Purpose Radiofrequency ablation (RFA) has been shown to elicit tumor-specific T cell immune responses but is not sufficient to prevent cancer progression. Here we investigated immune suppressive mechanisms limiting the efficacy of RFA. Experimental design We performed a retrospective case-controlled study on patients with synchronous colorectal cancer liver metastases who had received primary tumor resection with or without pre-operative RFA for liver metastases. Tumor infiltrating T cells and tumoral PD-L1 expression in human colorectal cancer tissues were analyzed by immunohistochemistry. T cell immune responses and PD-1/PD-L1 expression were also characterized in a RFA mouse model. In addition, the combined effect of RAF and PD-1 blockade was evaluated in the mouse RFA model. Results We found that RFA treatment of liver metastases increased not only T cell infiltration but also PD-L1 expression in primary human colorectal tumors. Using mouse tumor models, we demonstrated that RFA treatment of one tumor initially enhanced a strong T cell-mediated immune response in tumor. Nevertheless, tumor quickly overcame the immune responses by inhibiting the function of CD8+ and CD4+T cells, driving a shift to higher Treg to Teff ratio, and up-regulating of PD-L1/PD-1 expression. Furthermore, we established that the combined therapy of RFA and anti-PD-1 antibodies significantly enhanced T cell immune responses, resulting in stronger antitumor immunity and prolonged survival. Conclusions The PD-L1/PD-1 axis plays a critical role in dampening RFA-induced antitumor immune responses. And this study provides a strong rationale for combining RFA and the PD-L1/PD-1 blockade in the clinical setting.

ABSTRACT. Patterns of DNA methylation are established and maintained by a family of DNA methyltransferases (DNMTs). Aberrant promoter DNA methylation of tumor suppressor genes is found in breast cancer. Association studies between DNMT gene polymorphisms and breast cancer in various populations have reported inconsistent results. This study assessed the associations of single nucleotide polymorphisms (SNPs) in DNMT1, DNMT3A, DNMT3B, DNMT3L, and DNMT2 with breast cancer among Han Chinese women from South China. Sixteen SNPs (rs2114724, rs2228611, rs2228612, rs8101866, and rs16999593 in DNMT1; rs13420827, rs11887120, rs13428812, rs1550117, rs11695471, and rs6733301 in DNMT3A; rs2424908, rs2424913, and rs6087990 in DNMT3B; rs113593938 in DNMT3L, and rs11254413 in DNMT2) in 408 women with breast cancer and 469 controls were genotyped using a MassARRAY matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry platform. Two SNPs, Polymorphisms of DNMT1 and DNMT3B and breast cancer risk rs16999593 in DNMT1 and rs2424908 in DNMT3B, were significantly associated with breast cancer risk. The heterozygous genotype CT of rs16999593 was associated with increased breast cancer risk [odds ratio (OR) = 1.60; 95% confidence interval (95%CI) = 1.20-2.14; P = 0.0052], whereas rs2424908 was associated with decreased risk (OR = 0.62; 95%CI = 0.46-0.84; P = 0.0061). Other DNMT polymorphisms showed no significant associations with breast cancer risk in the study population. Haplotype CGTC of rs2114724, rs2228611, rs8101866, and rs16999593 in DNMT1 differed significantly as a risk factor between the case and control groups (OR = 1.51; 95%CI = 1.18-1.93; P = 0.0012). The heterozygous genotypes of rs16999593 in DNMT1 and rs2424908 in DNMT3B were strongly associated with breast cancer risk.

<div>Abstract<p><b>Purpose:</b> Radiofrequency ablation (RFA) has been shown to elicit tumor-specific T-cell immune responses, but is not sufficient to prevent cancer progression. Here, we investigated immune-suppressive mechanisms limiting the efficacy of RFA.</p><p><b>Experimental Design:</b> We performed a retrospective case-controlled study on patients with synchronous colorectal cancer liver metastases who had received primary tumor resection with or without preoperative RFA for liver metastases. Tumor-infiltrating T cells and tumoral PD-L1 expression in human colorectal cancer tissues were analyzed by immunohistochemistry. T-cell immune responses and PD-1/PD-L1 expression were also characterized in an RFA mouse model. In addition, the combined effect of RAF and PD-1 blockade was evaluated in the mouse RFA model.</p><p><b>Results:</b> We found that RFA treatment of liver metastases increased not only T-cell infiltration, but also PD-L1 expression in primary human colorectal tumors. Using mouse tumor models, we demonstrated that RFA treatment of one tumor initially enhanced a strong T-cell–mediated immune response in tumor. Nevertheless, tumor quickly overcame the immune responses by inhibiting the function of CD8⁺ and CD4⁺ T cells, driving a shift to higher regulatory T-cell to Teff ratio, and upregulating PD-L1/PD-1 expression. Furthermore, we established that the combined therapy of RFA and anti–PD-1 antibodies significantly enhanced T-cell immune responses, resulting in stronger antitumor immunity and prolonged survival.</p><p><b>Conclusions:</b> The PD-L1–PD-1 axis plays a critical role in dampening RFA-induced antitumor immune responses, and this study provides a strong rationale for combining RFA and the PD-L1/PD-1 blockade in the clinical setting. <i>Clin Cancer Res; 22(5); 1173–84. ©2016 AACR</i>.</p></div>

<p>Supplemental figure 1. Patient cohort and study design. Supplemental Figure 2. Immunochemical staining of PD-L1 antibody (1:500, clone: SP142) on the paraffin-embedded human melanoma and placenta tissues. Supplemental Figure 3. RFA of colorectal liver metastases increased PD-L1 expression on infiltrating immune cells. Supplemental Figure 4. Correlation between PD-L1 expression on tumor cells and the intensity of CD8+, CD4+ T cell infiltration in endoscopic biopsy (A, B) and resected tumor specimen (C, D) (all of 78 patients were included). Supplemental Figure 5. RFA increased CD45+ immune cells and T cells infiltrating into contralateral tumor. Supplemental Figure 6. RFA increased T cells infiltration and PD-1/PD-L1 expression in a disdant tumor in B16 tumor-bearing mice. Supplemental Figure 7. RFA did not upregulate PD-1/PD-L1 expression in splenic lymphocytes. Supplemental Figure 8. RFA increased Tim-3, CD160, CD244 and eomes expression in a disdant tumor in CT26 tumor-bearing mice. Supplemental Figure 9. RFA and the anti-PD-1 therapy increased CD8+TIL cell infiltration in distant tumor. Supplemental Figure 10. RFA and the anti-PD-1 therapy upregulated Tim-3 expression on T cells in a distant tumor. Supplemental Table 1 Primer sequences used for the detection of gene expression by RT-QPCR Supplemental Table 2 Patient characteristic Supplemental table 3. Multivariate analysis of factors related to increased PD-L1 expression in resected tumor specimen.</p>