BACKGROUND

Metformin is one of the biguanides commonly used in patients with type II Diabetes Mellitus. Apart from its hypoglycemic properties, metformin also inhibits the cell cycle by restricting protein synthesis and cell proliferation via regulating the LKB1/AMPL pathway. Furthermore, it also enhances the PD-1 blockade through a reduction of tumor hypoxia. Metformin has shown a significant favorable impact on treatment-related outcomes in solid tumors, but these outcomes have not been replicated in the limited clinical studies done on malignant melanoma. Moreover, none of these studies have reported on the efficacy of the combined use of metformin and immune checkpoint inhibitors (ICIs).

METHODS

This is a retrospective cohort study that includes patients diagnosed with metastatic malignant melanoma and treated with ipilimumab, nivolumab, and/or pembrolizumab (Cohort A); or ipilimumab, nivolumab, and/or pembrolizumab plus metformin (Cohort B) between January 1st 2011 through December 15th 2017. In this study, patients are stratified based on anti-PD-1 only and anti-CTLA4/anti-PD-1 combination therapies in each cohort. Objective response rate (ORR) is the primary endpoint. Disease control rate (DCR), overall survival (OS) and progression-free survival (PFS) are the secondary endpoints.

RESULTS

Cohort A had 33 patients (60%), while cohort B had 22 (40%). Overall patient characteristics were similar between both cohorts. ORR was higher in cohort B (68.2% vs. 54.5%, P = 0.31). The DCR was higher in cohort B as well (77.3% vs. 60.6%, P = 0.19). Median OS (46.7 months vs. 28 months), and median PFS (19.8 months vs. 5 months) were longer in cohort B. However, on univariate and multivariate analyses, none of these differences were statistically significant. The mean number of new metastatic sites which appeared during therapy were significantly higher in cohort A (A:1.51 vs. B:0.59, P = 0.009).

CONCLUSIONS

We have observed favorable treatment-related outcomes (ORR, DCR, median PFS and median OS) in patients who have received metformin in combination with ICIs without reaching significance, probably, due to small sample size. Hence, large prospective clinical trials are required to study the synergistic effect of metformin in combination with ICIs before it can be recommended as routine additive therapy.

BACKGROUND

It has been presumed that antibody-mediated selective costimulatory molecule blockade of CD28 is superior to cytotoxic T lymphocyte antigen 4 (CTLA4)-Ig. This is based on the premise that specifically blocking CD28 allows inhibitory signals through CTLA-4 to proceed, which furthermore suppresses T-cell function.

METHODS

The extracelluar domain of canine (ca)CD28 was cloned from dog peripheral blood mononuclear cells. Mice were immunized with a caCD28/murine IgG2a fusion protein. Hybridomas were produced by fusing splenocytes with mouse NSO cells and screened for caCD28 binding by ELISA. Agonistic and antagonistic activities of the monoclonal antibodies (mAb) were tested in mixed leukocyte reactions. Canine regulatory T cells were expanded using plate-bound anti-CD3 and an anti-CD28 agonist mAb.

RESULTS

One agonistic and seven antagonistic mAbs to canine (ca)CD28 were cloned. Binding studies indicated that an agonistic (5B8) and an antagonistic (1C6) mAb bound equally well to a caCD28/caIgG1 fusion protein and to CD28 expressed on CD4+ and CD8+ peripheral blood T cells. Antagonistic antibody blocked mixed lymphocyte reactions (MLR) in a dose-dependent manner similar to CTLA4-Ig, whereas the agonistic antibody to caCD28 enhanced MLR. The 5B8 was superior to 1C6 when either was combined with anti-caCD3 to stimulate lymphocyte proliferation. Furthermore, the agonistic mAb, 5B8, together with anti-CD3 mAb induced 100-fold proliferation of canine regulatory T cells. Relative to untreated control cells, anti-caCD28 (1C6) and CTLA4-Ig equivalently inhibited cytotoxic T lymphocyte-mediated killing of alloreactive target cells.

CONCLUSIONS

These studies demonstrated that mouse anti-caCD28 mAbs can be generated with agonistic or antagonistic function.

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.

The CTLA4 receptor (CD152) on activated T lymphocytes binds B7 molecules (CD80 and CD86) on APC and delivers a signal that inhibits T cell proliferation. Several regions involved in binding to B7 are known, but the relative importance of these is not clear. We have cloned porcine CTLA4 (pCTLA4). Although highly homologous to human CTLA4 (hCTLA4), the predicted protein sequence contains a leucine for methionine substitution at position 97 in the MYPPPY sequence. A fusion protein constructed from the extracellular regions of pCTLA4 and the constant regions of human IgG1 (pCTLA4-Ig) bound porcine CD86 with equivalent affinity to that of hCTLA4-Ig. However, pCTLA4-Ig bound poorly to human CD80 and CD86 expressed on transfectants and EBV-transformed human B cells. In functional assays with MHC class II-expressing porcine endothelial cells and human B cells, pCTLA4-Ig blocked human CD4+ T cell responses to pig but not human cells, whereas control hCTLA4-Ig inhibited responses to both. Comparison between mouse, human, and porcine CTLA4-Ig suggests that the selective binding of pCTLA4-Ig to porcine CD86 molecules is due to the L for M substitution at position 97. Our results indicate that pCTLA4-Ig may be a useful reagent to define the precise nature of the interaction between B7 and CTLA4. By failing to inhibit the delivery of costimulatory signals provided by human B7, it may also prove to be a relatively specific inhibitor of the direct human T cell response to immunogenic pig tissue.

BACKGROUND

Dendritic cells are potent antigen-presenting cells that bind allogeneic T cells. They are thus candidates for targeting immunoregulatory molecules to the alloreactive T cell compartment and suppressing the alloimmune response.

METHODS

A dendritic cell line derived from the BALB/c mouse (H2d) was genetically modified to express the immunoregulatory molecule CTLA4-Ig. The ability of these dendritic cell transfectants to downregulate the alloimmune response was tested in an islet transplant model. Allogeneic C57Bl/6 (H2b) mice were rendered diabetic with streptozocin, and they received BALB/c islet (H2d) transplants. Mice were administered 25 million untransfected or CTLA4-Ig-transfected D2SC/1 cells i.v. on the day of islet transplantation and 6 days later[fnc].

RESULTS

Mice treated with CTLA4-Ig-transfected D2SC/1 cells demonstrated prolonged allograft survival (mean = 20 days, median = 17 days, SD = 9.39) compared with mice treated with untransfected D2SC/1 cells (mean = 12 days, median = 11 days, SD=2.74) or untreated control mice (mean = 11 days, median = 11 days SD = 1.41). Third party allograft survival was not prolonged in mice receiving similar treatment.

CONCLUSIONS

These results demonstrate that a genetically modified dendritic cell line can suppress the alloimmune response and prolong islet allograft survival in an allospecific manner. The findings also suggest that genetically modified dendritic cells may be useful in targeting alloreactive T cells and prolonging allograft survival.

Radiation therapy controls local disease but also prompts the release of tumor-associated antigens and stress-related danger signals that primes T cells to promote tumor regression at unirradiated sites known as the abscopal effect. This may be enhanced by blocking inhibitory immune signals that modulate immune activity through a variety of mechanisms. Indeed, abscopal responses have occurred in patients with lung cancer or melanoma when given anti-CTLA4 antibody and radiation. Other approaches involve expanding and reinfusing T or NK cells or engineered T cells to express receptors that target specific tumor peptides. These approaches may be useful for immunocompromised patients receiving radiation. Preclinical and clinical studies are testing both immune checkpoint-based strategies and adoptive immunotherapies with radiation.

OBJECTIVE

The recent emergence of a system for distinguishing T-cell-mediated rejection (TCMR) from antibody-mediated rejection (ABMR), including C4d-negative ABMR, allows us to map the molecular features of these conditions.

RESULTS

The TCMR landscape is dominated by molecules expressed in effector T cells, antigen-presenting cells (macrophages, dendritic cells, B cells) and interferon-gamma (IFNG)-induced genes. A surprising finding is the association of transcripts for inhibitory molecules such as CTLA4 and PDL1 with TCMR, indicating that this tubulo-interstitial inflammatory compartment is actively controlled. ABMR is dominated by endothelial transcripts related to angiogenesis, reflecting endothelial injury; natural killer (NK)-cell transcripts; and selected IFNG-regulated transcripts. This suggests a cognate unit of NK cells engaging donor-specific antibody bound to donor human leukocyte antigen antigens through their CD16a (FCGR3A) Fc receptors, triggering IFNG release. TCMR and ABMR share many rejection-associated transcripts, mainly IFNG-induced genes and transcripts shared between NK cells and CD8 effector T cells (e.g., KLRD1). In addition, acute kidney injury transcripts, which reflect the parenchymal response to injury, are shared between different forms of rejection and are indicative of disease progression.

CONCLUSIONS

Microarray assessment provides a new dimension in biopsy assessment for diagnosis that offers mechanistic insights and sometimes challenges histology assessments.

An agonistic antibody DTA-1, to glucocorticoid-induced TNFR-related protein (GITR), induces T-cell activation and antitumor immunity. CD4(+) effector T cells are essential in initiating GITR-induced immune activation, and the sequentially activated cytolytic CD8(+) T cells are sufficient to induce tumor rejection. Administration of DTA-1 to a tumor-bearing mouse also induces B-cell activation illustrated by CD69 expression. Substantial evidence suggests that resting B cells are tumor promoting, which has prompted the idea of B-cell depletion by Rituximab, to be combined with other agents in the clinic to augment antitumor response. In this study, we have found that mature B cells are needed for the mechanism of anti-GITR agonist to kill tumors. The treatment of GITR agonist induces profound B-cell activation, differentiation, and antibody production. In a mature B-cell-deficient mouse (JHD), DTA-1 fails to induce tumor regression with a reduced early activation of CD4(+) and CD8(+) T cells. B-cell deficiency disables the capability of the DTA-1 in generating cytolytic CD8(+) T cells and significantly reduces the cytokine production in tumor bearing mice. The tumor-killing activities of DTA-1 are still present albeit reduced in the CD40(-/-) mice, in which IgG production is impaired. We have also shown that the dependence on B cells to kill tumors differentiates GITR costimulation from CTLA4 blockade and OX40 agonism in tumor immunotherapy. The findings underscore the reciprocal T-cell-B-cell interaction to enhance antitumor immunity upon GITR costimulation. The results provide the insight that attenuating B-cell functions may not be beneficial in cancer immunotherapy based on GITR agonism.

BACKGROUND

Several of the currently used anticancer drugs may variably affect thyroid function, with impairment ranging from modified total but not free concentration of thyroid hormones to overt thyroid disease.

CONCLUSIONS

Cytotoxic agents seem to alter thyroid function in a relatively small proportion of adult patients. Anticancer hormone drugs may mainly alter serum levels of thyroid hormone-binding proteins without clinically relevant thyroid dysfunction. Old immunomodulating drugs, such as interferon-α and interleukin-2, are known to induce variably high incidence of autoimmune thyroid dysfunction. Newer immune checkpoint inhibitors, such as anti-CTLA4 monoclonal antibodies, are responsible for a relatively low incidence of thyroiditis and may induce secondary hypothyroidism resulting from hypophysitis. Central hypothyroidism is a well-recognized side effect of bexarotene. Despite their inherent selectivity, tyrosine kinase inhibitors may cause high rates of thyroid dysfunction. Notably, thyroid toxicity seems to be restricted to tyrosine kinase inhibitors targeting key kinase-receptors in angiogenic pathways, but not other kinase-receptors (e.g., epidermal growth factor receptors family or c-KIT). In addition, a number of these agents may also increase the levothyroxine requirement in thyroidectomized patients.

CONCLUSIONS

The pathophysiology of thyroid toxicity induced by many anticancer agents is not fully clarified and for others it remains speculative. Thyroid dysfunction induced by anticancer agents is generally manageable and dose reduction or discontinuation of these agents is not required. The prognostic relevance of thyroid autoimmunity, overt and subclinical hypothyroidism induced by anticancer drugs, the value of thyroid hormone replacement in individuals with abnormal thyrotropin following anticancer systemic therapy, and the correct timing of replacement therapy in cancer patients need to be defined more accurately in well-powered prospective clinical trials.

Systemic administration of the checkpoint blockade antibody anti-CTLA4 results in severe autoimmune toxicity, limiting its clinical efficacy. Fransen and colleagues show here that peritumoral delivery of low doses of this immunomodulatory drug can trigger a systemic antitumor immune response while preventing the toxicity against other organs.

Regulatory T cells play a crucial role in the homeostasis of the immune response. In addition to CD4+Foxp3+ regulatory T cells, several subsets of Foxp3- regulatory T cells, such as T helper 3 (Th3) cells and type 1 regulatory T (Tr1) cells, have been described in mice and human. Accumulating evidence shows that naïve B cells contribute to tolerance and are able to promote regulatory T cell differentiation. Naïve B cells can convert CD4+CD25- T cells into CD25+Foxp3- regulatory T cells, named Treg-of-B cells by our group. Treg-of-B cells express LAG3, ICOS, GITR, OX40, PD1, and CTLA4 and secrete IL-10. Intriguingly, B-T cell-cell contact but not IL-10 is essential for Treg-of-B cells induction. Moreover, Treg-of-B cells possess both IL-10-dependent and IL-10-independent inhibitory functions. Treg-of-B cells exert suppressive activities in antigen-specific and non-antigen-specific manners in vitro and in vivo. Here, we review the phenotype and function of Foxp3+ regulatory T cells, Th3 cells, Tr1 cells, and Treg-of-B cells.

BALB/c mice are susceptible to progressive infection with Leishmania major due to the preferential development of CD4(+) T cells that secrete Th2 cytokines. Although Th2 cell development and susceptibility are disrupted by blockade of CD86 function early in infection, CD28-deficient BALB/c mice remain susceptible to leishmaniasis. We therefore examined whether the alternative CD86 ligand, CTLA4, contributes to the expression of susceptibility. BALB/c mice treated for 2 weeks of infection with anti-CTLA4 monoclonal antibody developed more rapidly progressive disease than sham-treated mice, whereas normally resistant C57BL/6 mice were unaffected. The draining lymph node cells of anti-CTLA4-treated BALB/c mice produced up to sixfold more interleukin-4 (IL-4) and IL-13 than control mice in the first 2 weeks of infection, but IFN-gamma synthesis was reciprocally decreased. Anti-CTLA4 treatment of BALB/c mice pretreated with neutralizing anti-IL-4 antibody or genetically deficient in IL-4 also caused significant worsening of leishmaniasis. Exacerbation in IL-4 KO mice was associated with increased IL-13 and decreased gamma interferon (IFN-gamma) and inducible nitric oxide synthase (iNOS) mRNA expression in vivo. These data indicate that anti-CTLA4 antibody induced earlier and more-polarized Th2 responses in susceptible BALB/c mice infected with L. major. The mechanism of disease worsening was partially IL-4 independent, indicating that increased IL-13 and/or decreased IFN-gamma production may have disrupted nitric oxide-based microbicidal responses. We conclude that CTLA4 significantly modulates Th2 development in murine leishmaniasis and that the Th2-polarizing effects of anti-CTLA4 treatment result in IL-4-independent exacerbation of disease.

Here, we report on the detection of a novel point mutation of the CTLA4 gene at nucleotide position 159 (C->>G) leading to amino acid substitution at position 53 (I->>M), as well as its association with type 1 diabetes in two ethnically distinct populations. Subjects included 182 unrelated type 1 diabetes children and 201 control subjects from Ghana, West Africa. The Chinese study population consisted of 350 type 1 diabetic children and 420 healthy control subjects from central China. Polymerase chain reaction-single-strand conformation polymorphism and sequence analysis were used to screen for polymorphisms in the CTLA4 gene. CTLA4 49 (A->>G) mutation conferred a risk of type 1 diabetes in the Chinese children (odds ratio 1.78, 95% CI 1.58-2.0), but not in the West African children (1.17, 0.84-1.64). On the other hand, the novel CTLA4 159 (C->>G) mutation conferred a risk of type 1 diabetes in the West African children (2.1, 1.54-2.86), but not in the Chinese type 1 diabetic children. The novel CTLA4 gene polymorphism at nucleotide position 159 significantly associated with type 1 diabetes in West Africans, but not in Chinese. On the other hand, the CTLA4 gene polymorphism at nucleotide position 49 significantly associated with type 1 diabetes in Chinese, but not in West Africans.

Targeted therapies require information on specific defective signaling pathways or mutations. Advances in genomic technologies and cell biology have led to identification of new therapeutic targets associated with signal-transduction pathways. Survival times of patients with colorectal cancer (CRC) can be extended with combinations of conventional cytotoxic agents and targeted therapies. Targeting EGFR- and VEGFR-signaling systems has been the major focus for treatment of metastatic CRC. However, there are still limitations in their clinical application, and new and better drug combinations are needed. This review provides information on EGFR and VEGF inhibitors, new therapeutic agents in the pipeline targeting EGFR and VEGFR pathways, and those targeting other signal-transduction pathways, such as MET, IGF1R, MEK, PI3K, Wnt, Notch, Hedgehog, and death-receptor signaling pathways for treatment of metastatic CRC. Additionally, multitargeted approaches in combination therapies targeting negative-feedback loops, compensatory networks, and cross talk between pathways are highlighted. Then, immunobased strategies to enhance antitumor immunity using specific monoclonal antibodies, such as the immune-checkpoint inhibitors anti-CTLA4 and anti-PD1, as well as the challenges that need to be overcome for increased efficacy of targeted therapies, including drug resistance, predictive markers of response, tumor subtypes, and cancer stem cells, are covered. The review concludes with a brief insight into the applications of next-generation sequencing, expression profiling for tumor subtyping, and the exciting progress made in in silico predictive analysis in the development of a prescription strategy for cancer therapy.

A better understanding of the relationships between vaccine, immunogenicity and protection from disease would greatly facilitate vaccine development. Modified vaccinia virus Ankara expressing antigen 85A (MVA85A) is a novel tuberculosis vaccine candidate designed to enhance responses induced by BCG. Antigen-specific interferon-γ (IFN-γ) production is greatly enhanced by MVA85A, however the variability between healthy individuals is extensive. In this study we have sought to characterize the early changes in gene expression in humans following vaccination with MVA85A and relate these to long-term immunogenicity. Two days post-vaccination, MVA85A induces a strong interferon and inflammatory response. Separating volunteers into high and low responders on the basis of T cell responses to 85A peptides measured during the trial, an expansion of circulating CD4+ CD25+ Foxp3+ cells is seen in low but not high responders. Additionally, high levels of Toll-like Receptor (TLR) 1 on day of vaccination are associated with an increased response to antigen 85A. In a classification model, combined expression levels of TLR1, TICAM2 and CD14 on day of vaccination and CTLA4 and IL2Rα two days post-vaccination can classify high and low responders with over 80% accuracy. Furthermore, administering MVA85A in mice with anti-TLR2 antibodies may abrogate high responses, and neutralising antibodies to TLRs 1, 2 or 6 or HMGB1 decrease CXCL2 production during in vitro stimulation with MVA85A. HMGB1 is released into the supernatant following atimulation with MVA85A and we propose this signal may be the trigger activating the TLR pathway. This study suggests an important role for an endogenous ligand in innate sensing of MVA and demonstrates the importance of pattern recognition receptors and regulatory T cell responses in determining the magnitude of the antigen specific immune response to vaccination with MVA85A in humans.

Intratumoral immunotherapies aim at reducing local immunosuppression, as well as reinstating and enhancing systemic anticancer T-cell functions, without inducing side effects. LTX-315 is a first-in-class oncolytic peptide-based local immunotherapy that meets these criteria by inducing a type of malignant cell death that elicits anticancer immune responses. Here, we show that LTX-315 rapidly reprograms the tumor microenvironment by decreasing the local abundance of immunosuppressive Tregs and myeloid-derived suppressor cells and by increasing the frequency of polyfunctional T helper type 1/type 1 cytotoxic T cells with a concomitant increase in cytotoxic T-lymphocyte antigen-4 (CTLA4) and drop in PD-1 expression levels. Logically, in tumors that were resistant to intratumoral or systemic CTLA4 blockade, subsequent local inoculation of LTX-315 cured the animals or caused tumor regressions with abscopal effects. This synergistic interaction between CTLA4 blockade and LTX-315 was reduced upon blockade of the β-chain of the interleukin-2 receptor (CD122). This preclinical study provides a strong rationale for administering the oncolytic peptide LTX-315 to patients who are receiving treatment with the CTLA4 blocking antibody ipilimumab.

Glioblastoma multiforme (GBM) is the most aggressive primary human brain tumor. The relatively high amount of T regulatory lymphocytes present in the tumor, contributes to the establishment of an immunosuppressive microenvironment. Samples of peripheral blood were collected from GBM patients and healthy controls and a purified population of Treg (CD4(+)/CD25(bright)) was isolated using flow cytometric cell sorting. Treg migrating capacities toward human glioma cell line conditioned medium were evaluated through an in vitro migration test. Our data show that supernatants collected from GBM cell lines were more attractant to Treg when compared to complete standard medium. The addition of an anti-CCL2 antibody to conditioned medium decreased conditioned medium-depending Treg migration, suggesting that CCL2 (also known as Monocyte Chemoattractant Protein, MCP-1) is implicated in the process. The number of circulating CD4(+)/μL or Treg/μL was similar in GBM patients and controls. Specific Treg markers (FOXP3; CD127; Helios; GITR; CTLA4; CD95; CCR2, CCR4; CCR7) were screened in peripheral blood and no differences could be detected between the two populations. These data confirm that the tumor microenvironment is attractive to Treg, which tend to migrate toward the tumor region changing the immunological response. Though we provide evidence that CCL2 is implicated in Treg migration, other factors are needed as well to provide such effect.

OBJECTIVE

From the published literature, we identified 23 germ line sequence variants in 17 genes from hypothesis-generating studies that were associated with prognosis of head and neck cancer, including sequence variants of DNA repair (ERCC1, ERCC4, ERCC5, MSH2, XPA, ERCC2, XRCC1, XRCC3), DNA methylation (DNMT3B), cell cycle and proliferation (CCND1, TP53), xenobiotic metabolism (GSTM1, GSTT1, CYP2D6), metastatic -potential (MMP3), immunologic (CTLA4), and growth factor pathways (FGFR4). The purpose of this study was to validate the role of these 23 sequence variants for overall (OS) and disease-free survival (DFS) in a large, comprehensive, well-annotated data set of patients with head and neck cancer.

METHODS

We genotyped these sequence variants in 531 patients with stage I and II radiation-treated head and neck cancer (originally recruited for an alpha-tocopherol/beta-carotene placebo-controlled secondary prevention study), and analyzed using Cox proportional hazards models, stratified by treatment arm, adjusting for clinical prognostic factors.

RESULTS

Two OS associations were statistically significant for each variant allele when compared with the wild-type: CTLA4: A49G [rs231775; adjusted HR (aHR), 1.32 (1.1-1.6); P = 0.01] and XRCC1: Arg339Gln [rs25487; aHR, 1.28 (1.05-1.57); P = 0.02]. Both of these sequence variants had significant results in the opposite direction as prior published literature. Two DFS associations were of borderline significance in the same direction as prior literature: ERCC2: Lys751Gln [rs13181; aHR, 0.80 (0.6-1.0); P = 0.05] and TP53: Arg72Pro [rs1042522; aHR, 1.28 (1.0-1.6); P = 0.03], comparing number of variant alleles with reference of zero variants.

CONCLUSIONS

None of the prognostic sequence variants previously published was validated for OS in our patients with early-stage radiation-treated head and neck cancer, though rs1381and rs1042522 had borderline significant association with DFS.

The success of immune checkpoint inhibitors in cancer immunotherapy has been widely heralded. However many cancer patients do not respond to immune checkpoint therapy and some relapse due to acquired tumor resistance. Epigenetic targeting may be beneficial in cancer immunotherapy by reversing immune avoidance and escape mechanisms employed by cancer cells, as well as by modulating immune cell differentiation and function. In this manuscript we review recent findings suggesting how epigenetics may be used to improve cancer immunotherapy. We focus on the inhibitors of the CTLA4 and PD1 immune checkpoints and epigenetic modifiers of histone acetylation and methylation and DNA methylation.

Although skin tumors are highly immunogenic, exposure to UV radiation is known to suppress immune responses via regulatory T cells. Specifically, the activity of cytotoxic lymphocyte-associated antigen-4 (CTLA-4) is integral in regulating the development of UV-induced tolerance and, concomitantly, skin cancers. Due to the inverse relationship between tumor surveillance and autoimmunity, we hypothesize that the same genetic variant in the CTLA4 locus that increases risk for autoimmune diseases is associated with decreased risk of non-melanoma skin cancer (NMSC). We analyzed whether the polymorphism CT60 or haplotypes of CTLA4 influence odds of developing the major types of NMSC, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), in a population-based case-control study of Caucasians in New Hampshire (849 controls, 930 BCC, and 713 SCC). The CTLA4 CT60 GG genotype was associated with decreased odds for BCC and SCC, controlling for age, sex, lifetime number of severe sunburns, and skin type [BCC: odds ratio (OR), 0.7; 95% confidence interval (95% CI), 0.5-0.9; SCC: OR, 0.7; 95% CI, 0.5-1.0]. For BCC, this decrease was apparent largely among those with a higher lifetime number of severe sunburns (P(interaction) = 0.0074). There were significantly decreased odds of disease associated with two haplotypes, which possess the CT60 G allele. Additionally, lifetime number of severe sunburns modestly altered the effects of the CTLA4 haplotypes in BCC, and the association seemed driven by the CT60 single nucleotide polymorphism. In conclusion, genetic variation at the CTLA4 locus may be etiologically important in NMSC, the most prevalent malignancy in the United States.

CTLA4 and CD28 are co-regulatory receptors with opposite roles in T-cell signaling. By RNA sequencing, we identified a fusion between the two genes from partial gene duplication in a case of angioimmunoblastic T-cell lymphoma. The fusion gene, which codes for the extracellular domain of CTLA4 and the cytoplasmic region of CD28, is likely capable of transforming inhibitory signals into stimulatory signals for T-cell activation. Ectopic expression of the fusion transcript in Jurkat and H9 cells resulted in enhanced proliferation and AKT and ERK phosphorylation, indicating activation of downstream oncogenic pathways. To estimate the frequency of this gene fusion in mature T-cell lymphomas, we examined 115 T-cell lymphoma samples of diverse subtypes using reverse transcriptase polymerase chain reaction analysis and Sanger sequencing. We identified the fusion in 26 of 45 cases of angioimmunoblastic T-cell lymphomas (58%), nine of 39 peripheral T-cell lymphomas, not otherwise specified (23%), and nine of 31 extranodal NK/T cell lymphomas (29%). We further investigated the mutation status of 70 lymphoma-associated genes using ultra-deep targeted resequencing for 74 mature T-cell lymphoma samples. The mutational landscape we obtained suggests that T-cell lymphoma results from diverse combinations of multiple gene mutations. The CTLA4-CD28 gene fusion is likely a major contributor to the pathogenesis of T-cell lymphomas and represents a potential target for anti-CTLA4 cancer immunotherapy.

Recent evidence has confirmed that a mutation of the isocitrate dehydrogenase (IDH) gene occurs early in gliomagenesis and contributes to suppressed immunity. The present study aimed to determine the candidate genes associated with IDH mutation status that could serve as biomarkers of immune suppression for improved prognosis prediction. Clinical information and RNA-seq gene expression data were collected for 932 glioma samples from the CGGA and TCGA databases, and differentially expressed genes in both lower-grade glioma (LGG) and glioblastoma (GBM) samples were identified according to IDH mutation status. Only one gene, interferon-stimulated exonuclease gene 20 (ISG20), with reduced expression in IDH mutant tumors, demonstrated significant prognostic value. ISG20 expression level significantly increased with increasing tumor grade, and its high expression was associated with a poor clinical outcome. Moreover, increased ISG20 expression was associated with increased infiltration of monocyte-derived macrophages and neutrophils, and suppressed adaptive immune response. ISG20 expression was also positively correlated with PD-1, PD-L1, and CTLA4 expression, along with the levels of several chemokines. We conclude that ISG20 is a useful biomarker to identify IDH-mediated immune processes in glioma and may serve as a potential therapeutic target.