Background
In the past decade, the use of immune checkpoint inhibitors (ICIs) has revolutionized the treatment of a variety of tumors, including lung cancer, melanoma, colorectal cancer, and renal cell carcinoma [
1,
2]. However, only some tumors, such as Hodgkin's lymphoma and tumors with high microsatellite instability (dMMR or MSI-H), respond well to ICIs with an objective response rate (ORR) of 53–87%; the ORR of most tumors, such as non-small cell lung cancer, head and neck tumors, gastroesophageal tumors, bladder urothelial tumors, renal cell carcinoma, and hepatocellular carcinoma, is much lower (15–25%) [
3,
4]. Meta analysis results showed that the incidence of grade 3–4 immunotoxicity was approximately 10–24% with programmed cell death protein 1( PD-1) or cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) monotherapy [
5,
6], and reached 59% with combined immunotherapy [
7]. Therefore, identifying prognostic biomarkers is urgently needed to optimize patient benefits, minimize toxicity risk, and guide clinical approaches. Among all prognostic biomarkers, the first FDA approved biomarker was PD-L1; however, PD-L1 shows low negative predictive value, dynamic changes in expression, and lack of a confirmed threshold between different detection method [
8]. Another FDA approved and promising biomarker was MSI-H detected by polymerase chain reaction or dMMR detected by immunohistochemistry, and patients with this biomarker showed an ORR rate of 53% to immunotherapy [
3]. Regretfully, MSI-H mainly occurred in endometrial cancer, colorectal cancer, and gastric cancer with rates of just 31.37%, 19.72%, and 19.09%, respectively [
9]. Subsequently, the FDA approved pembrolizumab for adults and children with TMB-H solid tumors; however this is applicable to only 13% of solid tumors and the ORR was limited to about 29% [
10]. Given these limitations, the aforementioned biomarkers do not guarantee therapeutic benefits. An increasing number of studies have shown that patients with somatic alterations to genes involved in DNA damage repair (DDR), such as BRCA1 DNA repair associated (
BRCA1), BRCA2 DNA repair associated (
BRCA2); mismatch repair (MMR) genes including
MSH2 (mutS homolog 2),
MSH6 (mutS homolog 6),
MLH1 (mutL homolog 1), and
PMS2 (PMS1 homolog 2); DNA polymerase epsilon, catalytic subunit (
POLE) and the DNA polymerase delta 1, catalytic subunit (
POLD1) are more likely to elicit a durable anti-tumor immune response from ICI treatment, demonstrating an important role or DDR-related genes as prognostic biomarkers [
4,
11‐
14].
The SET domain containing 2 histone lysine methyltransferase (
SETD2) gene encodes the functional domain of an enzyme that trimethylates histone H3 at lysine 36 (H3K36me3), which mediates MMR in a way that removes lesions associated with a persistently open chromatin structure in early replication, and preferentially safeguards active transcripts during replication by recruiting hMutSa which can quickly identify the mismatch to initiate the MMR reaction [
15,
16].
SETD2 also favors homologous recombination repair (HRR) via activation of ataxia-telangiesctasia mutated (
ATM) and formation of
RAD51 presynaptic filaments upon DNA double-strand breaks [
17]. Loss of
SETD2 function may afford an alternative mechanism for p53-mediated checkpoint inactivation and a strong reduction in DDR, resulting in alterations in DNA regulation, increased spontaneous mutations, and chromosomal instability [
18]. Deleterious mutations in
SETD2 have been implicated in a wide range of solid tumors, including renal cancer, lung cancer, melanoma, gastrointestinal cancer, and endometrial cancer [
19,
20]. Given the preclinical evidence, whether
SETD2 dysfunction was a complementary or independent factor to MSH-H and TMB to predict the prognostic of ICIs treatment needed to be figure out and whether this kind of dysfunction acted differently in different cancer types warrants further study. So this study was conducted. We inferred that patients with
SETD2 deficiency may benefit from immunotherapy through higher-TMB, more unstable microsatellites or other mechanism and we test this hypothesis in Chinese population from our corroborative GenePlus institute and the American population from TCGA [
21] and MSKCC [
22,
23] cohort.
Discussion
This study aimed to determine whether SETD2 dysfunction is a complementary or independent factor of MSH-H and TMB, and whether this type of dysfunction acts differently in various types of cancer.
In this study, we investigated the mutation distribution in detail and found that mutations were scattered throughout the SETD2 gene without accumulating in any specific area. To better understand the impact of SETD2 dysfunction, analysis of cases with deleterious SETD2 mutations were applied in this study.
As a DDR-related gene,
SETD2 ensures precise DNA replication. Accumulating evidence had shown that mutations in DDR-related genes, such as
MSH2, MSH6, PMS2, MLH1, BRCA1/2, and
POLE/D1, which function in MMR, HRR, and BER, are the main reasons for higher TMB and an immunogenic response [
43‐
46]. In our study, we found a parallel tendency of a TMB increase between
SETD2 deleterious mutations and DDR gene mutations including
BRCA1/2, MMR genes, and
POLE/D1 across seven cancer types
. Our multi-cox regression analysis showed that
SETD2 deleterious mutations (distinct from
BRCA1/2, MMR genes, and
POLE/D1) were an independent factor influencing OS with adjustment for TMB, and an even lower death hazard ratio following ICI treatment was found in the
SETD2 deleterious mutation group compared with TMB-H patients. The deletion of
SETD2 can result in accumulation of DNA damage and impaired cellular tolerance towards replication stress through either loss of
SETD2 function or dysregulation of recruitment and activation of early DDR factors like
ATM,
p53, and
RAD51, explaining the correlation between
SETD2 mutation and TMB elevation [
18].
FDA approved MSI-H detected by polymerase chain reaction or dMMR detected by immunohistochemistry with any deficiency of MSH2, MSH6, PMS2 and MLH1 as biomarker for immunocheckpoint treatments. Preclinical data showed that in Hela cells, H3K36me3 converted by
SETD2 from H3K36me2 recruits hMutSa onto chromatin via its interaction with the hMSH6 PWWP domain before DNA replication initiates. During DNA replication, H3K36me3-PWWP interaction was disrupted and released hMutSa which can quickly identify the mismatch to initiate the MMR reaction [
16]. We found that
MSH2,
MSH6,
MLH1,
PMS2 and
SETD2 independently influencing MSI-H in endometrial carcinoma, colorectal adenocarcinoma, and stomach adenocarcinoma patients. This finding was a confirmation of preclinical data and identify a distinct role of
SETD2 between endometrial carcinoma, colorectal adenocarcinoma, stomach adenocarcinoma and bladder urinary cancer, renal carcinoma, NSCLC, melanoma.
HRD is an index for measuring functional defects in homologous recombination DNA repair, deriving from germline or somatic mutations in BRCA1/2 function or other mechanisms [
47]. Preclinical data showed that
SETD2 is required for HRR, however we observed unimpaired HRR in certain types of cancers in
SETD2 deleterious mutant samples. For synthetical lethal was found in cells with deficient of both BER and HRR deficient patients we postulate that tumors cells with deficient in both MMR and HRR will undergo cell death but further scientific evidence is needed to confirm this conjecture [
48]. The remaining tumor cells that harbor mutations in one repair pathway would, consequently, have a lower HRD score. Meanwhile, the dysfunction of HRR is considered to be a biomarker for PARPi and platinum-based chemotherapy. Therefore, these results indicate that ICI treatment rather than chemotherapy or target therapy may be more suitable for endometrial carcinoma, colorectal adenocarcinoma, and stomach adenocarcinoma patients with
SETD2 mutations.
Extrinsic and intrinsic immune escape is crucial in tumorigenesis and cancer progression. Four reasons account for extrinsic immune escape: lack of immune cells, presence of immunoinhibitory cells, such as type 2 macrophages and regulatory T-cells (T
reg); high concentrations of immunoinhibitory cytokines, such as interleukin 10 (IL10) and transforming growth factor β (TGF-β); and fibrosis [
49]. Also, it is known that at least two aspects, including tumor immunogenicity and expression of immune checkpoint molecules, are responsible for intrinsic immune escape [
50]. When analyzing pooled data from patients with different cancer types, our results showed higher levels of infiltrating of CD8 cells, lower levels of infiltrating of M2 macrophages, higher TMB, and higher expression of immune checkpoint molecules in the
SETD2 deleterious mutation group. This may be the underlying mechanism for a favorable ICI response in this group. When comparing the differences across cancer types, our results showed that a more inflamed tumor microenvironment was present in the
SETD2 deleterious mutation group in colorectal carcinoma, endometrial carcinoma, and renal cell carcinoma. The data above illustrate the underlying mechanisms for the favored ICI response in the
SETD2 deleterious mutation group.
By analyzing our 13 patients with ICI treatment, we found a better ORR in these patients than in non-selective cancer patients treated with immunotherapy. Furthermore, we verified that SETD2 deleterious mutations may have the potential to serve as a biomarker for ICI therapy. Although the survival advantage has been shown in the MSKCC-IO cohort, there are limitations to using local cohorts for survival analysis like the insufficient sample size, lack of control group and the combined treatment which can’t rule out the possibility that the survival benefit of SETD2 mutation may result from chemotherapy or tyrosine kinase inhibitors; as such, a large randomized prospective study will be needed to further test our hypothesis. Additionally, the specific pathway through which SETD2 affects prognosis is not made clear in our study; therefore, in vivo and in vitro experiments are needed.
This study is of great importance. First, with respect to response, MSH-H/dMMR performed far better than other biomarkers, with ORR reaching 53% compared with just 29% in TMB-H patients. Based on our finding that SETD2 influences MSI-H with the adjustment of MMR related proteins, we recommend that SETD2 should be considered when testing MMR function by IHC (in addition to the classical MSH2, MSH6, MLH1, and PMS2 screening). This will screen for more groups that may benefit from ICI treatment. Second, TMB has been approved by the FDA as a tissue agnostic biomarker for ICI treatment. We found that SETD2 influences survival outcome independently of TMB following ICI treatment in pancancer patients, and also has a much lower death hazard compared to TMB. Third, although preclinical data has shown that SETD2 deficient cells have impaired MMR and HRR, we identified unimpaired HRR in endometrial carcinoma, colorectal adenocarcinoma, and stomach adenocarcinoma; this may be explained by possible synthetic lethality between impaired HRR and impaired MMR. Fourth, a SETD2 mutation rate greater than 5% was found in 10 cancer types, indicating that a large population of patients may benefit from ICI treatment. Taken together, our data demonstrate that SETD2 should be given more attention as a candidate biomarker for ICI treatment.
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