KRAS mutations testing in colorectal carcinoma patients in Italy: from guidelines to external quality assessment.
ABSTRACT Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients.
In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass.
The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers.
[show abstract] [hide abstract]
ABSTRACT: EGFR regulates cancer-cell proliferation, apoptosis and tumor-induced neoangiogenesis, and has been validated as a relevant therapeutic target in several human cancers, including metastatic colorectal cancer (mCRC). The anti-EGFR monoclonal antibodies cetuximab and panitumumab are available for the treatment of patients with mCRC. Although EGFR is expressed in approximately 85% of patients with mCRC, the clinical efficacy of treatment with anti-EGFR antibodies is limited to a subset of patients. A series of potential biomarkers that could be useful in predicting response to EGFR inhibitors has been investigated. In patients with mCRC, activating mutations within KRAS can predict resistance to anti-EGFR monoclonal antibodies. Activating mutations in KRAS, which could result in EGFR-independent intracellular signal transduction activation, are found in approximately 35-40% of patients with mCRC. These mutations are almost exclusively detected in codons 12 and 13 of exon 2. KRAS mutations have been significantly associated with lack of response to cetuximab or panitumumab therapy in patients with mCRC, which suggests that EGFR-independent, constitutive activation of the RAS signaling pathway could impair response to anti-EGFR drugs. We summarize the experimental and clinical evidence supporting the use of KRAS testing for the optimal selection of patients with mCRC to be treated with anti-EGFR monoclonal antibodies.Nature Reviews Clinical Oncology 08/2009; 6(9):519-27. · 11.96 Impact Factor
Cell 07/1990; 61(5):759-67. · 32.40 Impact Factor
Article: Fluorouracil, leucovorin, and oxaliplatin with and without cetuximab in the first-line treatment of metastatic colorectal cancer.[show abstract] [hide abstract]
ABSTRACT: This randomized study assessed whether the best overall response rate (ORR) of cetuximab combined with oxaliplatin, leucovorin, and fluorouracil (FOLFOX-4) was superior to that of FOLFOX-4 alone as first-line treatment for metastatic colorectal cancer. The influence of KRAS mutation status was investigated. Patients received cetuximab (400 mg/m(2) initial dose followed by 250 mg/m(2)/wk thereafter) plus FOLFOX-4 (oxaliplatin 85 mg/m(2) on day 1, plus leucovorin 200 mg/m(2) and fluorouracil as a 400 mg/m(2) bolus followed by a 600 mg/m(2) infusion during 22 hours on days 1 and 2; n = 169) or FOLFOX-4 alone (n = 168). Treatment was continued until disease progression or unacceptable toxicity. KRAS mutation status was assessed in the subset of patients with assessable tumor samples (n = 233). The confirmed ORR for cetuximab plus FOLFOX-4 was higher than with FOLFOX-4 alone (46% v 36%). A statistically significant increase in the odds for a response with the addition of cetuximab to FOLFOX-4 could not be established (odds ratio = 1.52; P = .064). In patients with KRAS wild-type tumors, the addition of cetuximab to FOLFOX-4 was associated with a clinically significant increased chance of response (ORR = 61% v 37%; odds ratio = 2.54; P = .011) and a lower risk of disease progression (hazard ratio = 0.57; P = .0163) compared with FOLFOX-4 alone. Cetuximab plus FOLFOX-4 was generally well tolerated. KRAS mutational status was shown to be a highly predictive selection criterion in relation to the treatment decision regarding the addition of cetuximab to FOLFOX-4 for previously untreated patients with metastatic colorectal cancer.Journal of Clinical Oncology 01/2009; 27(5):663-71. · 18.37 Impact Factor
KRAS Mutations Testing in Colorectal Carcinoma Patients
in Italy: From Guidelines to External Quality Assessment
Nicola Normanno1,2*, Carmine Pinto3, Francesca Castiglione4, Alberto Bardelli5, Marcello Gambacorta6,
Gerardo Botti7, Oscar Nappi8, Salvatore Siena9, Fortunato Ciardiello10, GianLuigi Taddei4, Antonio
1Cell Biology and Biotherapy Unit, INT Fondazione ‘‘G. Pascale’’, Naples, Italy, 2Pharmacogenomic Laboratory, CROM – Centro Ricerche Oncologiche di Mercogliano,
Avellino, Italy, 3Medical Oncology, S. Orsola-Malpighi Hospital, Bologna, Italy, 4Department of Human Pathology and Oncology, University of Florence, Florence, Italy,
5InstituteforCancerResearchandTreatment,Universita ` diTorino,Torino,Italy,6DivisionofPathology,OspedaleNiguardaCa’Granda,Milan,Italy,7Surgical PathologyUnit,
INT Fondazione ‘‘G. Pascale’’, Naples, Italy, 8Surgical Pathology and Cytopathology, Antonio Cardarelli Hospital, Naples, Italy, 9The Falck Division of Medical Oncology,
Department of Oncology, Ospedale Niguarda Ca’ Granda, Milano, Italy, 10Medical Oncology, Department of Experimental and Clinical Medicine and Surgery F. Magrassi and
A. Lanzara, Second University of Naples, Naples, Italy, 11Center of Predictive Molecular Medicine, Center of Excellence on Aging, University-Foundation, Chieti, Italy
Background: Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for
the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS
testing has become mandatory to chose the most appropriate therapy for these patients.
Methodology/Principal Findings: In order to guarantee the possibility for mCRC patients to receive an high quality KRAS
testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and
Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS
testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that
outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient
population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational
analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an
external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to
the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating
laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to
report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first
round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers
failed again to pass.
Conclusions: The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is
performed with good quality in the majority of Italian centers.
Citation: Normanno N, Pinto C, Castiglione F, Bardelli A, Gambacorta M, et al. (2011) KRAS Mutations Testing in Colorectal Carcinoma Patients in Italy: From
Guidelines to External Quality Assessment. PLoS ONE 6(12): e29146. doi:10.1371/journal.pone.0029146
Editor: Rakesh K. Srivastava, The University of Kansas Medical Center, United States of America
Received September 14, 2011; Accepted November 21, 2011; Published December 21, 2011
Copyright: ? 2011 Normanno et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The external quality assessment was funded by the Italian divisions of Merck and Amgen. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The Italian Association of Medical Oncology and the Italian Society of Pathology received an unrestricted grant from the Italian divisions
of Amgen and Merck that was used to pay the expenses of the program. No fee was given to the participants or the organizers of the program. This does not alter
the authors’ adherence to all the PLoS ONE policies on sharing data and materials. The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
* E-mail: firstname.lastname@example.org
Mutations of the KRAS gene occur in approximately 40% of
colorectal carcinomas (CRC), and about 90% of these mutations
affect codons 12 and 13 . KRAS mutations occur relatively
early in colorectal tumor progression, and therefore they are
usually present in the majority of the transformed cells within a
KRAS mutant tumor . The presence of the mutations in a
restricted and well defined region of the gene and the occurrence
of the mutations in an high percentage of tumor cells facilitates the
detection of KRAS mutations in tumor tissues.
A number of studies have demonstrated that anti-EGFR
monoclonal antibodies are active only in metastatic CRC (mCRC)
patients that do not carry mutations of the KRAS gene. In
particular, analysis of patients treated in phase II and III
randomized clinical trials with anti-EGFR antibodies alone or in
combination with chemotherapy, in any line of treatment, have
shown that anti-EGFR agents increase the response rate and
improve the progression free survival (PFS) only in mCRC patients
that do not carry KRAS mutations at codons 12 and 13
[3,4,5,6,7,8,9]. More recently, addition of cetuximab to FOLFIRI
in patients with KRAS wild-type disease was also found to result in
PLoS ONE | www.plosone.org1 December 2011 | Volume 6 | Issue 12 | e29146
significant improvements in overall survival (OS) . Following
these results, the European Medical Agency (EMEA) approved in
2009 the use of the anti-EGFR antibodies cetuximab and
panitumumab only in patients with mCRC carrying a wild type
KRAS gene. As a matter of fact, this was the first approval of a drug
for a solid tumor based on a genetic test.
Following the approval of anti-EGFR antibodies for KRAS wild
type CRC patients, KRAS testing has become mandatory to
choose the most appropriate therapeutic strategy in mCRC. In
this respect, both false-negative and false-positive results are
potentially harmful for patients. In fact, false positive findings will
deprive the patients of the possibility to benefit of an active
treatment. On the other hand, false-negative patients might be
treated with a drug that is not active. In addition, recent findings
suggest that administration of an anti-EGFR monoclonal antibody
in combination with a regimen containing oxaliplatin to patients
with a KRAS mutant tumor might significantly reduce progression
free survival [7,9].
The introduction of a mutational assay in clinical practice has
raised the issue to ensure a rapid and high quality KRAS testing to
all patients. Recommendations for KRAS testing in mCRC
patients were released by the European Society of Pathology (ESP)
in 2008 . A recent survey in 14 countries in Europe, Latin
America and Asia showed that the frequency of KRAS testing in
patients with mCRC increased from 3% in 2008 to 47% in 2009
and 69% in 2010 . In particular, the 2010 survey revealed that
test results were available within 15 days for 82%, 51% and 98%
of the tested patients in the European, Latin American and Asian
In Italy few surgical pathology laboratories were equipped to
run molecular diagnostics at the time KRAS testing became
mandatory for the prescription of anti-EGFR antibodies in mCRC
patients. In order to guarantee the possibility for mCRC patients
to receive an high quality KRAS testing in every Italian region, the
Italian Association of Medical Oncology (AIOM) and the Italian
Society of Pathology and Cytopathology -Italian division of the
International Academy of Pathology (SIAPEC-IAP) started a
program to improve KRAS testing. This program was based on
the development of guidelines for KRAS mutational analysis in
mCRC patients. Following the publication of the guidelines, the
scientific societies started an external quality assessment scheme
for KRAS testing, in order to evaluate the effects of guidelines on
molecular diagnostic for KRAS mutations in Italy. This paper
describes the development of this program that started in 2008 and
was completed in 2010.
Methodology for guidelines
AIOM and SIAPEC-IAP identified a large panel of Italian
medical oncologists, pathologists and molecular biologists that met
for the first time in September 2008. Following the meeting,
guidelines for KRAS testing were written by a restricted steering
committee and submitted to the panel of experts for their
comments. By the end of January 2009, the document was
integrated with all the comments. The guidelines were published
on the websites of both AIOM and SIAPEC-IAP in February
2009 (www.aiom.it, 2009; www.siapec.it, 2009). A revised version
of the guidelines was prepared in 2010 and it was published in
November 2010 (www.aiom.it, 2010; www.siapec.it, 2010).
External quality assessment scheme
AIOM and SIAPEC-IAP identified a board of experts who were
assigned to organize the external quality assessment scheme and that
are the co-authors of this paper. Within the group three referral
Oncology, University of Florence; Department of Pathology, Univer-
Granda, Milan) and three referral laboratories (Department of
Pathology, University-Foundation, Chieti; Institute for Cancer Re-
search and Treatment, University of Turin, Tuino; Pharmacogenomic
Laboratory, CROM, Avellino, Italy) were identified.
Formalin-fixed paraffin embedded (FFPE) colon carcinoma
specimens were collected at the referral surgical pathology
departments. For each specimen, 5m-thick slides were sent to the
three referral laboratories for mutational analysis in a blind
fashion. The referral laboratories analyzed the samples by using
two different methods: direct sequencing of the PCR product by
using in-house validated methods, and Real Time PCR with the
Therascreen KRAS kit (DxS, Manchester, UK) according to
Slides for each center participating to the quality assessment
were obtained from the selected samples. The scheme included
two rounds: the laboratories that failed the first round had the
chance to register for a second round.
The Italian guidelines for the mutational analysis of the KRAS
gene in CRC prepared by AIOM and SIAPEC-IAP were the
result of an open discussion that involved the Italian scientific
CRC community. The guidelines are available at the websites of
AIOM and SIAPEC-IAP (www.aiom.it; www.siapec.it) and the
main points are summarized in the following paragraphs.
Target patient population
Mutational screening of the KRAS gene should be performed in
patients with mCRC for which treatment with anti-EGFR
monoclonal antibodies might be indicated. Mutational analysis
can be performed by using tissue from either the primary tumor or
a metastatic site, since high concordance has been observed
between primary tumors and metastases for KRAS mutations in
the majority of the studies published up to now [13,14,15].
Biological material for molecular analysis
Either frozen or FFPE tissues can be used for mutational
analysis. Usually, testing for clinical purpose is performed with
FFPE tissues. The primary pathologist plays a fundamental role in
KRAS testing, since he has the responsibility to choose the most
appropriate specimen for mutational analysis (Table 1). In
particular, tissues for KRAS genotyping should contain an
adequate percentage of tumor cells to avoid false negative results.
However, this limit depends on the method that is used for
mutational analysis . International guidelines suggest that the
specimen should contain at least 70% of tumor cells if a low
sensitivity technique such as direct sequencing of the PCR product
is used . Based on the experience of our group in the last two
years, we recently set this limit to 50%. In any case, the specimen
should contain at least 100 tumor cells.
If the tumor specimen has a lower percentage of tumor cells, the
pathologist is requested to manually dissect the tissue in order to
isolate the region that contains the highest percentage of tumor
cells. Laser microdissection can also be used. However, this
approach is not feasible as a routine clinical procedure.
DNA can be extracted from FFPE tissues by using different
methods. Kits for DNA extraction from different sources including
Italian Quality Assessment for KRAS Testing
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FFPE tissues are commercially available, and are usually based on
the use of chromatographic columns. The use of kits has several
advantages, including the shorter time necessary for the extraction
and an easier standardization of the procedure. The quality and
the quantity of the extracted DNA should be assessed by
spectrophotometric analysis and/or agarose gel electrophoresis.
Different methods can be used to assess the mutational status of
KRAS in CRC patients (Table 2). These methods should be able
to detect the seven most common mutations of the KRAS gene in
codons 12 and 13: G35A (G12D), G35T (G12V), G34T (G12C),
G34A (G12S), G35C (G12A), G34C (G12R), and G38A (G13D)
. In fact, only these KRAS mutations were investigated in the
clinical trials that led to the registration of anti-EGFR monoclonal
antibodies in metastatic CRC patients with wild type KRAS.
Direct sequencing of the PCR product represents the golden
standard for mutational analysis. PCR primers should be designed
to amplify codons 12 and 13 of the KRAS gene. PCR reactions
should be prepared in a laminar flow hood by using gloves and
filter tips, and areas for pre- and post-PCR analysis should be kept
separated (Table 3).
An adequate amount of genomic DNA (80–100 ng) should be
amplified, in order to avoid artifacts that have been described to
occur when a low quantity of input DNA in the PCR reaction is
used. Positive and negative controls are required for each PCR
amplification. We also suggest to use for sequencing at least 40–
50 ng of PCR product. The products of two different PCR
reactions should be sequenced in forward and reverse in order to
obtain 3–4 sequences for each sample. A mutation can be called
when present in at least two different sequences (forward and
reverse) obtained from independent PCR amplifications.
One of the major limit of PCR/sequencing is the relatively low
sensitivity (Table 2). More sensitive techniques are available to
detect KRAS mutations. The Therascreen kit is a Real Time
PCR-based assay the uses ARMS primers and Scorpion probes to
detect the above listed seven most common mutations of KRAS in
codons 12 and 13. This method is highly sensitive being able to
detect KRAS mutations when they represent as low as 1% of the
Pyrosequencing and, more recently, methods based on
differential amplification of mutant DNA and hybridization with
probes immobilized on a membrane are quite sensitive assays to
detect KRAS mutations. However, comparative studies in large
cohorts of patients with these techniques are not available yet.
The general recommendations for the preparation of PCR
reactions for these latter methods are similar to PCR/sequencing.
The quantity of DNA to be amplified, the conditions of
amplification and the interpretation of the results are described
in details by the manufacturers.
External Quality Assessment scheme
Following the release of guidelines, AIOM and SIAPEC-IAP
decided to start an external quality assessment scheme for KRAS
testing in CRC that was mainly focused on genotyping. Italian
laboratories that perform mutational analysis of KRAS were
invited to participate to the quality assessment program. Sixty
centers registered to the program at a dedicated website (www.
krasquality.it). One of the centers declined, 59 centers participated
to the program that started on March 15, 2009.
Three referral surgical pathologies selected 16 primary FFPE
CRC with adequate content of tumor cells (.70%) (Table 4). The
mutational status of the 16 samples was assessed in the three
referral laboratories by using different techniques (Table 4). A
good agreement on KRAS mutational status of the selected
specimens was found among the three laboratories. A discordant
result was found only for sample N. 6 in which PCR/sequencing
detected only the G35C mutation, whereas Therascreen identified
also a G34A mutant clone (Table 4). The DCt for the G34A
mutation was significantly higher as compared with the G35C,
and this might explain the lack of detection with PCR/sequencing
that has a lower sensitivity as compared with Therascreen
(Table 2). A meeting was held in February 2009 to select 10
samples, 5 for the first round and 5 for the second round. Samples
for which a total concordance on the mutational status was found
between the three referral laboratories, and from which a good
yield of genomic DNA was obtained, were selected for the quality
assessment scheme (Table 4). For each round 3 mutant and 2 wild
type cases were chosen.
Five 5m-thick slides for each sample were sent to the laboratories
participating to the quality control. A random code, different for
Table 1. Characteristics of the specimens for KRAS
Tissues from the primary tumor or a metastatic site can be used
Testing for clinical purpose is usually performed with FFPE tissues
The specimen should contain at least 50% of tumor cells if a low sensitivity
technique is used
The region with the highest percentage of tumor cells can be isolated with
Laser microdissection should be limited to selected cases
Table 2. Sensitivity of the main methods used for KRAS
genotyping in Italy.
PCR with Stop primers and reverse dot blot 1–5
ARMS/scorpion probes (Therascreen)1
*lower level of mutant DNA that can be detected, expressed as % of total DNA.
Table 3. Recommendations for PCR/sequencing analysis.
PCR primers should amplify codons 12 and 13 of the KRAS gene
PCR reactions should be prepared in a laminar flow hood by using gloves and
Areas for pre- and post-PCR analysis must be kept separated
80–100 ng of genomic DNA should be amplified
Include positive and negative controls for each PCR amplification
The products of two different PCR reactions should be sequenced in forward
A mutation can be called when present in two different sequences (forward and
Italian Quality Assessment for KRAS Testing
PLoS ONE | www.plosone.org3 December 2011 | Volume 6 | Issue 12 | e29146
each center, was automatically assigned to the samples by an
application of the website, in order to avoid exchange of
information between the laboratories. The laboratories were given
4 weeks to complete the analyses and to submit the results through
the KRASquality website and by fax. The laboratories were asked
to submit only the results of genotyping.
All the centers submitted the results within the established
deadline. Of the 59 centers, 48 (81.3%) performed the mutational
analysis by using PCR sequencing, 5 (8.5%) with pyrosequencing,
3 (5.1%) with Real Time PCR (Therascreen kit), 2 (3.4%) with
RFLP analysis and 1 (1.7%) with the KRAS strip assay (Figure 1).
The limit to pass the quality assessment was set at 100% of true
responses. In the first round, only two centers failed to pass (3%).
Both centers did not manage to extract enough genomic DNA for
the mutational analyses. The samples from which the laboratories
failed to extract DNA were different, suggesting that this
phenomenon was not related to the quality of the specimens that
they received. According to the guidelines of the scheme, the two
centers were offered to participate to a second round. Again, both
centers failed to pass the second round due to inability to extract
genomic DNA from FFPE tissues.
The list of the centers that passed the external quality
assessment scheme was published on the websites of AIOM and
SIAPEC (www.aiom.it, 2009; www.siapec.it, 2009).
The approval of anti-EGFR agents for the treatment of mCRC
patients that do notcarry KRAS mutationsrepresented a significant
innovation for medical oncology. In fact, this was the first approval
of an anti-tumor agent for a solid tumor based on a mutational
analysis.More recently,theEGFR tyrosinekinaseinhibitorgefitinib
has been approved for treatment of non-small-cell lung cancer
(NSCLC)patientsthatcarrymutationsofthe EGFR gene.Itislikely
that in the next future other drugs will require the assessment of the
mutational statusof predictivebiomarkers, represented eitherbythe
target of the drug or by signaling proteins that can affect the efficacy
of the anti-tumor agent.
Different methods can be used to detect KRAS mutations in
CRC specimens, and each of them has advantages and limits .
The approval of anti-EGFR agents for CRC without mutations of
KRAS has not been linked by the regulatory agencies to a specific
mutational assay. In Italy approximately 18.000 new cases of
mCRC per year are expected. The Italian health system is
organized on a regional basis and, with few exceptions, the
majority of Italian regions did not set a limit in the number of
laboratories that can run mutational analysis. Therefore, several
different centers are offering KRAS testing in the different areas of
the country, and a wide array of methods are employed for such
analysis. For these reasons, external quality assessment is
mandatory to assure that mutational testing is performed with
high quality in every Italian center that provides this service.
Table 4. Mutational status of the samples used for the quality assessment.
Therascreen Sample code*
1 G35A (G12D)G35A (G12D)G35A (G12D) A1
2 G38A (G13D) G38A (G13D)G38A (G13D)A2
3 Wild type Wild typeWild type-
4 G35T (G12V) G35T (G12V)G35T (G12V)A3
5 Wild type Wild typeWild typeA4
6 G35C (G12A) G35C (G12A)G35C (G12A)-
7 Wild typeWild type Wild type-
8 Wild typeWild typeWild type A5
9 G35A (G12D)G35A (G12D) G35A (G12D)B1
10G34A (G12S) G34A (G12S)G34A (G12S) B2
11G35A (G12D)G35A (G12D) G35A (G12D)B3
12 Wild typeWild type Wild type-
13 Wild typeWild typeWild type B4
14Wild type Wild typeWild type-
15 Wild typeWild type Wild typeB5
16 G35A (G12D)G35A (G12D)G35A (G12D)-
*=samples chosen for the external quality assessment program: A1–A5: samples for the first round; B1–B5: samples for the second round.
Figure 1. Methods used for KRAS genotyping by the centers
participating to the Italian Quality assessment scheme.
Italian Quality Assessment for KRAS Testing
PLoS ONE | www.plosone.org4 December 2011 | Volume 6 | Issue 12 | e29146
The first external quality assessment scheme for KARS
mutation testing was exclusively focused on genotyping. In fact,
the aim of this first scheme was to assess the rate of false positive
and negative results. The results of the scheme were exciting. The
first success of the program was that the majority of the Italian
centers that perform KRAS mutational analysis accepted to
participate to the external quality assessment. This underlines the
need of the testing laboratories to confirm the quality of their
analysis through an external, independent and recognized system
of evaluation. In addition, the rate of laboratories that passed was
exceptionally high, since only 2 centers out of 59 failed. Although
the number of samples used was limited (n.5), the threshold to pass
the scheme was extremely high, since only the centers that
assigned the correct genotype to all the samples passed.
Nevertheless, a larger number of samples will be used in the next
external quality assessment that is planned for 2012. The two
laboratories that did not succeed did not manage to extract
enough genomic DNA from FFPE tissues. Interestingly, both
laboratories were not surgical pathologies, but genetics units that
were use to run tests on blood or fresh tissues. Therefore, we
hypothesize that they might have used procedures or kits for
genomic DNA extraction that are not specific for FFPE specimens.
The list of the centers that passed the external quality
assessment was published on the websites of AIOM and
SIAPEC-IAP. The publication of this list provides to both patients
and physicians the possibility to choose among a wide number of
certified laboratories that are localized in different regions and that
are able to provide KRAS mutational testing with adequate
This first Italian KRAS quality assessment did not reveal
difference in the ability to detect mutations between the techniques
used by the different laboratories. However, it must be emphasized
that CRC specimens with an high content of tumor cells (.70%)
were selected. Different reports suggest that direct sequencing of
PCR products should not be used when the specimens contains
30% or lower tumor cells [16,17]. Although the majority of CRC
specimens contains high numbers of tumor cells, a low tumor cell
content might occur in mucinous tumors, or following adjuvant
radio-chemotherapy for rectal tumors or in small biopsies.
The results of the Italian quality assessment scheme were
superior as compared with the German program . This latter
external quality assessment program run 7 ring trials between
2008 and 2011, with overall 319 participants. Of these, 90.9%
passed, with a failure rate of 9.1%. However, significant
differences between these programs exist. In the German quality
assessment, 10 samples were sent to each center and the response
was due in 10 working days. In addition, 2 points were assigned for
each sample (1 for the correct genotype and 1 for the specific
mutation) and 1 point was deducted for a maximum of two times
in case of technical failure (failure to extract or to amplify DNA).
The threshold to pass the test was set at 17 points corresponding to
85% of the total score. In contrast, the threshold to pass the Italian
quality control was 100% and technical failure were scored
negatively at the same extent of false-negative or false-positive
More recently, the results of a joined regional assessment round
for KRAS testing in Europe have been reported . The
assessment round included 59 laboratories from eight different
European countries. For each country, one regional scheme
organizer prepared and distributed the samples (n.10) for the
participants of their own country. The samples were centrally
validated by one of two reference laboratories. The results of this
assessment was that only 70% of laboratories correctly identified
the KRAS mutational status in all samples. Genotyping errors
were made by 22% of the laboratories, whereas 8% reported
technical failure. The majority of the genotyping errors were false
positive or false negative results. Mistakes were made using both
commercial kits and in-house validated methods.
Although direct comparison between these different quality
assessment schemes cannot be drawn, we hypothesize that the
educational program of AIOM and SIAPEC with the publication
of guidelines followed by their presentation in a number of
national meetings might have improved mutational testing in Italy.
The Italian external quality assessment scheme was only related to
genotyping, whereas the German and the European programs
included reporting. However, in these latter schemes only
genotyping was scored and, therefore, the outcomes are similar
to the Italian program.
In conclusion, the results of this first Italian quality assessment
for KRAS testing suggest that KRAS mutational analysis is
performed with good quality in the majority of Italian centers.
However, this conclusion is limited by the low number of samples
employed that will be increased in the next external quality
assessment, which will also include samples with low percentage of
tumor cells. The Italian KRAS quality assessment scheme might
represent a model for other national and international societies.
The following individuals participated to the panel of experts that outlined
the guidelines for KRAS testing: C. Barone (Roma), S. Cascinu (Ancona),
F. Di Costanzo (Firenze), A.P. Dei Tos (Treviso), A. Falcone (Livorno), G.
Gasparini (Roma), S. Iacobelli (Chieti), R. Labianca (Bergamo), E. Maiello
(S.Giovanni Rotondo), A. Russo (Palermo), and G. Viale (Milano).
Conceived and designed the experiments: NN CP FC AB MG GB ON SS
FC GT AM. Performed the experiments: NN CP FC AB MG GT AM.
Analyzed the data: NN CP FC GT AM. Contributed reagents/materials/
analysis tools: NN CP FC AB MG GB GT AM. Wrote the paper: NN CP
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Italian Quality Assessment for KRAS Testing
PLoS ONE | www.plosone.org5 December 2011 | Volume 6 | Issue 12 | e29146