Molecular Mosaics: Unveiling Heterogeneity in Synchronous Colorectal Cancers

doi:10.21203/rs.3.rs-4902441/v1

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Molecular Mosaics: Unveiling Heterogeneity in Synchronous Colorectal Cancers

https://doi.org/10.21203/rs.3.rs-4902441/v1

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Molecular characteristics of synchronous colorectal cancers (SCRCs) remain incompletely elucidated, despite their importance in targeted therapy selection. This retrospective study (2012–2014) compared the molecular characteristics and somatic mutations between SCRCs in 100 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 9 patients with at least one MSI-high (MSI-H) tumor. 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. Although synchronous cancers shared a mutated gene, the mutation subtypes differed. SCRCs exhibited 5% 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.

colorectal cancer

synchronous

microsatellite instability

tumor-infiltrating lymphocyte

whole-exome sequencing

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 [1], 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.

Study population and definition of clinical characteristics

This analysis included 200 synchronous tumor lesions from 100 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. The study was approved by the institutional review board of Asan Medical Center (approval number: 2022 − 0771).

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. Lynch syndrome was clinically diagnosed based on the Amsterdam II criteria [9]. Patients with a familial history of cancer due to inherited impairment in the deoxyribonucleic acid (DNA) mismatch repair system, such as endometrial, ovarian, or gastric cancers, were designated as having a history of Lynch syndrome-related cancers.

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 Supplementary Figure S1.

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, Hilden, Germany), per the manufacturer’s protocol. DNA quality was assessed with PicoGreen (Invitrogen, Waltham, MA, USA) 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 (MSI-L), and none as microsatellite stable (MSS) [11].

Twelve patients who had at least one MSI-H tumor out of a total of 100 patients with SCRC were selected for WES analysis. Two patient-paired SCRCs and normal samples were obtained via manual microdissection guided by archived H&E-stained slides. Thirty-six samples were collected by meticulous manual microdissection from FFPE tissues obtained from 12 patients with SCRC. One case did not satisfy the quality criteria; finally, DNA libraries for WES were constructed using 33 samples from 11 patients.

Genomic WES was performed using the SureSelect Human All Exon V8 FFPE library kit (Agilent Technologies, Santa Clara, CA, USA) on the NovaSeq 6000 platform (Illumina, San Diego, CA, USA), 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. Variant calling was performed by comparing the sequencing reads of the cancer and matched normal genomes to identify somatic mutations. Cancer-specific somatic single nucleotide variants and insertion–deletion mutations were determined to be present if they were absent in the normal counterpart.

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. Survival outcomes included recurrence-free survival (RFS) and overall survival (OS) rates. RFS and OS rates of groups stratified according to TIL density and MSI status were analyzed using the Kaplan–Meier method and compared using log-rank tests. 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 (version 25.0; IBM Statistics, Armonk, NY, USA), and statistical significance was set at a p < 0.05.

Clinicopathological characteristics of patients with SCRC

This study enrolled 100 patients with a mean age of 62.2 ± 13.3 years. The study population predominantly comprised men (72%) (Table 1). Forty-seven patients had ipsilateral SCRCs in the colon or rectum. The pT3 category was the most common (68%), and 46% of patients had regional lymph node metastasis. The initial staging based on the index tumor was as follows: stage I, 10% of patients; stage II, 42% of patients; stage III, n 32% of patients; and stage IV, 16.0% of patients. The median follow-up period was 59 months.

Table 1

Demographic, clinical, and pathological characteristics of the study population
Characteristics	Value
Sex
Male	72 (72.0)
Female	28 (28.0)
Age, years (mean)	62.2 ± 13.3
Location
Both right colon	21 (21.0)
Both left colon	23 (23.0)
Different colon	25 (25.0)
Colon and rectum	28 (28.0)
Both rectum	3 (3.0)
pT category
pT1	3 (3.0)
pT2	19 (19.0)
pT3	68 (68.0)
pT4	10 (10.0)
pN category
pN0	54 (54.0)
pN1	32 (32.0)
pN2	14 (14.0)
cM/pM category
cM0	84 (84.0)
c/pM1	16 (16.0)
AJCC Stage
I	10 (10.0)
II	42 (42.0)
III	32 (32.0)
IV	16 (16.0)
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)

Nineteen of 200 cancers from 100 patients with SCRC were MSI-H lesions (Supplementary Table S1). MSI-H cancers were more frequently located in the right colon (68.4% vs 33.7%, p = 0.011) 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).

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

Twelve of the 100 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 = 88), all MSI-H (n = 7), and MSI-H/MSS (n = 5). The representative microscopic images from the three groups are depicted in Supplementary Figure S2. Five patients were diagnosed with Lynch syndrome. 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 Lynch syndrome in the all-MSI-H group had a familial history of Lynch syndrome-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 63.3 ± 12.8 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.

Table 2

Comparison of clinicopathological characteristics between the groups stratified according to MSI status
Characteristics	All MSI-H (n = 7 patients, 14 tumors)	All MSS (n = 88 patients, 176 tumors)	p-value	MSI-H/MSS (n = 5 patients, 10 tumors)	All MSS (n = 88 patients, 176 tumors)	p-value	MSI-H/MSS (n = 5 patients, 10 tumors)	All MSI-H (n = 7 patients, 14 tumors)	p-value
Sex			0.083			0.613			0.417
Male	7 (100.0)	61 (69.3)		4 (80.0)	61 (69.3)		4 (80.0)	7 (100.0)
Female	0 (0)	27 (30.7)		1 (20.0)	27 (30.7)		1 (20.0)	0 (0)
Age (years)	49.3 ± 9.0	63.3 ± 12.8	0.006	59.6 ± 20.0	63.3 ± 12.8	0.536	59.6 ± 20.0	49.3 ± 9.0	0.216
Location type			0.127			0.974			0.359
Both right colon	4 (57.1)	16 (18.2)		1 (20.0)	16 (18.2)		1 (20.0)	4 (57.1)
Both left colon	0 (0)	22 (25.0)		1 (20.0)	22 (25.0)		1 (20.0)	0 (0)
Different colon	2 (28.6)	22 (25.0)		1 (20.0)	22 (25.0)		1 (20.0)	2 (28.6)
Colon and rectum	1 (14.3)	25 (28.4)		2 (40.0)	25 (28.4)		2 (40.0)	1 (14.3)
Both rectum	0 (0)	3 (3.4)		0 (0)	3 (3.4)		0 (0)	0 (0)
Evaluation for all tumors
Tumor location			0.018			0.477			0.504
Right colon	10 (71.4)	59 (33.5)		5 (50.0)	59 (33.5)		5 (50.0)	10 (71.4)
Left colon	3 (21.4)	86 (48.9)		3 (30.0)	86 (48.9)		3 (30.0)	3 (21.4)
Rectum	1 (7.1)	31 (17.6)		2 (20.0)	31 (17.6)		2 (20.0)	1 (7.1)
Histologic type			< 0.001			< 0.001			0.47
Tubular adenocarcinoma	12 (95.7)	176 (100.0)		8 (80.0)	176 (100.0)		8 (80.0)	12 (85.7)
Mucinous adenocarcinoma	2 (14.3)	0 (0)		1 (10.0)	0 (0)		1 (10.0)	2 (14.3)
Signet ring cell carcinoma	0 (0)	0 (0)		1 (10.0)	0 (0)		1 (10.0)	0 (0)
Differentiation ^a			0.024			0.617			0.267
Well	2 (16.7)	39 (22.2)		1 (12.5)	39 (22.2)		1 (12.5)	2 (16.7)
Moderately	7 (58.3)	128 (72.7)		7 (87.5)	128 (72.7)		7 (87.5)	7 (58.3)
Poorly	3 (25.0)	9 (5.1)		0 (0)	9 (5.1)		0 (0)	3 (25.0)
LVI present	5 (35.7)	62 (35.2)	0.971	3 (30.0)	62 (35.2)	0.736	3 (30.0)	5 (35.7)	0.77
PNI present	3 (21.4)	34 (19.3)	0.848	0 (0)	34 (19.3)	0.124	0 (0)	3 (21.4)	0.239
Tumor budding			0.19			0.39			0.803
Low	13 (92.9)	124 (70.5)		9 (90.0)	124 (70.5)		9 (90.0)	13 (92.9)
Intermediate	1 (7.1)	40 (22.7)		1 (10.0)	40 (22.7)		1 (10.0)	1 (7.1)
High	0 (0)	12 (6.8)		0 (0)	12 (6.8)		0 (0)	0 (0)
TILs			< 0.001			< 0.001			0.504
Weak (0–10%)	6 (42.9)	150 (85.2)		6 (60.0)	150 (85.2)		6 (60.0)	6 (42.9)
Moderate (20–40%)	2 (14.3)	24 (13.6)		2 (20.0)	24 (13.6)		2 (20.0)	2 (14.3)
Strong (50–90%)	6 (42.9)	2 (1.1)		2 (20.0)	2 (1.1)		2 (20.0)	6 (42.9)
LVI, lymphovascular invasion; PNI, perineural invasion; TILs, tumor-infiltrating lymphocytes; AJCC Stage, clinical or pathological staging according to the American Joint Committee on Cancer (8th edition)
^aOnly tubular adenocarcinomas were included, except for mucinous and signet ring cell carcinomas

The proportion of mucinous adenocarcinoma (14.3% vs 0%, p < 0.001) and density of TILs (67.2% vs 14.7%, 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 14.7%, p < 0.001) were significantly higher in the MSI-H/MSS group compared to the MSS group.

The distribution of pathologic stage was not different according to MSI status (Supplementary Table S2). The median RFS of three groups was as follows: all-MSS group, 58 months; all-MSI-H group, 68 months; and MSI-H/MSS group, 55 months (Supplementary Fig. S3a). The median OS was 59 months in the all-MSS group, 68 months in the all-MSI-H group, and 55 months in the MSI-H/MSS group, without significant between-group differences (Supplementary Fig. S3b). Since only the all-MSS group included stage IV CRC patients, survival analysis was performed by excluding stage IV patients (Supplementary Fig. S3c and S3d). RFS and OS did not differ significantly between any two MSI groups, even after excluding stage IV patients.

Mutation analysis of synchronous cancers using WES

The characteristics of 18 SCRCs analyzed with WES are shown in Supplementary Table S3. Five patients had only MSI-H cancers, and four patients had both MSI-H and MSS cancers. Three of nine patients were diagnosed with Lynch syndrome in accordance with the Amsterdam II criteria.

The median number of total mutations in each cancer identified by WES was 5,850 (range, 3,365–14,070). Supplementary Figure S4 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 (Supplementary Fig. S5). 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, 0.09–0.36%, 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 Lynch syndrome and those with sporadic SCRC (0.26 ± 0.09% vs 1.21 ± 2.59%, p = 0.56). Upon excluding case T02, patients diagnosed with Lynch syndrome 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).

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 T08-1 and T08-2 shared the same mutation site, KRAS G12D, while the other SCRCs had different mutation subtypes even if they shared the same mutated genes.

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, 95% of participants showed concordance in the MSI status, whereas only 5% 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 [12]. Notably, the prevalence of methylation of the MLH1 gene is significantly higher in the right colonic mucosa [13], 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 [14, 15]. In this study, MSI-H cancers showed higher TIL infiltration than MSS cancers. The frequent frameshift mutations and high tumor mutational burden in MSI-H cancers result in the production of a greater number of neoantigens, which increases T cell infiltration [16]. 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 [17, 18]. Several studies demonstrated that elevated levels of TILs are correlated with a more favorable prognosis and lower risk of metastasis [19, 20], emphasizing the significance of tumor immune infiltration.

The reported discordance rate in the MSI status in patients with SCRC ranges 6–13% [21–23], and our study found a discordance rate of 5%. 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.[21], 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.[23], 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 3 of the five MSI-H/MSS patients, while an MSS lesion was the index tumor in the other 2 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.

The MSI status is a prognostic factor for CRC, with the impact on prognosis varying by stage. MSI-H cancers have a better prognosis than MSS cancers in stage II and III CRCs [24–26]. On the contrary, stage IV disease with MSI-H has been linked to poor survival, although the prevalence of MSI-H in stage IV CRCs is only 3–4% [27, 28]. Few studies have performed survival analysis of patients with SCRCs according to the MSI status [22, 29]. Interestingly, Bae et al. found that patients with a discordant MSI status among their SCRCs had the worst clinical prognosis compared to those with a concordant MSI-H or MSS status [30]. In our cohorts, no statistically significant differences in RFS or OS rates were observed between groups, even when stage IV CRC patients were excluded. It may have been difficult to demonstrate statistical differences owing to the small sample size. Thus, the prognostic impact of MSI status discordance must be investigated through additional research with a larger sample population.

WES analysis revealed that SCRCs in a given patient shared only a few somatic mutations. Patients with Lynch syndrome also exhibited very low rates of shared somatic mutations between SCRCs, similar to those without Lynch syndrome. 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, 31]. Cereda et al. showed that SCRCs had independent genetic origins, somatic alterations, and clonal compositions [31]. 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 [31]. However, SCRCs showed a substantially higher proportion of shared variants in only one patient. In this case, the two lesions were located in the transverse colon at a distance of 1 cm, with intervening normal mucosa (Supplementary Fig. S6). A prior study investigating clonal relationships between different CRCs using molecular methods indicated that clonally related CRC is a frequent occurrence, with intraluminal spread being a probable cause [32]. The distance between lesions varied from 1.2 to 75 cm, and clonally related CRCs manifested synchronously or metachronously, indicating that clinicopathological characteristics may not always be helpful. However, since the distance between the lesions was small and they showed significant genetic similarity compared to other CRCs, 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 [32, 33].

In general, tumor heterogeneity affects the efficacy of therapies targeting specific key genes, such as KRAS, BRAF, or PIK3CA. 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 [34–37]. 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, the relatively small sample size, especially the small number of patients with MSI-H cancers, made it difficult to demonstrate significant differences in survival analysis according to the MSI status. 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. This study did not analyze germline mutation and 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% 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.

Acknowledgements: 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.

Author contributions

Conceptualization: Hyun Gu Lee, Yeseul Kim, In Ja Park, Seung-Mo Hong

Formal analysis: Hyun Gu Lee, Deokhoon Kim, Sun-Young Jun

Funding acquisition: In Ja Park, Seung-Mo Hong

Project administration: In Ja Park, Seung-Mo Hong

Resources: Hyun Gu Lee, In Ja Park, Mi-Ju Kim, Yeon Wook Kim, Sun-Young Jun

Supervision: In Ja Park, Seung-Mo Hong

Statistical analysis: Hyun Gu Lee, Deokhoon Kim.

Writing – original draft: Hyun Gu Lee, Yeseul Kim.

Writing – review & editing: Hyun Gu Lee, Yeseul Kim, Mi-Ju Kim, Yeon Wook Kim, Sun-Young Jun, Deokhoon Kim, In Ja Park, Seung-Mo Hong

Data curation: Hyun Gu Lee, Yeseul Kim

Data availability statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to conditions of the ethics committee of our university.

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).

Ethics declaration

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Asn 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.

Competing interests: The authors declare no competing interest.

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No competing interests reported.

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Molecular Mosaics: Unveiling Heterogeneity in Synchronous Colorectal Cancers

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Abstract

Figures

Introduction

Materials and methods

Study population and definition of clinical characteristics

Pathologic examination

Evaluation of MSI and WES

Statistical analysis

Results

Clinicopathological characteristics of patients with SCRC

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

Mutation analysis of synchronous cancers using WES

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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