scaffold and identify mutations.

Sample Tissue  Sample Type  Percent Aligned Reads Read Enrichment Uniformity of Coverage (Pct > 0.2*mean) Target Coverage at 1X Target Coverage at 20X
Breast cfDNA 99.70% 65.00% 95.40% 99.30% 98.80%
Prostate cfDNA 99.60% 64.60% 95.20% 99.40% 98.90%
Lung cfDNA 99.70% 65.20% 96.10% 99.80% 99.40%
Breast FFPE 99.70% 58.00% 97.90% 99.90% 99.60%
Prostate FFPE 99.70% 70.20% 96.80% 99.80% 99.60%
Lung FFPE 99.70% 56.50% 97.60% 99.60% 99.00%

Sequencing metrics: All samples ran well and coverage and aligned reads agreed well across samples, as shown by the quality control metrics listed above.

Comparison of cfDNA and FFPE Mutations

Genomics Poster Comparison Figure 7

Mutation matches: Mutations seen in matched samples of cfDNA and FFPE were compared. Depending on the cancer type and mutation type (indel or SNV), up to one third of mutations were only seen in one sample type. More indels were seen in cfDNA, whereas more SNVs were found in FFPE.

Genomic Location of Mismatches

Frequency of mismatches along chromosomes: All samples had similar areas of high mismatch and low mismatch, so samples were combined for analysis. Chromosome location was calculated as a percentage to account for different chromosome lengths. Mismatches were binned in groups of 10% of the chromosome.

Genomics Poster Mismatches Figure 8

Combined mismatches: Average mismatches across all samples are shown below. Samples were analyzed using a Student’s T test and grouped into different levels. The chromosomal regions analyzed have four different levels of sample mismatch shown below. Biomarkers in region D (the first 10 – 30%, 40 – 50%, 60-80%, or 90 – 100% of the chromosome) have the lowest number of mismatches between cfDNA and FFPE samples and would be most likely to be found by both sampling methods.

Genomics Poster Combined Mismatches Figure 9

Conclusions

Both FFPE and cfDNA detect at least two thirds of the observed indels and at least 90% of the observed SNVs. As SNVs are more likely to be found in both tissue types, they are more suitable for biomarker use if looking across different tissues. This was true for all three cancers tested.

Different regions of the chromosome have different rates of mismatches in mutation detection between plasma and FFPE tissue. The first 10-30%, 40-50%, 60-80%, and 90-100% of the chromosome have the lowest rates of mismatch and provide the best locations for biomarkers detectible in both tissues. Future work will focus in increasing the sample size to further narrow down the areas of the chromosome with the highest likelihood of good mutation detection in both plasma and FFPE.

 

Beckman Coulter makes no warranties of any kind whatsoever express or implied, with respect to these protocols, including but not limited to warranties of fitness for a particular purpose or merchantability or that the protocol is non-infringing. All warranties are expressly disclaimed. Your use of the method is solely at your own risk, without recourse to Beckman Coulter. Not intended or validated for use in the diagnosis of disease or other conditions. These protocols are for demonstration only, and are not validated by Beckman Coulter. © 2019 Beckman Coulter, Inc. All rights reserved. Beckman Coulter, the stylized logo, and the Beckman Coulter product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, Inc. in the United States and other countries. All other trademarks are the property of their respective owners.

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