
==== Front
Ann Med
Ann Med
Annals of Medicine
0785-3890
1365-2060
Taylor & Francis

39247989
10.1080/07853890.2024.2396559
2396559
Version of Record
Research Article
Oncology
Chromosomal instability is associated with prognosis and efficacy of bevacizumab after resection of colorectal cancer liver metastasis
W. LI et al.
Li Weihao a*
Lan Jin a*
Zhou Chi a*
Yang Rong b
Wang Jiayu c
He Jiahua a
Xiao Binyi a
Ou Qingjian a
Fang Yujing a
Fan Wenhua a
Lin Junzhong a
Pan Zhizhong a
Peng Jianhong a
Wu Xiaojun a
a Department of Colorectal Surgery, Sun Yat-Sen University Cancer Center, Guangzhou, China
b Department of Intensive Care Unit, Sun Yat-Sen University Cancer Center, Guangzhou, China
c Department of Pathology, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou, Guangdong, P. R. China
* These authors contributed equally to this work.

Supplemental data for this article can be accessed online at https://doi.org/10.1080/07853890.2024.2396559.

CONTACT Zhizhong Pan panzhzh@sysucc.org.cn Department of Colorectal Surgery, Sun Yat-sen University Cancer Center, Guangzhou, 510060, China;
Xiaojun Wu wuxj@sysucc.org.cn Department of Colorectal Surgery, Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine Guangzhou, 510060, P. R. China
Jianhong Peng pengjh@sysucc.org.cn Department of Colorectal Surgery, Sun Yat-sen University Cancer Center; State Key, Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine Guangzhou, 510060, P. R. China
9 9 2024
2024
9 9 2024
56 1 239655923 8 2023
18 3 2024
24 4 2024
KnowledgeWorks Global Ltd.6 9 2024
published online in a building issue6 9 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

Abstract

Introduction

Individualized treatment of colorectal cancer liver metastases (CRLM) remains challenging due to differences in the severity of metastatic disease and tumour biology. Exploring specific prognostic risk subgroups is urgently needed. The current study aimed to investigate the prognostic value of chromosomal instability (CIN) in patients with initially resectable CRLM and the predictive value of CIN for the efficacy of bevacizumab.

Methods

Ninety-one consecutive patients with initially resectable CRLM who underwent curative liver resection from 2006 to 2018 at Sun Yat-sen University Cancer Center were selected for analysis. CIN was evaluated by automated digital imaging systems. Immunohistochemistry (IHC) was performed to detect interleukin-6 (IL-6), vascular endothelial growth factor A (VEGFA) and CD31 expression in paraffin-embedded specimens. Recurrence-free survival (RFS) and overall survival (OS) were analysed using the Kaplan–Meier method and Cox regression models.

Results

Patients with high chromosomal instability (CIN-H) had a worse 3-year RFS rate (HR, 1.953; 95% CI, 1.001–3.810; p = 0.049) and a worse 3-year OS rate (HR, 2.449; 95% CI, 1.150–5.213; p = 0.016) than those with low chromosomal instability (CIN-L). CIN-H was identified as an independent prognostic factor for RFS (HR, 2.569; 95% CI, 1.078–6.121; p = 0.033) and OS (HR, 3.852; 95% CI, 1.173–12.645; p = 0.026) in the multivariate analysis. The protein levels of IL-6, VEGFA and CD31 were upregulated in patients in the CIN-H group compared to those in the CIN-L group in both primary tumour and liver metastases tissues. Among them, 22 patients with recurrent tumours were treated with first-line bevacizumab treatment and based on the clinical response assessment, disease control rates were adversely associated with chromosomal instability (p = 0.043).

Conclusions

Our study showed that high chromosomal instability is a negative prognostic factor for patients with initially resectable CRLM after liver resection. CIN may have positive correlations with angiogenesis through expression of IL-6–VEGFA axis and be used as a potential predictor of efficacy of bevacizumab.

Keywords

Colorectal cancer
liver metastases
chromosomal instability
prognosis
antiangiogenesis
Natural Science Foundation of Guangdong Province 10.13039/501100003453 2023A1515010417 Medical Scientific Research Foundation of Guangdong Province A2021130 Shenzhen Science and Technology Plan Projects This study was funded by grants from the Natural Science Foundation of Guangdong Province, China (no. 2023A1515010417), Medical Scientific Research Foundation of Guangdong Province, China (no. A2021130), and Shenzhen Science and Technology Plan Projects (no. JCYJ20220531094015034).
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pmcIntroduction

Colorectal cancer (CRC) is the third most common malignancy worldwide and the second most common cause of cancer-related death [1,2]. Recurrence and distant metastasis are still the leading causes of treatment failure and death among CRC patients [3]. And at diagnosis, 20%–30% of cases suffer from synchronous liver metastases and 50%–75% of all patients with CRC develop liver metastases [4,5]. Although patients with initially resectable colorectal cancer liver metastases (CRLM) achieve a 47.3%–50.2% five-year overall survival (OS) rate after hepatectomy, more than 80% of them relapsed and 50% of cases occurred within the first 2 years [6,7]. To date, the application of doublet or triplet chemotherapy regimens with targeted therapies including antiangiogenic therapy has increased the survival of patients with metastatic CRC [8,9], while intrinsic or acquired resistance seems inevitable and the overall benefit is still limited [10–12]. Therefore, exploring novel clinicopathological characteristics to distinguish various prognostic subgroups and guide personalized therapy is urgently needed.

Several vital clinicopathological factors have been consolidated into prognostic scoring systems for patients with CRLM receiving curative hepatectomy [13–15]. However, those scoring systems were mainly based on the gross level of the tumour. The combination of the characteristics of tumour growth and cell structure is expected to evaluate tumour biology and prognosis more accurately. Chromosomal instability (CIN), one of the essential types of genomic instability and common characteristics of cancer genomes recognized as an alternative mechanism of oncogenesis and progression in CRC, is present in approximately 65-70% of patients and is often inferred from DNA ploidy, whole chromatin structural organization, etc. [16–18]. As reported, CIN was found to accelerate the development of anticancer drug resistance [19], often leading to treatment failure and disease recurrence and also associated with a poor prognosis of patients with stage I–III CRC [20–22]. However, the prognostic value of CIN in patients with initially resectable CRLM having received curative hepatectomy is still unclear.

In recent years, it has been reported that in chromosomal instable cancers, errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol and then leads to the activation of the cGAS-STING cytosolic DNA-sensing pathway [23]. Interleukin-6 (IL-6) – IL-6R – STAT3 signalling induced by cGAS and STING is required for survival of CIN cancer cells [24]. IL-6 was found to trigger the malignancy of hemangioma cells via activation of HIF-1α/vascular endothelial growth factor A (VEGFA) signals and targeted inhibition of IL-6 can significantly suppress the proliferation and migration of cells [25]. In patients with metastatic CRC or unresectable hepatocellular carcinoma, high pretreatment IL-6 levels were associated with a poor outcome and bevacizumab resistance [26,27]. Therefore, we hypothesized that CIN might be a predictor of the efficacy of bevacizumab.

The present study applied automated digital imaging systems to evaluate CIN in patients with initially resectable CRLM. Accordingly, we aimed to (1) describe the characteristics of CIN in patients with initially resectable CRLM, (2) identify the prognostic value of CIN in liver metastases from patients undergoing liver resection, and (3) investigate the predictive value of CIN of the efficacy of bevacizumab after resection of colorectal cancer liver metastasis.

Materials and methods

Patient population

Clinical data from 583 consecutive patients with initially resectable CRLM who underwent primary tumour and liver resection from April 2006 to October 2018 at Sun Yat-sen University Cancer Centre were reviewed. Patients included in the final analysis satisfied the following inclusion criteria: (1) histologically confirmed colorectal adenocarcinoma, (2) metastases limited to the liver, (3) no preoperative chemotherapy before liver resection, (4) radical resection of both the colorectal primary tumour and liver metastases, (5) at least a 3-month follow-up period after resection, (6) available formalin-fixed and paraffin-embedded (FFPE) samples of primary tumour and liver metastases. Informed consent for the use of the tissue samples was obtained from the patients before tumour resection. The study was approved by the Institutional Research Ethics Committee of Sun Yat-sen University Cancer Centre (Guangzhou, China, approval number: B2020-294-01).

Tumour sampling and chromosomal instability evaluation

Tumour chromosome instability was evaluated by DNA ploidy and nucleotyping analyses. For DNA ploidy and nucleotyping analyses, a pathologist selected a tumour block considered representative from each patient and delineated the entire epithelial tumour region. These two pathological parameters were detected at Ningbo Meishan FTZ MBM Clinical Lab Co., Ltd as previously reported [28]. Patients with both nondiploid DNA ploidy and chromatin heterogeneous were identified as high chromosomal instability (CIN-H), otherwise, were identified as low chromosomal instability (CIN-L).

Immunohistochemistry

Immunohistochemistry (IHC) analysis using paraffin-embedded specimens was conducted following standard manufacturer’s protocols. The paraffin-embedded samples were sectioned continuously into 4 μm thick sections, which were dewaxed in xylene, rehydrated and rinsed in graded ethanol solutions. The antigens were retrieved by heating the tissue sections at 100 °C for 5 min in an ethylenediaminetetraacetic acid (EDTA) solution (1 mmol/L, pH 8.0). The sections were then immersed in a 0.3% hydrogen peroxide solution for 10 min and rinsed with phosphate-buffered saline (PBS) for 5 min. Primary antibodies anti-IL-6 (Proteintech, 21865-1-AP), anti-VEGFA (Proteintech, 19003-1-AP) and anti-CD31 (Proteintech, 11265-1-AP) were used for IHC staining. After washing with 1 × PBS, the sections were treated with an anti-rabbit secondary antibody (Zhongshan Golden Bridge Biotechnology, Beijing, China) at 37.5 °C for 30 min. Finally, the staining was developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Dako, Glostrup, Denmark).

IHC score

IHC staining was evaluated by two independent gastrointestinal pathologists blinded to the patients’ characteristics and clinical outcomes. The final IHC score was calculated based on both the extent and the intensity of staining. The staining intensity was divided into the following four groups: grade 0 with no staining, grade 1 with weak staining, grade 2 with medium staining and grade 3 with strong staining. The proportion of positively stained cells was determined by the percentage of positive stained area using the following 4 groups: grade 0 = 0, grade 1 = 1–25%, grade 2 = 26–50%, grade 3 = 51–75% and grade 4 ≥ 75%. The staining scores were calculated using the following formula: staining scores = staining intensity × proportion of positively stained pixels.

Follow-up

Patients were monitored at 3-month interval for the first 2 years and then biannually for 5 years after liver resection. Clinical examinations and CEA and carbohydrate antigen 19-9 (CA19-9) detection were performed every 3 months. Chest/abdominal/pelvic computed tomography (CT) and colonoscopy were performed annually. Recurrence-free survival (RFS) was defined as the interval from the date of resection to the date of disease recurrence, death, or the last follow-up. Overall survival (OS) was defined as the interval from the date of resection to the date of death from any cause or to the last follow-up. Random censoring was applied to patients without recurrence or death at the last follow-up date.

Statistical analysis

Statistical analyses were performed using SPSS 24.0 software (IBM, Chicago, IL, USA). Categorical variables are presented as percentages and were compared using the chi-square test or Fisher’s exact test. Kaplan–Meier survival curves with log-rank estimates were used to depict time-to-event parameters. Multivariate Cox proportional hazards analysis was performed using variables whose P value was less than 0.05 in the univariate analysis. Hazard ratios (HRs) and 95% confidence intervals (CIs) were subsequently calculated. A two-sided p < 0.05 was considered statistically significant.

Results

Patient demographics

The flowchart of the selection process is shown in Figure 1. Finally, 91 patients were selected for this study. The clinical and pathological characteristics of the cohort are presented in Table 1. The median age of all patients was 59.9 years old (range, 25–84 years old), and 44.0% of the patients were male. Sixty-four (70.3%) patients had colon cancer while 27 (29.7%) patients had rectum cancer. There were 26 (15.8%) patients achieving a clinical risk score (CRS) higher than 2 scores. A total of 75 (82.4%) patients underwent synchronous resection for both the primary tumour and metastases.

Figure 1. Flow chart of the total patient selection process.

Table 1. Characteristics of included CRLM patients and analysis of liver metastasis tumors.

Variables	Total patients (n = 91, %)	
Median age (years)	59.9 (25–84)	
Age, years	 	
 ≤ 60	46 (50.5)	
 > 60	45 (49.5)	
Sex	 	
 Male	40 (44.0)	
 Female	51 (56.0)	
Primary tumour location	 	
 Colon	64 (70.3)	
 Rectum	27 (29.7)	
Primary tumour differentiation	 	
 Well to moderate	64 (70.3)	
 Poor	27 (29.7)	
T stage	 	
 T1–3	71 (78)	
 T4	20 (22)	
N stage	 	
 N0	34 (37.4)	
 N1–2	57 (62.6)	
Baseline serum CEA - ng/mL (%)	 	
 ≤ 5	40 (44.0)	
 > 5	51 (56.0)	
Baseline serum CA19-9 - U/mL (%)	 	
 ≤ 35	65 (71.4)	
 > 35	23 (25.3)	
Timing of liver metastases	 	
 Synchronous	75 (82.4)	
 Metachronous	16 (17.6)	
Number of liver metastases	 	
 1	59 (64.8)	
 2	19 (20.9)	
 3	9 (9.9)	
 4	2 (2.2)	
 5	2 (2.2)	
Liver metastases diameter (cm)	 	
Median (range)	2 (0.5-7.8)	
 ≤ 3	69 (75.8)	
 > 3	22 (24.2)	
Distribution of liver metastases	 	
 Unilobar	77 (84.6)	
 Bilobar	12 (15.4)	
CRS score	 	
 ≤ 2	65 (84.2)	
 > 2	26 (15.8)	
Chromosomal instability	 	
 High	71 (78.0)	
 Low	20 (22.0)	
CRLM: colorectal cancer liver metastasis; CEA: carcinoembryonic antigen; CA19-9: carbohydrate antigen 19-9; CRS: clinical risk score.

Association of chromosomal instability and clinicopathologic characteristics

High chromosomal instability (CIN-H) was identified in 71 patients while 20 patients were identified as low chromosomal instability (CIN-L). The association between chromosomal instability and clinicopathological characteristics is listed in Table 2. There were no significant differences between the two groups in terms of age, sex, primary tumour location, primary tumour differentiation, T stage, N stage, baseline serum CEA level, baseline serum CA19-9 level, timing, number, diameter and distribution of liver metastases and CRS score.

Table 2. Characteristics of the 91 study patients with CRLM grouped by chromosomal instability.

Variables	CIN-L
(n = 20, %)	CIN-H
(n = 71, %)	P value	
Age (years)	 	 	0.115	
 ≤ 60	7 (35.0)	39 (54.9)	 	
 > 60	13 (65.0)	32 (45.1)	 	
Sex	 	 	0.687	
 Male	8 (40.0)	32 (45.1)	 	
 Female	12 (60.0)	39 (54.9)	 	
Primary tumour location	 	 	0.605	
 Colon	15 (75.0)	49 (69.0)	 	
 Rectum	5 (25.0)	22 (31.0)	 	
Primary tumour differentiation	 	 	0.971	
 Well to moderate	14 (70.0)	50 (70.4)	 	
 Poor	6 (30.0)	21 (29.6)	 	
T stage	 	 	0.809	
 T1–3	16 (80.0)	55 (77.5)	 	
 T4	4 (20.0)	16 (22.5)	 	
N stage	 	 	0.441	
 N0	6 (30.0)	28 (39.4)	 	
 N1–2	14 (70.0)	43 (60.6)	 	
Baseline serum CEA (ng/mL)	 	 	0.741	
 ≤ 5	9 (45.0)	34 (47.9)	 	
 > 5	11 (55.0)	37 (52.1)	 	
Baseline serum CA19-9 (U/mL)	 	 	0.569	
 ≤ 35	15 (75.0)	52 (73.2)	 	
 > 35	5 (25.0)	19 (26.8)	 	
Timing of liver metastases	 	 	0.313	
 Synchronous	18 (90.0)	57 (80.3)	 	
 Metachronous	2 (10.0)	14 (19.7)	 	
Number of liver metastases	 	 	0.584	
 1	14 (70.0)	45 (63.4)	 	
 2–5	6 (30.0)	26 (36.6)	 	
Liver metastases diameter (cm)	 	 	0.621	
 ≤ 3	16 (80.0)	53 (74.6)	 	
 > 3	4 (20.0)	18 (25.4)	 	
Distribution of liver metastases	 	 	0.145	
 Unilobar	19 (95.0)	58 (81.7)	 	
 Bilobar	1 (5.0)	13 (18.3)	 	
CRS score	 	 	0.337	
 ≤ 2	16 (80.0)	49 (69.0)	 	
 > 2	4 (20.0)	22 (31.0)	 	
CRLM: colorectal cancer liver metastasis; CIN-H: high chromosomal instability; CIN-L: low chromosomal instability; CEA: carcinoembryonic antigen; CA19-9: carbohydrate antigen 19-9; CRS: clinical risk score.

Survival analyses

After a median follow-up time of 44 months (25–75% quartiles: 28–57 months), 46 (50.5%) patients were alive with a tumour-free status, 45 (49.5%) patients experienced tumour recurrence. Among them, 53.3% (24/45) had intrahepatic recurrence, 13.3% (6/45) had lung metastasis, and 8.9% (4/45) had abdominal/pelvic metastasis. And 37 (40.7%) patients experienced cancer-related mortality. The 3-year RFS rate and OS rate were 55.0% and 69.1%, respectively. Patients with CIN-H had a worse 3-year RFS rate (HR, 1.953; 95% CI, 1.001–3.810; p = 0.049, Figure 2(A)) and a worse 3-year OS rate (HR, 2.449; 95% CI, 1.150–5.213; p = 0.016, Figure 2(B)) than those with CIN-L.

Figure 2. Kaplan–Meier Curves of patients with initially resectable colorectal cancer liver metastases (CRLM) grouped by chromosomal instability. (A) Comparison of recurrence-free survival (RFS) between the high chromosomal instability (CIN-H) group and the low chromosomal instability (CIN-L) group. (B) Comparison of overall survival (OS) between the CIN-H group and the CIN-L group.

The results of univariate and multivariate analyses of RFS are summarized in Table 3. The univariate analysis revealed that the primary rectal cancer, N1-2 stage, liver metastases diameter > 3 cm and CIN-H were associated with unfavourable RFS. The multivariate analysis showed that primary rectal cancer (HR, 2.891; 95% CI, 1.505–5.555; p = 0.001), N1-2 stage (HR, 3.687; 95% CI, 1.792–7.585; p < 0.001), CIN-H (HR, 2.569; 95% CI, 1.078–6.121; p = 0.033) were also independent predictive factors for an unfavourable RFS. The results of univariate and multivariate analyses of OS are summarized in Table 4. The univariate analysis revealed that primary rectal cancer, N1-2 stage, liver metastases diameter > 3 cm, and CIN-H were associated with unfavourable OS. The multivariate analysis showed that primary rectal cancer (HR, 2.067; 95% CI, 1.032–4.138; p = 0.040), N1-2 stage (HR, 3.020; 95% CI, 1.387–6.575; p = 0.005), CIN-H (HR, 3.852; 95% CI, 1.173–12.645; p = 0.026) were independent predictive factors for unfavourable OS.

Table 3. Univariate and multivariate analyses of risk factors influencing RFS after liver resection.

Variables	Univariate	Multivariate	
HR (95% CI)	P value	HR (95% CI)	P value	
Age (> 60 years vs ≤ 60 years)	0.683 (0.376–1.243)	0.212	 	 	
Sex (Male vs. Female)	0.808 (0.450–1.452)	0.476	 	 	
Primary tumour location (Rectum vs. Colon)	2.028 (1.121–3.670)	0.019	2.891 (1.505–5.555)	0.001	
Primary tumour differentiation (Poor vs. Well to moderate)	1.086 (0.569–2.073)	0.802	 	 	
T stage (T4 vs. T1–3)	1.408 (0.725–2.733)	0.312	 	 	
N stage (N1–2 vs. N0)	2.484 (1.276–4.838)	0.007	3.687 (1.792–7.585)	<0.001	
Baseline serum CEA (> 5 ng/mL vs. ≤ 5 ng/mL)	1.304 (0.710–2.396)	0.392	 	 	
Baseline serum CA19-9 (> 35 U/mL vs. ≤ 35 U/mL)	0.803 (0.395–1.630)	0.803	 	 	
Timing of liver metastasis (Synchronous vs. Metachronous)	0.592 (0.305–1.150)	0.112	 	 	
Number of liver metastases (2–5 vs. 1)	1.728(0.955–3.129)	0.071	 	 	
Liver metastases diameter (> 3 cm vs. ≤ 3 cm)	2.017 (1.078–3.774)	0.028	1.556 (0.814–2.975)	0.181	
Distribution of liver metastases (Bilobar vs. Unilobar)	2.312 (0.976–5.473)	0.057	 	 	
Chromosomal instability (High vs. Low)	2.200 (1.084–4.464)	0.029	2.569 (1.078–6.121)	0.033	
RFS: recurrence-free survival; HR: hazard ratio; CI: confidence interval; CEA: carcinoembryonic antigen; CA19-9: carbohydrate antigen 19-9.Bold values represents when the p-value result is less than 0.05.

Table 4. Univariate and multivariate analyses of risk factors influencing OS after liver resection.

Variables	Univariate	Multivariate	
HR (95% CI)	P value	HR (95% CI)	P value	
Age (> 60 years vs ≤ 60 years)	0.730 (0.377–1.411)	0.349	 	 	
Sex (Male vs. Female)	1.028 (0.534–1.978)	0.935	 	 	
Primary tumour location (Rectum vs. Colon)	1.970 (1.026–3.784)	0.042	2.067 (1.032–4.138)	0.040	
Primary tumour differentiation (Poor vs. Well to moderate)	1.196 (0.590–2.423)	0.619	 	 	
T stage (T4 vs. T1–3)	1.820 (0.897–3.690)	0.097	 	 	
N stage (N1–2 vs. N0)	2.593 (1.220–5.509)	0.013	3.020 (1.387–6.575)	0.005	
Baseline serum CEA (> 5 ng/mL vs. ≤ 5 ng/mL)	1.115 (0.577–2.158)	0.746	 	 	
Baseline serum CA19-9 (> 35 U/mL vs. ≤ 35 U/mL)	0.644 (0.282–1.471)	0.296	 	 	
Timing of liver metastasis (Synchronous vs. Metachronous)	0.614 (0.296–1.274)	0.190	 	 	
Number of liver metastases (2-5 vs. 1)	1.734(0.907–3.315)	0.096	 	 	
Liver metastases diameter (> 3 cm vs. ≤ 3 cm)	2.265 (1.162–4.414)	0.016	1.692 (0.830–3.450)	0.148	
Distribution of liver metastases (Bilobar vs. Unilobar)	1.833 (0.835–4.022)	0.131	 	 	
Chromosomal instability (High vs. Low)	3.701 (1.133–12.086)	0.030	3.852 (1.173–12.645)	0.026	
OS: overall survival; HR: hazard ratio; CI: confidence interval; CEA: carcinoembryonic antigen; CA19-9: carbohydrate antigen 19-9.Bold values represents when the p-value result is less than 0.05.

Association of chromosomal instability and IL-6 mediated angiogenesis

Using IHC staining, we detected the protein levels of IL-6, VEGFA, and CD31 in 91 paired CRLM primary tumour (Figure 3(A)) and liver metastases tissues (Figure 3(D)). Expression levels of all these proteins were upregulated in patients in the CIN-H group (n = 71) compared to those in the CIN-L group (n = 20) in both primary tumour (Figure 3(B)) and liver metastases tissues (Figure 3(E)). Expression levels of both VEGFA and CD31 showed significant positive correlation with expression level of IL-6 in primary tumour (VEGFA, r = 0.472, p < 0.001; CD31, r = 0.389, p < 0.001; Figure 3(C)) or in liver metastases (VEGFA, r = 0.461, p < 0.001; CD31, r = 0.518, p < 0.001; Figure 3(F)). The same results were identified based on the RNA-seq results in CRC patients from TCGA database (Figure S1).

Figure 3. Chromosomal instability showed positive correlations with angiogenesis through expression of Interleukin-6 (IL-6)–vascular endothelial growth factor A (VEGFA) axis in primary tumour and liver metastasis. (A) Two representative cases show expression of IL-6, VEGFA and CD31 in high compared with low levels of chromosomal instability human CRC tumour tissues analysed by immunohistochemistry (IHC) staining (scale bar: black, 200 μm; red, 100 μm;). (B) Comparison of IL-6, VEGFA and CD31 expression in primary tumour between the high chromosomal instability (CIN-H) group and the low chromosomal instability (CIN-L) group. (C) Correlation between IL-6 and VEGFA, CD31 expression levels in primary tumour. (D) Two representative cases show expression of IL-6, VEGFA and CD31 in high compared with low levels of chromosomal instability human CRC tumour liver metastasis tissues analysed by immunohistochemistry (IHC) staining (scale bar: black, 200 μm; red, 100 μm;). (E) Comparison of IL-6, VEGFA and CD31 expression in liver metastasis between the CIN-H group and the CIN-L group. (F) Correlation between IL-6 and VEGFA, CD31 expression levels in liver metastasis. *p ≤ 0.05, **p ≤ 0.01.

Among them, 22 patients with recurrent tumours were treated with first-line bevacizumab treatment. Disease control [partial response (PR) or stable disease (SD)] was achieved in nine patients (40.9%) and progressive disease (PD) was observed in 13 (59.1%) patients. Among patients with bevacizumab-treated PD, 12 of them were identified as CIN-H, while only one of them was CIN-L (Figure 4(A)). There were no significant differences between the two groups in terms of primary tumour location or the mutation status of KRAS/BRAF/NRAS (Figure S2). Based on the clinical response assessment, disease control rates were adversely associated with chromosomal instability (p = 0.043, Figure 4(B)).

Figure 4. Evaluation of bevacizumab treatment efficacy in CRLM grouped by chromosomal instability. (A) Best percentage change from baseline in the tumour burden (defined as the sum of the longest diameters of all target lesions) in all 22 patients with measurements of target lesions after baseline. (B) Number of CRLM patients with bevacizumab treated partial response (PR)/stable disease (SD) or bevacizumab-treated progressive disease (PD) in the high chromosomal instability (CIN-H) group or the low chromosomal instability (CIN-L) group.

Discussion

Due to the high morbidity and mortality, a precise prognostic prediction and personalized therapy guideline for patients with initially resectable CRLM having received curative hepatectomy is needed. In the current study, we first investigated the prognostic value of CIN in patients with initially resectable CRLM having received curative hepatectomy. Our data analyses showed that patients with CIN-H had a worse 3-year RFS and OS rate than those with CIN-L. The multivariate analysis showed that primary rectal cancer, N1-2 stage, CIN-H were also independent predictive factors for an unfavourable RFS and OS. Subsequently, we detected the protein levels of IL-6, VEGFA and CD31 in 91 paired CRLM primary tumour and liver metastases tissues through IHC staining. We found that expression levels of all these proteins were upregulated in the CIN-H group, which indicated that chromosomal instability may have positive correlations with angiogenesis through expression of IL-6–VEGFA axis. And based on the clinical response assessment in 22 patients with recurrent tumours treated with first-line bevacizumab treatment, disease control rates were adversely associated with chromosomal instability.

Chromosomal instability, a hallmark of cancer, has been reported to regulate multitudinous biological processes such as cancer cell evolution, metastasis and therapy resistance, and is a poor prognostic factor [19,29,30]. CIN drives intratumoural heterogeneity by potentially causing the simultaneous acquisition of whole-chromosome or segmental aneuploidy and structural chromosomal aberrations, which can be evaluated through the measurement of DNA content and nuclear texture analysis [20,31]. Although CIN has been reported to correlate with tumor metastasis [32,33], the role of it in patients with initially resectable CRLM having received curative hepatectomy is still unclear. Bakhoum and colleagues [23] identified that CIN promotes metastasis by sustaining a tumour-cell autonomous response to cytosolic DNA, leading to the activation of the cGAS-STING pathway and downstream noncanonical NF-κB signalling. Suppression of CIN significantly reduced spontaneous metastasis and prolonged survival in mice models. Several molecular mechanisms [34–37] of CRC cells have been reported to induce CIN, causing tumour progression and worse prognosis in CRC patients. A meta-analysis reported by Walther and colleagues [21] concluded that CIN is associated with a worse prognosis in CRC and should be evaluated as a prognostic marker. In the current study, we preliminarily identified the prognostic value of CIN in patients with initially resectable CRLM having received curative hepatectomy. Patients with CIN-H had a worse 3-year RFS and OS.

In decades, incorporating anti-angiogenic agents like bevacizumab into the therapeutic management of metastatic CRC has made a significant impact on survival. IL-6 exerts proangiogenic effects in the tumour microenvironment of several solid malignancies and there is emerging evidence that reveals significant relationships between IL-6 signalling and treatment failure with antibodies directed against VEGF [38]. Guo and colleagues [39] found that IL-6 is omnipresent in the inflammatory microenvironment of most solid tumours as a potent pro-angiogenic mediator. Treatment of various epithelial cell lines with IL-6 can significantly induce VEGF mRNA to a level comparable to the effects of hypoxia, an activator of hypoxia-induced genes [40]. Eldesoky and colleagues [41] measured preoperative serum VEGF and IL-6 in 35 colorectal cancer patients and 30 controls and found that CRC patients had significantly higher IL-6 and VEGF levels and were elevated further in those with advanced pathological tumour stage and metastatic disease. A phase-II study with bevacizumab combined with chemoradiation noted that elevated IL-6 was associated with worse prognosis [42]. We first detected the protein levels of IL-6, VEGFA and CD31 in 91 paired CRLM primary tumour and liver metastases tissues through IHC staining and found that expression levels of all these proteins were upregulated in patients with CIN-H, which indicated that CIN may have positive correlations with angiogenesis through expression of IL-6 – VEGFA axis. Our research confirmed the close relationship between CIN and angiogenesis and its use as a potential predictor of the efficacy of bevacizumab.

Several limitations to the current study should be acknowledged. First, this retrospective study included an uncontrolled methodology and a limited number of patients recruited from a single cohort. Selective bias exists, and the findings must be validated in external cohorts. Second, the 5-year survival data were unavailable for some patients due to insufficient follow-up duration. This issue may have led to the underestimation or overestimation of the prognostic effect of CIN on liver metastases. Third, the molecular mechanisms of CIN that promote tumour progression were not validated in the current study. Additionally, several tumour molecular markers were not included in the current study. RAS, BRAF, TP53, and SMAD4 mutations are significantly associated with the long-term survival of patients with CRLM after liver resection [43,44]. A confirmation of the association of CIN with potential driver gene mutations would help us further understand the effect of CIN on the postoperative recurrence of CRLM.

Conclusions

As shown in the present study, high chromosomal instability is a negative prognostic factor for patients with initially resectable CRLM after liver resection. CIN may have positive correlations with angiogenesis through expression of IL-6 – VEGFA axis and be used as a potential predictor of efficacy of bevacizumab.

Supplementary Material

Supplemental Material.zip

Acknowledgments

We greatly appreciate the help from all of our colleagues in the Department of Colorectal Surgery at Sun Yat-Sen University Cancer Center who were involved in administering the treatments in the current study.

Authors’ contributions

Conception and design: X.-J.W., J.-H.P., Z.-Z.P., W.-H.L., J.L., C.Z. Financial support: X.-J.W., W.-H.F., J.-Z.L. Provision of study materials or patients: X.-J.W., J.-Z.L., Z.-Z.P., J.-H.P. Collection and assembly of data: W.-H.L., J.L., C.Z., R.Y., J.-Y.W., J.-H.H., B.-Y.X, Q.-J.O., Y.-J.F., W.-H.F. Data analysis and interpretation: W.-H.L., J.-H.P., J.L., C.Z. Manuscript writing: All authors. Final approval of manuscript: All authors.

Ethics approval and consent to participate

The present study was performed according to the ethical standards of the World Medical Association Declaration of Helsinki and was approved by the Institutional Review Board and Independent Ethics Committees of Sun Yat-sen University Cancer Center. The informed consent requirement was waived by the ethics committees based on the nature of this retrospective study, in which patient data were kept confidential.

Consent for publication

Not applicable.

Disclosure statement

No potential conflict of interest was reported by the authors.

Availability of data and material

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. The authenticity of this article has been validated by uploading the key raw data onto the Research Data Deposit public platform (www. researchdata.org.cn), with the approval RDD number as RDDA2024606244.
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References

1 Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71 (3 ):209–249. doi: 10.3322/caac.21660.33538338
2 Siegel RL, Wagle NS, Cercek A, et al. Colorectal cancer statistics, 2023. CA Cancer J Clin. 2023;73 (3 ):233–254. doi: 10.3322/caac.21772.36856579
3 Van Cutsem E, Cervantes A, Adam R, et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann Oncol. 2016;27 (8 ):1386–1422. doi: 10.1093/annonc/mdw235.27380959
4 Jönsson K, Gröndahl G, Salö M, et al. Repeated liver ­resection for colorectal liver metastases: a comparison with primary liver resections concerning perioperative and long-term outcome. Gastroenterol Res Pract. 2012;2012 :568214–568214. doi: 10.1155/2012/568214.22973305
5 Rahbari NN, Reissfelder C, Schulze-Bergkamen H, et al. Adjuvant therapy after resection of colorectal liver ­metastases: the predictive value of the MSKCC clinical risk score in the era of modern chemotherapy. BMC Cancer. 2014;14 (1 ):174. doi: 10.1186/1471-2407-14-174.24612620
6 de Jong MC, Pulitano C, Ribero D, et al. Rates and patterns of recurrence following curative intent surgery for colorectal liver metastasis: an international multi-institutional analysis of 1669 patients. Ann Surg. 2009;250 (3 ):440–448. doi: 10.1097/SLA.0b013e3181b4539b.19730175
7 Imai K, Allard MA, Benitez CC, et al. Early recurrence after hepatectomy for colorectal liver metastases: what optimal definition and what predictive factors? Oncologist. 2016;21 (7 ):887–894. doi: 10.1634/theoncologist.2015-0468.27125753
8 Van Cutsem E, Köhne C-H, Láng I, et al. Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. J Clin Oncol. 2011;29 (15 ):2011–2019. doi: 10.1200/JCO.2010.33.5091.21502544
9 Venook AP, Niedzwiecki D, Lenz H-J, et al. Effect of first-line chemotherapy combined with cetuximab or bevacizumab on overall survival in patients with KRAS wild-type advanced or metastatic colorectal cancer: a randomized clinical trial. JAMA. 2017;317 (23 ):2392–2401. doi: 10.1001/jama.2017.7105.28632865
10 Tomasello G, Petrelli F, Ghidini M, et al. FOLFOXIRI plus bevacizumab as conversion therapy for patients with initially unresectable metastatic colorectal cancer: a systematic review and pooled analysis. JAMA Oncol. 2017;3 (7 ):e170278. doi: 10.1001/jamaoncol.2017.0278.28542671
11 Garcia J, Hurwitz HI, Sandler AB, et al. Bevacizumab (Avastin®) in cancer treatment: a review of 15 years of clinical experience and future outlook. Cancer Treat Rev. 2020;86 :102017. doi: 10.1016/j.ctrv.2020.102017.32335505
12 Vasudev NS, Reynolds AR. Anti-angiogenic therapy for cancer: current progress, unresolved questions and future directions. Angiogenesis. 2014;17 (3 ):471–494. Erratum in: Angiogenesis. 2014;17 (3 ):495–7. doi: 10.1007/s10456-014-9420-y.24482243
13 Nordlinger, Bernard, Guiguet, Marguerite, Vaillant, Jean-Christophe, et al. Surgical resection of colorectal carcinoma metastases to the liver. A prognostic scoring system to improve case selection, based on 1568 patients. Association Française de Chirurgie. Cancer. 1996;77 (7 ):1254–1262. doi: 10.1002/(SICI)1097-0142(19960401)77:7<1254::AID-CNCR5>3.0.CO;2-I.8608500
14 Fong Y, Fortner J, Sun RL, et al. Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer: analysis of 1001 consecutive cases. Ann Surg. 1999;230 (3 ):309–318. discussion 31821. doi: 10.1097/00000658-199909000-00004.10493478
15 Oshi M, Margonis GA, Sawada Y, et al. Higher tumor burden neutralizes negative margin status in hepatectomy for colorectal cancer liver metastasis. Ann Surg Oncol. 2019;26 (2 ):593–603. doi: 10.1245/s10434-018-6830-x.30483976
16 Ben-David U, Amon A. Context is everything: aneuploidy in cancer. Nat Rev Genet. 2020;21 (1 ):44–62. doi: 10.1038/s41576-019-0171-x.31548659
17 Bielski CM, Zehir A, Penson AV, et al. Genome doubling shapes the evolution and prognosis of advanced cancers. Nat Genet. 2018;50 (8 ):1189–1195. doi: 10.1038/s41588-018-0165-1.30013179
18 Schuster-Böckler B, Lehner B. Chromatin organization is a major influence on regional mutation rates in human cancer cells. Nature. 2012;488 (7412 ):504–507. doi: 10.1038/nature11273.22820252
19 Sansregret L, Vanhaesebroeck B, Swanton C. Determinants and clinical implications of chromosomal instability in cancer. Nat Rev Clin Oncol. 2018;15 (3 ):139–150. doi: 10.1038/nrclinonc.2017.198.29297505
20 Kleppe A, Albregtsen F, Vlatkovic L, et al. Chromatin ­organisation and cancer prognosis: a pan-cancer study. Lancet Oncol. 2018;19 (3 ):356–369. doi: 10.1016/S1470-2045(17)30899-9.29402700
21 Walther A, Houlston R, Tomlinson I. Association ­between chromosomal instability and prognosis in colorectal cancer: a meta-analysis. Gut. 2008;57 (7 ):941–950. doi: 10.1136/gut.2007.135004.18364437
22 Mouradov D, Domingo E, Gibbs P, et al. Survival in stage II/III colorectal cancer is independently predicted by chromosomal and microsatellite instability, but not by specific driver mutations. Am J Gastroenterol. 2013;108 (11 ):1785–1793. doi: 10.1038/ajg.2013.292.24042191
23 Bakhoum SF, Ngo B, Laughney AM, et al. Chromosomal instability drives metastasis through a cytosolic DNA response. Nature. 2018;553 (7689 ):467–472. doi: 10.1038/nature25432.29342134
24 Hong C, Schubert M, Tijhuis AE, et al. cGAS-STING drives the IL-6-dependent survival of chromosomally instable cancers. Nature. 2022;607 (7918 ):366–373. doi: 10.1038/s41586-022-04847-2.35705809
25 Fu X, Zhai S, Yuan J. Interleukin-6 (IL-6) triggers the ­malignancy of hemangioma cells via activation of HIF-1α/VEGFA signals. Eur J Pharmacol. 2018;841 :82–89. doi: 10.1016/j.ejphar.2018.10.022.30342949
26 Hara M, Nagasaki T, Shiga K, et al. High serum levels of interleukin-6 in patients with advanced or metastatic colorectal cancer: the effect on the outcome and the response to chemotherapy plus bevacizumab. Surg Today. 2017;47 (4 ):483–489. doi: 10.1007/s00595-016-1404-7.27549777
27 Yang H, Kang B, Ha Y, et al. High serum IL-6 correlates with reduced clinical benefit of atezolizumab and bevacizumab in unresectable hepatocellular carcinoma. JHEP Rep. 2023;5 (4 ):100672. doi: 10.1016/j.jhepr.2023.100672.36866388
28 Peng J, Li W, Fan W, et al. Prognostic value of a novel biomarker combining DNA ploidy and tumor burden score for initially resectable liver metastases from patients with colorectal cancer. Cancer Cell Int. 2021;21 (1 ):554. doi: 10.1186/s12935-021-02250-x.34688293
29 Kuznetsova AY, Seget K, Moeller GK, et al. Chromosomal instability, tolerance of mitotic errors and multidrug resistance are promoted by tetraploidization in human cells. Cell Cycle. 2015;14 (17 ):2810–2820. doi: 10.1080/15384101.2015.1068482.26151317
30 Pino MS, Chung DC. The chromosomal instability pathway in colon cancer. Gastroenterology. 2010;138 (6 ):2059–2072. doi: 10.1053/j.gastro.2009.12.065.20420946
31 Pradhan M, Abeler VM, Danielsen HE, et al. Prognostic importance of DNA ploidy and DNA index in stage I and II endometrioid adenocarcinoma of the endometrium. Ann Oncol. 2012;23 (5 ):1178–1184. doi: 10.1093/annonc/mdr368.21965471
32 Jamal-Hanjani M, Wilson GA, McGranahan N, et al. Tracking the evolution of non-small-cell lung cancer. N Engl J Med. 2017;376 (22 ):2109–2121. doi: 10.1056/NEJMoa1616288.28445112
33 Turajlic S, Swanton C. Metastasis as an evolutionary process. Science. 2016;352 (6282 ):169–175. doi: 10.1126/science.aaf2784.27124450
34 Chen B, Dragomir MP, Fabris L, et al. The long noncoding RNA CCAT2 induces chromosomal instability through BOP1-AURKB signaling. Gastroenterology. 2020;159 (6 ):2146–2162.e33. doi: 10.1053/j.gastro.2020.08.018.32805281
35 Khot M, Sreekumar D, Jahagirdar S, et al. Twist1 induces chromosomal instability (CIN) in colorectal cancer cells. Hum Mol Genet. 2020;29 (10 ):1673–1688. doi: 10.1093/hmg/ddaa076.32337580
36 Chou J, Kaller M, Jaeckel S, et al. AP4 suppresses DNA damage, chromosomal instability and senescence via inducing MDC1/mediator of DNA damage checkpoint 1 and repressing MIR22HG/miR-22-3p. Mol Cancer. 2022;21 (1 ):120. doi: 10.1186/s12943-022-01581-1.35624466
37 Vuaroqueaux V, Musch A, Kobelt D, et al. Elevated MACC1 expression in colorectal cancer is driven by chromosomal instability and is associated with molecular subtype and worse patient survival. Cancers (Basel). 2022;14 (7 ):1749. doi: 10.3390/cancers14071749.35406521
38 Middleton K, Jones J, Lwin Z, et al. Interleukin-6: an ­angiogenic target in solid tumours. Crit Rev Oncol Hematol. 2014;89 (1 ):129–139. doi: 10.1016/j.critrevonc.2013.08.004.24029605
39 Guo Y, Xu F, Lu T, et al. Interleukin-6 signaling pathway in targeted therapy for cancer. Cancer Treat Rev. 2012;38 (7 ):904–910. doi: 10.1016/j.ctrv.2012.04.007.22651903
40 Cohen T, Nahari D, Cerem LW, et al. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem. 1996;271 (2 ):736–741. doi: 10.1074/jbc.271.2.736.8557680
41 Eldesoky A, Shouma A, Mosaad Y, et al. Clinical relevance of serum vascular endothelial growth factor and interleukin-6 in patients with colorectal cancer. Saudi J Gastroenterol. 2011;17 (3 ):170–173. doi: 10.4103/1319-3767.80378.21546718
42 Willett CG, Duda DG, di Tomaso E, et al. Efficacy, safety, and biomarkers of neoadjuvant bevacizumab, radiation therapy, and fluorouracil in rectal cancer: a multidisciplinary phase II study. J Clin Oncol. 2009;27 (18 ):3020–3026. doi: 10.1200/JCO.2008.21.1771.19470921
43 Passiglia F, Bronte G, Bazan V, et al. Can KRAS and BRAF mutations limit the benefit of liver resection in metastatic colorectal cancer patients? A systematic review and meta-analysis. Crit Rev Oncol Hematol. 2016;99 :150–157. doi: 10.1016/j.critrevonc.2015.12.015.26775732
44 Kawaguchi Y, Kopetz S, Newhook TE, et al. Mutation Status of RAS, TP53, and SMAD4 is Superior to Mutation Status of RAS Alone for Predicting Prognosis after Resection of Colorectal Liver Metastases. Clin Cancer Res. 2019;25 (19 ):5843–5851. doi: 10.1158/1078-0432.CCR-19-0863.31221662
