Molecular characteristics of early-onset compared with late-onset colorectal cancer: a case controlled study

Abstract

Background:

Early-onset colorectal cancer (EOCRC) is associated with a poorer prognosis relative to late-onset colorectal cancer (LOCRC), and its incidence has witnessed a gradual escalation in recent years. This necessitates a comprehensive examination of the underlying pathogenesis and the identification of therapeutic targets specific to EOCRC patients. The present study aimed to delineate the distinct molecular landscape of EOCRC by juxtaposing it with that of LOCRC.

Methods:

A total of 11 344 colorectal cancer patients, diagnosed between 2003 and 2022, were enrolled in this study, comprising 578 EOCRC cases and 10 766 LOCRC cases. Next-generation sequencing technology was employed to assess the tumor-related mutation and tumor mutation burden (TMB) in these patients. PD-L1 expression was quantified using immunohistochemistry. Microsatellite instability (MSI) was determined via capillary electrophoresis (2B3D NCI Panel).

Results:

Upon comparing LOCRC with EOCRC patients, the latter group demonstrated a tendency towards advanced TNM stage, lower tumor differentiation, and less favorable histological types. Among LOCRC patients, those with MSI-H status were found to have an earlier TNM stage compared to those with MSI-L/MSS status. Significantly, the incidence of MSI-H was notably higher in EOCRC (10.2%) compared to LOCRC (2.2%). Mutations in the 7-gene panel (ARID1A, FANCI, CASP8, DGFRA, DPYD, TSHR, and PRKCI) were more prevalent in LOCRC. Within the EOCRC cohort, patients with the MSI-H subtype displayed an earlier TNM stage but concurrently exhibited poorer tissue differentiation and a higher frequency of mucinous adenocarcinoma. Among EOCRC patients, FBXW7, FAT1, ATM, ARID1A, and KMT2B mutations were significantly enriched in the MSI-H subgroup. A comparative analysis of MSI-H patients revealed heightened mutation frequencies of FGFBR2, PBRM1, RNF43, LRP1B, FBXW7, ATM, and ARID1A in the EOCRC group. Furthermore, EOCRC patients demonstrated a higher overall TMB, particularly in the MSI-H subtype. PD-L1 expression was elevated in EOCRC and positively associated with MSI status.

Conclusions:

This study revealed a significantly higher MSI-H distribution rate in EOCRC, and EOCRC exhibits a distinct mutational signature coupled with higher PD-L1 expression. These findings hold promise in guiding personalized therapeutic strategies for improved disease management in EOCRC patients.

Introduction

Highlights

• Early-onset colorectal cancer (EOCRC) patients show later TNM stages, lower tumor differentiation, and poorer histological types compared to late-onset colorectal cancer (LOCRC).

• EOCRC has a significantly higher frequency of MSI-H (10.2%) compared to LOCRC (2.2%), impacting TNM stage and histological differentiation.

• EOCRC patients with MSI-H exhibit increased mutation frequencies in FBXW7, FAT1, ATM, ARID1A, and KMT2B compared to LOCRC MSI-H patients.

• EOCRC patients, especially in the MSI-H type, show elevated tumor mutation burden and PD-L1 expression, suggesting potential for personalized treatment strategies.

Colorectal cancer (CRC) remains a significant global health challenge, with its treatment landscape continually evolving to address the diverse molecular and clinical features associated with the disease^1,2. Advances in therapeutic strategies, especially in the realm of immunotherapy, have reshaped the prognosis and management of CRC^3,4. The development of immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) and its ligand (PD-L1) has revolutionized cancer therapy, offering unprecedented improvements in patient outcomes across various malignancies, including CRC^5,6. Immunotherapy’s success in treating CRC is closely intertwined with understanding the intricate interplay between the tumor microenvironment, immune response, and molecular characteristics of the cancer⁷.

In recent years, the exploration of CRC has ventured into studying distinct subpopulations, such as early-onset colorectal cancer (EOCRC). EOCRC, characterized by its occurrence in individuals below the age of 50, presents unique challenges and opportunities for therapeutic interventions^8,9. EOCRC’s rising incidence and potentially aggressive behavior necessitate a comprehensive understanding of its clinical and molecular-pathological characteristics^10,11. This emerging field seeks to dissect the factors contributing to early-onset disease, investigate potential genetic predispositions, and delineate the molecular underpinnings that could guide targeted treatments. Despite the growing recognition of EOCRC as a distinct entity, there remains a paucity of research comprehensively unraveling its intricacies, especially in comparison to the more prevalent late-onset colorectal cancer (LOCRC).

This study aims to bridge the existing knowledge gap by conducting an exhaustive analysis of the clinical and molecular-pathological features distinguishing EOCRC from LOCRC. By meticulously examining a large patient cohort, we aspire to illuminate the salient characteristics that set EOCRC apart from its late-onset counterpart, encompassing factors such as age of onset, tumor differentiation, and genetic aberrations. Our investigation seeks to shed light on the mechanisms driving early-onset CRC and the potential implications for individualized therapies. Furthermore, we will delve into the rapidly evolving immunotherapy landscape in CRC, elucidating the current state of immunotherapeutic interventions and their relevance in enhancing prognosis for EOCRC patients. Ultimately, these findings could pave the way for informed therapeutic decision-making and optimized clinical outcomes in this distinct patient population.

In summary, the comparative exploration of EOCRC and LOCRC cohorts in this study offers a holistic understanding of the disparities and commonalities between these age-defined CRC subtypes. Insights into clinical variations, MSI status distribution, somatic mutations, and implications for immune therapy strategies collectively deepen our comprehension of CRC heterogeneity. Such revelations carry significant weight in refining treatment paradigms and enhancing patient outcomes.

Materials and methods

Patients

All procedures executed in research involving human participants adhere to the ethical standards set forth by institutional and/or national research committees, as well as the 1964 Declaration of Helsinki and its subsequent amendments, or analogous ethical standards. This cohort study follows the guidelines outlined in the “Strengthening the Reporting of Observational Studies in Epidemiology” (STROBE) statement for cohort studies and has received approval from the Ethics Committee. This work has been reported in line with the STROCSS criteria¹², Supplemental Digital Content 1, http://links.lww.com/JS9/C523. All patients were adequately informed and provided informed consent.

Included patients consisted of individuals diagnosed with colorectal cancer and receiving treatment at the Colorectal Center from January 2003 to December 2022, with histopathological confirmation of malignant colorectal tumors. All patients underwent comprehensive clinical evaluations upon admission, with treatment regimens aligned with the National Comprehensive Cancer Network (NCCN) guidelines for the management of colorectal cancer. Consequently, not all patients were subjected to immediate surgical intervention; rather, our patient cohort also encompassed cases that received neoadjuvant or conversion therapies prior to any operative procedures. All patients enrolled in our study underwent surgical treatment, either as a primary intervention or following neoadjuvant therapy.

Exclusion criteria encompassed (1): patients over the age of 80 (2); patients with recurrent colorectal cancer (3); patients with multiple primary tumors(4); patients with substantial missing data. In this study, early-onset colorectal cancer (EOCRC) patients were defined as those diagnosed with CRC under the age of 50. Data collected included patients’ clinical, pathological, and molecular variables, as well as tumor staging, differentiation, and molecular testing information. A total of 11 344 patients were included in this study (578 with EOCRC, 10 766 with late-onset CRC). The patient selection flowchart is illustrated in Supplemental Fig. 1, Supplemental Digital Content 2, http://links.lww.com/JS9/C524.

Figure 1.

Mutation spectrum in all lesions of EOCRC and LOCRC patients. (A) The detailed mutation type in EOCRC patients. (B) The detailed mutation type in LOCRC patients. The frequency of transition (Ti) or transversion (Tv) was compared. (C) The top 20 most frequently mutated genes and tumor mutation burden are shown in the histogram of EOCRC patients. (D) The top 20 most frequently mutated genes and tumor mutation burden in LOCRC patients. The number of mutations is illustrated in the right panel. The types of mutations are colored differentially and labeled. EOCRC, early-onset colorectal cancer; LOCRC, late-onset colorectal cancer.

MSI investigation

MSI status for each patient was determined using preoperative endoscopic biopsy specimens. The method primarily employed multiple fluorescent PCR combined with capillary electrophoresis. Specific microsatellite sequences were amplified through PCR, and differences in the lengths of microsatellite sequences between tumor and normal tissues were compared using capillary electrophoresis to determine the presence of MSI at a given locus. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples using a DNA isolation kit (AmoyDx 8.0223501X036G). An MSI detection kit (AmoyDx 8.0627301X024G) was used to analyze five mononucleotide repeat microsatellite markers (NR-24, BAT-25, CAT-25, BAT-26, MONO-27) in tumor and normal tissue samples. Two pentanucleotide markers (Penta D and Penta E) were simultaneously tested to confirm tissue homogeneity between tumor and normal samples. Capillary electrophoresis separation was performed using the ABI3500Dx genetic analyzer. The interpretation criteria were as follows, with the electropherogram of normal tissue serving as a reference: (I) MSI-H when the size alteration of two or more monomorphic mononucleotide products in tumor tissue was greater than or equal to 3 bp; (II) MSI-L when the size alteration of a single mononucleotide product in tumor tissue was greater than or equal to 3 bp; (III) MSS when no size alterations greater than or equal to 3 bp were observed in tumor tissue monomorphic mononucleotide products.

DNA extraction and targeted NGS

All patients underwent pathological tissue examination utilizing specimens obtained postoperatively following surgical resection. Genomic DNA from FFPE sections and whole blood control samples were extracted with the QIAamp DNA FFPE Tissue kit and DNeasy Blood and Tissue Kit (Qiagen, USA), respectively. Quantity and quality of the extracted DNA were evaluated by Qubit 3.0 fluorometer and Nanodrop 2000, respectively (Thermo Fisher Scientific).

Library preparations were performed using KAPA Hyper Prep kit (KAPA Biosystems) following manufacturer’s protocol. Hybridization-based target enrichment was carried out using the GeneseeqPrime pan-cancer gene panel with xGen Lockdown Hybridization and Wash Reagents Kit (Integrated DNA Technologies). Captured libraries by Dynabeads M-270 (Life Technologies) were amplified in KAPA HiFi HotStart ReadyMix (KAPA Biosystems) and quantified by qPCR using KAPA Library Quantification Kit (KAPA Biosystems). The target-enriched library was then sequenced on the HiSeq4000 NGS platform (Illumina) following the manufacturer’s instructions.

Sequence alignment and data processing

Trimmomatic was used for FASTQ file quality control. Leading/ trailing low quality (below 20) or N bases were removed. The sequencing data were aligned to the reference Human Genome (hg19) using Burrows-Wheeler Aligner (BWA-mem, v0.7.12)¹³. Alignment results underwent de-duplication by Sambamba (PMID: 25697820). Base quality recalibration and indel realignment were processed by Genome Analysis Toolkit (GATK 3.4.0)¹⁴. VarScan2 was employed for calling single-nucleotide variations (SNVs) and insertion/deletions (INDELs), which were identified with a minimum variant allele frequency threshold set at 0.01 and p value threshold for calling variants set at 0.05 to generate Variant Call Format files¹⁵. All SNVs/indels were annotated with ANNOVAR. The sequencing assay has been validated in compliance with the college of American pathologists (CAP) and clinical laboratory improvement amendments (CLIA) with a limit of detection of 1% VAF for tissue. Genomic fusions were identified by FACTERA with default parameters. Copy-number variations (CNVs) were detected using CNVkit with default parameters. Depth ratios of above 2.0 (tissue) and below 0.6 were considered as CNV gain and CNV loss, respectively¹⁶.

PD-L1 level

PD-L1 expression was evaluated using the Dako PD-L1 IHC 22C3 pharmDx kit (Agilent Technologies) in conjunction with the Dako Autostainer Link 48 system (Agilent Technologies). The assessment of PD-L1 expression included both the tumor proportion score (TPS) and the combined positive score (CPS).

Data analysis

Statistical analysis was performed using IBM SPSS Statistics version 26.0 and R version 4.0.4 for Windows (R Project for Statistical Computing). Two-tailed P values less than 0.05 were considered statistically significant. Continuous variables were presented as means with standard deviations (SD) or medians with interquartile ranges (IQR), depending on the distribution of data. For intergroup comparisons of continuous variables with a normal distribution, the t-test was used; otherwise, the Mann–Whitney U non-parametric test was employed. Categorical variable comparisons were conducted using the chi-square test or Fisher’s exact test, as appropriate. we employed the Kaplan–Meier (KM) method to visually illustrate the overall survival (OS) and disease-free survival (DFS) with logrank test.

Results

Clinical characteristics analysis

As depicted in Supplemental Fig. 1, Supplemental Digital Content 2, http://links.lww.com/JS9/C524, this study cohort included a total of 11 344 colorectal cancer patients, comprising 578 with EOCRC and 10 766 with LOCRC. The mean age of EOCRC patients was 30.7 years, contrasting with 54.2 years for LOCRC patients. No statistically significant differences were noted concerning patients’ sex or the proportion undergoing neoadjuvant or conversion therapy. Notably, EOCRC patients had a higher proportion of TNM stage I–II cases (44.1%) compared to LOCRC (35.1%). Furthermore, EOCRC displayed a higher incidence of poorly differentiated adenocarcinomas (26.8%), mucinous adenocarcinomas (25.8%), and signet-ring cell carcinomas (15.9%) compared to LOCRC (Table 1).

Table 1.

Clinical characteristics of patients with sporadic colorectal cancer.

	Patients No., n (%)
Characteristics	Early-onset (n=578)	Late-onset (n=10766)	P
Age
Mean (SD)	30.7 (7.34)	54.2 (15.7)	<0.001
Sex
Male	342 (59.2)	6439 (59.8)	0.76
Female	236 (40.8)	4327 (40.2)
TNM stage
I–II	255 (44.1)	3781 (35.1)	<0.001
III–IV	323 (55.9)	6985 (64.9)
Neoadjuvant/conversion
Yes	223 (38.6)	3791 (35.2)	0.09
No	355 (61.4)	6975 (64.8)
Tumor differentiation
Well differentiated	102 (17.6)	2997 (27.8)	<0.001
Moderately differentiated	321 (55.5)	6431 (59.7)
Poor differentiated	155 (26.8)	1338 (12.5)
Histological type
Adenocarcinoma	337 (31.3)	7569 (70.3)	<0.001
Mucinous adenocarcinoma	149 (25.8)	2019 (18.8)
Signet-ring cell carcinoma	92 (15.9)	1178 (10.9)

Distribution of MSI status in EOCRC and LOCRC

All 11,344 colorectal cancer patients underwent MSI testing and were classified into MSI-H, MSI-L, and MSS groups, with 298 cases in the MSI-H group and 11 046 cases in the combined MSI-L and MSS group, representing 2.6% and 97.4% respectively. Among EOCRC patients, 59 were identified as MSI-H (10.2% of total), while among LOCRC patients, 239 were MSI-H (2.2% of total). Within the EOCRC subgroup, MSI status did not significantly vary based on age or sex. However, EOCRC patients with MSI-H had a higher proportion of TNM stage I–II (66.1%) and elevated rates of poorly differentiated tumors (49.2%) and mucinous adenocarcinomas (52.5%). Similarly, the MSI-H subgroup in LOCRC also showed a higher proportion of TNM stage I–II. There were no significant associations between MSI status, age, sex distribution, tumor differentiation, or histological type in LOCRC (Table 2).

Table 2.

Clinical characteristics of sporadic colorectal cancer patients with different MSI status.

	Early-onset patients No. n=578, n (%)			Late-onset patients No. n=10 766, n (%)
Characteristics	MSI-H (n=59)	MSI-L/MSS (n=519)	P value	MSI-H (n=239)	MSI-L/MSS (n=10 527)	P
MSI status
MSI-H	59 (10.2)			239 (2.2)		<0.001
MSI-L/MSS	519 (89.8)			10 527 (97.8)
Age
Mean (SD)	31.3 (6.46)	29.2 (7.55)	0.11	56.7 (19.21)	58.1 (15.33)	0.23
Sex
Male	38 (64.4)	304 (58.6)	0.39	146 (61.1)	6293 (59.8)	0.68
Female	21 (35.6)	215 (41.3)		93 (38.9)	4234 (40.2)
TNM stage
I–II	39 (66.1)	216 (41.6)	<0.001	101 (42.3)	3680 (34.9)	0.02
III–IV	20 (33.9)	303 (58.4)		138 (57.7)	6847 (65.1)
Tumor differentiation
Well differentiated	20 (33.9)	82 (15.8)	<0.001	71 (29.7)	2926 (27.8)	0.48
Moderately differentiated	10 (16.9)	311 (59.9)		144 (60.3)	6287 (59.7)
Poor differentiated	29 (49.2)	126 (24.3)		24 (10.0)	1314 (12.5)
Histological type
Adenocarcinoma	20 (33.9)	317 (61.1)	<0.001	172 (72.0)	7397 (70.3)	0.78
Mucinous adenocarcinoma	31 (52.5)	118 (22.7)		44 (18.4)	1975 (18.8)
Signet-ring cell carcinoma	8 (13.6)	84 (16.2)		23 (9.6)	1155 (10.9)

MSI, microsatellite instability; MSI-H, high microsatellite instability; MSI-L, low microsatellite instability.

Somatic mutation comparison between EOCRC and LOCRC

NGS testing was performed on 180 EOCRC patients, with 15 identified as MSI-H, and 90 LOCRC patients, with 10 identified as MSI-H. To explore potential distinct mutation profiles in EOCRC and LOCRC, mutation analysis was conducted. As shown in Fig. 1A and B, no significant differences emerged in the detected mutation patterns between the two groups, affirming their comparability. The highest mutation frequencies in both EOCRC and LOCRC were observed in TP53 (77% and 76%), APC (68% and 68%), and KRAS (46% and 44%) (Fig. 1C and D). Additionally, comparing differential mutation profiles, significant high-frequency mutations were identified in LOCRC for genes ARID1A (14%), FANCI (6%), CASP8 (6%), DGFRA (7%), DPYD (7%), TSHR (6%), and PRKCI (6%) (Fig. 2A and B).

Figure 2.

Mutation landscape difference in EOCRC and LOCRC. (A) The detailed mutation frequency of genes in EOCRC and LOCRC. The types of mutations are colored differentially and labeled. (B) The mutation frequency difference was presented in forest maps with odds ratios (OR). EOCRC, early-onset colorectal cancer; LOCRC, late-onset colorectal cancer.

Somatic mutation distribution based on MSI subtypes

To further elucidate mutation spectrum distribution in different MSI subtypes of EOCRC, we compared 15 MSI-H EOCRC cases with 165 MSS EOCRC cases. In MSI-H EOCRC cases, the highest mutation frequencies were observed in ARID1A (60%), ATM (60%), FAT1 (60%), FBXW7 (60%), and KMT2B (60%), whereas in the MSS group, the highest mutation frequencies were observed in TP53 (75%), APC (68%), and KRAS (48%) (Supplemental Fig. 2A, Supplemental Digital Content 2, http://links.lww.com/JS9/C524 and Fig. 3A and B). Pathway enrichment analysis of these mutated genes revealed that RTK-RTS, PI3K, NOTCH, and WNT signaling pathways were most affected in MSI-H EOCRC, while in LOCRC, RTK-RTS, WNT, and TP53 signaling pathways were predominantly affected, indicating no significant differences between the two groups (Supplemental Fig. 2B, Supplemental Digital Content 2, http://links.lww.com/JS9/C524).

Figure 3.

Mutation landscape difference in EOCRC with different MSI status. (A) The detailed mutation frequency of genes in EOCRC with different MSI status. The types of mutations are colored differentially and labeled. (B) The mutation frequency difference was presented in forest maps with odds ratios (OR). EOCRC, early-onset colorectal cancer; MSI, microsatellite instability; MSI-H, high microsatellite instability.

Moreover, in LOCRC, comparison between MSI-H and MSS subgroups revealed the highest mutation frequencies in TP53 (80% vs. 75%), APC (60% vs. 69%), and KRAS (40% vs. 45%) (Supplemental Fig. 3A, Supplemental Digital Content 2, http://links.lww.com/JS9/C524). The pathway enrichment analysis suggested similar impacts on TP53, WNT, and RTK-RTS signaling pathways in both groups (Supplemental Fig. 3B, Supplemental Digital Content 2, http://links.lww.com/JS9/C524).

Given the progress in immune therapy for colorectal cancer, MSI-H tumors confront novel therapeutic strategies. To investigate molecular-pathological differences between EOCRC and LOCRC, we conducted a separate analysis on the MSI-H subgroups. As shown in Fig. 4A, compared to the LOCRC MSI-H subgroup, EOCRC MSI-H patients displayed significantly elevated mutation frequencies in genes such as FGFBR2 (47%), PBRM1 (47%), RNF43 (53%), LRP1B (47%), FBXW7 (60%), ATM (60%), and ARID1A (60%), whereas these genes exhibited minimal mutations in the MSI-H LOCRC subgroup.

Figure 4.

Somatic mutation in MSI-H EOCRC patients. (A) Mutation spectrum of special somatic mutation in MSI-H EOCRC patients. The frequency was labeled in left. (B) The mutation frequency difference was presented in forest maps with odds ratios (OR). EOCRC, early-onset colorectal cancer; MSI, microsatellite instability; MSI-H, high microsatellite instability.

Tumor mutation burden and PD-L1 expression

The prognosis of EOCRC is intimately tied to its specific molecular pathology. Existing evidence points to a correlation between tumor mutations and the efficacy of immune therapy. Further analysis of the tumor mutational burden (TMB) distribution in various subgroups using NGS data revealed significantly higher TMB in MSI-H patients in both EOCRC and LOCRC (Fig. 5A). Intriguingly, in the MSS subgroup of EOCRC, TMB was elevated compared to the MSS subgroup of LOCRC. Although PD-L1 therapy in colorectal cancer patients remains investigational, we assessed PD-L1 expression in tumor tissues using immunohistochemistry with both tumor proportion score (TPS) and combined positive score (CPS) evaluation methods. Results indicated that overall PD-L1 expression was higher in EOCRC than LOCRC, with the highest PD-L1 expression observed in MSI-H EOCRC patients (Fig. 5B).

Figure 5.

Tumor mutation burden (TMB) and PD-L1 value among each group of patients with early-onset colorectal cancer and late-onset colorectal cancer. (A) The TMB value of each patient was determined to be the highest TMB value of each lesion of the patient. mut/mB indicates mutations per megabase. B. PD-L1 expression was evaluated using tumor proportion score and combined positive score. * indicated P<0.05; ** indicated P <0.01. MSI, microsatellite instability; MSI-H, high microsatellite instability.

Prognosis analysis based on MSI status

In a deeper exploration of the relationship between molecular pathology and patient prognosis, we employed PCR-based methodology to determine the MSI (microsatellite instability) status in enrolled patients, subsequently dividing them into three categories: MSS (microsatellite stable), MSI-L (low microsatellite instability), and MSI-H (high microsatellite instability). This was carried out to investigate the correlation between different MSI statuses and patient outcomes. The study disclosed that, in LOCRC cohorts, patients in the MSS group had the poorest prognosis, whereas those in the MSI-L group demonstrated a marked improvement in prognosis relative to the MSS group. Importantly, patients categorized as MSI-H had the most favorable prognosis among the three classes, thereby indicating a significant link between MSI status and patient prognosis. Although no statistically significant differences were observed within the EOCRC cohort, a trend in the data suggests that patients in the MSS group display a relatively poorer prognosis compared to those in the MSI-H group, as evinced by their OS and progression-free survival (PFS) profiles (Fig. 6A and B).

Figure 6.

MSI status associated with overall survival (OS) and disease-free survival (DFS) of colorectal cancer patients. (A) Patients were divided into three groups including MSS, MSI-L and MSI-H. The OS and DFS were calculated in EOCRC patients. (B) Patients were divided into three groups including MSS, MSI-L and MSI-H. The overall survival and disease-free survival were calculated in LOCRC patients. EOCRC, early-onset colorectal cancer; LOCRC, late-onset colorectal cancer; MSI, microsatellite instability; MSI-H, high microsatellite instability; MSI-L, low microsatellite instability.

Discussion

This study provides comprehensive insights into the clinical and molecular characteristics of EOCRC and LOCRC through a meticulous analysis of a sizable cohort of patients. The investigation reveals intriguing patterns in terms of clinical features, MSI status distribution, somatic mutation profiles, and potential implications for immune therapy strategies. The discussion centers on the noteworthy findings within these domains.

The clinical analysis presented here underscores the distinct profiles of EOCRC and LOCRC. EOCRC patients exhibited a significantly younger mean age compared to their LOCRC counterparts, which aligns with the concept of an earlier disease onset in this subgroup. Moreover, the cohort’s sex distribution was comparable between EOCRC and LOCRC, suggesting that sex-based disparities may not play a substantial role in the observed differences. Importantly, the study found that EOCRC patients had a higher proportion of TNM stage I–II cases, indicating a potential trend toward earlier-stage diagnosis in this subgroup. This finding could reflect variations in disease progression dynamics between the two age-defined cohorts. Interestingly, a higher prevalence of poorly differentiated adenocarcinomas, mucinous adenocarcinomas, and signet-ring cell carcinomas was evident in EOCRC patients, hinting at potential differences in tumor biology and histopathological subtypes that could contribute to the clinical distinctions.

Over the past decade, researchers have paid significant attention to the epidemiological and pathological characteristics distinguishing EOCRC from LOCRC. Studies during this period have reported findings that highlight key differences in these two subsets of the disease. For instance, investigators have meticulously collated and synthesized epidemiological evidence on the prevalence of MSI-H/dMMR phenotypes, along with recurrent genetic alterations such as KRAS, NRAS, BRAF, PIK3CA, and TP53, in the context of EOCRC through the application of rigorous systematic review and meta-analysis methodologies¹⁷. Gao et al.¹⁸ have also published a retrospective cohort study encompassing over 34 000 cases, comparing various aspects of EOCRC and LOCRC in China, such as trends, clinicopathological characteristics, surgical treatment modalities, and patient outcomes. However, their investigation did not delve into the molecular intricacy achieved in the present work, particularly with regard to the assessment of immunotherapy-relevant biomarkers like TMB and PD-L1 expression. Meanwhile, a population-based study harnessing database resources has uncovered unique attributes in EOCRC cases concerning MSI, CpG island methylator phenotype (CIMP), and chromosome instability (CIN). Notably, a subset of these cases exhibits a microsatellite-stable, chromosomally-stable phenotype, coined MACS. It is crucial to acknowledge, though, that this research’s methodology is inherently constrained by its dependence on database-derived data¹⁹. More recently, a study has highlighted dissimilarities in the anatomical origins of EOCRC and LOCRC, revealing marked divergences in histopathology, primary tumor localization, and stage at diagnosis. Specifically, EOCRCs tend to arise more frequently in the distal colon and rectum among younger patients, often manifesting at advanced stages. This study also offered a panoramic view of the mutational landscape of KRAS, NRAS, PIK3CA, and TP53 across EOCRC instances. Nonetheless, it was founded on a meta-analysis of previously published literature²⁰. In contradistinction, the current study, rooted in a molecular-pathological interrogation of our institution’s specific patient cohort, strives to furnish potentially more exhaustive insights into the molecular pathology underlying EOCRC.

Microsatellite instability (MSI) analysis is a pivotal component of colorectal cancer assessment due to its prognostic and therapeutic implications²¹. The differential distribution of MSI subtypes in EOCRC and LOCRC points to varying underlying genetic alterations and potential etiological factors driving carcinogenesis^9,22. Notably, MSI-H cases were significantly higher in EOCRC compared to LOCRC, indicating a potential association between early-onset cases and MSI-high status. This observation supports previous studies suggesting a higher prevalence of MSI-H tumors in younger patients^23,24. Interestingly, within the EOCRC subgroup, MSI status exhibited a correlation with TNM stage and tumor differentiation, reinforcing the notion that MSI can influence clinical and pathological features^21,25. Besides, in the MSI-H population of LOCRC, we have observed a relatively favorable prognosis, which aligns with previous research findings²⁶. Although no statistically significant difference was discerned in EOCRC group, the overall trend indeed echoes the consistency seen in EOCRC studies. This lack of statistical significance could potentially be attributed to the relatively smaller sample size within our cohort.

The somatic mutation analysis conducted here sheds light on the genetic landscape of EOCRC and LOCRC, offering insights into potential therapeutic avenues. The comparison of mutation profiles between EOCRC and LOCRC revealed remarkable consistency in the distribution of high-frequency mutations, particularly in key genes such as TP53, APC, and KRAS. These genes have been extensively implicated in colorectal cancer pathogenesis, highlighting their significance across different age-defined subgroups^21,27. However, significant high-frequency mutations unique to LOCRC, including genes such as ARID1A, FANCI, CASP8, and others, suggest potential age-associated differences in mutational processes and tumor biology. This finding accentuates the complexity of colorectal cancer genetics and the need for targeted investigations in distinct patient cohorts.

The emergence of immune therapy has revolutionized cancer treatment paradigms, particularly for MSI-H tumors²⁸. Especially, the results from the NICHE or NICHE-2 study, which investigated the efficacy and safety of the combination therapy has ushered in a new era in immunotherapy^29,30. The employment of Nivolumab, a programmed cell death protein 1 (PD-1) inhibitor, in conjunction with Ipilimumab, a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor, as neoadjuvant therapy for locally advanced colorectal cancer (CRC) characterized by mismatch repair deficiency (dMMR), has yielded highly promising results, with a remarkable pathological complete response rate reaching 67%. This outcome has spurred significant optimism within the research community. Here, we focus on the MSI-H subgroup of both EOCRC and LOCRC assumes critical relevance. The observed elevated mutation frequencies in specific genes within EOCRC MSI-H patients compared to their LOCRC counterparts highlight the potential for novel therapeutic strategies tailored to early-onset cases. These genetic distinctions could influence treatment responses and resistance mechanisms, ultimately affecting patient outcomes. Furthermore, the exploration of TMB and PD-L1 expression provides valuable insights into the potential efficacy of immune checkpoint inhibitors³¹. The higher TMB observed in MSI-H patients aligns with existing evidence linking elevated mutation loads to enhanced response rates to immunotherapy³². This finding underscores the significance of TMB as a potential predictive biomarker for immune therapy in colorectal cancer. The higher PD-L1 expression in EOCRC, particularly in MSI-H cases, points toward the possible utility of immune checkpoint inhibitors in this subgroup, warranting further investigation in prospective clinical trials.

Currently, there is a lack of comparative studies on the efficacy of PD-1 immune therapy between EOCRC and LOCRC patients. The effectiveness of PD-1 therapy is closely related to the status of immune cells in the tumor microenvironment³³. In EOCRC, studies have reported immune cell infiltration, with a higher proportion of suppressive immune cells. Therefore, the combination of PD-1 immune therapy with other targets could potentially offer significant guidance to enhance the efficacy of EOCRC treatment.

Moreover, among the specific mutations detected in the EOCRC MSS cohort, several genes exhibiting high mutation frequencies are strongly linked to the tumor immune microenvironment or PD-1-targeted therapy. ARID1A, when mutated, has been demonstrated to possess heightened immunogenicity within the tumor milieu and is thus a promising candidate for monotherapy or first-line chemotherapy in combination with PD-1 inhibition³⁴. Loss of function in the ATM gene can trigger the cGAS/STING pathway through the leakage of mitochondrial DNA into the cytoplasm, instigating an immune response in certain malignancies. Pharmacological suppression of ATM has been shown to augment the efficacy of anti-PD-1 treatment³⁵. FBXW7, through a phosphorylation-dependent mechanism, ubiquitinates PD-1, ultimately promoting the degradation of PD-1 protein and augmenting cytotoxic lymphocyte infiltration within the tumor environment. Assessment of FBXW7 status has proven useful in predicting the clinical responsiveness of non-small cell lung cancer patients to anti-PD-1 immunotherapy³⁶. These immune microenvironment-related gene mutations collectively suggest the potential utility of PD-1-directed therapy in MSI-H EOCRC patients in the future.

The foremost limitation of this research lies in the relatively modest number of EOCRC cases with available NGS testing data, which restricts the power to draw definitive conclusions from clinical prognosis analyses based on mutational status. Furthermore, as a monocentric investigation, this study necessitates validation through independent or multi-center studies to enhance generalizability. Additionally, our study is subject to a conceptual limitation. Specifically, the analysis of genomic alterations detected herein was confined to surgical specimens rather than biopsy samples. This approach restricts the potential utility of the identified molecular targets for guiding neoadjuvant therapy, as biopsy profiling offers more timely information to inform upfront treatment decisions. While surgical specimen profiling can aid in devising adjuvant treatment strategies, it does not contribute to preoperative patient management. Lastly, while our study primarily focuses on describing the differential molecular mutations between EOCRC and LOCRC, the functional roles these mutated targets play in the pathogenesis or progression of colorectal cancer remain an area requiring further exploration in subsequent research endeavors. Another limitation of this study is the lack of information on potential confounding variables that could impact the observed associations. Despite the comprehensive analysis of clinical and molecular characteristics, unaccounted factors such as lifestyle, dietary habits, socioeconomic status, and genetic predispositions might contribute to the observed differences in EOCRC and LOCRC. These variables could potentially influence the prevalence of specific molecular alterations and patient outcomes, introducing bias and affecting the interpretation of the results. Addressing these confounding factors through multivariate analyses or matched cohort designs in future studies would enhance the accuracy of the conclusions and the clinical relevance of the findings.

Conclusion

To encapsulate, this study provides a comprehensive analysis of clinical and molecular characteristics in early-onset and late-onset colorectal cancer. The observed differences in clinical features, MSI distribution, somatic mutation profiles, and implications for immune therapy emphasize the intricate interplay between age, genetics, and disease pathogenesis. These findings contribute to our understanding of colorectal cancer heterogeneity and underscore the importance of tailored treatment strategies based on age-related molecular differences. As research in this field advances, unraveling the complexities underlying age-associated variations in colorectal cancer will offer novel avenues for targeted and effective therapeutic interventions.

Ethical approval

This cohort study follows the guidelines outlined in the "Strengthening the Reporting of Observational Studies in Epidemiology" (STROBE) statement for cohort studies and has received approval from the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University(2023-IRB-041).

Consent for publication

Not applicable.

Source of funding

This work was supported by the National Natural Science Foundation (Grant Number 82100617, 82273406); Nature Key Research and Development Program of Jiangsu Province (BE2021742); Basic Research Program of Jiangsu Province (BK20201491, BK20221415); National Key Research and Development Program of China (2017YFC0908200); and Jiangsu Key Medical Discipline (General Surgery; Grant No. ZDXK202222).

Author contribution

J.T., W.P. and C.T.: acquisition, analysis, or interpretation of data. Y.Z. and D.J.: drafting of the manuscript. L.W.: methodology, writing—reviewing and editing. K.J., F.W. and Y.S.: software, validation. Y.S. and X.W.: supervision, project administration and funding acquisition.

Conflicts of interest disclosure

The authors declare that they have no competing interests.

Research registration unique identifying number (UIN)

Our study was registered in Clinical Trail with the ID NCT06238193.

Guarantor

Yueming Sun.

Data availability statement

The sequencing data in this study are protected and restricted to be used with institutional permission and are therefore not publicly available due to data privacy policies. Requests will be assessed according to institutional policies to determine whether the data request is subject to patient privacy obligations. A user agreement will be required. All other relevant data supporting the key findings of this study are available from the corresponding authors upon request.

Provenance and peer review

Not commissioned, externally peer-reviewed.

Supplementary Material

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js9-110-4559-s002.docx ^{(1.2MB, docx)}