Analysis and Interpretation of Cell-free DNA

Sample to Insight

Analysis and interpretation of cell-free DNA 1

Analysis and interpretation of cell-free DNA

Anika Joecker, Ph. D.

Director Global Product Management, Clinical Program, QIAGEN Bioinformatics

Sample to Insight


Legal Disclaimer

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QIAGEN products shown here are intended for molecular biology

applications. These products are not intended for the diagnosis,

prevention, or treatment of a disease.

For up-to-date licensing information and product-specific

disclaimers, see the respective QIAGEN kit handbook or user

manual. QIAGEN kit handbooks and user manuals are available

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http://www.qiagen.com/

Sample to Insight

Agenda

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Why is cell-free DNA interesting?1

Challenges when analyzing cell-free DNA data from patients with cancer2

QIAGEN sample-to-insight offering for cell free DNA analysis3

QIAGEN Bioinformatics solutions4

Case story5

6 Summary and questions


Sample to Insight

Agenda

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Case story5



Sample to Insight

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Why is cell-free DNA interesting?

• Applications

Identification of tumoral heterogeneity

Tracking of the evolution of the tumor genome over time

Identification of tumor recurrence and upcoming resistance, which leads to a change

in treatment

Non-invasive prenatal testing

• Advantages:

◦ Avoid surgeries and be able to take samples when no surgery is possible

◦ Achieve accuracy in the detection of all pathogenic and actionable variants


Sample to Insight

Agenda

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Case story5



Sample to Insight


Challenges when analyzing cell-free DNA data from patients with cancer

Due to dilution from non-cancerous cells, variants are expected at allele percentages of as low as 0.01%

Most variant calling pipelines fail to call variants at this low percentage, which leads to many false negatives

At this level the separation between true variants and false positives is very challenging, so comparisons are

needed

Changes in coverage and allelic dropout makes it challenging to compare samples

Which variants are the ones driving the disease or are important for treatment decisions?

Sample to Insight


Challenges when analyzing cell-free DNA data from patients with cancer

Problems when variant calling done with software A is not well connected with software B, which is used for variant interpretation and software C, which is used for visualization

• Variant format available as export from software A is not supported in software B

• Information gets lost between software A and B

• Manual process: Variants have to be manually exported from software A and imported into software B and C.

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A lot of time and resources have to be invested in building up

the system, maintaining and

running it.

Software A(Variant calling)

Software B(Data

interpretation )

Software C (Result

visualization for validation)

$

Sample to Insight

Agenda

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Case story5



Sample to Insight

Sample to insight workflow

Title, Location, Date 10

Preparation

Collection

& stabilization

Detection

Data analysis

&Interpretation

• PAXgene Blood ccfDNA Tubes

• QIAamp Circulating

Nucleic Acid Kit

• QIAsymphony

Circulating Nucleic

Acid Kit

• QIAseq cfDNA All-in-

One Kit

• QIAseq 1-Step Amplicon Library Kit

• QIAseq Targeted DNA

Panels

• QIAseq FX DNA

Library Kit

• QIAseq Ultra Low

Input Library Kit

• Biomedical Genomics Workbench

• Biomedical Genomics Server Solution

• Ingenuity Variant Analysis

Sample to Insight

Agenda

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Case story5



Sample to Insight


>30,000 Publications

16+ yearsin bioinformatics

and literature curation

>13,000,000 Biological findings:• Disease relevant mutations• Gene/Mutation – Drug • Gene - Phenotype • Gene – pathways relationships

Powered by

QIAGEN Bioinformatics solutions are proven to scale

Sample to Insight


Biomedical Genomics Workbench – complex tasks, simply done

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Streamlined workflows and a rich toolbox to efficiently process data

Customize workflows

QC reports

History

Visualization and Validation

Sample to Insight


Specific functionality for target amplicon data

Remove amplicon primers after alignment and remove primer-dimer artefacts

Sample to Insight


Variant calling with Biomedical Genomics Workbench

Very sensitive and specific calling of variants at a very low level of 5% and even under 1%

Identification of known pathogenic mutations directly from mapped sequencing reads

Accuracy for calling 5% low-frequency variants using the default variant calling pipeline in Biomedical Genomics Workbench

Sample to Insight


Visualization of results

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Visualization of causal variants in Ingenuity Variant Analysis

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Interpret and understand variants

Details panel provides links to findings, viewers and assessment

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Pathway interpretation of variants and genes

Visualise variants on over-represented pathways, effects on genes and drugs that may directionally target and effect pathway

Sample to Insight

Agenda

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Case story5



Sample to Insight

Case story: Comparison between primary tumor and cell-free DNA

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Sample to Insight


Case story

Comparison between primary tumor and cell-free DNA

Sample to Insight


　

Primary tumor (FFPE) andbuffy coat

FFPE: QIAamp DNA FFPE Tissue KitDNA from buffy coat: QIAamp DNA Blood Mini Kit

Agilent SureSelectXT Human All Exon V4 + UTR

HiSeq 2000

Biomedical Genomics Workbench

Ingenuity Variant Analysis

cfDNA

QIAamp Circulating Nucleic Acid Kit

Agilent SureSelectXT Human All Exon V4 + UTR

HiSeq 2000

Biomedical Genomics Workbench

Ingenuity Variant Analysis

Sample extraction

Enrichment

NGS

Data Analysis


Data Interpretation

Sample to Insight


Step 1: Identification of somatic variants in primary tumor and cfDNA


• Run on primary tumor, buffy coat and cfDNA

• Conducts mapping of sequencing reads and low frequency variant calling

• cfDNA sample has to be run with a frequency cutoff of 1% or under and a read count of 5 reads at least supporting the variant

• Sends results from all three samples automatically to Ingenuity Variant Analysis, filters results and returns annotated and filtered variant list to Biomedical Genomics Workbench to be visualized in Genome Browser

• Variant Calling Results from primery tumor and cfDNA has to be stated as case and buffy coat as control

Sample to Insight


Step 1: Identification of somatic variants in primary tumor and cfDNA


Run on primary tumor, buffy coat and cfDNA

Conducts mapping of sequencing reads and low frequency variant calling

cfDNA sample has to be run with a frequency cutoff of 1% or under and a read count of 5 reads at least supporting the variant

Sends results from all 3 samples automatically to Ingenuity Variant Analysis, filters results and returns annotated and filtered variant list to Biomedical Genomics Workbench to be visualized in Genome Browser

Variant Calling Results from primary tumor and cfDNA has to be stated as case and buffy coat as control

Sample to Insight



Step 2: Modification of the filter cascade in Ingenuity Variant Analysis

1. Drag and drop the result from Ingenuity Variant Analysis (IVA) in an open Genome Browser

2. Open the filter cascade in Ingenuity Variant Analysis for modification

3. Change Filter Cascade in Ingenuity Variant Analysis

4. Fetch newest results from Ingenuity Variant Analysis

5. Drag and drop updated results from Ingenuity Variant Analysis in Genome Browser and compare results

Sample to Insight


Identified cancer driving variants by Ingenuity Variant Analysis for validation in Genome Browser


Identified actionable variant in ESR1, just identified in cfDNA

Sample to Insight


Problem: Some variants are not called, but are clearly present in primary tumor and/or buffy coat


Variant was identified in cfDNA, but not called in primary tumor, even when it is present. Reason was that only a low fraction of reads support this variant in the primary tumor

Sample to Insight


So, we are checking back into the sequencing reads to achieve a higher sensitivity


Variants identified in buffy coat

Variants identified in primary tumor

Variants identified in cfDNA

Read mapping

buffy coat

Read mapping primary tumor

Read mapping

cfDNA

Combined variant list from all samples

Check back into the read mappings of all samples to see if variants seen in one sample are also present in other samples

Sample to Insight


Now the ESR1 variant seems also to be present in the primary tumor, but supported by just one read. True or false positive?


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Number of potential cancer driver variants detected in cfDNA, primary tumor and buffy coat

41 3426

114 cancer drivers were detected in buffy coat, primary tumor and cfDNA together

26 variants and affected genes were directly associated with breast cancer

Primary Tumor cfDNA

Sample to Insight



Inspection of affected pathways

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...and drugs


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Not described in paper: Variant in SLC9A2 found in cfDNA


Sample to Insight

Agenda

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Case story5



Sample to Insight


Summary

Biomedical Genomics Workbench and Ingenuity Variant

Analysis as streamlined solutions enable the easy

comparison of variants in primary tumor and cfDNA as

well as their interpretation and validation

In the paper, validated variants in PIK3CA , ESR1 as well

as RNF144B in the breast cancer patient could be easily

detected, interpreted and confirmed together with

mapped sequencing reads

The actionable variant in ESR1 was also detected in

primary tumor, but only supported by one read. This

could indicate that the mutation was already in the tumor,

but only in a very small number of reads

Additional candidates were identified in the cfDNA, which

need to be validated

Sample to Insight


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Analysis and Interpretation of Cell-free DNA