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EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 17 to 20 October 2017

Collaborative study to evaluate the proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations

Pia Sanzone1, Ross Hawkins1, Eleanor Atkinson2, Peter Rigsby2, and Jennifer Boyle1,3 Divisions of Advanced Therapies1 and Biostatistics2, National Institute for Biological Standards and Control (NIBSC), Blanche Lane, South Mimms, Hertfordshire, EN6 3QG, United Kingdom.

3Principal Investigator (email: jennifer.boyle@nibsc.org; telephone: +44 (0)1707 641000)

NOTE:

This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on

Biological Standardization (ECBS). Comments MUST be received by 18 September 2017 and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:

Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to the Responsible Officer: Dr M. Nübling at email: nueblingc@who.int

© World Health Organization 2017

All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int).

Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press through the WHO web site:

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The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.

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All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication.

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Summary

An international collaborative study assessed the suitability of a panel of genomic DNA (gDNA) materials as the proposed World Health Organization 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations, NIBSC code 16/250, for use in the standardization of KRAS oncogene codons 12 and 13 mutation-based diagnostics. The panel comprised eight freeze-dried gDNA materials of the seven most-common colorectal cancer (CRC)-associated KRAS codons 12 and 13 mutations; NM_033360.3 (KRAS) c.35G>C (p.Gly12Ala, hereafter referred to as p.G12A; NIBSC material code 16/252), c.34G>T (p.Gly12Cys, p.G12C; 16/258), c.35G>A (p.Gly12Asp, p.G12D; 16/260), c.34G>C (p.Gly12Arg, p.G12R; 16/254), c.34G>A (p.Gly12Ser, p.G12S; 16/256), c.35G>T (p.Gly12Val, p.G12V; 16/264), c.38G>A (p.Gly13Asp, p.G13D; 16/262), plus a wild-type KRAS codons 12 and 13 material (16/266). Participants evaluated the materials using their routine diagnostic methods, and against in-house controls (previously characterized patient samples and cell line-derived gDNA) or commercial materials.

Where possible, results were reported quantitatively in order to assign consensus values to each of the materials. Fifty six laboratories in thirty four countries performed sixty eight testing methods on the panel, of which thirty six reported quantitative data.

Conclusions from this study indicated that all eight materials were suitable for use as reference materials in the genomic diagnosis of KRAS codons 12 and 13 mutations, with verified

performance in next-generation sequencing (NGS), Sanger sequencing, real-time PCR,

pyrosequencing, digital PCR (dPCR), Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometric analysis (MassARRAY®), KRAS StripAssay®, high

resolution melt analysis (HRM), Amplification Refractory Mutation System-PCR (ARMS-PCR), PCR-Reverse Sequence Specific Oligonucleotide probe technique (PCR-rSSO), minisequencing, and restriction fragment length polymorphism analysis (RFLP). The proposed consensus mutation percentage for each material is derived from the median value of NGS and dPCR methods as:

65.7% KRAS p.G12A (16/252), 99.98% p.G12C (16/258), 71.5% p.G12D (16/260), 85.6%

p.G12R (16/254), 99.7% p.G12S (16/256), 49.7% p.G12V (16/264), 66.9% p.G13D (16/262), and wild-type KRAS codons 12 and 13 (16/266). The collaborative study also analysed the response of these materials to dilution (with wild-type KRAS codons 12 and 13 material 16/266).

These dilution data were used to calculate the consensus KRAS mutant and total copy number for each material. These consensus copy number data can be applied to a mathematical formula, with which the end-user may calculate how to prepare further standards at lower KRAS codons 12 and 13 mutation percentages from each of the seven mutant KRAS materials (by dilution with wild-type KRAS codons 12 and 13 material 16/266, or another wild-type gDNA aligned to 16/266). These materials and their dilutions will enable the calibration of assays, kits, and secondary standards for the seven most-common CRC-associated KRAS codons 12 and 13 mutations. All collaborative study participants agreed with the proposed genotype and consensus mutation percentage for each material, along with its consensus KRAS copy number data (and associated dilution formula), and approved the panel as the proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations (NIBSC panel code 16/250).

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Introduction

Single nucleotide variants (SNVs) in the KRAS (Kirsten rat sarcoma 2 viral oncogene homolog) gene are present in approximately 30% of human cancers, and are particularly common in adenocarcinomas of lung, pancreas, and colon (COSMIC; Karnoub & Weinberg, 2008). Approximately 40% of CRCs are associated with KRAS mutations, with approximately 90% of these mutations occurring in codons 12 and 13 of KRAS exon 2 (COSMIC; Neumann et al., 2009).

KRAS is a downstream component of the epidermal growth factor receptor (EGFR)-led

RAS/MAPK (mitogen-activated protein kinase) signalling pathway, with EGFR regulating cell proliferation, apoptosis, and tumour-induced neoangiogenesis. KRAS mutations can lead to constitutive activation of KRAS (as GTP-bound KRAS), resulting in uncontrolled activation of downstream pathways. Anti-EGFR monoclonal antibodies (cetuximab and panitumumab) are available for the treatment of metastatic CRC, however, their treatment efficacy is limited to a subset of patients since EGFR-independent, constitutive activation of the RAS pathway impairs response to anti-EGFR treatment (Chan, 2015 and references within). Thus KRAS-activating mutations can predict resistance to anti-EGFR monoclonal antibody treatment, with KRAS mutation screening necessary prior to treatment (Amado et al., 2008; Douillard et al., 2013;

Peeters et al., 2015). Whilst there are currently no therapies which directly target mutant KRAS, it is an active area of development (Ostrem et al., 2013; Zimmermann et al., 2013). Of all KRAS mutations reported in human tumours, by far the most frequent are KRAS c.35G>A (p.G12D), c.35G>T (p.G12V), c.38G>A (p.G13D), c.34G>T (p.G12C), c.35G>C (p.G12A), c.34G>A (p.G12S), and c.34G>C (p.G12R; COSMIC; Table 1). Each of these seven mutations is represented in the proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations. Less frequent, CRC-associated mutations are not represented, including others in KRAS codons 12, 13, 59, 61, 117, and 146, and those in BRAF, PIK3CA, AKT1, SMAD4, PTEN, NRAS, and TGFBR2.

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KRAS mutation Percentage total reported incidence (%)

p.G12D 33.6

p.G12V 22.7

p.G13D 12.5

p.G12C 11.2

p.G12A 5.4

p.G12S 4.5

p.G12R 3.1

Other 7.0

Table 1. KRAS variant incidence. The percentage incidence of the most common KRAS

variants were calculated from variant counts on COSMIC; the seven most common mutations are represented in the proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations.

The availability of KRAS codons 12 and 13 mutation primary standards should improve the quality of CRC genomic diagnostics by enabling the calibration of assays and kits, and the derivation of secondary standards for routine diagnostic use in determining testing accuracy and sensitivity, thus providing inter-laboratory comparison towards the harmonisation of KRAS codons 12 and 13 testing.

The proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations is intended as a panel of primary standards or calibrants in DNA-based genotyping of KRAS codons 12 and 13 mutations, and has been validated in this collaborative study

encompassing NGS, Sanger sequencing, real-time PCR, pyrosequencing, dPCR, MassARRAY (Agena Bioscience, Hamburg, Germany), KRAS StripAssay (ViennaLab Diagnostics, Vienna, Austria; based on mutant-enriched PCR and reverse hybridization), HRM, ARMS-PCR, PCR- rSSO, minisequencing, and RFLP methods. The panel comprises eight freeze-dried human gDNA materials produced from mutant and wild-type KRAS codons 12 and 13 cell lines, providing standards for the seven most-common KRAS codons 12 and 13 mutations, plus a wild-type KRAS control or diluent.

The proposed consensus mutation percentages established from the international collaborative study involving 56 laboratories are the median values of all NGS and dPCR methods, the most

condordant of all quantitative methods: 65.7% KRAS p.G12A (16/252), 99.98% p.G12C (16/258), 71.5% p.G12D (16/260), 85.6% p.G12R (16/254), 99.7% p.G12S (16/256), 49.7% p.G12V

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(16/264), 66.9% p.G13D (16/262), and wild-type KRAS codons 12 and 13 (16/266). In expressing levels of somatic mutations in cancer, currently the overwhelmingly usual unit of clinical reporting is as percentage, and thus the consensus values for these reference materials are also reported as such (i.e. KRAS codon 12 or 13 mutant alleles as a percentage of total KRAS alleles). However, recently published data indicates the occurrence of KRAS allelic heterogeneity and mutant allele copy number gains in tumours (Birkeland et al., 2012; Kerr et al., 2016;

Mekenkamp et al., 2012; Sasaki et al., 2011). It is therefore probable that the use of ‘percentage’

to define mutational load is simplistic, with ploidy and gene copy number variation potentially meaning that samples with very different KRAS mutation content could be characterized as having similar mutation percentages. It is considered that the use of tumour-derived cell lines closely mimics this in vitro tumour genomic heterogeneity, thus achieving some commutability.

The collaborative study was able to derive mutant and total consensus KRAS copy numbers in the mutant KRAS materials, based upon their response to dilution with the wild-type KRAS codons 12 and 13 material 16/266; this information will also be provided to end-users. Moreover, a proposed dilution formula (based upon the calculated KRAS copy numbers) determines how the end-user may prepare standards at lower calculated KRAS codons 12 and 13 mutation

percentages for each mutant material (by dilution with wild-type KRAS codons 12 and 13

material 16/266, or another wild-type gDNA aligned to 16/266). The use of multiple standards at a range of mutation percentages, and for each of the seven variants, will enable assay calibration across a wide mutation percentage range.

A total of 2,057 panels of the proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations (panel code 16/250) are available from the National Institute for Biological Standards and Control (NIBSC, UK). These standards are intended for use in in vitro diagnostics and relate to BS EN ISO 17511:2003 Section 5.5.

Aims of the Collaborative Study

The study evaluated the panel of eight freeze-dried gDNA materials each of a different KRAS codons 12 and 13 genotype in an international collaborative study involving laboratories using a variety of diagnostic genotyping techniques, thereby assessing the panel’s suitability as the WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations, for use as a primary reference material in the calibration of secondary standards, kits, and assays.

All data were used to establish the genotypes; quantitative data were used to establish a consensus KRAS codons 12 or 13 mutation percentage for each of the materials. The materials were also evaluated at several dilutions (each material diluted in the nominal wild-type KRAS codons 12 and 13 material 16/266). These data were used to derive consensus mutant and total KRAS copy numbers for each of the mutant materials, and to establish a formula which

determines how a dilution should be performed (with the wild-type KRAS codons 12 and 13 material 16/266, or another wild-type gDNA aligned to 16/266) to generate standards at any specified lower KRAS codons 12 and 13 mutation percentage.

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Candidate Materials

Eight materials were evaluated as candidates for the proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations. All materials were of freeze-dried,

purified gDNA extracted from eight cell lines of either mutant or wild-type KRAS codons 12 and 13 genotypes.

Materials were freeze-dried in glass ampoules as an established format for ensuring long-term stability of gDNAs. Ideally, the formulation for reference materials should be as close as possible to the usual patient analyte, cover the entire analytical process, and be applicable to methods in use throughout the world. However, it is essential that the formulation be stable for many years, and that it is practically possible to produce batches of sufficient size to satisfy demand over a similar period of time. Additionally, it should ideally be possible to generate replacement standards from the same source material to ensure consistency in formulation and to minimize value drift. It would be impossible to obtain sufficient primary patient material to produce mutant KRAS codons 12 and 13 reference materials at both high quality and sufficient quantity.

Also, since the process of DNA extraction from cultured cells is different from that of solid tissue (typically formalin-fixed paraffin-embedded (FFPE) sections) or blood (if the analyte is circulating tumour DNA (ctDNA)), providing the materials as cultured cells would not provide standardization for this step of the process in the most optimal way. Materials were provided as high molecular weight gDNA rather than the fragmented DNA often obtained from FFPE sections, or present as ctDNA, due to stability concerns and the intent to provide materials applicable to potentially any substrate used for KRAS mutation detection.

The nominal wild-type KRAS ATDB102 lymphoblastoid cell line was established at NIBSC following Epstein-Barr virus (EBV) transformation of isolated monocytes from a whole blood sample provided by a consenting healthy donor, and was confirmed as having a diploid genomic content by karyotyping (data not shown). The nominal mutant KRAS codons 12 and 13 human cancer cell lines were derived from patient tumour tissue and obtained from the European Collection of Cell Cultures (ECACC; Public Health England, Salisbury, UK; Table 2).

All cell lines were tested and found negative for HIV1, HTLV1, Hepatitis B, and Hepatitis C by PCR; master and working cell banks were produced in-house to ensure a continual future cell supply. Large-scale cell culture was carried out (in-house and at ECACC), and frozen cell pellets at 1 x 108 cells prepared. Genomic DNA was extracted from the cell pellets using Gentra

Puregene chemistry with a Gentra Autopure LS robot (Qiagen, Manchester, UK). The DNA extraction process involved RNAse treatment, protein denaturation, protein removal, and 70%

ethanol washing. The use of 70% ethanol is an established method for viral inactivation (Roberts

& Lloyd, 2007). Additionally, gDNA extracted in-house using the same purification procedure from other EBV-transformed cell lines did not show EBV infectivity (Hawkins et al., 2010).

However, these materials should be handled with care, and according to local laboratory safety precautions for biological materials.

Many tumours exhibit high levels of genomic instability, including mutation mosaicism and variability in gene copy number, zygosity, and overall ploidy. Cell lines derived from tumours are believed to provide a snapshot of the tumour at the time of biopsy (Lansford et al., 1999),

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with evidence to support this including data from histopathology, molecular genetics, receptor expression, gene expression, and drug sensitivity (Masters, 2000). However, it is unclear as to what extent variability continues to occur within the cell line over time. Overall it is expected that the materials used in this study are a useful mimic to the in vivo genomic complexity and variability of a tumour sample, and thus some commutability is achieved. Furthermore, since these materials are each prepared as a large batch, they are a long-term source of an unchanging genomic content.

KRAS codon 12 and 13 genotypes for each of the eight materials were indicated by COSMIC and/or in-house droplet dPCR (ddPCR, BioRad, Hercules, CA, USA). Droplet dPCR was also used to confirm the presence of two copies of the wild-type KRAS allele in the ATDB102 cell line, which when used in the dilution of the mutant KRAS materials (as material 16/266) enabled the calculation of the KRAS allelic ratio (mutant: wild-type) and total KRAS copy number (mutant plus wild- type) in each of the mutant materials (see KRAS Copy Numbers: Establishment of a Dilution Formula, below).

Each of the gDNA materials was prepared at approximately 10 µg/ml DNA concentration in 2.0 mM Tris, 0.2 mM EDTA, with 5 mg/ml D-(+)-trehalose dehydrate (Sigma-Aldrich, St. Louis, MO, USA; Table 3). Aliquots of 0.5 ml (1.0 ml for nominal wild-type KRAS material 16/266) were dispensed into 3 ml autoclaved DIN glass ampoules (Schott, Pont-sur-Yonne, France) using an automated AFV5090 ampoule filling line (Bausch & Strobel, Ilfshofen, Germany) with the bulk continually stirred at a slow rate using a magnetic stirrer whilst at ambient temperature. The homogeneity of the fill was determined by on-line check-weighing of the wet weight of triplicate ampoules for every 90 ampoules filled, with ampoules outside the defined specification (0.5000 g to 0.5300 g; 1.0000 g to 1.0150 g for material 16/266) discarded. The ampoules were partially stoppered with 13 mm Igloo stoppers (West, St Austell, UK) before the materials were freeze- dried in a CS15 (Serail, Argenteuil, France) to ensure long-term stability: the ampoules were frozen to -50°C, with primary drying at -35°C, 50 µbar, for 30 hours (-40°C, 30 µbar, for 30 hours for material 16/266), followed by secondary drying at +30°C, 30 µbar, for 40 hours. The vacuum was then released and the ampoules back-filled using boil-off gas from high purity liquid nitrogen (99.99%), before stoppering in situ in the dryer and flame sealing of the ampoules.

Measurement of the mean oxygen head space after sealing served as a measure of ampoule integrity. This was measured non-invasively by frequency modulated spectroscopy (FMS 760, Lighthouse Instruments, Charlottesville, VA, USA), based upon the Near Infra-Red absorbance by oxygen at 760 nm when excited using a laser. Controls of 0% and 20% oxygen were tested before samples were analysed to verify the method. Twelve ampoules were tested at random from each material; oxygen should be less than 1.14%. Residual moisture content was measured for the same 12 ampoules per material using the coulorimetric Karl Fischer method in a dry box environment (Mitsubishi CA100, A1 Envirosciences, Cramlington, UK) with total moisture expressed as a percentage of the mean dry weight of the ampoule contents. Individual ampoules were opened in the dry box and reconstituted with approximately 1-3 ml Karl Fischer analyte reagent which was then injected back into the Karl Fischer reaction cell and the water present in the sample determined colourmetrically. Dry weight was determined for six ampoules per material weighed before and after drying, with the measured water expressed as a percentage of

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the dry weight. Residual moisture levels of less than 1% are typically obtained, but where the dry weight is low (as here) the moisture level can be higher, with the materials still expected to demonstrate long-term stability (as seen for the similarly prepared WHO 1st International Genetic Reference Panel for Prader Willi & Angelman Syndromes, NIBSC panel code 09/140, which continues to demonstrate high stability eight years post-manufacture). Ongoing stability will be confirmed by accelerated degradation studies (see Degradation Studies, below).

Upon reconstitution with 100 µl nuclease-free water, the DNA concentration was approximately 50 µg/ml in 10 mM Tris, 1 mM EDTA (1x TE buffer) with 25 mg/ml D-(+)-trehalose dehydrate, excepting nominal wild-type KRAS codons 12 and 13 material 16/266 which was reconstituted with 200 µl nuclease-free water to give a DNA concentration of approximately 125 µg/ml in 1x TE buffer with 25 mg/ml D-(+)-trehalose dehydrate (for further dilution with 1x TE buffer to achieve 50 µg/ml DNA concentration). Homogeneity of each fill was determined by analysis of ampoules from the beginning, middle, and end of the filling process with quality and quantity of the freeze-dried gDNAs confirmed by 260/280 nm absorbance (Nanodrop, Thermo Fisher Scientific, Wilmington, DE, USA), Qubit fluorometric DNA quantification (Thermo Fisher Scientific), TapeStation electrophoresis (Agilent, Santa Clara, CA, USA), and ddPCR, which also acted as a pilot study to determine the performance of the materials in this increasingly-used diagnostic technique (Table 3). Lower DNA integrity number (DIN) was noted for materials 16/252, 16/258, and 16/266, as compared with the other materials, although the DNA quality was still within the acceptable range. Microbiological results were negative for all eight materials.

The ampoules are stored at -20°C at NIBSC under continuous temperature monitoring for the lifetime of the product. Shipping will typically be at ambient temperature, as studies have indicated the retained stability of the materials at elevated temperatures (5 months at +56°C, see Degradation Studies, below).

NIBSC code

Nominal KRAS codon 12 or 13 mutation

Originating cell

line Human tissue source

16/252 p.G12A RPMI 8226 myeloma (blood)

16/258 p.G12C MIA-Pa-Ca-2 pancreatic carcinoma

16/260 p.G12D SK LU 1 lung adenocarcinoma

16/254 p.G12R PSN1 pancreatic adenocarcinoma

16/256 p.G12S A549 lung carcinoma

16/264 p.G12V SW 626 ovarian metastasis of primary colon adenocarcinoma

16/262 p.G13D LoVo supraclavicular lymph node

metastasis of colon adenocarcinoma 16/266 Wild-type ATDB102 EBV-transformed lymphocytes

(blood)

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Table 2. Source cell lines of the eight materials of the proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations. KRAS codon 12 and 13 genotypes were indicated by COSMIC and/or in-house ddPCR.

NIBSC code 16/252 16/258 16/260 16/254 16/256 16/264 16/262 16/266 Nominal KRAS

codons 12 and 13 mutation

p.G12A p.G12C p.G12D p.G12R p.G12S p.G12V p.G13D Wild- type

Date filled 30/09/16 24/11/16 28/10/16 30/9/16 14/10/16 14/10/16 28/10/16 10/11/16 Mean DNA

concentration upon filling (µg/ml; n=15 to

21)

11.04 (~5µg total; 21)

11.39 (~5µg total; 21)

10.74 (~5µg total; 21)

12.93 (~5µg total; 21)

10.98 (~5µg total; 15)

10.45 (~5µg total; 21)

11.87 (~5µg total; 21)

28.11 (~25µg total; 18)

Mean fill mass (g; n=78 to 133)

0.5246 (99)

0.5175 (107)

0.5254 (109)

0.5251 (81)

0.5238 (113)

0.5261 (102)

0.5233 (133)

1.0084 (78) Mean pH (n=

10-14)

7.0 (14)

6.5 (14)

7.0 (14)

7.0 (14)

6.5 (10)

6.5 (14)

6.5 (14)

7.0 (12) Coefficient of

variation of fill mass (%; n= 78

to 133)

0.33 (99)

0.99 (107)

0.48 (109)

0.56 (81)

0.87 (113)

0.22 (102)

0.93 (133)

0.15 (78)

Mean dry

weight (g; n= 6) 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.005 Coefficient of

variation of dry mass (%; n= 6)

11.00 5.10 6.43 4.47 4.01 4.15 3.57 1.89

Mean residual moisture after lyophilisation

(%; n= 12)

4.10346 2.13715 2.62897 2.96266 2.55461 2.56359 2.48148 1.90700

Coefficient of variation of

residual moisture (%;

n= 12)

27.64 30.10 24.44 24.14 12.88 38.37 25.14 29.48

Mean residual oxygen (%; n=

12)

0.44 0.49 0.35 0.54 0.40 0.28 0.40 0.64

Coefficient of variation of residual oxygen

(%; n= 12)

24.41 24.29 28.54 21.52 31.23 45.65 50.96 22.89

Mean DNA concentration

upon reconstitution

(µg/ml; n= 3)

51.80 60.30 57.40 60.00 57.80 57.20 68.07 118.67

Mean OD ratio (260/280 nm;

n= 3)

1.91 1.92 1.93 1.73 1.91 1.90 1.93 1.89

Mean 6.9 7.0 8.7 9.1 9.4 9.1 9.1 7.9

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TapeStation DIN (n= 3) Mean KRAS mutation % (ddPCR; n= 9)

67.00 99.97 71.53 85.80 99.95 51.47 67.90 0.00

Coefficient of variation of KRAS mutation

% (%; n=9)

0.39 0.02 0.43 0.55 0.05 1.43 0.77 N/C

Number of ampoules

available

2547 2526 2574 1997 2100 2572 2328 2057

Presentation Sealed, glass DIN ampoules, 3 ml

Excipient Trehalose, 5 mg/ml in 2.0 mM Tris, 0.2 mM EDTA buffer Address of

facility where material was

processed

NIBSC, South Mimms, Hertfordshire, UK

Present

custodian NIBSC, South Mimms, Hertfordshire, UK

Storage

temperature -20oC

Table 3. Production and testing summary of the eight materials of the proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations. N/C, not calculated as all values were zero.

Participants

Sixty laboratories were recruited to the collaborative study, predominantly through membership of the European Molecular Genetics Quality Network (EMQN; Manchester, UK) and UK National External Quality Assessment Service (UK NEQAS) for Molecular Genetics

(Edinburgh, UK), and also publications on KRAS genomic diagnostics, and personal contacts, ensuring maximal coverage of the principal KRAS codons 12 and 13 mutation diagnostic techniques. Four laboratories were unable to proceed with the study, either due to import constraints (n=1) or limited laboratory resources (n=3); all remaining fifty six laboratories participated in the study and returned data (Appendix I). Thirty four countries were represented by the participants returning results, encompassing Europe, Asia, North America, South

America, and Australia. Each laboratory was assigned a code number (1 to 60) which does not reflect the order of listing in Appendix I. Where laboratories submitted data from more than one method, each method is referred to by an alphabetical suffix, for example 2a and 2b for

laboratory 2 methods a and b. Data from a total of 68 methods were returned, including 36 quantitative data sets.

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Study Design

Triplicate coded samples of the panel of eight gDNA materials (n= 24) were sent to each

laboratory with instructions for reconstitution and storage. Overall the materials were each to be tested at four different dilutions (crude, 1:2, 1:2.5, and 1:5), by dilution with the nominal wild- type KRAS codons 12 and 13 material 16/266 (total n=96). However, since it was not reasonable to request each laboratory analyse such a high number of samples, the materials and their

dilutions were distributed amongst the participants based upon their method and reported assay sensitivity; details of the collaborative study design are provided in Appendices II and III.

Participants were asked to perform their routine testing method(s) for the investigation of KRAS codons 12 and 13 mutations by testing the 24 coded samples in groups of 8 at a single dilution (n=8), plus 1 of those samples at an additional dilution (n=1), on 3 separate days (in total, n=27).

Participants were requested to use different batches of reagents and/or different operators if possible, alongside in-house patient samples (or other control materials) if typically used.

Laboratories were asked to report quantitative results where possible, together with the clinical interpretation. Overall findings for each sample and raw data, for example Sanger sequencing traces or dPCR counts, were to be returned, together with full details of the techniques used, any reference samples used, and reasons for failure of any of the samples tested.

Collaborative Study Methods

Twelve principal methods were used by the fifty six participants of the collaborative study (Table 4). Ten laboratories used two methods or method modifications (laboratories 2, 15, 20, 24, 27, 30, 40, 52, 55, and 58), whilst laboratory 54 used three method modifications, thereby giving a total of sixty eight methods. Quantitative data were reported for 36 methods; 32 methods reported qualitative data only. Following the distribution of the collaborative study report, laboratory 13 withdrew their data from the study (see Comments from the Participants, below); subsequent analyses are of data from 55 participants, and 67 methods, of which 35 were quantitative. Details of each method are provided in Appendix IV.

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Table 4. Methods used by collaborative study participants. Principal testing methods used in the detection of KRAS codons 12 and 13 mutations by participants in the collaborative study.

Methods reporting quantitative data are in bold (n=36, including three MassARRAY methods which were noted to be semi-quantitative; green); all other methods reported qualitative data (n=32). Method 30a was HRM followed by Sanger sequencing; method 31 comprised wild-type allele-clamped PCR followed by Sanger sequencing of the amplified mutant allele; method 32 comprised peptide nucleic acid (PNA) wild-type allele-clamped PCR followed by Sanger sequencing; method 53 utilized selective cleavage of wild-type amplicons with restriction

endonucleases followed by Sanger sequencing (orange). Real time PCR methods 17, 40a, and 51 utilized the IdyllaTM KRAS mutation test (Biocartis, Mechelen, Belgium) which includes an integrated sample preparation method (yellow). Following the distribution of the collaborative study report, laboratory 13 withdrew their data from the study (red).

Principal Testing Method Total

Next-generation sequencing 1 7 4 25 29 30b 33 34 38 43 48 50 54a 54b 54c 58a 58b 17

Sanger sequencing 8 16 18 20b 27b 28 30a 31 32 40b 42 44 53 13

Real-time PCR 2b 3 9 17 19 24b 26 27a 40a 46 51 11

Pyrosequencing 5 6 10 23 37 49 59 7

digital PCR 60 21 52b 55b 4

MALDI-TOF mass spectrometry

(MassARRAY®) 2a 11 13 22 4

KRAS StripAssay® 24a 41 47 3

High Resolution Melt analysis 15a 20a 2

ARMS-PCR 35 55a 2

PCR-rSSO 39 52a 2

Minisequencing 36 56 2

RFLP 15b 1

68 Participating Laboratory/Method Number

Overall total

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Results

Expected Results

The 24 blinded materials comprised triplicate samples of each of the 8 materials; NIBSC material code 16/252 (nominal mutation KRAS p.G12A), 16/258 (p.G12C), 16/260 (p.G12D), 16/254 (p.G12R), 16/256 (p.G12S), 16/264 (p.G12V), 16/262 (p.G13D), and 16/266 (wild-type KRAS codons 12 and 13; Table 5). The nominal KRAS codons 12 and 13 genotypes were indicated by COSMIC and/or in-house ddPCR. Each material was tested at a single dilution (crude, 1:2, 1:2.5, or 1:5; n=24), plus one material was tested at an additional dilution (crude, 1:2, 1:2.5, or 1:5; n=3), distributed across three separate days (in total, n=27). The expected mutation percentages for each material/dilution are not shown as these were to be determined by the collaborative study.

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NIBSC

code 16/252 16/258 16/260 16/254 16/256 16/264 16/262 16/266 Nominal

KRAS codons 12

and 13 mutation

p.G12A p.G12C p.G12D p.G12R p.G12S p.G12V p.G13D Wild- type Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

Sample 9

Sample 10

Sample 11

Sample 12

Sample 13

Sample 14

Sample 15

Sample 16

Sample 17

Sample 18

Sample 19

Sample 20

Sample 21

Sample 22

Sample 23

Sample 24

Table 5. Collaborative study expected results. Participants tested 24 blinded samples which comprised triplicates of the 8 materials of the different nominal KRAS codons 12 and 13

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genotypes. Each of these materials was tested as crude, 1:2, 1:2.5, or 1:5 diluted with the

nominal wild-type KRAS codons 12 and 13 material (16/266); expected mutation percentages are not shown as they were to be established by the collaborative study.

Results returned by Participants Quantitative Data

Quantitative data were reported for 35 methods including NGS, Sanger sequencing, real-time PCR, pyrosequencing, dPCR, and MassARRAY (a semi-quantitative method), and the means of the triplicate sample values for each method and material calculated as mutation percentage (for full data see Appendix V).

Particular observations were:

a. method 1 reported KRAS p.G12A in sample 2 of material 16/254 (at 1:2.5 dilution);

b. method 7 reported 45% KRAS p.G12S for sample 23 of material 16/256 (at 1:5 dilution), which was considered an outlier and excluded from further analysis;

c. method 29 additionally reported STK11 p. P281L and p.F354L in all three samples of material 16/254 (at 1:5 dilution), STK11 p.Q37* in all three samples of material 16/256 (at 1:5 dilution), and EGFR p.R836R in all three samples of material 16/264 (at crude and 1:5 dilution);

d. method 33 reported 15% KRAS p.G12S for sample 14 of material 16/256 (at 1:2 dilution), which was considered an outlier and excluded from further analysis, and additionally reported IDH2 c.435delG (p.T146fs) in all three samples of material 16/262 (crude);

e. method 43 was unable to test samples 11 and 18 of material 16/262 at 1:2.5 dilution, due to resource availability;

f. method 58a reported KRAS p.G13D in sample 17 of material 16/260 (at 1:5 dilution);

g. method 46 reported >75% KRAS p.G12A for sample 1 of material 16/252 (at 1:2.5 dilution), >50% KRAS p.G12C for sample 20 of material 16/258 (at 1:2.5 dilution), and unknown percentage KRAS p.G12S for sample 3 of material 16/256 (at 1:2.5 dilution), all likely due to the absence of high percentage controls for quantification in these assays;

h. method 6 was unable to quantify KRAS c.G34 mutations, and thus the three samples of material 16/258 were reported as KRAS p.G12C of unknown percentage (at 1:5 dilution), the three samples of material 16/254 were reported as KRAS p.G12R of unknown

percentage (at 1:5 dilution), and the three samples of material 16/256 were reported as KRAS p.G12S of unknown percentage (at 1:5 dilution);

i. method 55b did not perform dPCR analysis (and therefore quantification) of materials 16/258 (crude), 16/254 (at 1:5 dilution), 16/256 (at 1:5 dilution), or 16/262 (at 1:5 dilution), although genotypes were identified in the laboratory’s other method, 55a.

Quantitative data from real-time PCR analysis were clearly distinct from those of other quantitative methods, were reported by only two methods (methods 3 and 46), and thus were excluded from further analysis to avoid the influence of these outlying data. It was furthermore apparent that data from all remaining quantitative methods (n=33) could not be considered as one

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dataset as there were clear differences between some of the principal methods’ results, meaning that the derivation of overall average values for each material would likely not be in agreement with any one method. Therefore, quantitative data from NGS, Sanger sequencing,

pyrosequencing, dPCR, and MassARRAY were considered separately (Table 6; Figure 1).

Summary statistics for each principal method and material were calculated as mean and median (Table 7). As there were insufficient data to confirm the assumption of a normal distribution of results within all methods for each material (see Figure 1), and to avoid the influence of any outliers, the median value was used as an appropriate summary statistic for each material (and dilution).

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Table 6. Summary data for quantitative methods in the collaborative study. Mean mutation percentages for triplicate tested samples in all quantitative methods were derived (n=33 methods; excluding real-time PCR methods 3 and 46). Method 7 reported 45%

KRAS p.G12S for one sample of material 16/256 at 1:5 dilution, which was considered to be an outlying value (compared with the other two samples of 20% and 23%), and so was excluded from the mean calculation (orange); method 33 reported 15% KRAS p.G12S for one sample of material 16/256 at 1:2 dilution, which was considered to be an outlying value (compared with the other two samples of 47% and 46%), and so was excluded from the mean calculation (orange); method 43 tested only one sample of material 16/262 (at 1:2.5 dilution; orange). Results are reported to one decimal place.

1 7 4 25 29 30b 33 34 38 43 48 50 54a 54b 54c 58a 58b 16 28 31 5 6 10 23 37 49 60 21 52b 55b 2a 11 22

crude 65.0 65.0 65.7 66.0 66.9 65.9 63.9 63.3 64.3 67.0 67.0 53.4 57.0

1:2 31.3 30.3 31.7 43.3 33.6 31.3 31.9

1:2.5 23.7 26.6 29.1 28.3 23.7 26.3 28.1 25.5 27.4

1:5 10.7 13.0 12.1 14.0 12.7 12.7 10.7 12.3 14.7 15.0 12.4 16.0 17.7

crude 100.0 100.0 99.7 99.9 99.4 100.0 95.0 97.1 95.0 100.0 100.0

1:2 59.4 58.7 53.7 55.9 80.0 59.9 64.0 59.1 57.3

1:2.5 46.3 37.5 53.2 48.7 47.0 55.5 49.2 42.9 61.3

1:5 28.7 29.2 24.7 42.0 29.0 20.7 22.0 42.3 29.4 29.0 48.3 44.7

crude 71.0 72.3 71.5 71.5 71.7 70.8 80.0 71.0 74.8 74.3 71.5 69.8

1:2 46.6 49.7 61.7 54.4 51.6 46.9

1:2.5 40.3 36.2 45.2 42.0 46.0 50.7 41.8 53.8

1:5 26.7 26.2 24.3 36.3 26.7 19.3 25.7 31.7 40.7 32.9 25.7 24.2 45.3 41.7

crude 85.0 86.7 85.6 85.0 80.9 86.5 86.0 85.0 89.0 83.0 85.8 72.2 63.3

1:2 78.1 78.3 75.5 77.9 75.6 75.1

1:2.5 72.0 73.5 71.6 69.7 62.0 70.3 71.9 49.2

1:5 62.0 58.2 57.6 54.3 59.3 51.7 58.0 58.4 40.3 43.0

crude 99.7 100.0 99.8 99.9 99.6 100.0 99.3 98.0 100.0 100.0 99.0 93.7 99.9 99.5

1:2 46.5 52.6 43.3 54.2 50.0 52.2

1:2.5 42.7 46.1 41.4 43.3 36.0 43.8 41.7 40.2

1:5 21.5 21.4 21.0 15.0 22.0 19.3 17.7 22.1 28.0 23.7 24.0

crude 46.3 51.0 49.7 49.9 49.3 47.1 47.4 40.0 50.4 43.7 51.5 50.3

1:2 26.0 40.0 27.5 29.9 27.6 25.4

1:2.5 20.7 23.1 24.9 26.7 30.0 29.0 25.0 23.8 37.5

1:5 15.0 11.0 12.0 14.3 12.0 18.0 11.7 10.7 8.8 17.7 13.7 12.5 24.7 26.5

crude 66.7 67.7 66.9 67.7 66.1 66.2 67.7 80.0 66.5 63.7 67.9 53.6

1:2 39.9 50.0 43.4 43.3 40.7

1:2.5 30.3 33.2 37.5 31.8 36.7 37.0 39.2 35.7 40.4

1:5 20.7 20.3 17.3 26.7 20.3 17.0 27.0 30.3 20.0 33.3 33.0

16/256

16/264

16/262 16/252

16/258

16/260

16/254

Principal method, Laboratory/method code

Material Dilution NGS Sanger Pyroseq dPCR MassARRAY

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Figure 1. Mean KRAS codons 12 and 13 mutations percentage values for triplicate tested materials in all quantitative methods in the collaborative study (n=33 methods; excluding quantitative real-time PCR methods 3 and 46). Data are shown for each material at crude, 1:2, 1:2.5, and 1:5 dilutions for each quantitative method.

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Table 7. Comparison of mean and median KRAS mutation percentages for quantitative methods in the collaborative study.

Subtraction of the overall median percentage from the overall mean percentage for each principal method and material/dilution showed minimal impact on the use of either median or mean as consensus values.

NGS Sanger Pyro dPCR MassARRAY NGS Sanger Pyro dPCR MassARRAY NGS Sanger Pyro dPCR MassARRAY

crude 65.5 63.3 65.7 60.2 57.0 65.7 63.3 65.7 60.2 57.0 -0.2 0.0 0.0 0.0 0.0

1:2 30.8 37.5 33.6 31.6 30.8 37.5 33.6 31.6 0.0 0.0 0.0 0.0

1:2.5 26.9 23.7 27.2 25.5 27.4 27.5 23.7 27.2 25.5 27.4 -0.5 0.0 0.0 0.0 0.0

1:5 12.3 13.5 13.7 16.8 12.7 13.5 13.7 16.8 -0.4 0.0 0.0 0.0

crude 99.8 95.0 96.1 100.0 100.0 95.0 96.1 100.0 -0.1 0.0 0.0 0.0

1:2 56.9 80.0 62.0 58.2 57.3 80.0 62.0 58.2 -0.4 0.0 0.0 0.0

1:2.5 46.4 47.0 55.5 46.1 61.3 47.5 47.0 55.5 46.1 61.3 -1.1 0.0 0.0 0.0 0.0

1:5 28.0 42.3 29.2 46.5 28.7 42.3 29.2 46.5 -0.6 0.0 0.0 0.0

crude 71.5 80.0 73.4 70.7 71.5 80.0 74.3 70.7 0.0 0.0 -1.0 0.0

1:2 48.1 61.7 54.4 49.2 48.1 61.7 54.4 49.2 0.0 0.0 0.0 0.0

1:2.5 40.9 46.0 50.7 41.8 53.8 41.2 46.0 50.7 41.8 53.8 -0.2 0.0 0.0 0.0 0.0

1:5 26.6 25.7 35.1 24.9 43.5 26.4 25.7 32.9 24.9 43.5 0.2 0.0 2.2 0.0 0.0

crude 85.1 85.0 86.0 79.0 63.3 85.6 85.0 86.0 79.0 63.3 -0.5 0.0 0.0 0.0 0.0

1:2 78.1 78.3 76.7 75.4 78.1 78.3 76.7 75.4 0.0 0.0 0.0 0.0

1:2.5 71.7 62.0 70.3 71.9 49.2 71.8 62.0 70.3 71.9 49.2 -0.1 0.0 0.0 0.0 0.0

1:5 57.2 58.0 58.4 41.7 57.9 58.0 58.4 41.7 -0.7 0.0 0.0 0.0

crude 99.5 100.0 97.6 99.7 99.7 100.0 99.0 99.7 -0.2 0.0 -1.4 0.0

1:2 49.5 43.3 54.2 51.1 49.5 43.3 54.2 51.1 0.0 0.0 0.0 0.0

1:2.5 43.4 36.0 43.8 41.7 40.2 43.0 36.0 43.8 41.7 40.2 0.4 0.0 0.0 0.0 0.0

1:5 20.0 17.7 22.1 25.2 21.2 17.7 22.1 24.0 -1.1 0.0 0.0 1.2

crude 48.7 40.0 47.0 50.9 49.3 40.0 47.0 50.9 -0.6 0.0 0.0 0.0

1:2 26.0 40.0 27.5 27.6 26.0 40.0 27.5 27.6 0.0 0.0 0.0 0.1

1:2.5 23.8 29.5 25.0 23.8 37.5 24.0 29.5 25.0 23.8 37.5 -0.2 0.0 0.0 0.0 0.0

1:5 13.1 13.3 13.1 25.6 12.0 13.3 13.1 25.6 1.1 0.0 0.0 0.0

crude 67.0 80.0 65.1 60.8 66.9 80.0 65.1 60.8 0.1 0.0 0.0 0.0

1:2 39.9 50.0 43.4 42.0 39.9 50.0 43.4 42.0 0.0 0.0 0.0 0.0

1:2.5 33.9 37.0 39.2 35.7 40.4 33.2 37.0 39.2 35.7 40.4 0.7 0.0 0.0 0.0 0.0

1:5 20.4 28.7 20.0 33.2 20.3 28.7 20.0 33.2 0.1 0.0 0.0 0.0

16/252

16/258

16/260

16/254

16/256

16/264

16/262

Principal method mean-median

Material Dilution Principal method mean Principal method median

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Next, agreement between quantitative principal methods was considered and Lin’s concordance correlation coefficient (ρC) was calculated for each pair of principal methods using the median values shown in Table 7 (Figure 2). MassARRAY showed poor concordance with the four quantitative methods (ρC < 0.80 in all cases), which may be attributed to the semi-quantitative reporting; NGS, Sanger sequencing, pyrosequencing, and dPCR showed good concordance with each other (ρC > 0.90 in all cases). Specifically, Sanger sequencing showed good concordance with NGS, pyrosequencing, and dPCR (0.90 < ρC < 0.95), whereas pyrosequencing showed stronger concordance with NGS and dPCR (ρC > 0.95), and dPCR showed the strongest concordance with NGS (ρC = 0.995).

Finally, to determine appropriate consensus mutation percentages for each material, only data from the three methods with the strongest concordance were considered (NGS, dPCR, and pyrosequencing; Table 8). If NGS data alone were used to assign the consensus mutation percentages, this would reflect the frequency at which this method appears to be used (n=17 of 67 methods in the collaborative study), and acknowledges the likely increasing use of NGS as more laboratories adopt this method, but it may be unwise to consider data from only one principal method. NGS and dPCR have very different underlying methodologies, yet have very strong concordance in this study. Thus the inclusion of dPCR data, with this technique gaining consideration as the new ‘gold standard’ high-sensitivity method in absolute quantification of molecular markers, strengthens the dataset (total n=21). Addition of the quantitative

pyrosequencing data (total n=27) would result in consensus mutation percentages derived from three technically-different principal methods, but since pyrosequencing has less concordance with NGS than does dPCR (Figure 2), its impact on the overall data must be considered. This is noted especially with the increase in inter-quartile ranges (IQRs) determined when

pyrosequencing is also included (for some materials/dilutions; Table 8). Therefore, consensus mutation percentages are presented as the overall median value for each material according to NGS and dPCR (Table 8).

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Figure 2. Agreement of median KRAS mutation percentages for each pair of principal methods. The median values for the seven mutant KRAS materials and their dilutions are shown for each quantitative principal method. Both axes are percentage mutant KRAS; solid lines represent perfect agreement; dashed lines indicate fitted Deming regression models. Lin’s concordance correlation coefficient (ρC) was calculated for the median KRAS mutation

percentages for each pair of principal methods. Good concordance is shown in light green (0.90

≤ ρC < 0.95); excellent concordance (ρC ≥ 0.95) is shown in dark green.

0.927

0.980

0.995

0.736

0.937

0.920 7

0.684

0.977

0.812 0.742

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Table 8. Overall median KRAS mutation percentages and inter-quartile ranges for

quantitative principal methods in excellent concordance; NGS, dPCR, and pyrosequencing.

The IQR is not shown where less than four mean values for triplicate tested samples are available for the principal method. Data are reported to 1 decimal place, except for crude material 16/258 which is reported to 2 decimal places to capture the apparent (low-level) presence of wild-type KRAS allele in this material (green).

Median IQR Median IQR Median IQR

crude 65.7 1.0 65.7 1.0 65.7 1.8

1:2 30.8 31.3 0.4 31.3 0.6

1:2.5 27.5 2.6 26.6 2.8 26.6 2.3

1:5 12.7 1.5 12.7 0.9 12.7 1.3

crude 100.0 0.3 99.98 0.1 100.0 0.5

1:2 57.3 3.5 58.0 2.8 58.9 2.6

1:2.5 47.5 5.7 47.5 5.3 48.7 6.6

1:5 28.7 5.8 29.0 4.5 29.0 3.7

crude 71.5 0.5 71.5 0.6 71.5 1.0

1:2 48.1 48.3 3.4 49.7 4.7

1:2.5 41.2 3.5 41.8 1.7 41.9 3.7

1:5 26.4 1.9 25.9 2.4 26.7 7.3

crude 85.6 1.3 85.6 1.0 85.6 2.3

1:2 78.1 75.6 75.6 2.4

1:2.5 71.8 1.3 71.9 0.4 71.7 1.3

1:5 57.9 3.9 58.2 2.9 58.1 1.9

crude 99.7 0.4 99.7 0.4 99.7 0.6

1:2 49.5 51.1 3.2 52.2 2.6

1:2.5 43.0 1.6 42.7 1.6 43.0 1.7

1:5 21.2 1.8 21.4 1.6 21.2 2.7

crude 49.3 2.6 49.7 2.9 49.7 3.1

1:2 26.0 26.8 2.3 27.5 1.6

1:2.5 24.0 2.9 23.8 1.8 24.4 1.7

1:5 12.0 3.0 12.2 2.4 12.2 3.0

crude 66.9 1.3 66.9 1.5 66.7 1.6

1:2 39.9 40.7 42.0 2.8

1:2.5 33.2 4.9 34.5 4.3 35.7 4.6

1:5 20.3 2.6 20.3 1.9 20.3 6.7

16/264

16/262 16/252

16/258

16/260

16/254

16/256

NGS only NGS & dPCR NGS, dPCR, & Pyro Principal methods

Material Dilution

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KRAS Copy Numbers: Establishment of a Dilution Formula

In this study, each crude material of the proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations was subjected to three dilutions (1:2; 1:2.5, and 1:5, by combination with the nominal wild-type KRAS codons 12 and 13 material 16/266). The presence of two copies of the wild-type KRAS allele in material 16/266 was determined by ddPCR by reference firstly to MRC-5, a primary diploid cell line derived from normal lung tissue of a 14 week-old male foetus (Jacobs et al., 1970) and commonly used in vaccine development, and for in vitro cytotoxicity testing, and secondly to a commercial human gDNA derived from multiple anonymous donors (catalogue number G3041, Promega, Madison, WI, USA). Use of the wild-type KRAS codons 12 and 13 material 16/266 in the dilution of the mutant KRAS materials enabled the calculation of the KRAS allelic ratio (mutant: wild-type) and total KRAS copy number (mutant plus wild-type) in the latter, based upon the dilution response for each of the materials, which varied from quasi-linear to hyperbolic, and reflected the differing genomic complexity of these materials (Figure 3). A model-fitting algorithm was performed using Python 2.7 SciPy, with the best fitting model given by:

y= x/(ax+b)

where a and b are the coefficients obtained for each of the mutant materials after the convergence of the fitting algorithm to the dilution response (Table 9).

To validate the fit of the dilution model for each of the mutant materials, particularly to lower mutation percentages, additional dilutions for each of the mutant materials were evaluated in- house with ddPCR. The resulting mutation percentages were consistent with those predicated by the model (Appendix VI), thereby demonstrating the accuracy of the model fitting. Due to the large number of samples already tested by the collaborative study laboratories, it was impractical to request they assess further dilutions. However, the strong agreement of the in-house ddPCR data with the collaborative study consensus mutation percentages and the model-derived data provide confirmation of the suitability of the in-house ddPCR in verifying the calculations (Appendix VI).

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Figure 3. Dilution responses of the seven mutant KRAS materials of the proposed WHO 1st International Reference Panel for genomic KRAS codons 12 and 13 mutations. Data shown are the consensus mutation percentages for each of the materials and their dilutions (crude, 1:2, 1:2.5, and 1:5). The blue lines represent the best fit dilution response.

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