Expression of ARID1A protein was associated with mRNA level
All the patients were divided into ARID1A negative group (n=58, 21.3%) and ARID1A positive group (n=214, 78.7%) by IHC (Fig.1a, b). The mRNA expression of ARID1A was detected in 236 samples by qRT-PCR and relative expression level of mRNA was calculated according to the formula 2-ΔΔCt. T test analysis showed that mRNA expression level of ARID1A protein negative group was significantly lower than that of protein positive group (Fig.1c).
Different protein expression levels of ARID1A in the five GC molecular subtypes
Among the 272 GC patients, 3 cases (1.1%) were EBV positive, 9 cases (3.3%) were MSI-H subtype, 33 cases (12.1%) were E-cadherin aberrant, 164 cases (60.3%) were p53 aberrant and 63 cases (23.2%) were p53 normal (Figure. 2a). The probability of ARID1A protein negative in MSI-H subtype (5/9, 55.6%) was significantly higher than that in p53 aberrant subtype (33/164, 20.1%, p=0.037) and p53 normal subtype (12/63, 19.0 %, p=0.046, Figure. 2b). Among the 3 patients with EBV positive GC, 1 patient was negative for ARID1A protein. There was no difference in the proportion of negative ARID1A protein between EBV positive GC and other subtypes due to the small sample size.
ARID1A expression pattern and clinicopathological characteristics of patients
Clinicopathological characteristics of 272 GC patients were summarized in the Table 1. Until now, no study has yet clearly established the grading standard for the relative expression level of ARID1A mRNA extracted from FFPE, and the loss of expression caused by ARID1A mutation has special value for research. Considering that the mutation rate of ARID1A in GC is 18.7%-23%, in this study, we arranged the relative expression level of ARID1A mRNA in ascending order, and the first 20% cases were defined as low expression of ARID1A mRNA (n = 47, 20%), and the remaining 80% cases were defined as high expression of ARID1A mRNA (n = 193, 80%). The protein or mRNA expression of ARID1A was not associated with gender, age, Body Mass Index (BMI), stage, depth of invasion, lymph node metastasis, distant metastasis, perineural invasion or vascular invasion. However, loss expression of ARID1A protein was significantly correlated with MSI-H subtype, PD-L1 CPS≥1 and CPS≥10 (p=0.033, p=0.005 and p=0.012, respectively). In addition, among 47 patients with low expression of ARID1A mRNA, there were 20 (42.6%) patients with PD-L1 CPS≥10 and only 29.6% (56/189) patients with high expression of ARID1A mRNA were PD-L1 CPS≥10, although the difference was not statistically significant. The low expression of ARID1A mRNA was correlated with abnormal E-cadherin (p =0.003). There was no correlation between the expression of p53, AFP or VEFGR2 and the expression of ARID1A protein or mRNA.
Relationship between ARID1A expression pattern and systemic inflammatory markers of patients
Both SIR and ARID1A were reported to have predictive value in the prognosis of immunotherapy(Formica et al. 2020; Okamura et al. 2020), so we investigated whether there is any association between ARID1A expression and systemic inflammatory markers. ARID1A negative protein group had a lower level of albumin (p=0.0064, Figure. 3e), while the low mRNA group had a higher level of PLR (p=0.0391, Figure. 3i). In addition, the LMR level appeared to be lower in the ARID1A negative protein group, but it was not statistically significant (p=0.0689, Figure. 3b). We also analyzed the relationship between systemic inflammatory markers and ARID1A expression by the chi-square test (Table S1), and found ARID1A protein negative was associated with high level of PLR and low level of albumin (p=0.038 and p=0.008, respectively, Table S1). Moreover, ARID1A mRNA was low in 17 (17.7%) of the 96 cases with low LMR, and in 30 (32.3%) of the 93 patients with high LMR, but the difference was not statistically significant (p=0.073, Table S1).
ARID1A expression pattern was significantly associated with overall survival
Follow-up of 172 patients was carried out up to the date of death from any cause or last contact, and the median survival time was 23 months. Both negative expression of ARID1A protein and low expression of ARID1A mRNA were significantly associated with poor prognosis (p=0.002 and p=0.013, respectively) (Figure 4a, b). Univariate and multivariate Cox proportional hazard models were performed to assess the value of ARID1A protein expression pattern and clinical variables on survival (Table 2). Univariate analysis demonstrated that negative expression of ARID1A protein (p=0.002), older age (p=0.003), higher T stage (p=0.002), and higher N stage (p=0.003) were associated with decreased OS. Multivariate Cox analysis indicated that negative expression of ARID1A protein (p=0.023), older age (p=0.004), higher T stage (p=0.009), or higher N stage (p=0.009) were independent risk factors for poor prognosis.
Development and validation of the nomogram
To predict the survival risk of patients with GC, a nomogram was established based on all independent risk factors selected by multivariate Cox analysis (Figure. 5a). The C-index of the nomogram was 0.719 (95% CI: 0.683-0.755), which was higher than 8th edition of the AJCC staging system (C-index=0.657, 95% CI: 0.621-0.692). In addition, the calibration plots of our nomogram showed high consistency between the nomogram-predicted outcomes and the observational outcomes (Figure. 5b, c).