Identification and Validation of Immune-Related Methylation Clusters for Predicting Immune Activity and Prognosis in Breast Cancer.

The role of DNA methylation of breast cancer-infiltrating immune cells has not been fully explored. We conducted a cohort-based retrospective study analyzing the genome-wide immune-related DNA methylation of 1057 breast cancer patients from the TCGA cohort and GSE72308 cohort. Based on patients' overall survival (OS), a prognostic risk score system using 18 immune-related methylation genes (IRMGs) was established and further validated in an independent cohort. Kaplan-Meier analysis showed a clear separation of OS between the low- and high-risk groups. Patients in the low-risk group had a higher immune score and stromal score compared with the high-risk group. Moreover, the characteristics based on 18-IRMGs signature were related to the tumor immune microenvironment and affected the abundance of tumor-infiltrating immune cells. Consistently, the 18-IRMGs signatures showed similar influences on immune modulation and survival in another external validation cohort (GSE72308). In conclusion, the proposed 18-IRMGs signature could be a potential marker for breast cancer prognostication.

The abscopal effect: a sense of DNA damage is in the air.

Tumor metastasis is a singularly important determinant of survival in most cancers. Historically, radiation therapy (RT) directed at a primary tumor mass was associated infrequently with remission of metastasis outside the field of irradiation. This away-from-target or "abscopal effect" received fringe attention because of its rarity. With the advent of immunotherapy, there are now increasing reports of abscopal effects upon RT in combination with immune checkpoint inhibition. This sparked investigation into underlying mechanisms and clinical trials aimed at enhancement of this effect. While these studies clearly attribute the abscopal effect to an antitumor immune response, the initial molecular triggers for its onset and specificity remain enigmatic. Here, we propose that DNA damage-induced inflammation coupled with neoantigen generation is essential during this intriguing phenomenon of systemic tumor regression and discuss the implications of this model for treatment aimed at triggering the abscopal effect in metastatic cancer.

Inducing regulated necrosis and shifting macrophage polarization with anti-EMMPRIN antibody (161-pAb) and complement factors.

Treatment of solid tumors is often hindered by an immunosuppressive tumor microenvironment (TME) that prevents effector immune cells from eradicating tumor cells and promotes tumor progression, angiogenesis, and metastasis. Therefore, targeting components of the TME to restore the ability of immune cells to drive anti-tumoral responses has become an important goal. One option is to induce an immunogenic cell death (ICD) of tumor cells that would trigger an adaptive anti-tumoral immune response. Here we show that incubating mouse renal cell carcinoma (RENCA) and colon carcinoma cell lines with an anti-extracellular matrix metalloproteinase inducer polyclonal antibody (161-pAb) together with complement factors can induce cell death that inhibits caspase-8 activity and enhances the phosphorylation of receptor-interacting protein kinase 3 (RIPK3) and mixed-lineage kinase-like domain (MLKL). This regulated necrotic death releases high levels of dsRNA molecules to the conditioned medium (CM) relative to the necrotic death of tumor cells induced by H2 O2 or the apoptotic death induced by etoposide. RAW 264.7 macrophages incubated with the CM derived from these dying cells markedly enhanced the secretion of IFNß, and enhanced their cytotoxicity. Furthermore, degradation of the dsRNA in the CM abolished the ability of RAW 264.7 macrophages to secrete IFNß, IFNγ-induced protein 10 (IP-10), and TRAIL. When mice bearing RENCA tumors were immunized with the 161-pAb, their lysates displayed elevated levels of phosphorylated RIPK3 and MLKL, as well as increased concentrations of dsRNA, IFNß, IP-10, and TRAIL. This shows that an antigen-targeted therapy using an antibody and complement factors that triggers ICD can shift the mode of macrophage activation by triggering regulated necrotic death of tumor cells.

DNA hypomethylation promotes transposable element expression and activation of immune signaling in renal cell cancer.

Recently, we reported that expression of endogenous retroviruses (ERVs) is associated with response to immune checkpoint blockade (ICB) in renal cell carcinoma (RCC). We show that decitabine, a DNA hypomethylating agent, activates transposable element (TE) expression (LINE1 and ERVs ERV3-2 and ERV4700) and antiviral signaling to potentially enhance response to ICB in kidney cancer cell lines and primary cells. KO of RIGI and MDA5 dsRNA sensors attenuated activation of antiviral signaling associated with DNA hypomethylation, and RIGI and MDA5 IPs showed increased ERV binding with decitabine treatment. Bioinformatic analyses showed the decitabine-induced signature could be associated with increased immune infiltration and response to ICB. Cytokine secretion induced by decitabine could modestly improve T cell activation and robustly enhanced T cell migration. In a small retrospective cohort of metastatic clear cell RCC (ccRCC) patients treated with anti-PD1/PDL1 blockade, activation of some antiviral genes was significantly higher in responders. Thus, we identified a potential strategy to induce TE expression through inhibition of DNA methylation in modulating T cell action via regulation of the innate antiviral pathway.

Monitoring minimal residual disease in the bone marrow using next generation sequencing.

Achieving minimal residual disease (MRD) negativity in the bone marrow is one of the strongest prognostic factors in multiple myeloma. Consequently, MRD testing is routinely performed in clinical trials and moving towards standard of care. This review focuses on the role of next generation sequencing (NGS) of tumor-specific immunoglobulin V(D)J sequences for MRD tracking. The immunoglobulin variable regions are ideal targets for tracking, because every tumor cell shares an identical gene sequence, which is stable over time and generally distinct from the immunoglobulin sequences of normal B-cells. Several excellent assays for NGS-based MRD testing are available, both commercial and community-based, including one that is FDA-approved. These assays can achieve the gold standard analytical sensitivity of one tumor cell per million (10-6), requiring a minimum input of 3 million bone marrow cells. On-going clinical trials will outline how MRD testing should be used to inform dynamic risk-adopted therapy.

Genomic profiling of multiple myeloma: New insights and modern technologies.

Advances in technologies for genomic profiling, primarily with next generation sequencing, have lead to a better understanding of the complex genomic landscape in multiple myeloma. Integrated analysis of whole genome, exome and transcriptome sequencing has lead to new insights on disease drivers including translocations, copy number alterations, somatic mutations, and altered gene expression. Disease progression in multiple myeloma is largely driven by structural variations including the traditional immunoglobulin heavy chain (IGH) translocations and hyperdiploidy which are early events in myelomagenesis as well as more complex events spanning over multiple chromosomes and involving amplifications and deletions. In this review, we will discuss recent insights on the genomic landscape of multiple myeloma and their implications for disease progression and personalized treatment. We will review how sequencing assays compare to current clinical methods and give an overview of modern technologies for interrogating genomic aberrations.

Methylation of immune synapse genes modulates tumor immunogenicity.

Cancer immune evasion is achieved through multiple layers of immune tolerance mechanisms including immune editing, recruitment of tolerogenic immune cells, and secretion of immunosuppressive cytokines. Recent success with immune checkpoint inhibitors in cancer immunotherapy suggests a dysfunctional immune synapse as a pivotal tolerogenic mechanism. Tumor cells express immune synapse proteins to suppress the immune system, which is often modulated by epigenetic mechanisms. When the methylation status of key immune synapse genes was interrogated, we observed disproportionately hypermethylated costimulatory genes and hypomethylation of immune checkpoint genes, which were negatively associated with functional T cell recruitment to the tumor microenvironment. Therefore, the methylation status of immune synapse genes reflects tumor immunogenicity and correlates with survival.

An immune-related gene signature predicts prognosis of gastric cancer.

BACKGROUND: Although the outcome of patients with gastric cancer (GC) has improved significantly with the recent implementation of annual screening programs. Reliable prognostic biomarkers are still needed due to the disease heterogeneity. Increasing pieces of evidence revealed an association between immune signature and GC prognosis. Thus, we aim to build an immune-related signature that can estimate prognosis for GC. METHODS: For identification of a prognostic immune-related gene signature (IRGS), gene expression profiles and clinical information of patients with GC were collected from 3 public cohorts, divided into training cohort (n = 300) and 2 independent validation cohorts (n = 277 and 433 respectively). RESULTS: Within 1811 immune genes, a prognostic IRGS consisting of 16 unique genes was constructed which was significantly associated with survival (hazard ratio [HR], 3.9 [2.78-5.47]; P < 1.0 × 10). In the validation cohorts, the IRGS significantly stratified patients into high- vs low-risk groups in terms of prognosis across (HR, 1.84 [1.47-2.30]; P = 6.59 × 10) and within subpopulations with stage I&II disease (HR, 1.96 [1.34-2.89]; P = 4.73 × 10) and was prognostic in univariate and multivariate analyses. Several biological processes, including TGF-ß and EMT signaling pathways, were enriched in the high-risk group. T cells CD4 memory resting and Macrophage M2 were significantly higher in the high-risk risk group compared with the low-risk group. CONCLUSION: In short, we developed a prognostic IRGS for estimating prognosis in GC, including stage I&II disease, providing new insights into the identification of patients with GC with a high risk of mortality.

DNA sensing by the cGAS-STING pathway in health and disease.

The detection of pathogens through nucleic acid sensors is a defining principle of innate immunity. RNA-sensing and DNA-sensing receptors sample subcellular compartments for foreign nucleic acids and, upon recognition, trigger immune signalling pathways for host defence. Over the past decade, our understanding of how the recognition of nucleic acids is coupled to immune gene expression has advanced considerably, particularly for the DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) and its downstream signalling effector stimulator of interferon genes (STING), as well as the molecular components and regulation of this pathway. Moreover, the ability of self-DNA to engage cGAS has emerged as an important mechanism fuelling the development of inflammation and implicating the cGAS-STING pathway in human inflammatory diseases and cancer. This detailed mechanistic and biological understanding is paving the way for the development and clinical application of pharmacological agonists and antagonists in the treatment of chronic inflammation and cancer.

Exosomes Shuttle TREX1-Sensitive IFN-Stimulatory dsDNA from Irradiated Cancer Cells to DCs.

Radiotherapy (RT) used at immunogenic doses leads to accumulation of cytosolic double-stranded DNA (dsDNA) in cancer cells, which activates type I IFN (IFN-I) via the cGAS/STING pathway. Cancer cell-derived IFN-I is required to recruit BATF3-dependent dendritic cells (DC) to poorly immunogenic tumors and trigger antitumor T-cell responses in combination with immune checkpoint blockade. We have previously demonstrated that the exonuclease TREX1 regulates radiation immunogenicity by degrading cytosolic dsDNA. Tumor-derived DNA can also activate cGAS/STING-mediated production of IFN-I by DCs infiltrating immunogenic tumors. However, how DNA from cancer cells is transferred to the cytoplasm of DCs remains unclear. Here, we showed that tumor-derived exosomes (TEX) produced by irradiated mouse breast cancer cells (RT-TEX) transfer dsDNA to DCs and stimulate DC upregulation of costimulatory molecules and STING-dependent activation of IFN-I. In vivo, RT-TEX elicited tumor-specific CD8+ T-cell responses and protected mice from tumor development significantly better than TEX from untreated cancer cells in a prophylactic vaccination experiment. We demonstrated that the IFN-stimulatory dsDNA cargo of RT-TEX is regulated by TREX1 expression in the parent cells. Overall, these results identify RT-TEX as a mechanism whereby IFN-stimulatory dsDNA is transferred from irradiated cancer cells to DCs. We have previously shown that the expression of TREX1 is dependent on the RT dose size. Thus, these data have important implications for the use of RT with immunotherapy. Cancer Immunol Res; 6(8); 910-20. ©2018 AACR.

Divergent evolutionary trajectories in transplanted tumor models.

Human-derived tumor models are becoming popular in the context of personalized medicine, but a new study shows that these models could be less representative of primary tumors than previously thought, particularly when using late passages.

Inhibiting DNA methylation activates cancer testis antigens and expression of the antigen processing and presentation machinery in colon and ovarian cancer cells.

Innovative therapies for solid tumors are urgently needed. Recently, therapies that harness the host immune system to fight cancer cells have successfully treated a subset of patients with solid tumors. These responses have been strong and durable but observed in subsets of patients. Work from our group and others has shown that epigenetic therapy, specifically inhibiting the silencing DNA methylation mark, activates immune signaling in tumor cells and can sensitize to immune therapy in murine models. Here we show that colon and ovarian cancer cell lines exhibit lower expression of transcripts involved in antigen processing and presentation to immune cells compared to normal tissues. In addition, treatment with clinically relevant low doses of DNMT inhibitors (that remove DNA methylation) increases expression of both antigen processing and presentation and Cancer Testis Antigens in these cell lines. We confirm that treatment with DNMT inhibitors upregulates expression of the antigen processing and presentation molecules B2M, CALR, CD58, PSMB8, PSMB9 at the RNA and protein level in a wider range of colon and ovarian cancer cell lines and treatment time points than had been described previously. In addition, we show that DNMTi treatment upregulates many Cancer Testis Antigens common to both colon and ovarian cancer. This increase of both antigens and antigen presentation by epigenetic therapy may be one mechanism to sensitize patients to immune therapies.

DNA sensing and immune responses in cancer therapy.

The identification of critical DNA sensors and their pathways has led to revealing the central role of DNA sensing in immune system. It has been initially demonstrated that DNA sensing and immune responses have high impacts on the development and prevention of infection and inflammatory. In addition to toll-like receptor pathways, there is now also emerging evidence that cytosolic enzyme cyclic GMP-AMP synthase (cGAS) is essential for the recognition of not only pathogen-derived DNA but also tumor DNA for innate sensing. The strategies through activating DNA sensing pathways toward enhancing antitumor immunity have shown promise and are further tested in clinical studies. Here, we highlight recent progresses in understanding mechanisms activated by DNA sensing mediated immune responses in cancer therapy.

DNA-Containing Exosomes Derived from Cancer Cells Treated with Topotecan Activate a STING-Dependent Pathway and Reinforce Antitumor Immunity.

Danger-associated molecular patterns derived from damaged or dying cells elicit inflammation and potentiate antitumor immune responses. In this article, we show that treatment of breast cancer cells with the antitumor agent topotecan (TPT), an inhibitor of topoisomerase I, induces danger-associated molecular pattern secretion that triggers dendritic cell (DC) activation and cytokine production. TPT administration inhibits tumor growth in tumor-bearing mice, which is accompanied by infiltration of activated DCs and CD8+ T cells. These effects are abrogated in mice lacking STING, an essential molecule in cytosolic DNA-mediated innate immune responses. Furthermore, TPT-treated cancer cells release exosomes that contain DNA that activate DCs via STING signaling. These findings suggest that a STING-dependent pathway drives antitumor immunity by responding to tumor cell-derived DNA.

Somatic hypermutation in immunity and cancer: Critical analysis of strand-biased and codon-context mutation signatures.

For 30 years two general mechanisms have competed to explain somatic hypermutation of immunoglobulin (Ig) genes. The first, the DNA-based model, is focused only on DNA substrates. The modern form is the Neuberger "DNA Deamination Model" based on activation-induced cytidine deaminase (AID) and short-patch error-prone DNA repair by DNA Polymerase-η operating around AID C-to-U lesions. The other is an RNA-based mechanism or the "Reverse Transcriptase Model" of SHM which produces strand-biased mutations at A:T and G:C base pairs. This involves error-prone cDNA synthesis via an RNA-dependent DNA polymerase copying the Ig pre-mRNA template and integrating the now error-filled cDNA copy back into the normal chromosomal site. The modern form of this mechanism depends on AID dC-to-dU lesions and long tract error-prone cDNA synthesis of the transcribed strand by DNA Polymerase-η acting as a reverse transcriptase. The evidence for and against each mechanism is critically evaluated. The conclusion is that all the SHM molecular data gathered since 1980 supports directly or indirectly the RNA/RT-based mechanism. All the data and critical analyses are systematically laid out so the reader can evaluate this conclusion for themselves. Recently we have investigated whether similar RNA/RT-based mutator mechanisms explain how de novo mutations arise in somatic tissues (cancer genomes). The data analyses indeed suggest that cancers arise via dysregulated "Ig-like SHM responses" involving rogue DNA and RNA deaminations coupled to genome-wide RT events. Further, Robyn Lindley has recently shown that the strand-biased mutations in cancer genome genes are also in "codon-context." This has been termed Targeted Somatic Mutation (TSM) to highlight that mutations are far more targeted than previously thought in somatic tissues associated with disease. The TSM process implies an "in-frame DNA reader" whereby DNA and RNA deaminases at transcribed regions are guided in their mutagenic action, by the codon reading frame of the DNA.

DNA Glycation from 3-Deoxyglucosone Leads to the Formation of AGEs: Potential Role in Cancer Auto-antibodies.

The non-enzymatic glycation reaction results in the generation of free radicals which play an important role in the pathophysiology of aging, diabetes, and cancer. 3-Deoxyglucosone (3-DG) is a dicarbonyl species which may lead to the formation of advanced glycation end products (AGEs). 3-DG also reacts with free amino group of nucleic acids resulting in the formation of DNA-AGEs. While the establishment of nucleoside AGEs has been revealed before, no extensive studies have been done to probe the role of 3-DG in the generation of immunogenicity and induction of cancer auto-antibodies. In this study, we report the immunogenicity of AGEs formed by 3-DG-Arg-Fe(3+) system. Spectroscopic analysis and melting temperature studies suggest structural perturbations in the DNA as a result of modification. Immunogenicity of native and 3-DG-Arg-Fe(3+) DNA was probed in female rabbits. The modified DNA was highly immunogenic eliciting high-titer immunogen-specific antibodies, while the unmodified form was almost non-immunogenic. We also report the presence of auto-antibodies against 3-DG-Arg-Fe(3+)-modified DNA in the sera of patients with different types of cancers. The glycoxidative lesions were also detected in the lymphocyte DNA isolated from selected cancer patients. The results show structural perturbations in 3-DG-Arg-Fe(3+)-DNA generating new epitopes that render the molecule immunogenic.

Exosomes and tumor-mediated immune suppression.

Tumor-derived exosomes (TEX) are harbingers of tumor-induced immune suppression: they carry immunosuppressive molecules and factors known to interfere with immune cell functions. By delivering suppressive cargos consisting of proteins similar to those in parent tumor cells to immune cells, TEX directly or indirectly influence the development, maturation, and antitumor activities of immune cells. TEX also deliver genomic DNA, mRNA, and microRNAs to immune cells, thereby reprogramming functions of responder cells to promote tumor progression. TEX carrying tumor-associated antigens can interfere with antitumor immunotherapies. TEX also have the potential to serve as noninvasive biomarkers of tumor progression. In the tumor microenvironment, TEX may be involved in operating numerous signaling pathways responsible for the downregulation of antitumor immunity.

A novel and easy FxCycle™ violet based flow cytometric method for simultaneous assessment of DNA ploidy and six-color immunophenotyping.

Abnormal DNA ploidy is a valuable prognostic factor in many neoplasms, especially in hematological neoplasms like B-cell acute lymphoblastic leukemia (B-ALL) and multiple myeloma (MM). Current methods of flow-cytometric (FC) DNA-ploidy evaluation are either technically difficult or limited to three- to four-color immunophenotyping and hence, challenging to evaluate DNA-ploidy in minute tumor population with background rich of its normal counterpart cells and other hematopoietic cells. We standardized a novel sensitive and easy method of simultaneous evaluation of six- to seven-color immunophenotyping and DNA-ploidy using a dye-FxCycle Violet (FCV). Linearity, resolution, and coefficient of variation (CV) for FCV were studied using chicken erythrocyte nuclei. Ploidy results of FCV were compared with Propidium iodide (PI) in 20 samples and intra-assay variation for FCV was studied. Using this six-color immunophenotyping & FCV-protocol DNA-ploidy was determined in bone-marrow samples from 124 B-ALL & 50 MM patients. Dilution experiment was also conducted to determine the sensitivity in detection of aneuploidy in minute tumor population. FCV revealed high linearity and resolution in 450/50 channel. On comparison with PI, CV of Go/G1-peak with FCV (mean-CV 4.1%) was slightly higher than PI (mean-CV 2.9%) but had complete agreement in ploidy results. Dilution experiment showed that aneuploidy could be accurately detected up to the limit of 0.01% tumor cells. Intra-assay variation was very low with CV of 0.005%. In B-ALL, hypodiploidy was noted in 4%, hyperdiploidy in 24%, near-hyperdiploidy in 13% and remaining 59% were diploid. In MM, hypodiploidy was in 2%, hyperdiploidy in 58%, near-hyperdiploidy in 8% and remaining 30% were diploid. FCV-based DNA-ploidy method is a sensitive and easy method for simultaneous evaluation of six-color immunophenotyping and DNA analysis. It is useful in DNA-ploidy evaluation of minute tumor population in cases like minimal residual disease and MM precursor conditions.