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Article

Clinical Characterization of Targetable Mutations (BRAF V600E and KRAS G12C) in Advanced Colorectal Cancer—A Nation-Wide Study

by
Paweł M. Potocki
1,*,
Piotr Wójcik
2,
Łukasz Chmura
2,
Bartłomiej Goc
3,4,
Marcin Fedewicz
3,5,
Zofia Bielańska
3,6,
Jakub Swadźba
7,
Kamil Konopka
1,
Łukasz Kwinta
1 and
Piotr J. Wysocki
1
1
Oncology Department, Faculty of Medicine, Jagiellonian University Medical College, 31-008 Cracow, Poland
2
Oncogene Diagnostics Sp. z o.o., 31-546 Cracow, Poland
3
Student Research Group, Oncology Department, Faculty of Medicine, Jagiellonian University Medical College, 31-008 Cracow, Poland
4
Radiotherapy Department, MSC National Research Institute of Oncology, 44-102 Gliwice, Poland
5
Józef Babiński Clinical Hospital, 30-393 Cracow, Poland
6
Klinik für Psychiatrie, Psychotherapie und Psychosomatik, Regio Klinikum, 25337 Elmshorn, Germany
7
Department of Laboratory Medicine, Faculty of Medicine and Health Sciences, Andrzej Frycz Modrzewski Krakow University, 30-705 Cracow, Poland
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(10), 9073; https://doi.org/10.3390/ijms24109073
Submission received: 23 April 2023 / Revised: 18 May 2023 / Accepted: 20 May 2023 / Published: 22 May 2023
(This article belongs to the Special Issue State-of-the-Art Molecular Oncology in Poland)
Browse Figures
Review Reports Versions Notes

Abstract

:
BRAF V600E and KRAS mutations that occur in colorectal cancer (CRC) define a subpopulation of patients with an inferior prognosis. Recently, the first BRAF V600E-targeting therapy has been approved and novel agents targeting KRAS G12C are being evaluated in CRC. A better understanding of the clinical characteristics of the populations defined by those mutations is needed. We created a retrospective database that collects clinical characteristics of patients with metastatic CRC evaluated for RAS and BRAF mutations in a single laboratory. A total of 7604 patients tested between October 2017 and December 2019 were included in the analysis. The prevalence of BRAF V600E was 6.77%. Female sex, primary in the right colon, high-grade, mucinous, signet cell, partially neuroendocrine histology, perineural and vascular invasion, and surgical tissue sample were factors associated with increased mutation rates. The prevalence of KRAS G12C was 3.11%. Cancer of primary origin in the left colon and in samples from brain metastases were associated with increased mutation rates. The high prevalence of the BRAF V600E mutation in cancers with a neuroendocrine component identifies a potential candidate population for BRAF inhibition. The association of KRAS G12C with the left part of the intestine and brain metastases of CRC are new findings and require further investigation.

1. Introduction

Metastatic colorectal cancer (CRC) is one of the leading causes of cancer-related mortality [1,2]. The mitogen-activated protein kinase (MAPK) cascade is the main pathway that transduces signalling from growth factor receptors. It is central in the regulation of proliferation, differentiation, and viability in healthy and malignant epithelial cells. Alterations increasing MAPK activity play a crucial role in cancer pathogenesis [3]. In addition to sustaining proliferative signalling, they are also closely related to the formation of metastases and drug resistance. Pathological MAPK activity is most commonly caused by activating mutations in genes in the RAS (Rat Sarcoma) family. Other pathomechanisms include epigenetic activation of growth factor receptors, activating mutations of genes for receptor tyrosine kinases, or activating mutations in signalling kinases downstream of RAS, i.e., RAF (rapidly accelerated fibrosarcoma) and MEK (mitogen-activated extracellular signal-related kinase) [3].
The RAS gene family consists of three isoforms, KRAS, NRAS, and HRAS, located on chromosomes 12, 1, and 11, respectively. They have a similar, evolutionarily conservative structure, consisting of four exons. RAS proteins play an important role in the regulation of multiple intracellular processes. By switching between their GTP-bound (Guanosine-5′-Triphosphate) active form and GDP-bound (Guanosine-5′-Diphosphate) inactive form, they control multiple signal cascades including those crucial to CRC pathogenesis: MAPK and PI3K (phosphatidylinositol 3-kinase). RAS itself can, in turn, be activated by multiple classes of signals including: by growth factor receptors through GRB2-SOS (Growth Factor Receptor-Bound Protein 2, Son of Sevenless) signal proteins; by intracellular AMP (Adenosine monophosphate) or calcium levels through GRF1 (Guanine Nucleotide Releasing Factor); by crosstalk with other signalling pathways through SHP2 (Src Homology Phosphatase 2) activity [3]. Activating mutations in RAS are found most frequently in pancreatic cancer (88.4% cases), colorectal cancer (55.1%), multiple myeloma (37.8%), lung adenocarcinoma (33.2%), follicular thyroid cancer (30.6%), cholangiocarcinoma (23.6%), endometrial cancer (20.0%), and skin melanoma (19.8%). In general, KRAS mutations occur more frequently than HRAS or NRAS mutations, although this is not true for all types of cancer [4].
In colorectal cancer, KRAS mutations occur in 50.4% of cases, NRAS mutations in 4.2%, and HRAS in 0.5% [4]. Their rate is higher in proximal colon tumours [5,6,7,8]. Hyperactive kinases resulting from mutated genes stimulate the MAPK pathway directly downstream of EGFR (Epithelial Growth Factor Receptor). Therefore, the KRAS and NRAS mutations are well-recognized negative predictors of EGFR inhibition. Their negative prognostic effect is also known, but due to a large heterogeneity of possible variations and the resulting kinase phenotypes, it is less well understood.
Several large efforts aimed at developing RAS inhibitors failed to produce clinically active therapies; thus, for a long time, RAS has been considered an undruggable target. The difficulty has stemmed from many reasons, but mainly from the complexity of the RAS function: heterogeneity of its mutated isoforms, different modes of activation, presence of inter-pathway crosstalk, and feedback loops reactivating the blocked cascades [3,9]. Recently, new small-molecule agents that specifically block the G12C-mutated form of KRAS have emerged and are being clinically evaluated. These include Adagrasib (form. MRTX849, Mirati Therapeutics Inc., San Diego CA, USA), Sotorasib (form. AMG510, Amgen, Thousand Oaks CA, USA), D-1553 (MSD, Rahway, NJ, USA), GDC-6036 (Genentech Inc., South San Francisco, CA, USA), JAB-21822 (Jacobio Pharm., Beijing, China), JDQ443 (Novartis Pharm., Basel, Switzerland), JNJ-74699157 (Janssen R&D, Rarita, NJ, USA), and LY3499446 (Eli Lilly, Indianapolis, IN, USA).
Within the MAPK cascade, RAF kinases transduce the signal from RAS, mainly through phosphorylative activation of downstream MEK kinases. Its three isoforms, ARAF, BRAF, and CRAF, occur in the inactive monomeric form. After activation, they bind to another RAF monomer, forming homodimers or heterodimers that exhibit kinase activity. The BRAF gene is located on chromosome 17 and can be affected by a variety of mutations. Those mutations typically occur in codon 600 (exon 15), substituting valine with another amino acid that results in a strongly hyperactive kinase. The most common allele is V600E. BRAF V600E mutations are commonly found in melanoma and lung, thyroid, and colorectal cancers, as well as gliomas and acute leukaemias [10]. Therapies targeting mutated BRAF are now a standard of care in a subset of patients with melanoma and lung cancer.
In colorectal cancer, the BRAF V600E mutation is associated with a distinct clinical and pathological phenotype: predominantly proximal colon tumour location, aggressive growth, adverse metastasizing patterns, resistance to the EGFR blockade, and an overall survival (OS) rate that is 1.5 to 3 times shorter as compared to the general population. Naturally, it has been investigated as a potential therapeutic target. Unlike the high efficacy of BRAF targeting strategies observed in melanoma, monotherapy with V600E-specific kinase inhibitors (vemurafenib, dabrafenib) has been proven to be ineffective in metastatic colorectal cancer (mCRC) [11]. It has been shown to trigger the feedback loop that facilitates alternative MAPK activation mechanisms, particularly through increased EGFR and MEK activity [11]. Simultaneous inhibition of this feedback loop through inhibition of EGFR or MEK has been shown to suppress the feedback, thus restoring control over BRAF V600E-activated MAPK [12]. The efficacy of such a strategy was confirmed in clinical trials, most recently in a phase III BEACON study that demonstrated the feasibility of a combined BRAF blockade in a randomized setting. The study also demonstrated that triple inhibition (anti-EGFR + anti-BRAF V600E + anti-MEK with cetuximab, encorafenib, binimetinib) appears to be not more effective than double inhibition (anti-EGFR + anti-BRAF V600E with cetuximab, encorafenib) [13]. As a result, the cetuximab-encorafenib combination is now a standard of care in patients with pretreated BRAF V600E-mutated mCRC. Trials evaluating the first-line efficacy, alternative, and derivative combinations are also under way. Noticeably, another double inhibition strategy (anti-BRAF V600E + anti-MEK) was subjected to an early clinical evaluation [14]. Agents with anti-BRAF activity that have been investigated in this setting include Vemurafenib (Genentech/Roche), Dabrafenib (GlaxoSmithKline), Encorafenib (Array BioPharma/Pierre Fabre), Regorafenib (Bayer), Sorafenib (Bayer), Lifirafenib (BeiGene), Agerafenib (Roche), Belvarafenib (Hanmi Pharmaceutical), Ro 5126766 (Chugai Pharmaceutical), and LY3009120 (Eli Lilly).
Antibodies targeting EGFR (cetuximab and panitumumab) have transformed systemic treatment for advanced CRC and are now an integral part of first-line regimens. As a result, molecular testing for KRAS, NRAS and BRAF mutations has become an obligatory part of the standard diagnostic routine. As MAPK pathway deregulation in CRC becomes better understood, novel, effective therapies targeting this signalling cascade, in unique, molecularly-defined mCRC patient subpopulations, are emerging. The aim of this study is to define the clinical characteristics of mCRC patients harbouring readily targetable mutations (KRAS G12C or BRAF V600E) and to screen for potential clinical factors predictive of the occurrence of the mutations.

2. Results

2.1. Population Characteristics

A total of 7798 cases tested between October 2017 and December 2019 were identified. After discarding 89 duplicates, 102 nondiagnostic results and 3 non-colorectal cancers, 7604 were included in the analysis (Figure 1). Men made up 58.8%. A total of 75.5% were over 60 and 4.91% were over 80 years old. The distal primary location was more prevalent, with the rectum being the most common one. The majority of the analysed samples originated in the primary tumour (87.9%). Of the 685 samples not originating from primaries, the most common sample source sites were the liver, peritoneal implants, and local recurrence. Surgical samples comprised the majority of the cases (73.49%), with endoscopic biopsy being the second most common sampling method (22.91%). The BRAF V600E mutation occurred in 515 cases (6.77%), KRAS G12C in 237 (3.12%), and any other KRAS in 3591 (47.23%). The detailed characteristics of the general population and the subpopulations are outlined in Table 1 and Table S1.

2.2. BRAF V600E Mutation

The prevalence of BRAF V600E was 6.77%. The characteristics of mutated cases compared to those without mutations are outlined in Table 2. BRAF V600E-mutated cancers exhibited higher T and N scores, as well as higher rates of vascular and perineural invasion compared to non-mutated ones.
The mutation was present more often in women (odds ratio (OR) 2.009, 95% confidence interval (CI) 1.677–2.408, p < 0.001). A strong yet nonsignificant trend towards greater mutation risk in older age was observed (Figure 2). The mutation was more likely to be found in high-grade cancers (OR 4.061, 95% CI 3.218–5.125, p < 0.001). It was more likely to occur in cancers with mucinous histology (OR 3.430, 95% CI 1.415–8.315, p = 0.006) or partially mucinous histology (OR 2.718, 95% CI 2.121–3.482, p < 0.001) or with the presence of signet cells (OR 1.544, 95% CI 1.104–2.160, p = 0.011). Mixed adeno-neuroendocrine cancers (MANEC) were more likely to carry the mutation (OR 9.211, 95% CI 3.071–27.626, p < 0.001). Proximal tumours, occurring in the caecum, ascending colon, hepatic flexure, or transverse colon, were more likely to carry the mutation as compared to distal ones occurring in the splenic flexure, descending colon, sigmoid colon, rectosigmoid junction, or rectum (OR 8.356, 95% CI 6.732–10.372, p < 0.001). Rectal/rectosigmoid tumours were less likely to carry the mutation as compared to the abdominal (proximal and distal combined) part of the colon (OR 0.231, 95% CI 0.179–0.299, p < 0.001), although the difference between rectal/rectosigmoid and distal abdominal tumours (occurring in splenic flexure or descending and sigmoid colon) was not significant (OR 1.32, 95% CI 0.928–1.876, p = 0.122). There was no significant difference in the mutation rate between primary, locally recurrent, and metastatic lesions, although a trend towards a higher rate was observed in primaries. The mutation was less likely to occur in samples from biopsies as compared with surgical ones. All samples secured during colonoscopy were 30.9% less likely to produce a positive result as compared to all samples secured during surgeries (OR 0.691, 95% CI 0.532–0.897, p = 0.006). All samples secured during needle biopsies were 58.7% less likely to produce a positive result as compared to all samples secured during surgeries (OR 0.691, 95% CI 0.532–0.897, p = 0.023). The same was true when restricted to metastatic lesions only. A biopsy was 57.1% less likely to produce a BRAF V600E-mutated sample than a surgery (OR 0.429, 95% CI 0.202–0.911, p = 0.028). Variability in the rate of BRAF V600E mutation was also observed according to geographical regions. Only the difference between the regions with the highest sample numbers (Lodzkie < Slaskie) was statistically significant (OR 0.669, 95% CI 0.481–0.930, p = 0.017). (Table 3 and Table S2, Figure 2 and Figure 3).

2.3. KRAS G12C Mutation

The prevalence of KRAS G12C in the overall population was 3.12% (6.60% of all KRAS mutations). The pathomorphological characteristics of the cases with the KRAS G12C mutation compared to the rest of the cohort and other KRAS-mutated cases are outlined in Table 4.
When comparing KRAS G12C mutant cases with the rest of the cohort, proximal tumours were less likely to carry the KRAS G12C mutation compared to distal tumours (OR 0.652, 95% CI 0.451–0.944, p = 0.024). Rectal/rectosigmoid tumours were more likely to carry the mutation as compared to those of the abdominal part of the colon (OR 1.470, 95% CI 1.122–1.925, p = 0.005), although the difference between the rectal/rectosigmoid and intraabdominal distal colon was borderline insignificant (OR 0.729, 95% CI 0.521–1.021, p = 0.066). CNS metastases were 21 times more likely to carry the G12C mutation than the rest of the cohort (OR 20.98, 95% CI 3.48–126.47, p = 0.001) (Table 5 and Table S3, Figure 4).
When comparing KRAS G12C-mutant cases with the rest of the KRAS-mutant cases, proximal tumours were less likely to carry the KRAS G12C mutation compared to distal tumours (OR 0.618, 95% CI 0.425–0.898, p = 0.012), whereas rectal/rectosigmoid tumours were more likely to carry the mutation as compared to the abdominal part of the colon (OR 1.425, 95% CI 1.083–1.874, p = 0.011). CNS metastases were 15.5 times more likely to carry the G12C mutation than the rest of the cohort (OR 15.529, 95% CI 2.173–110.997, p = 0.006). Higher rates of G12C mutation were observed in samples taken from needle biopsies but not endoscopic biopsies (OR 2.05, 95% CI 1.096–3.833, p = 0.025). This finding was not repeated when restricting the comparison to only metastatic cases and is not present when comparing G12C cases with the general population. (Table 6 and Table S4).

3. Discussion

Our study used one of the largest single-laboratory-generated databases comprising cases of advanced colorectal Caucasian patients worldwide. In the recent meta-analysis of 67 studies (32,407 patients) evaluating the clinical and pathological significance of the BRAF V600E mutation in colorectal cancer, the largest study collected less than 2000 cases. Our analysis comprises 7604 patients’ records, and this population would represent approximately 23% of all summarized records in the aforementioned meta-analysis. Moreover, there is not a single large-scale study published to date evaluating the clinicopathological features associated with KRAS G12C mutation, which is expected to shortly become the first clinically targeted KRAS mutation in CRC.
The prevalence of BRAF V600E mutations (below 7% (6.77%)) is lower than expected, based on previously published data. The observed mutation rate varies considerably when compared across other studied cohorts. The aforementioned meta-analysis (n = 32,407) reported a mean rate of 11.34% (3.14–23.14%) [15]. In contrast, previous, much smaller studies conducted on the Polish population showed values similar to the one reported in our analysis. A study on non-metastatic CRC patients found the mutation in 2/163 (1.22%) cases [16]. Two Polish studies on advanced CRC reported the incidence of BRAF V600E mutations in 24/500 (4.80%) and 7/102 (6.86%), respectively [17,18]. A recently published large study on the Russian population reported the incidence at 6.7% [19]. The observed variability may be due to a number of reasons, described below. However, it must be underscored that our analysis was unique in its homogeneity, in that all of the tested patients were Caucasian.
Most studies found that the BRAF V600E mutation was more likely to occur in women and in proximal tumours, which is also a finding in the current study. An association of mutation risk with age has also been described, although it did not reach the significance threshold in this study [20]. In some studies, a higher clinical stage at the time of diagnosis was associated with an increased risk of mutation [15,21,22]. The distribution of these and other risk factors among different cohorts may explain the variability.
Another reason for the lower-than-expected BRAF V600E mutation rates may be survivor bias. The V600E mutation is associated with an aggressive phenotype, adverse metastasizing patterns, and rapid progression. Therefore, it has been shown to negatively impact OS in patients with radically treated and metastatic colorectal cancer. Interestingly, the same studies did not show a significant impact on disease-free survival (DFS) or progression-free survival (PFS) [23,24,25,26,27]. Therefore, it can be concluded that it is the fast rate of disease progression that drives the decrease in OS, whereas the effectiveness of treatment (surgical or systemic as measured in recurrence rates and PFS) does not appear to be affected by the mutation. This indirectly hints that the mutation rate in the radically treated population should remain unchanged in the subpopulation of patients who ultimately recur. In this cohort, patients were only tested for the BRAF mutation when presenting with metastatic disease. Effectively, only patients who maintained a good general condition for long enough to be diagnosed, tested, and treated were included in the analysis. Therefore, subpopulations with adverse prognostic factors for metastatic disease (including BRAF V600E mut, older age, higher tumour burden, poor performance status) are likely to be underrepresented. This selection practice resulted directly from the national reimbursement policy. The authors are aware that most clinical practice guidelines for CRC treatment recommend testing at the time of first diagnosis.
In this study, the samples secured during surgery contained significantly higher numbers of BRAF V600E mutations than the samples secured by endoscopy. A similar relationship was not observed for the KRAS G12C mutation. Therefore, in the authors’ opinion, it is unlikely that the BRAF V600E mutation rate was affected by sample processing or the methodology for detecting mutations. The difference in the BRAF V600E rate between surgical and endoscopic samples may represent the differences in the possibilities of collecting material during colonoscopy. It is not always possible to reach the right side of the colon with the endoscope; therefore, the material from this location is less often collected during endoscopic examination. The BRAF-mutated tumours are associated with a right-sided location in the colon and such samples are less likely to be secured by endoscopy.
Tumour histology is another factor that was associated with the appearance of the BRAF mutation. This study confirmed the well-documented link between the risk of the BRAF mutation and mucinous histology, high tumour grade, and vascular and perineural invasion. The presence of signet cells or partial neuroendocrine components also increased the odds of the mutation. The last finding, although previously described [28], suggests a potential therapeutic target in this rare and clinically difficult cancer subtype. There are already case studies describing responses to dual inhibition of BRAF and MEK in this population [29,30]. Recently, a dabrafenib-trametinib combination has gained tissue-agnostic approval in the United States [31], which now allows any malignancy with the mutation to be treated with this regimen.
Tumour sidedness has also been shown to affect the rate of BRAF mutation in this study, with proximal (right-sided) tumours manifesting higher rates. Notably, rectal/rectosigmoid tumours were associated with significantly lower mutation rates than tumours located in the abdominal part of the colon. Because the occurrence of the mutation did not differ significantly between the rectal/rectosigmoid and distal abdominal colon (only a moderate trend was present), this difference is likely a manifestation of well-documented biological differences between the proximal and distal parts of the large intestine. A study on a larger dataset may resolve this doubt. This study also confirms the link between the risk of BRAF mutation and female sex, mucinous histology, high tumour grade, vascular and perineural invasion, and high T and N scores, which is also well documented in the literature [32].
In this study, some geographical variability is evidenced in the rates of BRAF mutations. Only the difference between the Śląskie and Łódzkie regions is significant, but notably, those were the ones with the highest number of samples. Nonsignificant trends for other regions are also present. In the authors’ opinion, this finding should be further evaluated in a larger study. This may provide insight into hereditary and environmental factors that influence the rates of BRAF mutations, but it may also be a manifestation of practice patterns that influenced patient selection for this cohort.
The frequency of KRAS G12C mutations in this study was 3.12%, which is similar to recently published datasets [33,34,35]. Surprisingly, the KRAS G12C mutation in the Polish population occurred more frequently in distal tumours, contrary to a known predisposition of RAS mutations for the proximal part of the colon [5,6,7,8] and data on KRAS G12C-mutated tumours in the Scandinavian population [35]. Additionally, samples from rectal/rectosigmoid cancers were more likely to carry the mutation compared to samples from the intraabdominal part of the intestine. These findings were true both in comparison to the rest of the cohort and in comparison to the rest of the KRAS-mutated cases. Given that there was only a borderline nonsignificant trend towards a higher mutation rate in the rectal/rectosigmoid tumours as compared to the left colon tumours, this should be interpreted with caution, as it may only be a manifestation of the aforementioned predisposition to the distal parts of the intestine. This predisposition for left-sidedness seems to be a new characteristic of the G12C mutation and should be confirmed in other datasets. The authors identified four recent publications that included sidedness in KRAS G12C vs. KRAS non-G12C population characteristics. In two of them, there was a trend towards a higher percentage of left-sided primaries in the KRAS G12C cohort compared to the cohort with the remaining RAS mutations [35,36]. In the remaining two, a higher percentage of right-sided primaries was reported in KRAS G12C-mutated cancers [33,37]. The aforementioned trends were not statistically significant.
Of the five samples originating from brain metastases in this study, two are KRAS G12C-mutated. Although the numbers are small, the rates of G12C occurrence in brain metastases differ significantly, both compared to the rest of the cohort and to the rest of the KRAS-mutated cases. Notably, another two of the brain metastasis samples harboured KRAS mutations (G12D and G13D). Evidence that KRAS mutations in general may play a role in the pathogenesis of CRC brain metastases has previously been described [38]. The finding in this study suggests that there may be a link between brain metastases and the G12C mutation, specifically. Recent studies involving brain metastases from lung cancer have reported similar findings [39,40]. If confirmed in further studies, the overrepresentation of the G12C mutation in brain metastases may be of clinical significance, in light of recent preliminary data on sotorasib being active in brain metastases [41].
A higher prevalence of KRAS G12C mutations in samples taken from needle biopsies is a puzzling finding. It only presented itself in the comparison between G12C and other KRAS mutations. A similar finding was not found for endoscopic biopsies and when the analysis was restricted to metastatic samples only. This may be a manifestation of selection bias, but the exact mechanisms are unclear and require further investigation.

4. Materials and Methods

We created a dedicated retrospective database of consecutive patients with metastatic colorectal cancer from 107 Polish institutions whose tissue samples had been tested for KRAS, NRAS, and BRAF mutations in a single laboratory. The cohort consisted of samples that were tested as part of standard practice. Although there was no active selection strategy, patients were passively selected, as testing was driven by the restrictive inclusion criteria of a national reimbursement policy. Clinical data were extracted from available records, including basic demographics (age, sex, geographic region), TNM scores (8th edition, 2016), histology (NOS, mucinous, partially mucinous, signet cell, cribriform, comedo-like, partially neuroendocrine, other), primary location (right or proximal colon including caecum, ascending colon, hepatic flexure and transverse colon; left or distal colon including splenic flexure, descending and sigmoid colon; rectum/rectosigmoid junction), sample origin (primary, local recurrence, metastatic, according to location of metastases), and sampling method (endoscopic biopsy, needle biopsy, surgery).
The suitability of the samples (amount of tissue, neoplastic cells content) was assessed by a pathologist. The appropriate tissue fragments were collected from microscopic slides by macrodissection. The minimum requirement for analysis was 20% of neoplastic cells, and in most cases (90% of samples) the percentage of neoplastic cells exceeded 50%. A validated Sanger sequencing technique with a detection limit of 10% of the mutant DNA (approx. 20% of cells) was used to test the mutations. The mutation analysis included selected gene fragments: KRAS exon 2 (including codons 10–22), KRAS exon 3 (including codons 58–64), KRAS exon 4 (including codons 117, 146), NRAS exon 2 (including codons 10–18), NRAS exon 3 (including codons 58–64), NRAS exon 4 (including codons 117, 146), BRAF exon 15 (including codons 594–601). The appropriate DNA fragments were amplified by the polymerase chain reaction and subsequently sequenced with the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) on a sequencer (3730 DNA Analyzer, Applied Biosystems). In parallel to the main Sanger sequencing technique, an independent screening method (SSCP or HRM) was used as an intra-laboratory control in each case. The mutational analysis was performed by a single laboratory that successfully participates in external quality assessment trials organized by ESP and GenQA.
Statistical analysis was performed in the R environment v 4.1.3 [42]. Qualitative variables were compared between groups using the chi-square test (with Yates correction for 2 × 2 tables). Fisher’s exact test was used when low expected quantities occurred in the tables. Quantitative variables were compared between groups using the Mann–Whitney test. A p-value of 0.05 was considered statistically significant.

5. Conclusions

Our nationwide study clearly demonstrates that BRAF V600E and KRAS G12C colorectal tumours show significantly detrimental clinicopathological features. On the other hand, the same mutations are highly predictive of the clinical benefit of novel targeted therapies.
The characteristics of the population with BRAF V600E-mutated CRC were generally consistent with the literature, apart from the lower overall prevalence of the mutation, the anomalies in its geographical distribution, and differences between sample types. These require further investigation. The increased prevalence of the BRAF V600E mutation in partially neuroendocrine cancers may identify a new subpopulation eligible for BRAF inhibition. The characteristics of the KRAS G12C mutation revealed two potentially novel findings: the higher prevalence of the KRAS G12C mutation in distal (left-sided tumours) contrary to other KRAS mutations and the higher prevalence of the KRAS G12C mutation in brain metastases compared to other KRAS mutations. Those require confirmation in other studies but may become clinically relevant with the ongoing development of specific KRAS G12C inhibitors.
With the rise of therapies targeting signal transduction in pathways central to CRC pathogenesis, new subpopulations defined by the ever-growing number of molecular biomarkers emerge. The clinical characterization of these subpopulations is both a requirement for the introduction of new drugs into clinical practice and a source of hypotheses that may affect the direction of future research. The authors present the first results from a newly created database, which hopefully will be expanded with more cases, survival data, and biomarkers to fuel future research.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24109073/s1.

Author Contributions

Conceptualization, P.M.P., P.W. and P.J.W.; methodology, P.M.P. and P.W.; software, P.M.P.; validation, P.W.; formal analysis, P.M.P., J.S., P.J.W., K.K. and Ł.K.; investigation, P.W., Ł.C., B.G., M.F. and Z.B.; resources, P.W. and P.M.P.; data curation, P.M.P., P.W., B.G., M.F. and Z.B.; writing—original draft preparation, P.M.P.; writing—review and editing, P.M.P., P.W., P.J.W., K.K. and Ł.K.; visualization, P.M.P.; supervision, P.M.P.; project administration, P.M.P. and P.W.; funding acquisition, P.W. and P.M.P. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Jagiellonian University Medical College (decision no 1072.61201.41.2022).

Informed Consent Statement

Patient consent was waived due to the retrospective nature of the study and local regulations.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to ethical and legal concerns.

Acknowledgments

The authors gratefully acknowledge the molecular pathology team of Oncogene Diagnostics, Cracow, Poland, for their dedication to quality in molecular testing; Łukasz Deryło for excellent support in statistical analysis; students of the Student Research Group, Oncology Department, Jagiellonian University Medical College for their unfading enthusiasm.

Conflicts of Interest

Paweł Potocki has received honoraria from Amgen, BMS, Merck, Pierre Fabre, and Roche. Piotr Wójcik has received honoraria from Amgen, AstraZeneca, Merck, Novartis, and Roche. Piotr J. Wysocki has received honoraria from Amgen, Merck, Roche, Servier, and Sanofi. The remaining authors declare no conflicts of interest.

References

  1. Sung, H.; Ferlay, J.; Siegel, R.L.; Laversanne, M.; Soerjomataram, I.; Jemal, A.; Bray, F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 2021, 71, 209–249. [Google Scholar] [CrossRef] [PubMed]
  2. Didkowska, J.; Wojciechowska, U.; Michałek, I.; Caetano dos Santos, F.; Olasek, P. NOWOTWORY ZŁOŚLIWE W POLSCE W 2019 ROKU (CANCER IN POLAND IN 2019); Polish National Cancer Registry: Warsaw, Poland, 2021; ISBN 0867–8251. [Google Scholar]
  3. Huang, L.; Guo, Z.; Wang, F.; Fu, L. KRAS Mutation: From Undruggable to Druggable in Cancer. Signal Transduct. Target. Ther. 2021, 6, 1–20. [Google Scholar] [CrossRef] [PubMed]
  4. Prior, I.A.; Hood, F.E.; Hartley, J.L. The Frequency of Ras Mutations in Cancer. Cancer Res. 2020, 80, 2669–2974. [Google Scholar] [CrossRef] [PubMed]
  5. Bylsma, L.C.; Gillezeau, C.; Garawin, T.A.; Kelsh, M.A.; Fryzek, J.P.; Sangaré, L.; Lowe, K.A.; Kimberly Lowe, C.A. Prevalence of RAS and BRAF Mutations in Metastatic Colorectal Cancer Patients by Tumor Sidedness: A Systematic Review and Meta-Analysis. Cancer Med. 2019, 9, 1044–1057. [Google Scholar] [CrossRef]
  6. Park, H.S.; Chun, Y.J.; Kim, H.S.; Kim, J.H.; Lee, C.K.; Beom, S.H.; Shin, S.J.; Ahn, J.B. Clinical Features and KRAS Mutation in Colorectal Cancer with Bone Metastasis. Sci. Rep. 2020, 10, 21180. [Google Scholar] [CrossRef]
  7. Kamphues, C.; Kadowaki, S.; Amini, N.; van den Berg, I.; Wang, J.; Andreatos, N.; Sakamoto, Y.; Ogura, T.; Kakuta, M.; Theochari, M.; et al. The Interplay of KRAS Mutational Status with Tumor Laterality in Non-Metastatic Colorectal Cancer: An International, Multi-Institutional Study in Patients with Known KRAS, BRAF, and MSI Status. J. Surg. Oncol. 2020, 123, 1005–1014. [Google Scholar] [CrossRef]
  8. Tran, C.G.; Goffredo, P.; Mott, S.L.; Hart, A.; You, Y.N.; Vauthey, J.N.; Weigel, R.J.; Hassan, I. The Impact of KRAS Mutation, Microsatellite Instability, and Tumor Laterality on the Prognosis of Nonmetastatic Colon Cancer. Surgery 2022, 171, 657–665. [Google Scholar] [CrossRef]
  9. Cox, A.D.; Fesik, S.W.; Kimmelman, A.C.; Luo, J.; Der, C.J. Drugging the Undruggable RAS: Mission Possible? Nat. Rev. Drug Discov. 2014, 13, 828–851. [Google Scholar] [CrossRef]
  10. Davies, H.; Bignell, G.R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M.J.; Bottomley, W.; et al. Mutations of the BRAF Gene in Human Cancer. Nature 2002, 417, 949–954. [Google Scholar] [CrossRef]
  11. Potocki, P.M.; Wysocki, P.J. BRAF—A New Therapeutic Target in Colorectal Cancer. Oncol. Clin. Pract. 2018, 14, 86–95. [Google Scholar]
  12. Yao, Z.; Torres, N.M.; Tao, A.; Gao, Y.; Luo, L.; Li, Q.; De, E.; Abdel-wahab, O.; Solit, D.B.; Poulikakos, P. BRAF Mutants Evade ERK Dependent Feedback by Different Mechanisms That Determine Their Sensitivity to Pharmacologic Inhibition Zhan. Cancer Cell 2015, 28, 370–383. [Google Scholar] [CrossRef] [PubMed]
  13. Kopetz, S.; Grothey, A.; Yaeger, R.; Van Cutsem, E.; Desai, J.; Yoshino, T.; Wasan, H.; Ciardiello, F.; Loupakis, F.; Hong, Y.S.; et al. Encorafenib, Binimetinib, and Cetuximab in BRAF V600E–Mutated Colorectal Cancer. N. Engl. J. Med. 2019, 381, 1632–1643. [Google Scholar] [CrossRef] [PubMed]
  14. Klute, K.; Garrett-Mayer, E.; Halabi, S.; Mangat, P.K.; Nazemzadeh, R.; Yost, K.J.; Butler, N.L.; Perla, V.; Schilsky, R.L. Cobimetinib plus Vemurafenib (C+V) in Patients (Pts) with Colorectal Cancer (CRC) with BRAF V600E Mutations: Results from the TAPUR Study. J. Clin. Oncol. 2020, 38, 122. [Google Scholar] [CrossRef]
  15. Wang, J.; Shen, J.; Huang, C.; Cao, M.; Shen, L. Clinicopathological Significance of BRAFV600E Mutation in Colorectal Cancer: An Updated Meta-Analysis. J. Cancer 2019, 10, 2332–2341. [Google Scholar] [CrossRef]
  16. Wójcik, P.; Okoń, K.; Osuch, C.; Klimkowska, A.; Tomaszewska, R. BRAF Mutations in Sporadic Colorectal Carcinoma from Polish Patients. Polish J. Pathol. 2010, 61, 23–26. [Google Scholar]
  17. Wojas-Krawczyk, K.; Kalinka-Warzocha, E.; Reszka, K.; Nicoś, M.; Szumiło, J.; Mańdziuk, S.; Szczepaniak, K.; Kupnicka, D.; Lewandowski, R.; Milanowski, J.; et al. Analysis of KRAS, NRAS, BRAF, and PIK3CA Mutations Could Predict Metastases in Colorectal Cancer: A Preliminary Study. Adv. Clin. Exp. Med. 2019, 28, 67–73. [Google Scholar] [CrossRef]
  18. Bożyk, A.; Krawczyk, P.; Reszka, K.; Krukowska, K.; Kolak, A.; Mańdziuk, S.; Wojas-Krawczyk, K.; Ramlau, R.; Milanowski, J. Correlation between KRAS, NRAS and BRAF Mutations and Tumor Localizations in Patients with Primary and Metastatic Colorectal Cancer. Arch. Med. Sci. 2022, 18, 1221. [Google Scholar] [CrossRef]
  19. Martianov, A.S.; Mitiushkina, N.V.; Ershova, A.N.; Martynenko, D.E.; Bubnov, M.G.; Amankwah, P.; Yanus, G.A.; Aleksakhina, S.N.; Tiurin, V.I.; Venina, A.R.; et al. KRAS, NRAS, BRAF, HER2 and MSI Status in a Large Consecutive Series of Colorectal Carcinomas. Int. J. Mol. Sci. 2023, 24, 4868. [Google Scholar] [CrossRef]
  20. Clancy, C.; Burke, J.P.; Kalady, M.F.; Coffey, J.C. BRAF Mutation Is Associated with Distinct Clinicopathological Characteristics in Colorectal Cancer: A Systematic Review and Meta-Analysis. Color. Dis. 2013, 15, e711–e718. [Google Scholar] [CrossRef]
  21. Cremolini, C.; Di Bartolomeo, M.; Amatu, A.; Antoniotti, C.; Moretto, R.; Berenato, R.; Perrone, F.; Tamborini, E.; Aprile, G.; Lonardi, S.; et al. BRAF Codons 594 and 596 Mutations Identify a New Molecular Subtype of Metastatic Colorectal Cancer at Favorable Prognosis. Ann. Oncol. 2015, 26, 2092–2097. [Google Scholar] [CrossRef]
  22. Jones, J.C.; Renfro, L.A.; Al-Shamsi, H.O.; Schrock, A.B.; Rankin, A.; Zhang, B.Y.; Kasi, P.M.; Voss, J.S.; Leal, A.D.; Sun, J.; et al. Non-V600 BRAF Mutations Define a Clinically Distinct Molecular Subtype of Metastatic Colorectal Cancer. J. Clin. Oncol. 2017, 35, 2624–2630. [Google Scholar] [CrossRef] [PubMed]
  23. Roth, A.D.; Tejpar, S.; Delorenzi, M.; Yan, P.; Fiocca, R.; Klingbiel, D.; Dietrich, D.; Biesmans, B.; Bodoky, G.; Barone, C.; et al. Prognostic Role of KRAS and BRAF in Stage II and III Resected Colon Cancer: Results of the Translational Study on the PETACC-3, EORTC 40993, SAKK 60-00 Trial. J. Clin. Oncol. 2010, 28, 466–474. [Google Scholar] [CrossRef] [PubMed]
  24. Fariña-Sarasqueta, A.; van Lijnschoten, G.; Moerland, E.; Creemers, G.; Lemmens, V.E.P.P.; Rutten, H.J.T.; van den Brule, A.J.C. The BRAF V600E Mutation Is an Independent Prognostic Factor for Survival in Stage II and Stage III Colon Cancer Patients. Ann. Oncol. 2010, 21, 2396–2402. [Google Scholar] [CrossRef] [PubMed]
  25. Venderbosch, S.; Nagtegaal, I.D.; Maughan, T.S.; Smith, C.G.; Cheadle, J.P.; Fisher, D.; Kaplan, R.; Quirke, P.; Seymour, M.T.; Richman, S.D.; et al. Mismatch Repair Status and BRAF Mutation Status in Metastatic Colorectal Cancer Patients: A Pooled Analysis of the CAIRO, CAIRO2, COIN, and FOCUS Studies. Clin. Cancer Res. 2014, 20, 5322–5330. [Google Scholar] [CrossRef] [PubMed]
  26. Kadowaki, S.; Ooki, A.; Yamaguchi, K.; Kakuta, M.; Takahashi, S.; Takahashi, A.; Arai, Y.; Akagi, K.; Shigenori Kadowaki, J.; Muro, K.; et al. Prognostic Value of KRAS and BRAF Mutations in Curatively Resected Colorectal Cancer Observational Study. World J. Gastroenterol. 2015, 21, 1275–1283. [Google Scholar] [CrossRef]
  27. Seligmann, J.F.; Fisher, D.; Smith, C.G.; Richman, S.D.; Elliott, F.; Brown, S.; Adams, R.; Maughan, T.; Quirke, P.; Cheadle, J.; et al. Investigating the Poor Outcomes of BRAF -Mutant Advanced Colorectal Cancer: Analysis from 2530 Patients in Randomised Clinical Trials. Ann. Oncol. 2017, 28, 562–568. [Google Scholar] [CrossRef]
  28. Lee, S.M.; Sung, C.O. Comprehensive Analysis of Mutational and Clinicopathologic Characteristics of Poorly Differentiated Colorectal Neuroendocrine Carcinomas. Sci. Rep. 2021, 11, 1–11. [Google Scholar] [CrossRef]
  29. Klempner, S.J.; Gershenhorn, B.; Tran, P.; Lee, T.K.; Erlander, M.G.; Gowen, K.; Schrock, A.B.; Morosini, D.; Ross, J.S.; Stephens, P.J.; et al. BRAFV600E Mutations in High-Grade Colorectal Neuroendocrine Tumors May Predict Responsiveness to BRAF-MEK Combination Therapy. Cancer Discov. 2016, 6, 594–600. [Google Scholar] [CrossRef]
  30. Nakano, M.; Shimada, Y.; Matsumoto, Y.; Saiki, T.; Zhou, Q.; Sasaki, K.; Moriyama, M.; Yoshihara, K.; Natsumeda, M.; Kuriyama, Y.; et al. Efficacy of BRAF Inhibitor and Anti-EGFR Antibody in Colorectal Neuroendocrine Carcinoma. Clin. J. Gastroenterol. 2022, 15, 413–418. [Google Scholar] [CrossRef]
  31. U.S. Food and Drug Afministration FDA Grants Accelerated Approval to Dabrafenib in Combination with Trametinib for Unresectable or Metastatic Solid Tumors with BRAF V600E Mutation. Available online: https://www.fda.gov/drugs/resources-information-approved-drugs/fda-grants-accelerated-approval-dabrafenib-combination-trametinib-unresectable-or-metastatic-solid (accessed on 7 May 2023).
  32. Scartozzi, M.; Giampieri, R.; Aprile, G.; Iacono, D.; Santini, D.; Dell’Aquila, E.; Silvestris, N.; Gnoni, A.; Bonotto, M.; Puzzoni, M.; et al. The Distinctive Molecular, Pathological and Clinical Characteristics of BRAF-Mutant Colorectal Tumors. Expert Rev. Mol. Diagn. 2015, 15, 979–987. [Google Scholar] [CrossRef] [PubMed]
  33. Chida, K.; Kotani, D.; Masuishi, T.; Kawakami, T.; Kawamoto, Y.; Kato, K.; Fushiki, K.; Sawada, K.; Kumanishi, R.; Shirasu, H.; et al. The Prognostic Impact of KRAS G12C Mutation in Patients with Metastatic Colorectal Cancer: A Multicenter Retrospective Observational Study. Oncologist 2021, 26, 845–853. [Google Scholar] [CrossRef] [PubMed]
  34. Henry, J.T.; Coker, O.; Chowdhury, S.; Shen, J.P.; Morris, V.K.; Dasari, A.; Raghav, K.; Nusrat, M.; Kee, B.; Parseghian, C.; et al. Comprehensive Clinical and Molecular Characterization of KRAS G12C -Mutant Colorectal Cancer. JCO Precis. Oncol. 2021, 5, 613–621. [Google Scholar] [CrossRef] [PubMed]
  35. Osterlund, E.; Ristimäki, A.; Kytölä, S.; Kuopio, T.; Heervä, E.; Muhonen, T.; Halonen, P.; Kallio, R.; Soveri, L.-M.; Sundström, J.; et al. KRAS-G12C Mutation in One Real-Life and Three Population-Based Nordic Cohorts of Metastatic Colorectal Cancer. Front. Oncol. 2022, 12, 826073. [Google Scholar] [CrossRef] [PubMed]
  36. Schirripa, M.; Nappo, F.; Cremolini, C.; Salvatore, L.; Rossini, D.; Bensi, M.; Businello, G.; Pietrantonio, F.; Randon, G.; Fucà, G.; et al. KRAS G12C Metastatic Colorectal Cancer: Specific Features of a New Emerging Target Population. Clin. Colorectal Cancer 2020, 19, 219–225. [Google Scholar] [CrossRef] [PubMed]
  37. Giampieri, R.; Lupi, A.; Ziranu, P.; Bittoni, A.; Pretta, A.; Pecci, F.; Persano, M.; Giglio, E.; Copparoni, C.; Crocetti, S.; et al. Retrospective Comparative Analysis of KRAS G12C vs. Other KRAS Mutations in MCRC Patients Treated with First-Line Chemotherapy Doublet + Bevacizumab. Front. Oncol. 2021, 11, 736104. [Google Scholar] [CrossRef]
  38. Roussille, P.; Tachon, G.; Villalva, C.; Milin, S.; Frouin, E.; Godet, J.; Berger, A.; Emambux, S.; Petropoulos, C.; Wager, M.; et al. Pathological and Molecular Characteristics of Colorectal Cancer with Brain Metastases. Cancers 2018, 10, 504. [Google Scholar] [CrossRef]
  39. Cui, W.; Franchini, F.; Alexander, M.; Officer, A.; Wong, H.L.; IJzerman, M.; Desai, J.; Solomon, B.J. Real World Outcomes in KRAS G12C Mutation Positive Non-Small Cell Lung Cancer. Lung Cancer 2020, 146, 310–317. [Google Scholar] [CrossRef]
  40. Vassella, E.; Kashani, E.; Zens, P.; Kündig, A.; Fung, C.; Scherz, A.; Herrmann, E.; Ermis, E.; Schmid, R.A.; Berezowska, S. Mutational Profiles of Primary Pulmonary Adenocarcinoma and Paired Brain Metastases Disclose the Importance of KRAS Mutations. Eur. J. Cancer 2021, 159, 227–236. [Google Scholar] [CrossRef]
  41. Skoulidis, F.; Li, B.T.; Dy, G.K.; Price, T.J.; Falchook, G.S.; Wolf, J.; Italiano, A.; Schuler, M.; Borghaei, H.; Barlesi, F.; et al. Sotorasib for Lung Cancers with KRAS p.G12C Mutation. N. Engl. J. Med. 2021, 384, 2371–2381. [Google Scholar] [CrossRef]
  42. R Core Team. R: A Language and Environment for Statistical Computing; R Core Team: Vienna, Austria, 2017. [Google Scholar]
Ijms 24 09073 g001 550
Figure 1. Data selection flowchart.
Figure 1. Data selection flowchart.
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Figure 2. Odds ratios for BRAF V600E mutation incidence according to selected factors: (a) According to patient’s age; (b) According to tumour histological type; (c) According to tumour location in the large intestine; (d) According to procedure resulting in the assessed sample.
Figure 2. Odds ratios for BRAF V600E mutation incidence according to selected factors: (a) According to patient’s age; (b) According to tumour histological type; (c) According to tumour location in the large intestine; (d) According to procedure resulting in the assessed sample.
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Figure 3. Geographical distribution of the incidence of (a) BRAF V600E and (b) KRAS G12C mutations. The lighter the colour the lower the mutation incidence.
Figure 3. Geographical distribution of the incidence of (a) BRAF V600E and (b) KRAS G12C mutations. The lighter the colour the lower the mutation incidence.
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Figure 4. Odds ratios for KRAS G12C mutation incidence according to selected factors: (a) According to patient’s age; (b) According to tumour histological type; (c) According to tumour location in the large intestine; (d) According to type of cancer lesion.
Figure 4. Odds ratios for KRAS G12C mutation incidence according to selected factors: (a) According to patient’s age; (b) According to tumour histological type; (c) According to tumour location in the large intestine; (d) According to type of cancer lesion.
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Table
Table 1. Clinical and pathological characteristics of the study population according to mutation status.
Table 1. Clinical and pathological characteristics of the study population according to mutation status.
ParameterPopulations Defined by Mutation Types
OverallBRAF V600EKRAS G12C Other KRAS
(N = 7604)(N = 515)(N = 237)(N = 3591)
SexFemale3132 (41.19%)295 (57.28%)101 (42.62%)1527 (42.52%)
Male4471 (58.81%)220 (42.72%)136 (57.38%)2064 (57.48%)
Age (years)<3021 (0.28%)0 (0.00%)0 (0.00%)10 (0.28%)
31–40115 (1.51%)6 (1.17%)6 (2.53%)49 (1.36%)
41–50432 (5.68%)22 (4.27%)13 (5.49%)190 (5.29%)
51–601298 (17.07%)74 (14.37%)36 (15.19%)588 (16.37%)
61–703192 (41.98%)222 (43.11%)111 (46.84%)1486 (41.38%)
71–802173 (28.58%)156 (30.29%)64 (27.00%)1070 (29.80%)
over 80373 (4.91%)35 (6.80%)7 (2.95%)198 (5.51%)
Tumour stageT139 (0.98%)0 (0.00%)1 (0.83%)25 (1.29%)
T2291 (7.29%)10 (3.33%)11 (9.09%)144 (7.44%)
T32611 (65.37%)175 (58.33%)81 (66.94%)1243 (64.20%)
T41039 (26.01%)115 (38.33%)28 (23.14%)516 (26.65%)
Tis14 (0.35%)0 (0.00%)0 (0.00%)8 (0.41%)
Nodal stage
(0 vs. 1 vs. 2)		N01006 (26.18%)57 (19.79%)37 (31.09%)486 (26.00%)
N11460 (37.99%)94 (32.64%)43 (36.13%)740 (39.59%)
N21377 (35.83%)137 (47.57%)39 (32.77%)643 (34.40%)
HistologyAdenocarcinoma NOS6364 (89.93%)362 (78.02%)196 (88.69%)3108 (88.52%)
Partially mucinous632 (8.93%)89 (58.17%)23 (56.10%)382 (56.26%)
Mucinous35 (0.49%)6 (3.92%)0 (0.00%)10 (1.47%)
Cribriform or comedo32 (0.45%)2 (1.31%)2 (4.88%)9 (1.33%)
MANEC14 (0.2%)5 (3.27%)0 (0.00%)2 (0.29%)
GradeG1590 (12.14%)26 (7.60%)17 (10.90%)299 (12.88%)
G23509 (72.22%)181 (52.92%)122 (78.21%)1708 (73.59%)
G3756 (15.56%)134 (39.18%)17 (10.90%)313 (13.49%)
G44 (0.08%)1 (0.29%)0 (0.00%)1 (0.04%)
Primary tumour
sidedness		Left colon or rectum4827 (75.46%)129 (30.57%)163 (82.32%)2255 (74.20%)
Right colon1570 (24.54%)293 (69.43%)35 (17.68%)784 (25.80%)
Sample typeEndoscopic biopsy1388 (22.91%)73 (17.76%)45 (22.84%)654 (22.58%)
Needle biopsy218 (3.6%)7 (1.70%)12 (6.09%)90 (3.11%)
Surgery4452 (73.49%)331 (80.54%)140 (71.07%)2152 (74.31%)
Sample sourcePrimary4968 (87.88%)346 (90.58%)153 (83.61%)2376 (88.43%)
Metastatic685 (12.12%)36 (9.42%)30 (16.39%)311 (11.57%)
Location of metastatic samplesLocal recurrence81 (11.82%)3 (0.79%)5 (2.73%)39 (1.45%)
Liver266 (38.83%)11 (2.88%)11 (6.01%)108 (4.02%)
Lung73 (10.66%)3 (0.79%)1 (0.55%)42 (1.56%)
Peritoneal148 (21.61%)12 (3.14%)5 (2.73%)76 (2.83%)
Nodal31 (4.53%)3 (0.79%)2 (1.09%)10 (0.37%)
Ovarian33 (4.82%)2 (0.52%)1 (0.55%)10 (0.37%)
Small intestine26 (3.8%)2 (0.52%)2 (1.09%)12 (0.45%)
CNS5 (0.73%)0 (0%)2 (1.09%)2 (0.07%)
Skin/subcutaneous22 (3.21%)0 (0%)1 (0.55%)12 (0.45%)
Expanded version available in the (Supplementary Data (Table S1)). Right colon (proximal) included caecum, ascending colon, hepatic flexure, and transverse colon. Left colon (distal) included splenic flexure and descending and sigmoid colon. CNS—central nervous system; MANEC—mixed adeno-neuroendocrine cancer; NOS—not otherwise specified.
Table
Table 2. Pathomorphological characteristics of primary tumours in BRAF V600E-mutated cancers.
Table 2. Pathomorphological characteristics of primary tumours in BRAF V600E-mutated cancers.
ParameterGroupp
BRAF V600E
Mutation (N = 515)		No BRAF V600E
Mutation (N = 7089)
Tumour stageT10 (0.00%)39 (1.06%)p < 0.001 *
T210 (3.33%)281 (7.61%)
T3175 (58.33%)2436 (65.94%)
T4115 (38.33%)924 (25.01%)
Tis0 (0.00%)14 (0.38%)
Nodal stageN057 (19.32%)949 (25.91%)p < 0.001 *
N1a40 (13.56%)476 (12.99%)
N1b43 (14.58%)692 (18.89%)
N1c11 (3.73%)198 (5.41%)
N2a55 (18.64%)594 (16.22%)
N2b82 (27.80%)646 (17.64%)
Nx7 (2.37%)108 (2.95%)
Nodal stage
(0 vs. 1 vs. 2)		N057 (19.79%)949 (26.69%)p < 0.001 *
N194 (32.64%)1366 (38.42%)
N2137 (47.57%)1240 (34.88%)
Nodal stage
(positive vs. negative)		Negative57 (19.79%)949 (26.69%)p = 0.013 *
Positive231 (80.21%)2606 (73.31%)
AngioinvasionNo41 (21.69%)775 (34.63%)p < 0.001 *
Yes148 (78.31%)1463 (65.37%)
Perineural invasionNo44 (38.94%)696 (51.98%)p = 0.01 *
Yes69 (61.06%)643 (48.02%)
Biopsy vs. surgery for
primary samples		Biopsy80 (19.46%)1526 (27.02%)p = 0.001 *
Surgery331 (80.54%)4121 (72.98%)
Right colon (proximal) included caecum, ascending colon, hepatic flexure, and transverse colon. Left colon (distal) included splenic flexure and descending and sigmoid colon. Rectum included rectosigmoid tumours. MANEC—mixed adeno-neuroendocrine cancer. p—for quantitative variables, Mann–Whitney test; for qualitative variables, chi-squared test or exact Fisher test. *—statistically significant difference (p < 0.05).
Table
Table 3. Clinical and pathological factors that affected the rate of BRAF V600E mutation.
Table 3. Clinical and pathological factors that affected the rate of BRAF V600E mutation.
TraitGroupBRAF V600EOR95% CIp
SexMale (N = 4471)220 (4.92%)1ref.
Female (N = 3132)295 (9.42%)2.0091.6772.408<0.001
Age (years)<40 (N = 136)6 (4.41%)1ref.
41–50 (N = 432)22 (5.09%)1.1630.4622.9290.749
51–60 (N = 1298)74 (5.70%)1.310.5593.0690.534
61–70 (N = 3192)222 (6.95%)1.620.7063.7130.255
71–80 (N = 2173)156 (7.18%)1.6760.7283.860.225
Over 80 (N = 373)35 (9.38%)2.2440.9225.4590.075
HistologyAdenocarcinoma NOS (N = 6364)362 (5.69%)1ref.
Partially mucinous (N = 632)89 (14.08%)2.7182.1213.482<0.001
Mucinous (N = 35)6 (17.14%)3.431.4158.3150.006
Cribriform or comedo (N = 32)2 (6.25%)1.1050.2634.6430.891
MANEC (N = 14)5 (35.71%)9.2113.07127.626<0.001
Mucous componentNo (N = 6555)385 (5.87%)1ref.
Yes (N = 1049)130 (12.39%)2.2671.8372.798<0.001
Signet cells presenceNo (N = 7187)474 (6.60%)1ref.
Yes (N = 417)41 (9.83%)1.5441.1042.160.011
GradeG1 (N = 590)26 (4.41%)1ref.
G2 (N = 3509)181 (5.16%)1.180.7751.7970.441
G3 (N = 756)134 (17.72%)4.6733.0247.221<0.001
G4 (N = 4)1 (25.00%)7.2310.72771.9090.091
Primary
tumour localization		Rectum/rectosigmoid (N = 2964)71 (2.40%)1ref.
Left colon (N = 1849)58 (3.14%)1.320.9281.8760.122
Right colon (N = 1570)293 (18.66%)9.3497.15512.216<0.001
Sample typeSurgery (N = 4452)331 (7.43%)1ref.
Endoscopy (N = 1388)73 (5.26%)0.6910.5320.8970.006
Needle (N = 218)7 (3.21%)0.4130.1930.8840.023
Sample originPrimary (N = 4968)346 (6.96%)1ref.
Metastatic (N = 685)36 (5.26%)0.7410.5211.0550.096
Biopsy vs. surgery for metastatic tissuesSurgery (N = 854)59 (6.91%)1ref.
Biopsy (N = 259)8 (3.09%)0.4290.2020.9110.028
Right colon (proximal) included caecum, ascending colon, hepatic flexure, and transverse colon. Left colon (distal) included splenic flexure and descending and sigmoid colon. Rectum included rectosigmoid tumours. MANEC—mixed adeno-neuroendocrine cancer. p—univariate logistic regressions. Expanded version available in the (Supplementary Data (Table S2)).
Table
Table 4. Pathomorphological characteristics of primary tumours in KRAS G12C-mutated cancers.
Table 4. Pathomorphological characteristics of primary tumours in KRAS G12C-mutated cancers.
ParameterGroup
KRAS G12C (N = 237)No KRAS G12C (N = 7367)pOther KRAS
(N = 3591)		p
Tumour stageT11 (0.83%)38 (0.98%)p = 0.835 p = 0.859
T211 (9.09%)280 (7.23%) 144 (7.44%)
T381 (66.94%)2530 (65.32%) 1243 (64.20%)
T428 (23.14%)1011 (26.10%) 516 (26.65%)
Tis0 (0.00%)14 (0.36%) 8 (0.41%)
Nodal stageN037 (30.33%)969 (25.26%)p = 0.051486 (25.34%)p = 0.062
N1a8 (6.56%)508 (13.24%) 272 (14.18%)
N1b27 (22.13%)708 (18.46%) 354 (18.46%)
N1c8 (6.56%)201 (5.24%) 114 (5.94%)
N2a26 (21.31%)623 (16.24%) 307 (16.01%)
N2b13 (10.66%)715 (18.64%) 336 (17.52%)
Nx3 (2.46%)112 (2.92%) 49 (2.55%)
Nodal stage
(0 vs. 1 vs. 2)		N037 (31.09%)969 (26.02%)p = 0.457486 (26.00%)p = 0.466
N143 (36.13%)1417 (38.05%) 740 (39.59%)
N239 (32.77%)1338 (35.93%) 643 (34.40%)
Nodal stage
(positive vs. negative)		Negative37 (31.09%)969 (26.02%)p = 0.257486 (26.00%)p = 0.265
Positive82 (68.91%)2755 (73.98%) 1383 (74.00%)
AngioinvasionNo26 (30.95%)790 (33.72%)p = 0.682434 (36.94%)p = 0.326
Yes58 (69.05%)1553 (66.28%) 741 (63.06%)
Perineural invasionNo25 (46.30%)715 (51.14%)p = 0.575375 (53.19%)p = 0.403
Yes29 (53.70%)683 (48.86%) 330 (46.81%)
Biopsy vs. surgery for
primary samples		Biopsy57 (28.93%)1549 (26.43%)p = 0.483744 (25.69%)p = 0.357
Surgery140 (71.07%)4312 (73.57%) 2152 (74.31%)
Right colon (proximal) included caecum, ascending colon, hepatic flexure, and transverse colon. Left colon (distal) included splenic flexure and descending and sigmoid colon. Rectum included rectosigmoid tumours. MANEC—mixed adeno-neuroendocrine cancer. p—for quantitative variables, Mann–Whitney test; for qualitative variables, chi-squared test or exact Fisher test. Expanded version available in the (Supplementary Data (Table S3)).
Table
Table 5. Clinical and pathological factors that affected the rate of KRAS G12C mutation.
Table 5. Clinical and pathological factors that affected the rate of KRAS G12C mutation.
TraitGroupKRAS G12COR95% CIp
SexMale (N = 4471)136 (3.04%)1ref.
Female (N = 3132)101 (3.22%)1.0620.8181.380.651
Age (years)<40 (N = 136)6 (4.41%)1ref.
41–50 (N = 432)13 (3.01%)0.6720.2511.8040.43
51–60 (N = 1298)36 (2.77%)0.6180.2561.4940.285
61–70 (N = 3192)111 (3.48%)0.7810.3371.8080.563
71–80 (N = 2173)64 (2.95%)0.6570.281.5470.337
Over 80 (N = 373)7 (1.88%)0.4140.1371.2560.119
HistologyPartially mucinous (N = 632)23 (3.64%)1ref.
Mucinous (N = 35)0 (0.00%)--------
Cribriform or comedo (N = 32)2 (6.25%)1.7650.3987.8380.455
MANEC (N = 14)0 (0.00%)--------
Other (N = 527)16 (3.04%)0.8290.4331.5860.571
Primary tumour
localization		Rectum (N = 2964)111 (3.74%)1ref.
Left colon (N = 1849)51 (2.76%)0.7290.5211.0210.066
Right colon (N = 1570)35 (2.23%)0.5860.3990.8610.007
Primary sidednessLeft colon or rectum (N = 4827)163 (3.38%)1ref.
Right colon (N = 1570)35 (2.23%)0.6520.4510.9440.024
Primary localization
(colon vs. rectum)		Colon (N = 4149)107 (2.58%)1ref.
Rectum (N = 2964)111 (3.74%)1.471.1221.9250.005
Sample typeSurgery (N = 4452)140 (3.14%)1ref.
Endoscopy (N = 1388)45 (3.24%)1.0320.7341.4520.856
Needle (N = 218)12 (5.50%)1.7940.9793.2890.059
Sample origin (primary vs. metastatic)Primary (N = 4968)153 (3.08%)1ref.
Metastatic (N = 685)30 (4.38%)1.4410.9662.150.073
Sample origin (detailed)Primary (N = 4968)153 (3.08%)1ref.
Metastatic—other (N = 598)23 (3.85%)1.2590.8051.9680.313
Metastatic—local (N = 82)5 (6.10%)2.0440.8155.1210.127
Metastatic—CNS (N = 5)2 (40.00%)20.983.48126.470.001
Biopsy vs. surgery for metastatic samplesSurgery (N = 854)32 (3.75%)1ref.
Biopsy (N = 259)14 (5.41%)1.4680.7712.7950.243
Right colon (proximal) included caecum, ascending colon, hepatic flexure, and transverse colon. Left colon (distal) included splenic flexure and descending and sigmoid colon. Rectum included rectosigmoid tumours. MANEC—mixed adeno-neuroendocrine cancer. p—for quantitative variables, Mann–Whitney test; for qualitative variables, chi-squared test or exact Fisher test. Expanded version available in the (Supplementary Data (Table S4)).
Table
Table 6. Clinical and pathological factors that affected the rate of KRAS G12C mutation among other KRAS mutations.
Table 6. Clinical and pathological factors that affected the rate of KRAS G12C mutation among other KRAS mutations.
TraitGroupKRAS G12COR95% CIp
SexMale (N = 2200)136 (6.18%)1ref.
Female (N = 1628)101 (6.20%)1.0040.771.3090.978
Age (years)<40 (N = 65)6 (9.23%)1ref.
41–50 (N = 203)13 (6.40%)0.6730.2451.8480.442
51–60 (N = 624)36 (5.77%)0.6020.2441.4880.272
61–70 (N = 1597)111 (6.95%)0.7350.311.7390.483
71–80 (N = 1134)64 (5.64%)0.5880.2451.4140.236
Over 80 (N = 205)7 (3.41%)0.3480.1131.0740.066
HistologyPartially mucinous (N = 405)23 (5.68%)1ref.
Mucinous (N = 10)0 (0.00%)--------
Cribriform or comedo (N = 11)2 (18.18%)3.6910.75318.0790.107
MANEC (N = 2)0 (0.00%)--------
Other (N = 292)16 (5.48%)0.9630.4991.8560.91
Primary tumour localizationRectum (N = 1533)111 (7.24%)1ref.
Left colon (N = 876)51 (5.82%)0.7920.5621.1150.182
Right colon (N = 819)35 (4.27%)0.5720.3870.8450.005
Primary sidednessLeft colon or rectum (N = 2418)163 (6.74%)1ref.
Right colon (N = 819)35 (4.27%)0.6180.4250.8980.012
Primary localization
(colon vs. rectum)		Colon (N = 2060)107 (5.19%)1ref.
Rectum (N = 1533)111 (7.24%)1.4251.0831.8740.011
Sample typeSurgery (N = 2292)140 (6.11%)1ref.
Endoscopy (N = 699)45 (6.44%)1.0580.7481.4970.752
Needle (N = 102)12 (11.76%)2.051.0963.8330.025
Sample origin (primary vs. metastatic)Primary (N = 2529)153 (6.05%)1ref.
Metastatic (N = 341)30 (8.80%)1.4980.9952.2550.053
Sample origin (detailed)Primary (N = 2529)153 (6.05%)1ref.
Metastatic—other (N = 293)23 (7.85%)1.3230.8382.0870.229
Metastatic—locoregional (N = 44)5 (11.36%)1.9910.7745.1240.153
Metastatic—CNS (N = 4)2 (50.00%)15.5292.173110.9970.006
Biopsy vs. surgery for metastatic samplesSurgery (N = 450)32 (7.11%)1ref.
Biopsy (N = 124)14 (11.29%)1.6620.8573.2240.132
Right colon (proximal) included caecum, ascending colon, hepatic flexure, and transverse colon. Left colon (distal) included splenic flexure, descending and sigmoid colon. MANEC—mixed adeno-neuroendocrine cancer. p—univariate logistic regressions.
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Potocki, P.M.; Wójcik, P.; Chmura, Ł.; Goc, B.; Fedewicz, M.; Bielańska, Z.; Swadźba, J.; Konopka, K.; Kwinta, Ł.; Wysocki, P.J. Clinical Characterization of Targetable Mutations (BRAF V600E and KRAS G12C) in Advanced Colorectal Cancer—A Nation-Wide Study. Int. J. Mol. Sci. 2023, 24, 9073. https://doi.org/10.3390/ijms24109073

AMA Style

Potocki PM, Wójcik P, Chmura Ł, Goc B, Fedewicz M, Bielańska Z, Swadźba J, Konopka K, Kwinta Ł, Wysocki PJ. Clinical Characterization of Targetable Mutations (BRAF V600E and KRAS G12C) in Advanced Colorectal Cancer—A Nation-Wide Study. International Journal of Molecular Sciences. 2023; 24(10):9073. https://doi.org/10.3390/ijms24109073

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Potocki, Paweł M., Piotr Wójcik, Łukasz Chmura, Bartłomiej Goc, Marcin Fedewicz, Zofia Bielańska, Jakub Swadźba, Kamil Konopka, Łukasz Kwinta, and Piotr J. Wysocki. 2023. "Clinical Characterization of Targetable Mutations (BRAF V600E and KRAS G12C) in Advanced Colorectal Cancer—A Nation-Wide Study" International Journal of Molecular Sciences 24, no. 10: 9073. https://doi.org/10.3390/ijms24109073

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