Addressing the Pre-PCR Analytical Variability of FFPE Samples

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Addressing the Pre-PCR Analytical Variability of FFPE Samples

Transcript of Addressing the Pre-PCR Analytical Variability of FFPE Samples

Addressing the Pre-PCR Analytical Variability of FFPE Samples

What is the impact of assay failure in your laboratory and how do you monitor for it?

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Addressing the Pre-PCR Analytical Variability of FFPE Samples

Pre-analytical Sample Handling and Processing

Biopsy DNA Quantification

StorageDNA Extraction

DNA Quantity & Quality

FFPE Processing

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Analytical Processing and Reporting

DNA Sample AnalysisActionable

Decision

Quality of Diagnostic Result

Sample preparation

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FFPE HDx™ Reference Standards to monitor your complete workflow

Mutant Wild type

FFPE Processing

FFPE Sections

Digital PCR

Sanger Sequencing

RT-PCR

SNP 6.0

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Characterization of FFPE HDx™ Reference Standards as External Controls

Specific Allelic Frequenciesi.e. 1%, 5%, 50%

Consistent FFPE Sections400ng - 700ng per vial

What is the impact of assay failure in your laboratory and how do you monitor for it?

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EGFR Sample Tested

EGFR Genotyping ErrorsExternal Quality Assessment 2014

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External Quality Assessment Proficiency Testing Scheme - 2014

Using HDx™ Reference Standards the EQA scheme was able to identify and recommend improvements

Pre-analytical FFPE challenges

Pre-analytical main challenges

Efficacy of DNA extraction• Small amount of DNA available –

mixture of tumour and normal DNA• Various extraction methods and yields

Sample collection and handling• Different labs follow different

protocols. Tumour sample

Diagnosis

Therapy

DNA extraction

Genotyping

Accuracy of DNA quantification• Various quantification methods

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PromegaMaxwell(n=12)

PromegaMagnesil (n=6)

PromegaReliaPrep

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Qiagen Dneasy(n=6)

Roche Cobas(n=6)

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Extraction Kit

DNA Recovery from Total Theoretical Yield

Comparison of DNA Extraction Methods (Internal data)Quantified using QuantiFluor assay

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Qiagen EZ1(n=3)

COBAS (n=1) LifetechRecover All

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QiagenDneasy (n=3)

QiagenQIAmp (n=4)

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Extraction Method

Kapp J R et al. J Clin Pathol doi:10.1136/jclinpath-2014-202644

Variation within Extraction Methods (External study)Quantified using Qubit

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N/Q

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Nanodrop:Qubit (N/Q Ratio)

Kapp J R et al. J Clin Pathol doi:10.1136/jclinpath-2014-202644

R2 = 0.4868, p<0.0001

DNA Quantitation

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Summary

Different extraction methods can result in varying DNA yields from the same starting material.

Nanodrop is very efficient at quantifying DNA at high concentrations

At low concentrations spectrophotometry methods overestimate the DNA concentration compared with fluorometry quantification methods

Important implications on diagnostic test – false negatives.

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Range of total DNA (ng) recovered from each sample by participants.

Kapp J R et al. J Clin Pathol doi:10.1136/jclinpath-2014-202644

Clinical Sample

Clinical samples cannot be utilised as external controls due to their huge variability

Cell Line Reference Standards

Validated Cell Line Reference Standards are ideal as External Controls

Validated Cell Line Reference Standards as External Controls

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Mild Formalin Fixation Severe Formalin Fixation

Formalin leads to over-quantitation of DNA

Impact of Formalin Treatment on DNA Quantification

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How to Test the Robustness and Sensitivity of your Workflow and Assay

Sensitivity of your Assay

HD701

Formalin Intensity

HD200

Robustness and Sensitivity of your Workflow

HD-C751

FFPE

DNA

Robustness of your Assay

HD-C750

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What are the Outstanding Questions?

What is the impact of assay failure in your laboratory and how do you monitor for it?

What extraction and quantification methods are you

using?

What is the limit of detection of your

workflow?

Is the impact of formalin treatment interesting to you?

Validated cell Line Reference Standards are Ideal as External Controls

References

Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial (2014)Kapp J, Diss T, Spicer J,Gandy M, Schrijver I, Jennings L, Li M, Tsongalis G, et al., Journal of Clinical Pathology, Volume 67

Assessing standardization of molecular testing for non-small-cell lung cancer: results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing (2014) Patton, S., Normanno, N., Murray, S., Kerr, K., Dietel, M., Filipits, M., et al. British Journal of Cancer, 413-420.

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