Article https://doi.org/10.1038/s41467-024-51536-x

Tislelizumab plus cetuximab and irinotecan

in refractory microsatellite stable and RAS

wild-type metastatic colorectal cancer: a

single-arm phase 2 study

Xiaojing Xu1,2,5, Luoyan Ai1,2,5, Keshu Hu1,2,5, Li Liang 1,2,5, Minzhi Lv2,5,

Yan Wang1,2, Yuehong Cui1,2, Wei Li1,2, Qian Li1,2, Shan Yu1,2, Yi Feng1,2, Qing Liu1,2,

Ying Yang3

, Jiao Zhang3

, Fei Xu3

, Yiyi Yu1,2 & Tianshu Liu 1,2,4

Immunotherapy confers little to no benefit in the treatment of microsatellite

stable (MSS) metastatic colorectal cancer (mCRC). Mechanistic insights suggested that epidermal growth factor receptor (EGFR) antibody plus irinotecan

might augment the tumor immune response in mCRC. Therefore, we

conducted a proof-of-concept, single-arm, phase 2 study (ChiCTR identifier:

ChiCTR2000035642) of a combination treatment regimen including tislelizumab

(anti-PD-1), cetuximab (anti-EGFR) and irinotecan in 33 patients with MSS and RAS

wild-type (WT) mCRC who were previously treated with ≥2 lines of therapy. The

primary endpoint was met, with a confirmed objective response rate of 33%. As

secondary endpoints, the disease control rate was 79%, and the median

progression-free survival and overall survival were 7.3 and 17.4 months respectively. Among the 33 patients, 32 (97.0%) had treatment-related adverse events

(AEs). Three (9.1%) reported grade ≥ 3 AEs, including rash (n = 1), neutropenia

(n = 2). The post-hoc evaluation of dynamic circulating tumor DNA using next

generation sequencing and the analysis of peripheral immune proteomics landscape using Olink revealed that lower variant allele frequency (VAF) at baseline,

greater reduction in VAF on treatment, and a hot peripheral macroenvironment

were associated with the treatment response independently. Our study showed

the antitumor activity of tislelizumab, cetuximab, and irinotecan combination

with a tolerable safety profile in previously treated MSS and RAS WT mCRC.

The combination of an anti-epidermal growth factor receptor (antiEGFR) antibody (cetuximab or panitumumab) with chemotherapy is a

standard treatment for patients with RAS and BRAF wild-type (WT)

metastatic colorectal cancer (mCRC)1,2

. Although these regimens are

highly effective in the first- or second-line setting, the clinical benefit

of the later-line standard therapies, such as regorafenib3 or trifluridine/tipiracil4

, is limited with a high incidence of adverse events

(AEs). Such later-line therapies reported an objective response rate

(ORR) of 1% to 4% and a median progression-free survival (mPFS) of

1.9 to 3.2 months. In this context, despite the lack of high-level

Received: 9 March 2024

Accepted: 12 August 2024

Check for updates

1

Department of Oncology, Zhongshan Hospital, Fudan University, 200032 Shanghai, China. 2

Cancer Center, Zhongshan Hospital, Fudan University, 200032

Shanghai, China. 3

Genecast Biotechnology Co., Ltd, 214104 Wuxi City, Jiangsu, China. 4

Center of Evidence-based medicine, Fudan University, 200032

Shanghai, China. 5

These authors contributed equally: Xiaojing Xu, Luoyan Ai, Keshu Hu, Li Liang, Minzhi Lv. e-mail: yu.yiyi@zs-hospital.sh.cn;

liu.tianshu@zs-hospital.sh.cn

Nature Communications | (2024) 15:7255 1

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evidence, re-treatment with previously administered drugs is often

adopted in clinical practice. For instance, the results from the

CRICKET study5 demonstrated an ORR of 21% (confirmed ORR 14%)

with the reintroduction of cetuximab plus irinotecan in the third-line

setting. CHRONOS study6 selected patients with ‘zero mutation

ctDNA triage’ for panitumumab rechallenge, and showed an exceptional ORR of 30% (confirmed ORR 22%) for mCRC patients with

RAS WT.

Immune checkpoint inhibitors (ICIs), such as anti-programmed

cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1)

monoclonal antibodies (mAbs), are established standards of care for

patients with mismatch repair deficient (dMMR) or microsatellite

instability-high (MSI-H) mCRC7,8

. However, ICI monotherapy offered

little to no effect for most of the proficient mismatch repair (pMMR)

or microsatellite stable (MSS) subtype9,10. Hence, a combination

strategy by adding tumor-targeting antibodies or cytotoxic agents to

ICIs is a logical next step through changing the immune microenvironment. Besides the aforementioned clinical use, cetuximab has

been reported to trigger the immunogenic cancer cell death and

augment the CRC immunogenicity11. Increased levels of cytotoxic

immune infiltrates, PD-L1, CXCR2, and LAG3 were observed after the

effective cetuximab treatment in patients with mCRC, potentially

providing opportunities to treat cetuximab-resistant CRCs with

immunotherapy12,13. Importantly, cetuximab is an IgG1 mAb that has a

strong antibody-dependent cell cytotoxicity (ADCC) and results in

cross talk among the immune cells, including natural killer (NK) cells

and dendritic cells14,15. This cross talk can prime the tumor antigenspecific cellular immunity and generate the antigen-specific T-lymphocyte responses16,17. In this scenario, cetuximab may have additive

or synergetic effects with PD-1 blockade. This has been proved in

patients with recurrent or metastatic head and neck squamous cell

carcinoma, with the longest median overall survival (mOS) of

18.4 months achieved by cetuximab plus pembrolizumab18. The phase

2 CAVE study (avelumab plus cetuximab) has demonstrated an active

clinical activity, with an mOS of 11.6 months and an mPFS of

3.6 months in pretreated MSS mCRCs19, highlighting the scientific

rationale of the combination. However, the result was not encouraging. Therefore, we considered that chemical agents may also play an

important role. Preclinical studies found that SN-38, an active metabolite of irinotecan, could sensitize unresponsive tumors responding

to anti-PD-1 therapy by engaging NK or CD8+ T cells to infiltrate the

tumor microenvironment (TME)20,21, suggesting that ICIs combined

with cetuximab and irinotecan may work synergically.

The circulating tumor DNA (ctDNA) can be used to detect tumorspecific alterations, and was highly consistent with tissues22,23. Moreover, ctDNA has been well documented to be correlated with response

and survival time in the field of chemotherapy or targeted therapy24–26.

Whether ctDNA monitoring could also predict the clinical benefit of

these ICI-based combination therapies in MSS mCRCs is yet to be

determined. Furthermore, tumor progression or regression is a consequence of the battle between tumor cells and host immunity. We

believe that factors reflecting the systemic immunity could also predict the response, especially in the context of immunotherapy. Serum

immune proteomics, with its high content, could be a suitable reflection of the global immunity27–29.

In this work, we conducted a study to investigate the efficacy and

safety of ICI combined with cetuximab and irinotecan (TEC, Tislelizumab plus Erbitux and CPT-11) in MSS and RAS/BRAF WT mCRC in

third or more lines settings. Furthermore, to identify which patients

may benefit from TEC, we performed ctDNA profiling and plasma

immune proteomics profiling using blood samples taken before and

during treatment. Here, we show that TEC has antitumor activity with

confirmed ORR of 33% and mOS of 17.4 months. Pretreatment ctDNA

levels and immune-oncology proteins levels are associated with

the response.

Results

Efficacy

Between February and September 2021, 36 patients were screened in

this study. One patient did not meet the inclusion criteria, and 2

patients withdrew the informed consent for personal reasons before

treatment. As a result, 33 patients had at least 1 cycle of treatment

(Fig. 1). Among them, 22 patients (67%) failed at least 3 lines of treatment. The median time from the initial diagnosis of metastasis to

enrollment was 30.2 months. The baseline characteristics are presented in Table 1 and Supplementary Table 1.

At the time of the data cutoff on December 12, 2022, the median

follow-up time was 17.6 months (1.1–21.8 months). All 33 patients had

at least 1 imaging assessment for efficacy. Radiographic partial

response (PR) was observed in 12 patients (36%), and 14 patients (42%)

achieved stable disease (SD). Confirmed PR was observed in 11 patients

(33%) (Supplementary Table 2). No patient achieved CR. Six patients

showed disease progression (PD) at the time of the first CT scan. One

patient died after 1 treatment cycle without CT assessment. The confirmed ORR was 33% (n = 11), and the disease control rate (DCR) was

79% (n = 26). Tumor shrinkage was observed in 24 patients (73%)

(Fig. 2A). For the 11 patients who achieved confirmed ORR, the median

time to response was 2.0 months and the median duration of response

was 6.2 months (Fig. 2B, C). The median PFS was 7.3 months (95% CI,

5.6–8.6), and the median overall survival (OS) reached 17.4 months

(95% CI, 15.9-not reached [NR]). The 6-month PFS rate was 60%, and the

1-year OS rate was 85% (Fig. 2D, E).

Twenty-nine (88%) patients have failed in the previous cetuximabcontaining therapy. Four patients who had never used cetuximab but

had failed on other second- or third-line treatment previously were

included in this study. Of the 4 cetuximab-naïve patients, only one

reached PR, while for the 29 cetuximab-exposed patients, 10 patients

reached PR. No statistical differences in PFS or OS were observed

between the two groups (PFS: 8.2 months for Cetuximab-naïve

patients vs 6.8 months for Cetuximab-exposed patients, P = 0.87; OS:

NR for Cetuximab-naïve patients vs 17.4 months for Cetuximabexposed patients, P = 0.41) (Supplementary Fig. 2A, B). Of the 29

Cetuximab-exposed patients, 7 experienced treatment failure on their

previous regimens containing Cetuximab and enrolled in our study

without a period of Cetuximab-free time. Of these 7 patients, 2

achieved PR after the addition of ICI, and 4 had SD, with one of them

controlling the disease for over 2 years.

Liver or lung metastasis may serve as a biomarker for ICIs, we hence

evaluated the efficacy of treatment between patients with or without

liver or lung metastasis. As shown in Supplementary Fig. 1, the ORR was

comparable between patients with liver metastasis (10/26, 38.5%) and

those without liver metastasis (2/6, 33.3%) using Fisher’s exact test

(P > 0.99). However, the PFS trended shorter in patients with liver

metastasis (6.1 months vs 8.6 months; P = 0.38), but no significance was

reached. The mPFS (6.8 with lung metastasis vs 7.3 months without lung

metastasis; P = 0.17) were also not significantly different between

patients with or without lung metastasis, although patients without lung

metastasis tended to have higher ORRs (4/19 [21.1%] with lung metastasis

vs 8/14 [57%] without lung metastasis, P = 0.07). More details of the

subgroup analysis are provided in Supplementary Fig. 1.

Safety

The median number of treatment cycles was 12 per patient (range,

1–42 cycles). The median exposed dose of tislelizumab, cetuximab,

and irinotecan was 2400, 8800, and 2400 mg per patient, respectively.

Among the 33 treated patients, 32 patients (97%) had treatmentrelated AEs (Fig. 2F and Supplementary Table 3). No grade 4/5 AEs were

reported. Immune-related AEs included 2 grade 1 hyperthyroidism, 3

grade 1 hypothyroidism, and 1 event each of grade 2 immune dermatitis and myocarditis, which improved after the treatment with thyroxine or corticosteroids.

Article https://doi.org/10.1038/s41467-024-51536-x

Nature Communications | (2024) 15:7255 2

Treatment was delayed in 68 cycles (17%) because of an AE, and it

was related to cetuximab and irinotecan in 25 and 39 cycles, respectively. The cetuximab dose was reduced in 9 patients (27%) due to rash,

and irinotecan dose was reduced in 11 patients (33%) due to diarrhea or

hematologic toxicity or vomiting. A total of 29 patients (88%) received

a further line of treatment after disease progression.

ctDNA metrics were associated with the clinical benefit

Similar ultimate outcomes were observed between the patients who

achieved PR or SD (Supplementary Fig. 2C, D). Therefore, we defined

patients with non-PD (SD or PR) as responders and sought to find

another way to identify the patients who would eventually benefit from

the TEC regimen. The post-hoc analysis of ctDNA used the mean VAF as

a continuous metric of ctDNA concentration. Overall, ctDNA was

detected in all patients (100%, 32 of 32) at baseline, with TP53 (81%),

APC (69%), and CARD11 (16%) being the most abundant alterations

(Supplementary Fig. 3A). Patients with a lower VAF (below the median)

had longer OS compared with those with a higher VAF (above the

median) at baseline (Fig. 3A, HR: 4.95, 95% CI, 1.71–14.29; P = 0.0058).

Patients with PD (n = 6) had a higher VAF than non-PDs (SD + PR, n = 26)

at all time points (Fig. 3B). The TEC treatment reduced the VAF 4 weeks

(C2D15) after initiation in most patients, particularly in responders,

regardless of PR or SD (Fig. 3B–D). By employing ROC curve and

Youden index analysis, we determined the optimal cutoff value for VAF

decrease from pretreatment and classified patients into two groups:

with or without ctDNA molecular response. Patients with ctDNA

molecular responses at T2 (Fig. 3E, HR = 3.12; P = 0.024) or T3 (Fig. 3F,

HR = 5.20; P = 0.042) both had significantly longer OS. Accordingly,

patients with lower ctDNA levels (VAF < 1%) and evaluated at every time

point (T2, T3, T4) had much longer OS than those with higher ctDNA

levels (VAF ≥ 1%) (Supplementary Fig. 4A). A similar trend for PFS was

also observed (Supplementary Fig. 4B).

The mean VAF was positively correlated with tumor size at similar

time points (Fig. 3G; Supplementary Fig. 4C, D). Moreover, the percent

change in the mean VAF from T1 (pretreatment) to T2 (C2D15) or T3

(C4D15) correlated with the tumor shrinkage from baseline to week 8

as assessed by CT (R = 0.54, P = 0.0013, Fig. 3H; Supplementary

Fig. 4E). Notably, in contrast to the overlapped Kaplan-Meier (KM)

curves for patients with conventional radiographic SD and PR (Supplementary Fig. 2C, D), the patients who achieved ctDNA molecular

response tended toward better OS compared with those who did not

(HR: 3.15; P = 0.059, Supplementary Fig. 4F), indicating ctDNA may be

more sensitive than radiographic tumor assessment for risk stratification among responders. For 13 responders who had blood samples

available for all 4 time points, visualizing ctDNA dynamics parallel to

the tumor size dynamics revealed that ctDNA changes mirror tumor

size changes (Fig. 3I, J). Moreover, plasma TMB at T2 and T3, but not T1,

showed a significant difference between PD and non-PD (Supplementary Fig. 4G).

Besides, we found 6 patients evolved into RAS mutation (n = 3) or

BRAF mutation (n = 3) in baseline ctDNA. One patient with KRAS

mutation detected at baseline even achieved partial response. This

specific mutation site was KRAS Q61H, but it is worth noting that the

mutation frequency in this case was quite low. Both the median OS

(12.0 months vs 18.0 months, HR: 4.60, 95% CI, 1.06–9.92; P = 0.026;

Fig. 3K) and median PFS (3.9 months vs 8.0 months, HR: 3.90, 95% CI,

1.25–12.17; P = 0.012; Supplementary Fig. 4H) were significantly shorter

in patients with RAS/BRAF MUT compared with patients with RAS/BRAF

Fig. 1 | Study flow. After excluding 1 patient who didn’t meet the inclusion criteria and 2 patients who withdrew their consent, 33 patients were treated with TEC regimen,

and included in the modified intention-to-treatment set (mITT) analysis.

Article https://doi.org/10.1038/s41467-024-51536-x

Nature Communications | (2024) 15:7255 3

WT. For ctDNA status evaluated at the time of progression for

responders, many secondary mutations, including EGFR (n = 6), KRAS

(n = 5), MAP2K1 (n = 3), HMCN1 (n = 2), and KDM5A (n = 2), were detected (Supplementary Fig. 3B).

Dynamics of plasma immune proteomics are associated with the

response to TEC

Post-hoc proteomic analysis revealed that a global increase of almost

all markers was found in responders (non-PD) at baseline (Fig. 4A, B

and Supplementary Fig. 5A). The 26 identified statistically significant

proteins (Fig. 4A, B) were inextricably interacted (Fig. 4C), and most of

them were correlated with PFS (Fig. 4D). Pathways involving innate and

adaptive immunity, such as “Positive regulation of lymphocytemediated immunity,” “Response to chemokines,” and “Neutrophil

migration,” were more enriched in responders (Fig. 4E).

Furthermore, TEC treatment also induced an increased expression

of most plasma proteins at T2 (C2D15) and T3 (C4D15), irrespective of

the response (Fig. 4F, G). However, at the time of progression, more than

half of the plasma proteins were decreased (Fig. 4H and Supplementary

Fig. 5B). Their functions were mainly enriched in pathways associated

with “cell chemotaxis,” “cytokine activity,” and “response to biotic stimulus” (Supplementary Fig. 5C–E). Pathways such as “Neoplasm,”

“Breast cancer basal up,” and “Abnormality of the lymphatic system”

were activated in comparisons between T4 (progression) and T3

(C4D15). We chose 10 proteins (Fig. 4I) that changed in both T2 and T3 in

comparison with T1 (indicating a robust and lasting activation after TEC

treatment) and found most of them also correlated with OS and PFS

(Supplementary Fig. 5F), especially at the time of T3 (C4D15). The level of

soluble PD-L1 expression was similar between non-PD and PD at all time

points that were assessed (Supplementary Fig. 5G).

We also endeavored to establish a score system to quantify the

peripheral immune proteomics in a global manner to reveal

the intensity of the PME’s “hot” or “cold” nature. As shown in Fig. 4J, the

survival probability of patients with high protein scores was

longer than those with low protein scores (HR: 0.306, 95% CI,

0.1–0.9; P = 0.031).

Negative correlation between plasma immune proteomics and

ctDNA metrics and prognosis for MSS mCRC

A negative correlation was observed between plasma proteomics and

ctDNA metrics after TEC treatment. All the 25 significant elevated

proteins in responders at baseline and key ctDNA metrics after TEC

treatment, including ctDNA levels at T2, ctDNA levels at T3, and

decreases in ctDNA levels at T2 or T3, were all negatively correlated

(Fig. 5A). In line with it, patients with ctDNA molecular responses at

T2 showed an increase of several immune markers compared with

baseline (Fig. 5B). Subsequently, we performed a univariate Cox

regression analysis of factors including clinicopathologic variables,

key ctDNA features, and peripheral immune protein levels. As shown in

Fig. 5C, ctDNA metrics, such as ctDNA levels or reduction in ctDNA

levels and proteomics scores at baseline and T3, were associated with

OS. Furthermore, we selected variables with a P-value < 0.1 in the univariate analysis to be included in the multivariate Cox regression

analysis. As illustrated in Fig. 5D, after adjusting for those confounding

factors, ΔctDNA (T2-T1) and proteomics scores at T1 were still independent factors related with OS.

Discussion

In this study, we reported that tislelizumab plus cetuximab and irinotecan (TEC) met its primary endpoint with an ORR of 33% in patients

with MSS and RAS WT refractory mCRC. The mPFS was 7.3 months and

the mOS was 17.4 months, which are exponentially favorable to the

historical standard salvage-line therapies including regorafenib3

,

fruquintinib30, or trifluridine-tipiracil4

. This study also included a

translational analysis of serial liquid biopsies. We found that pretreatment ctDNA levels (VAF) and on-treatment VAF reductions were

associated with the response of TEC. Moreover, the levels of most

immune-oncology proteins were higher in responders, and the

patients with high proteomics scores at baseline were associated with

better OS (Fig. 6). Importantly, a negative association between the

peripheral immune proteomics and ctDNA metrics was observed,

indicating the wax and wane of the host systemic immunity and tumor

dynamics.

Although only a limited number of relatively small size studies

have been published so far, the combination of EGFR antibody with

irinotecan in the third-line setting has shown an ORR ranging from 3%

to 23% and mPFS ranging from 2.4 months to 5 months5,31–33. For

instance, the CRICKET study5 showed that a rechallenge with cetuximab and irinotecan achieved an ORR of 21% (confirmed ORR, 14%),

mPFS of 3.4 months, and mOS of 9.8 months in the third-line setting

for mCRC (n = 28). In another cohort with larger sample size (n = 218),

patients with non-RAS-selected and irinotecan-refractory CRC, cetuximab plus irinotecan reported an ORR of 23% with PFS of 4.1 months31.

In this study, the confirmed ORR of the TEC regimen was 33%, the

median OS was 17.4 months, and the median PFS was 7.3 months. The

triplets in TEC exceeded the efficacy of the doublet of re-treatment

with cetuximab in combination with irinotecan. CHRONOS study6

showed an exceptional ORR of 30% (confirmed ORR 22%) for panitumumab rechallenge, but it specifically selected patients with ‘zero

mutation ctDNA triage’ at baseline. In our study, for the patients with

confirmed RAS/BRAF WT in the baseline ctDNA, the triplets (confirmed

ORR: 38%, mPFS: 8.0 months, and mOS: 18.0 months) exceeded the

single panitumumab in CHRONOS6 (confirmed ORR: 22% and mOS:

13.8 months). It also exceeded the doublets in the CRIKET study5 in the

RAS/BRAF WT population (cetuximab plus irinotecan; confirmed ORR:

Table 1 | Baseline and Demographic and Clinical Characteristics of Patients (n = 33)

n (%)

Median age, years (range) 58 (35–80)

≥65 years 10 (30%)

<65 years 23 (70%)

Sex

Male 24 (73%)

Female 9 (27%)

Primary tumor site

Left 32 (97%)

Right 1 (3%)

ECOG performance status

0 6 (18%)

1 27 (82%)

Number of metastatic organs

1–2 20 (61%)

≥3 13 (39%)

Liver metastasis

Yes 26 (79%)

No 7 (21%)

Lung metastasis

Yes 19 (58%)

No 14 (42%)

Time since diagnosis of the first metastasis, months, median (range)

30 (10–72)

Number of prior regimens

2 11 (33%)

3 17 (52%)

≥4 5 (15%)

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Nature Communications | (2024) 15:7255 4

31%, mPFS: 4.0 months, and mOS: 12.5 months). These data indicated

that the addition of ICIs into the combination may have better efficacy,

despite cross-trial comparisons require careful interpretation.

Notably, out of the 29 patients who had previously been exposed

to Cetuximab, 7 were promptly enrolled in this study following

Cetuximab resistance without any interval. Nevertheless, 6 of these 7

patients achieved disease control after the addition of ICI. Therefore,

PD-1 blockade may have additive or synergetic effects with Cetuximab,

even in patients with Cetuximab resistance. Theoretically, Cetuximab

may change the immune microenvironment through cross talk with

immune cells or increasing the expression of immune checkpoints11–17.

This has been proved in patients with recurrent or metastatic head and

neck squamous cell carcinoma18. In patients with mCRC, the CAVE

study19 (avelumab plus cetuximab; ORR: 7.8%, mPFS: 3.6 months, and

mOS: 11.6 months) or NCT0271337334 (pembrolizumab plus cetuximab; ORR: 2.6%, mPFS: 4.1 months, and mOS: 14.5 months) showed a

slight advantage; however, the results were not encouraging. Therefore, we hypothesized that chemical agents may exert a significant

effect on the combination, which could sensitize unresponsive tumors

by engaging the immune cells to infiltrate the TME20,21. Cohort B of the

phase 2 CRACK study35, along with our study, represented that

the triplets, including PD-1 antibody (camrelizumab or tislelizumab)

Fig. 2 | Tumor response. Waterfall plot shows the maximum percent change from

baseline in the sum of the longest diameters of target lesions in 32 patients who

were treated in the current study and underwent radiologic evaluation. A Spider

plot shows the change in sum of the target lesion diameters over time (n = 32).

B Swimmer plot shows the duration of treatment (n = 33). C Kaplan-Meier plot of

progression-free survival. D Kaplan-Meier plot of overall survival. NR not reached.

E Frequency of AEs. Source data are provided as a Source Data file.

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Nature Communications | (2024) 15:7255 5

Fig. 3 | Dynamic ctDNA levels associated with clinical outcomes. A Kaplan-Meier

analysis showing the prognostic value of pretreatment ctDNA for OS, where the

orange curve indicates patients with ctDNA levels greater than or equal to the

median ( ≥2.1%, n = 16), and the blue curve indicates patients with ctDNA less than

the median (n = 16). B Dynamic changes of ctDNA levels before and on treatment.

Mixed-effects model was used to derive p-values. (n = 12 for PR, n = 14 for SD, n = 6

for PD). C ctDNA level changes at T2 between non-PD (n = 26) and PD (n = 6). Twotailed Mann-Whitney U test was used to derive P-value. The exact P-value indicated

by ‘P < 0.0001’ is 6.6 × 10−5

. D ctDNA level changes at T3 between non-PD (n = 26)

and PD (n = 4). Two-tailed Mann-Whitney U test was used to derive P-value. E KM

curves showing OS for patients with ctDNA molecular response (n = 19) versus

patients without ctDNA molecular response (n = 13) at T2. The optimal cutoff value

for ctDNA decrease was calculated by ROC curve and Youden index analysis. And

the threshold is set at a 83.44% reduction from pretreatment. F KM curves showing

OS for patients with ctDNA molecular response (n = 14) versus patients without

ctDNA molecular response (n = 16) at T3. The optimal cutoff value for ctDNA

decrease was calculated by ROC and Youden index analysis. And the threshold at T3

is 82.57% reduction from pretreatment. G Correlation of ctDNA levels at T1 and

tumor size at baseline. Pearson correlation test was used (n = 32). H Correlation of

ctDNA level changes at T2 and radiographic assessment at week 8. The percent

change in tumor size was evaluated as the sum of longest diameters (SLD). Pearson

correlation test was used (n = 32). I Dynamic changes of ctDNA levels for patients

with available biopsies at all time points (n = 13). Two-tailed paired t-tests were used

to derive P-value. J The ctDNA landscape changes (upper panel) and the representative radiographic images of tumors (bottom panel) for patient #7 at

different time points. K Overall survival in patients with baseline ctDNA RAS/BRAF

mutational status. (n = 26 for RAS/BRAF WT, n = 6 for RAS/BRAF MUT). For

(A, E, F, K), a univariable Cox proportional hazards model was used to estimate the

HR and logrank test was used to report P-value. Source data are provided as a

Source Data file.

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Nature Communications | (2024) 15:7255 6

Fig. 4 | Dynamics of plasma immune proteomics are associated with the

response to TEC. A Heatmap showing the expression of 26 significantly changed

proteins between the non-PD group (n = 26) and PD group (n = 6) at baseline.

B Volcano plots showing differential expression of plasma proteins between the

non-PD (n = 26) and PD (n = 6) at baseline. C The interaction of 26 proteins in (A)

analyzed by Cytoscape. The color of the nodes shows the changes in protein levels

between the non-PD and PD (ΔNPX). The thickness of lines between the nodes

indicates the co-expression degree of 2 proteins linked. D The Spearman correlation between 26 significant proteins and PFS/OS (n = 32). E GSEA analysis of significant proteins identified in (A). F Volcano plots showing differential expression

of plasma proteins between T2 (on-treatment C2D15) and T1 (pretreatment), n = 31.

G Volcano plots showing differential expression of plasma proteins between T3

(on-treatment C4D15) and T1 (pretreatment), n = 30. H Volcano plots showing differential expression of plasma proteins between T4 (time of progression) and T3

(on-treatment C4D15), n = 6. I Dynamic changes of 10 plasma proteins significantly

changed both in T2 and T3. Dunnett’s multiple comparisons test was used. P-value

was adjusted, n = 30. J KM curves of OS for patients with high (n = 21) or low proteomics scores (n = 11). A univariable Cox proportional hazards model was used to

estimate the HR and logrank test was used to report P-value. For volcano plots, the

horizontal axis displays the magnitude of a protein’s change; the vertical axis displays the significance scale by the −log10 (P-value), which increases with statistical

significance. For (A), two-tailed Wilcoxon rank sum test was used. For (B, F, G, H),

two-tailed paired t-test was used. P-value was adjusted by Benjamini-Hochberg

method. The horizontal black dotted line corresponds to the P-value cutoff of 0.05.

Red dots are biomarkers with a significant upregulation and blue dots are biomarkers with a significant downregulation. Source data are provided as a Source

Data file.

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Nature Communications | (2024) 15:7255 7

Fig. 5 | Correlation between ctDNA metrics and PME. A The Pearson correlation

between the ctDNA metrics and plasma immune markers, n = 32. B Volcano plots

showing differential expression of plasma proteins between ΔctDNA (T2-T1) ≥ 50%

versus < 50%. Two-tailed paired t-test was used. P-value was adjusted by BenjaminiHochberg method, n = 32. C Univariate Cox regression analysis of different variables

for OS of patients. Note: bold indicates significant difference with P < 0.05.

Horizontal lines represent the 95% confidence interval of HR, n = 32. D Multivariate

Cox regression analysis of different variables for OS of patients. Note: bold indicates

significant difference with P < 0.05. Horizontal lines represent the 95% confidence

interval of HR, n = 32. Source data are provided as a Source Data file.

Article https://doi.org/10.1038/s41467-024-51536-x

Nature Communications | (2024) 15:7255 8

combined with cetuximab and irinotecan, may work synergically and

exceed the efficacy of the doublets of cetuximab in combination with

irinotecan or with ICIs. TEC has the potential to be a treatment option

for MSS and RAS/BRAF WT mCRCs in the later-line settings if validated

in larger trials.

Previous studies such as REGONIVO36, C800 trial37 and RIN38

therapy have shown that patients with liver metastasis tend to have a

weaker response to immunotherapy, while those with lung metastasis

may experience better efficacy. We also found the PFS trended shorter

in patients with liver metastasis than those without liver metastasis

despite a comparable ORR. Additionally, the mPFS was not significantly different between patients with or without lung metastasis,

although patients without lung metastasis tended to have higher

ORRs. When combined with EGFR antibody, ICIs may exhibit varying

responses compared to ICIs combined with anti-angiogenic agents in

patients with liver or lung metastasis. Further research using larger

sample sizes is necessary to fully understand the predictive role of liver

or lung metastasis on the effectiveness of immunotherapy.

The safety profiles for TEC were expected and manageable, based

on the toxicity profile of each drug, and no new AEs were identified for

the combination. Moreover, in contrast to the high incidence of grade

3/4 treatment-related AEs (ranging from 27% to 87%)36,39,40 with the

administration of antiangiogenic tyrosine kinase inhibitors with or

without ICIs, TEC was well tolerated. No serious or unexpected AEs

were reported. Notably, the recommended dose of tislelizumab is

200 mg every 3 weeks, but we used 200 mg tislelizumab every 2 weeks

in this study after deliberating the pharmacokinetics, safety, and

efficacy data for tislelizumab obtained from human studies. The findings revealed that the dose was safe and efficacious. Only seven mild

immune-related AEs were reported, which were manageable with the

treatment with thyroxine or corticosteroids. A total of 29 patients

(88%) received a further line of treatment after progression in this

study, indicating a good performance was still maintained after TEC

treatment.

By incorporating longitudinal ctDNA evaluation of paired pre- and

on-treatment liquid biopsies from nearly all patients, our study

demonstrated that the total amount of ctDNA levels were correlated

with total disease burden and that ctDNA metrics collected across

longitudinal time points can be used to risk stratify the patients and

predict the survival. On-treatment ctDNA dynamics may predict the OS

more accurately and earlier than traditional CT scans. This was consistent with the findings from prior studies showing that ctDNA can be

used effectively to monitor the response to cancer therapy25,26,41,42.

Another study showed that ctDNA dynamics after just a single infusion

can aid in the identification of patients who will achieve durable clinical

benefits42. Hence, early ctDNA molecular response appears to be a

promising approach for early response assessment and prediction of

ultimate clinical benefit more precisely. Another superiority and irreplaceability of ctDNA over radiographic imaging is that serial genomic

profiling of ctDNA could provide a real-time comprehensive characterization of clonal evolution, particularly the emergence of mutations of acquired resistance in patients treated with anti-EGFR therapy.

The concordance between the detection of mutations in tissue

and plasma has extensively been demonstrated. We also identified 6

Fig. 6 | Schematics of TEC administration and its efficacy. Tislelizumab plus

cetuximab and irinotecan (TEC) met its primary endpoint with an ORR of 33% in

patients with MSS and RAS WT refractory mCRC. The mPFS was 7.3 months and the

mOS was 17.4 months. Pretreatment ctDNA levels and immune-oncology proteins

levels were associated with the response. (Adobe Illustrator CS5 software).

Article https://doi.org/10.1038/s41467-024-51536-x

Nature Communications | (2024) 15:7255 9

patients with secondary RAS/BRAF mutations in ctDNA at baseline, and

most of these patients indeed had quite poor response to TEC treatment. With the rapid improvement of ctDNA sequencing technology,

ctDNA offers a promising option to characterize the course of mCRC in

a minimally invasive way to guide the treatment of the patient.

In contrast to the extensively studied local immunity in the TME,

little is known about the relationship between the systemic immunity

and immunotherapy response in mCRC, yet immunity is coordinated

across tissues. The localized antitumor immune response cannot exist

without continuous communication with systemic immunity. Therefore, a thorough understanding of immune responses to cancer should

include immunity across the peripheral immune system in addition to

within the TME. In this study, by performing parallel studies with

matched plasma biopsies from the same patients, we screened circulating immune protein expressions in the spectrum of responders and

nonresponders using a DNA oligonucleotide-based Olink immunoassay. We identified immune signatures in responders and over the

treatment continuum. It was found that responders had ubiquitously

higher expression of most immune proteins that belonged to the

pathways involved in both innate and adaptive immunity. TEC treatment increased the expression of the majority of plasma immune

proteins. The global immune environment in blood was defined as

PME, with the presumption that a hot PME may have the association

with the responses. Moreover, we developed a scoring system to

quantify the peripheral immune proteomics and demonstrated that

patients with high proteomics scores at baseline had better OS. Consistent with our observation, another study also found a significant

increase in almost all immune-oncology-related plasma proteins after

neoadjuvant immunotherapy in melanoma43.

Interestingly, we found a negative association between the peripheral immune proteomics and ctDNA levels, perhaps a reflection of

the “seesaw” effect between the tumor and host systemic immunity. By

using multivariate Cox regression analysis, we further integrated

clinical and translation data in our study to predict the response and

stratify the risk. This model was not limited only to static features of

pretreatment tumors but also encompassed dynamic biomarkers that

capture the tumor evolution under the selective pressure of ICI-based

therapy. Significant intratumoral heterogeneity of colorectal cancer

and limited biopsy depth largely impair the representation of biopsy

samples. Comparably, peripheral immune proteomics and ctDNA

metrics incorporate both systemic and local features for hosts and

tumors and are thus sensitive and informative.

This study was limited by its single-arm single-center design and

small sample size. The definition of PME would be better when taking

immune cells ratios and function into consideration. The value of

ctDNA and global immune proteomics deserve to be further explored

and validated. Nevertheless, the study showed encouraging antitumor

activity of TEC and could be a promising therapeutic option for

advanced mCRC. A randomized, multicenter, controlled clinical trial

(NCT05278351) of tislelizumab plus cetuximab and irinotecan versus

third-line standard of care in refractory MSS mCRC is currently

ongoing, in order to validate the results of this study.

In conclusion, tislelizumab in combination with cetuximab and

irinotecan showed an encouraging clinical efficacy and tolerable safety

profile in previously treated patients with MSS and RAS WT mCRC.

Peripheral immune profiling and ctDNA offer a promising, noninvasive

approach to monitor the therapeutic efficacy in real time.

Methods

Study design and participants

This was an open-label, single-arm, single-center, phase 2 study. The

study was approved by the ethics committee of the Zhongshan Hospital affiliated to Fudan University (Approval Number B2020-344) and

was conducted in accordance with the principles of the Declaration of

Helsinki and the International Conference on Harmonization and Good

Clinical Practice guidelines. The major inclusion criteria included (1)

histologically confirmed advanced or metastatic MSS and RAS WT

colorectal adenocarcinoma refractory to at least 2 previous lines of

therapy, (2) an Eastern Cooperative Oncology Group performance

status score of 0 or 1, and (3) adequate hematologic, hepatic, and renal

functions. The key exclusion criteria included (1) patients with dMMR

or MSI-H; (2) RAS or BRAF mutation detected in any previous tissue

samples; (3) previous treatment with immunotherapy, such as anti-PD1, anti-PD-L1, anti-cytotoxic T-lymphocyte–associated antigen 4, or any

immunocytes therapy; (4) active autoimmune diseases; or (5) serious

comorbidity. Sex or gender were not considered in the study design.

Tislelizumab was administered at a fixed dose of 200 mg in combination with cetuximab at a dose of 500 mg/m2 and irinotecan at a dose of

180 mg/m2 every 2 weeks until disease progression (PD) or the development of unacceptable toxicity. Dose modifications of tislelizumab

were not permitted. Modifications to the doses of cetuximab and irinotecan were made according to the drug instructions. Interruptions

in the doses were allowed according to the severity of AEs. All participants provided written informed consent for participating in this

study and publishing clinical information. There was no additional

compensation for patients. This trial was preregistered at https://www.

chictr.org.cn/ with identifier of ChiCTR2000035642 on August 15th,

2020, and retrospectively registered at ClinicalTrials.gov with identifier of NCT05143099 on November 22nd, 2021. The study protocol is

available in the Supplementary Information file.

Assessment

Tumor response was evaluated every 8 weeks using computed tomography (CT) or magnetic resonance imaging (MRI). Investigators performed the assessment using the Response Evaluation of Criteria in

Solid Tumor 1.1 (RECIST1.1). For patients who progressed for the first

time, another 2 cycles of treatment followed by a repeat CT assessment

were allowed according to the immune RECIST (iRECIST) criteria. AEs

were assessed from the date of initiation of protocol therapy until

28 days after the administration of the last dose according to the

National Cancer Institute’s Common Toxicity Criteria for Adverse

Events (version 5.0).

Outcomes

The primary endpoint was ORR, which was defined as the proportion

of patients with the best response of complete response (CR) or partial

response (PR). The PR was considered as confirmed only if PR was

maintained and observed on 2 consecutive scans. Patients whose

disease was not reassessed and those who were unavailable for the

follow-up were considered as nonresponders for the primary endpoint

analysis. The secondary endpoints included DCR, PFS, and OS. DCR

was defined as the proportion of patients achieving CR, PR or SD. PFS

was defined as the duration from the treatment initiation to PD or

death as a result of any cause, whichever occurred first. OS was defined

as the duration from the treatment initiation to death. ORR, DCR, and

PFS were assessed using the iRECIST criteria. The exploratory objective

was to examine the biomarkers for clinical activity through consecutive blood samples.

ctDNA evaluation by NGS

The consecutive blood samples were prospectively collected at baseline (T1, pretreatment), 4 weeks after the first administration (T2,

C2D15), first assessment at 8 weeks (T3, C4D15), and PD (T4, progression) for post hoc analysis. Approximately, 16 to 20 mL of blood was

collected in EDTA tubes and centrifuged at 4 °C for 10 minutes at 200 g

within 30 minutes after the collection of blood. Plasma samples were

stored at −80 °C until analysis. Circulating free DNA was extracted

using a MagMAX™ Cell-Free DNA Isolation Kit (Thermo Fisher Inc)

from plasma following the manufacturer’s protocol. Peripheral blood

mononuclear cell was treated as normal control to filter the germline

Article https://doi.org/10.1038/s41467-024-51536-x

Nature Communications | (2024) 15:7255 10

mutations, and DNA was extracted using TIANamp Blood DNA Kit

(TIANGEN). Genomic DNA was fragmented to a length of about 200 bp

using Covaris LE220, starting from an input amount of 30 to 300 ng.

The libraries were prepared by KAPA HyperPrep PCR-free Kit (KAPA).

The next-generation sequencing was performed using a panel

encompassing 769 cancer-related genes. The hybridized library was

subjected to sequencing on the Illumina NovaSeq 6000 platform using

paired-end 150 bp mode. Tumor mutational burden (TMB) was

reported as the number of somatic mutations per megabase (mut/Mb)

of the captured region. The mean variant allele frequency (VAF) was

calculated as the average VAF based on the identified mutations that

met the criteria for reliability, which was the representative of the

ctDNA levels in this study. Details about variant calling, somatic

mutation filtering, and monitoring are in the supplementary materials.

Plasma proteomics profiling using olink proximity extension

assay technology

Thirty-two pretreatment (T1), 31 on-treatment C2D15 (T2), 30 ontreatment C4D15 (T3), and 6 at progression (T4) plasma samples from

patients with TEC treatment were suitable for testing. A multiplex

assay to profile the plasma proteomics was performed using proximity

extension assay (PEA) technology (Olink Bioscience AB), according to

the manufacturer’s instructions. We selected the Olink® Target 96

Immuno-Oncology panel (Olink Proteomics AB, Uppsala, Sweden) that

included 92 key unique markers. The PEA technology used for the

Olink’s protocol has been well described27,44,45. In summary, the basis of

PEA is a dual-recognition immunoassay. Two matched antibodies

labeled with unique DNA oligonucleotides simultaneously bind to a

target protein in solution. Once the antibodies bind to a target protein,

the 2 antibodies would be placed in proximity, allowing their DNA

oligonucleotides to hybridize and serve as a template for a DNA

polymerase-dependent extension step. The hybridized DNA creates a

double-stranded DNA “barcode,” which is unique for the specific

antigen and quantitatively proportional to the initial concentration of

the target protein. The hybridization and extension are immediately

followed by PCR amplification and qualification. All assay validation

data (detection limits, intra-assay precision data, inter-assay precision

data, etc.) are available on the manufacturer’s website (www.olink.

com). The protein levels were presented in the form of normalized

protein expression (NPX). Proteins that were below the detection limit

in over 80% of the samples were excluded from further analysis. The

full plasma proteomics dataset is available in the Source Data file.

An attempt to quantify the peripheral immune proteomics globally was made to indicate the “hot” or “cold” extent of the peripheral

macroenvironment (PME). As no scoring system is available for proteomics evaluated by the Olink platform, insights from gene studies

were used to devise a scoring method in this study. We defined the

regression coefficient (β) for each protein, which was obtained from

the coef parameter in the DESeq2 function. A per-sample weighted

plasma immune proteins score was calculated by multiplying the

expression value of each protein with its corresponding β coefficient,

followed by z-score normalization46. The formula was as follows:

protein score = Xn

i=1

βi
proteini ð1Þ

where i ranges from 1 to number of proteins assessed and β corresponds to the beta coefficient of the respective protein obtained from

the coef parameter in the DESeq2 function. A 0.33 quantile cutoff was

used to divide patients into high or low expression groups based on

their corresponding weighted protein score.

Statistical analysis

According to Simon’s minimax two-stage design with a power of

0.9 and one-sided α of 0.05 and assuming that the poor ORR

(ineffectiveness, P0) was 10% compared with the good ORR (effectiveness, P1) of 30%, the sample size required for the first stage was 22.

If the number of patients achieving objective response was >2, the

study proceeded to the second stage. In stage 2, 11 more patients

were planned to be enrolled. A total of 33 patients was included, and

the primary endpoint was considered met if >6 patients achieved an

objective response. Considering a possible 5% dropout rate of patients,

a total of 35 patients were enrolled in the study.

Patients who received at least 1 cycle of treatment were included

in the primary efficacy and safety analysis, which was defined as a

modified intention-to-treat (ITT) set. Data are reported as of December

12, 2022. Descriptive statistics were used for categorical variables. PFS

and OS were estimated using the Kaplan-Meier method. The median

follow-up time was analyzed using the reverse Kaplan-Meier method.

The logrank test was performed to compare the differences across

groups. Hazard ratios (HRs) and corresponding 95% confidence

interval (CI) values were estimated using the Cox proportional hazards

regression model. The Fisher’s exact test or χ2 test (when appropriate)

was used for comparing the proportions across groups. The MannWhitney U (Wilcoxon rank sum) test or t-test (when appropriate) was

used to compare the means (and medians) between the 2 groups.

Statistical analyses were performed using SAS version 9.4 and GraphPad Prism (version 6.00) software. Further analyses were performed in

R (version 3.6.3) using the packages ComplexHeatmap (version 2.8.0),

OlinkAnalyze (version 3.4.1), DESeq2 (version 1.40.1), dplyr (version

1.1.2), tidyverse (version 2.0.0), survival (version 3.5), ggplot2 (version

3.4.2), survminer (version 0.4.9), stats (version 4.3.0), ggalluvial (version 0.12.3), corrplot (version 0.92), ggcorrplot (version 0.1.4), ggpubr

(version 0.6.0), and timeROC (version 0.4).

Reporting summary

Further information on research design is available in the Nature

Portfolio Reporting Summary linked to this article.

Data availability

The raw ctDNA sequencing data can be accessed through the Genome

Sequence Archive (GSA) under the accession code HRA005098.

Sequencing data are available under restricted access due to patient

privacy reasons. Access can be obtained by completing the application

form via GSA-Human System or by contacting the corresponding

authors. All requests for further data sharing will be reviewed by the

Ethics Review Committee of Zhongshan Hospital affiliated to Fudan

University, Shanghai, China to verify whether the request is subject to

any intellectual property or confidentiality obligations. Requests for

access to de-identified individual-level data from this trial can be

submitted via email to T.L. (liu.tianshu@zs-hospital.sh.cn) with

detailed proposals for approval and will be responded to within

30 days. The study protocol is available in the Supplementary Information file. The remaining data are available within the Article, Supplementary Information or Source Data file. Source data are provided

with this paper.

Code availability

Custom code for data processing and analysis is available at https://

doi.org/10.5281/zenodo.1279804247.

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Acknowledgements

We thank the patients, their families, and caregivers for participating in

the study, BeiGene Ltd. for free tislelizumab support, and Genecast

Biotechnology Co., Ltd. for NGS and Olink analysis. They did not participate in study design, clinical data collection and analysis or manuscript writing. The work was supported by the National Nature Science

Foundation of China [82373402 (T.L.), 82172925 (T.L.), 82272775 (L.A.)],

Clinical Research Foundation of Zhongshan Hospital Affiliated to Fudan

University (2020ZSLC02, T.L.), Funding from Science and Technology

Commission of Shanghai Municipality (19DZ1910102, T.L.), Shanghai

ShenKang Research Physicians Innovation and Transformation Ability

Training Project (SHDC2022CRT001, T.L.), Shanghai Rising-Star Program

(21QA1401600, L.A.) and Outstanding Youth of Zhongshan Hospital

Affiliated to Fudan University (2021ZSYQ01, L.A.).

Author contributions

X.X., L.A., K.H., L.L., and M.L. contributed equally to this work. X.X., L.A.,

L.L., M.L., Y.Yu., and T.L. conceived and designed the study. X.X., L.L.,

Y.W., Y.C., W.L., Q.Li., S.Y., Y.F., Q.Liu., Y.Yu., and T.L. provided study

material or treated patients. All authors collected and assembled the

data. X.X., L.A., K.H., M.L., Y.Yang., J.Z., and F.X. performed all the

bioinformatic analyses and developed the tables and figures. X.X., L.A.,

and K.H. conducted the literature search and wrote the manuscript. All

authors were involved in the critical review of the manuscript and

approved the final version.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains

supplementary material available at

https://doi.org/10.1038/s41467-024-51536-x.

Correspondence and requests for materials should be addressed to

Yiyi Yu or Tianshu Liu.

Peer review information Nature Communications thanks Matthew Reilley, Vivien Yin and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Nature Communications | (2024) 15:7255 13