Epigenetic MLH1 silencing concurs with mismatch repair deficiency in sporadic, naturally occurring colorectal cancer in rhesus macaques.

BACKGROUND: Naturally occurring colorectal cancers (CRC) in rhesus macaques share many features with their human counterparts and are useful models for cancer immunotherapy; but mechanistic data are lacking regarding the comparative molecular pathogenesis of these cancers. METHODS: We conducted state-of-the-art imaging including CT and PET, clinical assessments, and pathological review of 24 rhesus macaques with naturally occurring CRC. Additionally, we molecularly characterized these tumors utilizing immunohistochemistry (IHC), microsatellite instability assays, DNAseq, transcriptomics, and developed a DNA methylation-specific qPCR assay for MLH1, CACNA1G, CDKN2A, CRABP1, and NEUROG1, human markers for CpG island methylator phenotype (CIMP). We furthermore employed Monte-Carlo simulations to in-silico model alterations in DNA topology in transcription-factor binding site-rich promoter regions upon experimentally demonstrated DNA methylation. RESULTS: Similar cancer histology, progression patterns, and co-morbidities could be observed in rhesus as reported for human CRC patients. IHC identified loss of MLH1 and PMS2 in all cases, with functional microsatellite instability. DNA sequencing revealed the close genetic relatedness to human CRCs, including a similar mutational signature, chromosomal instability, and functionally-relevant mutations affecting KRAS (G12D), TP53 (R175H, R273*), APC, AMER1, ALK, and ARID1A. Interestingly, MLH1 mutations were rarely identified on a somatic or germline level. Transcriptomics not only corroborated the similarities of rhesus and human CRCs, but also demonstrated the significant downregulation of MLH1 but not MSH2, MSH6, or PMS2 in rhesus CRCs. Methylation-specific qPCR suggested CIMP-positivity in 9/16 rhesus CRCs, but all 16/16 exhibited significant MLH1 promoter hypermethylation. DNA hypermethylation was modelled to affect DNA topology, particularly propeller twist and roll profiles. Modelling the DNA topology of a transcription factor binding motif (TFAP2A) in the MLH1 promoter that overlapped with a methylation-specific probe, we observed significant differences in DNA topology upon experimentally shown DNA methylation. This suggests a role of transcription factor binding interference in epigenetic silencing of MLH1 in rhesus CRCs. CONCLUSIONS: These data indicate that epigenetic silencing suppresses MLH1 transcription, induces the loss of MLH1 protein, abrogates mismatch repair, and drives genomic instability in naturally occurring CRC in rhesus macaques. We consider this spontaneous, uninduced CRC in immunocompetent, treatment-naïve rhesus macaques to be a uniquely informative model for human CRC.

Visceral adipose microbial and inflammatory signatures in metabolically healthy and unhealthy nonhuman primates.

OBJECTIVE: Obesity is a key risk factor for metabolic syndrome (MetS); however, >10% of lean individuals meet MetS criteria. Visceral adipose tissue (VAT) disproportionately contributes to inflammation and insulin resistance compared with subcutaneous fat depots. The primary aim of this study was to profile tissue microbiome components in VAT over a wide range of metabolic statuses in a highly clinically relevant model. METHODS: VAT was profiled from nonhuman primates that naturally demonstrate four distinct health phenotypes despite consuming a healthy diet, namely metabolically healthy lean and obese and metabolically unhealthy lean and obese. RESULTS: VAT biopsied from unhealthy lean and obese nonhuman primates demonstrated upregulation of immune signaling pathways, a tissue microbiome enriched in gram-negative bacteria including Pseudomonas, and deficiencies in anti-inflammatory adipose tissue M2 macrophages. VAT microbiomes were distinct from fecal microbiomes, and fecal microbiomes did not differ by metabolic health group, which was in contrast to the VAT bacterial communities. CONCLUSIONS: Immune activation with gram-negative VAT microbial communities is a consistent feature in elevated MetS risk in both lean and obesity states.

Y-Chromosome-Linked Genes Are Associated With Sex-Related Head-Neck Squamous Cell Carcinoma Survival.

OBJECTIVE: To define novel gene biomarkers for prognosis of head and neck squamous cell carcinoma (HNSCC) patients' survival. STUDY DESIGN: Retrospective study. SETTING: The Cancer Genome Atlas (TCGA) HNSCC RNA-Seq dataset. METHODS: Coexpressed gene clusters were extracted from TCGA RNA-seq data using our previously published method (EPIG). Kaplan-Meier estimator was then used for overall survival-relevant analysis, with patients partitioned into 3 groups based on gene expression levels: female, male_low, and male_high. RESULTS: Male had better overall survival than female and male with higher expression level of Y-chromosome-linked (Y-linked) genes had significantly better survival than those with lower expression levels. In addition, male with a higher expression level of Y-linked genes showed even better survival when they have a higher level of coexpressed cluster of genes related to B or T cell immune response. Other clinical conditions related to immune responses also consistently showed favorable effects on the Y-linked genes for survival estimation. Male patients with higher expression level of Y-linked genes also have significantly higher tumor/normal tissue (T/N) ratio of those genes and higher level of several immune responses related clinical measurements (eg, lymphocyte and TCR related). Male patients with lower expression level of Y-linked genes benefited from radiation-only treatment. CONCLUSIONS: The favorable role of a cluster of coexpressed Y-linked genes in HNSCC patients' survival is potentially associated with elevated level of immune responses. These Y-linked genes could serve as useful prognostic biomarkers for HNSCC patients' survival estimation and treatment.

Macrophage Phenotypes and Gene Expression Patterns Are Unique in Naturally Occurring Metabolically Healthy Obesity.

Obesity impacts 650 million individuals globally, often co-occurring with metabolic syndrome. Though many obese individuals experience metabolic abnormalities (metabolically unhealthy obese [MUO]), ~30% do not (metabolically healthy obese [MHO]). Conversely, >10% of lean individuals are metabolically unhealthy (MUL). To evaluate the physiologic drivers of these phenotypes, a 44-animal African green monkey cohort was selected using metabolic syndrome risk criteria to represent these four clinically defined health groups. Body composition imaging and subcutaneous adipose tissue (SQ AT) biopsies were collected. Differences in adipocyte size, macrophage subtype distribution, gene expression, vascularity and fibrosis were analyzed using digital immunohistopathology, unbiased RNA-seq, endothelial CD31, and Masson's trichrome staining, respectively. MHO AT demonstrated significant increases in M2 macrophages (p = 0.02) and upregulation of fatty acid oxidation-related terms and transcripts, including FABP7 (p = 0.01). MUO AT demonstrated downregulation of these factors and co-occurring upregulation of immune responses. These changes occurred without differences in AT distributions, adipocyte size, AT endothelial cells, collagen I deposition, or circulating cytokine levels. Without unhealthy diet consumption, healthy obesity is defined by an increased SQ AT M2/M1 macrophage ratio and lipid handling gene expression. We highlight M2 macrophages and fatty acid oxidation as targets for improving metabolic health with obesity.

Prognostic attributes of immune signatures in soft tissue sarcomas show differential dependencies on tumor mutational burden.

BACKGROUND: Cellular and intrinsic markers of sarcoma immunogenicity are poorly understood. To gain insight into whether tumor-immune interactions correlate with clinical aggressiveness, the authors examined the prognostic significance of immune gene signatures in combination with tumor mutational burden (TMB) and cancer-testis antigen (CTA) expression. METHODS: RNA sequencing and clinical data of 259 soft tissue sarcomas from The Cancer Genome Atlas project were used to investigate associations between published immune gene signatures and patient overall survival (OS) in the contexts of TMB, as computed from whole-exome sequencing data, and CTA gene expression. Multivariate Cox proportional hazards regression models and log-rank tests were used to assess survival associations. RESULTS: Immune signature scores that reflected in part the intratumoral abundance of cytotoxic T cells showed significant positive associations with OS. However, the prognostic power of the T-cell signatures was highly dependent on TMB-high status, consistent with protective effects of tumor-infiltrating T cells in tumors with elevated antigenicity. In TMB-low tumors, a signature of infiltrating plasma B cells was significantly and positively associated with OS, independent of T-cell signature status. Although tumor subtypes based on differential expression patterns of CTA genes showed different survival associations within leiomyosarcoma and myxofibrosarcoma histologies, neither CTA nor histologic subtype interacted with the T-cell-survival association. CONCLUSIONS: Signatures of T-cell and plasma B-cell infiltrates were associated with a survival benefit in soft tissue sarcomas. TMB, but not CTA expression, influenced the prognostic power of T-cell-associated, but not plasma B-cell-associated, survival. LAY SUMMARY: Clinical data and RNA analysis of 259 soft tissue sarcomas from The Cancer Genome Atlas project were used to investigate associations between five published gene immune cell expression signatures and survival in the context of tumor mutations. Activated T cells had a significant positive association with patient survival. Although high tumor mutation burden was associated with good survival, the prognostic power of T-cell signatures was highly dependent on tumor mutational status, consistent with protective effects of tumor-infiltrating T cells in tumors with high levels of antigens. In low tumor mutation-bearing tumors, plasma B cells were positively associated with survival.

Phase II trial of cytarabine and mitoxantrone with devimistat in acute myeloid leukemia.

Devimistat is a TCA cycle inhibitor. A previously completed phase I study of devimistat in combination with cytarabine and mitoxantrone in patients with relapsed or refractory AML showed promising response rates. Here we report the results of a single arm phase II study (NCT02484391). The primary outcome of feasibility of maintenance devimistat following induction and consolidation with devimistat in combination with high dose cytarabine and mitoxantrone was not met, as maintenance devimistat was only administered in 2 of 21 responders. The secondary outcomes of response (CR + CRi) and median survival were 44% (21/48) and 5.9 months respectively. There were no unexpected toxicities observed. An unplanned, post-hoc analysis of the phase I and II datasets suggests a trend of a dose response in older but not younger patients. RNA sequencing data from patient samples reveals an age-related decline in mitochondrial gene sets. Devimistat impairs ATP synthesis and we find a correlation between mitochondrial membrane potential and sensitivity to chemotherapy. Devimistat also induces mitochondrial reactive oxygen species and turnover consistent with mitophagy. We find that pharmacological or genetic inhibition of mitochondrial fission or autophagy sensitizes cells to devimistat. These findings suggest that an age related decline in mitochondrial quality and autophagy may be associated with response to devimistat however this needs to be confirmed in larger cohorts with proper trial design.

Comprehensive gene cluster analysis of head and neck squamous cell carcinoma TCGA RNA-seq data defines B cell immunity-related genes as a robust survival predictor.

BACKGROUND: The authors aimed to define novel gene expression signatures that are associated with patients' survival with head and neck squamous cell carcinoma (HNSCC). METHODS: TCGA RNA-seq data were used for gene expression clusters extraction from 499 tumor samples by the "EPIG" method. Tumor samples were then partitioned into lower and higher than median level groups for survival relevant analysis by Kaplan-Meier estimator. RESULTS: We found that two gene clusters (_1, _2) are favorably, while two (_3, _4) are unfavorably, associated with patients' survival with HNSCC. Notably, most genes on the top lists of cluster_2 are associated with B cells. A gene expression signature with combined genes from cluster_2 and _4 was further determined to be associated with HNSCC survival rate. CONCLUSION: Our work strongly supported a favorable role of B cells in patients' survival with HNSCC and identified a novel coexpressed gene signature as prognostic biomarker for patients' survival with HNSCC estimation.

Bulk and Single-Cell Profiling of Breast Tumors Identifies TREM-1 as a Dominant Immune Suppressive Marker Associated With Poor Outcomes.

BACKGROUND: Triggering receptor expressed on myeloid cells (TREM)-1 is a key mediator of innate immunity previously associated with the severity of inflammatory disorders, and more recently, the inferior survival of lung and liver cancer patients. Here, we investigated the prognostic impact and immunological correlates of TREM1 expression in breast tumors. METHODS: Breast tumor microarray and RNAseq expression profiles (n=4,364 tumors) were analyzed for associations between gene expression, tumor immune subtypes, distant metastasis-free survival (DMFS) and clinical response to neoadjuvant chemotherapy (NAC). Single-cell (sc)RNAseq was performed using the 10X Genomics platform. Statistical associations were assessed by logistic regression, Cox regression, Kaplan-Meier analysis, Spearman correlation, Student's t-test and Chi-square test. RESULTS: In pre-treatment biopsies, TREM1 and known TREM-1 inducible cytokines (IL1B, IL8) were discovered by a statistical ranking procedure as top genes for which high expression was associated with reduced response to NAC, but only in the context of immunologically "hot" tumors otherwise associated with a high NAC response rate. In surgical specimens, TREM1 expression varied among tumor molecular subtypes, with highest expression in the more aggressive subtypes (Basal-like, HER2-E). High TREM1 significantly and reproducibly associated with inferior distant metastasis-free survival (DMFS), independent of conventional prognostic markers. Notably, the association between high TREM1 and inferior DMFS was most prominent in the subset of immunogenic tumors that exhibited the immunologically hot phenotype and otherwise associated with superior DMFS. Further observations from bulk and single-cell RNAseq analyses indicated that TREM1 expression was significantly enriched in polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and M2-like macrophages, and correlated with downstream transcriptional targets of TREM-1 (IL8, IL-1B, IL6, MCP-1, SPP1, IL1RN, INHBA) which have been previously associated with pro-tumorigenic and immunosuppressive functions. CONCLUSIONS: Together, these findings indicate that increased TREM1 expression is prognostic of inferior breast cancer outcomes and may contribute to myeloid-mediated breast cancer progression and immune suppression.

The Presence and Potential Role of ALDH1A2 in the Glioblastoma Microenvironment.

Glioblastoma (GBM) is the most aggressive malignant glioma. Therapeutic targeting of GBM is made more difficult due to its heterogeneity, resistance to treatment, and diffuse infiltration into the brain parenchyma. Better understanding of the tumor microenvironment should aid in finding more effective management of GBM. GBM-associated macrophages (GAM) comprise up to 30% of the GBM microenvironment. Therefore, exploration of GAM activity/function and their specific markers are important for developing new therapeutic agents. In this study, we identified and evaluated the expression of ALDH1A2 in the GBM microenvironment, and especially in M2 GAM, though it is also expressed in reactive astrocytes and multinucleated tumor cells. We demonstrated that M2 GAM highly express ALDH1A2 when compared to other ALDH1 family proteins. Additionally, GBM samples showed higher expression of ALDH1A2 when compared to low-grade gliomas (LGG), and this expression was increased upon tumor recurrence both at the gene and protein levels. We demonstrated that the enzymatic product of ALDH1A2, retinoic acid (RA), modulated the expression and activity of MMP-2 and MMP-9 in macrophages, but not in GBM tumor cells. Thus, the expression of ALDH1A2 may promote the progressive phenotype of GBM.

Skeletal muscle extracellular matrix remodeling with worsening glycemic control in nonhuman primates.

Type 2 diabetes (T2D) development may be mediated by skeletal muscle (SkM) function, which is responsible for >80% of circulating glucose uptake. The goals of this study were to assess changes in global- and location-level gene expression, remodeling proteins, fibrosis, and vascularity of SkM with worsening glycemic control, through RNA sequencing, immunoblotting, and immunostaining. We evaluated SkM samples from health-diverse African green monkeys (Cholorcebus aethiops sabaeus) to investigate these relationships. We assessed SkM remodeling at the molecular level by evaluating unbiased transcriptomics in age-, sex-, weight-, and waist circumference-matched metabolically healthy, prediabetic (PreT2D) and T2D monkeys (n = 13). Our analysis applied novel location-specific gene differences and shows that extracellular facing and cell membrane-associated genes and proteins are highly upregulated in metabolic disease. We verified transcript patterns using immunohistochemical staining and protein analyses of matrix metalloproteinase 16 (MMP16), tissue inhibitor of metalloproteinase 2 (TIMP2), and VEGF. Extracellular matrix (ECM) functions to support intercellular communications, including the coupling of capillaries to muscle cells, which was worsened with increasing blood glucose. Multiple regression modeling from age- and health-diverse monkeys (n = 33) revealed that capillary density was negatively predicted by only fasting blood glucose. The loss of vascularity in SkM co-occurred with reduced expression of hypoxia-sensing genes, which is indicative of a disconnect between altered ECM and reduced endothelial cells, and known perfusion deficiencies present in PreT2D and T2D. This report supports that rising blood glucose values incite ECM remodeling and reduce SkM capillarization, and that targeting ECM would be a rational approach to improve health with metabolic disease.

MAP3K7 and CHD1 Are Novel Mediators of Resistance to Oncolytic Vesicular Stomatitis Virus in Prostate Cancer Cells.

A key principle of oncolytic viral therapy is that many cancers develop defects in their antiviral responses, making them more susceptible to virus infection. However, some cancers display resistance to viral infection. Many of these resistant cancers constitutively express interferon-stimulated genes (ISGs). The goal of these experiments was to determine the role of two tumor suppressor genes, MAP3K7 and CHD1, in viral resistance and ISG expression in PC3 prostate cancer cells resistant to oncolytic vesicular stomatitis virus (VSV). MAP3K7 and CHD1 are often co-deleted in aggressive prostate cancers. Silencing expression of MAP3K7 and CHD1 in PC3 cells increased susceptibility to the matrix (M) gene mutant M51R-VSV, as shown by increased expression of viral genes, increased yield of progeny virus, and reduction of tumor growth in nude mice. Silencing MAP3K7 alone had a greater effect on virus susceptibility than did silencing CHD1. Silencing MAP3K7 and CHD1 decreased constitutive expression of ISG mRNAs and proteins, whereas silencing MAP3K7 alone decreased expression of ISG proteins, but actually increased expression of ISG mRNAs. These results suggest a role for the protein product of MAP3K7, transforming growth factor ß-activated kinase 1 (TAK1), in regulating translation of ISG mRNAs and a role of CHD1 in maintaining the transcription of ISGs.

APOL1 Kidney-Risk Variants Induce Mitochondrial Fission.

INTRODUCTION: APOL1 G1 and G2 nephropathy-risk variants cause mitochondrial dysfunction and contribute to kidney disease. Analyses were performed to determine the genetic regulation of APOL1 and elucidate potential mechanisms in APOL1-nephropathy. METHODS: A global gene expression analysis was performed in human primary renal tubule cell lines derived from 50 African American individuals. Follow-up gene knock out, cell-based rescue, and microscopy experiments were performed. RESULTS: APOL1 genotypes did not alter APOL1 expression levels in the global gene expression analysis. Expression quantitative trait locus (eQTL) analysis in polyinosinic-polycytidylic acid (poly IC)-stimulated renal tubule cells revealed that single nucleotide polymorphism (SNP) rs513349 adjacent to BAK1 was a trans eQTL for APOL1 and a cis eQTL for BAK1; APOL1 and BAK1 were co-expressed in cells. BAK1 knockout in a human podocyte cell line resulted in diminished APOL1 protein, supporting a pivotal effect for BAK1 on APOL1 expression. Because BAK1 is involved in mitochondrial dynamics, mitochondrial morphology was examined in primary renal tubule cells and HEK293 Tet-on cells of various APOL1 genotypes. Mitochondria in APOL1 wild-type (G0G0) tubule cells maintained elongated morphology when stimulated by low-dose poly IC, whereas those with G1G1, G2G2, and G1G2 genotypes appeared to fragment. HEK293 Tet-on cells overexpressing APOL1 G0, G1, and G2 were created; G0 cells appeared to promote mitochondrial fusion, whereas G1 and G2 induced mitochondrial fission. The mitochondrial dynamic regulator Mdivi-1 significantly preserved cell viability and mitochondrial cristae structure and reversed mitochondrial fission induced by overexpression of G1 and G2. CONCLUSION: Results suggest the mitochondrial fusion/fission pathway may be a therapeutic target in APOL1-nephropathy.

Solute Carrier Family 37 Member 2 (SLC37A2) Negatively Regulates Murine Macrophage Inflammation by Controlling Glycolysis.

Increased flux of glucose through glycolysis is a hallmark of inflammatory macrophages and is essential for optimal effector functions. Solute carrier (SLC) 37A2 is an endoplasmic reticulum-anchored phosphate-linked glucose-6-phosphate transporter that is highly expressed in macrophages and neutrophils. We demonstrate that SLC37A2 plays a pivotal role in murine macrophage inflammatory activation and cellular metabolic rewiring. Toll-like receptor (TLR) 4 stimulation by lipopolysaccharide (LPS) rapidly increases macrophage SLC37A2 protein expression. SLC37A2 deletion reprograms macrophages to a hyper-glycolytic process and accelerates LPS-induced inflammatory cytokine production, which partially depends on nicotinamide adenine dinucleotide (NAD+) biosynthesis. Blockade of glycolysis normalizes the differential expression of pro-inflammatory cytokines between control and SLC37A2 deficient macrophages. Conversely, overexpression of SLC37A2 lowers macrophage glycolysis and significantly reduces LPS-induced pro-inflammatory cytokine expression. In conclusion, our study suggests that SLC37A2 dampens murine macrophage inflammation by down-regulating glycolytic reprogramming as a part of macrophage negative feedback system to curtail acute innate activation.

Dysregulated Pyrimidine Biosynthesis Contributes to 5-FU Resistance in SCLC Patient-Derived Organoids but Response to a Novel Polymeric Fluoropyrimidine, CF10.

Chemo-immunotherapy is central to the treatment of small cell lung cancer (SCLC). Despite modest progress made with the addition of immunotherapy, current cytotoxic regimens display minimal survival benefit and new treatments are needed. Thymidylate synthase (TS) is a well-validated anti-cancer drug target, but conventional TS inhibitors display limited clinical efficacy in refractory or recurrent SCLC. We performed RNA-Seq analysis to identify gene expression changes in SCLC biopsy samples to provide mechanistic insight into the potential utility of targeting pyrimidine biosynthesis to treat SCLC. We identified systematic dysregulation of pyrimidine biosynthesis, including elevated TYMS expression that likely contributes to the lack of efficacy for current TS inhibitors in SCLC. We also identified E2F1-3 upregulation in SCLC as a potential driver of TYMS expression that may contribute to tumor aggressiveness. To test if TS inhibition could be a viable strategy for SCLC treatment, we developed patient-derived organoids (PDOs) from human SCLC biopsy samples and used these to evaluate both conventional fluoropyrimidine drugs (e.g., 5-fluorouracil), platinum-based drugs, and CF10, a novel fluoropyrimidine polymer with enhanced TS inhibition activity. PDOs were relatively resistant to 5-FU and while moderately sensitive to the front-line agent cisplatin, were relatively more sensitive to CF10. Our studies demonstrate dysregulated pyrimidine biosynthesis contributes to drug resistance in SCLC and indicate that a novel approach to target these pathways may improve outcomes.

Transcriptomic Features of T Cell-Barren Tumors Are Conserved Across Diverse Tumor Types.

Background: Understanding how tumors subvert immune destruction is essential to the development of cancer immunotherapies. New evidence suggests that tumors limit anti-tumor immunity by exploiting transcriptional programs that regulate intratumoral trafficking and accumulation of effector cells. Here, we investigated the gene expression profiles that distinguish immunologically "cold" and "hot" tumors across diverse tumor types. Methods: RNAseq profiles of tumors (n = 8,920) representing 23 solid tumor types were analyzed using immune gene signatures that quantify CD8+ T cell abundance. Genes and pathways associated with a low CD8+ T cell infiltration profile (CD8-Low) were identified by correlation, differential expression, and statistical ranking methods. Gene subsets were evaluated in immunotherapy treatment cohorts and functionally characterized in cell lines and mouse tumor models. Results: Among different cancer types, we observed highly significant overlap of genes enriched in CD8-Low tumors, which included known immunomodulatory genes (e.g., BMP7, CMTM4, KDM5B, RCOR2) and exhibited significant associations with Wnt signaling, neurogenesis, cell-cell junctions, lipid biosynthesis, epidermal development, and cancer-testis antigens. Analysis of mutually exclusive gene clusters demonstrated that different transcriptional programs may converge on the T cell-cold phenotype as well as predict for response and survival of patients to Nivo treatment. Furthermore, we confirmed that a top-ranking candidate belonging to the TGF-ß superfamily, BMP7, negatively regulates CD8+ T cell abundance in immunocompetent murine tumor models, with and without anti-PD-L1 treatment. Conclusions: This study presents the first evidence that solid tumors of diverse anatomical origin acquire conserved transcriptional alterations that may be operative in the T cell-cold state. Our findings demonstrate the potential clinical utility of CD8-Low tumor-associated genes for predicting patient immunotherapy outcomes and point to novel mechanisms with potential for broad therapeutic exploitation.

Prognostic Molecular Classification of Appendiceal Mucinous Neoplasms Treated with Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy.

BACKGROUND: Appendiceal mucinous neoplasm (AMN) with peritoneal metastasis is a rare but deadly disease with few prognostic or therapy-predictive biomarkers to guide treatment decisions. Here, we investigated the prognostic and biological attributes of gene expression-based AMN molecular subtypes. METHODS: AMN specimens (n = 138) derived from a population-based subseries of patients treated at our institution with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) between 05/2000 and 05/2013 were analyzed for gene expression using a custom-designed NanoString 148-gene panel. Signed non-negative matrix factorization (sNMF) was used to define a gene signature capable of delineating robustly-classified AMN molecular subtypes. The sNMF class assignments were evaluated by topology learning, reverse-graph embedding and cross-cohort performance analysis. RESULTS: Three molecular subtypes of AMN were discerned by the expression patterns of 17 genes with roles in cancer progression or anti-tumor immunity. Tumor subtype assignments were confirmed by topology learning. AMN subtypes were termed immune-enriched (IE), oncogene-enriched (OE) and mixed (M) as evidenced by their gene expression patterns, and exhibited significantly different post-treatment survival outcomes. Genes with specialized immune functions, including markers of T-cells, natural killer cells, B-cells, and cytolytic activity showed increased expression in the low-risk IE subtype, while genes implicated in the promotion of cancer growth and progression were more highly expressed in the high-risk OE subtype. In multivariate analysis, the subtypes demonstrated independent prediction power for post-treatment survival. CONCLUSIONS: Our findings suggest a greater role for the immune system in AMN than previously recognized. AMN subtypes may have clinical utility for predicting CRS/HIPEC treatment outcomes.

Whole-Genome and Segmental Homozygosity Confirm Errors in Meiosis as Etiology of Struma Ovarii.

Strumae ovarii are neoplasms composed of normal-appearing thyroid tissue that occur within the ovary and rarely spread to extraovarian sites. A unique case of struma ovarii with widespread dissemination detected 48 years after removal of a pelvic dermoid provided the opportunity to reexamine the molecular nature of this form of neoplasm. One tumor, from the heart, consisting of benign thyroid tissue was found to have whole-genome homozygosity. Another tumor from the right mandible composed of malignant-appearing thyroid tissue showed whole-genome homozygosity and a deletion of 7p, presumably the second hit that transformed it into a cancerous tumor. Specimens from 2 other cases of extraovarian struma confined to the abdomen and 8 of 9 cases of intraovarian struma showed genome-wide segmental homozygosity. These findings confirm errors in meiosis as the origin of struma ovarii. The histological and molecular findings further demonstrate that even when outside the ovary, strumae ovarii can behave nonaggressively until they receive a second hit, thereafter behaving like cancer.

Tumor mutational burden is a determinant of immune-mediated survival in breast cancer.

Mounting evidence supports a role for the immune system in breast cancer outcomes. The ability to distinguish highly immunogenic tumors susceptible to anti-tumor immunity from weakly immunogenic or inherently immune-resistant tumors would guide development of therapeutic strategies in breast cancer. Genomic, transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohorts were used to examine statistical associations between tumor mutational burden (TMB) and the survival of patients whose tumors were assigned to previously-described prognostic immune subclasses reflecting favorable, weak or poor immune-infiltrate dispositions (FID, WID or PID, respectively). Tumor immune subclasses were associated with survival in patients with high TMB (TMB-Hi, P < 0.001) but not in those with low TMB (TMB-Lo, P = 0.44). This statistical relationship was confirmed in the METABRIC cohort (TMB-Hi, P = 0.047; TMB-Lo, P = 0.39), and also found to hold true in the more-indolent Luminal A tumor subtype (TMB-Hi, P = 0.011; TMB-Lo, P = 0.91). In TMB-Hi tumors, the FID subclass was associated with prolonged survival independent of tumor stage, molecular subtype, age and treatment. Copy number analysis revealed the reproducible, preferential amplification of chromosome 1q immune-regulatory genes in the PID immune subclass. These findings demonstrate a previously unappreciated role for TMB as a determinant of immune-mediated survival of breast cancer patients and identify candidate immune-regulatory mechanisms associated with immunologically cold tumors. Immune subtyping of breast cancers may offer opportunities for therapeutic stratification.

An assessment of prognostic immunity markers in breast cancer.

Tumor-infiltrating lymphocytes (TIL) and immunity gene signatures have been reported to be significantly prognostic in breast cancer but have not yet been applied for calculation of risk of recurrence in clinical assays. A compact set of 17 immunity genes was derived herein from an Affymetrix-derived gene expression dataset including 1951 patients (AFFY1951). The 17 immunity genes demonstrated significant prognostic stratification of estrogen receptor (ER)-negative breast cancer patients with high proliferation gene expression. Further analysis of blood and breast cancer single-cell RNA-seq datasets revealed that the 17 immunity genes were derived from TIL that were inactive in the blood and became active in tumor tissue. Expression of the 17 immunity genes was significantly (p < 2.2E-16, n = 91) correlated with TILs percentage on H&E in triple negative breast cancer. To demonstrate the impact of tumor immunity genes on prognosis, we built a Cox model to incorporate breast cancer subtypes, proliferation score and immunity score (72 gene panel) with significant prediction of outcomes (p < 0.0001, n = 1951). The 72 gene panel and its risk evaluation model were validated in two other published gene expression datasets including Illumina beads array data METABRIC (p < 0.0001, n = 1997) and whole transcriptomic mRNA-seq data TCGA (p = 0.00019, n = 996) and in our own targeted RNA-seq data TARGETSEQ (p < 0.0001, n = 303). Further examination of the 72 gene panel in single cell RNA-seq of tumors demonstrated tumor heterogeneity with more than two subtypes observed in each tumor. In conclusion, immunity gene expression was an important parameter for prognosis and should be incorporated into current multi-gene assays to improve assessment of risk of distant metastasis in breast cancer.

SOX7 Target Genes and Their Contribution to Its Tumor Suppressive Function.

SOX7 is a transcription factor and acts as a tumor suppressor, but its target genes in cancers are poorly explored. We revealed SOX7-mediated gene expression profile in breast cancer cells using microarray chips and discovered multiple altered signaling pathways. When combinatorially analyzing the microarray data with a gene array dataset from 759 breast cancer patients, we identified four genes as potential targets of SOX7 and validated them by quantitative PCR and chromatin immunoprecipitation assays. Among these four genes, we determined that SOX7-activated SPRY1 and SLIT2, and SOX7-repressed TRIB3 and MTHFD2 could all differentially contribute to SOX7-mediated tumor suppression. Overall, we identified multiple cancer-related pathways mediated by SOX7 and for the first time revealed SOX7-regulated target genes in a cancer-relevant context.