Association of RAS mutations with early progression after rst-line systemic therapy in patients with initially unresectable colorectal cancer liver metastasis

Purpose Patients with initially unresectable colorectal cancer with liver metastases (IU-CRLM) need to undergo rst-line systemic therapy with the aid of chemotherapy. However, the driver gene attributed to early progression in IU-CRLM patients after rst-line systemic therapy remains unclear. Our study explored the RAS mutation status related to early progression in IU-CRLM patients. Methods A total of 193 IU-CRLM patients with RAS status detection were retrospectively enrolled from December to 2020. We dened early progression as tumour progression within 6 months after rst-line systemic therapy. Univariate and multivariate logistic regression for early progression were implemented to identify the risk factors.

nearly 15% higher chance of long-term survival for initially unresectable CRLM (IU-CRLM) patients than for those without complete resection [7] . At present, the conversion rate in IU-CRLM varies between 22% and 57% [8] . Nevertheless, only 28% of IU-CRLM patients who receive R0 resection can achieve long-term survival (over 5 years) [9] . One of the most important factors of poor survival after treatment is disease progression, which occurs in approximately 50% of all patients, among whom approximately 30% develop early disease progression. Patients with early progression have extremely poor survival [10,11] . Therefore, it is necessary to identify risk factors for early progression that can be used to guide clinical decisions.
There are various risk clinicopathological factors that have been associated with poor prognosis, such as poorly differentiated primary tumours, metastatic tumour size over 5 cm, positive resection margins, and high preoperative carcinoembryonic antigen (CEA) levels [12][13][14][15] . Distant metastasis is mainly caused by a variety of gene mutations, and the epidermal growth factor receptor (EGFR) signalling pathway activated by KRAS mutation has been found to be crucial for CRC progression and metastasis [16][17][18][19] . There have been several studies reporting RAS mutation as an independent predictor of poor prognosis in patients with CRLM undergoing liver resection. [16,[20][21][22] Our previous study showed that patients with KRAS mutations had worse progression-free survival (PFS) than those with wild-type KRAS (4.9 vs. 13.1 months, P=0.017). [10] However, whether there is a correlation between RAS mutation and early progression after rst-line systemic therapy in IU-CRLM patients remains unclear. Elucidating the relationship between RAS mutation and early progression in CRLM patients can help to establish and optimize therapeutic strategies.
To address this issue, we performed this retrospective study. First, we describe the RAS mutation spectrum of IU-CRLM patients. Second, we focus on identifying the relationships among RAS mutation, conversion therapy outcomes and early disease progression.

Source of data
A total of 193 patients diagnosed with CRLM metastases who underwent rst-line treatment were retrospectively sourced from the Sun Yat-sen University Center between December 2012 and January 2020. All patients in both cohorts satis ed the following inclusion criteria: (1) histologically con rmed colorectal adenocarcinoma, (2) metastases limited to the liver, (3) initially unresectable liver metastases, (4) no previous liver resection or interventional therapy, (5) available information on RAS status, and (6) an explicit conversion outcome. Informed consent for the use of the imaging and clinical data was obtained from the patients before rst-line treatment. All procedures were performed based on the Declaration of Helsinki. The study was approved by the Institutional Research Ethics Committee of Sun Yat-sen University Cancer Center (approval number: B2020-309-01).

Clinical parameter assessment
The TNM stages of primary tumours were classi ed according to the American Joint Committee on Cancer (AJCC) eight edition criteria. The pre-treatment characteristics of liver metastases were assessed by enhanced abdominal computed tomography (CT) and nuclear magnetic resonance imaging (MRI), including tumour numbers, the size of the largest tumour, and distribution. The CEA and cancer antigen (CA) 19-9 levels were measured before rst-line treatment was administered.
RAS mutation pro ling RAS mutations were assessed in DNA from resected primary tumour specimens of CRLM. A routine polymerase chain reaction-based primer extension assay was performed to screen for mutations in KRAS codons 12, 13 61 and 146 and NRAS codons 12, 13, and 61 in all patients. Mutations in the various codons of KRAS and NRAS were reported and analysed together as RAS mutations.

Treatment outcomes
The treatment strategy and operability of the liver metastases of each patient were determined based on the nal agreement of the multidisciplinary team (MDT), including staff from the Department of Colorectal Surgery, Hepatobiliary Surgery, Medical Oncology, Medical Imaging, and Invasive Technology.
Tumour response or progression after rst-line treatment was determined according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 [23] . Successful conversion was de ned as liver metastases deemed to be resectable after rst-line systemic treatment, and patients achieving no evidence of disease (NED) status contributed by local treatment, including surgery and radiofrequency ablation (RFA), while conversion failure was de ned as liver metastases remaining unresectable after rst-line systemic treatment and patients failing to receive curative local treatment. According to previous data, early progression was de ned as disease recurrence or death within 6 months after rst-line systemic treatment [10,[24][25][26][27] .

Statistical analysis
The statistical analyses were performed using IBM SPSS Statistics 26 software (IBM, NY, USA) and GraphPad Prism version 6.01 (GraphPad Software, Inc., USA). Variables with a value of p < 0.05 in univariate analysis were further assessed with a logistic regression model for multivariate analysis to identify independent factors associated with early progression, and odds ratios (ORs) and 95% con dence intervals (CIs) were subsequently calculated.

Patient characteristics and systemic therapy
The demographic and clinicopathological characteristics and treatment approaches of the 193 patients are summarized in Table 1. The median age of the patients was 54.5 years (range 26-80 years), comprising 141 (73.1%) males and 52 (26.9%) females. The median diameter of the largest liver metastasis was 6 cm (range 1-18 cm), and the median number of liver metastases was 8 (range 1-100).
In the patients achieving successful conversion outcomes, the early progression rate was higher in RASmutated patients than in those with wild-type RAS (53.3% vs. 24.4%, P=0.004, Fig. 2B). However, the early progression rate was not signi cantly different between the RAS mutated and RAS wild-type patients with failed conversion outcomes (85.7% vs. 79.7%, P=0.771, Fig. 2C). As shown in

Discussion
The occurrence and development of CRC metastases involve a number of signalling pathways, such as transforming growth factor β (TGF-β), Wnt, Notch, Hedgehog, phosphatidylinositol 3-kinase and protein kinase B (PI3K/Akt), nuclear factor kappa light chain enhancer of activated B cells (NF-κB) and microtubule associated protein kinase (MAPK) [28] . KRAS mutation is the most common mutated driver gene in CRC, with an incidence of approximately 40% [29,30] . Our study showed the incidence of total RAS mutations in 26.0% of IU-CRLM patients. There was a signi cant association between RAS mutation and early progression of CRLM after rst-line systemic therapy. The results indicated that RAS mutation IU-CRLM represented more malignant biological behaviour than RAS wild-type IU-CRLM.
The epidermal growth factor receptor (EGFR) signalling pathway, which is activated by mutated KRAS, plays a crucial role in CRLM progression. In the EGFR pathway, the RAS protein acts as a gating switch, and the activation of RAS and MEK, downstream molecules of the RAS protein, depends on RAS mutation. Once RAS is mutated, it will constantly activate downstream pathways involving MAPK and PI3K-AKT as well as the downstream transcription factors of these pathways. Ultimately, mutated RAS regulates the expression level of proteins related to tumour invasion and metastasis. [31,32] Animal experiments also con rmed that CRC with mutated KRAS could promote the occurrence of lung metastasis with cells from liver metastases by activating the MAPK pathway. [33] These results suggest that KRAS mutation enhances malignant invasion and metastasis in CRC cells, which accelerates the progression of CRLM. In addition, glycolysis metabolism in cancer cells is elevated by mutated KRAS. [34] Previous studies reported that KRAS mutation was able to upregulate the expression levels of GLUT1, HK and LDH, which increased the process of tumour glucose uptake. Recently, research from Yun et al.
revealed an increase in the expression level of GLUT1 and higher glucose uptake in CRC cell lines with mutated KRAS [35,36] . Such alterations in metabolism confer CRC cells with a survival advantage, as they can gain long-term survival advantages in low-glucose environments, thereby creating favourable conditions for tumour metastasis.
Interestingly, our results showed that RAS mutation was a risk factor for early progression only in patients with successful conversion therapy outcomes. In fact, in recent years, a growing number of studies have indicated that RAS mutation is a negative prognostic factor for patients with CRLM after surgery. A phase II clinical study from the Medical University of Vienna enrolled 60 patients with initially resectable CRLM, all of whom had been administered oxaliplatin combined with bevacizumab before hepatectomy. The results demonstrated that in patients with KRAS mutations, the overall survival (OS) and relapse-free survival (RFS) were shorter. [37] Another study from the M.D. Anderson Cancer Center enrolled a total of 524 CRLM patients who underwent both curative liver section and RAS mutant detection. The results suggested that the median overall survival was remarkably shorter in patients with KRAS codons 12 and 13 mutations than in wild-type patients. [20] A study conducted by Brudvik KW et al indicated that although the size of tumours was similar, liver metastases with mutational KRAS in ltrated more widely than wild-type metastases, increasing the rate of margin positivity, which may be the leading cause of poor prognosis. [31] However, in patients experiencing conversion therapy failure, tumours may develop acquired resistance during treatment. At present, one of the mechanisms of acquired resistance is secondary mutations in driver genes. In metastatic CRC, gene mutations in RAS/RAF pathways are the most common molecular mechanism causing acquired resistance. [ with bevacizumab can maximize the e cacy of conversion therapy [39] . Additionally, surgeons should ensure adequate liver resection to reduce the rates of positive surgical margins in these patients. Meanwhile, a su cient number of preoperative chemotherapy cycles and more frequent postoperative follow-up visits may effectively prevent early disease progression.
There are some limitations to this retrospective study. First, the number of included patients may be inadequate, and selection bias may exist. Hence, larger numbers of patients are required for external validation. Second, several biomarkers were identi ed as crucial prognostic factors for survival and recurrence, including BRAF, PI3K and TP53, which are still under debate [40][41][42] . In this study, we included only RAS in the analysis; future studies should take more gene status into consideration to gain a better understanding of the in uence of these biomarkers on early disease progression. The 5-year survival data were unavailable for some patients due to an insu cient follow-up duration. This issue may have led to the underestimation or overestimation of the effect of RAS mutation on conversion therapy outcomes.

Conclusions
Our study revealed that RAS mutation was a risk factor for early disease progression in patients with IU-CRLM after rst-line systemic therapy. More attention should be given to treatment decisions for IU-CRLM