Table 1.

Immunoepigenetic-based studies in bladder cancer.

Epigenetic Target/mechanism	Immune Therapy/Target	Major Finding	Methods	Sample Type and Size	Author
Methylation (decitabine) and acetylation (TSA)	9 CTAs	The most expressed antigens are the MAGE-A families Expression of CTAs is induced by decitabine	RT-PCR WB	Cell lines BlCa and normal urothelium tissues (n = 56)	Fradet Y, 2006 [54]
Methylation (25 tumor-suppressor genes)	Response to BCG-therapy	Methylation status of several targets predicted response to BCG-therapy and disease recurrence in T1 G3 NMIBC	MS-MLPA	91 BlCa tissues	Agundez M, 2011 [55]
PMF-1 methylation	Response to BCG-therapy	Unmethylated PMF-1 associated with failure of BCG therapy (disease recurrence in T1 high-risk NMIBC)	qMSP	108 BlCa tissues	Alvarez-Múgica M, 2013 [56]
Methylation (57 targets)	Response to BCG-therapy	Methylation status of several targets predicted response to BCG-therapy and disease recurrence in high-grade NMIBC	MS-MLPA	82 BlCa and 13 normal urothelium tissues	Husek P, 2017 [57]
DNA methylation-derived index	Neutrophil-to-lymphocyte ratio	Higher methylation index associated with disease outcome in BlCa	Bioinformatics	DNA methylation data from leukocyte subtypes	Koestler DC, 2017 [61]
Methylation (decitabine)	IL-6	Decitabine leads to NOTCH1 demethylation and expression, leading to IL-6 release	WB RT-qPCR Methylation array RNA-seq Pyrosequencing ELISA FACS-sorting	Tissues (BlCa + normal urothelium, n = 174) + in vitro (cell lines)	Ramakrishnan S, 2019 [64]
FOXP3, IFNG, IL13, IL17A (methylation)	CD4+ T cells in BlCa	↑ CD4+ T cell lineage commitment assessed by CpG methylation associates with better prognosis Hypomethylation of the four targets in CD4+ T cells associated with complete response to CT	FACS-sorting Pyrosequencing 5mC ELISA	BlCa tissues (n = 22), LNs (n = 76) and blood (n = 48)	Bergman EA, 2018 [58]
PRF1 methylation	Tissue-resident memory CD8+ T cells in BlCa	These cells are epigenetically cytotoxic and show signs of exhaustion (show ↓ methylation levels of PRF1 and ↑ PD-L1 expression)	FACS-sorting Pyrosequencing	BlCa tissues, LNs and blood (n = 53 patients)	Hartana CA, 2018 [59]
Knockout of lncRNA UCA1	Knockout of PD-1	Combined UCA1 and PD-1 knockout resulted in synergistic antitumor effect by favoring an immunostimulatory microenvironment	CRISPR-Cas9 FACS-sorting RT-PCR WB	In vitro (cell lines) + in vivo (mouse)	Zhen S, 2018 [60]
EZH2 inhibition (EPZ011989)	NK cells	BlCa cells with KDM6A and SWI/SNF mutations are frequent and show overactivation of EZH2 EZH2 inhibition in these cells resulted in ↑ immune signature (IFN-γ) and activated NK signaling, resulting in MIBC cell death	WB RNA-sequencing IHC	In vitro (cell lines) + in vivo + BlCa tissues	Ramakrishnan S, 2019 [62]
EHMT2/DNMT inhibition (CM-272, A-366, decitabine)	Anti-PD-L1	Dual targeting of EHMT2/DNMT leads to immunogenic cell death (conversion into a “hot tumor”), and this is potentiated by combining with anti-PD-L1 ↑ EHMT2 expression leads to resistance to anti-PD-L1	RT-qPCR WB/Dot blot ChIP IF/IHC FACS-sorting ELISA Whole transcriptome analyses Pyrosequencing	In vitro (cell lines) + in vivo (mouse) + BlCa tissues (n = 87 patients)	Segovia C, 2019 [63]

Abbreviations: 5mC—5-methylcytosine; BCG - Bacillus Calmette-Guérin; BlCa—bladder cancer; ChIP—chromatin immunoprecipitation; CT—chemotherapy; CTA—cancer testis antigen; ELISA -enzyme-linked immunosorbent assay; FACS—fluorescence-activated cell sorting; IF—immunofluorescence; IFN-γ—interferon gamma; IHC—immunohistochemistry; IL-6—interleukin 6; LN—lymph node; lnCRNA—long non-coding RNA; MIBC—muscle-invasive bladder cancer; MS-MLPA—methylation-sensitive multiplex ligation-dependent probe amplification; NMIBC—non muscle-invasive bladder cancer; PD-1—programmed cell death protein 1; PD-L1—Programmed death-ligand 1; qMSP—quantitative methylation-specific PCR; RT-(q)PCR—real-time quantitative polymerase chain reaction; TSA—trichostatin A; WB—Western Blot.