These authors have contributed equally to this work
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Gastric adenocarcinoma (GAC) is a common malignant tumor, and patients with advanced GAC have a poor prognosis. At present, clinical trials of anti-PD-1 (programmed cell death protein-1)/PD-L1(Programmed Cell Death-Ligand 1) therapy for GAC have achieved good results. Microsatellite instability-high (MSI-H), high tumor mutational burden (TMB) or PD-L1 expression are the main indicators to predict its efficacy. However, the response to treatment remains highly variable, and some patients remain unresponsive to immunotherapy (
Whole-exome sequencing revealed that genes encoding subunits of the SWItch/Sucrose Nonfermentable (SWI/SNF) complex are mutated in more than 20% of cancers, involving multiple cancer types (
This study sought to explore the relationship between PBRM1 gene abnormalities and the occurrence and development of GAC and tumor immune activity and to provide evidence for its potential use as a predictive marker for GAC immunotherapy. In this paper, cBioPortal, LinkedOmics and TISIDB datasets were used to analyze the abnormality of the PBRM1 gene in GAC and its relationship with prognosis, related molecular changes and tumor-infiltrating lymphocytes (TIL). GAC cases were collected to further verify and study the effect of PBRM1 loss/attenuation on clinicopathology and prognosis and analyze its relationship with microsatellite stability, PD-L1 expression and TILs in GAC.
Overall, 198 patients with advanced GAC who underwent radical gastrectomy from July 2015 to December 2016 in our department with follow-up information were retrospectively collected. Patient clinical information included age, sex, tumor site, histological differentiation, lymph node metastasis status, and TNM stage. This study was approved by the Ethics Committee of the Affiliated Wuxi People’s Hospital of Nanjing Medical University (No. KS202017). Overall survival (OS) was determined by telephone follow-up until April 2020. Clinical information of these cases was summarized in
The cBioPortal online platform (
The LinkedOmics (
The TISIDB database (
Using the EnVision method, antibodies included PBRM1 (polyclonal antibody, A301-591A, 1:4000; Bethyl Laboratories), MLH1, MSH2, MSH6, PMS2 (clone numbers are ES05, FE11, EP49 and EP51, Dako company), and PD-L1 (mouse mAb 22C3, concentrated solution, 1:50 dilution, Dako company). Expression results were analyzed using a double-blind method. The primary antibody was replaced with PBS as a negative control.
According to the literature, normal epithelial cells, inflammatory cells and fibroblasts were used as internal positive controls; if the nuclear staining intensity was lost or significantly reduced (including the heterogeneous staining), it was classified as PBRM1 expression loss/attenuation (
Referring to the International Immuno-Oncology Biomarkers Working Group (IIOBWG) standards and related literature (
Genomic DNA from formalin-fixed paraffin-embedded (FFPE) samples was extracted using QIAamp DNA FFPE Tissue Kit (Qiagen)and fragmented by ultrasonication (Covaris). Libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) (
Paired-end sequencing data were aligned to the reference human genome hg19) using the Burrows-Wheeler Aligner (bwa-mem), and then were subjected to de-duplication, base quality recalibration and indel realignment using Picard (
A chi-square test was used to analyze the relationship between PBRM1 deletion/mutation (or loss/attenuation of PBRM1 expression) and various clinicopathological and molecular parameters. Survival analysis was performed using Kaplan–Meier to draw survival curves, and the log-rank test was used to compare differences in survival time. Cox regression was used to determine independent prognostic factors. SPSS 23.0 statistical software was used, and
According to cBioPortal data, 7.32% of GACs had PBRM1 deletions/mutations (Shown in
PBRM1 deletion/mutation in GAC and its relationship with various molecular alterations in cBioPortal data (CIN, chromosomal instability; EBV, Epstein-Barr virus type; GS, genome stability; MSI, microsatellite instability; MSH-H, microsatellite instability-High; MSH-L, microsatellite instability-Low; MSS, microsatellite stability; 0 = No, 1 = Yes).
Correlation of PBRM1 deletion/mutation with various molecular alterations in 287 TCGA GC cases.
Parameters | PBRM1 Deletion/mutation | χ2 value |
|
|
---|---|---|---|---|
Yes | No | |||
Molecular Subtype | 35.993 | 0.000 | ||
EBV | 3 | 22 | ||
MSI | 15 | 48 | ||
GS | 1 | 53 | ||
CIN | 2 | 143 | ||
MSI Status | 32.656 | 0.000 | ||
MSS | 4 | 176 | ||
MSI-L | 2 | 42 | ||
MSI-H | 15 | 48 | ||
Hyper-mutated | 33.214 | 0.000 | ||
Yes | 15 | 47 | ||
No | 6 | 219 | ||
CIMP Category | 20.582 | 0.000 | ||
CIMP | 13 | 62 | ||
EBV-CIMP | 4 | 23 | ||
Other | 4 | 181 | ||
|
21.618 | 0.000 | ||
Yes | 16 | 73 | ||
No | 5 | 193 | ||
|
31.188 | 0.000 | ||
Yes | 14 | 43 | ||
No | 7 | 223 |
CIN, chromosomal instability; EBV, Epstein-Barr virus type; GS, genome stability; MSI, microsatellite instability; MSH-H, microsatellite instability-High; MSH-L, microsatellite instability-Low; MSS, microsatellite stability; CIMP, CpG island methylator phenotype.
The LinkedOmics dataset showed that PBRM1 mRNA expression was significantly lower in PBRM1 mutant cases (
The LinkedOmics dataset shows that PBRM1 mRNA expression is significantly lower in PBRM1 mutant cases (Wilcoxon Test)
The relationship between PD-L1 (CD274)
The prognosis of GAC patients with PBRM1 abnormalities. In the 354 cases of the LinkedOmics dataset, the prognosis of GAC patients with PBRM1 mutation is relatively better than that of PBRM1 wild-type patients (Cox Regression Test,
Analysis of the TISIDB database showed that PBRM1 abnormalities (including gene expression, mutation, CNA and gene methylation) were significantly associated with a variety of TILs, including activated CD4 (
The TISIDB database shows that PBRM1 mutation is significantly associated with activated CD4
The relationship between various types of PBRM1 Abnormality and TIL in GAC analyzed by the TISIDB database.
type |
Mutation |
CNA |
Gene methylation |
Gene expression |
---|---|---|---|---|
Activated CD4 T cells | 6.41e−07 | 0.13 (rho = 0.075) | 0.532 (rho = −0.032) | 0.00288 (rho = 0.146) |
Activated CD8 T cells | 7.19e−05 | 0.366 (rho = 0.045) | 0.706 (rho = −0.02) | 0.00129 (rho = −0.158) |
Activated dendritic cells | 0.0382 | 0.000749 (rho = 0.165) | 0.408 (rho = −0.043) | 0.222 (rho = −0.06) |
Monocytes | 0.0171 | 0.0182 (rho = 0.116) | 0.000329 (rho = 0.186) | 1.01e-05 (rho = −0.215) |
NK cells | 0.0745 | 0.0488 (rho = 0.099) | 0.14 (rho = 0.077) | 0.897 (rho = −0.006) |
Based on Wilcoxon Test. There is a positive correlation between TIL and PBRM1 mutation status.
Based on Spearman Correlation Test. The positive or negative of the correlation coefficient rho indicated the positive or negative of the correlation.
Of the 198 cases in our group, PBRM1 expression was lost/attenuated in 29 (14.6%) (
PBRM1, MMR, and PD-L1 expression and iTIL of GAC in our group. PBRM1
Correlation between PBRM1 expression loss/attenuation and clinicopathological features in 198 GC cases in our group.
Clinicopathological parameters | Loss/attenuation of PBRM1 expression | χ2 value |
|
|
---|---|---|---|---|
Yes | No | |||
Age | 0.866 | 0.352 | ||
≤60 | 4 | 36 | ||
>60 | 25 | 133 | ||
Sex | 0.050 | 0.824 | ||
Male | 20 | 113 | ||
Female | 9 | 56 | ||
Tumor site | 0.340 | 0.844 | ||
Upper 1/3 | 14 | 72 | ||
Middle 1/3 | 7 | 47 | ||
Lower 1/3 | 8 | 50 | ||
Tumor size (cm) | 0.809 | 0.369 | ||
≤3 | 11 | 50 | ||
>3 | 18 | 119 | ||
Lauren type | 4.961 | 0.084 | ||
Intestinal type | 23 | 101 | ||
Diffuse type | 1 | 29 | ||
Mixed type | 5 | 39 | ||
Histological differentiation | 3.507 | 0.173 | ||
Well differentiated | 2 | 3 | ||
Moderately differentiated | 16 | 82 | ||
Poorly differentiated | 11 | 84 | ||
T stage | 1.946 | 0.163 | ||
T1-2 | 11 | 43 | ||
T3-4 | 18 | 126 | ||
Lymph node metastasis | 0.540 | 0.463 | ||
No | 12 | 58 | ||
Yes | 17 | 111 | ||
TNM stage | 3.063 | 0.080 | ||
1-2 | 19 | 81 | ||
3-4 | 10 | 88 | ||
MMR protein expression | 6.084 | 0.019 | ||
Intact | 20 | 146 | ||
Loss | 9 | 23 | ||
PD-L1 expression | 4.081 | 0.043 | ||
Positive (CPS ≥ 1) | 7 | 18 | ||
Negative (CPS < 1) | 22 | 151 | ||
sTIL | 2.018 | 0.155 | ||
Less | 12 | 94 | ||
More | 17 | 75 | ||
iTIL | 5.433 | 0.020 | ||
No | 17 | 133 | ||
Yes | 12 | 36 |
As of the end of follow-up, the 3-year OS rate of 29 GAC patients with loss/attenuation of PBRM1 expression was 89.7%, while the 3-year OS rate of 169 GAC patients without PBRM1 expression loss/attenuation was 63.9%. Kaplan–Meier survival analysis showed (
In addition to loss/attenuation of PBRM1 expression, tumor size, depth of invasion, histological grade, lymph node metastasis, clinical stage, MSI, sTIL, and iTIL were statistically significant factors for survival time. The above 9 factors were included in Cox multivariate regression analysis, and the results showed that lymph node metastasis, clinical stage and iTIL were independent factors affecting the prognosis of GAC (
The variables closely related to the overall survival rate of GAC in Cox multivariate regression analysis.
Variable |
|
SE | Wald | df |
|
Exp( |
95% |
|
---|---|---|---|---|---|---|---|---|
Lower | Upper | |||||||
Lymph node metastasis | 1.317 | 0.646 | 4.161 | 1 | 0.041 | 3.732 | 1.053 | 13.229 |
Clinical stage | 1.405 | 0.430 | 10.689 | 1 | 0.001 | 4.074 | 1.755 | 9.458 |
iTIL | −0.836 | 0.400 | 4.363 | 1 | 0.037 | 0.433 | 0.198 | 0.950 |
Among the 7 GAC cases with loss of PBRM1 expression in our group, 3 cases showed PBRM1 mutations, including missense mutations and splicing mutations, and 2 cases had chromosome 3p21.1 deletions (the locus of the PBRM1 gene). Mutations in other SWI/SNF chromatin remodeling subunits are often detected simultaneously in the same tumor (
Mutation of SWI/SNF subunit genes in 7 GC cases with loss of PBRM1 expression in our group.
Case no. | Gene | HGVS | Variant classification |
---|---|---|---|
1 |
|
c.996-5dupT | Splice region |
|
c.3283C > T (p.R1095C) | Missense nutation | |
|
c.3274_3276delCCT (p.P1092del) | In frame del | |
|
c.291-6G > A | Splice region | |
|
c.3999_4001delGCA (p.Q1334del) | In frame del | |
|
c.384_386delGCA (p.Q131del) | In frame del | |
|
c.31G > C (p.G11R) | Missense mutation | |
|
c.2761A > C (p.T921P) | Missense mutation | |
2 |
|
c.996-4G > T | Splice region |
|
c.3971G > C (p.G1324A) | Missense mutation | |
|
c.2239G > A (p.A747T) | Missense mutation | |
|
c.3999_4001delGCA (p.Q1334del) | In frame del | |
|
c.285_287delCCA(p.H96del) | In frame del | |
|
c.3832G > A (p.A1278T) | Missense mutation | |
3 |
|
c.142G > T (p.A48S) | Missense mutation |
|
c.2416-7C > T | Splice region | |
|
c.4347C > A (p.F1449L) | Missense mutation | |
|
c.3274_3276delCCT (p.P1092del) | In frame del | |
|
c.3274_3276delCCT (p.P1092del) | In frame del | |
|
c.544-4C > T | Splice region | |
|
c.1489delC (p.Q497Nfs*122) | Frame shift del | |
|
c.3999_4001delGCA (p.Q1334del) | In frame del | |
|
c.1489C > G (p.Q497E) | Missense mutation | |
|
c.2594A > C (p.Q865P) | Missense mutation | |
|
c.2761A > C (p.T921P) | Missense mutation | |
4 |
|
c.3422A > C (p.N1141T) | Missense mutation |
|
c.932C > A (p.A311E) | Missense mutation | |
|
c.2081G > A (p.S694N) | Missense mutation | |
|
c.4855+1G > A (p.X1619_splice) | Splice site | |
|
c.2594A > C (p.Q865P) | Missense mutation | |
|
c.2761A > C (p.T921P) | Missense mutation | |
|
c.2777T > C (p.L926P) | Missense mutation | |
5 |
|
c.544-5dupT | Splice region |
|
c.3248C > T (p.P1083L) | Missense mutation | |
|
c.384_386dupGCA (p.Q131dup) | In frame ins | |
|
c.178C > T (p.R60*) | Nonsense mutation | |
6 |
|
c.3203G > A (p.R1068H) | Missense mutation |
|
c.3274_3276delCCT (p.P1092del) | In frame del | |
|
c.988G > A (p.E330K) | Missense mutation | |
|
c.3999_4001delGCA (p.Q1334del) | In frame del | |
|
c.5554_5555insTTGAG (p.T1852Ifs*33) | Frame shift ins | |
7 |
|
c.3115G > A (p.A1039T) | Missense mutation |
KEGG signaling pathways enriched in GACs with PBRM1 expression loss mainly included lysine degradation, antigen processing and presentation, the Notch signaling pathway and phagosome-related genes (
KEGG signaling pathways
Analysis of public databases and the data of GAC cases collected in our group showed that the PBRM1 gene was significantly abnormal in a small number of GACs (approximately 10%), including gene deletion/mutation, methylation and loss/attenuation of expression. According to the literature, PBRM1 inactivating mutation or expression loss exists in approximately 40% of clear cell renal carcinoma, 32% of cholangiocarcinoma and 83% of epithelioid sarcoma (
Interestingly, prognostic analysis showed that abnormality of the PBRM1 gene was a favorable prognostic factor, and multivariate regression analysis showed that iTILs, which were closely positively related to PBRM1 expression loss/attenuation, were an independent prognostic factor. Comprehensive public databases and the data in our group found that the abnormal SWI/SNF subunit PBRM1 was closely related to tumor immune microenvironment-related factors, such as high expression of the immunosuppressive molecules PD-L1 and CTLA4, high sTIL, and iTIL. This indicates that there may be a balance between carcinogenesis and tumor suppressor immune activity of PBRM1 abnormalities (
Studies have shown that the mRNA and protein expression levels of the immune checkpoint molecules PD-L1 and CTLA4 are also closely related to high TILs, an active immune response, and a “hot” immune microenvironment, which is beneficial for the tumor-targeted immune response or immunotherapy (
There is a significant correlation between abnormal SWI/SNF subunit and MSI-H, and both are related to PD-L1 expression, high tumor genome mutation rate, and high TIL, resulting in high tumor immune activity. There are several hypotheses regarding the causal affiliation of the two: 1) SWI/SNF complex subunit gene mutations may be caused by MSI; 2) the two may be a reflection of genome-wide hypermethylation, that is, a CpG island methylation phenotype; and 3) SWI/SNF complex deficiency leads to impaired MMR, mutation or MLH1 promoter methylation and epigenetic alterations (
In clinical practice, it is difficult to recover from the abnormal deletion of gene products, but abnormal SWI/SNF subunits often co-occur with abnormalities of other molecular signaling pathways. The use of specific synthetic lethal relationships is an effective approach to its treatment and has now become a hot field of precision therapy (
In conclusion, PBRM1 gene abnormalities may play an important carcinogenic role in some gastric cancer subgroups and may affect their tumor immune activity, thereby influencing the clinicopathological and overall prognosis of GAC. The detection of PBRM1 gene abnormalities may be an effective predictive marker in immunotherapy, and harnessing the synthetic lethal relationship associated with PBRM1 gene abnormalities may also be a potential novel therapeutic strategy in GAC.
The original contributions presented in the study are included in the article/
The studies involving human participants were reviewed and approved by the Ethics Committee of the Affiliated Wuxi People’s Hospital of Nanjing Medical University (No. KS202017). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.
Conception or design of the work: ZZ and QR. Data collection: DH, SY, JL, and XW. Data analysis and interpretation: ZZ, DH, and QR. Drafting the article: ZZ. Critical revision of the article: ZZ, DH, SY, and QR. Final approval of the version to be published: ZZ, DH, SY, JL, XW, and QR.
This work was supported by Wuxi Science and Technology Development Fund (N20192016).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: