Clinical significance of the KRAS G13D mutation in anastomotic recurrence of colorectal cancer
- Authors:
- Published online on: March 28, 2023 https://doi.org/10.3892/ol.2023.13778
- Article Number: 192
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Copyright: © Matsunaga et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Colorectal cancer (CRC) is the third most common type of cancer worldwide in terms of the number of patients affected and the second most common in terms of the number of deaths, and its prevalence is increasing (1). Although a number of CRC cases can be cured by surgery, it is reported worldwide that recurrence occurs in ~30% of cases, even after curative resection (2), and prevention and early detection of recurrence are still major issues.
Local recurrence (LR) after resection for CRC is a serious issue. LR is defined as a recurrent lesion in or around the primary tumor site, including the pericolic tissue, the adjacent mesentery, lymph nodes or the suture line of anastomosis. Anastomotic recurrence (AR) has often been considered a type of LR, and its incidence has been reported to be 1–2% worldwide (3). AR has been shown to have a poorer prognosis and can progress to more advanced pathological stages than primary tumors (3). AR is thought to be caused by the implantation of exfoliated tumor cells into the anastomotic line, and the risk of AR is high in rectal cancer (3,4); however, to the best of our knowledge, the relationship with other clinicopathological factors has not been established. At present, there have been two reports on the genetic analysis of AR cases, but none of them have presented with any specific genetic features of AR (5,6). Our previous study reported on two cases of repeated AR following curative resection of CRC and both were revealed to have the KRAS G13D mutation (7). Although only two cases were reported, it may be hypothesized that the KRAS G13D mutation could contribute to the development of AR, as well as other aspects of recurrence, such as resistance and dormancy. The present study aimed to clarify the relationship between the KRAS G13D mutation and AR in CRC.
Patients and methods
Patients
The present study assessed 21 patients who underwent curative resection for CRC at the Department of Surgical Oncology, Faculty of Medicine, The University of Tokyo (Tokyo, Japan) between January 2005 and December 2019, and were diagnosed with AR. A total of 67 patients who were diagnosed with non-anastomotic LR (NALR) after curative resection were also included. Patients with hereditary CRC and colitis-associated cancer were excluded from the study. In the present study, AR was defined as ‘recurrence on the anastomotic line’; the recurrence site must be located on the anastomotic line and pathologically proven with a resection specimen or endoscopic biopsy. Recurrent lesions that were in contact with the anastomotic line, but mainly located outside of the bowel wall were not considered as AR and were classified as NALR. NALR did not include pelvic peritoneal dissemination or lateral lymph node recurrence in the present study. NALR was observed in 67 cases, and 21 cases of NALR matched to 21 cases of AR were used as a control group to compare the prevalence of the KRAS G13D mutation. In addition to the prevalence of the KRAS G13D mutation, the following clinicopathological findings were retrospectively evaluated: Sex, age, gross appearance type (classification of gross appearance), tumor size, histopathological type, tumor depth, lymph node metastasis, venous invasion, lymphatic invasion, preoperative carcinoembryonic antigen and carbohydrate antigen 19-9 levels and the association of the KRAS G13D mutation with prognosis (Table I). The clinicopathological findings were described according to the American Joint Committee on Cancer/International Union Against Cancer TNM classification, 8th edition (8).
The present study was conducted according to The Declaration of Helsinki and the study protocol was approved by the ethics committee of The University of Tokyo [approval no. 3252-(13)]. Informed consent was obtained in the form of an opt-out option on the website (http://all-1su.umin.jp/research/files/04_2.pdf).
DNA extraction from formalin-fixed, paraffin-embedded (FFPE) specimens
DNA was extracted from 10-µm FFPE specimens obtained from the University of Tokyo. A Maxwell® RSC DNA FFPE Kit (Promega Corporation) was used for DNA extraction. All samples were extracted after an overnight proteinase K digestion step at 70°C, and all extractions were performed according to the manufacturer's protocol.
KRAS G13D mutation detection by droplet digital polymerase chain reaction (ddPCR)
Mutation of the KRAS gene was examined by ddPCR using the QX200™ Droplet Digital™ PCR system (Bio-Rad Laboratories, Inc.). Each DNA sample was diluted to 3,000 ng/ml, as measured by a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Inc.). PCR reaction mixtures contained 12 µl ddPCR Supermix for Probes (Bio-Rad Laboratories, Inc.), 1.2 µl PrimePCR for ddPCR, 1.2 µl Uracil-DNA Glycosylase (UDG; New England BioLabs, Inc.) and 9.6 µl diluted DNA sample; 20 µl of the 24-µl reaction mixture was loaded in a DG8™ Cartridges for QX200™/QX100™ Droplet Generator (Bio-Rad Laboratories, Inc.) and droplets were generated. The entire droplet emulsion volume was further loaded in ddPCR™ 96-Well Plates (Bio-Rad Laboratories, Inc.). The loaded 96-well PCR plate was then heat-sealed with pierceable foil in the PX1™ PCR Plate Sealer and placed in a T100™ Thermal Cycler (both from Bio-Rad Laboratories, Inc.). Amplification was conducted as follows: 95°C for 1 min, followed by 40 cycles at 55°C for 10 min, 94°C for 30 sec and a final extension step at 98°C for 10 min. Commercial primers (PrimePCR for ddPCR KRAS G13D, assay ID: dHsaMDV2510598; Bio-Rad Laboratories, Inc.) were used. UDG was used to limit the chances of artifacts due to formalin-fixation (9).
KRAS mutation detection in clinical practice
RAS mutation status (KRAS exon 2, 3, or 4 mutation, NRAS exon 2, 3, or 4 mutation) was evaluated using the PCR-reverse sequence-specific oligonucleotide method (BML, Inc.).
Statistical analysis
All statistical analyses were carried out using EZR version 4.1.2 (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R version 3.0.2 (The R Foundation) (10). Comparisons were performed using χ2 test or Fisher's exact test for categorical variables. In univariate analysis using Fisher's exact test, odds ratio and their 95% confidence intervals were estimated. Survival curves were drawn using the Kaplan-Meier method and compared using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.
To reduce potential confounding effects and treatment selection bias, case matching was conducted in the comparative analysis of AR and NALR. The following five factors that could affect KRAS status were selected: Age, sex, cancer location, histological type and cancer stage. R version 3.0.2 package ‘optmatch (https://cran.r-project.org/web/packages/optmatch)’ was used for the matching.
Results
The study population comprised 21 AR cases and 21 matched NALR cases. The clinicopathological characteristics of patients with AR and NALR before matching are shown in Table SI. The median follow-up period was 52.3 months. The clinicopathological characteristics of patients with AR or NALR after matching are shown in Table I.
There was no significant difference in the clinicopathological characteristics between the matched AR and the NALR groups, whereas the KRAS G13D mutation rate was significantly higher in the AR group; AR 33.3% (7/21) vs. NALR 4.8% (1/21) (P=0.047; Fig. 1).
The pathological findings on the recurrent lesions and details of treatment and prognosis in the AR group are shown in Table II. Of the 14 patients who tested negative for the KRAS G13D mutation in the present study, the KRAS status of 8 cases was evaluated for clinical purposes; 2 cases were positive for the KRAS G12D mutation, and the remaining 6 cases presented with wild-type KRAS. All of the 7 patients who tested positive for the KRAS G13D mutation were evaluated for KRAS status in the clinical setting, and there were no cases with double KRAS mutations. The RAS status of the matched NALR group is shown in Table SII. Of the 20 patients who tested negative for the KRAS G13D mutation, the KRAS status of 10 cases was evaluated for clinical purposes; three cases were positive for the KRAS G12D mutation, two cases were positive for the KRAS G12V mutation, one case was positive for the KRAS G12A mutation and the remaining four cases presented with wild-type KRAS.
Comparing the KRAS G13D mutation-positive (KRAS G13D+) and KRAS G13D mutation-negative (KRAS G13D−) patients in the AR group, there was no significant difference in the clinicopathological background (Table III), interval from initial surgery to AR (Fig. 2A) and recurrence resection rate (Table III). On analyzing the 17 cases who underwent surgical resection of AR, even though there was no significant difference in recurrence-free survival (RFS) after resection (2-year RFS after resection: KRAS G13D+ 0% vs. KRAS G13D− 33.3%; P=0.10), all KRAS G13D+ patients experienced subsequent recurrence within 2 years (Fig. 2B). Notably, KRAS G13D+ patients had a significantly poorer overall survival (OS) (3-year OS: KRAS G13D+ 68.6% vs. KRAS G13D− 90.9%; P=0.02) (Fig. 2C). The rate of synchronous recurrence at the diagnosis of AR was 28.6% (2/7) in KRAS G13D+ patients and 42.9% (6/14) in KRAS G13D− patients. There was no significant difference in the synchronous recurrence patterns between patients with and without the KRAS G13D mutation (Table IV). By contrast, KRAS G13D+ patients tended to experience more instances of metachronous locoregional recurrences (P=0.10), and repetitive AR was observed in KRAS G13D+ patients alone (P=0.07) (Table V); 80.0% (4/5) of the KRAS G13D+ patients experienced subsequent distant metastatic recurrences.
Discussion
The present study observed a high KRAS G13D mutation rate in patients with AR (AR 33.3% vs. matched NALR 4.8%; P=0.047). All KRAS G13D+ patients who underwent curative resection for AR had subsequent recurrence within 2 years, and the OS was poorer than that of KRAS G13D− patients (3-year OS: 68.6% vs. 90.9%; P=0.02).
KRAS is one of the key driver genes in CRC and is detected early in the carcinogenesis of CRC. The KRAS mutation rate in CRC is reported to be 30–40% worldwide, and KRAS codon 13 mutations, including KRAS G13D, are reported to occur in 6–8% of cases worldwide (11–13). In a large-scale study in Japan, the total KRAS mutation rate was 37.6%, and the KRAS codon 13 mutation rate was 7.7%, which was comparable to the results reported in Western countries (14).
The 33.3% KRAS G13D mutation rate among patients with AR in the present study was higher than that reported in previous studies, suggesting that there may be a certain association between the KRAS G13D mutation and AR. In addition, there was a significant difference in the KRAS G13D mutation rate between AR and NALR, suggesting that this is a specific characteristic of AR rather than of LR. Andreyev et al (11) reported that G to A mutations in the KRAS gene, which include the KRAS G13D mutation, were more frequent (58.3%) among patients with AR than among patients with other types of recurrence (~22%) (P=0.02), and the results of this previous study are consistent with the current findings.
Although several reports have stated that CRC with KRAS mutations has a poor prognosis (14–16), there are few reports on the association between prognosis and each KRAS subtype, and consistent results have not been obtained (17–20). Notably, the association between KRAS subtypes and the clinical significance of CRC has been reported in few studies. Kodaz et al (21) reported that the KRAS G13D mutation was more frequent in the left colon and in patients <70 years old, whereas the KRAS G12D mutation was more frequent in the right colon and in patients >50 years old. Bazan et al (18) reported that codon 12 KRAS mutations were associated with mucinous histology, whereas codon 13 KRAS mutations were associated with lymph node metastasis and advanced Dukes' stage.
In contrast to previous reports, the present study focused on AR cases, which may be responsible for the difference in prognosis between the cases with KRAS G13D mutation and those without the mutation. Several in vitro analyses have shown the characteristics of KRAS subtypes using colon cancer cell lines. Organ et al (22) reported that DLD1, a colon cancer cell line that is positive for the KRAS G13D mutation, showed a higher adhesion ability to the extracellular matrix and migration, in contrast to DKO4, a cell line in which KRAS G13D was knocked out of DLD1 cells. It was hypothesized that the KRAS G13D mutant may have an enhanced adhesion ability to the extracellular matrix and migration compared with wild-type KRAS, and these characteristics may contribute to the implantation of tumor cells into the anastomotic line, which is the pathogenic mechanism of AR. Stolze et al (23) reported that KRAS G13D, unlike other subtypes of KRAS mutation, showed a high expression of epidermal growth factor receptors (EGFRs) and high activation of proliferative signaling in the presence of EGF. In the tissue repair process at the anastomotic site, the role of growth factors is important; therefore, these characteristics of the KRAS G13D mutation may be responsible for AR.
In the present study, KRAS G13D+ patients with AR had a poor prognosis, probably because all KRAS G13D+ patients with AR experienced a subsequent recurrence after undergoing resection for AR. Margonis et al (24) reported that the KRAS codon 13 mutation was a risk factor for extrahepatic and pulmonary recurrence after curative resection of liver metastasis of CRC, and this was not observed for all KRAS mutations. Owing to the small number of AR cases in the present study, statistically significant difference was not be observed; however, it was observed that 80% (4/5) of the KRAS G13D+ patients experienced subsequent distant metastatic recurrence after curative resection for AR. As aforementioned, it has been reported that the KRAS G13D mutation enhances the adhesion and migratory ability, and is associated with increased proliferative signaling. These characteristics may be responsible for the pathogenesis of AR, in addition to the subsequent recurrences and poor prognoses.
The poor prognosis of KRAS G13D+ patients could also be attributed to the absence of an indication for a regimen including anti-EGFR antibodies (21). However, only 2 of the 14 patients with AR diagnosed as KRAS G13D− received chemotherapy including anti-EGFR antibodies in the present study; therefore, the effect of anti-EGFR antibodies may be limited. Few reports have suggested that the KRAS G13D mutation differs from other KRAS subtypes and may benefit from treatment with the anti-EGFR antibody cetuximab (25,26), but this has been doubted by some reports (27) and no conclusion has been reached. The therapeutic efficacy of anti-EGFR antibody therapy in KRAS G13D+ requires further study.
There were several limitations to the present study. First, it was a single-center, retrospective study with a small number of patients. Second, the study only analyzed the KRAS G13D mutation and did not consider other KRAS subtypes or BRAF mutations. Although 8 KRAS G13D− patients were tested for the RAS status in the clinical setting, 6 KRAS G13D− patients were not tested. Therefore, the KRAS G13D− group may have included patients with other KRAS subtypes or BRAF mutations. Additionally, 2 cases belonging to the KRAS G13D− group possessed the KRAS G12D mutation. Third, there was no analysis of the effect of AR on patient survival compared with the patients with NALR.
In conclusion, the KRAS G13D mutation rate was significantly higher in patients with AR, and patients with AR and the KRAS G13D mutation had a poorer prognosis than KRAS G13D− patients with AR. Although the role of the KRAS G13D mutation in the development of AR requires further investigation, postoperative surveillance and treatment strategies should be considered with attention to the possibility of AR and subsequent recurrence in KRAS G13D mutation-positive patients. Although a definitive conclusion could not be reached due to the small sample size and the fact that it was not considered that mutations other than KRAS G13D may affect the outcome, the present results may be worth confirming in future studies containing a larger number of patients.
Supplementary Material
Supporting Data
Acknowledgements
The authors would like to thank Dr Shinya Abe and Dr Yuzo Nagai (Department of Surgical Oncology, The University of Tokyo, Tokyo, Japan) for their advisory assistance.
Funding
The present study was supported by Grants-in-Aid for Scientific Research (grant nos. 21H02778, 18K07194, 19K09114, 19K09115 and 20K09051) and Challenging Research (Exploratory; grant no. 20K21626) from the Japan Society for the Promotion of Science and by the Project for Cancer Research and Therapeutic Evolution (grant no. JP 19cm0106502) from the Japan Agency for Medical Research and Development.
Availability of data and materials
The datasets generated and/or analyzed during the current study are not publicly available due to a license agreement with the University of Tokyo, but are available from the corresponding author on reasonable request.
Authors' contributions
KeM, KS and KH substantially contributed to the study conception, design and analysis of data. KeM, KU, SK and TY substantially contributed to the acquisition of laboratory data. HN, KK, KoM, SE, YY, HS substantially contributed to the acquisition of clinical data. HY and SI substantially contributed to the interpretation of data. KeM and KS confirm the authenticity of all the raw data. HY and SI gave final approval of the version to be published. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study protocol was approved by the ethics committee of The University of Tokyo [approval no. 3252-(13); Tokyo, Japan]. This study was conducted in accordance with The Declaration of Helsinki.
Patient consent for publication
Informed consent was obtained in the form of an opt-out option on the website for the participation in the research (http://all-1su.umin.jp/custom8.html).
Competing interests
The authors declare that they have no competing interests.
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