Publications by authors named "Paul T Spellman"

83 Publications

Causal interactions from proteomic profiles: Molecular data meet pathway knowledge.

Patterns (N Y) 2021 Jun 12;2(6):100257. Epub 2021 May 12.

Computational Biology Program, Oregon Health and Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.

We present a computational method to infer causal mechanisms in cell biology by analyzing changes in high-throughput proteomic profiles on the background of prior knowledge captured in biochemical reaction knowledge bases. The method mimics a biologist's traditional approach of explaining changes in data using prior knowledge but does this at the scale of hundreds of thousands of reactions. This is a specific example of how to automate scientific reasoning processes and illustrates the power of mapping from experimental data to prior knowledge via logic programming. The identified mechanisms can explain how experimental and physiological perturbations, propagating in a network of reactions, affect cellular responses and their phenotypic consequences. Causal pathway analysis is a powerful and flexible discovery tool for a wide range of cellular profiling data types and biological questions. The automated causation inference tool, as well as the source code, are freely available at http://causalpath.org.
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http://dx.doi.org/10.1016/j.patter.2021.100257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8212145PMC
June 2021

A community challenge to evaluate RNA-seq, fusion detection, and isoform quantification methods for cancer discovery.

Cell Syst 2021 Jun 16. Epub 2021 Jun 16.

Biomedical Engineering, Oregon Health and Science University, Portland, OR 97239, USA. Electronic address:

The accurate identification and quantitation of RNA isoforms present in the cancer transcriptome is key for analyses ranging from the inference of the impacts of somatic variants to pathway analysis to biomarker development and subtype discovery. The ICGC-TCGA DREAM Somatic Mutation Calling in RNA (SMC-RNA) challenge was a crowd-sourced effort to benchmark methods for RNA isoform quantification and fusion detection from bulk cancer RNA sequencing (RNA-seq) data. It concluded in 2018 with a comparison of 77 fusion detection entries and 65 isoform quantification entries on 51 synthetic tumors and 32 cell lines with spiked-in fusion constructs. We report the entries used to build this benchmark, the leaderboard results, and the experimental features associated with the accurate prediction of RNA species. This challenge required submissions to be in the form of containerized workflows, meaning each of the entries described is easily reusable through CWL and Docker containers at https://github.com/SMC-RNA-challenge. A record of this paper's transparent peer review process is included in the supplemental information.
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http://dx.doi.org/10.1016/j.cels.2021.05.021DOI Listing
June 2021

The impact of tumor epithelial and microenvironmental heterogeneity on treatment responses in HER2+ breast cancer.

JCI Insight 2021 Jun 8;6(11). Epub 2021 Jun 8.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

Despite the availability of multiple human epidermal growth factor receptor 2-targeted (HER2-targeted) treatments, therapeutic resistance in HER2+ breast cancer remains a clinical challenge. Intratumor heterogeneity for HER2 and resistance-conferring mutations in the PIK3CA gene (encoding PI3K catalytic subunit α) have been investigated in response and resistance to HER2-targeting agents, while the role of divergent cellular phenotypes and tumor epithelial-stromal cell interactions is less well understood. Here, we assessed the effect of intratumor cellular genetic heterogeneity for ERBB2 (encoding HER2) copy number and PIK3CA mutation on different types of neoadjuvant HER2-targeting therapies and clinical outcome in HER2+ breast cancer. We found that the frequency of cells lacking HER2 was a better predictor of response to HER2-targeted treatment than intratumor heterogeneity. We also compared the efficacy of different therapies in the same tumor using patient-derived xenograft models of heterogeneous HER2+ breast cancer and single-cell approaches. Stromal determinants were better predictors of response than tumor epithelial cells, and we identified alveolar epithelial and fibroblastic reticular cells as well as lymphatic vessel endothelial hyaluronan receptor 1-positive (Lyve1+) macrophages as putative drivers of therapeutic resistance. Our results demonstrate that both preexisting and acquired resistance to HER2-targeting agents involve multiple mechanisms including the tumor microenvironment. Furthermore, our data suggest that intratumor heterogeneity for HER2 should be incorporated into treatment design.
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http://dx.doi.org/10.1172/jci.insight.147617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262355PMC
June 2021

Characterizing genetic intra-tumor heterogeneity across 2,658 human cancer genomes.

Cell 2021 Apr 7;184(8):2239-2254.e39. Epub 2021 Apr 7.

Wellcome Trust Sanger Institute, Cambridge CB10 1SA, UK.

Intra-tumor heterogeneity (ITH) is a mechanism of therapeutic resistance and therefore an important clinical challenge. However, the extent, origin, and drivers of ITH across cancer types are poorly understood. To address this, we extensively characterize ITH across whole-genome sequences of 2,658 cancer samples spanning 38 cancer types. Nearly all informative samples (95.1%) contain evidence of distinct subclonal expansions with frequent branching relationships between subclones. We observe positive selection of subclonal driver mutations across most cancer types and identify cancer type-specific subclonal patterns of driver gene mutations, fusions, structural variants, and copy number alterations as well as dynamic changes in mutational processes between subclonal expansions. Our results underline the importance of ITH and its drivers in tumor evolution and provide a pan-cancer resource of comprehensively annotated subclonal events from whole-genome sequencing data.
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http://dx.doi.org/10.1016/j.cell.2021.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054914PMC
April 2021

The Feasibility of Patient-Specific Circulating Tumor DNA Monitoring throughout Multi-Modality Therapy for Locally Advanced Esophageal and Rectal Cancer: A Potential Biomarker for Early Detection of Subclinical Disease.

Diagnostics (Basel) 2021 Jan 5;11(1). Epub 2021 Jan 5.

Department of Radiation Medicine, Oregon Health & Science University (OHSU), 3181 SW Sam Jackson Park Rd, KPV4, Portland, OR 97239, USA.

As non-operative management (NOM) of esophageal and rectal cancer is becoming more prevalent, blood-biomarkers such as circulating tumor DNA (ctDNA) may provide clinical information in addition to endoscopy and imaging to aid in treatment decisions following chemotherapy and radiation therapy. In this feasibility study, we prospectively collected plasma samples from locally advanced esophageal ( = 3) and rectal cancer ( = 2) patients undergoing multimodal neoadjuvant therapy to assess the feasibility of serial ctDNA monitoring throughout neoadjuvant therapy. Using the Dual-Index Degenerate Adaptor-Sequencing (DIDA-Seq) error-correction method, we serially interrogated plasma cell-free DNA at 28-41 tumor-specific genomic loci throughout therapy and in surveillance with an average limit of detection of 0.016% mutant allele frequency. In both rectal cancer patients, ctDNA levels were persistently elevated following total neoadjuvant therapy with eventual detection of clinical recurrence prior to salvage surgery. Among the esophageal cancer patients, ctDNA levels closely correlated with tumor burden throughout and following neoadjuvant therapy, which was associated with a pathologic complete response in one patient. In this feasibility study, patient- and tumor-specific ctDNA levels correlated with clinical outcomes throughout multi-modality therapy suggesting that serial monitoring of patient ctDNA has the potential to serve as a highly sensitive and specific biomarker to risk-stratify esophageal and rectal cancer patients eligible for NOM. Further prospective investigation is warranted.
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http://dx.doi.org/10.3390/diagnostics11010073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824834PMC
January 2021

Large-Scale Characterization of Drug Responses of Clinically Relevant Proteins in Cancer Cell Lines.

Cancer Cell 2020 12 5;38(6):829-843.e4. Epub 2020 Nov 5.

Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Graduate Program in Quantitative and Computational Biosciences, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

Perturbation biology is a powerful approach to modeling quantitative cellular behaviors and understanding detailed disease mechanisms. However, large-scale protein response resources of cancer cell lines to perturbations are not available, resulting in a critical knowledge gap. Here we generated and compiled perturbed expression profiles of ∼210 clinically relevant proteins in >12,000 cancer cell line samples in response to ∼170 drug compounds using reverse-phase protein arrays. We show that integrating perturbed protein response signals provides mechanistic insights into drug resistance, increases the predictive power for drug sensitivity, and helps identify effective drug combinations. We build a systematic map of "protein-drug" connectivity and develop a user-friendly data portal for community use. Our study provides a rich resource to investigate the behaviors of cancer cells and the dependencies of treatment responses, thereby enabling a broad range of biomedical applications.
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http://dx.doi.org/10.1016/j.ccell.2020.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738392PMC
December 2020

Diurnal stability of cell-free DNA and cell-free RNA in human plasma samples.

Sci Rep 2020 10 5;10(1):16456. Epub 2020 Oct 5.

Knight Cancer Institute, Cancer Early Detection Advanced Research Center (CEDAR), Oregon Health & Science University, Portland, OR, 97201, USA.

Many emerging technologies are reliant on circulating cell-free DNA (cfDNA) and cell-free RNA (cfRNA) applications in the clinic. However, the impact of diurnal cycles or daily meals on circulating analytes are poorly understood and may be confounding factors when developing diagnostic platforms. To begin addressing this knowledge gap, we obtained plasma from four healthy donors serially sampled five times during 12 h in a single day. For all samples, we measured concentrations of cfDNA and cfRNA using both bulk measurements and gene-specific digital droplet PCR. We found no significant variation attributed to blood draw number for the cfDNA or cfRNA. This indicated that natural diurnal cycles and meal consumption do not appear to significantly affect abundance of total cfDNA, total cfRNA, or our two selected cfRNA transcripts. Conversely, we observed significant variation between individual donors for cfDNA and one of the cfRNA transcripts. The results of this work suggest that it will be important to consider patient-specific baselines when designing reliable circulating cfDNA or cfRNA clinical assays.
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http://dx.doi.org/10.1038/s41598-020-73350-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536441PMC
October 2020

The evolutionary history of 2,658 cancers.

Nature 2020 02 6;578(7793):122-128. Epub 2020 Feb 6.

University of Toronto, Toronto, Ontario, Canada.

Cancer develops through a process of somatic evolution. Sequencing data from a single biopsy represent a snapshot of this process that can reveal the timing of specific genomic aberrations and the changing influence of mutational processes. Here, by whole-genome sequencing analysis of 2,658 cancers as part of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), we reconstruct the life history and evolution of mutational processes and driver mutation sequences of 38 types of cancer. Early oncogenesis is characterized by mutations in a constrained set of driver genes, and specific copy number gains, such as trisomy 7 in glioblastoma and isochromosome 17q in medulloblastoma. The mutational spectrum changes significantly throughout tumour evolution in 40% of samples. A nearly fourfold diversification of driver genes and increased genomic instability are features of later stages. Copy number alterations often occur in mitotic crises, and lead to simultaneous gains of chromosomal segments. Timing analyses suggest that driver mutations often precede diagnosis by many years, if not decades. Together, these results determine the evolutionary trajectories of cancer, and highlight opportunities for early cancer detection.
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http://dx.doi.org/10.1038/s41586-019-1907-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054212PMC
February 2020

Multiregion exome sequencing of ovarian immature teratomas reveals 2N near-diploid genomes, paucity of somatic mutations, and extensive allelic imbalances shared across mature, immature, and disseminated components.

Mod Pathol 2020 06 7;33(6):1193-1206. Epub 2020 Jan 7.

Department of Dermatology, University of California, San Francisco, CA, USA.

Immature teratoma is a subtype of malignant germ cell tumor of the ovary that occurs most commonly in the first three decades of life, frequently with bilateral ovarian disease. Despite being the second most common malignant germ cell tumor of the ovary, little is known about its genetic underpinnings. Here we performed multiregion whole-exome sequencing to interrogate the genetic zygosity, clonal relationship, DNA copy number, and mutational status of 52 pathologically distinct tumor components from ten females with ovarian immature teratomas, with bilateral tumors present in five cases and peritoneal dissemination in seven cases. We found that ovarian immature teratomas are genetically characterized by 2N near-diploid genomes with extensive loss of heterozygosity and an absence of genes harboring recurrent somatic mutations or known oncogenic variants. All components within a single ovarian tumor (immature teratoma, mature teratoma with different histologic patterns of differentiation, and yolk sac tumor) were found to harbor an identical pattern of loss of heterozygosity across the genome, indicating a shared clonal origin. In contrast, the four analyzed bilateral teratomas showed distinct patterns of zygosity changes in the right versus left sided tumors, indicating independent clonal origins. All disseminated teratoma components within the peritoneum (including gliomatosis peritonei) shared a clonal pattern of loss of heterozygosity with either the right or left primary ovarian tumor. The observed genomic loss of heterozygosity patterns indicate that diverse meiotic errors contribute to the formation of ovarian immature teratomas, with 11 out of the 15 genetically distinct clones determined to result from nondisjunction errors during meiosis I or II. Overall, these findings suggest that copy-neutral loss of heterozygosity resulting from meiotic abnormalities may be sufficient to generate ovarian immature teratomas from germ cells.
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http://dx.doi.org/10.1038/s41379-019-0446-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286805PMC
June 2020

Human Tumor-Associated Macrophage and Monocyte Transcriptional Landscapes Reveal Cancer-Specific Reprogramming, Biomarkers, and Therapeutic Targets.

Cancer Cell 2019 04 28;35(4):588-602.e10. Epub 2019 Mar 28.

MRC Centre for Reproductive Health, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh EH16 4TJ, UK; Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, New York 10461, USA. Electronic address:

The roles of tumor-associated macrophages (TAMs) and circulating monocytes in human cancer are poorly understood. Here, we show that monocyte subpopulation distribution and transcriptomes are significantly altered by the presence of endometrial and breast cancer. Furthermore, TAMs from endometrial and breast cancers are transcriptionally distinct from monocytes and their respective tissue-resident macrophages. We identified a breast TAM signature that is highly enriched in aggressive breast cancer subtypes and associated with shorter disease-specific survival. We also identified an auto-regulatory loop between TAMs and cancer cells driven by tumor necrosis factor alpha involving SIGLEC1 and CCL8, which is self-reinforcing through the production of CSF1. Together these data provide direct evidence that monocyte and macrophage transcriptional landscapes are perturbed by cancer, reflecting patient outcomes.
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http://dx.doi.org/10.1016/j.ccell.2019.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472943PMC
April 2019

Circulating tumor DNA dynamics using patient-customized assays are associated with outcome in neoadjuvantly treated breast cancer.

Cold Spring Harb Mol Case Stud 2019 04 1;5(2). Epub 2019 Apr 1.

Department of Molecular and Medical Genetics, Oregon Health and Science University (OHSU) Portland, Oregon 97201, USA.

Pathological complete response (pCR) is an accurate predictor of good outcome following neoadjuvant chemotherapy (NAC) for locally advanced breast cancer. The presence of circulating-tumor DNA (ctDNA) has recently been reported to be strongly predictive of poor outcome in similar patient groups. We monitored ctDNA levels from 10 women undergoing NAC for locally advanced breast cancer using a patient-specific, hybrid-capture sequencing technique sensitive to the level of one altered allele in 10,000. Plasma was collected prior to the start of NAC, prior to each infusion of NAC, and during follow-up for between 350 and 1150 d after the start of NAC. Prior to the start of NAC, ctDNA was detectable in 3/3 triple negative, 3/3 HER2, and 2/4 HER2, ER breast cancer patients. Total cell-free DNA levels were considerably higher when patients were on NAC than at other times. ctDNA dynamics during NAC showed that patients with pCR experienced rapid declines in ctDNA levels, whereas patients without pCR typically showed evidence of residual ctDNA after initiation of treatment. Intriguingly, two of three patients that showed marked increases in ctDNA while on NAC experienced rapid recurrences (<2 yr following start of NAC). The third patient that had increases in ctDNA levels while on NAC had low-grade ER disease and showed residual ctDNA after surgery, which became undetectable after local radiation. Taken together, these results demonstrate the ability of our approach to sensitively serially monitor ctDNA during NAC, and identifies a need to further investigate the possibility of stratifying patients who need additional treatment or identify therapies that are ineffective.
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http://dx.doi.org/10.1101/mcs.a003772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549569PMC
April 2019

Alternative Splicing in Tumors - A Path to Immunogenicity?

N Engl J Med 2019 02;380(9):877-880

From the Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora (J.E.S.); and the Department of Molecular and Medical Genetics, Oregon Health and Science University, and the Cancer Early Detection Advanced Research Center, Knight Cancer Institute, Portland (P.T.S.).

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http://dx.doi.org/10.1056/NEJMcibr1814237DOI Listing
February 2019

Differentiation-state plasticity is a targetable resistance mechanism in basal-like breast cancer.

Nat Commun 2018 09 19;9(1):3815. Epub 2018 Sep 19.

Department of Molecular and Medical Genetics, Oregon Health & Science University, 3181 SW Sam Jackson Park Road L103, Portland, OR, 97239, USA.

Intratumoral heterogeneity in cancers arises from genomic instability and epigenomic plasticity and is associated with resistance to cytotoxic and targeted therapies. We show here that cell-state heterogeneity, defined by differentiation-state marker expression, is high in triple-negative and basal-like breast cancer subtypes, and that drug tolerant persister (DTP) cell populations with altered marker expression emerge during treatment with a wide range of pathway-targeted therapeutic compounds. We show that MEK and PI3K/mTOR inhibitor-driven DTP states arise through distinct cell-state transitions rather than by Darwinian selection of preexisting subpopulations, and that these transitions involve dynamic remodeling of open chromatin architecture. Increased activity of many chromatin modifier enzymes, including BRD4, is observed in DTP cells. Co-treatment with the PI3K/mTOR inhibitor BEZ235 and the BET inhibitor JQ1 prevents changes to the open chromatin architecture, inhibits the acquisition of a DTP state, and results in robust cell death in vitro and xenograft regression in vivo.
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http://dx.doi.org/10.1038/s41467-018-05729-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145927PMC
September 2018

The Cancer Genome Atlas Comprehensive Molecular Characterization of Renal Cell Carcinoma.

Cell Rep 2018 04;23(1):313-326.e5

Leukemia Therapeutics LLC., Hull, MA 02045, USA.

Renal cell carcinoma (RCC) is not a single disease, but several histologically defined cancers with different genetic drivers, clinical courses, and therapeutic responses. The current study evaluated 843 RCC from the three major histologic subtypes, including 488 clear cell RCC, 274 papillary RCC, and 81 chromophobe RCC. Comprehensive genomic and phenotypic analysis of the RCC subtypes reveals distinctive features of each subtype that provide the foundation for the development of subtype-specific therapeutic and management strategies for patients affected with these cancers. Somatic alteration of BAP1, PBRM1, and PTEN and altered metabolic pathways correlated with subtype-specific decreased survival, while CDKN2A alteration, increased DNA hypermethylation, and increases in the immune-related Th2 gene expression signature correlated with decreased survival within all major histologic subtypes. CIMP-RCC demonstrated an increased immune signature, and a uniform and distinct metabolic expression pattern identified a subset of metabolically divergent (MD) ChRCC that associated with extremely poor survival.
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http://dx.doi.org/10.1016/j.celrep.2018.03.075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075733PMC
April 2018

MHC class I loaded ligands from breast cancer cell lines: A potential HLA-I-typed antigen collection.

J Proteomics 2018 03 10;176:13-23. Epub 2018 Jan 10.

Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR, United States.

To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cells. We determined the sequence of 3196 MHC class I ligands representing 1921 proteins from a panel of 20 breast cancer cell lines. After removing duplicate peptides, i.e., the same peptide eluted from more than one cell line, the total number of unique peptides was 2740. Of the unique peptides eluted, more than 1750 had been previously identified, and of these, sixteen have been shown to be immunogenic. Importantly, half of these immunogenic peptides were shared between different breast cancer cell lines. MHC class I binding probability was used to plot the distribution of the eluted peptides in accordance with the binding score for each breast cancer cell line. We also determined that the tested breast cancer cells presented 89 mutation-containing peptides and peptides derived from aberrantly translated genes, 7 of which were shared between four or two different cell lines. Overall, the high throughput identification of MHC class I-loaded peptides is an effective strategy for systematic characterization of cancer peptides, and could be employed for design of multi-peptide anticancer vaccines.

Significance: By employing proteomic analyses of eluted peptides from breast cancer cells, the current study has built an initial HLA-I-typed antigen collection for breast cancer research. It was also determined that immunogenic epitopes can be identified using established cell lines and that shared immunogenic peptides can be found in different cancer types such as breast cancer and leukemia. Importantly, out of 3196 eluted peptides that included duplicate peptides in different cells 89 peptides either contained mutation in their sequence or were derived from aberrant translation suggesting that mutation-containing epitopes are on the order of 2-3% in breast cancer cells. Finally, our results suggest that interfering with MHC class I function is one of the mechanisms of how tumor cells escape immune system attack.
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http://dx.doi.org/10.1016/j.jprot.2018.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5999401PMC
March 2018

Circulating-tumor DNA as an early detection and diagnostic tool.

Curr Opin Genet Dev 2017 Feb 23;42:14-21. Epub 2017 Jan 23.

Oregon Health and Science University Department of Biomedical Engineering, Mail Code CL3G, 2730 SW Moody Ave., Portland, OR 97201-5042, United States; Oregon Health and Science University Knight Cancer Institute, Mail Code CR145, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098, United States; Oregon Health and Science University Center for Spatial System Biomedicine, Collaborative Life Sciences Building, 2730 SW Moody Ave, Mail Code CL3G, Portland, OR 97201-5042, United States. Electronic address:

The development of new circulating-tumor DNA (ctDNA) analysis techniques has led to an explosion of studies demonstrating exciting clinical applications. Non-invasive genotyping can characterize mutations of interest without the need for an invasive biopsy. Serial ctDNA monitoring can assess response to treatment, and potentially identify mechanisms of resistance. Perhaps most excitingly, sensitive ctDNA analysis methods allow for detection of minimally residual disease, predicting recurrence months before clinical presentation. In this review, we highlight several recent, key studies in the ctDNA field and discuss future advances which would further improve patient care.
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http://dx.doi.org/10.1016/j.gde.2016.12.003DOI Listing
February 2017

Context Specificity in Causal Signaling Networks Revealed by Phosphoprotein Profiling.

Cell Syst 2017 01 22;4(1):73-83.e10. Epub 2016 Dec 22.

Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97201, USA. Electronic address:

Signaling networks downstream of receptor tyrosine kinases are among the most extensively studied biological networks, but new approaches are needed to elucidate causal relationships between network components and understand how such relationships are influenced by biological context and disease. Here, we investigate the context specificity of signaling networks within a causal conceptual framework using reverse-phase protein array time-course assays and network analysis approaches. We focus on a well-defined set of signaling proteins profiled under inhibition with five kinase inhibitors in 32 contexts: four breast cancer cell lines (MCF7, UACC812, BT20, and BT549) under eight stimulus conditions. The data, spanning multiple pathways and comprising ∼70,000 phosphoprotein and ∼260,000 protein measurements, provide a wealth of testable, context-specific hypotheses, several of which we experimentally validate. Furthermore, the data provide a unique resource for computational methods development, permitting empirical assessment of causal network learning in a complex, mammalian setting.
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http://dx.doi.org/10.1016/j.cels.2016.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5279869PMC
January 2017

FOXA1 overexpression mediates endocrine resistance by altering the ER transcriptome and IL-8 expression in ER-positive breast cancer.

Proc Natl Acad Sci U S A 2016 10 6;113(43):E6600-E6609. Epub 2016 Oct 6.

Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030; Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; Department of Medicine, Baylor College of Medicine, Houston, TX 77030;

Forkhead box protein A1 (FOXA1) is a pioneer factor of estrogen receptor α (ER)-chromatin binding and function, yet its aberration in endocrine-resistant (Endo-R) breast cancer is unknown. Here, we report preclinical evidence for a role of FOXA1 in Endo-R breast cancer as well as evidence for its clinical significance. FOXA1 is gene-amplified and/or overexpressed in Endo-R derivatives of several breast cancer cell line models. Induced FOXA1 triggers oncogenic gene signatures and proteomic profiles highly associated with endocrine resistance. Integrated omics data reveal IL8 as one of the most perturbed genes regulated by FOXA1 and ER transcriptional reprogramming in Endo-R cells. IL-8 knockdown inhibits tamoxifen-resistant cell growth and invasion and partially attenuates the effect of overexpressed FOXA1. Our study highlights a role of FOXA1 via IL-8 signaling as a potential therapeutic target in FOXA1-overexpressing ER-positive tumors.
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http://dx.doi.org/10.1073/pnas.1612835113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087040PMC
October 2016

Genome co-amplification upregulates a mitotic gene network activity that predicts outcome and response to mitotic protein inhibitors in breast cancer.

Breast Cancer Res 2016 07 1;18(1):70. Epub 2016 Jul 1.

Department of Biomedical Engineering, School of Medicine, Oregon Health & Science University, 3303 SW Bond Ave., CH13B, Portland, OR, 97239, USA.

Background: High mitotic activity is associated with the genesis and progression of many cancers. Small molecule inhibitors of mitotic apparatus proteins are now being developed and evaluated clinically as anticancer agents. With clinical trials of several of these experimental compounds underway, it is important to understand the molecular mechanisms that determine high mitotic activity, identify tumor subtypes that carry molecular aberrations that confer high mitotic activity, and to develop molecular markers that distinguish which tumors will be most responsive to mitotic apparatus inhibitors.

Methods: We identified a coordinately regulated mitotic apparatus network by analyzing gene expression profiles for 53 malignant and non-malignant human breast cancer cell lines and two separate primary breast tumor datasets. We defined the mitotic network activity index (MNAI) as the sum of the transcriptional levels of the 54 coordinately regulated mitotic apparatus genes. The effect of those genes on cell growth was evaluated by small interfering RNA (siRNA).

Results: High MNAI was enriched in basal-like breast tumors and was associated with reduced survival duration and preferential sensitivity to inhibitors of the mitotic apparatus proteins, polo-like kinase, centromere associated protein E and aurora kinase designated GSK462364, GSK923295 and GSK1070916, respectively. Co-amplification of regions of chromosomes 8q24, 10p15-p12, 12p13, and 17q24-q25 was associated with the transcriptional upregulation of this network of 54 mitotic apparatus genes, and we identify transcription factors that localize to these regions and putatively regulate mitotic activity. Knockdown of the mitotic network by siRNA identified 22 genes that might be considered as additional therapeutic targets for this clinically relevant patient subgroup.

Conclusions: We define a molecular signature which may guide therapeutic approaches for tumors with high mitotic network activity.
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http://dx.doi.org/10.1186/s13058-016-0728-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4930593PMC
July 2016

Identification of shared TCR sequences from T cells in human breast cancer using emulsion RT-PCR.

Proc Natl Acad Sci U S A 2016 07 15;113(29):8272-7. Epub 2016 Jun 15.

Department of Immunology and Microbiology, University of Colorado School of Medicine, Aurora, CO 80045;

Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.
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http://dx.doi.org/10.1073/pnas.1606994113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961128PMC
July 2016

Patient-specific factors influence somatic variation patterns in von Hippel-Lindau disease renal tumours.

Nat Commun 2016 05 13;7:11588. Epub 2016 May 13.

Department of Molecular &Medical Genetics, Oregon Health &Science University, Mail Code: CL6S, 2730 SW Moody St, Portland, Oregon 97201, USA.

Cancer development is presumed to be an evolutionary process that is influenced by genetic background and environment. In laboratory animals, genetics and environment are variables that can largely be held constant. In humans, it is possible to compare independent tumours that have developed in the same patient, effectively constraining genetic and environmental variation and leaving only stochastic processes. Patients affected with von Hippel-Lindau disease are at risk of developing multiple independent clear cell renal carcinomas. Here we perform whole-genome sequencing on 40 tumours from six von Hippel-Lindau patients. We confirm that the tumours are clonally independent, having distinct somatic single-nucleotide variants. Although tumours from the same patient show many differences, within-patient patterns are discernible. Single-nucleotide substitution type rates are significantly different between patients and show biases in trinucleotide mutation context. We also observe biases in chromosome copy number aberrations. These results show that genetic background and/or environment can influence the types of mutations that occur.
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http://dx.doi.org/10.1038/ncomms11588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869254PMC
May 2016

Inferring causal molecular networks: empirical assessment through a community-based effort.

Nat Methods 2016 Apr 22;13(4):310-8. Epub 2016 Feb 22.

Department of Bioengineering, Rice University, Houston, Texas, USA.

It remains unclear whether causal, rather than merely correlational, relationships in molecular networks can be inferred in complex biological settings. Here we describe the HPN-DREAM network inference challenge, which focused on learning causal influences in signaling networks. We used phosphoprotein data from cancer cell lines as well as in silico data from a nonlinear dynamical model. Using the phosphoprotein data, we scored more than 2,000 networks submitted by challenge participants. The networks spanned 32 biological contexts and were scored in terms of causal validity with respect to unseen interventional data. A number of approaches were effective, and incorporating known biology was generally advantageous. Additional sub-challenges considered time-course prediction and visualization. Our results suggest that learning causal relationships may be feasible in complex settings such as disease states. Furthermore, our scoring approach provides a practical way to empirically assess inferred molecular networks in a causal sense.
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http://dx.doi.org/10.1038/nmeth.3773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854847PMC
April 2016

ARTIK-52 induces replication-dependent DNA damage and p53 activation exclusively in cells of prostate and breast cancer origin.

Cell Cycle 2016 22;15(3):455-70. Epub 2015 Dec 22.

a Department of Cell Stress Biology , Roswell Park Cancer Institute , Buffalo , NY , USA.

The realization, that the androgen receptor (AR) is essential for prostate cancer (PC) even after relapse following androgen deprivation therapy motivated the search for novel types of AR inhibitors. We proposed that targeting AR expression versus its function would work in cells having either wild type or mutant AR as well as be independent of androgen synthesis pathways. Previously, using a phenotypic screen in androgen-independent PC cells we identified a small molecule inhibitor of AR, ARTIK-52. Treatment with ARTIK-52 caused the loss of AR protein and death of AR-positive, but not AR-negative, PC cells. Here we present data that ARTIK-52 induces degradation of AR mRNA through a mechanism that we were unable to establish. However, we found that ARTIK-52 is toxic to breast cancer (BC) cells expressing AR, although they were not sensitive to AR knockdown, suggesting an AR-independent mechanism of toxicity. Using different approaches we detected that ARTIK-52 induces replication-dependent double strand DNA breaks exclusively in cancer cells of prostate and breast origin, while not causing DNA damage, or any toxicity, in normal cells, as well as in non-PC and non-BC tumor cells, independent of their proliferation status. This amazing specificity, combined with such a basic mechanism of toxicity, makes ARTIK-52 a potentially useful tool to discover novel attractive targets for the treatment of BC and PC. Thus, phenotypic screening allowed us to identify a compound, whose properties cannot be predicted based on existing knowledge and moreover, uncover a barely known link between AR and DNA damage response in PC and BC epithelial cells.
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http://dx.doi.org/10.1080/15384101.2015.1127478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943693PMC
December 2016

Src and STAT3 inhibitors synergize to promote tumor inhibition in renal cell carcinoma.

Oncotarget 2015 Dec;6(42):44675-87

OHSU Knight Cancer Institute, Oregon Health and Science University, Portland, OR 97239, USA.

The intracytoplasmic tyrosine kinase Src serves both as a conduit and a regulator for multiple processes required for the proliferation and survival cancer cells. In some cancers, Src engages with receptor tyrosine kinases to mediate downstream signaling and in other cancers, it regulates gene expression. Src therefore represents a viable oncologic target. However, clinical responses to Src inhibitors, such as dasatinib have been disappointing to date. We identified Stat3 signaling as a potential bypass mechanism that enables renal cell carcinoma (RCC) cells to escape dasatinib treatment. Combined Src-Stat3 inhibition using dasatinib and CYT387 (a JAK/STAT inhibitor) synergistically reduced cell proliferation and increased apoptosis in RCC cells. Moreover, dasatinib and CYT387 combine to suppress YAP1, a transcriptional co-activator that promotes cell proliferation, survival and organ size. Importantly, this combination was well tolerated, and caused marked tumor inhibition in RCC xenografts. These results suggest that combination therapy with inhibitors of Stat3 signaling may be a useful therapeutic approach to increase the efficacy of Src inhibitors.
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http://dx.doi.org/10.18632/oncotarget.5971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792584PMC
December 2015

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma.

N Engl J Med 2016 Jan 4;374(2):135-45. Epub 2015 Nov 4.

Background: Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist.

Methods: We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis.

Results: Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH).

Conclusions: Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).
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http://dx.doi.org/10.1056/NEJMoa1505917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775252PMC
January 2016

Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease.

PLoS One 2015 28;10(8):e0136407. Epub 2015 Aug 28.

Knight Cancer Institute, Oregon Health and Sciences University, Portland, Oregon, United States of America.

The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome. In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient's resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0136407PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552879PMC
May 2016