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Rev Invest Clin. 2020;72(6):337-43 BRIEF COMMUNICATIONS

Complete Screening of Exons 2, 3,

and 4 of KRAS and NRAS Genes Reveals
a Higher Number of Clinically
Relevant Mutations than Food
and Drug Administration Quantitative
Polymerase Chain Reaction-Based
Commercial Kits
Héctor E. Sánchez-Ibarra, Luisa M. Reyes-Cortes, Esteban López-Tavera, Claudia M. Luna-Aguirre,
and Hugo A. Barrera-Saldaña*
Genetics Laboratory, Vitagénesis SA de CV., Monterrey, NL, Mexico

ABSTRACT

Background: The presence of clinically relevant mutations in KRAS and NRAS genes determines the response of anti-epidermal
growth factor receptor antibody therapy for metastatic colorectal cancer (mCRC). The only quantitative polymerase chain
reaction (qPCR)-based diagnostic tests approved by the Food and Drug Administration (FDA) screen merely for mutations in
codons 12 and 13 of KRAS. Objective: The objective of the study was to study the frequency of clinically relevant mutations
in KRAS and NRAS genes that are not included in FDA-approved qPCR tests. Methods: Formalin-fixed paraffin-embedded tumor
specimens from 1113 mCRC Mexican patients from different health institutions across the country were analyzed by Sanger
sequencing for KRAS mutations in exons 2, 3, and 4. Furthermore, 83 were analyzed in exons 2, 3, and 4 of NRAS. Results:
From the specimens tested for KRAS, 33.69% harbored a mutation. From these, 71.77% were in codon 12 and 27.69% in codon
13 (both located in exon 2). Codons 59 (exon 3) and 146 (exon 4) accounted for the remaining 0.54%. From the 83 specimens,
in which NRAS was analyzed, three mutations were found in codon 12 (3.61%). Approximately 6% of RAS mutated specimens
would have been falsely reported as RAS wild type if an FDA-approved qPCR diagnostic test had been used. Conclusions: While
these kits based on qPCR can be very practical and highly sensitive, their mutation coverage ignores mutations from poorly
genetically characterized populations. (REV INVEST CLIN. 2020;72(6):337-43)

Key words: Colorectal cancer. Molecular diagnostics. RAS genes. Real-time polymerase chain reaction. Sanger sequencing.

*Corresponding author: Received for publication: 19-03-2020

Hugo A. Barrera-Saldaña Approved for publication: 18-05-2020
E-mail: habarrera@[Link] DOI: 10.24875/RIC.20000111
0034-8376 / © 2020 Revista de Investigación Clínica. Published by Permanyer. This is an open access article under the
CC BY-NC-ND license ([Link]

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INTRODUCTION level, the above-mentioned KRAS and NRAS muta-

tions result in reduced intrinsic GTPase activity, which,
Colorectal cancer (CRC) is the third most common in turn, leads to permanent activation of KRAS or
neoplasia in men and the second in women (10.9% NRAS itself and of its downstream signaling path-
and 9.5% of all cancers, respectively) worldwide. In ways, thereby mediating malignant transformation.
North America, CRC is the fourth most common can- KRAS gene is in the short arm of chromosome 12, in
cer with an estimated incidence in 2018 of 49.9 for p12.1, and is composed of 46,148 nucleotides di-
every 100,000 individuals1. Precursor lesions of the vided into six exons. In contrast, the NRAS gene is in
colonic mucosa known as adenomatous polyps (ad- 1p13.2 of chromosome 1 and consists of seven exons
enomas) may develop into colorectal tumors; how- spread over 12,431 nucleotides. Of the three human
ever, < 10% of colorectal adenomas develop into in- RAS isoforms, KRAS (Kirsten ras or Ki-ras) is the most
vasive cancers. The progression from adenoma to frequently mutated oncogene in mCRC11,12, from
adenocarcinoma has been associated with many dif- which codons 12 and 13 of the second exon and 61
ferent molecular mechanisms, including epigenetic of the third exon have been found to harbor muta-
events, DNA mismatch repair defects, chromosomal tions in roughly 40-50% of tumors, with approxi-
rearrangements, and mutations in key oncogenes and mately 85-90% of mutations occurring in codons 12
tumor suppressor genes2,3. and 134. Mutations involving codon 61 in the third
exon and codon 146 in the fourth exon of the KRAS
Colon cancer is one of the main diseases treated by gene have also been described, with frequencies rang-
targeted therapy as there is extensive information ing from 1 to 6.6%13. An approximately 10% of the
about the genes affected by cancer-causing muta- remaining KRAS negative tumors have been found
tions, their normal functions, and their carcinogenic mutated, mostly in NRAS codon 6114.
effects when mutated2,4. Epidermal growth factor re-
ceptor (EGFR) triggers a signaling pathway related to Around the world, several research groups are study-
the proliferation of tumor cells. The EGFR signaling ing and publishing data on the relation of RAS genes
cascade promotes cell proliferation by activation of with mCRC15, revealing that mutations in these genes
Ras-GTPase and Erk/Map kinase. Because of this, might be associated with an increase of tumor ag-
medical treatment consists of blocking EGFR with gressiveness and poor survival (prognostic value), as
monoclonal antibodies (mAbs), such as cetuximab well as with resistance to gene-targeted therapies
and panitumumab5,6. However, mutational screening (predictive value)16.
is necessary to identify resistant patients and is man-
datory as a companion diagnostic test7. Even though A number of methods to detect KRAS mutations are
EFGR is commonly overexpressed in all human can- available, including real-time or quantitative poly-
cers, its abnormality fails to predict the success of merase chain reaction (qPCR) with or without melting
anti-EGFR therapy. On the other hand, the RAS gene curve analysis, pyrosequencing, and Sanger sequenc-
family has become widely accepted as a biomarker ing (SS), the latter being the “gold standard” in clinical
for the response to treatment of several types of mutation testing17. Sanger sequencing can detect es-
cancer, such as metastatic CRC (mCRC). Correspond- sentially all base substitutions, small insertions, and
ingly, a patient with mCRC not harboring a KRAS or deletions; however, its modest limit of detection (≥
NRAS mutation could benefit from the treatment 10% mutated DNA in a wild-type background) could
with anti-EGFR mAbs8,9 and vice versa. be highly variable depending on the DNA extraction
method and the laboratory performing the test18.
RAS is one of the most studied and best characterized
mCRC-associated gene families. Oncogenic mutations The only qPCR-based companion diagnostic tests ap-
of three of its members, H, K, and NRAS, were among proved by the U.S. Food and Drug Administration
the first genetic changes identified in human tumors. (FDA) are the Cobas® KRAS Mutation Test from Roche
These three members encode membrane-associated (Pleasanton, CA, USA) and the therascreen KRAS RGQ
guanine nucleotide-binding proteins (p21ras) and are PCR Kit from QIAGEN (Manchester, UK), which screen
closely related, having 85% amino acid sequence only for mutations in codon 12 and 13 of KRAS. This
identity and very similar functions10. At the molecular study aims to state the frequency of mutations that

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 H.E. Sánchez-Ibarra, et al.: RAS screening by Sanger seq versus qPCR

are not included in FDA-approved qPCR-based com- DNA extraction and mutation analysis
mercial companion diagnostic devices.
DNA extraction from scrapped tumor areas per-
formed with the QIAamp DNA FFPE Tissue Kit (QIA-
GEN, Mexico City, Mexico) was used as a template to
METHODS
amplify the exons of interest of RAS genes by PCR
followed by SS using an automated capillary electro-
Tumor samples phoresis sequencer from Applied Biosystems (Mexico
City, Mexico) as described in our previous study9.
Tumor samples consisted of formalin-fixed paraffin-
Primer pairs for amplification of exons 2, 3, and 4 of
embedded (FFPE) tissue of primary tumor lesions from
KRAS and NRAS genes are described in table S1.
1030 consecutive mCRC patients received in our labo-
ratory for routine KRAS gene mutation screening from
Commercial kits
April 2010 to December 2013. This study also in-
cluded 83 consecutive mCRC patient tumor speci-
Conceptual analysis of the sequencing revealed muta-
mens received from 2013 to 2016 that were rou-
tions was performed based on the information in the
tinely analyzed for both, KRAS and NRAS genes. The
fact sheets from Cobas KRAS Mutation Test and the
patients, 646 men and 447 women, were residents
therascreen KRAS RGQ PCR Kit describing the muta-
from Mexico and had Stage IV carcinoma. Patients
tions that can be detected by them, as well as the
signed informed consent before the performance of
reported limits of detection.
tests where it is agreed that their results may be used
for research purposes. The study was approved by the
Public and patient involvement
Ethics and Research Committee from Hospital La Mis-
statement
ión private hospital (17CI19039096). The specimens
came from High Specialty Regional Hospitals of Bajío
Patients signed informed consent before the perfor-
(HRAEB), Oaxaca (HRAEO), Yucatan’s peninsula
mance of tests where it is agreed that their results
(HRAEPY), Chiapas (HRAEC), Ciudad Victoria
may be used for research purposes. This study was
(HRAECV), and Ixtapaluca (HRAEI), as well as from
carried out as a result of the finding of uncommon
Doctor’s Hospital, Opción Oncología, ONCARE, OCA
mutations in routine tests. This is a retrospective
Hospital, Hospital San José, Hospital Zambrano Hel-
study; patients were not specifically recruited to per-
lion, and Hospital La Misión, Monterrey, NL, Mexico.
form this analysis. The information obtained will not
Information on previous or current treatments re-
be notified to patients unless it represents a risk to
ceived was not available.
their health.

Biological samples
RESULTS
In most cases, the FFPE blocks were accompanied with
their respective slide stained with hematoxylin and eo- In our laboratory, of the 1113 samples tested for
sin; the tumor area was bounded by the pathologist of KRAS, 375 (~33.69%) harbored a mutation. From
the institution where the sample came from. For FFPE these, 267 were in codon 12 (~72%) and 103 in co-
blocks that had < 20% of tumoral tissue, the tumoral don 13 (~28%) (exon 2). Codons 59 and 146 (exon
area was microdissected until reaching an area of ap- 3 and 4, respectively) accounted for the remaining
proximately 20 mm² of tumoral tissue since the limit ~0.1%, with incidences of one case for each codon. In
of detection of SS mutation screening is equal or great- KRAS, we found c.36T > A and c.39C > T mutations
er than 10% of mutated cells in a background of wild- in codons 12 and 13, respectively, which are synony-
type cells. When only blocks were received, one section mous mutations not available for screening by the
was obtained and stained with hematoxylin and eosin; FDA-approved qPCR kits.
a certified pathologist was on hand to identify the
tumor region of interest. Additional histological infor- These devices can detect all non-synonymous muta-
mation about the tumors was not available. tions in codon 12 (Table 1). However, they cannot

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Table 1. Comparison of Sanger sequencing versus commercial kits for detecting RAS mutations

Mutationa COSMIC ID Type of Sanger sequencing Commercial kits

mutationb mutation coveragee

Incidencec %d % p/codon 1 2

KRAS Codon 12
(n = 1113)

c.35G > A p.(G12D) 55497369 NS 129 11.59 48.31 ✓ ✓

c.35G > T p.(G12V) 55497419 NS 66 5.93 24.72 ✓ ✓
c.34G > T p.(G12C) 55497469 NS 28 2.52 10.49 ✓ ✓
c.35G > C p.(G12A) 55497479 NS 25 2.25 9.36 ✓ ✓
c.34G > A p.(G12S) 55497461 NS 12 1.08 4.49 ✓ ✓
c.34G > C p.(G12R) 55497582 NS 5 0.45 1.87 ✓ ✓
c.36T > A p.(G12=) 55510792 S 2 0.18 0.75
267 23.99 100.00

KRAS Codon 13
(n = 1113)

c.38G > A p.(G13D) 55497388 NS 80 7.19 77.67 ✓ ✓

c.39C > T p.(G13=) 55756103 S 8 0.72 7.77
c.37G > T p.(G13C) 55497378 NS 9 0.81 8.74
c.37G > A p.(G13S) 55509530 NS 4 0.36 3.88
c.37G > C p.(G13R) 55502117 NS 1 0.09 0.97
c.38G > C p.(G13A) 55497357 NS 1 0.09 0.97
103 9.25 100.00

KRAS Codon 61
(n = 1113)

c.183A > C p.(Q61H) 55498802 NS 1 0.09 100.00

1 0.09 100.00

KRAS Codon 146

(n = 1113)

c.436G > A p.(A146T) 55501778 NS 1 1.20 100.00

1 0.09 100.00

NRAS Codon 12
(n = 83)

c.34G > T p.(G12C) 54736487 NS 1 1.20 33.33

c.35G > A p.(G12D) 54736383 NS 1 1.20 33.33
c.35G > C p.(G12A) 54736555 NS 1 1.20 33.33
3 3.61 100.00
Total 375 33.69 94.0% 94.0%
aMutations found by Sanger sequencing.
bSynonymous (S) or non-synonymous (NS).
cTotal number of patients harboring each mutation.
dPercentage of each mutation over total samples analyzed by SS.
eCommercial kits numbered as follows: 1, Cobas® KRAS Mutation Test; 2, TheraScreen®: KRAS Mutation Kit. Percentages on bottom represent

the proportion of mutations covered by each kit, calculated as the sum of the incidence of all covered mutations divided by the total of mutations
in codons 12 and 13.

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detect the mutations c.37G > A (p.G13S) and c.38G hematopoietic and lymphoid tissue, pancreas, endo-
> C (p.G13A), which account for 0.45% of false neg- metrium, and biliary tract carcinomas23.
atives according to our results. In addition, we found
unique cases of the mutations c.176C > T (p.A59V) Mutations c.37G > A (p.G13S) and c.38G > C (p.G13A),
and c.183A > C (p.Q61H) in exon 3 and c.436G > A despite being in codon 13, are not covered by FDA-
(p.A146T) in exon 4. approved qPCR-based kits but were found in our study.
These are known predictive biomarkers to determine
Of the 83 tumors analyzed for NRAS, the mutations anti-EGFR therapies. The first has been found distrib-
c.34G > T, c.35G > A, and c.35G > C in codon 12 uted in the large intestine, thyroid, stomach, pancreas,
(~4%) were found. None of the kits included in this and lung carcinomas, while the latter, in the large in-
study include mutation screening in the NRAS gene; testine, lung, ovary, pancreas, and thyroid carcinomas
the commercial kits included would have reported 6% (COSMIC mutation IDs COSV55509530 and
of false negatives. COSV55497357, respectively).

There were no significant differences in the frequency In exon 3 of KRAS, we found the c.176C > T (p.A59V)
of mutations by age or sex. mutation that has not been reported in mCRC, but
in the breast (accession RCV000119371.1 in NCBI
ClinVar database) and thyroid cancers (COSMIC mu-
tation ID COSV55904854). In contrast, the c.183A
DISCUSSION > C (pQ61H) mutation (also in exon 3 of KRAS) has
been found distributed in the large intestine, hema-
Our findings agree with the previous studies, in which topoietic and lymphoid tissue, lung, and biliary tract
35-40% incidence has been reported for KRAS muta- cancer lesions (COSMIC mutation ID COSV55498802).
tions4,8,14,19. Nonetheless, recent studies have un- Both mutations are predicted to be pathogenic by
veiled the importance and potential risk of synony- FATHMM. We also found the c.436G > A (p.A146T)
mous mutations in disease causation, suggesting that mutation in exon 4 of KRAS, which is predicted as
these mutations could affect the predictive value of pathogenic by FATHMM and has been reported to
different malignancies20,21. Physiological effects of be present in the large intestine, hematopoietic and
synonymous mutations could be related to cancer lymphoid tissue, stomach, biliary tract, and pan-
through diverse mechanisms, such as altering splicing, creas carcinomas. Further research should explore
RNA structure and stability, the translational rate, and the effect of these mutations on mCRC proliferation
other unknown mechanisms21. Moreover, according to and treatment, and whether they imply a clinical risk
a study based on a data set of 292,405 missense or interfere with anti-EGFR antibody treatments.
mutations and 123,193 synonymous mutations iden-
tified in the exomes of 3851 cancer samples, synony- The mutations found in codon 12 of NRAS in this
mous mutations tend to be enriched in oncogenes study have been well documented to predict resis-
(but not in tumor suppressor genes) in a cancer type- tance to anti-EGFR therapy24.
specific manner and often affect splicing motifs22.
This suggests a greater relevance for synonymous It is worth mentioning that all specimens in this study
mutations in cancer than previously thought, hence corresponded to Stage IV carcinomas. The previous
highlighting the importance of detecting and reporting studies have shown that the frequency of mutations
these “silent” mutations. The KRAS c.36T > A (p.G12=) varies according to the stages25, and for a wider anal-
and c.39C > T (p.G13=) synonymous mutations found ysis, further studies should include samples from all
in this study are predicted to be pathogenic by Func- stages.
tional Analysis through Hidden Markov Models
(FATHMM) (COSMIC mutation IDs COSV55573999 Despite 6% of false negatives that would be reported
and COSV55510792, respectively). The first one has if FDA-approved companion diagnostic tests were
been reported as a somatic mutation in the biliary used, SS has important constraints when compared
tract, large intestine, and pancreas carcinomas, while to these kits: the processing time is much longer in
the latter has been found in the large intestine, SS due to the additional steps required (amplification,

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REV INVEST CLIN. 2020;72(6):337-43

cycle sequencing, purification of products, and capil- It should be noted that NRAS should also be included
lary electrophoresis) and the time needed to analyze in every companion diagnostic for mCRC, at least in
results. In addition, some problems can arise in the the Mexican population, as we found that ~4% of the
process due to degradation of DNA and the presence 83 specimens analyzed for this gene contained a mu-
of inhibitors as a result of formalin fixation and de- tation. Given the non-neglectable frequency of KRAS
waxing, respectively26. Furthermore, the limit of de- mutations in mCRC revealed here, to understand their
tection of SS is considerably lower than that of kits: contribution to tumor proliferation, further studies
a mutation can be detected by SS in a specimen with should explore their possible association with the out-
at least 20% mutant DNA in a background of wild- come of anti-EGFR therapies.
type DNA, which means ~20-40% heterozygous mu-
tant cells27. On the other hand, the commercial kits
here discussed have reported sensitivities of <5% of ACKNOWLEDGMENTS
mutated DNA in a wild-type background. Despite its
modest limit of detection and relative complexity, SS We thank all the current and previous Vitagénesis
is currently one of the most reliable techniques for personnel that made it possible to obtain and process
mutation discovery and validation, especially helpful all the specimens included in this study. We also thank
in molecular genetic studies of genetically poorly all patients’ advisers for participating in this study.
characterized populations18. Studies have reported an This research was supported in part by the National
overall concordance of 97.5% between SS and qPCR Council on Science and Technology, Mexico (CONA-
for KRAS mutation detection28. Nevertheless, the CYT) (Grant Nos. 185427, ECO-2015-C01-260826,
drawbacks must be considered in the selection of the 294875, and 280114).
technique to be used for a certain application. A no-
table limitation of SS is that large deletion/duplica-
tions could not be detected. An alternative for the SUPPLEMENTARY DATA
detection of low-frequency mutations that are not
covered by commercial kits could be next-generation Supplementary data are available at Revista de Inves-
sequencing, which can cover the whole sequence of tigación Clínica online ([Link]-
target genes with an even higher sensitivity than [Link]). These data are provided by the
SS29,30, although its cost still prevents it from compet- corresponding author and published online for the
ing with SS in a commercial laboratory. benefit of the reader. The contents of supplementary
data are the sole responsibility of the authors.
Similar studies analyzing additional mutations to
those found in KRAS codons 12 and 13 in American
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