Molecular Mosaics: Unveiling Heterogeneity in Synchronous Colorectal Cancers

Hyun Gu Lee; Yeseul Kim; Mi-Ju Kim; Yeon Wook Kim; Sun-Young Jun; Deokhoon Kim; In Ja Park; Seung-Mo Hong

doi:10.4143/crt.2024.947

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Original Article Gastrointestinal cancer Molecular Mosaics: Unveiling Heterogeneity in Synchronous Colorectal Cancers: Hyun Gu Lee^1,a), Yeseul Kim², Mi-Ju Kim³, Yeon Wook Kim³, Sun-Young Jun⁴, Deokhoon Kim⁵, In Ja Park¹, Seung-Mo Hong⁵; Cancer Research and Treatment : Official Journal of Korean Cancer Association 2026;58(1):264-274.
DOI: https://doi.org/10.4143/crt.2024.947
Published online: February 18, 2025

¹Division of Colon and Rectal Surgery, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea

²Department of Pathology, Korea University Anam Hospital, Korea University College of Medicine, Seoul, Korea

³Asan Institute for Life Sciences, Asan Medical Center, Seoul, Korea

⁴Department of Pathology, Incheon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea

⁵Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea

Correspondence: In Ja Park, Division of Colon and Rectal Surgery, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea
Tel: 82-2-3010-3938 E-mail: ipark@amc.seoul.kr

Co-correspondence: Seung-Mo Hong, Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 05505, Korea
Tel: 82-2-3010-4558 E-mail: smhong28@gmail.com

^*Hyun Gu Lee and Yeseul Kim contributed equally to this work.
^a)Present address: Department of Surgery, Kyung Hee University Hospital at Gangdong, Kyung Hee University College of Medicine, Seoul, Korea

• Received: September 30, 2024 • Accepted: February 17, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Abstract

Purpose
Molecular characteristics of synchronous colorectal cancers (SCRCs) remain incompletely elucidated, despite their importance in targeted therapy selection. We compared the molecular characteristics and somatic mutations between SCRCs.
Materials and Methods
This retrospective study (2012-2014) included 98 consecutive patients with surgically resected SCRCs. Molecular characteristics, including microsatellite instability (MSI) and tumor-infiltrating lymphocytes (TILs), were analyzed for all cancer lesions. The intertumoral heterogeneity of SCRCs was evaluated using whole-exome sequencing (WES) for 18 cancers from nine patients with at least one MSI-high (MSI-H) tumor.
Results
Twelve patients had at least one MSI-H tumor; five showed discordant MSI status. Mucinous adenocarcinoma frequency and TIL density were higher in patients with at least one MSI-H tumor than in those with only microsatellite-stable tumors. WES revealed that, except one patient (6.5%), most synchronous cancers shared few variants in each patient (0.09%-0.36%). The concordance rates for BRAF, KRAS, NRAS, and PIK3CA in synchronous cancers from each patient were 66.7%, 66.7%, 66.7%, and 55.6%, respectively.
Conclusion
Although synchronous cancers shared a mutated gene, the mutation subtypes differed. SCRCs exhibited 5.1% MSI status discordance rate and a high discordance rate in somatic mutational variants. As intertumoral heterogeneity may affect the targeted therapy response, molecular analysis of all tumors is recommended for patients with SCRCs.
Key words: Colorectal neoplasms, Synchronous, Microsatellite instability, Tumor-infiltrating lymphocytes, Exome sequencing

Introduction

Synchronous colorectal cancers (SCRCs) are characterized by the presence of more than one primary colorectal carcinoma (CRC) in the same individual at the time of the initial diagnosis. Patients with hereditary CRC syndromes or inflammatory bowel disease face a higher risk of SCRCs [1,2]. However, these predisposing conditions account for only approximately 10% of CRCs [2], the majority of which are caused by other genetic and environmental factors. SCRCs arise from very similar or identical background genetic or environmental factors, providing a unique model to investigate multistep carcinogenesis.

CRC is a highly heterogeneous disease, which is caused by the accumulation of a variety of genetic and epigenetic changes in the colonic mucosa. Currently, molecular subtyping based on gene expression is widely accepted and facilitates in-depth understanding of the etiology and features of CRC as well as subtype-based targeted therapies [3]. Despite advances in the understanding of molecular pathways involved in CRC carcinogenesis, the molecular mechanisms underlying SCRC remain unclear. Several hypotheses have been posited to explain the occurrence of SCRCs. According to the clonal hypothesis, multifocal tumors arise from a founder tumor and are disseminated via intraluminal or intraepithelial spread [4]. In contrast, the field effect suggests that a large area of cells is transformed by a carcinogen, followed by neoplastic transformation through subsequent genetic mutations, highlighting the importance of the tumor microenvironment in the development of synchronous cancers [5,6]. Given that cancer is caused by the sequential accumulation of genetic and epigenetic mutations, each hypothesis would result in a distinct genetic signature.

The current therapeutic trends focus on individualized treatment strategies for CRC based on the molecular characteristics of each cancer lesion. Immunotherapy has demonstrated outstanding success as a treatment paradigm for CRC with high microsatellite instability (MSI-H) [7,8]. In contrast to solitary CRC, for which treatment strategies can be determined based on the characteristics of a single lesion, SCRCs have different characteristics of multiple cancers, and the manner in which each cancer affects the response to treatment is unclear. Therefore, we aimed to assess intertumoral heterogeneity in individuals with SCRCs by comparing the clinical, pathological, and molecular characteristics of SCRCs, including MSI status and whole-exome sequencing (WES) results.

Materials and Methods

1. Study population and definition of clinical characteristics

This analysis included 196 synchronous tumor lesions from 98 consecutive patients with SCRC who underwent surgical resection at Asan Medical Center between 2012 and 2014. Patients with more than one primary adenocarcinoma in the colon or rectum at the time of first diagnosis were included. All patients underwent surgical resection of the tumors without receiving any neoadjuvant treatment. Patients with colon cancer associated with inflammatory bowel disease, familial adenomatous polyposis, or concurrent cancers from other organs were excluded.

Clinical and pathological data were extracted retrospectively from the electronic medical records stored in the institutional database. In each patient, the tumor with the most advanced pT category was defined as the index tumor (labeled as tumor subnumber 1, T-1), irrespective of tumor size or location. If the tumors belonged to the same pT category, the tumor with the largest diameter was designated as T-1. The second-most advanced pT category or second-largest tumor from the same patient was designated as T-2. Cancer location was classified into three groups, viz., the right colon, left colon, and rectum. The right colon included the cecum, ascending colon, hepatic flexure, and transverse colon, while the left colon included the splenic flexure, descending colon, and sigmoid colon. Hereditary nonpolyposis colorectal cancer (HNPCC) was clinically diagnosed based on the Amsterdam II criteria [9]. Patients with a familial history of cancer due to inherited impairment in the DNA mismatch repair (MMR) system, such as endometrial, ovarian, or gastric cancers, were designated as having a history of HNPCC-related cancers. Lynch syndrome was defined as the presence of a pathogenic germline mutation identified through WES, as described later.

2. Pathologic examination

Tumor-infiltrating lymphocytes (TILs) were assessed in tissue specimens stained with hematoxylin and eosin (H&E) and evaluated by two independent pathologists (S.M. Hong and Y. S. Kim) blinded to the patients’ clinical information. The density of TILs was determined based on the recommendation by the International TILs Working Group (ITWG) [10]. The density of TILs was calculated as a percentage of all mononuclear inflammatory cells (e.g., lymphocytes and plasma cells), and other inflammatory cells (i.e., neutrophils and granulocytes) were excluded. The density of TILs was assessed within the intratumoral stromal area and counted in five areas (total magnification, ×200-400) on average (not hotspots). Only TILs within the border of invasive tumors were assessed, so that tumor areas with dysplasia, in-situ area, crush artefacts, necrosis, or regressive hyalinization were excluded. For statistical analysis, the level of stromal infiltration by the TILs was stratified as follows: weak (0%-10% of stromal TILs), moderate (20%-40% of stromal TILs), and strong (50%-90% of stromal TILs). Examples of TILs assessment using the ITWG system are shown in S1 Fig.

3. Evaluation of MSI and WES

For MSI and WES analyses, DNA was extracted using the QIAamp DNA formalin-fixed paraffin-embedded (FFPE) tissue kit (Qiagen), per the manufacturer’s protocol. DNA quality was assessed with PicoGreen (Invitrogen) using Victor 3 fluorometry and gel electrophoresis methods.

The Bethesda panel was used to assess the MSI status. The presence of two or more altered microsatellite markers was classified as MSI-H, only one altered marker as low MSI, and none as microsatellite stable (MSS) [11].

Out of 98 patients with SCRC, 12 patients who had at least one MSI-H tumor were selected for whole exome sequencing (WES) analysis. For these patients, two SCRCs and one normal tissue sample were obtained via manual microdissection guided by archived H&E-stained slides, resulting in 36 samples collected from FFPE tissues. However, one case did not meet the quality criteria. Consequently, DNA libraries for WES were successfully constructed from 33 samples representing 11 patients.

Genomic WES was performed using the SureSelect Human All Exon V8 FFPE library kit (Agilent Technologies) on the NovaSeq 6000 platform (Illumina), in accordance with the manufacturer’s protocol. Briefly, genomic DNA samples were randomly fragmented and ligated to the 5′ and 3′ molecular barcoded adapters. The adapter-ligated DNA fragments were amplified with SureSelect Human All Exon V8 primers, which anneal to the ends of each adapter. The quality and quantity of the 33 library templates were assessed using 2100 Bioanalyzer (Agilent Technologies) and Illumina qPCR Quantification (Illumina). Since two samples of capture libraries were not amplified, 27 samples from nine patients were hybridized and amplified. The samples were subjected to paired-end sequencing using the NovaSeq 6000 sequencer with a 101-base pair read length. The base calling files were converted into FASTQ files using the bcl2fastq package built into Illumina. Quality control (QC) metrics for sequencing data were assessed to ensure high-quality sequencing results. The mean sequencing depth across samples was 56.4× (range, 49.6× to 67.4×), with a duplication rate of 25.3% on average (range, 20.6% to 38.1%). All samples met the QC criteria for mean depth (≥ 30×) and acceptable duplication rates (≤ 38.1%), and no samples were excluded prior to further analysis.

The sequenced reads were aligned to the GRCh38 release of the human genome using bwa [12]. Somatic mutations were identified using MuTect2 [13]. For the final variant selection, we chose variants with a total depth of at least 30x and a variant allele depth of at least 3×. The final mutations were annotated using vcf2maf tool [14]. Copy number variation (CNV) data were analyzed with CNVkit [15].

4. Statistical analysis

Statistical analyses comparing the characteristics of the patients and cancers were performed using Fisher’s exact or Pearson’s chi-squared tests for categorical variables and Student’s t test for continuous variables. To assess the genetic heterogeneity of paired cancers evaluated by WES, the Jaccard coefficient was calculated as the proportion of shared variants over the total number of variants. The Jaccard coefficient of each pair was compared using Student’s t test. All statistical analyses were performed using SPSS software ver. 25.0 (IBM Corp.), and statistical significance was set at a p < 0.05.

Results

1. Clinicopathological characteristics of patients with SCRC

This study enrolled 98 patients with a mean age of 62.4±12.9 years. The study population predominantly comprised men (71.4%) (Table 1). Forty-five patients had ipsilateral SCRCs in the colon or rectum. The pT3 category was the most common (69.4%), and 44.9% of patients had regional lymph node metastasis. The pT category of index tumors and concurrent tumors is shown in the S2 Table. The initial staging based on the index tumor was as follows: stage I, 10.2% of patients; stage II, 42.9% of patients; stage III, 30.6% of patients; and stage IV, 16.3% of patients. The median follow-up period was 59 months.

Nineteen of 196 cancers from 98 patients with SCRC were MSI-H lesions (S3 Table). MSI-H cancers were more frequently located in the right colon (68.4% vs. 32.2%, p=0.007) and had a higher frequency of mucinous adenocarcinomas (15.8% vs. 0%, p < 0.001) than MSS cancers. MSI-H cancers exhibited significantly higher densities of TILs than MSS cancers (p < 0.001).

2. Comparison of clinicopathological characteristics and oncologic outcomes in the groups stratified according to MSI status

Twelve of the 98 enrolled patients had at least one MSI-H tumor, and five exhibited a discordant MSI status. Patients were divided into three groups according to the MSI status of SCRCs: all MSS (n=86), all MSI-H (n=7), and MSI-H/MSS (n=5). The representative microscopic images from the three groups are depicted in S4 Fig. Five patients were diagnosed with HNPCC. Three of these patients had two MSI-H cancers and two patients had a discordant MSI status (Fig. 1). Two of the four patients without HNPCC in the all-MSI-H group had a familial history of HNPCC-related cancers, while the other two had a past medical history of CRCs. All seven patients in the all-MSI-H group were men, and the all-MSI-H group was significantly younger than the all-MSS group (49.3±9.0 vs. 63.5±12.6 years, p=0.006) (Table 2). In the all-MSI-H group, four patients (57.1%) had SCRCs in the right colon. However, the MSI-H/MSS group showed a similar cancer localization pattern to the all-MSS group.

The proportion of mucinous adenocarcinoma (14.3% vs. 0%, p < 0.001) and moderate or strong density of TILs (67.2% vs. 14.0%, p < 0.001) were higher tumors in the all-MSI-H group compared to the all-MSS group. Similarly, the proportion of mucinous adenocarcinoma (10.0% vs. 0%, p < 0.001) and moderate or strong density TILs (40.0% vs. 14.0%, p < 0.001) were significantly higher in the MSI-H/MSS group compared to the MSS group.

3. Mutation analysis of synchronous cancers using WES

The characteristics of 18 SCRCs analyzed with WES are shown in S5 Table. Five patients had only MSI-H cancers, and four patients had both MSI-H and MSS cancers. Three of nine patients were diagnosed with HNPCC in accordance with the Amsterdam II criteria. Among these, pathogenic germline mutations in MMR genes were identified in two cases, while no such mutations were detected in the remaining patient (S6 Table). Conversely, the presence of pathogenic germline mutations did not always correlate with an HNPCC diagnosis. For instance, some individuals with nonsense or frameshift mutations in MMR genes, including MSH2 and MSH6, did not meet the clinical criteria for HNPCC diagnosis.

The median number of total somatic mutations in each cancer identified by WES was 5,850 (range, 3,365 to 14,070). S7 Fig. shows the numbers of different mutation types in each cancer. To evaluate the heterogeneity of mutations between two cancers in the same patient, variants simultaneously found in both cancers were identified. T02-1 and T02-2 shared 6.5% of variants (S8 Fig.). Except for T02, the other synchronous cancers in the same patient shared only a few variants, and the proportion of shared mutated genes relative to the total number of mutated genes was < 1% (range, 0.09% to 0.36%) (Fig. 2). Nevertheless, synchronous lesions from the same patient showed a significantly greater proportion of shared variants than pairs of cancers from different patients (0.89%±2.1% vs. 0.13%±0.04%, p < 0.001). This statistical significance was retained after excluding T02, which showed a notably high proportion of shared variants (0.19%±0.08% vs. 0.13%±0.04%, p < 0.001). The proportion of shared variants did not differ significantly between patients with HNPCC and those with sporadic SCRC (0.26%±0.09% vs. 1.21%±2.59%, p=0.56). Upon excluding case T02, patients diagnosed with HNPCC exhibited a greater proportion of shared variants compared to those with sporadic SCRCs; however, this difference did not attain statistical significance (0.26%±0.09% vs. 0.15%±0.06%, p=0.087). To further explore the genetic similarities between synchronous cancers, CNV profiles were analyzed. S9 Fig. shows the CNV patterns of each cancer, highlighting amplification and deletion regions across the genome. While some overlapping CNV regions were observed, distinct differences in CNV patterns were also noted, indicating significant divergence in genomic alterations between the two lesions.

We mapped the driver genes reported in the literature or known to be associated with oncogenic transformation from the cancer gene census list in the Catalogue of Somatic Mutations in Cancer (Fig. 3). Mutations in APC were identified in samples from eight patients, except T09. However, five of these patients had different mutation types, and all eight patients had different mutation sites in the SCRCs, even if the SCRCs harbored the same APC mutation. The concordance rates for BRAF, KRAS, NRAS, and PIK3CA were 66.7%, 66.7%, 66.7%, and 55.6%, respectively. However, only T06-1 and T06-2 shared the same mutation site, KRAS G12D, while the other SCRCs had different mutation subtypes even if they shared the same mutated genes (S10 Table).

Discussion

The importance of an individualized approach rooted in the molecular characteristics of each cancer for the treatment of CRC has been emphasized. Assessing the degree of similarity in molecular characteristics between SCRCs to ensure appropriate selection of treatment strategies is needed. In this study, 94.9% of participants showed concordance in the MSI status, whereas only 5.1% showed a discordant MSI status. Interestingly, WES analysis of patients with SCRCs revealed that synchronous cancers in each individual shared only a few somatic variants, indicating substantial intertumoral heterogeneity.

MSI-H cancers showed a predilection for the right side of the colon compared to MSS cancers in our SCRC cohort. The right and left sides of the colon have distinct embryologic origins, resulting in differences in the underlying biology. The patterns of gene methylation differ significantly between the right and left colons [16]. Notably, the prevalence of methylation of the MLH1 gene is significantly higher in the right colonic mucosa [17], suggesting that the right colon is more susceptible to MMR deficiency. Environmental factors, such as colonic microbiota, exposure to carcinogens, or bile acid levels, may also differ between the right and left colon [18,19]. In this study, MSI-H cancers showed higher TIL infiltration than MSS cancers. The frequent frameshift mutations and high tumor mutational burden (TMB) in MSI-H cancers result in the production of a greater number of neoantigens, which increases T cell infiltration [20]. CD8+ cytotoxic T lymphocytes, which play an essential role in the adaptive immune system, mediate tumor rejection via the recognition of tumor antigens and the production of several substances, such as granulysin, granzymes, and tumor necrosis factor-α, leading to tumor cell killing [21,22]. Several studies demonstrated that elevated levels of TILs are correlated with a more favorable prognosis and lower risk of metastasis [23,24], emphasizing the significance of tumor immune infiltration.

The reported discordance rate in the MSI status in patients with SCRC ranges 6%-13% [25-27], and our study found a discordance rate of 5.1%. The variation in discordance rates among studies may be attributed to the proportion of patients with Lynch syndrome included in each study cohort. In a study conducted by Dykes et al. [27], the proportion of patients with Lynch syndrome was 27%, the proportion of MSI-H cancers was high at 32%, and the discordance rate was also high at 13%. In contrast, in the study by Arriba et al. [25], which did not include Lynch syndrome patients, the proportion of MSI-H cancers was as low as 5.1%, and the discordance rate was 6.2%. The low rates of MSI-H tumors and low discordance rate may be attributed to the relatively small number of Lynch syndrome cases in this study population.

The all-MSI-H group was associated with younger age, higher proportion of mucinous adenocarcinoma, and moderate or strong density of TILs compared to the all-MSS group. These results correspond to the comparison between MSI-H and MSS cancers. The MSI discordant group also showed a higher proportion of mucinous adenocarcinomas and moderate or strong concentration of TILs compared to the all-MSS group, similar to the result of the all-MSI-H group. The index tumor was the MSI-H lesion in three of the five MSI-H/MSS patients, while an MSS lesion was the index tumor in the other two patients. In other words, treatment decisions may be based on the misclassification of MSI-H/MSS cancers as MSI-H or MSS cancers if the MSI test is exclusively conducted with the index tumor. The MSI status is closely linked to the response to adjuvant chemotherapy as well as immunotherapy. How the discordant MSI status affects the response to treatment in SCRCs remains unclear, necessitating further investigation.

Although our study did not include an analysis of promoter methylation, patients with pathogenic germline mutations in MMR genes identified through WES can be diagnosed with Lynch syndrome. However, the observed discrepancy between HNPCC diagnosis and pathogenic germline mutations in MMR genes highlights the limitations of the Amsterdam II criteria, which rely primarily on family history rather than molecular genetic data. This discrepancy may be attributed to the incomplete penetrance or variable expressivity of these mutations [28]. Interestingly, among the four patients with pathogenic germline mutations in MMR genes, two cases (T06 and T07) exhibited MSI discordance among their cancers. Previous studies have also reported that patients with Lynch syndrome can develop MSS cancers [29]. These findings suggest that, even in patients with germline mutations in MMR genes, cancers may not arise solely due to germline mutations but could also be influenced by somatic mutations or other mechanisms driving carcinogenesis.

WES analysis revealed that SCRCs in a given patient shared only a few somatic mutations. Patients with HNPCC also exhibited very low rates of shared somatic mutations between SCRCs, similar to those without HNPCC. This finding indicates that most SCRCs may have independent clonal origins, consistent with previous studies that demonstrated the intertumoral heterogeneity of SCRCs using WES [4,30]. Cereda et al. [30] showed that SCRCs had independent genetic origins, somatic alterations, and clonal compositions. A WES-based analysis of 32 tumor lesions from 15 patients with SCRCs identified very few ubiquitously mutated genes, ranging 0.34%-4.22% in nonhypermutated tumors and 0.8%-7.0% in hypermutated tumors [4]. These findings support the field effect theory of colorectal carcinogenesis [5,6]. The molecular basis of this theory may be genetic susceptibility to CRC development or extensive exposure to carcinogens. Inherited mutations in immune-related genes may increase the frequency of independent events of cancer initiation, implying that an inflammatory microenvironment promotes carcinogenesis via cytokine secretion or genomic instability [30].

However, only one SCRC patient (case T02) showed a substantially higher proportion of shared variants. In this case, the two lesions were located in the transverse colon at a distance of 1 cm, with intervening normal mucosa (S11 Fig.). Given the proximity and notable genetic similarity of these lesions, we postulated that these cancers may have had a common origin and may have spread to adjacent areas via monoclonal seeding, which is one of the possible explanations for multifocal colorectal carcinogenesis [31,32]. Some studies have included the intertumoral distance (e.g., 5 cm) in the definition of SCRC, based on the hypothesis that intertumoral distance may influence independent carcinogenesis [33]. Although the proximity of tumors might suggest that the concurrent lesion originated from the index lesion via intraluminal spread, distance alone is insufficient to determine the mechanism underlying multifocal cancer development. A prior molecular study has frequently observed clonally related CRCs, likely due to intraluminal spread, even with lesion distances ranging from 1.2 to 75 cm [32]. These findings indicate that clinicopathological characteristics, such as intertumoral distance, may not reliably predict clonal origin of SCRCs. In this study, 25 of the 98 patients had an inter-tumor distance of less than 5 cm (S12 Fig.), and all had pathologically distinct cancers with clearly observable normal mucosa between the two tumors. Although the genetic characteristics could not be analyzed in all patients, case of T09 presented a notable example: despite a short intertumoral distance of 3 cm, the MSI status differed between the tumors, and the shared somatic mutation rate was extremely low, consistent with other cases except T02. These findings further support the notion that genetic independence cannot be assessed based solely on intertumoral distance, underscoring the potential need for genetic testing of each tumor.

In general, MSI-H cancers are associated with high TMB due to MMR deficiency, leading to the accumulation of mutations. However, in our study, there was no significant correlation between MSI status and TMB. This could be due to the limited sample size and the presence of MSS cancers with mutations in the POLE gene, suggesting the possibility of a hypermutator phenotype [34]. Such MSS cancers could exhibit high TMB similar to MSI-H tumors, potentially obscuring the difference between the groups. Since high TMB, like MSI-H, is known to be a determinant of response to immunotherapy, future studies should include a larger sample size and investigate the relationship between TMB and treatment response in patients with SCRC.

In general, tumor heterogeneity affects the efficacy of therapies targeting specific key genes, such as KRAS, NRAS, or BRAF. The management of SCRCs is currently challenged by the existence of genetically different cancers; consequently, no definitive guidelines are available. The concordance rates of TP53, KRAS, and BRAF mutations have been reported to be < 40% in previous studies [35-37]. In contrast, the concordance rates in our study were higher than those reported previously; however, many of the concordant variants differed in mutation type and site. The efficacy of targeted agents and prognosis may vary depending on the specific mutation subtypes [38]. Therefore, we recommend that all SCRCs within a single individual should be assessed to formulate appropriate treatment decisions.

This study has several limitations. First, its retrospective design may have engendered selection bias. Second, Patients with MSS cancers only were not included in WES analysis. Since MSS cancers have different clinical, pathologic, and molecular characteristics compared to MSI-H cancers, they should also be analyzed using WES in further studies. We did not perform immunohistochemical staining for MMR genes in each tumor from the same individual, nor did we analyze DNA methylation. Moreover, we did not validate WES using polymerase chain reaction and Sanger sequencing to assess the accuracy of variant calling.

We identified a discordance rate of 5.1% in the MSI status between concurrent cancers in patients with SCRC, which could be missed if all SCRCs are not analyzed. Moreover, WES analysis revealed that SCRCs in an individual patient shared only a few somatic mutations, indicating substantial intertumoral heterogeneity of SCRCs. This intertumoral heterogeneity was evident in known druggable mutations, including KRAS, BRAF, or PIK3CA. Although the relationship between intertumoral heterogeneity and the response to treatment has not yet been studied in SCRCs, molecular analysis of all cancers in patients with SCRC is recommended while determining the treatment strategy for SCRCs.

Electronic Supplementary Material

Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).

crt-2024-947_S1_Fig.pdf

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NOTES

Ethical Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Asan Medical Center (approval number; 2022-0771). Patient consent was waived because of the retrospective observational nature of this study, which involves the analysis of previously collected data.

Author Contributions

Conceived and designed the analysis: Lee HG, Kim Y, Park IJ, Hong SM.

Collected the data: Lee HG, Kim Y.

Contributed data or analysis tools: Lee HG, Kim Y, Kim MJ, Kim YW, Jun SY.

Performed the analysis: Lee HG, Jun SY, Kim D.

Wrote the paper: Lee HG, Kim Y, Park IJ, Hong SM.

Data curation: Lee HG, Kim Y.

Conflicts of Interest

Conflict of interest relevant to this article was not reported.

Funding

This work was supported by a National Research Foundation of Korea grant funded by the Korean government (MSIT) (grant number: 2021R1A2C1003898, awarded to Jun SY), Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea (grant number: 2022IF0028-1), and Asan Institute for Life Sciences, Asan Medical Center (grant number: 2024IP0080-1).

Acknowledgments

We would like to express our gratitude to Professor Sung-Min Ahn (Department of Genome Medicine and Science, College of Medicine, Gachon University, Incheon, Republic of Korea) for his insightful analysis and comments from the perspective of genetic immunology, although they were not included in this research’s outcome.

Fig. 1.

Associated family history of 98 patients with synchronous colorectal cancer. CRC, colorectal carcinoma; HNPCC, hereditary nonopolyposis colorectal cancer; MSI-H, high microsatellite instability; MSS, microsatellite stable.

Fig. 2.

Similarity matrix of all mutated genes across the 18 synchronous colorectal cancers. The Jaccard coefficient was calculated for each combination of tumors from the same or different patients.

Fig. 3.

Types of mutations in synchronous colorectal cancers. Each square represents a mutated cancer gene in a single tumor, with the color representing the mutation type. The upper panel indicates the total mutation burden of each tumor. The blue bars on the right panel indicate the proportions of lesions with each mutated cancer gene. HNPCC, hereditary nonopolyposis colorectal cancer; TMB, tumor mutational burden.

Table 1.

Demographic, clinical, and pathological characteristics of the study population

Characteristic	Value
Sex
Male	70 (71.4)
Female	28 (28.6)
Age (yr)	62.4±12.9
Location
Both right colon	19 (19.4)
Both left colon	23 (23.5)
Different colon	25 (25.5)
Colon and rectum	28 (28.6)
Both rectum	3 (3.1)
pT category
pT1	3 (3.1)
pT2	17 (17.3)
pT3	68 (69.4)
pT4	10 (10.2)
pN category
pN0	54 (55.1)
pN1	31 (31.6)
pN2	13 (13.3)
cM/pM category
cM0	82 (83.7)
c/pM1	16 (16.3)
AJCC stage
I	10 (10.2)
II	42 (42.9)
III	30 (30.6)
IV	16 (16.3)

Values are presented as number (%) or mean±standard deviation unless otherwise indicated. AJCC stage, clinical or pathological staging according to the American Joint Committee on Cancer (8th edition).

Table 2.

Comparison of clinicopathological characteristics between the groups stratified according to MSI status

Characteristic	All MSI-H (7 patients, 14 tumors)	All MSS (86 patients, 172 tumors)	p-value	MSI-H/MSS (5 patients, 10 tumors)	All MSS (86 patients, 172 tumors)	p-value	MSI-H/MSS (5 patients, 10 tumors)	All MSI-H (7 patients, 14 tumors)	p-value
Sex
Male	7 (100)	59 (68.6)	0.079	4 (80.0)	59 (68.6)	0.591	4 (80.0)	7 (100)	0.417
Female	0	27 (31.4)		1 (20.0)	27 (31.4)		1 (20.0)	0
Age (yr)	49.3±9.0	63.5±12.6	0.006	59.6±20.0	63.5±12.6	0.524	59.6±20.0	49.3±9.0	0.216
Location type
Both right colon	4 (57.1)	14 (16.3)	0.089	1 (20.0)	14 (16.3)	0.472	1 (20.0)	4 (57.1)	0.359
Both left colon	0	22 (25.6)		1 (20.0)	22 (25.6)		1 (20.0)	0
Different colon	2 (28.6)	22 (25.6)		1 (20.0)	22 (25.6)		1 (20.0)	2 (28.6)
Colon and rectum	1 (14.3)	25 (29.1)		2 (40.0)	25 (29.1)		2 (40.0)	1 (14.3)
Both rectum	0	3 (3.5)		0	3 (3.5)		0	0
Evaluation for all tumors
Tumor location
Right colon	10 (71.4)	55 (32.0)	0.012	5 (50.0)	55 (32.0)	0.192	5 (50.0)	10 (71.4)	0.504
Left colon	3 (21.4)	86 (50.0)		3 (30.0)	86 (50.0)		3 (30.0)	3 (21.4)
Rectum	1 (7.1)	31 (18.0)		2 (20.0)	31 (18.0)		2 (20.0)	1 (7.1)
Histologic type
Tubular adenocarcinoma	12 (95.7)	172 (100)	< 0.001	8 (80.0)	172 (100)	< 0.001	8 (80.0)	12 (85.7)	0.470
Mucinous adenocarcinoma	2 (14.3)	0		1 (10.0)	0		1 (10.0)	2 (14.3)
Signet ring cell carcinoma	0	0		1 (10.0)	0		1 (10.0)	0
Differentiation^a)
Well	2 (16.7)	37 (21.5)	0.027	1 (12.5)	37 (21.5)	0.340	1 (12.5)	2 (16.7)	0.267
Moderately	7 (58.3)	126 (73.3)		7 (87.5)	126 (73.3)		7 (87.5)	7 (58.3)
Poorly	3 (25.0)	9 (5.2)		0	9 (5.2)		0	3 (25.0)
LVI present	5 (35.7)	61 (35.5)	0.985	3 (30.0)	61 (35.5)	0.725	3 (30.0)	5 (35.7)	0.770
PNI present	3 (21.4)	34 (19.8)	0.881	0	34 (19.8)	0.119	0	3 (21.4)	0.239
Tumor budding
Low	13 (92.9)	120 (69.8)	0.062	9 (90.0)	120 (69.8)	0.161	9 (90.0)	13 (92.9)	0.803
Intermediate	1 (7.1)	40 (23.2)		1 (10.0)	40 (23.2)		1 (10.0)	1 (7.1)
High	0	12 (7.0)		0	12 (7.0)		0	0
TILs (%)
Weak (0-10)	6 (42.9)	148 (86.0)	< 0.001	6 (60.0)	148 (86.0)	< 0.001	6 (60.0)	6 (42.9)	0.504
Moderate (20-40)	2 (14.3)	22 (12.8)		2 (20.0)	22 (12.8)		2 (20.0)	2 (14.3)
Strong (50-90)	6 (42.9)	2 (1.2)		2 (20.0)	2 (1.2)		2 (20.0)	6 (42.9)

Values are presented as number (%) or mean±standard deviation. LVI, lymphovascular invasion; MSI-H, high microsatellite instability; MSS, microsatellite stable; PNI, perineural invasion; TILs, tumor-infiltrating lymphocytes.

^a) Only tubular adenocarcinomas were included, except for mucinous and signet ring cell carcinomas.

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Molecular Mosaics: Unveiling Heterogeneity in Synchronous Colorectal Cancers

Cancer Res Treat. 2026;58(1):264-274. Published online February 18, 2025

DOI: https://doi.org/10.4143/crt.2024.947

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Abstract
Introduction
Materials and Methods
Results
Discussion
Electronic Supplementary Material
NOTES
REFERENCES

Evaluation of Molecular Residual Disease by a Fixed Panel in Resectable Colorectal Cancer

Molecular Mosaics: Unveiling Heterogeneity in Synchronous Colorectal Cancers

Fig. 1. Associated family history of 98 patients with synchronous colorectal cancer. CRC, colorectal carcinoma; HNPCC, hereditary nonopolyposis colorectal cancer; MSI-H, high microsatellite instability; MSS, microsatellite stable.

Fig. 2. Similarity matrix of all mutated genes across the 18 synchronous colorectal cancers. The Jaccard coefficient was calculated for each combination of tumors from the same or different patients.

Fig. 3. Types of mutations in synchronous colorectal cancers. Each square represents a mutated cancer gene in a single tumor, with the color representing the mutation type. The upper panel indicates the total mutation burden of each tumor. The blue bars on the right panel indicate the proportions of lesions with each mutated cancer gene. HNPCC, hereditary nonopolyposis colorectal cancer; TMB, tumor mutational burden.

Fig. 1.

Fig. 2.

Fig. 3.

Molecular Mosaics: Unveiling Heterogeneity in Synchronous Colorectal Cancers

Characteristic	Value
Sex
Male	70 (71.4)
Female	28 (28.6)
Age (yr)	62.4±12.9
Location
Both right colon	19 (19.4)
Both left colon	23 (23.5)
Different colon	25 (25.5)
Colon and rectum	28 (28.6)
Both rectum	3 (3.1)
pT category
pT1	3 (3.1)
pT2	17 (17.3)
pT3	68 (69.4)
pT4	10 (10.2)
pN category
pN0	54 (55.1)
pN1	31 (31.6)
pN2	13 (13.3)
cM/pM category
cM0	82 (83.7)
c/pM1	16 (16.3)
AJCC stage
I	10 (10.2)
II	42 (42.9)
III	30 (30.6)
IV	16 (16.3)

Characteristic	All MSI-H (7 patients, 14 tumors)	All MSS (86 patients, 172 tumors)	p-value	MSI-H/MSS (5 patients, 10 tumors)	All MSS (86 patients, 172 tumors)	p-value	MSI-H/MSS (5 patients, 10 tumors)	All MSI-H (7 patients, 14 tumors)	p-value
Sex
Male	7 (100)	59 (68.6)	0.079	4 (80.0)	59 (68.6)	0.591	4 (80.0)	7 (100)	0.417
Female	0	27 (31.4)		1 (20.0)	27 (31.4)		1 (20.0)	0
Age (yr)	49.3±9.0	63.5±12.6	0.006	59.6±20.0	63.5±12.6	0.524	59.6±20.0	49.3±9.0	0.216
Location type
Both right colon	4 (57.1)	14 (16.3)	0.089	1 (20.0)	14 (16.3)	0.472	1 (20.0)	4 (57.1)	0.359
Both left colon	0	22 (25.6)		1 (20.0)	22 (25.6)		1 (20.0)	0
Different colon	2 (28.6)	22 (25.6)		1 (20.0)	22 (25.6)		1 (20.0)	2 (28.6)
Colon and rectum	1 (14.3)	25 (29.1)		2 (40.0)	25 (29.1)		2 (40.0)	1 (14.3)
Both rectum	0	3 (3.5)		0	3 (3.5)		0	0
Evaluation for all tumors
Tumor location
Right colon	10 (71.4)	55 (32.0)	0.012	5 (50.0)	55 (32.0)	0.192	5 (50.0)	10 (71.4)	0.504
Left colon	3 (21.4)	86 (50.0)		3 (30.0)	86 (50.0)		3 (30.0)	3 (21.4)
Rectum	1 (7.1)	31 (18.0)		2 (20.0)	31 (18.0)		2 (20.0)	1 (7.1)
Histologic type
Tubular adenocarcinoma	12 (95.7)	172 (100)	< 0.001	8 (80.0)	172 (100)	< 0.001	8 (80.0)	12 (85.7)	0.470
Mucinous adenocarcinoma	2 (14.3)	0		1 (10.0)	0		1 (10.0)	2 (14.3)
Signet ring cell carcinoma	0	0		1 (10.0)	0		1 (10.0)	0
Differentiation^a)
Well	2 (16.7)	37 (21.5)	0.027	1 (12.5)	37 (21.5)	0.340	1 (12.5)	2 (16.7)	0.267
Moderately	7 (58.3)	126 (73.3)		7 (87.5)	126 (73.3)		7 (87.5)	7 (58.3)
Poorly	3 (25.0)	9 (5.2)		0	9 (5.2)		0	3 (25.0)
LVI present	5 (35.7)	61 (35.5)	0.985	3 (30.0)	61 (35.5)	0.725	3 (30.0)	5 (35.7)	0.770
PNI present	3 (21.4)	34 (19.8)	0.881	0	34 (19.8)	0.119	0	3 (21.4)	0.239
Tumor budding
Low	13 (92.9)	120 (69.8)	0.062	9 (90.0)	120 (69.8)	0.161	9 (90.0)	13 (92.9)	0.803
Intermediate	1 (7.1)	40 (23.2)		1 (10.0)	40 (23.2)		1 (10.0)	1 (7.1)
High	0	12 (7.0)		0	12 (7.0)		0	0
TILs (%)
Weak (0-10)	6 (42.9)	148 (86.0)	< 0.001	6 (60.0)	148 (86.0)	< 0.001	6 (60.0)	6 (42.9)	0.504
Moderate (20-40)	2 (14.3)	22 (12.8)		2 (20.0)	22 (12.8)		2 (20.0)	2 (14.3)
Strong (50-90)	6 (42.9)	2 (1.2)		2 (20.0)	2 (1.2)		2 (20.0)	6 (42.9)

Table 1. Demographic, clinical, and pathological characteristics of the study population

Table 2. Comparison of clinicopathological characteristics between the groups stratified according to MSI status

Only tubular adenocarcinomas were included, except for mucinous and signet ring cell carcinomas.