KRAS and NRAS mutational gene profile of metastatic colorectal cancer patients in Jordan: Molecular spectrum, frequency and distribution pattern

doi:10.21203/rs.2.11767/v1

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KRAS and NRAS mutational gene profile of metastatic colorectal cancer patients in Jordan: Molecular spectrum, frequency and distribution pattern

https://doi.org/10.21203/rs.2.11767/v1

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Background

A constitutively active RAS protein in the absence of stimulation of the epidermal growth factor receptor (EGFR) is the result of mutations in KRAS and NRAS genes. Mutations in the KRAS exon 2 and outside exon 2 have been found to predict the resistance to anti-EGFR monoclonal therapy. A substantial proportion of metastatic colorectal cancer cases (mCRC) exhibit RAS mutations outside KRAS exon 2, particularly in KRAS exon 3 and 4 and NRAS exons 2, 3. No data about RAS mutations outside KRAS exon 2 are available for Jordanian patients with mCRC. We aim to study the molecular spectrum, frequency, and distribution pattern of KRAS and NRAS mutations in Jordanian patients with mCRC.

Methods

A cohort of 190 Jordanian metastatic colorectal cancer patients were enrolled in the trial. We detected mutations in exon 2 of the KRAS and NRAS gene as well as mutations outside of exon 2 using the StripAssay technique. The KRAS StripAssay covered 29 mutations and 22 NRAS mutations.

Results

Mutations were observed in 92 (48.42%) cases, and KRAS exon 2 accounted for 76 cases (83.69%). KRAS G12D was the most common mutation, occurring in 18 cases, followed by KRAS G12A in 16 cases, and G12T in 13 cases. Mutations outside of KRAS exon 2 represented 16.3% of the mutated cases. Among those, 6 cases (6.48%) carried mutations in NRAS exon 2, 3 and 10 cases (10.87%) in KRAS exon 3 and 4.

Conclusion

The frequency of NRAS and KRAS mutations outside of exon 2 appears to be higher in Jordanian patients in comparison with patients from western countries. KRAS mutations outside of exon 2 should be tested routinely to identify patients who should not be treated with anti-EGFR antibodies.

Oncology

Cancer Biology

KRAS exon 2

NRAS

KRAS outside exon 2

mutations

Jordan

metastatic colorectal cancer

Colorectal cancer (CRC) is considered the most common type of cancer among males and the second most common type among females in the Jordanian population.¹ Recent significant advancements in the treatment of CRC have been achieved with new therapeutic approaches, which result from improved understanding of the molecular pathways involved in the development and progression of CRC.

Following ligand binding to the transmembrane receptor, the epidermal growth factor receptor (EGFR) forms a dimer that signals within the cell by activating the receptor auto-phosphorylation through its tyrosine kinase activity ². This intracellular signaling results in cancer-cell proliferation, enabling invasion, metastasis and stimulating tumor-induced neovascularization ²^,³.

The v-Ki-Ras2 Kirsten rat sarcoma (KRAS) gene, first identified as an oncogene in the Kirsten rat sarcoma virus, is a member of the RAS gene family ⁴; it is a downstream component of the EGFR signaling pathway ⁵. KRAS acts as an intracellular signal transducer by coupling the signal from the cell surface receptor with different intracellular targets. Mutations in the RAS family are frequently found in many human tumors. Mutant RAS proteins are constitutively active in the absence of any upstream stimulation of the EGFR receptor ⁶; this is due to the reduced intrinsic GTPase activity and insensitivity to GTPase activation proteins.

Mutations in the RAS gene occur in approximately 20% of all human cancers. ⁷^,⁸ KRAS mutations account for about 85% of all RAS mutations in human cancers, while NRAS account for about 15%. ⁹ In CRC, mutant KRAS is found in about 35-45% of cases ¹⁰^,¹¹. Codon 12 and 13 on exon 2 of the KRAS gene are considered the two main 'hotspots,' together accounting for nearly 95% of all mutation types, with approximately 80% occurring in codon 12 and 15% in codon 13. Other mutations outside of exon 2 occurring in codon 61, 146 and 154 are less frequent in CRC and account for the remaining 5% of all mutation types. ¹²

The anti-EGFR monoclonal antibodies cetuximab and panitumumab bind to the extracellular domain of EGFR when it is in the inactive configuration. The antibodies compete for the receptor binding by occluding the ligand-binding region, thereby blocking the ligand-induced EGFR tyrosine kinase activation.³^,¹³^,¹⁴

Mutation in the KRAS gene results in the continuous activation of signaling pathways without any upstream stimulation of the EGFR/HER receptors. ⁶ This mutation mediates the resistance to the anti-EGFR therapy, thus mandating RAS (KRAS exon 2, codon 12, 13) testing before the treatment with anti-EGFR therapy ¹⁵. Recent studies revealed that despite having wild-type RAS, some patients with metastatic CRC (mCRC) had a reduced response to anti-EGFR therapy¹⁶. These data suggest that NRAS and KRAS mutations outside of exon 2 are also associated with a reduced response to anti-EGFR antibodies. ¹⁷

This leads to the recommendation that mutational analysis of KRAS exon 2, as well as outside exon 2 and NRAS gene should be introduced as a routine screening test for mCRC patients who intend to receive cetuximab and panitumumab, in order to minimize drug toxicity and improve cost-effectiveness.¹⁸

In Jordan, patients with mCRC are routinely investigated for RAS mutations when considered for anti-EGFR therapy, but no data have been reported on KRAS mutations outside of exon 2. This work aimed to investigate the genotyping of KRAS mutations in a Jordanian population and to study the RAS mutations in exon 2 of KRAS and outside of exon 2.

DNA Extraction:

DNA was extracted using QIAamp FFPE Tissue Kit (QIAGEN, Germany) according to the manufacturer instructions with few modifications. The paraffin tissue samples were cut into eight sections (5-10 µm thick), then washed in xylene deparaffinization and absolute ethanol (99%) solution to remove the paraffin. After removing the residual ethanol, each pellet was re-suspended in 180 µl ATL buffer, then treated with 20 µl proteinase K and subjected to incubation at 56 ◦C for two hours. The lysed samples were then incubated at 90^◦C for one hour to reverse formaldehyde cross-linking.

After a brief spin down, 200 µl AL buffer and 200 µl absolute ethanol (99%) were added directly to the samples and vortexed thoroughly. The whole lysate was then transferred to a QIAamp MinElute column placed in a 2 ml collection tube. Samples were centrifuged, and the QIAamp MinElute column was placed into a clean 2 ml collection tube.

Thereafter, 500 µl AW1 buffer was added to the column, centrifuged, and the column was placed in a clean 2 ml collection tube. This step was also repeated using 500 µl AW2 buffer. After centrifugation, the column was placed in a new 1.5 ml Eppendorf tube, and 100µl ATE buffer was added to the center of the membrane. Following incubation at room temperature for 1 minute, the samples were centrifuged, and the DNA was collected and stored at -20^◦C for further use.

KRAS and NRAS mutation analysis:

The KRAS StripAssay (ViennaLab, Austria) covers 29 mutations in codons 12 & 13 (Exon 2), codons 59, 60 & 61 (Exon 3), and codons 117 & 146 (Exon 4). The NRAS StripAssay covers 22 mutations in codons 12 & 13 (Exon 2), codons 59, 60 & 61 (Exon 3), and codon 146 (Exon 4).

PCR amplification and hybridization:

Briefly, the KRAS gene sequence was amplified using a mixture of 15 µl amplification mix, 5 µl diluted Taq DNA polymerase (1U) and 5 µl DNA template (10 µg/ml). KRAS gene sequence was amplified using the following cycling conditions: initial incubation step at 37˚C for 10 minutes and 94 ˚C for 2 minutes, followed by 35 cycles of 94˚C for 1 minute, 70˚C for 50 seconds, 56˚C for 50 seconds and 60˚C for 1 minute, with a final extension step at 60˚C for 3 minutes.

Finally, the amplification products were selectively hybridized to a test strip containing allele-specific oligonucleotide probes immobilized as an array of parallel lines. Bound biotinylated sequences were detected using streptavidin-alkaline phosphatase and color substrates. For each polymorphic position, one of the two possible patterns was obtained: either the presence of KRAS mutations hybridization bands or the absence of KRAS mutations.

In this study, 190 patient samples with metastatic colorectal cancer were analyzed for the presence of RAS gene mutations. The median age at testing was 58 with a range of 19-83 years. Male patients accounted for more than half of the cases (n=114, 60%). The mean age of female patients was slightly lower than that of male patients (55 ±12.36 vs. 58.48 ±12.31). The colon was the most likely primary tumor site (n=182, 95.78%). There was a significantly higher frequency of left-sided colon cancer (n=107, 56.32%) than right-sided (n=62, 32.63%). Furthermore, the right-side tumors were more common in males (n=37, 19.47%) compared to females (n=25, 13.16%). The general characteristics of the patients tested are summarized in Table 1.

Table 1
Characteristics of the 190 patients

Gender, N (%)
Male	114, (60)
Female	76, (40)

Median age at testing (range), years	58, (19-83)

Primary tumor site, N (%)
Colon
Right	62, (32.63)
Left	107, (56.32)
Transverse	9, (4.74)
Sigmoid	2, (1.05)
NOS^a	2, (1.05)
Rectosigmoid	3, (1.58)
Rectum	5 (2.63)

Mutational Status, N(%)
Wild-type	98, (51.57)
Mutated	92, (48.42)
^a NOS: Not otherwise specified

All 190 patients had KRAS mutational analysis performed by Immunostrip technique. A total of 92 (48.42%) mutations in the RAS gene were identified. The KRAS mutations were divided by mutations occurring in either exon 2 (n=76, 83.69%) or outside of exon 2 (n=16, 16.30%). Within the 76 KRAS exon 2 mutations identified, the frequencies of mutation at codon 12, 13 and 117 were 81.57%, 17.1%, and 1.3% respectively. The majority of mutations occurred at codons 12 and 13 which accounted for more than 81% of the total mutated cases (98.68% of mutated cases on exon 2)

The glycine to aspartate on codon 12 (G12D) mutation was most common, accounting for 18 (19.56%) of all the mutations identified. Mutations of glycine to alanine (G12A) was the second most common mutation (n=16, 17.39%). Mutation from glycine to threonine (G12T) and glycine to valine (G12V) constituted 13 and 10 (14.13% and 10.87%) of all mutated cases, respectively.

Mutation from glycine to aspartate (G13D) was the most common mutation on codon 13 (n=7, 7.60%) followed by mutation of glycine to alanine (G13A) (n=6, 6.52%). One case demonstrated a lysine to asparagine (K117N) mutation on codon 117 (n=1, 1.08%).

Ten different mutations were observed outside of KRAS exon 2; of those, four were KRAS mutations and six were NRAS mutations. The two most common KRAS mutations outside exon 2 were mutations from alanine to threonine on codon 146 (A146T) (n=6, 6.52%) followed by lysine to asparagine (K117N) mutation on codon 117 (n=2, 2.17%). One case harbored a KRAS mutation on codon 117 (K117N) and codon 146 (A146V) (n=1, 1.08%) simultaneously.

NRAS testing was only done if the KRAS mutation result was negative. Of the 104 samples analyzed by Immunostrip technique, six were positive for NRAS mutation (6.52%). All six mutations occurred once, with two located in exon 3, and the rest located in exon 2. A summary of all KRAS and NRAS mutations is shown in Table 2.

Table 2
Mutational status and detailed mutation classes found

Mutational Status	N= 92, (%)
KRAS exon 2	76, (83.69)
Outside of KRAS exon 2	16, (16.3)

KRAS exon 2	N, (% of total mutated cases)
KRAS, exon 2, codon 12, G12D	18, (19.56)
KRAS, exon 2, codon 12, G12A	16, (17.39)
KRAS, exon 2, codon 12, G12T	13, (14.13)
KRAS, exon 2, codon 12, G12V	10, (10.87)
KRAS, exon 2, codon 13, G13D	7, (7.60)
KRAS, exon 2, codon 13, G13A	6, (6.52)
KRAS, exon 2, codon 12, G12S	3, (3.26)
KRAS, exon 2, codon 12, G12C	2, (2.17)
KRAS, exon 2, codon 117, K117N	1, (1.08)

Outside of KRAS exon 2	N, (% of total mutated cases)
KRAS, exon 4, codon 146, A146T	6, (6.52)
KRAS, exon 4, codon 117, K117N	2, (2.17)
KRAS, exon 4, codon 117, K117N & KRAS, exon 4, codon 146, A146V	1, (1.08)
	1, (1.08)
KRAS, exon 3, codon 61, Q61H	1, (1.08)
NRAS, exon 3, codon 61, Q61R	1, (1.08)
NRAS, exon 3, codon 61, Q61P	1, (1.08)
NRAS, exon 2, codon 13, G13A	1, (1.08)
NRAS, exon 2, codon 13, G12C	1, (1.08)
NRAS, exon 2, codon 61, A61T	1, (1.08)
NRAS, exon 2, codon 12, G12V	1, (1.08)

Cetuximab and panitumumab are monoclonal antibodies that work by blocking the EGFR receptor, thus inhibiting its downstream signaling pathway. Mutations in any component of this pathway can make the treatment with cetuximab and panitumumab ineffective. Tumors with mutations in the KRAS gene, commonly in codon 12 and 13 of exon 2 or outside of exon 2 are virtually insensitive to cetuximab and panitumumab.¹¹^,¹⁹

In this study, we analyzed KRAS mutations in patients with metastatic CRC: those occurring in exon 2 and outside of exon 2. The incidence of KRAS mutations in our study was approximately 48%, which is in line with what has been reported in a previous study in Jordan ²⁰. The majority of mutations fell in the KRAS exon 2 and constituted about 84% of the mutated cases. The frequencies of mutations in codon 12 and 13 were approximately 82% and 17% respectively. This suggests that the frequency and spectrum of KRAS mutations in our study are similar to what is reported in other studies²¹^,²².

A prospective-retrospective analysis was published which assessed the efficacy and safety of panitumumab together with oxaliplatin, fluorouracil, and leucovorin (FOLFOX4), compared with FOLFOX4 alone stratified according to RAS (KRAS or NRAS), or BRAF mutational status. This study showed that patients with mutations in either NRAS, BRAF, or KRAS outside exon 2 genes had inferior progression-free survival (PFS) and overall survival (OS) with panitumumab-FOLFOX4 treatment.¹⁷

A systematic review and meta-analysis were carried out on nine randomized controlled trials to evaluate the anti-EGFR therapy on PFS and OS of tumors with KRAS exon 2 mutations compared to tumors without any RAS mutations and tumors with KRAS mutation in either exon 3 or 4 or an NRAS mutation in either exon 2, 3 or 4. The study demonstrated that tumors with no RAS mutation showed a significantly superior anti-EGFR PFS and OS treatment effect compared with tumors with a mutation in RAS genes. Further, no difference was observed in PFS and OS benefit between tumors with KRAS exon 2 mutations and tumors with KRAS exon 3 or 4 mutations or NRAS exon 2, 3 or 4 mutations. These results indicated that no PFS or OS benefit was obtained with the use of anti-EGFR therapy for tumors harboring any RAS mutation.¹⁸

The studies mentioned above also confirmed that although NRAS and KRAS outside of exon 2 mutations are less frequent than KRAS exon 2 mutations, they still predict the lack of response to cetuximab and panitumumab.

In our study, we found that mutations outside exon 2 occurred in 16 (16.3%) cases, and of that, 37.5% were due to NRAS mutations on exon 2 and 3, and 62.5% were KRAS exon 3 and 4 mutations. To the best of our knowledge, this has not been investigated previously among Jordanian CRC patients.

Of interest, KRAS exon 4, codon 146 (A146T) mutation was the most frequently detected mutation outside exon 2, constituting about 6% of the mutated cases. This is significantly higher than the frequency of the same mutation reported in the literature¹⁶^,²³. The KRAS mutations on codon 146 have been described in human colorectal cell lines²⁴. Two studies from Hong Kong and the US detected codon 146 mutations in 9 out of 220 cases, giving a combined frequency of 4% ²¹. The second most common mutation observed in our study, outside exon 2, was KRAS exon 4 codon 117 (K117N) mutation. This mutation constituted about a 2% mutation rate, which was significantly higher than what was reported in the literature²⁵.

NRAS mutations are considered rare in CRC, with one study detecting NRAS mutations in 2.2% of the 225 colorectal cancer cases.²⁶. In our research, we found that NRAS mutations constituted approximately 6% (6/92) of all mutated cases.

The clinical significance of the KRAS mutations, except those of codons 12 and 13, remains unclear. Loupakis et al. reported that a patient with mCRC and KRAS 146 mutation was resistant to cetuximab²⁷. In another study, they found that NRAS mutation carriers showed a significantly lower response rate than patients with wild-type KRAS when treated with cetuximab. ¹⁶

This study suggests that Jordanian patients with metastatic colorectal cancer have a higher rate of KRAS outside exon 2 and NRAS mutations when compared to the literature.

In summary, widening the KRAS mutational and subtyping analysis of colorectal cancer patients beyond the KRAS ‘hotspot’ codons 12 and 13 is useful in identifying patients who should not be treated with anti-EGFR antibodies, either alone or in combination with other anticancer agents. Because they are unlikely to benefit, and the exposure to toxicity and expense cannot be justified. A well designed prospective study to determine the full therapeutic implication of NRAS and KRAS outside of exon 2 mutation and validate the observational data is needed.

NRAS, KRAS, CRC, EGFR, mCRC

Ethics approval and consent to participate

This study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects/patients were approved by the University of Jordan Hospital Institutional Review Board.

Consent for publication

Written informed consent was obtained from all study participants.

Availability of data and materials

Access to the data can be obtained by contacting the department of hematology at JUH. The dataset supporting the conclusions of this article is included within the article (and its additional file)

Competing interests

The authors declare no conflict of interest

Funding

This research received no specific grant from any funding agency in the commercial or not-for-profit sectors

Author’s contribution

M.A: Was involved in data entry, data curation, data analysis, contributed to the writing and editing of the manuscript
N.A: Was involved in data entry and capturing the patients, analyzing patient samples, and contributed to the writing of the manuscript.
M.S: Was involved in data entry and capturing the patients, contributed to the writing of the manuscript.
F.F: Was involved in data entry and capturing the patients, contributed to the writing of the manuscript.
M.A: Was involved in the data curation, analyzing patient samples, contributed to writing and editing the manuscript.
L.O: Was involved in the data curation, analyzing patient samples, contributed to writing and editing the manuscript.
B.T: Was involved in the data curation, analyzing patient samples, contributed to writing and editing the manuscript.
A.A: Was involved in the data curation, analyzing patient samples and supervision, contributed to the writing and editing the manuscript.

Acknowledgment

The authors would like to thank the patients’ advisers and the oncology nursing staff at JUH for their contribution.

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KRAS and NRAS mutational gene profile of metastatic colorectal cancer patients in Jordan: Molecular spectrum, frequency and distribution pattern

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