A Tumor and Immune-Related Micro-RNA Signature Predicts Relapse-Free Survival of Melanoma Patients Treated with Ipilimumab.

Despite the unprecedented advances in the treatment of melanoma with immunotherapy, there continues to be a major need for biomarkers of clinical benefits and immune resistance associated with immune checkpoint inhibitors; microRNA could play a vital role in these efforts. This study planned to identify differentially expressed miRNA molecules that may have prognostic value for clinical benefits. Patients with surgically operable regionally advanced melanoma were treated with neoadjuvant ipilimumab (10 mg/kg intravenously every 3 weeks × two doses) bracketing surgery. Tumor biospecimens were obtained at baseline and surgery, and microRNA (miRNA) expression profiling was performed on the tumor biopsies. We found that an expression profile consisting of a 4-miRNA signature was significantly associated with improved relapse-free survival (RFS). The signature consisted of biologically relevant molecules previously reported to have prognostic value in melanoma and other malignancies, including miR-34c, miR-711, miR-641, and miR-22. Functional annotation analysis of target genes for the 4-miRNA signature was significantly enriched for various cancer-related pathways, including cell proliferation regulation, apoptosis, the MAPK signaling pathway, and the positive regulation of T cell activation. Our results presented miRNAs as potential biomarkers that can guide the treatment of melanoma with immune checkpoint inhibitors. These findings warrant further investigation in relation to CTLA4 blockade and other immune checkpoint inhibitors. ClinicalTrials.gov NCT00972933.

Enhanced immune activation within the tumor microenvironment and circulation of female high-risk melanoma patients and improved survival with adjuvant CTLA4 blockade compared to males.

BACKGROUND: We hypothesized that a gender difference in clinical response may exist to adjuvant CTLA4 blockade with ipilimumab versus high-dose IFNα (HDI). We investigated differences in candidate immune biomarkers in the circulation and tumor microenvironment (TME). PATIENTS AND METHODS: This gender-based analysis was nested within the E1609 trial that tested adjuvant therapy with ipilimumab 3 mg/kg (ipi3) and 10 mg/kg (ipi10) versus HDI in high risk resected melanoma. We investigated gender differences in treatment efficacy with ipi3 and ipi10 versus HDI while adjusting for age, stage, ECOG performance (PS), ulceration, primary tumor status and lymph node number. Forest plots were created to compare overall survival (OS) and relapse free survival (RFS) between ipi and HDI. Gene expression profiling (GEP) was performed on tumors of 718 (454 male, 264 female) patients. Similarly, serum and peripheral blood mononuclear cells (PBMC) samples were tested for soluble and cellular biomarkers (N = 321 patients; 109 female and 212 male). RESULTS: The subgroups of female, stage IIIC, PS = 1, ulcerated primary, in-transit metastasis demonstrated significant improvement in RFS and/or OS with ipi3 versus HDI. Female gender was significant for both OS and RFS and was further explored. In the RFS comparison, a multivariate Cox regression model including significant variables indicated a significant interaction between gender and treatment (P = 0.024). In peripheral blood, percentages of CD3+ T cells (P = 0.024) and CD3+ CD4+ helper T cells (P = 0.0001) were higher in females compared to males. Trends toward higher circulating levels of IL1ß (P = 0.07) and IL6 (P = 0.06) were also found in females. Males had higher percentages of monocytes (P = 0.03) with trends toward higher percentages of regulatory T cells (T-reg). Tumor GEP analysis supported enhanced infiltration with immune cells including gammadelta T cells (P = 0.005), NK cells (P = 0.01), dendritic cells (P = 0.01), CD4+ T cells (P = 0.03), CD8+ T cells (P = 0.03) and T-reg (P = 0.008) in the tumors of females compared to males and a higher T-effector and IFNγ gene signature score (P = 0.0244). CONCLUSION: Female gender was associated with adjuvant CTLA4 blockade clinical benefits and female patients were more likely to have evidence of type1 immune activation within the TME and the circulation. Trial registration ClinicalTrials.gov NCT01274338. Registered 11 January 2011, https://www. CLINICALTRIALS: gov/ct2/show/NCT01274338.

Absolute quantitative proteomics using the total protein approach to identify novel clinical immunohistochemical markers in renal neoplasms.

BACKGROUND: Renal neoplasms encompass a variety of malignant and benign tumors, including many with shared characteristics. The diagnosis of these renal neoplasms remains challenging with currently available tools. In this work, we demonstrate the total protein approach (TPA) based on high-resolution mass spectrometry (MS) as a tool to improve the accuracy of renal neoplasm diagnosis. METHODS: Frozen tissue biopsies of human renal tissues [clear cell renal cell carcinoma (n = 7), papillary renal cell carcinoma (n = 5), chromophobe renal cell carcinoma (n = 5), and renal oncocytoma (n = 5)] were collected for proteome analysis. Normal adjacent renal tissue (NAT, n = 5) was used as a control. Proteins were extracted and digested using trypsin, and the digested proteomes were analyzed by label-free high-resolution MS (nanoLC-ESI-HR-MS/MS). Quantitative analysis was performed by comparison between protein abundances of tumors and NAT specimens, and the label-free and standard-free TPA was used to obtain absolute protein concentrations. RESULTS: A total of 205 differentially expressed proteins with the potential to distinguish the renal neoplasms were found. Of these proteins, a TPA-based panel of 24, including known and new biomarkers, was selected as the best candidates to differentiate the neoplasms. As proof of concept, the diagnostic potential of PLIN2, TUBB3, LAMP1, and HK1 was validated using semi-quantitative immunohistochemistry with a total of 128 samples assessed on tissue micro-arrays. CONCLUSIONS: We demonstrate the utility of combining high-resolution MS and the TPA as potential new diagnostic tool in the pathology of renal neoplasms. A similar TPA approach may be implemented in any cancer study with solid biopsies.

Development of a Robust Ultrasonic-Based Sample Treatment To Unravel the Proteome of OCT-Embedded Solid Tumor Biopsies.

An effective three-step proteomics workflow is proposed to overcome the pitfalls caused by polymers present in optimum cutting temperature (OCT)-embedded tissue during its preparation for mass spectrometry analysis. First, the OCT-embedded tissue biopsies are cleaned using ethanol and water in a sequential series of ultrasonic washes in an ultrasound bath (35 kHz ultrasonic frequency, 100% ultrasonic amplitude, 2 min of ultrasonic duty time). Second, a fast ultrasonic-assisted extraction of proteins is done using an ultrasonic probe (30 kHz ultrasonic frequency, 50% ultrasonic amplitude, 2 min of ultrasonic duty time, 1 mm diameter tip). Third, a rapid ultrasonic digestion of complex proteomes is performed using a microplate horn assembly device (20 kHz ultrasonic frequency, 25% ultrasonic amplitude, 4 min of ultrasonic duty time). As a proof of concept, the new workflow was applied to human normal and tumor kidney biopsies including chromophobe renal cell carcinomas (chRCCs) and renal oncocytomas (ROs). A successful cluster of proteomics profiles was obtained comprising 511 and 172 unique proteins found in chRCC and RO samples, respectively. The new method provides high sample throughput and comprehensive protein recovery from OCT samples.

Loss of Chromatin-Remodeling Proteins and/or CDKN2A Associates With Metastasis of Pancreatic Neuroendocrine Tumors and Reduced Patient Survival Times.

Despite prognostic grading and staging systems, it is a challenge to predict outcomes for patients with pancreatic neuroendocrine tumors (PanNETs). Sequencing studies of PanNETs have identified alterations in death domain-associated protein (DAXX) and alpha-thalassemia/mental retardation X-linked chromatin remodeler (ATRX). In tumors, mutations in DAXX or ATRX and corresponding loss of protein expression correlate with shorter times of disease-free survival and disease-specific survival of patients. However, DAXX or ATRX proteins were lost in only 50% of distant metastases analyzed. We performed whole-exome sequencing analyses of 20 distant metastases from 20 patients with a single nonsyndrome, nonfunctional PanNET. We found distant metastases contained alterations in multiple endocrine neoplasia type 1 (MEN1) (n = 8), ATRX (n = 5), DAXX (n = 5), TSC2 (n = 3), and DEP domain containing 5 (DEPDC5) (n = 3). We found copy number loss of cyclin dependent kinase inhibitor 2A (CDKN2A) in 15 metastases (75%) and alterations in genes that regulate chromatin remodeling, including set domain containing 2 (SETD2) (n = 4), AT-rich interaction domain 1A (ARID1A) (n = 2), chromodomain helicase DNA binding protein 8 (CHD8) (n = 2), and DNA methyl transferase 1 (DNMT1) (n = 2). In a separate analysis of 347 primary PanNETs, we found loss or deletion of DAXX and ATRX, disruption of SETD2 function (based on loss of H3 lysine 36 trimethylation), loss of ARID1A expression or deletions in CDKN2A in 81% of primary PanNETs with distant metastases. Among patients with loss or deletion of at least 1 of these proteins or genes, 39% survived disease-free for 5 years and 44% had disease-specific survival times of 10 years. Among patients without any of these alterations, 98% survived disease-free for 5 years and 95% had disease-specific survival times of 10 years. Therefore, primary PanNETs with loss of DAXX, ATRX, H3 lysine 36 trimethylation, ARID1A, and/or CDKN2A associate with shorter survival times of patients. Our findings indicate that alterations in chromatin-remodeling genes and CDKN2A contribute to metastasis of PanNETs.

Discrimination of low- and high-grade appendiceal mucinous neoplasms by targeted sequencing of cancer-related variants.

DNA was obtained from matching micro-dissected, primary tumor cells, paired metastases, and peripheral blood mononuclear cells (germline) from patients with appendiceal mucinous neoplasms. We compared specimens from patient cohorts comprising low-grade adenomucinous neoplasm versus high-grade mucinous adenocarcinoma using a targeted, amplicon sequencing panel of 409 cancer related genes (Ion Torrent Comprehensive Cancer Panel, Thermo-Fisher, Waltham, MA). Copy number variants, single nucleotide variants and small insertions/deletions were identified using a multiplex algorithm pipeline (GATK, VarScan2, MuTect2, SIFT, SIFT-INDEL, PolyPhen-2, Provean). There were significantly more damaging variants in high-grade versus low-grade tumor cohorts. Both cohorts contained damaging, heterozygous germline variants (catenin ß1; notch receptor 1 and 4) in pathways associated with cell-lineage specification (WNT, NOTCH). Damaging, somatic KRAS proto-oncogene, GTPase mutations were present in both cohorts, while somatic GNAS complex locus mutations were confined to low-grade neoplasms. Variants predominantly affected transcription factors, kinases, and stem cell signaling molecules in canonical pathways including epithelial to mesenchymal transition, stem cell pluripotency, p53, PTEN, and NF-қB signaling pathways. High-grade tumors demonstrated MYC proto-oncogene, bHLH transcription factor (MYC) and death domain associated protein (DAXX) amplification and damaging somatic variants in tumor protein p53 (TP53), likely to amplify an aggressive phenotype. Damaging APC, WNT signaling pathway regulator (APC) deletions were identified in metastatic tissue of both cohorts suggesting a role in invasive disease. Our data suggest that germline dysregulation of WNT and/or NOTCH pathways predisposes patients toward a secretory cell phenotype (i.e., goblet-like cells) upon acquisition of somatic KRAS mutations. Additional somatically acquired variants activating oncogenes MYC and DAXX and inhibiting the critical tumor suppressor, tumor protein TP53, were consistent with manifestation of a high-grade phenotype. These additional changes within the epithelial to mesenchymal transition signaling network (WNT, NOTCH, RAS/ERK/PI3K, PTEN, NF-қB), produce aggressive high-grade tumor characteristics by actively driving cells towards dedifferentiation, proliferation, and migration.

KRAS mutation is predictive of outcome in patients with pulmonary sarcomatoid carcinoma.

AIMS: Pulmonary sarcomatoid carcinoma (PSC) is a poorly differentiated non-small-cell lung carcinoma (NSCLC) with aggressive behaviour. This study aimed to evaluate the prognostic clinicopathological and genetic characteristics of PSCs. METHODS AND RESULTS: Fifty-three cases of surgically treated PSCs were selected, 23 of which were subjected to mutation and copy number variation analysis using the 50-gene Ion AmpliSeq Cancer Panel. The majority of the patients were male (32 of 53, 60.3%) and smokers (51 of 53, 96.2%). Overall, 25 (47.1%) patients died within 2-105 months (mean = 22.7 months, median = 15 months) after diagnosis, and 28 were alive 3-141 months (mean = 38.7 months, median = 21.5 months) after diagnosis. Five-year overall survival was 12.5%. KRAS codon 12/13 mutation in adenocarcinomas (P = 0.01), age more than 70 years (P = 0.008) and tumour size ≥4.0 cm (P = 0.02) were associated strongly with worse outcome. TP53 (17 of 23, 74.0%) and KRAS codon 12 of 13 mutations (10 of 23, 43.4%) were the most common genetic alterations. Potentially actionable variants were identified including ATM (four of 23, 17.3%), MET, FBXW7 and EGFR (two of 23, 8.7%), AKT1, KIT, PDGFRA, HRAS, JAK3 and SMAD4 (one of 23, 4.3%). MET exon 14 skipping and missense mutations were identified in two (11.1%) cases with adenocarcinoma histology. Copy number analysis showed loss of RB1 (three of 23, 13%) and ATM (two of 23, 8.7%). Copy number gains were seen in EGFR (two of 23, 13.0%) and in one (4.3%) of each PIK3CA, KRAS, MET and STK11. CONCLUSIONS: Potentially targetable mutations can be identified in a subset of PSC, although most tumours harbour currently untargetable prognostically adverse TP53 and KRAS mutations.

A MicroRNA Cluster miR-23-24-27 Is Upregulated by Aldosterone in the Distal Kidney Nephron Where it Alters Sodium Transport.

The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules, and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na+ ) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na+ regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu-miR-23-24-27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na+ transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR-27a/b was verified to bind to the 3'-untranslated region of intersectin-2, a multi-domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC-mediated Na+ transport, while Itsn2 overexpression reduced ENaC's function. These findings reinforce a role for miRs in aldosterone regulation of Na+ transport, and implicate miR-27 in aldosterone's action via a novel target. J. Cell. Physiol. 232: 1306-1317, 2017. © 2016 Wiley Periodicals, Inc.

Atrial Fibrillation Associated Genetic Variants and Left Atrial Histology: Evaluation for Molecular Sub-Phenotypes.

INTRODUCTION: Genome wide association studies have identified several single nucleotide polymorphisms (SNPs) associated with atrial fibrillation (AF), but the mechanisms underlying these relationships have not yet been elucidated. Inflammation and fibrosis have been posited as important mechanisms responsible for AF. We sought to investigate the impact of SNP carrier status on inflammation and fibrosis in left atrial appendage tissue. METHODS AND RESULTS: Carrier status of 10 AF-associated SNPs was evaluated on DNA extracted from left atrial appendage tissue in 176 individuals (120 with AF). The presence of inflammation was evaluated through visual quantification of leukocyte infiltration following hematoxylin and eosin staining, while fibrosis was quantified using picrosirius red with fast green staining. Unadjusted and adjusted linear and logistic regression models were utilized to evaluate for an association between SNP carrier status and inflammation and fibrosis. On adjusted logistic regression analysis, the rs7164883 SNP (intronic within HCN4) was associated with a reduced odds of inflammation (odds ratio: 0.42; 95% CI: 0.22-0.81, P = 0.01), and was not associated with fibrosis on adjusted linear regression analysis (ß-coefficient: -0.31; 95% CI: -1.03-0.40, P = 0.40). None of the remaining SNPs exhibited significant associations with left atrial inflammation or fibrosis. CONCLUSIONS: Among 10 AF-associated SNPs, a single genetic variant was associated with reduced left atrial inflammation, while no histologic differences were observed in the remaining 9. The known AF-associated SNPs do not appear to predispose to the development of pro-inflammatory or pro-fibrotic AF sub-phenotypes.

Aldosterone regulates microRNAs in the cortical collecting duct to alter sodium transport.

A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein. A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3' untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.

A four-marker signature of TNF-RII, TGF-α, TIMP-1 and CRP is prognostic of worse survival in high-risk surgically resected melanoma.

BACKGROUND: E1694 tested GM2-KLH-QS21 vaccine versus high-dose interferon-α2b (HDI) as adjuvant therapy for operable stage IIB-III melanoma. We tested banked serum specimens from patients in the vaccine arm of E1694 for prognostic biomarkers. METHODS: Aushon Multiplex Platform was used to quantitate baseline serum levels of 115 analytes from 40 patients. Least absolute shrinkage and selection operator proportional hazard regression (Lasso PH) was used to select markers that are most informative for relapse-free survival (RFS) and overall survival (OS). Regular Cox PH models were then fit with the markers selected by the Lasso PH. Survival receiver operating characteristic (ROC) analysis was used to evaluate the ability of the models to predict 1-year RFS and 5-year OS. RESULTS: Four markers that include Tumor Necrosis Factor alpha Receptor II (TNF-RII), Transforming Growth Factor alpha (TGF-α), Tissue Inhibitor of Metalloproteinases 1 (TIMP-1), and C-reactive protein (CRP) were found to be most informative for the prediction of OS (high levels correlate with worse prognosis). The dichotomized risk score based on the four markers could significantly separate the OS curves (p = 0.0005). When using the four-marker PH model to predict 5-year OS, we achieved an area under the curve (AUC) of 89% (cross validated AUC = 72%). High baseline TNF-RII was also significantly associated with worse RFS. The RFS with high (above median) TNF-RII was significantly lower than low TNF-RII (p = 0.01). CONCLUSIONS: The biomarker signature consisting of TNFR-II, TGF-α, TIMP-1 and CRP is significantly prognostic of survival in patients with high-risk melanoma and warrants further investigation.

Modeling the Effect of Spatial Structure on Solid Tumor Evolution and Circulating Tumor DNA Composition.

Circulating tumor DNA (ctDNA) monitoring, while sufficiently advanced to reflect tumor evolution in real time and inform cancer diagnosis, treatment, and prognosis, mainly relies on DNA that originates from cell death via apoptosis or necrosis. In solid tumors, chemotherapy and immune infiltration can induce spatially variable rates of cell death, with the potential to bias and distort the clonal composition of ctDNA. Using a stochastic evolutionary model of boundary-driven growth, we study how elevated cell death on the edge of a tumor can simultaneously impact driver mutation accumulation and the representation of tumor clones and mutation detectability in ctDNA. We describe conditions in which invasive clones are over-represented in ctDNA, clonal diversity can appear elevated in the blood, and spatial bias in shedding can inflate subclonal variant allele frequencies (VAFs). Additionally, we find that tumors that are mostly quiescent can display similar biases but are far less detectable, and the extent of perceptible spatial bias strongly depends on sequence detection limits. Overall, we show that spatially structured shedding might cause liquid biopsies to provide highly biased profiles of tumor state. While this may enable more sensitive detection of expanding clones, it could also increase the risk of targeting a subclonal variant for treatment. Our results indicate that the effects and clinical consequences of spatially variable cell death on ctDNA composition present an important area for future work.

Marker selection strategies for circulating tumor DNA guided by phylogenetic inference.

Motivation: Blood-based profiling of tumor DNA ("liquid biopsy") has offered great prospects for non-invasive early cancer diagnosis, treatment monitoring, and clinical guidance, but require further advances in computational methods to become a robust quantitative assay of tumor clonal evolution. We propose new methods to better characterize tumor clonal dynamics from circulating tumor DNA (ctDNA), through application to two specific questions: 1) How to apply longitudinal ctDNA data to refine phylogeny models of clonal evolution, and 2) how to quantify changes in clonal frequencies that may be indicative of treatment response or tumor progression. We pose these questions through a probabilistic framework for optimally identifying maximum likelihood markers and applying them to characterizing clonal evolution. Results: We first estimate a distribution over plausible clonal lineage models, using bootstrap samples over pre-treatment tissue-based sequence data. We then refine these lineage models and the clonal frequencies they imply over successive longitudinal samples. We use the resulting framework for modeling and refining tree distributions to pose a set of optimization problems to select ctDNA markers to maximize measures of utility capturing ability to solve the two questions of reducing uncertain in phylogeny models or quantifying clonal frequencies given the models. We tested our methods on synthetic data and showed them to be effective at refining distributions of tree models and clonal frequencies so as to minimize measures of tree distance relative to the ground truth. Application of the tree refinement methods to real tumor data further demonstrated their effectiveness in refining a clonal lineage model and assessing its clonal frequencies. The work shows the power of computational methods to improve marker selection, clonal lineage reconstruction, and clonal dynamics profiling for more precise and quantitative assays of tumor progression. Availability: https://github.com/CMUSchwartzLab/Mase-phi.git. Contact: russells@andrew.cmu.edu.

Navigating Precision Oncology: Insights from an Integrated Clinical Data and Biobank Repository Initiative across a Network Cancer Program.

Advancing cancer treatment relies on the rapid translation of new scientific discoveries to patient care. To facilitate this, an oncology biobank and data repository program, also referred to as the "Moonshot" program, was launched in 2021 within the Integrated Network Cancer Program of the Allegheny Health Network. A clinical data program (CDP) and biospecimen repository were established, and patient data and blood and tissue samples have been collected prospectively. To date, the study has accrued 2920 patients, predominantly female (61%) and Caucasian (90%), with a mean age of 64 ± 13 years. The most common cancer sites were the endometrium/uterus (12%), lung/bronchus (12%), breast (11%), and colon/rectum (11%). Of patients diagnosed with cancer, 34% were diagnosed at stage I, 25% at stage II, 26% at stage III, and 15% at stage IV. The CDP is designed to support our initiative in advancing personalized cancer research by providing a comprehensive array of patient data, encompassing demographic characteristics, diagnostic details, and treatment responses. The "Moonshot" initiative aims to predict therapy responses and clinical outcomes through cancer-related biomarkers. The CDP facilitates this initiative by fostering data sharing, enabling comparative analyses, and informing the development of novel diagnostic and therapeutic methods.

Genome abnormalities precede prostate cancer and predict clinical relapse.

The prediction of prostate cancer clinical outcome remains a major challenge after the diagnosis, even with improved early detection by prostate-specific antigen (PSA) monitoring. To evaluate whether copy number variation (CNV) of the genomes in prostate cancer tumor, in benign prostate tissues adjacent to the tumor (AT), and in the blood of patients with prostate cancer predicts biochemical (PSA) relapse and the kinetics of relapse, 241 samples (104 tumor, 49 matched AT, 85 matched blood, and 3 cell lines) were analyzed using Affymetrix SNP 6.0 chips. By using gene-specific CNV from tumor, the genome model correctly predicted 73% (receiver operating characteristic P = 0.003) cases for relapse and 75% (P < 0.001) cases for short PSA doubling time (PSADT, <4 months). The gene-specific CNV model from AT correctly predicted 67% (P = 0.041) cases for relapse and 77% (P = 0.015) cases for short PSADT. By using median-sized CNV from blood, the genome model correctly predicted 81% (P < 0.001) cases for relapse and 69% (P = 0.001) cases for short PSADT. By using median-sized CNV from tumor, the genome model correctly predicted 75% (P < 0.001) cases for relapse and 80% (P < 0.001) cases for short PSADT. For the first time, our analysis indicates that genomic abnormalities in either benign or malignant tissues are predictive of the clinical outcome of a malignancy.

Renal cell neoplasms contain shared tumor type-specific copy number variations.

Copy number variant (CNV) analysis was performed on renal cell carcinoma (RCC) specimens (chromophobe, clear cell, oncocytoma, papillary type 1, and papillary type 2) using high-resolution arrays (1.85 million probes). The RCC samples exhibited diverse genomic changes within and across tumor types, ranging from 106 to 2238 CNV segments in a clear-cell specimen and in a papillary type 2 specimen, respectively. Despite this heterogeneity, distinct CNV segments were common within each tumor classification: chromophobe (seven segments), clear cell (three segments), oncocytoma (nine segments), and papillary type 2 (two segments). Shared segments ranged from a 6.1-kb deletion (oncocytomas) to a 208.3-kb deletion (chromophobes). Among common tumor type-specific variations, chromophobes, clear-cell tumors, and oncocytomas were composed exclusively of noncoding DNA. No CNV regions were common to papillary type 1 specimens, although there were 12 amplifications and 12 deletions in five of six samples. Three microRNAs and 12 mRNA genes had a ≥98% coding region contained within CNV regions, including multiple gene families (chromophobe: amylases 1A, 1B, and 1C; oncocytoma: general transcription factors 2H2, 2B, 2C, and 2D). Gene deletions involved in histone modification and chromatin remodeling affected individual subtypes (clear cell: SFMBT and SETD2; papillary type 2: BAZ1A) and the collective RCC group (KDM4C). The genomic amplifications/deletions identified herein represent potential diagnostic and/or prognostic biomarkers.

Modeling the effect of spatial structure on solid tumor evolution and ctDNA composition.

Circulating tumor DNA (ctDNA) monitoring, while sufficiently advanced to reflect tumor evolution in real time and inform on cancer diagnosis, treatment, and prognosis, mainly relies on DNA that originates from cell death via apoptosis or necrosis. In solid tumors, chemotherapy and immune infiltration can induce spatially variable rates of cell death, with the potential to bias and distort the clonal composition of ctDNA. Using a stochastic evolutionary model of boundary-driven growth, we study how elevated cell death on the edge of a tumor can simultaneously impact driver mutation accumulation and the representation of tumor clones and mutation detectability in ctDNA. We describe conditions in which invasive clones end up over-represented in ctDNA, clonal diversity can appear elevated in the blood, and spatial bias in shedding can inflate subclonal variant allele frequencies (VAFs). Additionally, we find that tumors that are mostly quiescent can display similar biases, but are far less detectable, and the extent of perceptible spatial bias strongly depends on sequence detection limits. Overall, we show that spatially structured shedding might cause liquid biopsies to provide highly biased profiles of tumor state. While this may enable more sensitive detection of expanding clones, it could also increase the risk of targeting a subclonal variant for treatment. Our results indicate that the effects and clinical consequences of spatially variable cell death on ctDNA composition present an important area for future work.

Serum protein profiles predict coronary artery disease in symptomatic patients referred for coronary angiography.

BACKGROUND: More than a million diagnostic cardiac catheterizations are performed annually in the US for evaluation of coronary artery anatomy and the presence of atherosclerosis. Nearly half of these patients have no significant coronary lesions or do not require mechanical or surgical revascularization. Consequently, the ability to rule out clinically significant coronary artery disease (CAD) using low cost, low risk tests of serum biomarkers in even a small percentage of patients with normal coronary arteries could be highly beneficial. METHODS: Serum from 359 symptomatic subjects referred for catheterization was interrogated for proteins involved in atherogenesis, atherosclerosis, and plaque vulnerability. Coronary angiography classified 150 patients without flow-limiting CAD who did not require percutaneous intervention (PCI) while 209 required coronary revascularization (stents, angioplasty, or coronary artery bypass graft surgery). Continuous variables were compared across the two patient groups for each analyte including calculation of false discovery rate (FDR ≤ 1%) and Q value (P value for statistical significance adjusted to ≤ 0.01). RESULTS: Significant differences were detected in circulating proteins from patients requiring revascularization including increased apolipoprotein B100 (APO-B100), C-reactive protein (CRP), fibrinogen, vascular cell adhesion molecule 1 (VCAM-1), myeloperoxidase (MPO), resistin, osteopontin, interleukin (IL)-1ß, IL-6, IL-10 and N-terminal fragment protein precursor brain natriuretic peptide (NT-pBNP) and decreased apolipoprotein A1 (APO-A1). Biomarker classification signatures comprising up to 5 analytes were identified using a tunable scoring function trained against 239 samples and validated with 120 additional samples. A total of 14 overlapping signatures classified patients without significant coronary disease (38% to 59% specificity) while maintaining 95% sensitivity for patients requiring revascularization. Osteopontin (14 times) and resistin (10 times) were most frequently represented among these diagnostic signatures. The most efficacious protein signature in validation studies comprised osteopontin (OPN), resistin, matrix metalloproteinase 7 (MMP7) and interferon γ (IFNγ) as a four-marker panel while the addition of either CRP or adiponectin (ACRP-30) yielded comparable results in five protein signatures. CONCLUSIONS: Proteins in the serum of CAD patients predominantly reflected (1) a positive acute phase, inflammatory response and (2) alterations in lipid metabolism, transport, peroxidation and accumulation. There were surprisingly few indicators of growth factor activation or extracellular matrix remodeling in the serum of CAD patients except for elevated OPN. These data suggest that many symptomatic patients without significant CAD could be identified by a targeted multiplex serum protein test without cardiac catheterization thereby eliminating exposure to ionizing radiation and decreasing the economic burden of angiographic testing for these patients.

Identification and expansion of a unique stem cell population from adult mouse gallbladder.

UNLABELLED: The identification of resident stem cells in the mouse gallbladder is, to date, unexplored. In addition, the relationship between adult gallbladder stem cells and intrahepatic bile duct (IHBD) cells is not well understood. The aim of this study was to isolate stem cells from an adult mouse gallbladder and determine whether they were unique, compared to IHBD cells. By limiting dilution analyses and index sorts, we found that an EpCAM(+) CD49f(hi) epithelial cell subpopulation from primary gallbladder is enriched in colony-forming cells, compared to EpCAM(+) CD49f(lo) cells. EpCAM(+) CD49f(hi) cells expressed cluster of differentiation (CD)29, CD133, and stem cell antigen-1, but were negative for lineage markers CD31, CD45, and F4/80. Using a novel feeder cell-culture system, we observed long-term (>passage 20) and clonal expansion of the EpCAM(+) CD49f(hi) cells in vitro. In a matrigel differentiation assay, EpCAM(+) CD49f(+) cells expanding in vitro underwent organotypic morphogenesis forming ductular structures and cysts. These structures are similar to, and recapitulate a transport function of, primary gallbladder. EpCAM(+) CD49f(+) cells also engraft into the subcutaneous space of recipient mice. We compared primary gallbladder and IHBD cells by flow cytometry and found phenotypic differences in the expression of CD49f, CD49e, CD81, CD26, CD54, and CD166. In addition, oligonucleotide microarrays showed that the expanded EpCAM(+) CD49f(+) gallbladder cells and IHBD cells exhibit differences related to lipid and drug metabolism. Notable genes that were different are cytochrome P450, glutathione S-transferase, Indian hedgehog, and solute carrier family genes. CONCLUSION: We have isolated an epithelial cell population from primary mouse gallbladder with stem cell characteristics and found it to be unique, compared to IHBD cells.

Improved prognosis and evidence of enhanced immunogenicity in tumor and circulation of high-risk melanoma patients with unknown primary.

BACKGROUND: Melanoma of unknown primary (MUP) represents a poorly understood group of patients both clinically and immunologically. We investigated differences in prognosis and candidate immune biomarkers in patients with unknown compared with those with known primary melanoma enrolled in the E1609 adjuvant trial that tested ipilimumab at 3 and 10 mg/kg vs high-dose interferon-alfa (HDI). PATIENTS AND METHODS: MUP status was defined as initial presentation with cutaneous, nodal or distant metastasis without a known primary. Relapse-free survival (RFS) and overall survival (OS) rates were estimated by the Kaplan-Meier method. Stratified (by stage) log-rank test was used to compare RFS and OS by primary tumor status. Gene expression profiling (GEP) was performed on the tumor biopsies of a subset of patients. Similarly, peripheral blood samples were tested for candidate soluble and cellular immune biomarkers. RESULTS: MUP cases represented 12.8% of the total population (N=1699) including 11.7% on the ipilimumab arms and 14.7% on the HDI arm. Stratifying by stage, RFS (p=0.001) and overall survival (OS) (p=0.009) showed outcomes significantly better for patients with unknown primary. The primary tumor status remained prognostically significant after adjusting for treatment and stage in multivariate Cox proportional hazards models. Including only ipilimumab-treated patients, RFS (p=0.005) and OS (p=0.023) were significantly better in favor of those with unknown primary. Among patients with GEP data (n=718; 102 MUP, 616 known), GEP identified pathways and genes related to autoimmunity, inflammation, immune cell infiltration and immune activation that were significantly enriched in the MUP tumors compared with known primaries. Further investigation into infiltrating immune cell types estimated significant enrichment with CD8 +and CD4+T cells, B cells and NK cells as well as significantly higher major histocompatibility complex (MHC)-I and MHC-II scores in MUP compared with known primary. Among patients tested for circulating biomarkers (n=321; 66 unknown and 255 known), patients with MUP had significantly higher circulating levels of IL-2R (p=0.04). CONCLUSION: Patients with MUP and high-risk melanoma had significantly better prognosis and evidence of significantly enhanced immune activation within the TME and the circulation, supporting the designation of MUP as a distinct prognostic marker in patients with high-risk melanoma.