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Objective: The genomic landscapes of metastatic colorectal cancer (mCRC) have been extensively studied; however, the genetic mechanisms underlying the locoregional recurrence (LR) of CRC remain unclear. The objective of our study was to investigate genomic evolution during LR in CRC using high-throughput sequencing. Methods: Twenty-three CRC patients with matched primary and LR tissues were recruited from Nanfang Hospital and Zhejiang Cancer Hospital between January 2011 and December 2018. The last date of follow-up was March 2020. Tissue samples were analyzed by whole-exome sequencing and the genomic profiles were depicted by single nucleotide variation, mutational signature, copy number variation, clonal architecture, and other features. The evolutionary process was speculated with comparison of the genetic variations between primary and LR lesions. The disseminating clusters from primary to LR lesions were identified by variant allele frequency dynamics. Furthermore, the early-recurrent biomarker was explored by comparing the indel signature between early- and late-recurrent patients. The study was approved by the Institutional Review Board of Nanfang Hospital of Southern Medical University (approval No. 2020010) on September 11, 2020. Results: The results highlighted distinct origins of LR between patients with high microsatellite instability and microsatellite stability. LR lesions evolved independently in patients with high microsatellite instability, while LR lesions were highly clonally related to the primary lesions in patients with microsatellite stability. Late-acquired variations in LR lesions encompassed a wide range of driver genes involved in histone methylation, DNA replication, T cell activation, PDCD1 gain, and LMNA loss. Furthermore, clonal analysis of the disseminating cells identified a dominant polyclonal seeding pattern during LR. The indel signature ID4 was associated with significantly shorter disease-free survival in patients with relapsed CRC according to a public dataset. Conclusion: These findings pose a challenge for the development of new approaches targeting the interactions of multiple clones in the establishment of LR and in terms of optimizing the clinical management of susceptible patients. Keywords: biomarker, colorectal cancer, locoregional recurrence, polyclonal seeding, tumor evolution XL, XxW, CZ, and GW contributed equally to the writing of the article. a b Department of Pathology, Nanfang Hospital and Basic Medical College, Department of General Surgery & Guangdong Provincial Key Laboratory of Precision Medicine for Gastrointestinal Tumor, Nanfang Hospital, The First School of Clinical Medicine, Southern Medical University, Guangzhou, Guangdong Province, c d Department of Pathology, Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), Institute of Basic and Cancer Medicine e f Geneplus-Shenzhen, Shenzhen, Huiqiao Medical Center, Nanfang Hospital, Southern Medical (IBMC), Chinese Academy of Sciences, Hangzhou, Zhejiang Province, g h Guangdong Province Key Laboratory of Molecular Tumor Pathology, Guangzhou, Department of Pathology, Molecular Pathology Research University, Guangzhou, Center, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China * Corresponding authors: Li Liang, Department of Pathology, Nanfang Hospital and Basic Medical College, Southern Medical University; Guangdong Province Key Laboratory of Molecular Tumor Pathology, Guangzhou 510515, Guangdong Province, China. E-mail: firstname.lastname@example.org; Dan Su, Department of Pathology, Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital); Institute of Basic and Cancer Medicine (IBMC), Chinese Academy of Sciences, Hangzhou 310022, Zhejiang Province, China. E-mail: email@example.com; Zhiyong Liang, Department of Pathology, Molecular Pathology Research Center, Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing 100000, China. E-mail: firstname.lastname@example.org. Copyright © 2022 The Chinese Medical Association, Published by Wolters Kluwer Health, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Journal of Bio-X Research (2022) 5:171–180 Received: 5 August 2021; Accepted: 2021 November 12 Published online 9 December 2021 http://dx.doi.org/10.1097/JBR.0000000000000116 171 RESEARCH ARTICLE Journal of Bio-X Research Introduction The study was approved by the Institutional Review Board of Nanfang Hospital of Southern Medical University (approval No. Colorectal cancer (CRC) is the third leading cause of cancer-re- 2020010) on September 11, 2020 and conducted in accordance  lated deaths worldwide. Although distant metastasis remains with the 1964 Declaration of Helsinki, as revised in 2013. the most common cause of death, locoregional recurrence (LR) of CRC after surgery is also an issue of increasing concern. Library construction and high-throughput sequencing The reported incidence of locally recurrent colon cancer var- [2–5] ied from 10% to 29%. The incidence of LR rectal cancer Genomic DNA prepared as above was fragmented using an before the adoption of total mesorectal excision was 40% to ultra-sonicator UCD-200 (Diagenode, Seraing, Belgium) and  50% in patients with advanced-stage disease, but the broad then purified and size-selected with magnetic beads (Beckman,  adoption of this approach has reduced the incidence to <10%. MA, USA). The quality of the DNA was determined using a In addition, preoperative neoadjuvant chemoradiotherapy with Qubit 2.0 Fluorometer with Quanti-IT dsDNA HS Assay Kit subsequent total mesorectal excision has further reduced the (Thermo Fisher Scientific, Waltham, MA, USA). Library con- [8,9] incidence of LR of rectal cancer in several clinical trials. struction was then performed using a custom 53 M whole- LR in patients with CRC is always associated with a poor exon capturing probe (IDT, Coralville, IA, USA). The captured prognosis. A previous report revealed 5-year overall survival libraries were then pair-end sequenced in 100-bp lengths using rates of 9% and 13% for patients with LR of colon cancer and the Geneplus-2000 sequencing platform (Geneplus, Beijing,  rectal cancer, respectively. The patterns and predictors of LR China), following the manufacturer’s instructions. Raw data after curative resection have been investigated to optimize the from next-generation sequencing was then filtered to remove  therapeutic regimen and postoperative surveillance. Liska et al low-quality reads and adaptor sequences. Reads were further proved that tumor stage, bowel obstruction, margin involvement, aligned to the reference human genome (hg19) utilizing BWA lymphovascular invasion, and local tumor invasion were signifi-  aligner software (version 0.7.10) for mutation calling.  cantly associated with colon cancer LR, and Rokke et al further demonstrated that reoperation for postoperative complications, Genomic data analysis other than anastomotic leakage, was a significant predictor of LR Single nucleotide variants (SNVs) were called by MuTect (ver- of CRC. Although recent advances in molecular characterization  sion 1.1.4). For quality control, somatic mutations were of CRC have identified prognostic and predictive biomarkers for [13–15] identified if they met the following criteria: (1) present in disease recurrence, the pattern of genomic evolution between <1% of the population in the 1000 Genomes Project (https:// paired primary and LR CRC tissues and molecular evidence for www.internationalgenome.org/), the Exome Aggregation the origin of recurrence are still unclear. Consortium (ExAC), and the Genome Aggregation Database In this study, we used whole-exome sequencing to scruti- (gnomAD) (https://gnomad.broadinstitute.org); (2) not pres- nize the molecular events occurring during genomic evolution ent in paired germline DNA from normal tissues; and (3) in a unique cohort of 23 patients with CRC, for whom paired detected in at least three high-quality reads containing the par- primary and LR tumor samples were available. With the cut- ticular base, where high-quality reads were defined as a Phred ting-edge analysis tools, we aim to compare the LR patterns score >30, mapping quality >30, and without paired-end reads between high microsatellite instability (MSI-H) and microsat- bias. Germline mutations were called using GATK software ellite stability (MSS) patients, identify the disseminating mecha-  (version 4.0) and an in-house script. The germline variations nism of the primary tumor, and explore the potential biomarker were validated in the ClinVar database (https://www.ncbi.nlm. for LR early detection and precise clinical management. nih.gov/clinvar/), to confirm its pathogenicity. Mutational sig- nature analysis was performed with unfiltered somatic muta-  Materials and methods tions using R package YAPSA and matched to the COSMIC signature database (https://cancer.sanger.ac.uk/cosmic/signa- Patient specimen acquisition tures). Somatic CNVs (SCNVs) were identified using GATK In this retrospective observational study, 23 patients with CRC (version 4.0) and the frequency of larger fragmental CNVs was who completed standard initial treatment were recruited from  detected using R package Copy number. The focal level of January 2011 to December 2018. Clinicopathological character- SCNV was detected using an in-house script with BAM files. istics of patients were obtained from the hospital’s case manage- The clonal architectures of the somatic mutations were inferred ment system. The inclusion criteria include that patients were  by ABSOLUTE software considering tumor purity and copy diagnosed with colon or rectal cancer, patients had not received number alterations. Events with an estimated upper 95% con- any treatment before surgery, both primary tumor and LR tumor fidence interval for the cancer cell fraction (CCF) of 1 were can be obtained, patients were metastasis-free. The included defined as clonal, whereas the rest were defined as sub-clonal. 13 patients with high sequencing read coverage enrolled from A list of driver genes was referenced from previous reports Nanfang Hospital of Southern Medical University, Guangzhou [22,23] and databases (https://cancer.sanger.ac.uk/census). The City and 10 patients with low sequencing read coverage pathway enrichment was performed with an in-house script. recruited from Zhejiang Cancer Hospital, Hangzhou City, whose Clusters of seeding clones were calculated using PyClone-VI sequencing data were used to validate chromosome arm copy   software and visualized by ClonEvol software. The public number variation (CNV). All patients underwent radical surgery. dataset of CRC which consists of 276 cases, was downloaded Primary tumor, matched LR tumor, and matched normal tissue from the cbioportal database (https://www.cbioportal.org/). samples were collected, and formalin-fixed, paraffin-embedded samples were prepared. Genomic DNA was extracted using a Statistical analysis TIANamp Genomic DNA kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions. Written informed Data were analyzed by two-sided Mann-Whitney U test, consent was obtained from all patients during sample collection. Fisher exact test, Wilcoxon test and Pearson’s correlation using 172 Journal of Bio-X Research RESEARCH ARTICLE GraphPad Prism (version 7.01; GraphPad, San Diego, CA, Driver mutations in primary and LR lesions  USA) or R (version 3.6.1). For all tests, a P value < 0.05 was Coding driver mutations were identified to construct a mutational considered statistically significant. The enrolled patients were landscape. The most frequently mutated drivers such as KRAS, selected according to inclusion and exclusion criteria, because APC, and TP53, have been well-characterized in the progression it is extremely difficult to collect three samples from the same of CRC (Fig. 1C). In addition, KRAS mutation status was concor- patient, which might take many years, thus 23 patients in this dant between primary and matched LR lesions in MSS patients, cohort are the maximum number we can recruit. but KRAS mutation only occurred in the primary tumor in one MSI-H patient. TP53 mutations were concordant between the pri- mary and LR samples in most (8/10) MSS patients but differed Results between the two samples in two of the three MSI-H patients. These Distribution of somatic mutations between primary and results suggest that KRAS and TP53 mutations might be common LR lesions in patients with CRC clonal mutations promoting recurrence of the primary tumor in MSS patients but may not be associated with recurrence in MSI-H Thirteen patients were recruited and 39 formalin-fixed, paraf- patients. Moreover, several driver mutations with high frequencies, fin-embedded samples were subjected to whole-exome sequenc- such as BRCA2, INTS1, KMT2B, KMT2D, and RNF43, were ing (13 sets of primary tumors, LR tumors, and adjacent normal found exclusively in LR lesions in MSS patients and might thus tissues). The clinical characteristics of this cohort are summarized play significant roles in the progression of LR. We further inves- in Table 1. We detected averages of 291 (range 22-1642) and 383 tigated the distributions of KRAS and TP53 mutations in MSI-H (range 40-2451) non-synonymous somatic mutations and indels  patients in The Cancer Genome Atlas (TCGA) dataset. Notably, in the primary and LR tumor samples, respectively. We calcu- KRAS (Additional Fig. 1B, http://links.lww.com/JR9/A36) and lated the mutation distributions across each patient (Fig. 1A) TP53 (Additional Fig. 1C, http://links.lww.com/JR9/A36) muta- and showed that 10 patients had 12% to 76% shared SNVs and tions were significantly less frequent in MSI-H compared with MSS indels across both primary and LR lesions, while the other three patients. These results emphasized the different impacts of KRAS patients had very few shared SNVs or indels. Notably, both the and TP53 mutations on LR between MSI-H and MSS patients. primary and LR tumor samples in the 10 patients were MSS, while both samples in the other three patients were MSI-H. We examined the germline variations of the three MSI-H patients to Mutational signatures in disease progression of CRC clarify the etiology of the MSI-H status. MLH1 nonsense muta- We examined the mutational signature to identify the processes tion c.5C > A, splice site mutation c.208-1G > A, and PMS2 mis- contributing to the early initiation and late evolution of CRC. The sense mutation c.2570G > C were identified in patients P1 to higher mutation rates in LR lesions suggested that the patterns P3, respectively (Additional Fig. 1A, http://links.lww.com/JR9/ could potentially be indicative of disease progression. The muta- A36). Based on this molecular evidence, these three patients were tion signatures were stratified as mutations exclusive to primary identified with non-sporadic familial CRC with MSI-H status. In and LR lesions, respectively, and shared mutations. In light of their contrast, the MSS patients shared an average of 41.61% of all discrepant mutational mechanisms, the signatures of MSI-H and mutations between the primary and LR lesions, indicating the MSS patients were considered separately. A total of 23 single-base existence of common ancestral clones between the two lesions. In substitution (SBS) signatures were matched with the COSMIC addition, 22.99% (5.15%-60.9%) and 35.4% (8.43%-82.39%) database (https://cancer.sanger.ac.uk/cosmic/signatures) in all 13 of mutations in the MSS patients were exclusive to the primary patients (Fig. 2A and B). SBS1, SBS6, and SBS15 occurred in most and LR lesions, respectively. LR lesions included significantly of these patients. SBS1 resulted from an endogenous mutational more mutations than the primary lesions (P< 0.05; Fig. 1B), sug- process initiated by spontaneous deami-nation of 5-methylcyto- gesting that the mutation rate increased in LR tumors compared sine and correlated with age at cancer diagnosis, while SBS6 and with their matched primary tumors after divergence. These data SBS15 were associated with defective DNA mismatch repair. The suggest a difference in the origin of LRs between patients with three MSI-H patients had no shared SBS signatures between the MSI-H and MSS CRC. individual primary lesion and LR lesion, while each MSS patient had a high contribution of shared SBS signatures. Strikingly, the Table 1 signature heterogeneity across each patient was greater than Patients’ demographic information that across each evolutionary stage. According to their treatment records, six patients (P1, P2, P5, P7, P8, and P9) underwent post- Characteristics No. of cases Proportion (%) operative chemoradiotherapy. However, the LR lesions in these Total number 23 patients showed a lack of new signatures, although few shifts were Age [yr, mean (range)] 58 (37-86) observed in the relative contributions of SBS signatures over dif- Sex ferent evolutionary stages. These results suggest that chemoradio- Male 16 70 therapy is unlikely to be a major cause of driver accumulation. Female 7 30 Smoking history Smoker 9 39 Frequent focal CNVs were enriched in LR lesions Non-smoker 14 61 Drinking 7 30 Regarding chromosomal segment distribution, large CNVs were Diagnosed site infrequent in MSI-H patients (Additional Fig. 2, http://links.lww. Colon cancer 15 65  com/JR9/A36), consistent with a previous report. However, Rectal cancer 8 35 there was obvious segmental variation between primary and Stage at diagnosis I-II 7 30 LR lesions in MSS patients. We compared the CNV frequencies III-IV 16 30 calculated by 2M segment as a unit between primary and LR Chemotherapy 10 43 lesions for all patients (Fig. 3A). LR lesions included losses in 173 RESEARCH ARTICLE Journal of Bio-X Research Figure 1. Distribution of somatic mutations between primary and LR lesions in CRC. (A) Percentages of somatic mutations in matched primary and LR lesions in 13 patients. (B) Percentages of exclusive and shared mutations between primary and LR lesions in 10 microsatellite stability (MSS) patients. Data are expressed as the mea±SD. P=0.0227 (Mann-Whitney U test). (C) Mutational landscapes in 13 patients with matched primary and LR lesions, showing number of somatic mutations in each patient (top), mutation frequency of each gene (right), and other clinical information including tumor site, microsatellite status, variation types (bottom). CRC=Colorectal cancer, LR = locoregional recurrence, P1–P3 = Patient 1–Patient 13. segments located in 1q, 5p, and 6q and gains in segments in 2q, functional impact on disease progression. Significant copy num- 5q, and 6q. We validated large segmental variations in the addi- ber gain of PDCD1 (2q37.3) and loss of LMNA(1q22) were tional 10 pairs of primary and LR samples with lower sequenc- enriched in LR lesions (Fig. 3B and C). ing depth (12–123 X). Compared with the primary lesions, the major fragment variations with lower sequencing depth were Dynamics of mutation clonality between primary and LR consistent with the previous 13 pairs of samples (Additional lesions Fig. 3, http://links.lww.com/JR9/A36). Although limited by the small sample size, we sought to identify the CNVs between pri- We further investigated the evolution of LR by calculating mary and LR lesions at the driver gene level to emphasize the the CCF value and defined clonal and subclonal mutations. 174 Journal of Bio-X Research RESEARCH ARTICLE Figure 2. Mutation signatures of disease progression in patients with CRC. (A) Signature analysis of exclusive mutations in primary and LR lesions in three microsatellite instability (MSI-H) patients, the various color block represents different SBS signature. (B) Signature analysis of exclusive and shared mutations in primary and LR lesions for 10 microsatellite stability (MSS) patients, the various color block represents different SBS signature. The number represents dif- ferent signatures derived from COSMIC database. CRC=Colorectal cancer, LR = locoregional recurrence, P1–P13 = Patient 1–Patient 13, SBS = single base substitution signature. We created a CCF scatter plot to exhibit dynamic clonal-sub- attributes of the evolutionary stages (Fig. 4A and Additional clonal transitions between primary and LR lesions. Most of the Fig. 4A, http://links.lww.com/JR9/A36). In contrast, dynamic clonal and subclonal mutations were exclusive to either primary clonal transition was observed in MSS patients, suggesting or LR lesions in MSI-H patients, indicating the independent the existence of a common ancestor between the primary and 175 RESEARCH ARTICLE Journal of Bio-X Research Figure 3. Frequent focal copy numbervariations (CNVs) were enriched in LR lesions. (A) Comparison of fragment variations between primary and LR lesions. X-axis indicates chromosome number and Y-axis indicates the percentage of copy gain (red) or loss (blue), the star indicates the significant gain or loss in LR lesions compare with primary lesions. (B) Distribution of PDCD1 gain in 13 patients, *P=0.039 indicates the PDCD1 gain significantly enriched in LR lesions. (C) Distribution of LMNA loss in 13 patients, *P=0.03 indicates the LMNA loss significantly enriched in LR lesions. LR = locoregional recurrence, P1–P13 = Patient 1–Patient 13. LR lesions (Fig. 4B and Additional Fig. 4B, http://links.lww. showed unambiguous truncal mutations and divergence com/JR9/A36). In addition, we inferred the divergence points points (Fig. 5B). Notably, the LR tumors had an average of between the relapsed seeding clone and primary tumor, and a 63% greater mutation burden than the primary tumor in showed a “molecular time,” representing the relapse divergence MSS patients (Additional Fig. 5A, http://links.lww.com/JR9/ from the primary tumor, of 33% to 89% (Fig. 4C). These results A36), indicating that the rate of mutation accumulation suggest that the matched samples in MSS patients were clonally increased during late LR evolution. The recurrence-specific related. increase in mutations was also loosely correlated with the time interval between first surgery and the diagnosis of LR (Pearson’s correlation r=0.313; Additional Fig. 5B, http:// Evolutionary pattern of primary CRC to LR links.lww.com/JR9/A36). In addition, mutations exclusive We created a phylogenetic tree for each patient based on the to LR lesions were significantly enriched in broad pathways, mutation sites. The three MSI-H patients with no truncal such as histone methylation, DNA replication, regulation mutations (Shared clonal mutations between the primary and of the MAPK pathway, and T cell activation (Additional LR lesion) were considered to follow independent evolution- Fig. 5C, http://links.lww.com/JR9/A36). The mutational ary trajectories (Fig. 5A). In contrast, the 10 MSS patients landscape of significantly enriched pathways is shown in 176 Journal of Bio-X Research RESEARCH ARTICLE Figure 4. Dynamics of mutation clonality between primary and LR lesions. The CCF dynamics in a representative patient. (A) MSI-H and (B) MSS patients, the X-axis indicates the CCF of primary lesion, Y-axis indicates the CCF of LR lesion, the red dots indicate the clonal mutations, the green dots indicate the subclonal mutation, and the purple dots indicate the mutations transition from subclonal to clonal. (C) Divergence time in each MSS patient, the different shape on the top of the column represents a different patient. CCF=Cancer cell fraction, LR = locoregional recurrence, MSI-H = microsatellite instability-high, MSS = microsatellite stable, P3–P13 = Patient 3–Patient 13. (Additional Figure 5D, http://links.lww.com/JR9/A36). These the 10 MSS patients consisted of more than one cluster and results suggest that relapsing clones continually acquired showed variant allele frequency (VAF) dynamics between the driver mutations with diverse functions after dissemination primary and LR lesions (Additional Fig. 6, http://links.lww. from the primary tumor. To understand the seeding pat- com/JR9/A36). We also visualized the evolutionary models tern between the primary tumor and LR lesion, we identi- for representative patients using ClonEvol (Additional Fig. 7, fied somatic mutations shared by matched samples to detect http://links.lww.com/JR9/A36) and showed that LR required the disseminating cell clones, using PyClone-VI, based on a interactions among multiple distinct clones (polyclonal seed- Bayesian clustering method. All the disseminating clones in ing) in MSS CRC patients. Figure 5. Phylogenetic trees of 13 patients with CRC and LR. Phylogenetic trees of (A) three MSI-H patients and (B) 10 MSS patients. The blue branch indicates exclusive mutations of primary lesions; The red branch indicates exclusive mutations of LR lesions; The black branch indicates the shared clonal mutations between primary and LR lesions; The purple branch indicates the subclonal mutations in primary lesion and present in LR lesion; Representative driver genes for LR lesions indicated with orange color. CRC = Colorectal cancer, LR = locoregional recurrence, MSI-H = microsatellite instability-high, MSS = microsatellite stable, P1–P13 = Patient 1–Patient 13. 177 RESEARCH ARTICLE Journal of Bio-X Research Figure 6. Biomarkers for early recurrence of CRC. (A) Mutation distribution of disseminating clone between patients with early (three patients) and late (seven patients) recurrence, the number indicates the mutation counts of each group (B) Comparison of SBS signatures between patients with early (three patients) and late recurrence (seven patients), the Y-axis indicates the relative SBS signature contribution. (C) Comparison of indel signatures between patients with early (three patients) and late (seven patients) recurrence, the Y-axis indicates the relative Indel signature contribution. (D) Comparison of DFS between ID4-positive (12 patients) and -negative (15 patients) patients from TCGA. P=0.04 (Wilcoxon test). CRC = Colorectal cancer, DFS = disease-free survival, SBS = single base substitution signature. Clinical identification of biomarkers for early recurrence patients. According to the COSMIC database, the etiology of of CRC ID4 is unknown and ID8 is involved in the repair of DNA doublestrand breaks by non-homologous DNA end-joining Analysis of the clinical information for the MSS patients mechanisms. revealed recurrence intervals of <12 months in three patients To confirm if CRC patients with a positive ID4 or ID8 sig- and 21 to 48 months in the other seven patients. We investi- nature relapsed earlier than those negative for ID4 and ID8, gated biomarkers of early recurrence in MSS patients using we downloaded the mutational and clinical data for 594 CRC shared mutations between the primary and LR lesions as a patients in TCGA pan-cancer cohort from cbioportal. This surrogate for the disseminating clone. We assumed that the cohort included 27 MSS patients who relapsed, for whom spe- specific genomic variations in the disseminating cells could cific indel signature information was available. Disease-free sur - be used to discriminate between early and late recurrences. vival (DFS) was significantly shorter in relapsed patients with an Comparison of the disseminating mutations in patients with ID4 signature compared with those without ID4 (Wilcoxon P = early and late relapses revealed only one shared mutation 0.04) (Fig. 6D). Moreover, patients with an ID8 signature also (KRAS c.38G> A), 151 mutations exclusive to early-relapsed had a shorter DFS, but the difference was not significant because patients, and 446 exclusive to late-relapsed patients (Fig. 6A). of the small number of positive patients (Additional Fig. 8C, However, the host genes at the mutation site showed high http://links.lww.com/JR9/A36). In addition, DFS was shorter overlap in these two groups. Enrichment analysis further in patients with either an ID4 or ID8 signature compared with revealed that the host genes were distributed in similar path- patients without either signature (Additional Fig. 8D, http:// ways, including pathways in cancer, predominantly including links.lww.com/JR9/A36). APC, KRAS, TP53, TCF7L2, and other cancer-related genes (Additional Fig. 8A and B, http://links.lww.com/JR9/A36). We also wondered if the mutation signature of the disseminat- Discussion ing cell could be used to differentiate between early and late recurrences. Although the SBS signatures were almost iden- To the best of our knowledge, this was the first study to sys- tical (Fig. 6B), there were distinct indel signatures between tematically elucidate the genomic differences between primary the early- and late-recurrence groups (Fig. 6C). Interestingly, and LR CRC tissues. Importantly, we revealed distinct patterns ID4 and ID8 were specifically detected in early-relapse of disease progression between MSS and MSI-H patients, with 178 Journal of Bio-X Research RESEARCH ARTICLE few shared SNVs or indels in MSI-H patients, compared with including histone methylation, DNA replication, regulation 41.61% of somatic SNVs and indels shared between the primary of the MAPK pathway, and T cell activation. These findings and matched LR lesions in MSS patients. An average of 23% expanded the range of potential therapeutic targets for relapsed of mutations was specific to the primary lesions and 35.4% to CRC, such as PRMT, KDM, ATR, and KRAS inhibitors and [37–39] the LR lesions, indicating that the mutation rate was higher in cytokines. Notably, CNV analysis identified copy number the LR compared with the primary clone. We also demonstrated alterations in two driver genes that were enriched in LR lesions. more mutations in MSI-H than in MSS patients, consistent with PDCD1 (PD-1) encodes a cell surface membrane protein of the  a previous report. immunoglobulin superfamily and associates with CD3-TCR The most frequently mutated genes in the current cohort were in the immunological synapse and directly inhibits T-cell acti- [30,31]  genes with well-established roles in CRC, such as KARS, va-tion. It may thus be possible to develop immunotherapy TP53,and APC. However, the distributions of mutated KRAS for relapsed CRC patients whose genome harbors increased and TP53 differed between MSS and MSI-H patients; mutated copies of PDCD1 . Moreover, loss of LMNA, which encodes KRAS and TP53 were significantly less common in MSI-H com- a two-dimensional matrix of proteins located next to the inner pared with MSS patients, as confirmed in TCGA dataset. These nuclear membrane and which contributes to nuclear stability, results suggested that KRAS and TP53 mutations might follow was also enriched in LR lesions. Loss of LMNA expression was a definite pattern in MSI-H patients. BRAF and KRAS mutation previously reported to be associated with disease recurrence in  has been reported to be mutually exclusive, due to negative selec- stage II and III colon cancer. These lines of evidence suggest   tion driven by oncogene-induced senescence. Kumar et al that loss of LMNA may be a risk biomarker for LR in CRC. demonstrated that BRAF mutations were mainly distributed in In line with oncological practice, we aimed to stratify patients MSI-H tumors, suggesting that KRAS mutations might also be with LR. The mutational signatures ID4 and ID8 were identified predominantly distributed in MSS tumors. In addition, Lin et as biomarkers for early recurrence. ID4-positivity was signifi- al. confirmed that the incidence of TP53 mutations was lower cantly associated with shorter DFS in patients who experienced  in MSI-H than in MSS tumors. However, the mechanism recurrence. However, the low incidence of ID8-positivity in remains unclear and requires further intensive investigations. relapsed patients meant that it only showed a loose correlation The relatively low incidence of LR means that the evolu- with shorter DFS. It is important for doctors to be able to dis- tionary pattern from primary to LR CRC remains unclear. We, tinguish patients susceptible to LR and to thus implement the therefore, explored the progression to LR. Analyses including appropriate clinical management. mutation distribution, clonal dynamics, and phylogenetic trees demonstrated that LR occurred as a result of germline varia- Limitations tions in mismatch repair genes in MSI-H patients, and evolved Several limitations of this study should be indicated. First, the independently; that is, matched primary and LR lesions in sample size of this cohort is small, and the relatively small sam- MSI-H patients could be regarded as two metachronous pri- ple size may reduce the persuasiveness of the study. The second mary tumors. This strongly supports the use of high-through- aspect to be emphasized is certainly the difficulty to implement put sequencing to clarify the nature of the “recurrence,” the biomarker detection in clinical practice due to the cost of especially in individuals with a genetic predisposition and in time and money, therefore not very feasible for prognostic pur- relation to decisions regarding systematic therapy in the adju- poses in clinical practice. Despite these limitations, we still elab- vant setting. orately described the dynamics and disseminating types of LR, In contrast, the matched primary and LR lesions were clon- which provided a new perspective for further research. ally related in MSS patients, as indicated by the persistence of the mutational signature in the primary lesion through to the LR lesion, indicating that the mutation signature could be extended Conclusion from the primary tumor to the disseminating cells. In addi- LR of CRC is a complex process that may require cooperation tion, clonal transition analysis showed the presence of shared among multiple subclones or may occur via multiple rounds of subclonal mutations between primary and LR lesions in each seeding involving distinct clones. These findings pose a challenge patient. Finally, the phylogenetic trees also showed shared trun- to the development of new therapeutic approaches targeting cal mutations with distinct numbers. Taken together, these dif- these interactions to inhibit LR. ferent lines of evidence indicate distinct origins of LRs between MSS and MSI-H patients. Acknowledgments This study also clarified the seeding pattern of LR. The pri- mary tumors in all 10 MSS demonstrated polyclonal seeding None. of the disseminating clones. This shows some conflict with the [35,36] reported dissemination patterns in metastatic CRC, with Author contributions a monoclonal seeding pattern between primary CRC and met- astatic lesions being relatively common. It is speculated that XL participated in data collection and manuscript drafting. metastasis of the disseminating clone to distant organs is asso- XxW participated in investigation and manuscript drafting. CZ ciated with greater selective pressure due to the longer trans- participated in data analysis and manuscript drafting. XhW was mission distance, meaning that only the best-adapted clones responsible for investigation. GW was responsible for data val- survive. In contrast, the relatively shorter transmission distance idation. BL, WQ, and DL were responsible for project admin- required for LR allowed multiple clones to evade selection pres- istration. HW implemented the study. YD was responsible for sure and establish new lesions near the primary site. funding acquisition. JY, ZT, and ZY participated in data analy- We further explored the progression of relapsed lesions after sis. ZL and DS were responsible for conceptualization. LL was divergence from the primary tumor. Late-acquired mutations responsible for supervision, manuscript review, and editing. All encompassed a wide range of driver genes enriched in functions authors approved the final version of the manuscript. 179 RESEARCH ARTICLE Journal of Bio-X Research  Erstad DJ, Tumusiime G, Cusack JC, Jr. Prognostic and predictive bio- Financial support markers in colorectal cancer: implications for the clinical surgeon. Ann This work was supported by the National Key R&D Program Surg Oncol 2015;22:3433–3450. of China (No. 2017YFC1309002), National Natural Science  Li H, Durbin R. Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 2009;25:1754–1760. 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Journal of Bio-X Research – Wolters Kluwer Health
Published: Dec 9, 2022
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