Oncologic relevance of genetic alterations in sporadic synchronous and solitary colorectal cancer: a retrospective multicenter study

Background Oncologic impact of genetic alteration across synchronous colorectal cancer (CRC) still remains unclear. This study aimed to compare the oncologic relevance according to genetic alteration between synchronous and solitary CRC with performing systematic review. Methods Multicenter retrospective analysis was performed for CRC patients with curative resection. Genetic profiling was consisted of microsatellite instability (MSI) testing, RAS (K-ras, and N-ras), and BRAF (v-Raf murine sarcoma viral oncogene homolog B1) V600E mutation. Multivariate analyses were conducted using logistic regression for synchronicity, and Cox proportional hazard model with stage-adjusting for overall survival (OS) and disease-free survival (DFS). Results It was identified synchronous (n = 36) and solitary (n = 579) CRC with similar base line characteristics. RAS mutation was associated to synchronous CRC with no relations of MSI and BRAF. During median follow up of 77.8 month, Kaplan–meier curves showed significant differences according to MSI-high for OS, and in RAS, and BRAF mutation for DFS, respectively. In multivariable analyses, RAS and BRAF mutation were independent factors (RAS, HR = 1.808, 95% CI = 1.18–2.77, p = 0.007; BRAF, HR = 2.417, 95% CI = 1.32–4.41, p = 0.004). Old age was independent factor for OS (HR = 3.626, 95% CI = 1.09–12.00, p = 0.035). Conclusion This study showed that oncologic outcomes might differ according to mutation burden characterized by RAS, BRAF, and MSI between synchronous CRC and solitary CRC. In addition, our systematic review highlighted a lack of data and much heterogeneity in genetic characteristics and survival outcomes of synchronous CRC relative to that of solitary CRC.


Introduction
Multiplicity in colorectal cancer (CRC) is defined as a synchronous cancer in which at least one additional colorectal tumor is detected simultaneously to the initially diagnosed primary CRC in a single individual.Although synchronous CRC accounts for a small proportion of cases, approximately 2%-10% of all CRCs [1,2], it differs from solitary CRC in terms of the extent of curative resection required for the tumor location [3] and molecular genomic heterogeneity [4,5] because tumor carcinogenesis is influenced by genetic, epigenetic, and environmental factors [6].Those complexities can affect the management and prognosis of synchronous CRC.
Previous studies have compared the clinicopathologic features and prognosis between synchronous and solitary CRC [3,[7][8][9][10][11][12][13][14].However, some studies included CRC patients with a metachronous tumor or stage IV cancer and others lacked data for synchronous CRCrelated gene information or survival outcomes in large populations.In our prior literature reviews [15,16], we identified heterogeneity in the oncologic outcomes in studies comparing both cancers [1, 6, 9, 11-14, 17, 18].Study populations that include patients with Lynch syndrome or familial adenomatous polyposis might exhibit selection bias, as synchronous CRCs harboring multiple genes associated with hereditary tumors rather than sporadic tumors can show heterogeneous prognosis [6,16,[19][20][21].Synchronous CRCs are frequently characterized by microsatellite instability (MSI), which is caused by epigenetic inactivation of the MLH1 gene via promoter methylation, whereas Lynch syndrome is caused by germline mutations in the mismatch repair (MMR) genes [22].Furthermore, the V600E mutation in BRAF (v-Raf murine sarcoma viral oncogene homolog B1) in MSIhigh CRC with methylation of MLH1 promoters is associated with poor prognosis and decreased likelihood of Lynch syndrome [23].Previous studies have established the prognostic roles of MSI status, BRAF mutations, and RAS mutations in solitary CRC with concordance for MSI-deficient status [23][24][25][26].However, the oncologic impact of these genetic factors in synchronous CRC, relative to solitary CRC, remains unclear [27].
Therefore, the aims of this study were to compare the oncologic relevance of BRAF mutations, KRAS mutations, and MSI status between synchronous and solitary CRC.In addition, we performed a systematic review of prior studies that compared the genetic status and survival outcomes between synchronous and solitary CRC.

Methods
We retrospectively reviewed the medical records of CRC patients who underwent curative surgery at four tertiary hospitals (Hallym Sacred Heart Hospital, Dontan Sacred Heart Hospital, Kangnam Sacred Heart Hospital, and Kangdong Sacred Heart Hospital) between March 2014 and December 2020.Patients diagnosed with familial adenomatous polyposis, hereditary non-polyposis colorectal cancer (HNPCC), and patients with inflammatory bowel disease combined with a metachronous malignancy were excluded from this study.Patients diagnosed with clinical or pathological stage IV CRC and patients without genetic information regarding their MSI status, BRAF mutations, and RAS mutations were also excluded.The study protocol was approved by the institutional review board (a central IRB No. 2022-12-022).The review board waived the requirement for informed consent because this study involved retrospective analyses.
Synchronous CRC was defined as follows: each lesion must be diagnosed as malignant, separate entities and must not be metastases of another tumor; and the synchronous lesions must be diagnosed simultaneously or within 6 months of diagnosis of the first tumor.For synchronous cancer, the most pathologically advanced lesion was defined as the index tumor.The tumor location and pathological status were defined relative to the index tumor.Asymptomatic patients were diagnosed in healthcare screening.Surgical resection was classified into three categories.Single segmental resection was defined as radical resection.Multiple segmental resections were defined as two radical resections with two anastomoses.Extended resection included total colectomy, subtotal collection, or total proctocolectomy.The surgical procedure, use of adjuvant/neoadjuvant therapy, and postoperative surveillance were determined by the attending physician based on the pathologic stage and the general condition of the patient in accordance with the National Comprehensive Cancer Network guideline [28].
After histologic examination, we performed real-time polymerase chain reaction analysis of BRAF codon 600 and MSI status, and peptide nucleic acid clamp of KRAS (mutation in codons 12 and 13 of exon 2, codon 61 of exon 3, and codon 146 of exon 4), and NRAS (mutation of exons 2, 3, and 4) [29][30][31][32].RAS mutations were defined as mutations in KRAS or NRAS.MSI status was determined using the markers BAT25, BAT26, NR21, NR24, and NR27 and classified as MSI-high (two or more unstable markers) or microsatellite stable (MSS; one or no unstable marker).
Categorical variables were compared using Pearson's χ 2 test, and continuous variables were compared using Student's t test.Multivariable binomial logistic regression analysis was performed to evaluate associations between the genetic factors and CRC synchronicity.The Kaplan-Meier method with the log-rank test was used to assess disease-free survival (DFS) and overall survival (OS) in patients stratified by RAS mutations, BRAS mutations, and MSI status.The proportional hazard ratio model with adjustment for pathological stage was used for multivariable analyses of DFS and OS.In all analyses, p-values of < 0.05 were considered statistically significant.All statistical analyses were performed using SPSS version 25.0 (SPSS Inc., Chicago, IL, USA).
We also performed systematic searches of PubMed, Cochrane Library, EMBASE, Medline, and Web of Science to identify all of the available studies on synchronous CRC that had been published and indexed up to October 31, 2022.We searched for comparative studies of solitary and synchronous or multiple CRC and excluded studies of metachronous CRC and noncomparative studies.Medical Subject Headings (MeSH) terms and Emtree terms were used in PubMed and EMBASE, respectively, together with separate words or word combinations to search the title or abstract.

Results
Of 615 patients available in the medical records, 36 patients (5.9%) were diagnosed for with synchronous CRC.The clinical characteristics were similar between the solitary CRC and synchronous CRC patients (Table 1).Multiple segmental resection or extended resection was performed in a greater proportion of synchronous CRC patients than solitary CRC patients (p < 0.001).Lymph node metastasis was more common (p = 0.037), and the number of harvested and retrieved lymph nodes was significantly greater (p = 0.002) in synchronous CRC patients (Table 2).Regarding genetic mutations, RAS mutations were more frequent in solitary CRC patients than in synchronous CRC patients (51.6% vs. 16.7,p < 0.001; Table 3).A multivariable analysis showed that the presence of RAS mutations was significantly associated with reduced risk of synchronous CRC, with an odds ratio (OR) of 0.184 (95% confidence interval [CI] 0.07-0.45,p = 0.001).In addition, when we assessed genetic mutations stratified by tumor location, RAS mutations were more frequently detected in solitary CRC for both right-sided (56.6% vs. 6.3%, p < 0.001) and leftsided (52.2% vs. 21.4%,p = 0.026) tumors, whereas no differences were found for MSI status or BRAF mutations.The multivariable analyses showed that synchronous CRCs were less frequently associated with RAS mutations for both right-sided (OR = 0.05, 95% CI 0.01-0.39,p = 0.004) and left-sided (OR = 0.025, 95% CI 0.07-0.92,p = 0.038) tumors.MSI status and BRAF mutations were not associated with tumor location (Table 4).

Discussion
This study showed that DFS might be influenced by the mutation burden, independent to clinical factors, or tumor burden in synchronous and solitary CRC.The genetic profiles revealed that RAS and BRAF mutations were associated with more pronounced effects than MSI on DFS.In this study, old age was the only risk factor for OS, regardless of the mutation profile and pathologic stage, and OS was not associated with MSI status in synchronous CRC.By contrast, a previous study suggested that, due to the concordance between MSI status and synchronicity, older individuals are more likely to develop multiple cancers through the MSI pathway, secondary to a widespread CpG island methylator phenotype (CIMP) and silencing of the MMR gene MLH1 by genetic and or environmental factors [6,33].However, the systematic review of studies comparing synchronous and solitary CRC revealed a lack of published data, as well as much heterogeneity in genetic and survival information, with unclear associations of clinical factors and genetic profiles with the prognosis of synchronous CRC (Table 6).Thus, we consider that the association between genetic mutations and the prognosis of synchronous CRC patients remains unclear and is open to debate.Undetermined association might implicate a complex hypothetical predisposition of development to synchronous CRC.Field effect as cancerization concept of molecular alterations induced by global DNA methylation such as LINE-1 methylation, MGMT promoter methylation or other CpG island methylation, has been proposed to explain the development of multiple primary malignancies in the same organ [34].The epigenetic field effect can drive concordant or discordant genetic patterns in synchronous cancer pairs, which also exhibit different phenotypes depending on the MSI status, as a confounding effect that can lead to a worse prognosis than the corresponding solitary tumor [33].Therefore, we suggest that many contributing factors to the controversy over the prognosis for synchronous CRC patients should be addressed in a controlled dataset of clinical, pathological, genetic, and survival information, accompanied by intensive surveillance.
As a commonly identified genotype in multiple CRCs, MSI arising from promoter methylation of the biallelic hMLH1 gene differs to that arising from the HNPCC pathway [37].In sporadic CRC, the risk of synchronicity was higher (2.14-fold) in patients with MSI than in patients with MSS, but there were no relationships between clinical features and the MSI genotype [38].Global hypermethylation of colorectal epithelium, as     independent events, could increase the frequency of multiple CRCs in older age instead of developing from a predisposition to cancer in patients with sporadic MSI CRC [20].In terms of the oncologic outcomes, the association between MSI status and greater tumor burden in synchronous CRC suggests that synchronicity and BRAF mutations are risk factors for OS in patients with MSS CRC, but the disease-specific survival of MSI CRC patients was unaffected by synchronicity in a stageadjusted analysis [33].By contrast, a prospective cohort study found that the overall mortality was greater in synchronous CRC patients than in solitary CRC patients, and the authors reported that multiple colon cancers arose through the serrated pathway, which is characterized by high frequencies of BRAF mutations, CIMP-high, and MSI-high [6].Another study reported contradictory results regarding the rarity of BRAF c.1799T4A mutation in synchronous advanced malignancies as a stage-independent predictor of poor prognosis in association with MSS, which is incompatible with the various epigenetic defects of synchronous CRCs [33].
Poor oncologic outcomes of synchronous CRC, in terms of the OS, DFS, and cancer-specific survival (CSS), were reported in a previous study using a matched-pairs analysis [36] (Table 6).Following disease relapse, it is important to select the most appropriate moleculartargeted drug by performing biomarker analysis [12].However, the greater and heterogenous mutation burden of paired tumors makes it difficult to identify the most appropriate target, resulting in poor prognosis of patients with relapsed synchronous CRC.Adjuvant therapeutic strategies have not yet been established for relapsed synchronous CRC, and we are still dependent on the clinical guidelines for solitary CRC.For synchronous CRCs within the same patient, it has been reported that paired lesions display heterogeneity in canonical genes, including APC, KRAS, TP53, and PIK3CA, together with a high frequency of mutations, compared with solitary CRC.Therefore, when drugs such as vemurafenib and dabrafenib, which target the BRAF mutation pV600E, are used to treat one lesion, the other lesion might be unresponsive due to the heterogeneous mutation profile of paired synchronous CRCs [39].Those molecular profiles develop independently, and lesions present with different gene copy numbers resulting in unique gene signatures in each lesion, combined with clonal mutations at different loci and accumulated timing [4].When treating patients with BRAF-mutated synchronous CRC, in particular, the MSI status and genetic heterogenicity of the paired tumors should be considered rather than the tumor burden or clinical stage.According to a systematic review of patients with BRAF-mutated CRC, MSS was associated with worse prognosis than MSI, but the clinical stratification by MSI testing and heterogeneity of genetic mutations have not been established for patients with BRAF-mutated synchronous CRC [40].Physician should also be aware that the poor prognosis of synchronous cancers might be independent of genetic factors such as BRAF mutations, MSI-high, and CIMP-high due to unidentified molecular events caused by the genetic or environmental background [6].Several markers, such as the transcriptional effector RPL22, a candidate gene involved in nodal/transforming growth factor-β and the ribosomal protein-murine double minute 2 (MDM2)-p53 signaling pathway [4], as well as different methylation rates of CACNA1G, NEUROG1, and CDKN2A (p16) [1], might confound analyses of the prognosis of synchronous CRC.Some studies have also demonstrated similar or better prognosis of synchronous CRC patients compared with solitary CRC patients, regardless of CIMP status and KRAS or BRAF mutations [1].This prognostic pattern was observed in several studies that lacked genetic information [8,9,11,13] (Table 6).Although there was no clear explanation for this finding, intensive perioperative colonoscopy detected associated adenomas that are more prone to progress into multiple colorectal cancers in old patients and slow growing tumors with an uncharacterized predisposition [11,13,35].In addition, advanced surgical procedures could achieve comparable long-term outcomes for synchronous CRC patients, regardless of whether they underwent resection of more than two regions or extensive resection of a single region [3].
This study has some limitations, including a small sample size due to the exclusion of synchronous CRC patients without genetic information, which reduced the statistical power.The absence of data regarding CIMP status and germline mutation of MMR genes might also introduce bias in terms of assessing whether the synchronous CRCs were sporadic or Lynch-associated tumors.Furthermore, there were no data for palliative therapy for synchronous CRC patients with distant recurrence.Genetic information for BRAF mutation in included patients was not available in solitary CRC patients (n = 169, 29.1%), but all genetic information was available in synchronous CRC patients.
In conclusion, this study showed that the oncologic outcomes might differ according to the mutation burden characterized by RAS, BRAF, and MSI between synchronous CRC and solitary CRC.Furthermore, RAS and BRAF mutations were associated with worse DFS compared with MSI status, independently of clinical factors, stage, and tumor burden.Our systematic review highlighted a lack of data and much heterogeneity in the genetic characteristics and survival outcomes of synchronous CRC relative to that of solitary CRC.These factors make it difficult to predict the prognosis of synchronous CRC and complicate the decision-making process when selecting the most appropriate target drug following relapse of synchronous CRC.

Fig. 1 Fig. 2 Fig. 3
Fig. 1 The Kaplan-Meier curves stratified by RAS mutation status and tumor type.(a) disease-free survival and (b) overall survival

Table 1
Clinical characteristics of the patients between solitary and synchronous CRC patients CRC Colorectal cancer, SD Standard deviation, BMI Body mass index, ASA American society of anesthesiologist, CEA Carcinoembryonic antigen

Table 2
Pathologic features of the patients between solitary and synchronous CRC patients CRC Colorectal cancer, LN Lymph node, SD Standard deviation, LVI Lymphovascular invasion, PNI Perineural invasion, MSI Microsatellite instability, MSI-H Microsatellite instability high, MSS Microsatellite stable

Table 3
Distribution of gene mutation and risk for synchronicity of colorectal cancer CRC Colorectal cancer a Not available data b binomial logistic regression model for synchronicity of CRC

Table 4
Genetic profiling for solitary and synchronous colorectal cancer according to tumor location HR Hazard ratio, CI Confidence interval, MSI Microsatellite instability, MSS Microsatellite stable, MSI-H Microsatellite instability high

Table 5
Univariate and multivariate analyses for disease-free survival and overall survival HR Hazard ratio, CI Confidence interval, MSI Microsatellite instability, MSI-H Microsatellite instability high, MSS Microsatellite stable a Pathologic stage-adjusted regression model

Table 6
A systematic review of synchronous CRC compared with solitary CRC

Table 6
(continued) Synch Synchronous, PCS Prospective cohort study, RCS Retrospective cohort study, RPS Retrospective population-based study, PSM Propensity score-matched analysis; Study including patients with hereditary colorectal cancer, or Lynch syndrome or, inflammatory bowel disease or, familial adenomatous polyposis or, metachronous cancer CRC Colorectal cancer, * valuse with a statistical significance