Circulating microRNA Panel for Prediction of Recurrence and Survival in Early-Stage Lung Adenocarcinoma.

Early-stage lung adenocarcinoma (LUAD) patients remain at substantial risk for recurrence and disease-related death, highlighting the unmet need of biomarkers for the assessment and identification of those in an early stage who would likely benefit from adjuvant chemotherapy. To identify circulating miRNAs useful for predicting recurrence in early-stage LUAD, we performed miRNA microarray analysis with pools of pretreatment plasma samples from patients with stage I LUAD who developed recurrence or remained recurrence-free during the follow-up period. Subsequent validation in 85 patients with stage I LUAD resulted in the development of a circulating miRNA panel comprising miR-23a-3p, miR-320c, and miR-125b-5p and yielding an area under the curve (AUC) of 0.776 in predicting recurrence. Furthermore, the three-miRNA panel yielded an AUC of 0.804, with a sensitivity of 45.8% at 95% specificity in the independent test set of 57 stage I and II LUAD patients. The miRNA panel score was a significant and independent factor for predicting disease-free survival (p < 0.001, hazard ratio [HR] = 1.64, 95% confidence interval [CI] = 1.51-4.22) and overall survival (p = 0.001, HR = 1.51, 95% CI = 1.17-1.94). This circulating miRNA panel is a useful noninvasive tool to stratify early-stage LUAD patients and determine an appropriate treatment plan with maximal efficacy.

Long non-coding RNA TILR constitutively represses TP53 and apoptosis in lung cancer.

Non-coding RNAs have an integral regulatory role in numerous functions related to lung cancer development. Here, we report identification of a novel lncRNA, termed TP53-inhibiting lncRNA (TILR), which was found to function as a constitutive negative regulator of p53 expression, including activation of downstream genes such as p21 and MDM2, and induction of apoptosis. A proteomic search for TILR-associated proteins revealed an association with PCBP2, while the mid-portion of TILR was found to be required for both PCBP2 and p53 mRNA binding. In addition, depletion of PCBP2 resulted in phenocopied effects of TILR silencing. TILR was also shown to suppress p53 expression in a post-transcriptional manner, as well as via a positive feedback loop involving p53 and Fanconi anemia pathway genes. Taken together, the present findings clearly demonstrate that TILR constitutively inhibits p53 expression in cooperation with PCBP2, thus maintaining p53 transcriptional activity at a level sufficiently low for avoidance of spurious apoptosis induction.

A Comprehensive Search of Non-Canonical Proteins in Non-Small Cell Lung Cancer and Their Impact on the Immune Response.

There is substantial interest in mining neoantigens for cancer applications. Non-canonical proteins resulting from frameshift mutations have been identified as neoantigens in cancer. We investigated the landscape of non-canonical proteins in non-small cell lung cancer (NSCLC) and their induced immune response in the form of autoantibodies. A database of cryptoproteins was computationally constructed and comprised all alternate open reading frames (altORFs) and ORFs identified in pseudogenes, noncoding RNAs, and untranslated regions of mRNAs that did not align with known canonical proteins. Proteomic profiles of seventeen lung adenocarcinoma (LUAD) cell lines were searched to evaluate the occurrence of cryptoproteins. To assess the immunogenicity, immunoglobulin (Ig)-bound cryptoproteins in plasmas were profiled by mass spectrometry. The specimen set consisted of plasmas from 30 newly diagnosed NSCLC cases, pre-diagnostic plasmas from 51 NSCLC cases, and 102 control plasmas. An analysis of LUAD cell lines identified 420 cryptoproteins. Plasma Ig-bound analyses revealed 90 cryptoproteins uniquely found in cases and 14 cryptoproteins that had a fold-change >2 compared to controls. In pre-diagnostic samples, 17 Ig-bound cryptoproteins yielded an odds ratio ≥2. Eight Ig-bound cryptoproteins were elevated in both pre-diagnostic and newly diagnosed cases compared to controls. Cryptoproteins represent a class of neoantigens that induce an autoantibody response in NSCLC.

Mutational Activation of the NRF2 Pathway Upregulates Kynureninase Resulting in Tumor Immunosuppression and Poor Outcome in Lung Adenocarcinoma.

Activation of the NRF2 pathway through gain-of-function mutations or loss-of-function of its suppressor KEAP1 is a frequent finding in lung cancer. NRF2 activation has been reported to alter the tumor microenvironment. Here, we demonstrated that NRF2 alters tryptophan metabolism through the kynurenine pathway that is associated with a tumor-promoting, immune suppressed microenvironment. Specifically, proteomic profiles of 47 lung adenocarcinoma (LUAD) cell lines (11 KEAP1 mutant and 36 KEAP1 wild-type) revealed the tryptophan-kynurenine enzyme kynureninase (KYNU) as a top overexpressed protein associated with activated NRF2. The siRNA-mediated knockdown of NFE2L2, the gene encoding for NRF2, or activation of the NRF2 pathway through siRNA-mediated knockdown of KEAP1 or via chemical induction with the NRF2-activator CDDO-Me confirmed that NRF2 is a regulator of KYNU expression in LUAD. Metabolomic analyses confirmed KYNU to be enzymatically functional. Analysis of multiple independent gene expression datasets of LUAD, as well as a LUAD tumor microarray demonstrated that elevated KYNU was associated with immunosuppression, including potent induction of T-regulatory cells, increased levels of PD1 and PD-L1, and resulted in poorer survival. Our findings indicate a novel mechanism of NRF2 tumoral immunosuppression through upregulation of KYNU.

SRGN-Triggered Aggressive and Immunosuppressive Phenotype in a Subset of TTF-1-Negative Lung Adenocarcinomas.

BACKGROUND: Approximately 20% of lung adenocarcinoma (LUAD) is negative for the lineage-specific oncogene Thyroid transcription factor 1 (TTF-1) and exhibits worse clinical outcome with a low frequency of actionable genomic alterations. To identify molecular features associated with TTF-1-negative LUAD, we compared the transcriptomic and proteomic profiles of LUAD cell lines. SRGN , a chondroitin sulfate proteoglycan Serglycin, was identified as a markedly overexpressed gene in TTF-1-negative LUAD. We therefore investigated the roles and regulation of SRGN in TTF-1-negative LUAD. METHODS: Proteomic and metabolomic analyses of 41 LUAD cell lines were done using mass spectrometry. The function of SRGN was investigated in 3 TTF-1-negative and 4 TTF-1-positive LUAD cell lines and in a syngeneic mouse model (n = 5 to 8 mice per group). Expression of SRGN was evaluated in 94 and 105 surgically resected LUAD tumor specimens using immunohistochemistry. All statistical tests were 2-sided. RESULTS: SRGN was markedly overexpressed at mRNA and protein levels in TTF-1-negative LUAD cell lines (P < .001 for both mRNA and protein levels). Expression of SRGN in LUAD tumor tissue was associated with poor outcome (hazard ratio = 4.22, 95% confidence interval = 1.12 to 15.86, likelihood ratio test, P = .03), and with higher expression of Programmed cell death 1 ligand 1 (PD-L1) in tumor cells and higher infiltration of Programmed cell death protein 1-positive lymphocytes. SRGN regulated expression of PD-L1 as well as proinflammatory cytokines, including Interleukin-6, Interleukin-8, and C-X-C motif chemokine 1 in LUAD cell lines; increased migratory and invasive properties of LUAD cells and fibroblasts; and enhanced angiogenesis. SRGN was induced by DNA demethylation resulting from Nicotinamide N-methyltransferase-mediated impairment of methionine metabolism. CONCLUSIONS: Our findings suggest that SRGN plays a pivotal role in tumor-stromal interaction and reprogramming into an aggressive and immunosuppressive tumor microenvironment in TTF-1-negative LUAD.

Identification of Blood-Based Biomarkers for the Prediction of the Response to Neoadjuvant Chemoradiation in Rectal Cancer.

The current standard of care for patients with locally advanced rectal cancer (LARC) is neoadjuvant chemoradiation (nCRT) followed by total mesorectal excision surgery. However, the response to nCRT varies among patients and only about 20% of LARC patients achieve a pathologic complete response (pCR) at the time of surgery. Therefore, there is an unmet need for biomarkers that could predict the response to nCRT at an early time point, allowing for the selection of LARC patients who would or would not benefit from nCRT. To identify blood-based biomarkers for prediction of nCRT response, we performed in-depth quantitative proteomic analysis of pretreatment plasma from mice bearing rectal tumors treated with concurrent chemoradiation, resulting in the quantification of 567 proteins. Among the plasma proteins that increased in mice with residual rectal tumor after chemoradiation compared to mice that achieved regression, we selected three proteins (Vascular endothelial growth factor receptor 3 [VEGFR3], Insulin like growth factor binding protein 4 [IGFBP4], and Cathepsin B [CTSB]) for validation in human plasma samples. In addition, we explored whether four tissue protein biomarkers previously shown to predict response to nCRT (Epidermal growth factor receptor [EGFR], Ki-67, E-cadherin, and Prostaglandin G/H synthase 2 [COX2]) also act as potential blood biomarkers. Using immunoassays for these seven biomarker candidates as well as Carcinoembryonic antigen [CEA] levels on plasma collected before nCRT from 34 patients with LARC (6 pCR and 28 non-pCR), we observed that levels of VEGFR3 (p = 0.0451, AUC = 0.720), EGFR (p = 0.0128, AUC = 0.679), and COX2 (p = 0.0397, AUC = 0.679) were significantly increased in the plasma of non-pCR LARC patients compared to those of pCR LARC patients. The performance of the logistic regression model combining VEGFR3, EGFR, and COX2 was significantly improved compared with the performance of each biomarker, yielding an AUC of 0.869 (sensitivity 43% at 95% specificity). Levels of VEGFR3 and EGFR were significantly decreased 5 to 7 months after tumor resection in plasma from 18 surgically resected rectal cancer patients, suggesting that VEGFR3 and EGFR may emanate from tumors. These findings suggest that circulating VEGFR3 can contribute to the prediction of the nCRT response in LARC patients together with circulating EGFR and COX2.

Bioinformatics and Functional Analyses Implicate Potential Roles for EOGT and L-fringe in Pancreatic Cancers.

Notch signaling receptors, ligands, and their downstream target genes are dysregulated in pancreatic ductal adenocarcinoma (PDAC), suggesting a role of Notch signaling in pancreatic tumor development and progression. However, dysregulation of Notch signaling by post-translational modification of Notch receptors remains poorly understood. Here, we analyzed the Notch-modifying glycosyltransferase involved in the regulation of the ligand-dependent Notch signaling pathway. Bioinformatic analysis revealed that the expression of epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) and Lunatic fringe (LFNG) positively correlates with a subset of Notch signaling genes in PDAC. The lack of EOGT or LFNG expression inhibited the proliferation and migration of Panc-1 cells, as observed by the inhibition of Notch activation. EOGT expression is significantly increased in the basal subtype, and low expression of both EOGT and LFNG predicts better overall survival in PDAC patients. These results imply potential roles for EOGT- and LFNG-dependent Notch signaling in PDAC.

Conditional Ror1 knockout reveals crucial involvement in lung adenocarcinoma development and identifies novel HIF-1α regulator.

We previously reported that ROR1 is a crucial downstream gene for the TTF-1/NKX2-1 lineage-survival oncogene in lung adenocarcinoma, while others have found altered expression of ROR1 in multiple cancer types. Accumulated evidence therefore indicates ROR1 as an attractive molecular target, though it has yet to be determined whether targeting Ror1 can inhibit tumor development and growth in vivo. To this end, genetically engineered mice carrying homozygously floxed Ror1 alleles and an SP-C promoter-driven human mutant EGFR transgene were generated. Ror1 ablation resulted in marked retardation of tumor development and progression in association with reduced malignant characteristics and significantly better survival. Interestingly, gene set enrichment analysis identified a hypoxia-induced gene set (HALLMARK_HYPOXIA) as most significantly downregulated by Ror1 ablation in vivo, which led to findings showing that ROR1 knockdown diminished HIF-1α expression under normoxia and clearly hampered HIF-1α induction in response to hypoxia in human lung adenocarcinoma cell lines. The present results directly demonstrate the importance of Ror1 for in vivo development and progression of lung adenocarcinoma, and also identify Ror1 as a novel regulator of Hif-1α. Thus, a future study aimed at the development of a novel therapeutic targeting ROR1 for treatment of solid tumors such as seen in lung cancer, which are frequently accompanied with a hypoxic tumor microenvironment, is warranted.

Inhibition of heat shock protein 90 destabilizes receptor tyrosine kinase ROR1 in lung adenocarcinoma.

We have previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a direct transcriptional target of TTF-1/NKX2-1, a lineage-survival oncogene in lung adenocarcinoma. ROR1 sustains prosurvival signaling from multiple receptor tyrosine kinases including epidermal growth factor receptor, MET, and insulin-like growth factor 1 receptor in part by maintaining the caveolae structure as a scaffold protein of cavin-1 and caveolin-1. In this study, a high throughput screening of the natural product library containing 2560 compounds was undertaken using a cell-based FluoPPI assay detecting ROR1-cavin-1 interaction. As a result, geldanamycin (GA), a known inhibitor of heat shock protein 90 (HSP90), was identified as a potential inhibitor of ROR1. Geldanamycin, as well as two GA derivatives tested in the clinic, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreased ROR1 protein expression. We found that ROR1 physically interacted with HSP90α, but not with other HSP90 paralogs, HSP90ß or GRP94. Geldanamycin in turn destabilized and degraded ROR1 protein in a dose- and time-dependent manner through the ubiquitin/proteasome pathway, resulting in a significant suppression of cell proliferation in lung adenocarcinoma cell lines, for which the kinase domain of ROR1, but not its kinase activity or N-glycosylation, was required. Our findings indicate that HSP90 is required to sustain expression of ROR1 crucial for lung adenosarcoma survival, suggesting that inhibition of HSP90 could be a promising therapeutic strategy in ROR1-positive lung adenocarcinoma.

ST6GAL1 Is a Novel Serum Biomarker for Lenvatinib-Susceptible FGF19-Driven Hepatocellular Carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is characterized by high intertumor heterogeneity of genetic drivers. Two multitarget tyrosine kinase inhibitors (TKI), lenvatinib and sorafenib, are used as standard-of-care chemotherapeutics in patients with advanced HCC, but a stratification strategy has not been established because of a lack of efficacious biomarkers. Therefore, we sought biomarkers that indicate lenvatinib-susceptible HCC. EXPERIMENTAL DESIGN: We performed genetic screening of HCC driver genes involved in TKI susceptibility using a novel HCC mouse model in which tumor diversity of genetic drivers was recapitulated. A biomarker candidate was evaluated in human HCC cell lines. Secreted proteins from HCC cells were then screened using mass spectrometry. Serum and tumor levels of the biomarker candidates were analyzed for their association and prediction of overall survival in patients with HCC. RESULTS: We found that lenvatinib selectively eliminated FGF19-expressing tumors, whereas sorafenib eliminated MET- and NRAS-expressing tumors. FGF19 levels and lenvatinib susceptibility were correlated in HCC cell lines, and FGF19 inhibition eliminated lenvatinib susceptibility. Lenvatinib-resistant HCC cell lines, generated by long-term exposure to lenvatinib, showed FGF19 downregulation but were resensitized to lenvatinib by FGF19 reexpression. Thus, FGF19 is a tumor biomarker of lenvatinib-susceptible HCC. Proteome and secretome analyses identified ST6GAL1 as a tumor-derived secreted protein positively regulated by FGF19 in HCC cells. Serum ST6GAL1 levels were positively correlated with tumor FGF19 expression in patients with surgically resected HCC. Among patients with serum ST6GAL1-high HCC who underwent TKI therapy, lenvatinib therapy showed significantly better survival than sorafenib. CONCLUSIONS: Serum ST6GAL may be a novel biomarker that identifies lenvatinib-susceptible FGF19-driven HCC.

Novel urinary protein biomarker panel for early diagnosis of gastric cancer.

BACKGROUND: With the goal of discovering non-invasive biomarkers for early diagnosis of GC, we conducted a case-control study utilising urine samples from individuals with predominantly early GC vs. healthy control (HC). METHODS: Among urine samples from 372 patients, age- and sex-matched 282 patients were randomly divided into three groups: 18 patients in a discovery cohort; 176 patients in a training cohort and 88 patients in a validation cohort. RESULTS: Among urinary proteins identified in the comprehensive quantitative proteomics analysis, urinary levels of TFF1 (uTFF1) and ADAM12 (uADAM12) were significantly independent diagnostic biomarkers for GC, in addition to Helicobacter pylori status. A urinary biomarker panel combining uTFF1, uADAM12 and H. pylori significantly distinguished between HC and GC patients in both training and validation cohorts. On the analysis for sex-specific biomarkers, this combination panel demonstrated a good AUC of 0.858 for male GC, whereas another combination panel of uTFF1, uBARD1 and H. pylori also provided a good AUC of 0.893 for female GC. Notably, each panel could distinguish even stage I GC patients from HC patients (AUC = 0.850 for males; AUC = 0.845 for females). CONCLUSIONS: Novel urinary protein biomarker panels represent promising non-invasive biomarkers for GC, including early-stage disease.

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling.

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic. METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, γ-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients. RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients. CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

A Promising CPS1 Inhibitor Keeping Ammonia from Fueling Cancer.

Carbamoyl phosphate synthetase 1 (CPS1) drives ammonia conversion to carbamoyl phosphate, and its overexpression supports pyrimidine synthesis and tumor growth, highlighting the potential of CPS1 inhibition as a therapeutic target. In this issue of Cell Chemical Biology, Yao et al. (2020) introduce H3B-120 as a promising novel inhibitor of CPS1.

Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer.

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

Syndecan 1 is a critical mediator of macropinocytosis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains recalcitrant to all forms of cancer treatment and carries a five-year survival rate of only 8%1. Inhibition of oncogenic KRAS (hereafter KRAS*), the earliest lesion in disease development that is present in more than 90% of PDACs, and its signalling surrogates has yielded encouraging preclinical results with experimental agents2-4. However, KRAS*-independent disease recurrence following genetic extinction of Kras* in mouse models anticipates the need for co-extinction strategies5,6. Multiple oncogenic processes are initiated at the cell surface, where KRAS* physically and functionally interacts to direct signalling that is essential for malignant transformation and tumour maintenance. Insights into the complexity of the functional cell-surface-protein repertoire (surfaceome) have been technologically limited until recently and-in the case of PDAC-the genetic control of the function and composition of the PDAC surfaceome in the context of KRAS* signalling remains largely unknown. Here we develop an unbiased, functional target-discovery platform to query KRAS*-dependent changes of the PDAC surfaceome, which reveals syndecan 1 (SDC1, also known as CD138) as a protein that is upregulated at the cell surface by KRAS*. Localization of SDC1 at the cell surface-where it regulates macropinocytosis, an essential metabolic pathway that fuels PDAC cell growth-is essential for disease maintenance and progression. Thus, our study forges a mechanistic link between KRAS* signalling and a targetable molecule driving nutrient salvage pathways in PDAC and validates oncogene-driven surfaceome annotation as a strategy to identify cancer-specific vulnerabilities.

Exosomes harbor B cell targets in pancreatic adenocarcinoma and exert decoy function against complement-mediated cytotoxicity.

Although B cell response is frequently found in cancer, there is little evidence that it alters tumor development or progression. The process through which tumor-associated antigens trigger humoral response is not well delineated. We investigate the repertoire of antigens associated with humoral immune response in pancreatic ductal adenocarcinoma (PDAC) using in-depth proteomic profiling of immunoglobulin-bound proteins from PDAC patient plasmas and identify tumor antigens that induce antibody response together with exosome hallmark proteins. Additional profiling of PDAC cell-derived exosomes reveals significant overlap in their protein content with immunoglobulin-bound proteins in PDAC plasmas, and significant autoantibody reactivity is observed between PDAC cell-derived exosomes and patient plasmas compared to healthy controls. Importantly, PDAC-derived exosomes induce a dose-dependent inhibition of PDAC serum-mediated complement-dependent cytotoxicity towards cancer cells. In summary, we provide evidence that exosomes display a large repertoire of tumor antigens that induce autoantibodies and exert a decoy function against complement-mediated cytotoxicity.

A Plasma-Derived Protein-Metabolite Multiplexed Panel for Early-Stage Pancreatic Cancer.

BACKGROUND: We applied a training and testing approach to develop and validate a plasma metabolite panel for the detection of early-stage pancreatic ductal adenocarcinoma (PDAC) alone and in combination with a previously validated protein panel for early-stage PDAC. METHODS: A comprehensive metabolomics platform was initially applied to plasmas collected from 20 PDAC cases and 80 controls. Candidate markers were filtered based on a second independent cohort that included nine invasive intraductal papillary mucinous neoplasm cases and 51 benign pancreatic cysts. Blinded validation of the resulting metabolite panel was performed in an independent test cohort consisting of 39 resectable PDAC cases and 82 matched healthy controls. The additive value of combining the metabolite panel with a previously validated protein panel was evaluated. RESULTS: Five metabolites (acetylspermidine, diacetylspermine, an indole-derivative, and two lysophosphatidylcholines) were selected as a panel based on filtering criteria. A combination rule was developed for distinguishing between PDAC and healthy controls using the Training Set. In the blinded validation study with early-stage PDAC samples and controls, the five metabolites yielded areas under the curve (AUCs) ranging from 0.726 to 0.842, and the combined metabolite model yielded an AUC of 0.892 (95% confidence interval [CI] = 0.828 to 0.956). Performance was further statistically significantly improved by combining the metabolite panel with a previously validated protein marker panel consisting of CA 19-9, LRG1, and TIMP1 (AUC = 0.924, 95% CI = 0.864 to 0.983, comparison DeLong test one-sided P= .02). CONCLUSIONS: A metabolite panel in combination with CA19-9, TIMP1, and LRG1 exhibited substantially improved performance in the detection of early-stage PDAC compared with a protein panel alone.

Assessment of Lung Cancer Risk on the Basis of a Biomarker Panel of Circulating Proteins.

Importance: There is an urgent need to improve lung cancer risk assessment because current screening criteria miss a large proportion of cases. Objective: To investigate whether a lung cancer risk prediction model based on a panel of selected circulating protein biomarkers can outperform a traditional risk prediction model and current US screening criteria. Design, Setting, and Participants: Prediagnostic samples from 108 ever-smoking patients with lung cancer diagnosed within 1 year after blood collection and samples from 216 smoking-matched controls from the Carotene and Retinol Efficacy Trial (CARET) cohort were used to develop a biomarker risk score based on 4 proteins (cancer antigen 125 [CA125], carcinoembryonic antigen [CEA], cytokeratin-19 fragment [CYFRA 21-1], and the precursor form of surfactant protein B [Pro-SFTPB]). The biomarker score was subsequently validated blindly using absolute risk estimates among 63 ever-smoking patients with lung cancer diagnosed within 1 year after blood collection and 90 matched controls from 2 large European population-based cohorts, the European Prospective Investigation into Cancer and Nutrition (EPIC) and the Northern Sweden Health and Disease Study (NSHDS). Main Outcomes and Measures: Model validity in discriminating between future lung cancer cases and controls. Discrimination estimates were weighted to reflect the background populations of EPIC and NSHDS validation studies (area under the receiver-operating characteristics curve [AUC], sensitivity, and specificity). Results: In the validation study of 63 ever-smoking patients with lung cancer and 90 matched controls (mean [SD] age, 57.7 [8.7] years; 68.6% men) from EPIC and NSHDS, an integrated risk prediction model that combined smoking exposure with the biomarker score yielded an AUC of 0.83 (95% CI, 0.76-0.90) compared with 0.73 (95% CI, 0.64-0.82) for a model based on smoking exposure alone (P = .003 for difference in AUC). At an overall specificity of 0.83, based on the US Preventive Services Task Force screening criteria, the sensitivity of the integrated risk prediction (biomarker) model was 0.63 compared with 0.43 for the smoking model. Conversely, at an overall sensitivity of 0.42, based on the US Preventive Services Task Force screening criteria, the integrated risk prediction model yielded a specificity of 0.95 compared with 0.86 for the smoking model. Conclusions and Relevance: This study provided a proof of principle in showing that a panel of circulating protein biomarkers may improve lung cancer risk assessment and may be used to define eligibility for computed tomography screening.