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Reliability of KRAS mutation testing in metastatic colorectal cancer patients across five laboratories.
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MedLine Citation:
PMID:  22534075     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
BACKGROUND: Mutations in the KRAS gene are associated with poor response to epidermal growth factor receptor inhibitors used in the treatment of metastatic colorectal cancer. Factors influencing KRAS test results in tumor specimens include: tumor heterogeneity, sample handling, slide preparation, techniques for tumor enrichment, DNA preparation, assay design and sensitivity. We evaluated comparability and consistency of KRAS test results among five laboratories currently being used to determine KRAS mutation status of metastatic colorectal cancer specimens in a large, multi-center observational study.
FINDINGS: Twenty formalin-fixed paraffin-embedded human colorectal cancer samples from colon resections previously tested for KRAS mutations were selected based on mutation status (6 wild type, 8 codon 12 mutations, and 6 codon 13 mutations). We found good agreement across laboratories despite differences in mutation detection methods. Eighteen of twenty samples (90%) were concordant across all five labs. Discordant results are likely not due to laboratory error, but instead to tumor heterogeneity, contamination of the tumor sample with normal tissue, or analytic factors affecting assay sensitivity.
CONCLUSIONS: Our results indicate commercial and academic laboratories provide reliable results for the common KRAS gene mutations at codons 12 and 13 when an adequate percentage of tumor cells is present in the sample.
Authors:
Heather Spencer Feigelson; Katrina A B Goddard; Monique A Johnson; Kellyan C Funk; Alanna Kulchak Rahm; Tia L Kauffman; Dhananjay A Chitale; Loic Le Marchand; C Sue Richards
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Publication Detail:
Type:  Journal Article; Multicenter Study; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-04-25
Journal Detail:
Title:  BMC research notes     Volume:  5     ISSN:  1756-0500     ISO Abbreviation:  BMC Res Notes     Publication Date:  2012  
Date Detail:
Created Date:  2012-09-13     Completed Date:  2013-01-31     Revised Date:  2013-06-25    
Medline Journal Info:
Nlm Unique ID:  101462768     Medline TA:  BMC Res Notes     Country:  England    
Other Details:
Languages:  eng     Pagination:  196     Citation Subset:  IM    
Affiliation:
Institute for Health Research Legacy Highlands, Kaiser Permanente Colorado, Denver, CO 80237-8066, USA. heather.s.feigelson@kp.org
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MeSH Terms
Descriptor/Qualifier:
Adenocarcinoma / genetics*,  secondary
Aged
Aged, 80 and over
Carcinoma / genetics*,  secondary
Clinical Laboratory Techniques / standards*
Codon
Colorectal Neoplasms / genetics*,  pathology
DNA Mutational Analysis / standards*
Guidelines as Topic / standards
Humans
Middle Aged
Mutation*
Observer Variation
Paraffin Embedding
Predictive Value of Tests
Proto-Oncogene Proteins / genetics*
Quality Control
Real-Time Polymerase Chain Reaction / standards
Reproducibility of Results
Tissue Fixation
United States
ras Proteins / genetics*
Grant Support
ID/Acronym/Agency:
U19 CA79689/CA/NCI NIH HHS; UC2-CA148471/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Codon; 0/KRAS protein, human; 0/Proto-Oncogene Proteins; EC 3.6.5.2/ras Proteins
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): BMC Res Notes
Journal ID (iso-abbrev): BMC Res Notes
ISSN: 1756-0500
Publisher: BioMed Central
Article Information
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Copyright ©2012 Feigelson et al.; licensee BioMed Central Ltd.
open-access:
Received Day: 17 Month: 1 Year: 2012
Accepted Day: 25 Month: 4 Year: 2012
collection publication date: Year: 2012
Electronic publication date: Day: 25 Month: 4 Year: 2012
Volume: 5First Page: 196 Last Page: 196
ID: 3441241
Publisher Id: 1756-0500-5-196
PubMed Id: 22534075
DOI: 10.1186/1756-0500-5-196

Reliability of KRAS mutation testing in metastatic colorectal cancer patients across five laboratories
Heather Spencer Feigelson1 Email: heather.s.feigelson@kp.org
Katrina AB Goddard2 Email: Katrina.Goddard@kpchr.org
Monique A Johnson3 Email: johnsonmon@ohsu.edu
Kellyan C Funk1 Email: Kellyan.C.Funk@kp.org
Alanna Kulchak Rahm1 Email: Alanna.K.Rahm@kp.org
Tia L Kauffman2 Email: Tia.l.kauffman@kpchr.org
Dhananjay A Chitale4 Email: DCHITAL1@hfhs.org
Loic Le Marchand5 Email: loic@crch.hawaii.edu
C Sue Richards3 Email: richarsu@ohsu.edu
1Institute for Health Research Legacy Highlands, Kaiser Permanente Colorado, Suite 300, P.O. Box 378066, Denver, CO, 80237-8066, USA
2Kaiser Permanente Northwest, Center for Health Research, 3800 N. Interstate Avenue, Portland, OR, 97227, USA
3Molecular and Medical Genetics, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR, 97239-3098, USA
4Department of Pathology and Laboratory Medicine, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI, 48202, USA
5University of Hawaii Cancer Center, 677 Ala Moana Boulevard, Suite 901, Honolulu, HI, 96813, USA

Findings
Background and hypothesis

Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies are approved for the treatment of metastatic colorectal cancer (CRC). However, these anti-EGFR therapies do not benefit patients whose tumors harbor a KRAS mutation [1]. Genetic testing for the presence of KRAS mutations has been recommended to guide treatment for these patients [2].

Several factors can influence KRAS mutation testing results in CRC specimens [3-5]. The purpose of this study was to evaluate comparability and consistency of clinical KRAS test results among laboratories used to determine KRAS mutation status in a large multi-center study. Three commercial laboratories (Genzyme, Clarient, Quest Diagnostics), one clinical academic laboratory (Henry Ford Health System), and one academic research laboratory (Molecular and Medical Genetics, Oregon Health and Science University) were contracted to analyze KRAS mutation status for comparison with previous clinical results. While all five laboratories are Clinical Laboratories Improvement Act (CLIA) certified, they have different sample preparation and mutation detection methods. Our aim was not to certify these laboratories, but to ensure that we could combine data from previously tested clinical samples in our research study.

Methods

Twenty surgical specimens from colon resections were used; eighteen specimens were adenocarcinomas, two were carcinomas. Blocks were reviewed by a pathologist to determine whether the samples were of sufficient quality and quantity for testing, then the samples were de-identified and slides were prepared per individual laboratory specifications. Our intention was to replicate routine sample testing of clinical specimens as much as possible, so sample handling and shipping procedures varied slightly by laboratory. All laboratories used microdissection for tumor enrichment when necessary, but mutation detection methods differed. Methods for each laboratory are described in Table 1.

KRAS test results were compared across labs, and discrepancies were evaluated further. This study was approved by the Institutional Review Boards (IRB) at Kaiser Permanente Colorado and Kaiser Permanente Northwest (the Oregon Health and Science University IRB ceded authority to Kaiser Permanente Northwest).

Results

Twenty formalin-fixed paraffin-embedded (FFPE) CRC samples previously tested clinically for KRAS mutations by sequencing were selected based on mutation status (6 wild-type samples, 8 with codon 12 mutations, and 6 with codon 13 mutations) from two study sites (Kaiser Permanente Colorado and Northwest). Patients ranged in age from 46–85 years; specimens were collected between 2005–2009. We found good agreement in KRAS test results with prior clinical results despite differences in mutation detection methods (Table 2). Eighteen of twenty samples (90%) were concordant across all five laboratories, and the mutation type was always consistent.

One laboratory reported a wild-type result for sample 14 which was actually a p.Gly12Asp (G12D) mutation. While this sample was confirmed to contain an acceptable 40% tumor cells, tumor heterogeneity in this sample may have resulted in the variant being present below the pre-determined 20% threshold. A very small electropherogram peak indicating a c.35 G > A change was visible by sequencing indicating p.Gly12Asp mutation, but was not reported because it was below acceptable level of confidence per laboratory protocol.

We also found a discrepancy in sample 6. The initial clinical result was wild-type; two labs reported a mutation in exon 12 p.Gly12Asp, and three labs were consistent with the clinical results (wild-type). We evaluated this discrepancy first by sending additional slides (from the same tumor block) to the two laboratories that reported the mutation. These slides were assigned a new study number to blind the laboratory to the re-testing. Laboratory #2 found the same result (p.Gly12Asp) in the second set of slides, but there was not enough tumor tissue for laboratory #3 to reliably genotype. Next, we used a second FFPE block from the same patient to send a third set of slides with a new study number to laboratories 1–3. Results from this block were concordant at all three laboratories (KRAS wild-type). Finally, we asked laboratories #1 and #2 to “swap” aliquots of the extracted DNA from their original sample 6 FFPE slides. This re-analysis confirmed the initial (discrepant) results at each laboratory. Thus, we conclude that the laboratory results, while different, are accurate for the sample of tissue received at each laboratory. The discrepant results could be due to either true tumor heterogeneity or contamination of the tumor sample with normal tissue. We cannot conclusively determine which of these two scenarios is responsible for our observed results, nor eliminate the possibility that a laboratory error resulting in sample mix-up lead to the discrepant results.

Discussion

We found high concordance of KRAS test results with previously received clinical results across five laboratories, despite differences in laboratory methods. The discordant results observed in two samples are most likely due to sample characteristics rather than to laboratory error. Our study focused only on mutations in codons 12 and 13 of KRAS. These are the most common mutations and are often the only mutations targeted in clinical testing. However, other mutations may have clinical implications and were not included in our study [6,7].

We limited our study to samples from colon resections. Samples with smaller volume, such as from metastatic sites or biopsy, may not perform as reliably as colectomy specimens if they contain only a small percentage of tumor cells. Several new methods of mutation detection have been reported [3,8-13] and may be better suited to samples with a low percentage of tumor cells.

Our results are in agreement with previous studies [8-12], including a recent report by Oliner et al. [13] who evaluated five commercial laboratories, one of which (Genzyme) was also included in our study. They tested forty FFPE samples from several tissue procurement providers, whereas our samples were obtained from colectomies performed at our own clinic facilities, previously tested, and used to guide clinical care. Because we were able to select our samples based on mutation status, and thus oversample for the KRAS mutations of interest, our estimate of the agreement across laboratories corresponds to an estimate from an effectively larger sample size. It is reassuring that, while both studies evaluated different commercial laboratories and used slightly different methodologies, both found good agreement across testing facilities.

The commercial and academic-based laboratories included in this study provide reliable test results for common mutations in the KRAS gene from samples with an adequate percentage of tumor cells. Discrepancies observed are likely due to either tumor heterogeneity, or contamination of the tumor sample with normal tissue. Both of these sources of variability are likely to be encountered in the clinical setting, and may have important consequences for treatment decisions.


Abbreviations

CLIA, Clinical Laboratories Improvement Act; CRC, colorectal cancer; EGFR, epidermal growth factor receptor; FFPE, formalin-fixed paraffin-embedded; H&E, hematoxylin and eosin; IRB, Institutional Review Board; WT, wild-type.


Competing interests

The authors declare that they have no competing interests.


Authors’ contributions

HSF conceived of the study, participated in its design and drafted the manuscript. KABG designed the larger study under which this research was carried out and contributed to the design of this study. MAJ participated in the laboratory analysis. KCF coordinated collection of samples for analysis and collated results. AKR managed the study, participated in the study design, and participated in manuscript preparation. TLK managed the larger study and assisted in coordinating sample collection and results. DAC participated in the laboratory analysis of samples. LLM conceived of the study and participated in its design and manuscript preparation. CSR participated in laboratory analysis, interpretation of results, and participated in manuscript preparation. All authors read and approved the final manuscript.


Acknowledgements

This work was funded by the National Cancer Institute grant UC2-CA148471, and was performed in affiliation with the Cancer Research Network (CRN). The Cancer Research Network (NCI cooperative agreement # U19 CA79689, PI: Edward H. Wagner) is a collaboration of 14 research centers based in organized health care delivery systems.


References
Dahabreh IJ,Terasawa T,Castaldi PJ,Trikalinos TA,Systematic review: Anti-epidermal growth factor receptor treatment effect modification by KRAS mutations in advanced colorectal cancerAnn Intern MedYear: 2011154374921200037
Allegra CJ,Jessup JM,Somerfield MR,Hamilton SR,Hammond EH,Hayes DF,et al. American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapyJ Clin OncolYear: 2009272091209610.1200/JCO.2009.21.917019188670
Franklin WA,Haney J,Sugita M,Bemis L,Jimeno A,Messersmith WA,KRAS mutation: comparison of testing methods and tissue sampling techniques in colon cancerJ Mol DiagnYear: 201012435010.2353/jmoldx.2010.08013120007845
Holdhoff M,Schmidt K,Donehower R,Diaz LA,Analysis of circulating tumor DNA to confirm somatic KRAS mutationsJ Natl Cancer InstYear: 20091011284128510.1093/jnci/djp24019641175
Jimeno A,Messersmith WA,Hirsch FR,Franklin WA,Eckhardt SG,KRAS mutations and sensitivity to epidermal growth factor receptor inhibitors in colorectal cancer: practical application of patient selectionJ Clin OncolYear: 2009271130113610.1200/JCO.2008.19.816819124802
Loupakis F,Ruzzo A,Cremolini C,Vincenzi B,Salvatore L,Santini D,et al. KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancerBr J CancerYear: 200910171572110.1038/sj.bjc.660517719603018
Janakiraman M,Vakiani E,Zeng Z,Pratilas CA,Taylor BS,Chitale D,et al. Genomic and biological characterization of exon 4 KRAS mutations in human cancerCancer ResYear: 2010705901591110.1158/0008-5472.CAN-10-019220570890
Carotenuto P,Roma C,Rachiglio AM,Tatangelo F,Pinto C,Ciardiello F,et al. Detection of KRAS mutations in colorectal carcinoma patients with an integrated PCR/sequencing and real-time PCR approachPharmacogenomicsYear: 2010111169117910.2217/pgs.10.8620712532
Whitehall V,Tran K,Umapathy A,Grieu F,Hewitt C,Evans TJ,et al. A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical settingJ Mol DiagnYear: 20091154355210.2353/jmoldx.2009.09005719815694
Weichert W,Schewe C,Lehmann A,Sers C,Denkert C,Budczies J,et al. KRAS genotyping of paraffin-embedded colorectal cancer tissue in routine diagnostics: comparison of methods and impact of histologyJ Mol DiagnYear: 201012354210.2353/jmoldx.2010.09007920007841
Tsiatis AC,Norris-Kirby A,Rich RG,Hafez MJ,Gocke CD,Eshleman JR,et al. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: Diagnostic and clinical implicationsJ Mol DiagnYear: 2010121810.2353/jmoldx.2010.090194
Angulo B,Garcia-Garcia E,Martinez R,Suarez-Gauthier A,Conde E,Hidalgo M,et al. A commercial real-time PCR kit provides greater sensitivity than direct sequencing to detect KRAS mutations: a morphology-based approach in colorectal carcinomaJ Mol DiagnYear: 20101229229910.2353/jmoldx.2010.09013920203003
Oliner K,Juan T,Suggs S,Wolf M,Sarosi I,Freeman DJ,et al. A comparability study of 5 commercial KRAS testsDiagn PatholYear: 201052310.1186/1746-1596-5-2320398393

Tables
[TableWrap ID: T1] Table 1 

Specimen requirements and assay specifications of KRAS genotyping by laboratories


  Lab #1 (Sequencing) Lab #2 (Sequencing) Lab #3 (Sequencing) Lab #4 (Primer Extension) Lab #5 (Real Time PCR)
Specimen Requirements
Preferred sample type*:
Slides from FFPE block
1  H&E stained slide sections with tumor circled; 4 matching unstained slides,
10 microns each.
Preferred sample type: Archival FFPE or frozen surgical biopsies confirmed to contain >50% tumor by a surgical pathologist.
1 H&E slide;
5 unstained sections,
10 microns each.
Preferred sample type: FFPE tissue
6 unstained sections,
10 microns each.
Preferred sample type: Pre-cut slides from FFPE. Send all slides within 5–7 days of cutting. Air dry. Do not oven dry. Store specimen at room temperature (20–23.5°C).
5 unstained sections,
7 microns each
Preferred sample type: FFPE block, unstained slides, or fresh snap frozen biopsy
5 unstained sections,
7 microns each
Genotyping
Method: PCR amplification followed
by Direct Sanger sequencing
(Big Dye v. 1.1)
Detected mutations: KRAS codons 12 and 13
Method: PCR amplification followed by standard bidirectional sequencing on ABI 3100.
Detected mutations: KRAS codons 12 and 13
Method: PCR amplification followed by sequencing.
Detected mutations: KRAS codons 12,
13 and 61
Method: Single nucleotide primer extension with fragment analysis by capillary electrophoresis using a modified SNaPshot assay.
Detected mutations:
KRAS codons 12 and 13
Methods are propietary: qualitative real time PCR
Detected mutations: KRAS codons 12 and 13
Lower Limit of Detection 20% when ≥ 40% tumor cells present 20% 15-20% 10% when ≥ 2% tumor cells present 1-5%

*For this study, slides prepared from Formalin-fixed paraffin embedded (FFPE) blocks were sent to each lab.


[TableWrap ID: T2] Table 2 

Results of KRAS testing by five CLIA-certified laboratories


Sample ID Clinical Result* Sequencing Lab 1 Sequencing Lab 2 Sequencing Lab 3 Primer Extension
Lab 4
Real-time PCR
Lab 5
1
WT**
WT
WT
WT
WT
WT
2
WT
WT
WT
WT
WT
WT
3
WT
WT
WT
WT
WT
WT
4
WT
WT
WT
WT
WT
WT
5
WT
WT
WT
WT
WT
WT
6
WT
WT
G12D
G12D
WT
WT
7
G12V
G12V
G12V
G12V
G12V
G12V
8
G12D
G12D
G12D
G12D
G12D
G12D
9
G12V
G12V
G12V
G12V
G12V
G12V
10
G12S
G12S
G12S
G12S
G12S
G12S
11
G12V
G12V
G12V
G12V
G12V
G12V
12
G12C
G12C
G12C
G12C
G12C
G12C
13
G12V
G12V
G12V
G12V
G12V
G12V
14
G12D
WT
G12D
G12D
G12D
G12D
15
G13D
G13D
G13D
G13D
G13D
G13D
16
G13D
G13D
G13D
G13D
G13D
G13D
17
G13D
G13D
G13D
G13D
G13D
G13D
18
G13D
G13D
G13D
G13D
G13D
G13D
19
G13D
G13D
G13D
G13D
G13D
G13D
20 G13D G13D G13D G13D G13D G13D

*Samples were selected for testing based on these prior clinical results.

**WT: wildtype; G12D: p.Gly12Asp; G12V: p.Gly12Val; G12S: p.Gly12Ser;

G13D: p.Gly13Asp.

This laboratory did see some evidence that the sample had a mutation, but was below the confidence threshold. This specimen showed tumor enrichment of approximately 40%, which is at the lower level of detection for this laboratory.



Article Categories:
  • Short Report

Keywords: KRAS, Colorectal cancer, EGFR, Laboratory error.

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