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Purpose: Immunotherapy with checkpoint inhibitors is improving the outcomes of several cancers. However, only a subset of patients respond. Therefore, predictive biomarkers are critically needed to guide treatment decisions and develop approaches to the treatment of therapeutic resistance. Experimental Design: We compared bioenergetics of circulating immune cells and metabolomic profiles of plasma obtained at baseline from patients with melanoma treated with anti–PD-1 therapy. We also performed single-cell RNA sequencing (scRNAseq) to correlate transcriptional changes associated with metabolic changes observed in peripheral blood mononuclear cells (PBMC) and patient plasma. Results: Pretreatment PBMC from responders had a higher reserve respiratory capacity and higher basal glycolytic activity compared with nonresponders. Metabolomic analysis revealed that responder and nonresponder patient samples cluster differently, suggesting differences in metabolic signatures at baseline. Differential levels of specific lipid, amino acid, and glycolytic pathway metabolites were observed by response. Further, scRNAseq analysis revealed upregulation of T-cell genes regulating glycolysis. Our analysis showed that SLC2A14 (Glut-14; a glucose transporter) was the most significant gene upregulated in responder patients' T-cell population. Flow cytometry analysis confirmed significantly elevated cell surface expression of the Glut-14 in CD3+, CD8+, and CD4+ circulating populations in responder patients. Moreover, LDHC was also upregulated in the responder population. Conclusions: Our results suggest a glycolytic signature characterizes checkpoint inhibitor responders; consistently, both ECAR and lactate-to-pyruvate ratio were significantly associated with overall survival. Together, these findings support the use of blood bioenergetics and metabolomics as predictive biomarkers of patient response to immune checkpoint inhibitor therapy.

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.

Ethnicity is considered to be one of the major risk factors in certain subtypes of breast cancer. However, the mechanism of this racial disparity remains poorly understood. Here, we demonstrate that SOS1, a key regulator of Ras pathway, is highly expressed in African-American (AA) patients with breast cancer compared with Caucasian-American patients. Because of the higher obesity rate in AA women, increased levels of SOS1 facilitated signal transduction of the c-Met pathway, which was highly activated in AA patients with breast cancer via hepatocyte growth factor secreted from adipocytes. Elevated expression of SOS1 also enhanced cancer stemness through upregulation of PTTG1 and promoted M2 polarization of macrophages by CCL2 in metastatic sites. SOS1 was epigenetically regulated by a super-enhancer identified by H3K27ac in AA patients. Knockout of the super-enhancer by CRISPR in AA cell lines significantly reduced SOS1 expression. Furthermore, SOS1 was posttranscriptionally regulated by miR-483 whose expression is reduced in AA patients through histone trimethylation (H3K27me3) on its promoter. The natural compound, taxifolin, suppressed signaling transduction of SOS1 by blocking the interaction between SOS1 and Grb2, suggesting a potential utility of this compound as a therapeutic agent for AA patients with breast cancer. Significance: These findings elucidate the signaling network of SOS1-mediated metastasis in African-American patients, from the epigenetic upregulation of SOS1 to the identification of taxifolin as a potential therapeutic strategy against SOS1-driven tumor progression.

In this work, we study the survival outcomes of patients with lung adenocarcinoma in four independent cohorts. By classifying patients with KRAS mutations into three subgroups based on their mutation status of TP53 and SMARCA4, our analysis indicates that patients harboring both KRAS and SMARCA4 mutations do not benefit from the treatment with nonimmunotherapy or immune checkpoint inhibitor‐based immunotherapy. Alternative treatment strategy is requested for this subset of patients., KRAS is a key oncogenic driver in lung adenocarcinoma (LUAD). Chromatin‐remodeling gene SMARCA4 is comutated with KRAS in LUAD; however, the impact of SMARCA4 mutations on clinical outcome has not been adequately established. This study sought to shed light on the clinical significance of SMARCA4 mutations in LUAD. The association of SMARCA4 mutations with survival outcomes was interrogated in four independent cohorts totaling 564 patients: KRAS‐mutant patients with LUAD who received nonimmunotherapy treatment from (a) The Cancer Genome Atlas (TCGA) and (b) the MSK‐IMPACT Clinical Sequencing (MSK‐CT) cohorts; and KRAS‐mutant patients with LUAD who received immune checkpoint inhibitor‐based immunotherapy treatment from (c) the MSK‐IMPACT (MSK‐IO) and (d) the Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) immunotherapy cohorts. Of the patients receiving nonimmunotherapy treatment, in the TCGA cohort (n = 155), KRAS‐mutant patients harboring SMARCA4 mutations (KS) showed poorer clinical outcome [P = 6e‐04 for disease‐free survival (DFS) and 0.031 for overall survival (OS), respectively], compared to KRAS‐TP53 comutant (KP) and KRAS‐only mutant (K) patients; in the MSK‐CT cohort (n = 314), KS patients also exhibited shorter OS than KP (P = 0.03) or K (P = 0.022) patients. Of patients receiving immunotherapy, KS patients consistently exhibited the shortest progression‐free survival (PFS; P = 0.0091) in the MSK‐IO (n = 77), and the shortest PFS (P = 0.0026) and OS (P = 0.0014) in the WFBCCC (n = 18) cohorts, respectively. Therefore, mutations of SMARCA4 represent a genetic factor leading to adverse clinical outcome in lung adenocarcinoma treated by either nonimmunotherapy or immunotherapy.

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.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.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.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.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.

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), oncogeneenriched (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.

There is a lack of robust generalizable predictive biomarkers of response to immune checkpoint blockade in multiple types of cancer. We develop hDirect-MAP, an algorithm that maps T cells into a shared high-dimensional (HD) expression space of diverse T cell functional signatures in which cells group by the common T cell phenotypes rather than dimensional reduced features or a distorted view of these features. Using projection-free single-cell modeling, hDirect-MAP first removed a large group of cells that did not contribute to response and then clearly distinguished T cells into response-specific subpopulations that were defined by critical T cell functional markers of strong differential expression patterns. We found that these grouped cells cannot be distinguished by dimensional-reduction algorithms but are blended by diluted expression patterns. Moreover, these identified response-specific T cell subpopulations enabled a generalizable prediction by their HD metrics. Tested using five single-cell RNA-seq or mass cytometry datasets from basal cell carcinoma, squamous cell carcinoma and melanoma, hDirect-MAP demonstrated common response-specific T cell phenotypes that defined a generalizable and accurate predictive biomarker.

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy.

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.

BackgroundCD47 is an integral membrane protein that alters adaptive immunosurveillance when bound to the matricellular glycoprotein thrombospondin-1 (TSP1). We examined the impact of the CD47/TSP1 signaling axis on melanoma patient response to anti-PD-1 therapy due to alterations in T cell activation, proliferation, effector function, and bioenergetics.MethodsA syngeneic B16 mouse melanoma model was performed to determine if targeting CD47 as monotherapy or in combination with anti-PD-1 impacted tumor burden. Cytotoxic (CD8+) T cells from Pmel-1 transgenic mice were used for T cell activation, cytotoxic T lymphocyte, and cellular bioenergetic assays. Single-cell RNA-sequencing, ELISA, and flow cytometry was performed on peripheral blood mononuclear cells and plasma of melanoma patients receiving anti-PD-1 therapy to examine CD47/TSP1 expression.ResultsHuman malignant melanoma tissue had increased CD47 and TSP1 expression within the tumor microenvironment compared with benign tissue. Due to the negative implications CD47/TSP1 can have on antitumor immune responses, we targeted CD47 in a melanoma model and observed a decrease in tumor burden due to increased tumor oxygen saturation and granzyme B secreting CD8+ T cells compared with wild-type tumors. Additionally, Pmel-1 CD8+ T cells exposed to TSP1 had reduced activation, proliferation, and effector function against B16 melanoma cells. Targeting CD47 allowed CD8+ T cells to overcome this TSP1 interaction to sustain these functions. TSP1 exposed CD8+ T cells have a decreased rate of glycolysis; however, targeting CD47 restored glycolysis when CD8+ T cells were exposed to TSP1, suggesting CD47 mediated metabolic reprogramming of T cells. Additionally, non-responding patients to anti-PD-1 therapy had increased T cells expressing CD47 and circulating levels of TSP1 compared with responding patients. Since CD47/TSP1 signaling axis negatively impacts CD8+ T cells and non-responding patients to anti-PD-1 therapy have increased CD47/TSP1 expression, we targeted CD47 in combination with anti-PD-1 in a melanoma model. Targeting CD47 in combination with anti-PD-1 treatment further decreased tumor burden compared with monotherapy and control.ConclusionCD47/TSP1 expression could serve as a marker to predict patient response to immune checkpoint blockade treatment, and targeting this pathway may preserve T cell activation, proliferation, effector function, and bioenergetics to reduce tumor burden as a monotherapy or in combination with anti-PD-1.

Tumor‐infiltrating myeloid cells are the most abundant leukocyte population within tumors. Molecular cues from the tumor microenvironment promote the differentiation of immature myeloid cells toward an immunosuppressive phenotype. However, the in situ dynamics of the transcriptional reprogramming underlying this process are poorly understood. Therefore, we applied single cell RNA‐seq (scRNA‐seq) to computationally investigate the cellular composition and transcriptional dynamics of tumor and adjacent normal tissues from 4 early‐stage non‐small cell lung cancer (NSCLC) patients. Our scRNA‐seq analyses identified 11 485 cells that varied in identity and gene expression traits between normal and tumor tissues. Among these, myeloid cell populations exhibited the most diverse changes between tumor and normal tissues, consistent with tumor‐mediated reprogramming. Through trajectory analysis, we identified a differentiation path from CD14+ monocytes to M2 macrophages (monocyte‐to‐M2). This differentiation path was reproducible across patients, accompanied by increased expression of genes (eg, MRC1/CD206, MSR1/CD204, PPARG, TREM2) with significantly enriched functions (Oxidative phosphorylation and P53 pathway) and decreased expression of genes (eg, CXCL2, IL1B) with significantly enriched functions (TNF‐α signaling via NF‐κB and inflammatory response). Our analysis further identified a co‐regulatory network implicating upstream transcription factors (JUN, NFKBIA) in monocyte‐to‐M2 differentiation, and activated ligand‐receptor interactions (eg, SFTPA1‐TLR2, ICAM1‐ITGAM) suggesting intratumoral mechanisms whereby epithelial cells stimulate monocyte‐to‐M2 differentiation. Overall, our study identified the prevalent monocyte‐to‐M2 differentiation in NSCLC, accompanied by an intricate transcriptional reprogramming mediated by specific transcriptional activators and intercellular crosstalk involving ligand‐receptor interactions.

Purpose/Objectives: We aimed to assess the predictive value of a lung cancer gene panel for the development of brain metastases. Materials/Methods: Between 2011 and 2015, 102 patients with lung cancer were prospectively enrolled in a clinical trial in which a diagnostic fine-needle aspirate was obtained. Gene expression was conducted on all samples that rendered a diagnosis of non-small cell lung cancer (NSCLC). Subsequent retrospective analysis of brain metastases-related outcomes was performed by reviewing patient electronic medical records. A competing risk multivariable regression was performed to estimate the adjusted hazard ratio for the development of brain metastases and non-brain metastases from NSCLC. Results: A total of 49 of 102 patients had died by the last follow-up. Median time of follow-up was 13 months (range 0.23–67 months). A total of 17 patients developed brain metastases. Median survival time after diagnosis of brain metastases was 3.58 months (95% confidence interval (CI) 2.17, not available). A total of 30 patients developed metastases without any evidence of brain metastases until the time of death or last follow-up. Competing risk analysis identified three genes that were downregulated differentially in the patients with brain metastases versus non-brain metastatic disease: CD37 (0.017), cystatin A (0.022), and IL-23A (0.027). Other factors associated with brain metastases include: stage T ( P ⩽ 8.3e-6) and stage N ( P= 6.8e-4). Conclusions: We have identified three genes, CD37, cystatin A, and IL-23A, for which downregulation of gene expression was associated with a greater propensity for developing brain metastases. Validation of these biomarkers could have implications on surveillance patterns in patients with brain metastases from NSCLC.

P53-binding protein 1 (53BP1) mediates DNA repair pathway choice and promotes checkpoint activation. Chromatin marks induced by DNA double-strand breaks and recognized by 53BP1 enable focal accumulation of this multifunctional repair factor at damaged chromatin. Here, we unveil an additional level of regulation of 53BP1 outside repair foci. 53BP1 movements are constrained throughout the nucleoplasm and increase in response to DNA damage. 53BP1 interacts with the structural protein NuMA, which controls 53BP1 diffusion. This interaction, and colocalization between the two proteins in vitro and in breast tissues, is reduced after DNA damage. In cell lines and breast carcinoma NuMA prevents 53BP1 accumulation at DNA breaks, and high NuMA expression predicts better patient outcomes. Manipulating NuMA expression alters PARP inhibitor sensitivity of BRCA1-null cells, end-joining activity, and immunoglobulin class switching that rely on 53BP1. We propose a mechanism involving the sequestration of 53BP1 by NuMA in the absence of DNA damage. Such a mechanism may have evolved to disable repair functions and may be a decisive factor for tumor responses to genotoxic treatments.

Introduction RNA isolation from tumor tissue is used for biomarker analyses and validation. Limited diagnostic material from small volume biopsies combined with an increasing demand for standard histologic, molecular characterization, and next generation sequencing applications often leads to limited material for research. We sought to evaluate small volume sampling of lung cancer tissue collected from a single needle pass during a diagnostic procedure and determine if it can provide RNA of acceptable quantity and quality. Methods We enrolled 140 patients with probable primary bronchogenic carcinoma and collected RNA from a dedicated FNA aspiration. Total RNA (ηg), RNA integrity number (RIN), and %Mass in base pairs were evaluated from each patient sample. A customized nanoString nCounter® 95-gene panel was used to profile the expression patterns of feature NSCLC genes. We compared gene expression patterns that distinguish lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) in our cohort with a corresponding Cancer Genome Atlas (TCGA) NSCLC datasets. Results Of the 149 patients consented. RNA-extraction was performed in 101 eligible patients. A satisfactory total RNA mass and RIN was quantified for all samples with a similar distribution among cellular subtypes. Mean %-Mass over 300 base pairs was noted for all specimens and 96% of samples met criteria to perform genetic evaluation with our commercialized gene expression assay. The FNA-derived transcriptomic results showed excellent consistency with the TCGA counterparts, and the differential expression pattern of LUAD vs LUSC subtypes were highly similar. Discussion In this study, RNA retrieval from a single-pass FNA regardless of procedural approach showed equivalence and suitability for gene expression assessments. RNA extraction from small volume samples has the potential to provide valuable material for genetic profiling.

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.

Increased regulatory T cells (Treg) after radiotherapy have been reported, but the mechanisms of their induction remain incompletely understood. TGFβ is known to foster Treg differentiation within tumors and is activated following radiotherapy. Thus, we hypothesized that TGFβ blockade would result in decreased Tregs within the irradiated tumor microenvironment. We found increased Tregs in the tumors of mice treated with focal radiotherapy and TGFβ blockade. This increase was mediated by upregulation of another TGFβ family member, activin A. In vitro, activin A secretion was increased following irradiation of mouse and human breast cancer cells, and its expression was further enhanced upon TGFβ blockade. In vivo, dual blockade of activin A and TGFβ was required to decrease intratumoral Tregs in the context of radiotherapy. This resulted in an increase in CD8+ T-cell priming and was associated with a reduced tumor recurrence rate. Combination of immune checkpoint inhibitors with the dual blockade of activin A and TGFβ led to the development of tumor-specific memory responses in irradiated breast cancer. Supporting the translational value of activin A targeting to reduce Treg-mediated immunosuppression, retrospective analysis of a public dataset of patients with breast cancer revealed a positive correlation between activin A gene expression and Treg abundance. Overall, these results shed light on an immune escape mechanism driven by activin A and suggest that dual targeting of activin A and TGFβ may be required to optimally unleash radiation-induced antitumor immunity against breast cancer.

Tumor cells require increased rates of cell metabolism to generate the macromolecules necessary to sustain proliferation. They rely heavily on NAD+ as a cofactor for multiple metabolic enzymes in anabolic and catabolic reactions. NAD+ also serves as a substrate for PARPs, sirtuins, and cyclic ADP-ribose synthases. Dysregulation of the cyclic ADP-ribose synthase CD38, the main NAD'ase in cells, is reported in multiple cancer types. This study demonstrates a novel connection between CD38, modulation of NAD+, and tumor cell metabolism in prostate cancer. CD38 expression inversely correlates with prostate cancer progression. Expressing CD38 in prostate cancer cells lowered intracellular NAD+, resulting in cell-cycle arrest and expression of p21Cip1 (CDKNA1). In parallel, CD38 diminishes glycolytic and mitochondrial metabolism, activates AMP-activated protein kinase (AMPK), and inhibits fatty acid and lipid synthesis. Pharmacologic inhibition of nicotinamide phosphoribosyltransferase (NAMPT) mimicked the metabolic consequences of CD38 expression, demonstrating similarity between CD38 expression and NAMPT inhibition. Modulation of NAD+ by CD38 also induces significant differential expression of the transcriptome, producing a gene expression signature indicative of a nonproliferative phenotype. Altogether, in the context of prostate cancer, the data establish a novel role for the CD38–NAD+ axis in the regulation of cell metabolism and development. Implications: This research establishes a mechanistic connection between CD38 and metabolic control. It also provides the foundation for the translation of agents that modulate NAD+ levels in cancer cells as therapeutics. Mol Cancer Res; 16(11); 1687–700. ©2018 AACR.

Purpose: CPI-613, a lipoate analogue that inhibits pyruvate dehydrogenase (PDH) and α-ketogluterate dehydrogenase (KGDH), has activity in patients with myeloid malignancies. This study explored the role of mitochondrial metabolism in chemotherapy response and determined the MTD, efficacy, and safety of CPI-613 combined with high-dose cytarabine and mitoxantrone in patients with relapsed or refractory acute myeloid leukemia. Experimental Design: The role of mitochondrial response to chemotherapy was assessed in cell lines and animal models. A phase I study of CPI-613 plus cytarabine and mitoxantrone was conducted in patients with relapsed or refractory AML. Results: Exposure to chemotherapy induced mitochondrial oxygen consumption that depended on PDH. CPI-613 sensitized AML cells to chemotherapy indicating that mitochondrial metabolism is a source of resistance. Loss of p53 did not alter response to CPI-613. The phase I study enrolled 67 patients and 62 were evaluable for response. The overall response rate was 50% (26CR+5CRi/62). Median survival was 6.7 months. In patients over 60 years old, the CR/CRi rate was 47% (15/32) with a median survival of 6.9 months. The response rate for patients with poor-risk cytogenetics also was encouraging with 46% (11/24 patients) achieving a CR or CRi. RNA sequencing analysis of a subset of baseline bone marrow samples revealed a gene expression signature consistent with the presence of B cells in the pretreatment marrow of responders. Conclusions: The addition of CPI-613 to chemotherapy is a promising approach in older patients and those with poor-risk cytogenetics. Clin Cancer Res; 24(9); 2060–73. ©2018 AACR.

Tissue biopsy is the standard diagnostic procedure for cancer. Biopsy may also provide material for genotyping, which can assist in the diagnosis and selection of targeted therapies but may fall short in cases of inadequate sampling, particularly from highly heterogeneous tumors. Traditional tissue biopsy suffers greater limitations in its prognostic capability over the course of disease, most obviously as an invasive procedure with potential complications, but also with respect to probable tumor clonal evolution and metastasis over time from initial biopsy evaluation. Recent work highlights circulating tumor DNA (ctDNA) present in the blood as a supplemental, or perhaps an alternative, source of DNA to identify the clinically relevant cancer mutational landscape. Indeed, this noninvasive approach may facilitate repeated monitoring of disease progression and treatment response, serving as a means to guide targeted therapies based on detected actionable mutations in patients with advanced or metastatic solid tumors. Notably, ctDNA is heralding a revolution in the range of genomic profiling and molecular mechanisms to be utilized in the battle against cancer. This review will discuss the biology of ctDNA, current methods of detection and potential applications of this information in tumor diagnosis, treatment, and disease prognosis. Conventional classification of tumors to describe cancer stage follow the TNM notation system, heavily weighting local tumor extent (T), lymph node invasion (N), and detectable metastasis (M). With recent advancements in genomics and bioinformatics, it is conceivable that routine analysis of ctDNA from liquid biopsy (B) may make cancer diagnosis, treatment, and prognosis more accurate for individual patients. We put forward the futuristic concept of TNMB tumor classification, opening a new horizon for precision medicine with the hope of creating better outcomes for cancer patients.

Background Metaplastic breast cancer (MBC) is a rare disease subtype characterized by an aggressive clinical course. MBC is commonly triple negative (TN), although hormone receptor (HR) positive and human epidermal growth receptor 2 (HER2) positive cases do occur. Previous studies have reported similar outcomes for MBC with regard to HR status. Less is known about outcomes for HER2 positive MBC. Materials and Methods Surveillance, Epidemiology, and End Results Program data were used to identify women diagnosed 2010–2014 with MBC or invasive ductal carcinoma (IDC). Kaplan-Meier curves estimated overall survival (OS) and multivariate Cox models were fitted. For survival analyses, only first cancers were included, and 2014 diagnoses were excluded to allow for sufficient follow-up. Results Our MBC sample included 1,516 women. Relative to women with IDC, women with MBC were more likely to be older (63 vs. 61 years), black (16.0% vs. 11.1%), and present with stage III disease (15.6% vs. 10.8%). HER2 positive and HER2 negative/HR positive MBC tumors represented 5.2% and 23.0% of cases. For MBC overall, 3-year OS was greatest for women with HER2 positive MBC (91.8%), relative to women with TN (75.4%) and HER2 negative/HR positive MBC (77.1%). This difference was more pronounced for stage III MBC, for which 3-year OS was 92.9%, 47.1%, and 42.2% for women with HER2 positive, TN, and HER2 negative/HR positive MBC, respectively. A multivariate Cox model of MBC demonstrated that HER2 positive tumors (relative to TN) were associated with improved survival (hazard ratio = 0.32, 95% confidence interval [CI] 0.13–0.79). In a second Cox model of exclusively HER2 positive tumors, OS did not differ between MBC and IDC disease subtypes (hazard ratio = 1.16, 95% CI 0.48–2.81). Conclusion In this contemporary, population-based study of women with MBC, HER2 but not HR status was associated with improved survival. Survival was similar between HER2 positive MBC and HER2 positive IDC. This suggests HER2 positive MBC is responsive to HER2-directed therapy, a finding that may offer insights for additional therapeutic approaches to MBC. Implications for Practice This population-based study reports recent outcomes, by receptor status, for women with metaplastic breast cancer. Survival in metaplastic breast cancer is not impacted by hormone receptor status. To the authors' knowledge, this is the first report indicating that women with human epidermal growth receptor 2 (HER2) positive metaplastic breast cancer have survival superior to women with HER2 negative metaplastic breast cancer and survival similar to women with HER2 positive invasive ductal carcinoma. This information can be used for counseling patients diagnosed with metaplastic breast cancer. Further understanding of HER2 positive metaplastic breast cancer could offer insights for the development of therapeutic approaches to metaplastic breast cancer more broadly.

Summary Background Pancreatic cancer statistics are dismal, with a 5-year survival of less than 10%, and more than 50% of patients presenting with metastatic disease. Metabolic reprogramming is an emerging hallmark of pancreatic adenocarcinoma. CPI-613 is a novel anticancer agent that selectively targets the altered form of mitochondrial energy metabolism in tumour cells, causing changes in mitochondrial enzyme activities and redox status that lead to apoptosis, necrosis, and autophagy of tumour cells. We aimed to establish the maximum tolerated dose of CPI-613 when used in combination with modified FOLFIRINOX chemotherapy (comprising oxaliplatin, leucovorin, irinotecan, and fluorouracil) in patients with metastatic pancreatic cancer. Methods In this single-centre, open-label, dose-escalation phase 1 trial, we recruited adult patients (aged ≥18 years) with newly diagnosed metastatic pancreatic adenocarcinoma from the Comprehensive Cancer Center of Wake Forest Baptist Medical Center (Winston-Salem, NC, USA). Patients had good bone marrow, liver and kidney function, and good performance status (Eastern Cooperative Oncology Group [ECOG] performance status 0–1). We studied CPI-613 in combination with modified FOLFIRINOX (oxaliplatin at 65 mg/m 2 , leucovorin at 400 mg/m 2 , irinotecan at 140 mg/m 2 , and fluorouracil 400 mg/m 2 bolus followed by 2400 mg/m 2 over 46 h). We applied a two-stage dose-escalation scheme (single patient and traditional 3+3 design). In the single-patient stage, one patient was accrued per dose level. The starting dose of CPI-613 was 500 mg/m 2 per day; the dose level was then escalated by doubling the previous dose if there were no adverse events worse than grade 2 within 4 weeks attributed as probably or definitely related to CPI-613. The traditional 3+3 dose-escalation stage was triggered if toxic effects attributed as probably or definitely related to CPI-613 were grade 2 or worse. The dose level for CPI-613 for the first cohort in the traditional dose-escalation stage was the same as that used in the last cohort of the single-patient dose-escalation stage. The primary objective was to establish the maximum tolerated dose of CPI-613 (as assessed by dose-limiting toxicities). This trial is registered with ClinicalTrials.gov, number NCT01835041, and is closed to recruitment. Findings Between April 22, 2013, and Jan 8, 2016, we enrolled 20 patients. The maximum tolerated dose of CPI-613 was 500 mg/m 2 . The median number of treatment cycles given at the maximum tolerated dose was 11 (IQR 4–19). Median follow-up of the 18 patients treated at the maximum tolerated dose was 378 days (IQR 250–602). Two patients enrolled at a higher dose of 1000 mg/m 2 , and both had a dose-limiting toxicity. Two unexpected serious adverse events occurred, both for the first patient enrolled. Expected serious adverse events were: thrombocytopenia, anaemia, and lymphopenia (all for patient number 2; anaemia and lymphopenia were dose-limiting toxicities); hyperglycaemia (in patient number 7); hypokalaemia, hypoalbuminaemia, and sepsis (patient number 11); and neutropenia (patient number 20). No deaths due to adverse events were reported. For the 18 patients given the maximum tolerated dose, the most common grade 3–4 non-haematological adverse events were hyperglycaemia (ten [55%] patients), hypokalaemia (six [33%]), peripheral sensory neuropathy (five [28%]), diarrhoea (five [28%]), and abdominal pain (four [22%]). The most common grade 3–4 haematological adverse events were neutropenia (five [28%] of 18 patients), lymphopenia (five [28%]), anaemia (four [22%], and thrombocytopenia in three [17%]). Sensory neuropathy (all grade 1–3) was recorded in 17 (94%) of the 18 patients and was managed with dose de-escalation or discontinuation per standard of care. No patients died while on active treatment; 11 study participants died, with cause of death as terminal pancreatic cancer. Of the 18 patients given the maximum tolerated dose, 11 (61%) achieved an objective (complete or partial) response. Interpretation A maximum tolerated dose of CPI-613 was established at 500 mg/m 2 when used in combination with modified FOLFIRINOX in patients with metastatic pancreatic cancer. The findings of clinical activity will require validation in a phase 2 trial. Funding Comprehensive Cancer Center of Wake Forest Baptist Medical Center.

As cancer strikes, individuals vary not only in terms of factors that contribute to its occurrence and development, but as importantly, in their capacity to respond to treatment. While exciting new therapeutic options that mobilize the immune system against cancer have led to breakthroughs for a variety of malignancies, success is limited to a subset of patients. Pre-existing immunological features of both the host and the tumor may contribute to how patients will eventually fare with immunotherapy. A broad understanding of baseline immunity, both in the periphery and in the tumor microenvironment, is needed in order to fully realize the potential of cancer immunotherapy. Such interrogation of the tumor, blood, and host immune parameters prior to treatment is expected to identify biomarkers predictive of clinical outcome as well as to elucidate why some patients fail to respond to immunotherapy. To approach these opportunities for progress, the Society for Immunotherapy of Cancer (SITC) reconvened the Immune Biomarkers Task Force. Comprised of an international multidisciplinary panel of experts, Working Group 4 sought to make recommendations that focus on the complexity of the tumor microenvironment, with its diversity of immune genes, proteins, cells, and pathways naturally present at baseline and in circulation, and novel tools to aid in such broad analyses.

Ovarian cancer is a lethal malignancy that has not seen a major therapeutic advance in over 30 years. We demonstrate that ovarian cancer exhibits a targetable alteration in iron metabolism. Ferroportin (FPN), the iron efflux pump, is decreased, and transferrin receptor (TFR1), the iron importer, is increased in tumor tissue from patients with high grade but not low grade serous ovarian cancer. A similar profile of decreased FPN and increased TFR1 is observed in a genetic model of ovarian cancer tumor initiating cells (TICs). The net result of these changes is an accumulation of excess intracellular iron and an augmented dependence on iron for proliferation. A forced reduction in intracellular iron reduces the proliferation of ovarian cancer TICs in vitro, and inhibits both tumor growth and intraperitoneal dissemination of tumor cells in vivo. Mechanistic studies demonstrate that iron increases metastatic spread by facilitating invasion through expression of matrix metalloproteases and synthesis of IL6. We show that the iron dependence of ovarian cancer tumor initiating cells renders them exquisitely sensitive in vivo to agents that induce iron-dependent cell death (ferroptosis) as well as iron chelators, and thus creates a metabolic vulnerability that can be exploited therapeutically.

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-β 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.

We sought to identify candidate biomarkers for early brain metastasis (BM) recurrence in patients who underwent craniotomy followed by adjuvant stereotactic radiosurgery. RNA sequencing was performed on eight resected brain metastasis tissue samples and revealed B-cell related genes to be highly expressed in patients who did not experience a distant brain failure and had prolonged overall survival. To translate the findings from RNA sequencing data, we performed immunohistochemistry to stain for B and T cell markers from formalin-fixed parffin-embedded tissue blocks on 13 patients. CD138 expressing plasma cells were identified and quantitatively assessed for each tumor sample. Patients’ tumor tissues that expressed high levels of CD138 plasma cells (N = 4) had a statistically significant improvement in OS compared to low levels of CD138 (N = 9) (p = 0.01). Although these findings are preliminary, the significance of CD138 expressing plasma cells within BM specimens should be investigated in a larger cohort. Immunologic markers based on resection cavity analysis could be predictive for determining patient outcomes following cavity-directed SRS.

Administration of amplitude modulated 27·12 MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown.Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF.Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by CaIntrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. FUND: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117.

INTRODUCTION. We hypothesized that engineering a combined lymph node/melanoma organoid from the same patient would allow tumor, stroma, and immune system to remain viable for personalized immunotherapy screening. METHODS. Surgically obtained matched melanoma and lymph node biospecimens from the same patient were transferred to the laboratory and washed with saline, antibiotic, and red blood cell lysis buffer. Biospecimens were dissociated, incorporated into an extracellular matrix (ECM)-based hydrogel system, and biofabricated into three dimensional (3D) mixed melanoma/node organoids. Cells were not sorted, so as to preserve tumor heterogeneity, including stroma and immune cell components, resulting in immune-enhanced patient tumor organoids (iPTOs). Organoid sets were screened in parallel with nivolumab, pembrolizumab, ipilimumab, and dabrafenib/trametinib for 72 h. LIVE/DEAD staining and quantitative metabolism assays recorded relative drug efficacy. Histology and immunohistochemistry were used to compare tumor melanoma cells with organoid melanoma cells. Lastly, node-enhanced iPTOs were employed to activate patient-matched peripheral blood T cells for killing of tumor cells in naïve PTOs. RESULTS. Ten biospecimen sets obtained from eight stage III and IV melanoma patients were reconstructed as symbiotic immune/tumor organoids between September 2017 and June 2018. Successful establishment of viable organoid sets was 90% (9/10), although organoid yield varied with biospecimen size. Average time from organoid development to initiation of immunotherapy testing was 7 days. In three patients for whom a node was not available, it was substituted with peripheral blood mononuclear cells. iPTO response to immunotherapy was similar to specimen clinical response in 85% (6/7) patients. In an additional pilot study, peripheral T cells were circulated through iPTOs, and subsequently transferred to naïve PTOs from the same patient, resulting in tumor killing, suggesting a possible role of iPTOs in generating adaptive immunity. CONCLUSION. Development of 3D mixed immune-enhanced tumor/node organoids is a feasible platform, allowing individual patient immune system and tumor cells to remain viable for studying of personalized immunotherapy response.

Correlative studies in humans have demonstrated that an active immune microenvironment characterized by the presence of a T-helper 1 immune response typifies a tumor phenotype associated with better outcome and increased responsiveness to immune manipulation. This phenotype also signifies the counter activation of immune-regulatory mechanisms. Variables modulating the development of an effective anti-tumor immune response are increasingly scrutinized as potential therapeutic targets. Genetic alterations of cancer cells that functionally influence intratumoral immune response include mutational load, specific mutations of genes involved in oncogenic pathways and copy number aberrations involving chemokine and cytokine genes. Inhibiting oncogenic pathways that prevent the development of the immune-favorable cancer phenotype may complement modern immunotherapeutic approaches.

PURPOSE: For glioblastoma (GBM), imaging response (IR) or pseudoprogression (PSP) is frequently observed after chemoradiation and may connote a favorable prognosis. With tumors categorized by the Cancer Genome Atlas Project (mesenchymal, classical, neural, and proneural) and by methylguanine-methyltransferase (MGMT) methylation status, we attempted to determine if certain genomic or molecular subtypes of GBM were specifically associated with IR or PSP. METHODS: Patients with GBM treated at two institutions were reviewed. Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS). Mantel-cox test determined effect of IR and PSP on OS and PFS. Fisher’s exact test was utilized to correlate IR and PSP with genomic subtypes and MGMT status. RESULTS: Eighty-two patients with GBM were reviewed. The median OS and PFS were 17.9 months and 8.9 months. IR was observed in 28 (40%) and was associated with improved OS (median 29.4 vs 14.5 months p < 0.01) and PFS (median 17.7 vs 5.5 months, p < 0.01). PSP was observed in 14 (19.2%) and trended towards improved PFS (15.0 vs 7.7 months p = 0.08). Tumors with a proneural component had a higher rate of IR compared to those without a proneural component (IR 60% vs 28%; p = 0.03). MGMT methylation was associated with IR (58% vs 24%, p = 0.032), but not PSP (34%, p = 0.10). CONCLUSION: IR is associated with improved OS and PFS. The proneural subtype and MGMT methylated tumors had higher rates of IR.

Yin Yang 1 (YY1) is a multifunctional protein with regulatory potential in tumorigenesis. Ample studies demonstrated the activities of YY1 in regulating gene expression and mediating differential protein modifications. However, the mechanisms underlying YY1 gene expression are relatively understudied. G-quadruplexes (G4s) are four-stranded structures or motifs formed by guanine-rich DNA or RNA domains. The presence of G4 structures in a gene promoter or the 5'-UTR of its mRNA can markedly affect its expression. In this report, we provide strong evidence showing the presence of G4 structures in the promoter and the 5'-UTR of YY1. In reporter assays, mutations in these G4 structure forming sequences increased the expression of Gaussia luciferase (Gluc) downstream of either YY1 promoter or 5'-UTR. We also discovered that G4 Resolvase 1 (G4R1) enhanced the Gluc expression mediated by the YY1 promoter, but not the YY1 5'-UTR. Consistently, G4R1 binds the G4 motif of the YY1 promoter in vitro and ectopically expressed G4R1 increased endogenous YY1 levels. In addition, the analysis of a gene array data consisting of the breast cancer samples of 258 patients also indicates a significant, positive correlation between G4R1 and YY1 expression.

Background: Cancers related to tobacco use and African-American ancestry are under-characterized by genomics. This gap in precision oncology research represents a major challenge in the health disparities in the United States. Methods: The Precision Oncology trial at the Wake Forest Baptist Comprehensive Cancer Center enrolled 431 cancer patients from March 2015 to May 2016. The composition of these patients consists of a high representation of tobacco-related cancers (e.g., lung, colorectal, and bladder) and African-American ancestry (13.5%). Tumors were sequenced to identify mutations to gain insight into genetic alterations associated with smoking and/or African-American ancestry. Results: Tobacco-related cancers exhibit a high mutational load. These tumors are characterized by high-frequency mutations in TP53, DNA damage repair genes (BRCA2 and ATM), and chromatin remodeling genes (the lysine methyltransferases KMT2D or MLL2, and KMT2C or MLL3). These tobacco-related cancers also exhibit augmented tumor heterogeneities. Smoking related genetic mutations were validated by The Cancer Genome Atlas dataset that includes 2,821 cases with known smoking status. The Wake Forest and The Cancer Genome Atlas cohorts (431 and 7,991 cases, respectively) revealed a significantly increased mutation rate in the TP53 gene in the African-American subgroup studied. Both cohorts also revealed 5 genes (e.g. CDK8) significantly amplified in the African-American population. Conclusions: These results provide strong evidence that tobacco is a major cause of genomic instability and heterogeneity in cancer. TP53 mutations and key oncogene amplifications emerge as key factors contributing to cancer outcome disparities among different racial/ethnic groups.

Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β–activated kinase-1 (TAK1) phosphorylation of NF-κB–activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB–mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.

Experimental and epidemiologic evidence suggests that dysregulation of proteins involved in iron metabolism plays a critical role in cancer. The mechanisms by which cancer cells alter homeostatic iron regulation are just beginning to be understood. Here, we demonstrate that iron regulatory protein 2 (IRP2) plays a key role in iron accumulation in breast cancer. Although both IRP1 and IRP2 are overexpressed in breast cancer, the overexpression of IRP2, but not IRP1, is associated with decreased ferritin H and increased transferrin receptor 1 (TfR1). Knockdown of IRP2 in triple-negative MDA-MB-231 human breast cancer cells increases ferritin H expression and decreases TfR1 expression, resulting in a decrease in the labile iron pool. Further, IRP2 knockdown reduces growth of MDA-MB-231 cells in the mouse mammary fat pad. Gene expression microarray profiles of patients with breast cancer demonstrate that increased IRP2 expression is associated with high-grade cancer. Increased IRP2 expression is observed in luminal A, luminal B, and basal breast cancer subtypes, but not in breast tumors of the ERBB2 molecular subtype. These results suggest that dysregulation of IRP2 is an early nodal point underlying altered iron metabolism in breast cancer and may contribute to poor outcome of some patients with breast cancer. Cancer Res; 74(2); 497–507. ©2013 AACR.

Background Molecular markers have transformed our understanding of the heterogeneity of breast cancer and have allowed the identification of genomic profiles of estrogen receptor (ER)-α signaling. However, our understanding of the transcriptional profiles of ER signaling remains inadequate. Therefore, we sought to identify the genomic indicators of ER pathway activity that could supplement traditional immunohistochemical (IHC) assessments of ER status to better understand ER signaling in the breast tumors of individual patients. Materials and Methods We reduced ESR1 (gene encoding the ER-α protein) mRNA levels using small interfering RNA in ER+ MCF7 breast cancer cells and assayed for transcriptional changes using Affymetrix HG U133 Plus 2.0 arrays. We also compared 1034 ER+ and ER− breast tumors from publicly available microarray data. The principal components of ER activity generated from these analyses and from other published estrogen signatures were compared with ESR1 expression, ER-α IHC, and patient survival. Results Genes differentially expressed in both analyses were associated with ER-α IHC and ESR1 mRNA expression. They were also significantly enriched for estrogen-driven molecular pathways associated with ESR1, cyclin D1 (CCND1), MYC (v-myc avian myelocytomatosis viral oncogene homolog), and NFKB (nuclear factor kappa B). Despite their differing constituent genes, the principal components generated from these new analyses and from previously published ER-associated gene lists were all associated with each other and with the survival of patients with breast cancer treated with endocrine therapies. Conclusion A biomarker of ER-α pathway activity, generated using ESR1-responsive mRNAs in MCF7 cells, when used alongside ER-α IHC and ESR1 mRNA expression, could provide a method for further stratification of patients and add insight into ER pathway activity in these patients.

The abundance and functional orientation of tumor-infiltrating lymphocytes in breast cancer is associated with distant metastasis-free survival, yet how this association is influenced by tumor phenotypic heterogeneity is poorly understood. Here, a bioinformatics approach defined tumor biologic attributes that influence this association and delineated tumor subtypes that may differ in their ability to sustain durable antitumor immune responses. A large database of breast tumor expression profiles and associated clinical data was compiled, from which the ability of phenotypic markers to significantly influence the prognostic performance of a classification model that incorporates immune cell–specific gene signatures was ascertained. Markers of cell proliferation and intrinsic molecular subtype reproducibly distinguished two breast cancer subtypes that we refer to as immune benefit-enabled (IBE) and immune benefit-disabled (IBD). The IBE tumors, comprised mostly of highly proliferative tumors of the basal-like, HER2-enriched, and luminal B subtypes, could be stratified by the immune classifier into significantly different prognostic groups, while IBD tumors could not, indicating the potential for productive engagement of metastasis-protective immunity in IBE tumors, but not in IBD tumors. The prognostic stratification in IBE was independent of conventional variables. Gene network analysis predicted the activation of TNFα/IFNγ signaling pathways in IBE tumors and the activation of the transforming growth factor-β pathway in IBD tumors. This prediction supports a model in which breast tumors can be distinguished on the basis of their potential for metastasis-protective immune responsiveness. Whether IBE and IBD represent clinically relevant contexts for evaluating sensitivity to immunotherapeutic agents warrants further investigation. Cancer Immunol Res; 4(7); 600–10. ©2016 AACR.

Cyclin E2, but not cyclin E1, is included in several gene signatures that predict disease progression in either tamoxifen-resistant or metastatic breast cancer. We therefore examined the role of cyclin E2 in antiestrogen resistance in vitro and its potential for therapeutic targeting through cyclin-dependent kinase (CDK) inhibition. High expression of CCNE2, but not CCNE1, was characteristic of the luminal B and HER2 subtypes of breast cancer and was strongly predictive of shorter distant metastasis-free survival following endocrine therapy. After antiestrogen treatment of MCF-7 breast cancer cells, cyclin E2 mRNA and protein were downregulated and cyclin E2–CDK2 activity decreased. However, this regulation was lost in tamoxifen-resistant (MCF-7 TAMR) cells, which overexpressed cyclin E2. Expression of either cyclin E1 or E2 in T-47D breast cancer cells conferred acute antiestrogen resistance, suggesting that cyclin E overexpression contributes to the antiestrogen resistance of tamoxifen-resistant cells. Ectopic expression of cyclin E1 or E2 also reduced sensitivity to CDK4, but not CDK2, inhibition. Proliferation of tamoxifen-resistant cells was inhibited by RNAi-mediated knockdown of cyclin E1, cyclin E2, or CDK2. Furthermore, CDK2 inhibition of E-cyclin overexpressing cells and tamoxifen-resistant cells restored sensitivity to tamoxifen or CDK4 inhibition. Cyclin E2 overexpression is therefore a potential mechanism of resistance to both endocrine therapy and CDK4 inhibition. CDK2 inhibitors hold promise as a component of combination therapies in endocrine-resistant disease as they effectively inhibit cyclin E1 and E2 overexpressing cells and enhance the efficacy of other therapeutics. Mol Cancer Ther; 11(7); 1488–99. ©2012 AACR.

Background Appendiceal cancer (AC) patients treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) often demonstrate an unpredictable variability in their survival outcomes. Biomarkers predictive of CRS/HIPEC efficacy could better guide treatment decisions. We hypothesized that variation in the transcriptional programming of AC tumors might distinguish molecular subtypes with differential outcomes after CRS/HIPEC. Study Design Gene expression profiles of 2 AC cohorts were analyzed using Affymetrix whole-genome expression microarrays. Hierarchical clustering methods, Kaplan-Meier analysis, and Cox regression models were used to discover and validate prognostic molecular subtypes of AC. Gene set enrichment analysis was used to infer pathologic attributes of the molecular subtypes. Results Unsupervised hierarchical clustering analysis of tumor expression profiles revealed a 139-gene cassette that distinguished 2 molecular subtypes (based on low vs high expression of the gene cassette) with statistically significant survival differences (disease-specific survival, p = 0.0075; progression-free survival, p = 0.0072). In a second AC cohort, the 139-gene cassette reproducibly partitioned tumors into subtypes with significant survival differences. Tumors showing high relative expression of the genes comprising the cassette associated with poor survival outcomes (disease-specific survival, p = 0.047; progression-free survival, p = 0.0079), and exhibited gene expression patterns enriched for oncogenic processes and pathways. The prognostic value of the molecular subtypes was specific for low-grade appendiceal tumors (disease-specific survival, p = 0.028; progression-free survival, p = 0.0016), and remained significant in the presence of conventional prognostic markers, including grade, surgical resection score, Eastern Cooperative Oncology Group status, and age. Conclusions The 139-gene cassette can have actionable clinical utility for identifying low-grade appendiceal tumor molecular subtypes predictive of therapeutic efficacy of CRS/HIPEC.

BACKGROUND Y-box binding factor 1 (YB-1) has been associated with prognosis in many tumor types. Reduced YB-1 expression inhibits tumor cell growth, but the mechanism is unclear. METHODS YB-1 mRNA levels were compared with tumor grade and histology using microarray data from 771 breast cancer patients and with disease-free survival and distant metastasis-free survival using data from 375 of those patients who did not receive adjuvant therapy. Microarrays were further searched for genes that had correlated expression with YB-1 mRNA. Small interfering RNA (siRNA) was used to study the effects of reduced YB-1 expression on growth of three tumor cell lines (MCF-7 breast, HCT116 colon, and A549 lung cancer cells), on tumorigenesis by A549 cells in nude mice, and on global transcription in the three cancer cell lines. Reporter gene assays were used to determine whether YB-1 siRNAs affected the expression of E2F1, and chromatin immunoprecipitation was used to determine whether YB-1 bound to various E2F promoters as well as E2F1-regulated promoters. All P values were from two-sided tests. RESULTS YB-1 levels were elevated in more aggressive tumors and were strongly associated with poor disease-free survival and distant metastasis-free survival. YB-1 expression was often associated with the expression of genes with E2F sites in their promoters. Cells expressing YB-1 siRNA grew substantially more slowly than control cells and formed tumors less readily in nude mice. Transcripts that were altered in cancer cell lines with YB-1 siRNA included 32 genes that are components of prognostic gene expression signatures. YB-1 regulated expression of an E2F1 promoter-reporter construct in A549 cells (eg, relative E2F1 promoter activity with control siRNA = 4.04; with YB-1 siRNA = 1.40, difference= -2.64, 95% confidence interval = -3.57 to -1.71, P < .001) and bound to the promoters of several well-defined E2F1 target genes. CONCLUSION YB-1 expression is associated with the activity of E2F transcription factors and may control tumor cell growth by this mechanism.

Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole-genome data-mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal-type 1 (SPINK1) to be associated with poor survival in estrogen receptor-positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER- cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down-regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug-induced apoptosis due to reduced caspase-3 levels and high expression of Bcl2 and phospho-Bcl2 proteins. Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.

Purpose: East-Asian (EA) patients with non–small-cell lung cancer (NSCLC) are associated with a high proportion of nonsmoking women, epidermal growth factor receptor (EGFR)-activating somatic mutations, and clinical responses to tyrosine kinase inhibitors. We sought to identify novel molecular differences between NSCLCs from EA and Western European (WE) patients. Experimental Design: A total of 226 lung adenocarcinoma samples from EA (n = 90) and WE (n = 136) patients were analyzed for copy number aberrations (CNA) by using a common high-resolution SNP (single nucleotide polymorphism) microarray platform. Univariate and multivariate analyses were carried out to identify CNAs specifically related to smoking history, EGFR mutation status, and ethnicity. Results: The overall genomic profiles of adenocarcinomas from EA and WE patients were highly similar. Univariate analyses revealed several CNAs significantly associated with ethnicity, EGFR mutation, and smoking, but not to gender, and KRAS or p53 mutations. A multivariate model identified four ethnic-specific recurrent CNAs—significantly higher rates of copy number gain were observed on 16p13.13 and 16p13.11 in EA tumors, whereas higher rates of genomic loss on 19p13.3 and 19p13.11 were observed in tumors from WE patients. We identified several potential driver genes in these regions, showing a positive correlation between cis-localized copy number changes and transcriptomic changes. Conclusion: 16p copy number gains (EA) and 19p losses (WE) are ethnic-specific chromosomal aberrations in lung adenocarcinoma. Patient ethnicity should be considered when evaluating future NSCLC therapies targeting genes located on these areas. Clin Cancer Res; 17(11); 3542–50. ©2011 AACR.

We have previously identified an oncogenic role of artemin (ARTN), a member of glial cell derived neurotrophic factor family of ligands, in mammary carcinoma. We herein report that ARTN is an estrogen-inducible gene. Meta-analysis of gene expression data sets showed that ARTN expression is positively correlated to estrogen receptor (ER) status in human mammary carcinoma. Furthermore, in patients with ER-positive mammary carcinoma treated with tamoxifen, high ARTN expression is significantly correlated with decreased survival. Forced expression of ARTN in ER-positive human mammary carcinoma cells increased ER transcriptional activity, promoted estrogen-independent growth and produced resistance to tamoxifen and fulvestrant in vitro and to tamoxifen in xenograft models. ARTN-stimulated resistance to tamoxifen and fulvestrant is mediated by increased BCL-2 expression. Conversely, depletion of endogenous ARTN by small-interfering RNA or functional antagonism of ARTN by antibody enhanced the efficacy of antiestrogens. Tamoxifen decreased ARTN expression in tamoxifen-sensitive mammary carcinoma cells whereas ARTN expression was increased in tamoxifen-resistant cells and not affected by tamoxifen treatment. Antibody inhibition of ARTN in tamoxifen-resistant cells improved tamoxifen sensitivity. Functional antagonism of ARTN therefore warrants consideration as an adjuvant therapy to enhance antiestrogen efficacy in ER-positive mammary carcinoma.

Estrogen receptors (ER) and nuclear factor-kappaB (NF-kappaB) are known to play important roles in breast cancer, but these factors are generally thought to repress each other's activity. However, we have recently found that ER and NF-kappaB can also act together in a positive manner to synergistically increase gene transcription. To examine the extent of cross-talk between ER and NF-kappaB, a microarray study was conducted in which MCF-7 breast cancer cells were treated with 17beta-estradiol (E(2)), tumor necrosis factor alpha (TNFalpha), or both. Follow-up studies with an ER antagonist and NF-kappaB inhibitors show that cross-talk between E(2) and TNFalpha is mediated by these two factors. We find that although transrepression between ER and NF-kappaB does occur, positive cross-talk is more prominent with three gene-specific patterns of regulation: (a) TNFalpha enhances E(2) action on approximately 30% of E(2)-upregulated genes; (b) E(2) enhances TNFalpha activity on approximately 15% of TNFalpha-upregulated genes; and (c) E(2) + TNFalpha causes a more than additive upregulation of approximately 60 genes. Consistent with their prosurvival roles, ER and NF-kappaB and their target gene, BIRC3, are involved in protecting breast cancer cells against apoptosis. Furthermore, genes positively regulated by E(2) + TNFalpha are clinically relevant because they are enriched in luminal B breast tumors and their expression profiles can distinguish a cohort of patients with poor outcome following endocrine treatment. Taken together, our findings suggest that positive cross-talk between ER and NF-kappaB is more extensive than anticipated and that these factors may act together to promote survival of breast cancer cells and progression to a more aggressive phenotype.

Cancer/testis (CT) genes are predominantly expressed in human germ line cells, but not somatic tissues, and frequently become activated in different cancer types. Several CT antigens have already proved to be useful biomarkers and are promising targets for therapeutic cancer vaccines. The aim of the present study was to investigate the expression of CT antigens in breast cancer. Using previously generated massively parallel signature sequencing (MPSS) data, together with 9 publicly available gene expression datasets, the expression pattern of CT antigens located on the X chromosome (CT-X) was interrogated. Whereas a minority of unselected breast cancers was found to contain CT-X transcripts, a significantly higher expression frequency was detected in estrogen and progesterone receptor (ER) negative breast cancer cell lines and primary breast carcinomas. A coordinated pattern of CT-X antigen expression was observed, with MAGEA and NY-ESO-1/CTAG1B being the most prevalent antigens. Immunohistochemical staining confirmed the correlation of CT-X antigen expression and ER negativity in breast tumors and demonstrated a trend for their coexpression with basal cell markers. Because of the limited therapeutic options for ER-negative breast cancers, vaccines based on CT-X antigens might prove to be useful.

Previous studies showed that expression of the novel candidate tumor suppressor gene, DEC1 (Deleted in Esophageal Cancer 1), is reduced in esophageal carcinoma and suppresses cancer cell growth in vitro and tumor growth in vivo in nude mice. This study shows that DEC1 gene expression was downregulated in 100% of 16 esophageal squamous cell carcinoma (ESCC) cell lines and 52 and 45%, respectively, of esophageal tumor specimens from Hong Kong and a high-risk ESCC region of Henan, China. Using epitope tagging, the DEC1 protein was localized to both the cytoplasm and nucleus of the cell. In 3D Matrigel culture, no significant difference in colony numbers formed was observed for DEC1 stable transfectants, as compared to vector-alone transfectant controls. However, significantly smaller colony sizes were observed for the DEC1 transfectants. In in vitro cell migration, invasion and soft agar assays of DEC1 transfectants, only the soft agar assay showed statistically significant differences in colony numbers with the vector-alone controls, indicating that DEC1 may be involved in anchorage-independent cell growth. In addition, the global gene expression affected by DEC1 in tumor-suppressive stable transfectants was investigated using cDNA oligonucleotide microarray hybridization. Three candidate genes, TFPI-2, GDF15 and DUSP6, were identified through this approach; they are downregulated in tumor segregants of DEC1 stable transfectants, ESCC cell lines and esophageal tumors and have a potential role in tumor growth and progression. These studies show that DEC1 is involved in esophageal cancer development and help elucidate its functional role in tumor development.

Purpose: Women with metastatic triple negative breast cancer (TNBC) can have a poor prognosis with treatment limited to cytotoxic chemotherapy. The identification of effective therapies that may limit exposure to cytotoxic chemotherapy and lead to prolonged survival is an unmet medical need. We tested an inhibitor of the epidermal growth factor receptor, panitumumab in combination with chemotherapy. Methods: We conducted a single arm clinical trial in women with metastatic or locally advanced TNBC to paclitaxel 80 mg/m2 and carboplatin AUC of 2 on days 1, 8, and 15 and panitumumab 6 mg/kg on days 1 and 15 for a cycle length of 28 days. The objectives were to evaluate the response rate and safety of the combination in comparison to historical controls. Results: Fourteen patients with TNBC were enrolled with a median age of 53 years. The majority of women were African American (64.3%) with visceral metastasis (64.2%). Hematologic toxicities, particularly neutropenia and thrombocytopenia, were a major cause of missed chemotherapy and delayed treatment in this study. The overall response rate (complete and partial response) of the 13 evaluable patients was 46%. The median time to best response was 2.4 months and the median time to disease progression was 3.6 months. We were able to perform the PAM50 analysis on tumors from 7 of our subjects. All the samples tested clustered within the basal-like subtype. Conclusions: In our experience the response rate of carboplatin, paclitaxel and panitumumab was consistent with other reports of response for cytotoxic chemotherapy in metastatic TNBC.

Purpose: Estrogen-related receptor alpha (ERRα) signaling has recently been implicated in breast cancer. We investigated the clinical value of ERRα in randomized cohorts of tamoxifen-treated and adjuvant-untreated patients. Experimental Design: Cox proportional hazards regression was used to evaluate the significance of associations between ERRα gene expression levels and patient DMFS in a previously published microarray dataset representing 2,000 breast tumor cases derived from multiple medical centers worldwide. The 912 tumors used for immunostaining were from a tamoxifen-randomized primary breast cancer trial conducted in Stockholm, Sweden, during 1976–1990. Mouse model was used to study the effect of tamoxifen treatment on lung colonization of MDA-MB-231 control cells and MDA-MB-231 cells with stable knockdown of ERRα. The phenotypic effects associated with ERRα modulation were studied using immunoblotting analyses and wound-healing assay. Results: We found that in ER-negative and triple-negative breast cancer (TNBC) adjuvant-untreated patients, ERRα expression indicated worse prognosis and correlated with poor outcome predictors. However, in tamoxifen-treated patients, an improved outcome was observed with high ERRα gene and protein expression. Reduced ERRα expression was oncogenic in the presence of tamoxifen, measured by in vitro proliferation and migration assays and in vivo metastasis studies. Conclusions: Taken together, these data show that ERRα expression predicts response to tamoxifen treatment, and ERRα could be a biomarker of tamoxifen sensitivity and a prognostic factor in TNBC. Clin Cancer Res; 22(6); 1421–31. ©2015 AACR.

Hepcidin is a circulating peptide hormone made by the liver that is a central regulator of systemic iron uptake and recycling. Here, we report that prostate epithelial cells also synthesize hepcidin, and that synthesis and secretion of hepcidin are markedly increased in prostate cancer cells and tissue. Prostatic hepcidin functions as an autocrine hormone, decreasing cell surface ferroportin, an iron exporter, increasing intracellular iron retention, and promoting prostate cancer cell survival. Synthesis of hepcidin in prostate cancer is controlled by a unique intersection of pathways that involves BMP4/7, IL6, Wnt, and the dual BMP and Wnt antagonist, SOSTDC1. Epigenetic silencing of SOSTDC1 through methylation is increased in prostate cancer and is associated with accelerated disease progression in patients with prostate cancer. These results establish a new connection between iron metabolism and prostate cancer, and suggest that prostatic dysregulation of hepcidin contributes to prostate cancer growth and progression. Cancer Res; 75(11); 2254–63. ©2015 AACR.

Yin Yang 1 (YY1) regulates both gene expression and protein modifications, and has shown a proliferative role in cancers. In this study, we demonstrate that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. YY1 expression positively correlated with AKT(S473) phosphorylation in a tissue microarray and cultured cells of breast cancer, but negatively associated with the distant metastasis-free survival of 166 breast cancer patients. YY1 promotes AKT phosphorylation at S473 through direct interaction with AKT, and the AKT-binding site is mapped to the residues G201-S226 on YY1. These residues are also involved in YY1 interaction with Mdm2, Ezh2, and E1A, and thus are designated as the oncogene protein binding (OPB) domain. YY1-promoted AKT phosphorylation relies on the OPB domain but is independent of either transcriptional activity of YY1 or the activity of phosphoinositide-3-kinases. We also determine that YY1-promoted mTORC2 access to AKT leads to its phosphorylation at S473. Importantly, a peptide based on the OPB domain blocks YY1 interaction with AKT and reduces AKT phosphorylation and cell proliferation. Thus, we demonstrate for the first time that YY1 promotes mTORC2-mediated AKT activation and disrupting YY1-AKT interaction by OPB domain-based peptide may represent a potential strategy for cancer therapy.

We present a retrospective investigation of the role of genomics in the prediction of central versus marginal disease progression patterns for glioblastoma (GBM). Between August 2000 and May 2010, 41 patients with GBM and gene expression and methylation data available were treated with radiotherapy with or without concurrent temozolomide. Location of disease progression was categorized as within the high dose (60 Gy) or low dose (46 Gy) volume. Samples were grouped into previously described TCGA genomic groupings: Mesenchymal (m), classical (c), proneural (pn), and neural (n); and were also classified by MGMT-Methylation status and G-Cimp methylation phenotype. Genomic groupings and methylation status were investigated as a possible predictor of disease progression in the high dose region, progression in the low dose region, and time to progression. Based on TCGA category there was no difference in OS (p = 0.26), 60 Gy progression (PN: 71 %, N: 60 %, M: 89 %, C: 83 %, p = 0.19), 46 Gy progression (PN: 57 %, N: 40 %, M: 61 %, C: 50 %, p = 0.8) or time to progression (PN: 9 months, N:15 months, M: 9 months, C: 7 months, p = 0.58). MGMT methylation predicted for improved OS (median 25 vs. 13 months, p = 0.01), improved DFS (median 13 vs. 8 months, p = 0.007) and decreased 60 Gy (p = 0.003) and 46 Gy (p = 0.006) progression. There was a cohort of MGMT methylated patients with late marginal disease progression (4/22 patients, 18 %). TCGA groups demonstrated no difference in survival or progression patterns. MGMT methylation predicted for a statistically significant decrease in in-field and marginal disease progression. There was a cohort of MGMT methylated patients with late marginal progression. Validations of these findings would have implications that could affect radiation field size.