Publications by authors named "Joel S Parker"

194 Publications

Characterization of the CpG island hypermethylated phenotype (CIMP) subclass in primary melanomas.

J Invest Dermatol 2021 Nov 26. Epub 2021 Nov 26.

Department of Dermatology, School of Medicine, University of North Carolina, Chapel Hill, NC; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.

Cutaneous melanoma can be lethal even if detected at an early stage. Epigenetic profiling may facilitate the identification of aggressive primary melanomas with unfavorable outcomes. We performed clustering of whole-genome methylation data to identify subclasses that were then assessed for survival, clinical features, methylation patterns and biological pathways. Among 89 cutaneous primary invasive melanomas, we identified three methylation subclasses exhibiting low-methylation (LM), intermediate-methylation (IM), or hypermethylation of CpG islands, known as the CpG island methylator phenotype (CIMP). CIMP melanomas occurred as early as T-stage 1b; and, compared with LM melanomas, were associated with age at diagnosis >65 years, lentigo maligna melanoma histologic subtype, presence of ulceration, higher AJCC stage and T-stage, and lower tumor infiltrating lymphocyte (TIL) grade (all p<0.05). Patients with CIMP melanomas had worse melanoma-specific survival (HR=11.84; CI=4.65-30.20) than those with LM melanomas, adjusted for age, sex, AJCC stage, and TIL grade. Genes hypermethylated in CIMP compared with LM melanomas included PTEN, VDR, PD-L1, TET2 and gene sets related to development/differentiation, the extracellular matrix and immunity. CIMP melanomas exhibited hypermethylation of genes important in melanoma progression and tumor immunity; and, although present in some early melanomas, CIMP was associated with worse survival independent of known prognostic factors.
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http://dx.doi.org/10.1016/j.jid.2021.11.017DOI Listing
November 2021

Genome-wide cancer-specific chromatin accessibility patterns derived from archival processed xenograft tumors.

Genome Res 2021 Nov 23. Epub 2021 Nov 23.

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples.
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http://dx.doi.org/10.1101/gr.275219.121DOI Listing
November 2021

A multi-omic single-cell landscape of human gynecologic malignancies.

Mol Cell 2021 Nov 1. Epub 2021 Nov 1.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Genetics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Bioinformatics and Computational Biology Graduate Program, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Deconvolution of regulatory mechanisms that drive transcriptional programs in cancer cells is key to understanding tumor biology. Herein, we present matched transcriptome (scRNA-seq) and chromatin accessibility (scATAC-seq) profiles at single-cell resolution from human ovarian and endometrial tumors processed immediately following surgical resection. This dataset reveals the complex cellular heterogeneity of these tumors and enabled us to quantitatively link variation in chromatin accessibility to gene expression. We show that malignant cells acquire previously unannotated regulatory elements to drive hallmark cancer pathways. Moreover, malignant cells from within the same patients show substantial variation in chromatin accessibility linked to transcriptional output, highlighting the importance of intratumoral heterogeneity. Finally, we infer the malignant cell type-specific activity of transcription factors. By defining the regulatory logic of cancer cells, this work reveals an important reliance on oncogenic regulatory elements and highlights the ability of matched scRNA-seq/scATAC-seq to uncover clinically relevant mechanisms of tumorigenesis in gynecologic cancers.
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http://dx.doi.org/10.1016/j.molcel.2021.10.013DOI Listing
November 2021

UNMASC: tumor-only variant calling with unmatched normal controls.

NAR Cancer 2021 Dec 6;3(4):zcab040. Epub 2021 Oct 6.

Center for Cancer Research, University of Tennessee Health Science Center, 19 South Manassas, Memphis, TN 38163, USA.

Despite years of progress, mutation detection in cancer samples continues to require significant manual review as a final step. Expert review is particularly challenging in cases where tumors are sequenced without matched normal control DNA. Attempts have been made to call somatic point mutations without a matched normal sample by removing well-known germline variants, utilizing unmatched normal controls, and constructing decision rules to classify sequencing errors and private germline variants. With budgetary constraints related to computational and sequencing costs, finding the appropriate number of controls is a crucial step to identifying somatic variants. Our approach utilizes public databases for canonical somatic variants as well as germline variants and leverages information gathered about nearby positions in the normal controls. Drawing from our cohort of targeted capture panel sequencing of tumor and normal samples with varying tumortypes and demographics, these served as a benchmark for our tumor-only variant calling pipeline to observe the relationship between our ability to correctly classify variants against a number of unmatched normals. With our benchmarked samples, approximately ten normal controls were needed to maintain 94% sensitivity, 99% specificity and 76% positive predictive value, far outperforming comparable methods. Our approach, called UNMASC, also serves as a supplement to traditional tumor with matched normal variant calling workflows and can potentially extend to other concerns arising from analyzing next generation sequencing data.
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http://dx.doi.org/10.1093/narcan/zcab040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8494212PMC
December 2021

CPT1A and fatty acid β-oxidation are essential for tumor cell growth and survival in hormone receptor-positive breast cancer.

NAR Cancer 2021 Sep 9;3(3):zcab035. Epub 2021 Sep 9.

Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, 08903, USA.

Chromosome 11q13-14 amplification is a defining feature of high-risk hormone receptor-positive (HR+) breast cancer; however, the mechanism(s) by which this amplicon contributes to breast tumorigenesis remains unclear. In the current study, proteogenomic analyses of >3000 breast tumors from the TCGA, METABRIC and CPTAC studies demonstrated that carnitine palmitoyltransferase 1A (), which is localized to this amplicon, is overexpressed at the mRNA and protein level in aggressive luminal tumors, strongly associated with indicators of tumor proliferation and a predictor of poor prognosis. genetic studies demonstrated that is required for and can promote luminal breast cancer proliferation, survival, as well as colony and mammosphere formation. Since is the rate-limiting enzyme during fatty acid oxidation (FAO), our data indicate that FAO may be essential for these tumors. Pharmacologic inhibition of FAO prevented and tumor growth and cell proliferation as well as promoted apoptosis in luminal breast cancer cells and orthotopic xenograft tumor models. Collectively, our data establish an oncogenic role for and FAO in HR+ luminal tumors and provide preclinical evidence and rationale supporting further investigation of FAO as a potential therapeutic opportunity for the treatment of HR+ breast cancer.
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http://dx.doi.org/10.1093/narcan/zcab035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8428294PMC
September 2021

Validation of reference genes for whole blood gene expression analysis in cord blood of preterm and full-term neonates and peripheral blood of healthy adults.

BMC Genomics 2021 Jun 30;22(1):489. Epub 2021 Jun 30.

Department of Pediatrics, University Hospital Carl Gustav Carus, Technische Universität Dresden, Fetscherstraße 74, 01307, Dresden, Germany.

Background: Preterm birth is the leading cause of neonatal morbidity and mortality, but research efforts in neonatology are complicated due to the unavailability of large volume blood samples. Whole blood assays can be used to overcome this problem by performing both functional and gene expression studies using small amounts of blood. Gene expression studies using RT-qPCR estimate mRNA-levels of target genes normalized to reference genes. The goal of this study was to identify and validate stable reference genes applicable to cord blood samples obtained from developing neonates of different gestational age groups as well as to adult peripheral blood samples. Eight reference gene candidates (ACTB, B2M, GAPDH, GUSB, HPRT, PPIB, RPLP0, RPL13) were analyzed using the three published software algorithms Bestkeeper, GeNorm and NormFinder.

Results: A normalization factor consisting of ACTB and PPIB allows for comparative expression analyses of neonatal samples from different gestational age groups. Normalization factors consisting of GAPDH and PPIB or ACTB and GAPDH are suitable when samples from preterm and full-term neonates and adults are compared. However, all candidate reference genes except RPLP0 exhibited significant intergroup gene expression variance and a higher gene expression towards an older age which resulted in a small but statistically significant systematic bias. Systematic analysis of RNA-seq data revealed new reference gene candidates with potentially superior stability.

Conclusions: The current study identified suitable normalization factors and proposed the use of the additional single gene RPLP0 to avoid systematic bias. This combination will enable comparative analyses not only between neonates of different gestational ages, but also between neonates and adults, as it facilitates more detailed investigations of developmental gene expression changes. The use of software algorithms did not prevent unintended systematic bias. This generally highlights the need for careful validation of such results to prevent false interpretation of potential age-dependent changes in gene expression. To identify the most stable reference genes in the future, RNA-seq based global approaches are recommended.
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http://dx.doi.org/10.1186/s12864-021-07801-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8244134PMC
June 2021

FOXA1 and adaptive response determinants to HER2 targeted therapy in TBCRC 036.

NPJ Breast Cancer 2021 May 12;7(1):51. Epub 2021 May 12.

Department of Pharmacology, UNC Chapel Hill, Chapel Hill, NC, USA.

Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.
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http://dx.doi.org/10.1038/s41523-021-00258-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8115531PMC
May 2021

Radiation-related genomic profile of papillary thyroid carcinoma after the Chernobyl accident.

Science 2021 05 22;372(6543). Epub 2021 Apr 22.

Radiation Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD 20892, USA.

The 1986 Chernobyl nuclear power plant accident increased papillary thyroid carcinoma (PTC) incidence in surrounding regions, particularly for radioactive iodine (I)-exposed children. We analyzed genomic, transcriptomic, and epigenomic characteristics of 440 PTCs from Ukraine (from 359 individuals with estimated childhood I exposure and 81 unexposed children born after 1986). PTCs displayed radiation dose-dependent enrichment of fusion drivers, nearly all in the mitogen-activated protein kinase pathway, and increases in small deletions and simple/balanced structural variants that were clonal and bore hallmarks of nonhomologous end-joining repair. Radiation-related genomic alterations were more pronounced for individuals who were younger at exposure. Transcriptomic and epigenomic features were strongly associated with driver events but not radiation dose. Our results point to DNA double-strand breaks as early carcinogenic events that subsequently enable PTC growth after environmental radiation exposure.
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http://dx.doi.org/10.1126/science.abg2538DOI Listing
May 2021

Cistrome analysis of YY1 uncovers a regulatory axis of YY1:BRD2/4-PFKP during tumorigenesis of advanced prostate cancer.

Nucleic Acids Res 2021 05;49(9):4971-4988

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA.

Castration-resistant prostate cancer (CRPC) is a terminal disease and the molecular underpinnings of CRPC development need to be better understood in order to improve its treatment. Here, we report that a transcription factor Yin Yang 1 (YY1) is significantly overexpressed during prostate cancer progression. Functional and cistrome studies of YY1 uncover its roles in promoting prostate oncogenesis in vitro and in vivo, as well as sustaining tumor metabolism including the Warburg effect and mitochondria respiration. Additionally, our integrated genomics and interactome profiling in prostate tumor show that YY1 and bromodomain-containing proteins (BRD2/4) co-occupy a majority of gene-regulatory elements, coactivating downstream targets. Via gene loss-of-function and rescue studies and mutagenesis of YY1-bound cis-elements, we unveil an oncogenic pathway in which YY1 directly binds and activates PFKP, a gene encoding the rate-limiting enzyme for glycolysis, significantly contributing to the YY1-enforced Warburg effect and malignant growth. Altogether, this study supports a master regulator role for YY1 in prostate tumorigenesis and reveals a YY1:BRD2/4-PFKP axis operating in advanced prostate cancer with implications for therapy.
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http://dx.doi.org/10.1093/nar/gkab252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136773PMC
May 2021

CRTC1/MAML2 directs a PGC-1α-IGF-1 circuit that confers vulnerability to PPARγ inhibition.

Cell Rep 2021 02;34(8):108768

Division of Oral and Craniofacial Health Sciences, UNC Adams School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Cell Biology and Physiology, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Biomedical Research Imaging Center, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Lineberger Comprehensive Cancer Center, Cancer Cell Biology Program, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address:

Mucoepidermoid carcinoma (MEC) is a life-threatening salivary gland cancer that is driven primarily by a transcriptional coactivator fusion composed of cyclic AMP-regulated transcriptional coactivator 1 (CRTC1) and mastermind-like 2 (MAML2). The mechanisms by which the chimeric CRTC1/MAML2 (C1/M2) oncoprotein rewires gene expression programs that promote tumorigenesis remain poorly understood. Here, we show that C1/M2 induces transcriptional activation of the non-canonical peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) splice variant PGC-1α4, which regulates peroxisome proliferator-activated receptor gamma (PPARγ)-mediated insulin-like growth factor 1 (IGF-1) expression. This mitogenic transcriptional circuitry is consistent across cell lines and primary tumors. C1/M2-positive tumors exhibit IGF-1 pathway activation, and small-molecule drug screens reveal that tumor cells harboring the fusion gene are selectively sensitive to IGF-1 receptor (IGF-1R) inhibition. Furthermore, this dependence on autocrine regulation of IGF-1 transcription renders MEC cells susceptible to PPARγ inhibition with inverse agonists. These results yield insights into the aberrant coregulatory functions of C1/M2 and identify a specific vulnerability that can be exploited for precision therapy.
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http://dx.doi.org/10.1016/j.celrep.2021.108768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955229PMC
February 2021

Rad18 mediates specific mutational signatures and shapes the genomic landscape of carcinogen-induced tumors .

NAR Cancer 2021 Mar 6;3(1):zcaa037. Epub 2021 Jan 6.

Department of Biostatistics, University of North Carolina at Chapel Hill, 135 Dauer Drive, 3101 McGavran-Greenberg Hall, Chapel Hill, NC 27599, USA.

The E3 ubiquitin ligase Rad18 promotes a damage-tolerant and error-prone mode of DNA replication termed trans-lesion synthesis that is pathologically activated in cancer. However, the impact of vertebrate on cancer genomes is not known. To determine how Rad18 affects mutagenesis , we have developed and implemented a novel computational pipeline to analyze genomes of carcinogen (7, 12-Dimethylbenz[a]anthracene, DMBA)-induced skin tumors from and mice. We show that mediates specific mutational signatures characterized by high levels of A(T)>T(A) single nucleotide variations (SNVs). In tumors, an alternative mutation pattern arises, which is characterized by increased numbers of deletions >4 bp. Comparison with annotated human mutational signatures shows that COSMIC signature 22 predominates in tumors whereas tumors are characterized by increased contribution of COSMIC signature 3 (a hallmark of BRCA-mutant tumors). Analysis of The Cancer Genome Atlas shows that expression is strongly associated with high SNV burdens, suggesting RAD18 also promotes mutagenesis in human cancers. Taken together, our results show Rad18 promotes mutagenesis , modulates DNA repair pathway choice in neoplastic cells, and mediates specific mutational signatures that are present in human tumors.
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http://dx.doi.org/10.1093/narcan/zcaa037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787264PMC
March 2021

Re-expression of SMARCA4/BRG1 in small cell carcinoma of ovary, hypercalcemic type (SCCOHT) promotes an epithelial-like gene signature through an AP-1-dependent mechanism.

Elife 2020 12 23;9. Epub 2020 Dec 23.

Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, United States.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive form of ovarian cancer. SCCOHT tumors have inactivating mutations in (BRG1), one of the two mutually exclusive ATPases of the SWI/SNF chromatin remodeling complex. To address the role that BRG1 loss plays in SCCOHT tumorigenesis, we performed integrative multi-omic analyses in SCCOHT cell lines +/- BRG1 reexpression. BRG1 reexpression induced a gene and protein signature similar to an epithelial cell and gained chromatin accessibility sites correlated with other epithelial originating TCGA tumors. Gained chromatin accessibility and BRG1 recruited sites were strongly enriched for transcription-factor-binding motifs of AP-1 family members. Furthermore, AP-1 motifs were enriched at the promoters of highly upregulated epithelial genes. Using a dominant-negative AP-1 cell line, we found that both AP-1 DNA-binding activity and BRG1 reexpression are necessary for the gene and protein expression of epithelial genes. Our study demonstrates that BRG1 reexpression drives an epithelial-like gene and protein signature in SCCOHT cells that depends upon by AP-1 activity.
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http://dx.doi.org/10.7554/eLife.59073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7813545PMC
December 2020

Development and validation of a NanoString BASE47 bladder cancer gene classifier.

PLoS One 2020 17;15(12):e0243935. Epub 2020 Dec 17.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

Background: Recent molecular characterization of urothelial cancer (UC) has suggested potential pathways in which to direct treatment, leading to a host of targeted therapies in development for UC. In parallel, gene expression profiling has demonstrated that high-grade UC is a heterogeneous disease. Prognostic basal-like and luminal-like subtypes have been identified and an accurate transcriptome BASE47 classifier has been developed. However, these phenotypes cannot be broadly investigated due to the lack of a clinically viable diagnostic assay. We sought to develop and evaluate a diagnostic classifier of UC subtype with the goal of accurate classification from clinically available specimens.

Methods: Tumor samples from 52 patients with high-grade UC were profiled for BASE47 genes concurrently by RNAseq as well as NanoString. After design and technical validation of a BASE47 NanoString probeset, results from the RNAseq and NanoString were used to translate diagnostic criteria to the Nanostring platform. Evaluation of repeatability and accuracy was performed to derive a final Nanostring based classifier. Diagnostic classification resulting from the NanoString BASE47 classifier was validated on an independent dataset (n = 30). The training and validation datasets accurately classified 87% and 93% of samples, respectively.

Results: Here we have derived a NanoString-platform BASE47 classifier that accurately predicts basal-like and luminal-like subtypes in high grade urothelial cancer. We have further validated our new NanoString BASE47 classifier on an independent dataset and confirmed high accuracy when compared with our original Transcriptome BASE47 classifier.

Conclusions: The NanoString BASE47 classifier provides a faster turnaround time, a lower cost per sample to process, and maintains the accuracy of the original subtype classifier for better clinical implementation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243935PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7745986PMC
February 2021

Neoadjuvant pazopanib and molecular analysis of tissue response in renal cell carcinoma.

JCI Insight 2020 11 19;5(22). Epub 2020 Nov 19.

Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina, USA.

BACKGROUNDSurgery remains the frontline therapy for patients with localized clear cell renal cell carcinoma (ccRCC); however, 20%-40% recur. Angiogenesis inhibitors have improved survival in metastatic patients and may result in responses in the neoadjuvant setting. The impact of these agents on the tumor genetic heterogeneity or the immune milieu is largely unknown. This phase II study was designed to evaluate safety, response, and effect on tumor tissue of neoadjuvant pazopanib.METHODSccRCC patients with localized disease received pazopanib (800 mg daily; median 8 weeks), followed by nephrectomy. Five tumors were examined for mutations by whole exome sequencing from samples collected before therapy and at nephrectomy. These samples underwent RNA sequencing; 17 samples were available for posttreatment assessment.RESULTSTwenty-one patients were enrolled. The overall response rate was 8 of 21 (38%). No patients with progressive disease. At 1-year, response-free survival and overall survival was 83% and 89%, respectively. The most frequent grade 3 toxicity was hypertension (33%, 7 of 21). Sequencing revealed strong concordance between pre- and posttreatment samples within individual tumors, suggesting tumors harbor stable core profiles. However, a reduction in private mutations followed treatment, suggesting a selective process favoring enrichment of driver mutations.CONCLUSIONNeoadjuvant pazopanib is safe and active in ccRCC. Future genomic analyses may enable the segregation of driver and passenger mutations. Furthermore, tumor infiltrating immune cells persist during therapy, suggesting that pazopanib can be combined with immune checkpoint inhibitors without dampening the immune response.FUNDINGSupport was provided by Novartis and GlaxoSmithKline as part of an investigator-initiated study.
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http://dx.doi.org/10.1172/jci.insight.132852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710285PMC
November 2020

Improved T-cell Receptor Diversity Estimates Associate with Survival and Response to Anti-PD-1 Therapy.

Cancer Immunol Res 2021 01 11;9(1):103-112. Epub 2020 Nov 11.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

T-cell receptor (TCR) repertoire profiling has emerged as a powerful tool for biological discovery and biomarker development in cancer immunology and immunotherapy. A key statistic derived from repertoire profiling data is , which summarizes the frequency distribution of TCRs within a mixed population. Despite the growing use of TCR diversity metrics in clinical trial correlative studies in oncology, their accuracy has not been validated using published ground-truth datasets. Here, we reported the performance characteristics of methods for TCR repertoire profiling from RNA-sequencing data, showed undersampling as a prominent source of bias in diversity estimates, and derived a model via statistical learning that attenuates bias to produce corrected diversity estimates. This modeled diversity improved discrimination in The Cancer Genome Atlas data and associated with survival and treatment response in patients with melanoma treated with anti-PD-1 therapy, where the commonly used diversity normalizations did not. These findings have the potential to increase our understanding of the tumor immune microenvironment and improve the accuracy of predictions of patient responses to immunotherapy.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0398DOI Listing
January 2021

A multivariable prognostic score to guide systemic therapy in early-stage HER2-positive breast cancer: a retrospective study with an external evaluation.

Lancet Oncol 2020 11;21(11):1455-1464

SOLTI Breast Cancer Research Group, Barcelona, Spain; Department of Medical Oncology, Hospital 12 de Octubre, Madrid, Spain.

Background: In early-stage HER2-positive breast cancer, escalation or de-escalation of systemic therapy is a controversial topic. As an aid to treatment decisions, we aimed to develop a prognostic assay that integrates multiple data types for predicting survival outcome in patients with newly diagnosed HER2-positive breast cancer.

Methods: We derived a combined prognostic model using retrospective clinical-pathological data on stromal tumour-infiltrating lymphocytes, PAM50 subtypes, and expression of 55 genes obtained from patients who participated in the Short-HER phase 3 trial. The trial enrolled patients with newly diagnosed, node-positive, HER2-positive breast cancer or, if node negative, with at least one risk factor (ie, tumour size >2 cm, histological grade 3, lymphovascular invasion, Ki67 >20%, age ≤35 years, or hormone receptor negativity), and randomly assigned them to adjuvant anthracycline plus taxane-based combinations with either 9 weeks or 1 year of trastuzumab. Trastuzumab was administered intravenously every 3 weeks (8 mg/kg loading dose at first cycle, and 6 mg/kg thereafter) for 18 doses or weekly (4 mg/kg loading dose in the first week, and 2 mg/kg thereafter) for 9 weeks, starting concomitantly with the first taxane dose. Median follow-up was 91·4 months (IQR 75·1-105·6). The primary objective of our study was to derive and evaluate a combined prognostic score associated with distant metastasis-free survival (the time between randomisation and distant recurrence or death before recurrence), an exploratory endpoint in Short-HER. Patient samples in the training dataset were split into a training set (n=290) and a testing set (n=145), balancing for event and treatment group. The training set was further stratified into 100 iterations of Monte-Carlo cross validation (MCCV). Cox proportional hazard models were fit to MCCV training samples using Elastic-Net. A maximum of 92 features were assessed. The final prognostic model was evaluated in an independent combined dataset of 267 patients with early-stage HER2-positive breast cancer treated with different neoadjuvant and adjuvant anti-HER2-based combinations and from four other studies (PAMELA, CHER-LOB, Hospital Clinic, and Padova) with disease-free survival outcome data.

Findings: From Short-HER, data from 435 (35%) of 1254 patients for tumour size (T1 vs rest), nodal status (N0 vs rest), number of tumour-infiltrating lymphocytes (continuous variable), subtype (HER2-enriched and basal-like vs rest), and 13 genes composed the final model (named HER2DX). HER2DX was significantly associated with distant metastasis-free survival as a continuous variable (p<0·0001). HER2DX median score for quartiles 1-2 was identified as the cutoff to identify low-risk patients; and the score that distinguished quartile 3 from quartile 4 was the cutoff to distinguish medium-risk and high-risk populations. The 5-year distant metastasis-free survival of the low-risk, medium-risk, and high-risk populations were 98·1% (95% CI 96·3-99·9), 88·9% (83·2-95·0), and 73·9% (66·0-82·7), respectively (low-risk vs high-risk hazard ratio [HR] 0·04, 95% CI 0·0-0·1, p<0·0001). In the evaluation cohort, HER2DX was significantly associated with disease-free survival as a continuous variable (HR 2·77, 95% CI 1·4-5·6, p=0·0040) and as group categories (low-risk vs high-risk HR 0·27, 0·1-0·7, p=0·005). 5-year disease-free survival in the HER2DX low-risk group was 93·5% (89·0-98·3%) and in the high-risk group was 81·1% (71·5-92·1).

Interpretation: The HER2DX combined prognostic score identifies patients with early-stage, HER2-positive breast cancer who might be candidates for escalated or de-escalated systemic treatment. Future clinical validation of HER2DX seems warranted to establish its use in different scenarios, especially in the neoadjuvant setting.

Funding: Instituto Salud Carlos III, Save the Mama, Pas a Pas, Fundación Científica, Asociación Española Contra el Cáncer, Fundación SEOM, National Institutes of Health, Agenzia Italiana del Farmaco, International Agency for Research on Cancer, and the Veneto Institute of Oncology, and Italian Association for Cancer Research.
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http://dx.doi.org/10.1016/S1470-2045(20)30450-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140650PMC
November 2020

Survival, Pathologic Response, and Genomics in CALGB 40601 (Alliance), a Neoadjuvant Phase III Trial of Paclitaxel-Trastuzumab With or Without Lapatinib in HER2-Positive Breast Cancer.

J Clin Oncol 2020 12 23;38(35):4184-4193. Epub 2020 Oct 23.

Lineberger Comprehensive Center, University of North Carolina, Chapel Hill, NC.

Purpose: CALGB 40601 assessed whether dual versus single human epidermal growth factor receptor 2 (HER2) -targeting drugs added to neoadjuvant chemotherapy increased pathologic complete response (pCR). Here, we report relapse-free survival (RFS), overall survival (OS), and gene expression signatures that predict pCR and survival.

Patients And Methods: Three hundred five women with untreated stage II and III HER2-positive breast cancer were randomly assigned to receive weekly paclitaxel combined with trastuzumab plus lapatinib (THL), trastuzumab (TH), or lapatinib (TL). The primary end point was pCR, and secondary end points included RFS, OS, and gene expression analyses. mRNA sequencing was performed on 264 pretreatment samples.

Results: One hundred eighteen patients were randomly allocated to THL, 120 to TH, and 67 to TL. At more than 7 years of follow-up, THL had significantly better RFS and OS than did TH (RFS hazard ratio, 0.32; 95% CI, 0.14 to 0.71; = .005; OS hazard ratio, 0.34; 95% CI, 0.12 to 0.94; = .037), with no difference between TH and TL. Of 688 previously described gene expression signatures, significant associations were found in 215 with pCR, 45 with RFS, and only 22 with both pCR and RFS (3.2%). Specifically, eight immune signatures were significantly correlated with a higher pCR rate and better RFS. Among patients with residual disease, the immunoglobulin G signature was an independent, good prognostic factor, whereas the HER2-enriched signature, which was associated with a higher pCR rate, showed a significantly shorter RFS.

Conclusion: In CALGB 40601, dual HER2-targeting resulted in significant RFS and OS benefits. Integration of intrinsic subtype and immune signatures allowed for the prediction of pCR and RFS, both overall and within the residual disease group. These approaches may provide means for rational escalation and de-escalation treatment strategies in HER2-positive breast cancer.
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http://dx.doi.org/10.1200/JCO.20.01276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723687PMC
December 2020

Subependymal giant cell astrocytomas are characterized by mTORC1 hyperactivation, a very low somatic mutation rate, and a unique gene expression profile.

Mod Pathol 2021 02 13;34(2):264-279. Epub 2020 Oct 13.

Cancer Genetics Laboratory, Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5-10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2-46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0-7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation.
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http://dx.doi.org/10.1038/s41379-020-00659-9DOI Listing
February 2021

A Prognostic Model Based on PAM50 and Clinical Variables (PAM50MET) for Metastatic Hormone Receptor-positive HER2-negative Breast Cancer.

Clin Cancer Res 2020 12 22;26(23):6141-6148. Epub 2020 Sep 22.

Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

Purpose: Predicting prognosis in HR/HER2 metastatic breast cancer (MBC) might be clinically useful; however, no validated prognostic biomarkers exist in this setting to date.

Patients And Methods: In phase III, EGF30008 trial, 484 patients with HER2 MBC who received letrozole and placebo or lapatinib were selected. PAM50 data, ECOG performance status, visceral disease, number of metastasis, biopsy type, and age were evaluated. A progression-free survival (PFS) Cox model was evaluated. The final model (PAM50MET) with a prespecified cutoff was validated in patients ( = 261) with HR/HER2 advanced breast cancer (aBC) from BOLERO-2 (phase III trial that evaluated exemestane and placebo or everolimus).

Results: In EGF30008, prognostic models with PAM50 plus clinical variables yielded higher C-index values versus models with only PAM50 or clinical variables. The PAM50MET model combined 21 variables: 2 PAM50 subtypes, basal signature, 14 genes, and 4 clinical variables. In EGF30008, the optimized cutoff was associated with PFS [HR = 0.37; 95% confidence interval (CI), 0.29-0.47; < 0.0001] and overall survival (OS; HR = 0.37; 95% CI, 0.27-0.51; < 0.0001). The median (months; 95% CI) PFS and OS were 22.24 (19.0-24.9) and not reached in PAM50MET-low versus 9.13 (8.15-11.0) and 33.0 (28.0-40.0) in PAM50MET-high groups, respectively. In BOLERO-2, the PAM50MET-low was associated with better PFS (HR = 0.72; 95% CI, 0.53-0.96; = 0.028) and OS (HR = 0.51; 95% CI, 0.35-0.69; < 0.0001). The median (months) (95% CI) PFS and OS were 6.93 (5.57-11.0) and 36.9 (33.4-NA) in PAM50MET-low versus 5.23 (4.2-6.8) and 23.5 (20.2-28.3) in PAM50MET-high groups, respectively.

Conclusions: PAM50MET is prognostic in HR/HER2 MBC, and further evaluation might help identify candidates for endocrine therapy only or novel therapies.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2793DOI Listing
December 2020

Genomic Analysis of Germline Variation Associated with Survival of Patients with Colorectal Cancer Treated with Chemotherapy Plus Biologics in CALGB/SWOG 80405 (Alliance).

Clin Cancer Res 2021 01 21;27(1):267-275. Epub 2020 Sep 21.

Duke Cancer Institute, Duke University, Durham, North Carolina.

Purpose: Irinotecan/5-fluorouracil (5-FU; FOLFIRI) or oxaliplatin/5-FU (FOLFOX), combined with bevacizumab or cetuximab, are approved, first-line treatments for metastatic colorectal cancer (mCRC). We aimed at identifying germline variants associated with survival in patients with mCRC treated with these regimens in Cancer and Leukemia Group B/SWOG 80405.

Experimental Design: Patients with mCRC receiving either FOLFOX or FOLFIRI were randomized to either cetuximab or bevacizumab. DNA from peripheral blood was genotyped for approximately 700,000 SNPs. The association between SNPs and overall survival (OS) was tested in 613 patients of genetically estimated European ancestry using Cox proportional hazards models.

Results: The four most significant SNPs associated with OS were three haplotypic SNPs between microsomal glutathione S-transferase 1 () and LIM domain only 3 (, representative HR, 1.56; = 1.30 × 10), and rs11644916 in (HR, 1.39, = 4.26 × 10). is a well-established tumor suppressor gene in colorectal cancer, and rs11644916 (G>A) conferred shorter OS. Median OS for patients with the AA, AG, or GG genotypes was 18.4, 25.6, or 36.4 months, respectively. In 90 patients with stage IV colorectal cancer from The Cancer Genome Atlas (TCGA), rs11649255 in [in almost complete linkage disequilibrium (LD) with rs11644916], was associated with shorter OS (HR, 2.24, = 0.0096). Using rs11648673 in (in very high LD with rs11644916 and with functional evidence), luciferase activity in three colorectal cancer cell lines was reduced.

Conclusions: This is the first large genome-wide association study ever conducted in patients with mCRC treated with first-line standard treatment in a randomized phase III trial. A common SNP in conferred worse OS and the effect was replicated in TCGA. Further studies in colorectal cancer experimental models are required.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785628PMC
January 2021

Tn-Seq Analysis Identifies Genes Important for Yersinia pestis Adherence during Primary Pneumonic Plague.

mSphere 2020 08 5;5(4). Epub 2020 Aug 5.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Following inhalation, rapidly colonizes the lung to establish infection during primary pneumonic plague. Although several adhesins have been identified in spp., the factors mediating early adherence in the lung remain unknown. To identify genes important for adherence during primary pneumonic plague, we used transposon insertion sequencing (Tn-seq). Wild-type and capsule mutant (Δ) transposon mutant libraries were serially passaged to enrich for nonadherent mutants in the lung using a mouse model of primary pneumonic plague. Sequencing of the passaged libraries revealed six mutants that were significantly enriched in both the wild-type and Δ backgrounds. The enriched mutants had insertions in genes that encode transcriptional regulators, chaperones, an endoribonuclease, and YPO3903, a hypothetical protein. Using single-strain infections and a transcriptional analysis, we identified a significant role for in adherence in the lung and showed that YPO3903 regulated transcript levels of which encodes a fimbria previously implicated in adherence Deletion of had a minor effect on adherence in the lung, suggesting that YPO3903 regulates other adhesins in addition to By enriching for mutations in genes that regulate the expression or assembly of multiple genes or proteins, we obtained screen results indicating that there may be not just one dominant adhesin but rather several factors that contribute to early adherence during primary pneumonic plague. Colonization of the lung by is a critical first step in establishing infection during primary pneumonic plague, a disease characterized by high lethality. However, the mechanisms by which adheres in the lung after inhalation remain elusive. Here, we used Tn-seq to identify genes important for adherence early during primary pneumonic plague. Our mutant enrichment strategy resulted in the identification of genes important for regulation and assembly of genes and proteins rather than adhesin genes themselves. These results reveal that there may be multiple adhesins or redundancy among adhesins. Identifying the adhesins regulated by the genes identified in our enrichment screen may reveal novel therapeutic targets for preventing adherence and the subsequent development of pneumonic plague.
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http://dx.doi.org/10.1128/mSphere.00715-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407073PMC
August 2020

A pan-cancer analysis of the frequency of DNA alterations across cell cycle activity levels.

Oncogene 2020 08 24;39(32):5430-5440. Epub 2020 Jun 24.

Department of Oncology and Pathology, Karolinska Institutet and University Hospital, Stockholm, Sweden.

Pan-cancer genomic analyses based on the magnitude of pathway activity are currently lacking. Focusing on the cell cycle, we examined the DNA mutations and chromosome arm-level aneuploidy within tumours with low, intermediate and high cell-cycle activity in 9515 pan-cancer patients with 32 different tumour types. Boxplots showed that cell-cycle activity varied broadly across and within all cancers. TP53 and PIK3CA mutations were common in all cell cycle score (CCS) tertiles but with increasing frequency as cell-cycle activity levels increased (P < 0.001). Mutations in BRAF and gains in 16p were less frequent in CCS High tumours (P < 0.001). In Kaplan-Meier analysis, patients whose tumours were CCS Low had a longer Progression Free Interval (PFI) relative to Intermediate or High (P < 0.001) and this significance remained in multivariable analysis (CCS Intermediate: HR = 1.37; 95% CI 1.17-1.60, CCS High: 1.54; 1.29-1.84, CCS Low = Ref). These results demonstrate that whilst similar DNA alterations can be found at all cell-cycle activity levels, some notable exceptions exist. Moreover, independent prognostic information can be derived on a pan-cancer level from a simple measure of cell-cycle activity.
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http://dx.doi.org/10.1038/s41388-020-1367-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159764PMC
August 2020

FGFR4 regulates tumor subtype differentiation in luminal breast cancer and metastatic disease.

J Clin Invest 2020 09;130(9):4871-4887

Lineberger Comprehensive Center and.

Mechanisms driving tumor progression from less aggressive subtypes to more aggressive states represent key targets for therapy. We identified a subset of luminal A primary breast tumors that give rise to HER2-enriched (HER2E) subtype metastases, but remain clinically HER2 negative (cHER2-). By testing the unique genetic and transcriptomic features of these cases, we developed the hypothesis that FGFR4 likely participates in this subtype switching. To evaluate this, we developed 2 FGFR4 genomic signatures using a patient-derived xenograft (PDX) model treated with an FGFR4 inhibitor, which inhibited PDX growth in vivo. Bulk tumor gene expression analysis and single-cell RNA sequencing demonstrated that the inhibition of FGFR4 signaling caused molecular switching. In the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohort, FGFR4-induced and FGFR4-repressed signatures each predicted overall survival. Additionally, the FGFR4-induced signature was an independent prognostic factor beyond subtype and stage. Supervised analysis of 77 primary tumors with paired metastases revealed that the FGFR4-induced signature was significantly higher in luminal/ER+ tumor metastases compared with their primaries. Finally, multivariate analysis demonstrated that the FGFR4-induced signature also predicted site-specific metastasis for lung, liver, and brain, but not for bone or lymph nodes. These data identify a link between FGFR4-regulated genes and metastasis, suggesting treatment options for FGFR4-positive patients, whose high expression is not caused by mutation or amplification.
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http://dx.doi.org/10.1172/JCI130323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456247PMC
September 2020

Identifying transcriptional profiles and evaluating prognostic biomarkers of HIV-associated diffuse large B-cell lymphoma from Malawi.

Mod Pathol 2020 08 20;33(8):1482-1491. Epub 2020 Feb 20.

University of North Carolina, Chapel Hill, NC, USA.

Lymphoma incidence in sub-Saharan Africa (SSA) is increasing due to HIV and population aging. Diffuse Large B-cell lymphoma (DLBCL), the most common lymphoma in SSA and worldwide, is highly associated with HIV, but molecular studies of HIV-associated DLBCL are scarce globally. We describe profiling of DLBCL from Malawi, aiming to elucidate tumor biology and identify clinically meaningful biomarkers specifically for SSA. Between June 1, 2013 and June 1, 2016, 59 cases of DLBCL (32 HIV+/27 HIV-) enrolled in the Kamuzu Central Hospital Lymphoma Study were characterized, of which 54 (92%) were negative for Epstein-Barr virus. Gene expression profiling (GEP) by whole transcriptome sequencing was performed on the first 36 cases (22 HIV+/14 HIV-). Immunohistochemistry (IHC) and GEP results were compared with published data and correlated to clinical outcome and pathologic features. Unsupervised clustering strongly segregated DLBCL by HIV status (p = 0.0003, Chi-squared test), indicating a marked contribution of HIV to expression phenotype. Pathway analysis identified that HIV-associated tumors were enriched in hypoxia, oxidative stress, and metabolism related gene expression patterns. Cell-of-origin subtype, determined by sequencing and IHC, did not associate with differences in overall survival (OS), while Ki-67 proliferation index ≥80% was associated with inferior OS in HIV+ DLBCL only (p = 0.03) and cMYC/BCL2 co-expression by IHC was negatively prognostic across the entire cohort (p = 0.01). This study provides among the first molecular characterizations of DLBCL from SSA, demonstrates marked gene expression differences by HIV status, and identifies genomic and immunophenotypic characteristics that can inform future basic and clinical investigations.
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http://dx.doi.org/10.1038/s41379-020-0506-3DOI Listing
August 2020

A P53-Independent DNA Damage Response Suppresses Oncogenic Proliferation and Genome Instability.

Cell Rep 2020 02;30(5):1385-1399.e7

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

The Mre11-Rad50-Nbs1 complex is a DNA double-strand break sensor that mediates a tumor-suppressive DNA damage response (DDR) in cells undergoing oncogenic stress, yet the mechanisms underlying this effect are poorly understood. Using a genetically inducible primary mammary epithelial cell model, we demonstrate that Mre11 suppresses proliferation and DNA damage induced by diverse oncogenic drivers through a p53-independent mechanism. Breast tumorigenesis models engineered to express a hypomorphic Mre11 allele exhibit increased levels of oncogene-induced DNA damage, R-loop accumulation, and chromosomal instability with a characteristic copy number loss phenotype. Mre11 complex dysfunction is identified in a subset of human triple-negative breast cancers and is associated with increased sensitivity to DNA-damaging therapy and inhibitors of ataxia telangiectasia and Rad3 related (ATR) and poly (ADP-ribose) polymerase (PARP). Thus, deficiencies in the Mre11-dependent DDR drive proliferation and genome instability patterns in p53-deficient breast cancers and represent an opportunity for therapeutic exploitation.
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http://dx.doi.org/10.1016/j.celrep.2020.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361372PMC
February 2020

Virus expression detection reveals RNA-sequencing contamination in TCGA.

BMC Genomics 2020 Jan 28;21(1):79. Epub 2020 Jan 28.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, USA.

Background: Contamination of reagents and cross contamination across samples is a long-recognized issue in molecular biology laboratories. While often innocuous, contamination can lead to inaccurate results. Cantalupo et al., for example, found HeLa-derived human papillomavirus 18 (H-HPV18) in several of The Cancer Genome Atlas (TCGA) RNA-sequencing samples. This work motivated us to assess a greater number of samples and determine the origin of possible contaminations using viral sequences. To detect viruses with high specificity, we developed the publicly available workflow, VirDetect, that detects virus and laboratory vector sequences in RNA-seq samples. We applied VirDetect to 9143 RNA-seq samples sequenced at one TCGA sequencing center (28/33 cancer types) over 5 years.

Results: We confirmed that H-HPV18 was present in many samples and determined that viral transcripts from H-HPV18 significantly co-occurred with those from xenotropic mouse leukemia virus-related virus (XMRV). Using laboratory metadata and viral transcription, we determined that the likely contaminant was a pool of cell lines known as the "common reference", which was sequenced alongside TCGA RNA-seq samples as a control to monitor quality across technology transitions (i.e. microarray to GAII to HiSeq), and to link RNA-seq to previous generation microarrays that standardly used the "common reference". One of the cell lines in the pool was a laboratory isolate of MCF-7, which we discovered was infected with XMRV; another constituent of the pool was likely HeLa cells.

Conclusions: Altogether, this indicates a multi-step contamination process. First, MCF-7 was infected with an XMRV. Second, this infected cell line was added to a pool of cell lines, which contained HeLa. Finally, RNA from this pool of cell lines contaminated several TCGA tumor samples most-likely during library construction. Thus, these human tumors with H-HPV or XMRV reads were likely not infected with H-HPV 18 or XMRV.
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http://dx.doi.org/10.1186/s12864-020-6483-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986043PMC
January 2020

Leveraging gene expression subgroups to classify DLBCL patients and select for clinical benefit from a novel agent.

Blood 2020 03;135(13):1008-1018

Celgene Institute for Translational Research Europe, a Bristol-Myers Squibb Company, Seville, Spain.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, commonly described by cell-of-origin (COO) molecular subtypes. We sought to identify novel patient subgroups through an unsupervised analysis of a large public dataset of gene expression profiles from newly diagnosed de novo DLBCL patients, yielding 2 biologically distinct subgroups characterized by differences in the tumor microenvironment. Pathway analysis and immune deconvolution algorithms identified higher B-cell content and a strong proliferative signal in subgroup A and enriched T-cell, macrophage, and immune/inflammatory signals in subgroup B, reflecting similar biology to published DLBCL stratification research. A gene expression classifier, featuring 26 gene expression scores, was derived from the public dataset to discriminate subgroup A (classifier-negative, immune-low) and subgroup B (classifier-positive, immune-high) patients. Subsequent application to an independent series of diagnostic biopsies replicated the subgroups, with immune cell composition confirmed via immunohistochemistry. Avadomide, a CRL4CRBN E3 ubiquitin ligase modulator, demonstrated clinical activity in relapsed/refractory DLBCL patients, independent of COO subtypes. Given the immunomodulatory activity of avadomide and the need for a patient-selection strategy, we applied the gene expression classifier to pretreatment biopsies from relapsed/refractory DLBCL patients receiving avadomide (NCT01421524). Classifier-positive patients exhibited an enrichment in response rate and progression-free survival of 44% and 6.2 months vs 19% and 1.6 months for classifier-negative patients (hazard ratio, 0.49; 95% confidence interval, 0.280-0.86; P = .0096). The classifier was not prognostic for rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone or salvage immunochemotherapy. The classifier described here discriminates DLBCL tumors based on tumor and nontumor composition and has potential utility to enrich for clinical response to immunomodulatory agents, including avadomide.
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http://dx.doi.org/10.1182/blood.2019002414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099333PMC
March 2020

Genetic determinants of the molecular portraits of epithelial cancers.

Nat Commun 2019 12 11;10(1):5666. Epub 2019 Dec 11.

Curriculum in Bioinformatics and Computational Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

The ability to characterize and predict tumor phenotypes is crucial to precision medicine. In this study, we present an integrative computational approach using a genome-wide association analysis and an Elastic Net prediction method to analyze the relationship between DNA copy number alterations and an archive of gene expression signatures. Across breast cancers, we are able to quantitatively predict many gene signatures levels within individual tumors with high accuracy based upon DNA copy number features alone, including proliferation status and Estrogen-signaling pathway activity. We can also predict many other key phenotypes, including intrinsic molecular subtypes, estrogen receptor status, and TP53 mutation. This approach is also applied to TCGA Pan-Cancer, which identify repeatedly predictable signatures across tumor types including immune features in lung squamous and basal-like breast cancers. These Elastic Net DNA predictors could also be called from DNA-based gene panels, thus facilitating their use as biomarkers to guide therapeutic decision making.
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http://dx.doi.org/10.1038/s41467-019-13588-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906458PMC
December 2019

Interaction between androgen receptor and coregulator SLIRP is regulated by Ack1 tyrosine kinase and androgen.

Sci Rep 2019 12 9;9(1):18637. Epub 2019 Dec 9.

Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA.

Aberrant activation of the androgen receptor (AR) may play a critical role in castration resistant prostate cancer. After ligand binding, AR is recruited to the androgen responsive element (ARE) sequences on the DNA where AR interaction with coactivators and corepressors modulates transcription. We demonstrated that phosphorylation of AR at Tyr-267 by Ack1/TNK2 tyrosine kinase results in nuclear translocation, DNA binding, and androgen-dependent gene transcription in a low androgen environment. In order to dissect downstream mechanisms, we searched for proteins whose interaction with AR was regulated by Ack1. SLIRP (SRA stem-loop interacting RNA binding protein) was identified as a candidate protein. Interaction between AR and SLIRP was disrupted by Ack1 kinase activity as well as androgen or heregulin treatment. The noncoding RNA, SRA, was required for AR-SLIRP interaction. SLIRP was bound to ARE's of AR target genes in the absence of androgen. Treatment with androgen or heregulin led to dissociation of SLIRP from the ARE. Whole transcriptome analysis of SLIRP knockdown in androgen responsive LNCaP cells showed that SLIRP affects a significant subset of androgen-regulated genes. Our data suggest that Ack1 kinase and androgen regulate interaction between AR and SLIRP and that SLIRP functions as a coregulator of AR with properties of a corepressor in a context-dependent manner.
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http://dx.doi.org/10.1038/s41598-019-55057-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901447PMC
December 2019

PIK3CA Mutation in HPV-Associated OPSCC Patients Receiving Deintensified Chemoradiation.

J Natl Cancer Inst 2020 08;112(8):855-858

Department of Radiation Oncology, University of North Carolina Hospitals, Chapel Hill, NC.

PIK3CA is the most frequently mutated gene in human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC). Prognostic implications of such mutations remain unknown. We sought to elucidate the clinical significance of PIK3CA mutations in HPV-associated OPSCC patients treated with definitive chemoradiation (CRT). Seventy-seven patients with HPV-associated OPSCC were enrolled on two phase II clinical trials of deintensified CRT (60 Gy intensity-modulated radiotherapy with concurrent weekly cisplatin). Targeted next-generation sequencing was performed. Of the 77 patients, nine had disease recurrence (two regional, four distant, three regional and distant). Thirty-four patients had mutation(s) identified; 16 had PIK3CA mutations. Patients with wild-type-PIK3CA had statistically significantly higher 3-year disease-free survival than PIK3CA-mutant patients (93.4%, 95% confidence interval [CI] = 85.0% to 99.9% vs 68.8%, 95% CI = 26.7% to 89.8%; P = .004). On multivariate analysis, PIK3CA mutation was the only variable statistically significantly associated with disease recurrence (hazard ratio = 5.71, 95% CI = 1.53 to 21.3; P = .01). PIK3CA mutation is associated with worse disease-free survival in a prospective cohort of newly diagnosed HPV-associated OPSCC patients treated with deintensified CRT.
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http://dx.doi.org/10.1093/jnci/djz224DOI Listing
August 2020
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