Publications by authors named "Lodewyk Wessels"

208 Publications

Epigenetic and transcriptional analysis reveals a core transcriptional program conserved in clonal prostate cancer metastases.

Mol Oncol 2021 Feb 11. Epub 2021 Feb 11.

Division of Oncogenomics, Oncode Institute, Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands.

The epigenomic regulation of transcriptional programs in metastatic prostate cancer is poorly understood. We studied the epigenomic landscape of prostate cancer drivers using transcriptional profiling and chromatin immunoprecipitation sequencing (ChIP-seq) in four clonal metastatic tumors derived from a single prostate cancer patient. Our epigenomic analyses focused on Androgen Receptor (AR), which is a key oncogenic driver in prostate cancer , the AR pioneer factor FOXA1, chromatin insulator CTCF, as well as for modified histones H3K27ac and H3K27me3. The vast majority of AR binding sites were shared among healthy prostate, primary prostate cancer and metastatic tumor samples, signifying core AR-driven transcriptional regulation within the prostate cell lineage. Genes associated with core AR-binding events were significantly enriched for essential genes in prostate cancer cell proliferation. Remarkably, the metastasis-specific active AR binding sites showed no differential transcriptional output, indicating a robust transcriptional program across metastatic samples. Combined, our data reveal a core transcriptional program in clonal metastatic prostate cancer, despite epigenomic differences in the AR cistrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/1878-0261.12923DOI Listing
February 2021

Correction to: A role for the unfolded protein response stress sensor ERN1 in regulating the response to MEK inhibitors in KRAS mutant colon cancers.

Genome Med 2021 Feb 10;13(1):24. Epub 2021 Feb 10.

Division of Molecular Carcinogenesis, Oncode Institute, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13073-020-00815-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7877087PMC
February 2021

Preoperative ipilimumab plus nivolumab in locoregionally advanced urothelial cancer: the NABUCCO trial.

Nat Med 2020 12 12;26(12):1839-1844. Epub 2020 Oct 12.

Department of Medical Oncology, Netherlands Cancer Institute, Amsterdam, the Netherlands.

Preoperative immunotherapy with anti-PD1 plus anti-CTLA4 antibodies has shown remarkable pathological responses in melanoma and colorectal cancer. In NABUCCO (ClinicalTrials.gov: NCT03387761 ), a single-arm feasibility trial, 24 patients with stage III urothelial cancer (UC) received two doses of ipilimumab and two doses of nivolumab, followed by resection. The primary endpoint was feasibility to resect within 12 weeks from treatment start. All patients were evaluable for the study endpoints and underwent resection, 23 (96%) within 12 weeks. Grade 3-4 immune-related adverse events occurred in 55% of patients and in 41% of patients when excluding clinically insignificant laboratory abnormalities. Eleven patients (46%) had a pathological complete response (pCR), meeting the secondary efficacy endpoint. Fourteen patients (58%) had no remaining invasive disease (pCR or pTisN0/pTaN0). In contrast to studies with anti-PD1/PD-L1 monotherapy, complete response to ipilimumab plus nivolumab was independent of baseline CD8 presence or T-effector signatures. Induction of tertiary lymphoid structures upon treatment was observed in responding patients. Our data indicate that combined CTLA-4 plus PD-1 blockade might provide an effective preoperative treatment strategy in locoregionally advanced UC, irrespective of pre-existing CD8 T cell activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-020-1085-zDOI Listing
December 2020

Multiple low dose therapy as an effective strategy to treat EGFR inhibitor-resistant NSCLC tumours.

Nat Commun 2020 06 22;11(1):3157. Epub 2020 Jun 22.

Division of Molecular Carcinogenesis and Oncode Institute. The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

Resistance to targeted cancer drugs is thought to result from selective pressure exerted by a high drug dose. Partial inhibition of multiple components in the same oncogenic signalling pathway may add up to complete pathway inhibition, while decreasing the selective pressure on each component to acquire a resistance mutation. We report here testing of this Multiple Low Dose (MLD) therapy model in EGFR mutant NSCLC. We show that as little as 20% of the individual effective drug doses is sufficient to completely block MAPK signalling and proliferation when used in 3D (RAF + MEK + ERK) or 4D (EGFR + RAF + MEK + ERK) inhibitor combinations. Importantly, EGFR mutant NSCLC cells treated with MLD therapy do not develop resistance. Using several animal models, we find durable responses to MLD therapy without associated toxicity. Our data support the notion that MLD therapy could deliver clinical benefit, even for those having acquired resistance to third generation EGFR inhibitor therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16952-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308397PMC
June 2020

Functional Categorization of Variants of Uncertain Clinical Significance in Homologous Recombination Repair Complementation Assays.

Clin Cancer Res 2020 Sep 16;26(17):4559-4568. Epub 2020 Jun 16.

Oncode Institute and Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

Purpose: Because is a high-risk breast/ovarian cancer susceptibility gene, sequence variants of uncertain clinical significance (VUS) complicate genetic counseling. As most VUS are rare, reliable classification based on clinical and genetic data is often impossible. However, all pathogenic variants analyzed result in defective homologous recombination DNA repair (HRR). Thus, VUS may be categorized based on their functional impact on this pathway.

Experimental Design: Two hundred thirty-eight VUS-comprising most VUS known in the Netherlands and Belgium-were tested for their ability to complement deficient mouse embryonic stem cells in HRR, using cisplatin and olaparib sensitivity assays and a direct repeat GFP (DR-GFP) HRR assay. Assays were validated using 25 known benign and 25 known pathogenic variants. For assessment of pathogenicity by a multifactorial likelihood analysis method, we collected clinical and genetic data for functionally deleterious VUS and VUS occurring in three or more families.

Results: All three assays showed 100% sensitivity and specificity (95% confidence interval, 83%-100%). Out of 238 VUS, 45 showed functional defects, 26 of which were deleterious in all three assays. For 13 of these 26 variants, we could calculate the probability of pathogenicity using clinical and genetic data, resulting in the identification of 7 (likely) pathogenic variants.

Conclusions: We have functionally categorized 238 VUS using three different HRR-related assays. Classification based on clinical and genetic data alone for a subset of these variants confirmed the high sensitivity and specificity of our functional assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-20-0255DOI Listing
September 2020

Radiogenomic Analysis of Breast Cancer by Linking MRI Phenotypes with Tumor Gene Expression.

Radiology 2020 08 26;296(2):277-287. Epub 2020 May 26.

From the Division of Molecular Carcinogenesis, Oncode Institute (T.B., S.C., L.F.A.W.), Division of Molecular Pathology (S.C., E.H.L., J.W.), Department of Radiology (C.E.L.), and Department of Pathology (J.W.), the Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands; Image Sciences Institute, University Medical Center Utrecht, Utrecht, the Netherlands (B.H.M.v.d.V., M.A.V., K.G.A.G.); and Faculty of Electrical Engineering, Mathematics, and Computer Science, Delft University of Technology, Delft, the Netherlands (L.F.A.W.).

Background Better understanding of the molecular biology associated with MRI phenotypes may aid in the diagnosis and treatment of breast cancer. Purpose To discover the associations between MRI phenotypes of breast cancer and their underlying molecular biology derived from gene expression data. Materials and Methods This is a secondary analysis of the Multimodality Analysis and Radiologic Guidance in Breast-Conserving Therapy, or MARGINS, study. MARGINS included patients eligible for breast-conserving therapy between November 2000 and December 2008 for preoperative breast MRI. Tumor RNA was collected for sequencing from surgical specimen. Twenty-one computer-generated MRI features of tumors were condensed into seven MRI factors related to tumor size, shape, initial enhancement, late enhancement, smoothness of enhancement, sharpness, and sharpness variation. These factors were associated with gene expression levels from RNA sequencing by using gene set enrichment analysis. Statistical significance of these associations was evaluated by using a sample permutation test and the false discovery rate. Results Gene expression and MRI data were obtained for 295 patients (mean age, 56 years ± 10.3 [standard deviation]). Larger and more irregular tumors showed increased expression of cell cycle and DNA damage checkpoint genes (false discovery rate <0.25; normalized enrichment statistic [NES], 2.15). Enhancement and sharpness of the tumor margin were associated with expression of ribosomal proteins (false discovery rate <0.25; NES, 1.95). Smoothness of enhancement, tumor size, and tumor shape were associated with expression of genes involved in the extracellular matrix (false discovery rate <0.25; NES, 2.25). Conclusion Breast cancer MRI phenotypes were related to their underlying molecular biology revealed by using RNA sequencing. The association between enhancements and sharpness of the tumor margin with the ribosome suggests that these MRI features may be imaging biomarkers for drugs targeting the ribosome. © RSNA, 2020 See also the editorial by Cho in this issue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1148/radiol.2020191453DOI Listing
August 2020

Integrative analysis of genomic amplification-dependent expression and loss-of-function screen identifies ASAP1 as a driver gene in triple-negative breast cancer progression.

Oncogene 2020 05 31;39(20):4118-4131. Epub 2020 Mar 31.

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University, 2300 RA, Leiden, The Netherlands.

The genetically heterogeneous triple-negative breast cancer (TNBC) continues to be an intractable disease, due to lack of effective targeted therapies. Gene amplification is a major event in tumorigenesis. Genes with amplification-dependent expression are being explored as therapeutic targets for cancer treatment. In this study, we have applied Analytical Multi-scale Identification of Recurring Events analysis and transcript quantification in the TNBC genome across 222 TNBC tumors and identified 138 candidate genes with positive correlation in copy number gain (CNG) and gene expression. siRNA-based loss-of-function screen of the candidate genes has validated EGFR, MYC, ASAP1, IRF2BP2, and CCT5 genes as drivers promoting proliferation in different TNBC cells. MYC, ASAP1, IRF2BP2, and CCT5 display frequent CNG and concurrent expression over 2173 breast cancer tumors (cBioPortal dataset). More frequently are MYC and ASAP1 amplified in TNBC tumors (>30%, n = 320). In particular, high expression of ASAP1, the ADP-ribosylation factor GTPase-activating protein, is significantly related to poor metastatic relapse-free survival of TNBC patients (n = 257, bc-GenExMiner). Furthermore, we have revealed that silencing of ASAP1 modulates numerous cytokine and apoptosis signaling components, such as IL1B, TRAF1, AIFM2, and MAP3K11 that are clinically relevant to survival outcomes of TNBC patients. ASAP1 has been reported to promote invasion and metastasis in various cancer cells. Our findings that ASAP1 is an amplification-dependent TNBC driver gene promoting TNBC cell proliferation, functioning upstream apoptosis components, and correlating to clinical outcomes of TNBC patients, support ASAP1 as a potential actionable target for TNBC treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-020-1279-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7220851PMC
May 2020

Endonuclease FEN1 Coregulates ERα Activity and Provides a Novel Drug Interface in Tamoxifen-Resistant Breast Cancer.

Cancer Res 2020 05 19;80(10):1914-1926. Epub 2020 Mar 19.

Division of Oncogenomics, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

Estrogen receptor α (ERα) is a key transcriptional regulator in the majority of breast cancers. ERα-positive patients are frequently treated with tamoxifen, but resistance is common. In this study, we refined a previously identified 111-gene outcome prediction-classifier, revealing FEN1 as the strongest determining factor in ERα-positive patient prognostication. FEN1 levels were predictive of outcome in tamoxifen-treated patients, and FEN1 played a causal role in ERα-driven cell growth. FEN1 impacted the transcriptional activity of ERα by facilitating coactivator recruitment to the ERα transcriptional complex. FEN1 blockade induced proteasome-mediated degradation of activated ERα, resulting in loss of ERα-driven gene expression and eradicated tumor cell proliferation. Finally, a high-throughput 465,195 compound screen identified a novel FEN1 inhibitor, which effectively blocked ERα function and inhibited proliferation of tamoxifen-resistant cell lines as well as -cultured ERα-positive breast tumors. Collectively, these results provide therapeutic proof of principle for FEN1 blockade in tamoxifen-resistant breast cancer. SIGNIFICANCE: These findings show that pharmacologic inhibition of FEN1, which is predictive of outcome in tamoxifen-treated patients, effectively blocks ERα function and inhibits proliferation of tamoxifen-resistant tumor cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-19-2207DOI Listing
May 2020

Approximating multivariate posterior distribution functions from Monte Carlo samples for sequential Bayesian inference.

PLoS One 2020 13;15(3):e0230101. Epub 2020 Mar 13.

Division of Molecular Carcinogenesis, Netherlands Cancer Institute, Amsterdam, The Netherlands.

An important feature of Bayesian statistics is the opportunity to do sequential inference: the posterior distribution obtained after seeing a dataset can be used as prior for a second inference. However, when Monte Carlo sampling methods are used for inference, we only have a set of samples from the posterior distribution. To do sequential inference, we then either have to evaluate the second posterior at only these locations and reweight the samples accordingly, or we can estimate a functional description of the posterior probability distribution from the samples and use that as prior for the second inference. Here, we investigated to what extent we can obtain an accurate joint posterior from two datasets if the inference is done sequentially rather than jointly, under the condition that each inference step is done using Monte Carlo sampling. To test this, we evaluated the accuracy of kernel density estimates, Gaussian mixtures, mixtures of factor analyzers, vine copulas and Gaussian processes in approximating posterior distributions, and then tested whether these approximations can be used in sequential inference. In low dimensionality, Gaussian processes are more accurate, whereas in higher dimensionality Gaussian mixtures, mixtures of factor analyzers or vine copulas perform better. In our test cases of sequential inference, using posterior approximations gives more accurate results than direct sample reweighting, but joint inference is still preferable over sequential inference whenever possible. Since the performance is case-specific, we provide an R package mvdens with a unified interface for the density approximation methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230101PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069631PMC
June 2020

Response of metastatic mouse invasive lobular carcinoma to mTOR inhibition is partly mediated by the adaptive immune system.

Oncoimmunology 2020 12;9(1):1724049. Epub 2020 Feb 12.

Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Effective treatment of invasive lobular carcinoma (ILC) of the breast is hampered by late detection, invasive growth, distant metastasis, and poor response to chemotherapy. Phosphoinositide 3-kinase (PI3K) signaling, one of the major druggable oncogenic signaling networks, is frequently activated in ILC. We investigated treatment response and resistance to AZD8055, an inhibitor of mammalian target of rapamycin (mTOR), in the (KEP) mouse model of metastatic ILC. Inhibition of mTOR signaling blocked the growth of primary KEP tumors as well as the progression of metastatic disease. However, primary tumors and distant metastases eventually acquired resistance after long-term AZD8055 treatment, despite continued effective suppression of mTOR signaling in cancer cells. Interestingly, therapeutic responses were associated with increased expression of genes related to antigen presentation. Consistent with this observation, increased numbers of tumor-infiltrating major histocompatibility complex class II-positive (MHCII+) immune cells were observed in treatment-responsive KEP tumors. Acquisition of treatment resistance was associated with loss of MHCII+ cells and reduced expression of genes related to the adaptive immune system. The therapeutic efficacy of mTOR inhibition was reduced in mice lacking mature T and B lymphocytes, compared to immunocompetent mice. Furthermore, therapy responsiveness could be partially rescued by transplanting AZD8055-resistant KEP tumors into treatment-naïve immunocompetent hosts. Collectively, these data indicate that the PI3K signaling pathway is an attractive therapeutic target in invasive lobular carcinoma, and that part of the therapeutic effect of mTOR inhibition is mediated by the adaptive immune system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/2162402X.2020.1724049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028325PMC
February 2020

A network analysis to identify mediators of germline-driven differences in breast cancer prognosis.

Authors:
Maria Escala-Garcia Jean Abraham Irene L Andrulis Hoda Anton-Culver Volker Arndt Alan Ashworth Paul L Auer Päivi Auvinen Matthias W Beckmann Jonathan Beesley Sabine Behrens Javier Benitez Marina Bermisheva Carl Blomqvist William Blot Natalia V Bogdanova Stig E Bojesen Manjeet K Bolla Anne-Lise Børresen-Dale Hiltrud Brauch Hermann Brenner Sara Y Brucker Barbara Burwinkel Carlos Caldas Federico Canzian Jenny Chang-Claude Stephen J Chanock Suet-Feung Chin Christine L Clarke Fergus J Couch Angela Cox Simon S Cross Kamila Czene Mary B Daly Joe Dennis Peter Devilee Janet A Dunn Alison M Dunning Miriam Dwek Helena M Earl Diana M Eccles A Heather Eliassen Carolina Ellberg D Gareth Evans Peter A Fasching Jonine Figueroa Henrik Flyger Manuela Gago-Dominguez Susan M Gapstur Montserrat García-Closas José A García-Sáenz Mia M Gaudet Angela George Graham G Giles David E Goldgar Anna González-Neira Mervi Grip Pascal Guénel Qi Guo Christopher A Haiman Niclas Håkansson Ute Hamann Patricia A Harrington Louise Hiller Maartje J Hooning John L Hopper Anthony Howell Chiun-Sheng Huang Guanmengqian Huang David J Hunter Anna Jakubowska Esther M John Rudolf Kaaks Pooja Middha Kapoor Renske Keeman Cari M Kitahara Linetta B Koppert Peter Kraft Vessela N Kristensen Diether Lambrechts Loic Le Marchand Flavio Lejbkowicz Annika Lindblom Jan Lubiński Arto Mannermaa Mehdi Manoochehri Siranoush Manoukian Sara Margolin Maria Elena Martinez Tabea Maurer Dimitrios Mavroudis Alfons Meindl Roger L Milne Anna Marie Mulligan Susan L Neuhausen Heli Nevanlinna William G Newman Andrew F Olshan Janet E Olson Håkan Olsson Nick Orr Paolo Peterlongo Christos Petridis Ross L Prentice Nadege Presneau Kevin Punie Dhanya Ramachandran Gad Rennert Atocha Romero Mythily Sachchithananthan Emmanouil Saloustros Elinor J Sawyer Rita K Schmutzler Lukas Schwentner Christopher Scott Jacques Simard Christof Sohn Melissa C Southey Anthony J Swerdlow Rulla M Tamimi William J Tapper Manuel R Teixeira Mary Beth Terry Heather Thorne Rob A E M Tollenaar Ian Tomlinson Melissa A Troester Thérèse Truong Clare Turnbull Celine M Vachon Lizet E van der Kolk Qin Wang Robert Winqvist Alicja Wolk Xiaohong R Yang Argyrios Ziogas Paul D P Pharoah Per Hall Lodewyk F A Wessels Georgia Chenevix-Trench Gary D Bader Thilo Dörk Douglas F Easton Sander Canisius Marjanka K Schmidt

Nat Commun 2020 01 16;11(1):312. Epub 2020 Jan 16.

Division of Molecular Pathology, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands.

Identifying the underlying genetic drivers of the heritability of breast cancer prognosis remains elusive. We adapt a network-based approach to handle underpowered complex datasets to provide new insights into the potential function of germline variants in breast cancer prognosis. This network-based analysis studies ~7.3 million variants in 84,457 breast cancer patients in relation to breast cancer survival and confirms the results on 12,381 independent patients. Aggregating the prognostic effects of genetic variants across multiple genes, we identify four gene modules associated with survival in estrogen receptor (ER)-negative and one in ER-positive disease. The modules show biological enrichment for cancer-related processes such as G-alpha signaling, circadian clock, angiogenesis, and Rho-GTPases in apoptosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-14100-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965101PMC
January 2020

In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer.

EMBO J 2020 03 13;39(5):e102169. Epub 2020 Jan 13.

Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Genetically engineered mouse models (GEMMs) of cancer have proven to be of great value for basic and translational research. Although CRISPR-based gene disruption offers a fast-track approach for perturbing gene function and circumvents certain limitations of standard GEMM development, it does not provide a flexible platform for recapitulating clinically relevant missense mutations in vivo. To this end, we generated knock-in mice with Cre-conditional expression of a cytidine base editor and tested their utility for precise somatic engineering of missense mutations in key cancer drivers. Upon intraductal delivery of sgRNA-encoding vectors, we could install point mutations with high efficiency in one or multiple endogenous genes in situ and assess the effect of defined allelic variants on mammary tumorigenesis. While the system also produces bystander insertions and deletions that can stochastically be selected for when targeting a tumor suppressor gene, we could effectively recapitulate oncogenic nonsense mutations. We successfully applied this system in a model of triple-negative breast cancer, providing the proof of concept for extending this flexible somatic base editing platform to other tissues and tumor types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.2019102169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049816PMC
March 2020

RUNX2/CBFB modulates the response to MEK inhibitors through activation of receptor tyrosine kinases in KRAS-mutant colorectal cancer.

Transl Oncol 2020 Feb 20;13(2):201-211. Epub 2019 Dec 20.

Division of Molecular Carcinogenesis, Oncode Institute, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066, CX, the Netherlands. Electronic address:

Intrinsic and acquired resistances are major hurdles preventing the effective use of MEK inhibitors for treatment of colorectal cancer (CRC). Some 35-45% of colorectal cancers are KRAS-mutant and their treatment remains challenging as these cancers are refractory to MEK inhibitor treatment, because of feedback activation of receptor tyrosine kinases (RTKs). We reported previously that loss of ERN1 sensitizes a subset of KRAS-mutant colon cancer cells to MEK inhibition. Here we show that the loss of RUNX2 or its cofactor CBFB can confer MEK inhibitor resistance in CRC cells. Mechanistically, we find that cells with genetically ablated RUNX2 or CBFB activate multiple RTKs, which coincides with high SHP2 phosphatase activity, a phosphatase that relays signals from the cell membrane to downstream pathways governing growth and proliferation. Moreover, we show that high activity of SHP2 is causal to loss of RUNX2-induced MEK inhibitor resistance, as a small molecule SHP2 inhibitor reinstates sensitivity to MEK inhibitor in RUNX2 knockout cells. Our results reveal an unexpected role for loss of RUNX2/CBFB in regulating RTK activity in colon cancer, resulting in reduced sensitivity to MEK inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tranon.2019.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6931198PMC
February 2020

TLE3 loss confers AR inhibitor resistance by facilitating GR-mediated human prostate cancer cell growth.

Elife 2019 12 19;8. Epub 2019 Dec 19.

Division of Molecular Carcinogenesis, Netherlands Cancer Institute, Amsterdam, Netherlands.

Androgen receptor (AR) inhibitors represent the mainstay of prostate cancer treatment. In a genome-wide CRISPR-Cas9 screen using LNCaP prostate cancer cells, loss of co-repressor conferred resistance to AR antagonists apalutamide and enzalutamide. Genes differentially expressed upon loss share AR as the top transcriptional regulator, and loss rescued the expression of a subset of androgen-responsive genes upon enzalutamide treatment. GR expression was strongly upregulated upon AR inhibition in a -negative background. This was consistent with binding of TLE3 and AR at the locus. Furthermore, GR binding was observed proximal to TLE3/AR-shared genes. GR inhibition resensitized cells to enzalutamide. Analyses of patient samples revealed an association between TLE3 and GR levels that reflected our findings in LNCaP cells, of which the clinical relevance is yet to be determined. Together, our findings reveal a mechanistic link between TLE3 and GR-mediated resistance to AR inhibitors in human prostate cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.47430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6968917PMC
December 2019

Pancreatic cancer organoids recapitulate disease and allow personalized drug screening.

Proc Natl Acad Sci U S A 2019 Dec 9. Epub 2019 Dec 9.

Oncode Institute, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands;

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1911273116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936689PMC
December 2019

Androgen modulation of XBP1 is functionally driving part of the AR transcriptional program.

Endocr Relat Cancer 2020 Feb;27(2):67-79

Division of Oncogenomics, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Prostate cancer development and progression is largely dependent on androgen receptor (AR) signaling. AR is a hormone-dependent transcription factor, which binds to thousands of sites throughout the human genome to regulate expression of directly responsive genes, including pro-survival genes that enable tumor cells to cope with increased cellular stress. ERN1 and XBP1 - two key players of the unfolded protein response (UPR) - are among such stress-associated genes. Here, we show that XBP1 levels in primary prostate cancer are associated with biochemical recurrence in five independent cohorts. Patients who received AR-targeted therapies had significantly lower XBP1 expression, whereas expression of the active form of XBP1 (XBP1s) was elevated. In vitro results show that AR-induced ERN1 expression led to increased XBP1s mRNA and protein levels. Furthermore, ChIP-seq analysis revealed that XBP1s binds enhancers upon stress stimuli regulating genes involved in UPR processes, eIF2 signaling and protein ubiquitination. We further demonstrate genomic overlap of AR- and XBP1s-binding sites, suggesting genomic conversion of the two signaling cascades. Transcriptomic effects of XBP1 were further studied by knockdown experiments, which lead to decreased expression of androgen-responsive genes and UPR genes. These results suggest a two-step mechanism of gene regulation, which involves androgen-induced expression of ERN1, thereby enhancing XBP1 splicing and transcriptional activity. This signaling cascade may prepare the cells for the increased protein folding, mRNA decay and translation that accompanies AR-regulated tumor cell proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1530/ERC-19-0181DOI Listing
February 2020

CD4 T cell help creates memory CD8 T cells with innate and help-independent recall capacities.

Nat Commun 2019 12 4;10(1):5531. Epub 2019 Dec 4.

Division of Tumor Biology and Immunology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, 1066 CX, Amsterdam, The Netherlands.

CD4 T cell help is required for the generation of CD8 cytotoxic T lymphocyte (CTL) memory. Here, we use genome-wide analyses to show how CD4 T cell help delivered during priming promotes memory differentiation of CTLs. Help signals enhance IL-15-dependent maintenance of central memory T (T) cells. More importantly, help signals regulate the size and function of the effector memory T (T) cell pool. Helped T cells produce Granzyme B and IFNγ upon antigen-independent, innate-like recall by IL-12 and IL-18. In addition, helped memory CTLs express the effector program characteristic of helped primary CTLs upon recall with MHC class I-restricted antigens, likely due to epigenetic imprinting and sustained mRNA expression of effector genes. Our data thus indicate that during priming, CD4 T cell help optimizes CTL memory by creating T cells with innate and help-independent antigen-specific recall capacities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-13438-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892909PMC
December 2019

Genomic data integration by WON-PARAFAC identifies interpretable factors for predicting drug-sensitivity in vivo.

Nat Commun 2019 11 6;10(1):5034. Epub 2019 Nov 6.

Division of Molecular Carcinogenesis, Oncode Institute, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Integrative analyses that summarize and link molecular data to treatment sensitivity are crucial to capture the biological complexity which is essential to further precision medicine. We introduce Weighted Orthogonal Nonnegative parallel factor analysis (WON-PARAFAC), a data integration method that identifies sparse and interpretable factors. WON-PARAFAC summarizes the GDSC1000 cell line compendium in 130 factors. We interpret the factors based on their association with recurrent molecular alterations, pathway enrichment, cancer type, and drug-response. Crucially, the cell line derived factors capture the majority of the relevant biological variation in Patient-Derived Xenograft (PDX) models, strongly suggesting our factors capture invariant and generalizable aspects of cancer biology. Furthermore, drug response in cell lines is better and more consistently translated to PDXs using factor-based predictors as compared to raw feature-based predictors. WON-PARAFAC efficiently summarizes and integrates multiway high-dimensional genomic data and enhances translatability of drug response prediction from cell lines to patient-derived xenografts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-13027-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834616PMC
November 2019

Are contralateral parenchymal enhancement on dynamic contrast-enhanced MRI and genomic ER-pathway activity in ER-positive/HER2-negative breast cancer related?

Eur J Radiol 2019 Dec 16;121:108705. Epub 2019 Oct 16.

Image Sciences Institute, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands.

Purpose: To retrospectively explore the relation between parenchymal enhancement of the healthy contralateral breast on dynamic contrast-enhanced magnetic resonance imaging (MRI) and genomic tests for estrogen receptor (ER)-pathway activity in patients with ER-positive/HER2-negative cancer.

Methods: A subset of 227 consecutively included patients with unilateral invasive ER-positive/HER2-negative breast cancer underwent dynamic contrast-enhanced MRI prior to breast-conserving therapy between 2000 and 2008. Perfusion of the parenchyma in the healthy breast was assessed using a previously reported measure of contralateral parenchymal enhancement (CPE), consisting of the mean of the top-10% late enhancement. ER-pathway activity was assessed from the surgical resection specimen by the previously reported sensitivity to endocrine therapy (SET)-index and ER-factor. The SET-index is a genetic test to estimate survival benefit from endocrine therapy, consisting of genes related to the ESR1 gene. The ER-factor examines other factors as well including protein expression. The relation between CPE and ER-pathway activity was modeled using linear regression.

Results: Patients had a median age of 59 years. CPE was not significantly associated with the SET-index (R-squared = 0.005) nor the ER-factor (R-squared = 0.0002). The only variable significantly different between low and high CPE was age at diagnosis (P < 0.001).

Conclusions: Contralateral parenchymal enhancement on dynamic contrast-enhanced MRI was not associated with tumor-derived estrogen receptor pathway activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejrad.2019.108705DOI Listing
December 2019

Patient-derived organoids can predict response to chemotherapy in metastatic colorectal cancer patients.

Sci Transl Med 2019 10;11(513)

Department of Molecular Oncology and Immunology, Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands.

There is a clear and unmet clinical need for biomarkers to predict responsiveness to chemotherapy for cancer. We developed an in vitro test based on patient-derived tumor organoids (PDOs) from metastatic lesions to identify nonresponders to standard-of-care chemotherapy in colorectal cancer (CRC). In a prospective clinical study, we show the feasibility of generating and testing PDOs for evaluation of sensitivity to chemotherapy. Our PDO test predicted response of the biopsied lesion in more than 80% of patients treated with irinotecan-based therapies without misclassifying patients who would have benefited from treatment. This correlation was specific to irinotecan-based chemotherapy, however, and the PDOs failed to predict outcome for treatment with 5-fluorouracil plus oxaliplatin. Our data suggest that PDOs could be used to prevent cancer patients from undergoing ineffective irinotecan-based chemotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aay2574DOI Listing
October 2019

PRECISE: a domain adaptation approach to transfer predictors of drug response from pre-clinical models to tumors.

Bioinformatics 2019 07;35(14):i510-i519

Computational Cancer Biology, Division of Molecular Carcinogenesis, Oncode Institute, the Netherlands Cancer Institute, Amsterdam CX, The Netherlands.

Motivation: Cell lines and patient-derived xenografts (PDXs) have been used extensively to understand the molecular underpinnings of cancer. While core biological processes are typically conserved, these models also show important differences compared to human tumors, hampering the translation of findings from pre-clinical models to the human setting. In particular, employing drug response predictors generated on data derived from pre-clinical models to predict patient response remains a challenging task. As very large drug response datasets have been collected for pre-clinical models, and patient drug response data are often lacking, there is an urgent need for methods that efficiently transfer drug response predictors from pre-clinical models to the human setting.

Results: We show that cell lines and PDXs share common characteristics and processes with human tumors. We quantify this similarity and show that a regression model cannot simply be trained on cell lines or PDXs and then applied on tumors. We developed PRECISE, a novel methodology based on domain adaptation that captures the common information shared amongst pre-clinical models and human tumors in a consensus representation. Employing this representation, we train predictors of drug response on pre-clinical data and apply these predictors to stratify human tumors. We show that the resulting domain-invariant predictors show a small reduction in predictive performance in the pre-clinical domain but, importantly, reliably recover known associations between independent biomarkers and their companion drugs on human tumors.

Availability And Implementation: PRECISE and the scripts for running our experiments are available on our GitHub page (https://github.com/NKI-CCB/PRECISE).

Supplementary Information: Supplementary data are available at Bioinformatics online.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bioinformatics/btz372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612899PMC
July 2019

Loss of p53 triggers WNT-dependent systemic inflammation to drive breast cancer metastasis.

Nature 2019 08 31;572(7770):538-542. Epub 2019 Jul 31.

Division of Tumour Biology & Immunology, Oncode Institute, Netherlands Cancer Institute, Amsterdam, The Netherlands.

Cancer-associated systemic inflammation is strongly linked to poor disease outcome in patients with cancer. For most human epithelial tumour types, high systemic neutrophil-to-lymphocyte ratios are associated with poor overall survival, and experimental studies have demonstrated a causal relationship between neutrophils and metastasis. However, the cancer-cell-intrinsic mechanisms that dictate the substantial heterogeneity in systemic neutrophilic inflammation between tumour-bearing hosts are largely unresolved. Here, using a panel of 16 distinct genetically engineered mouse models for breast cancer, we uncover a role for cancer-cell-intrinsic p53 as a key regulator of pro-metastatic neutrophils. Mechanistically, loss of p53 in cancer cells induced the secretion of WNT ligands that stimulate tumour-associated macrophages to produce IL-1β, thus driving systemic inflammation. Pharmacological and genetic blockade of WNT secretion in p53-null cancer cells reverses macrophage production of IL-1β and subsequent neutrophilic inflammation, resulting in reduced metastasis formation. Collectively, we demonstrate a mechanistic link between the loss of p53 in cancer cells, secretion of WNT ligands and systemic neutrophilia that potentiates metastatic progression. These insights illustrate the importance of the genetic makeup of breast tumours in dictating pro-metastatic systemic inflammation, and set the stage for personalized immune intervention strategies for patients with cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1450-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707815PMC
August 2019

Publisher Correction: Immune induction strategies in metastatic triple-negative breast cancer to enhance the sensitivity to PD-1 blockade: the TONIC trial.

Nat Med 2019 Jul;25(7):1175

Division of Molecular Oncology & Immunology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

In the version of this article originally published, there was an error in Fig. 3j. A label on the heatmap read "TGF-α signaling via NF-κB". It should have read "TNF-α signaling via NF-κB". The error has been corrected in the HTML and PDF versions of this article.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-019-0520-5DOI Listing
July 2019

Identifying Epistasis in Cancer Genomes: A Delicate Affair.

Cell 2019 05;177(6):1375-1383

Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA; Program in Bioinformatics, University of California, San Diego, La Jolla, CA 92093, USA; Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA; Cancer Cell Map Initiative, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Recent studies of the tumor genome seek to identify cancer pathways as groups of genes in which mutations are epistatic with one another or, specifically, "mutually exclusive." Here, we show that most mutations are mutually exclusive not due to pathway structure but to interactions with disease subtype and tumor mutation load. In particular, many cancer driver genes are mutated preferentially in tumors with few mutations overall, causing mutations in these cancer genes to appear mutually exclusive with numerous others. Researchers should view current epistasis maps with caution until we better understand the multiple cause-and-effect relationships among factors such as tumor subtype, positive selection for mutations, and gross tumor characteristics including mutational signatures and load.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816465PMC
May 2019

Immune induction strategies in metastatic triple-negative breast cancer to enhance the sensitivity to PD-1 blockade: the TONIC trial.

Nat Med 2019 06 13;25(6):920-928. Epub 2019 May 13.

Division of Molecular Oncology & Immunology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

The efficacy of programmed cell death protein 1 (PD-1) blockade in metastatic triple-negative breast cancer (TNBC) is low, highlighting a need for strategies that render the tumor microenvironment more sensitive to PD-1 blockade. Preclinical research has suggested immunomodulatory properties for chemotherapy and irradiation. In the first stage of this adaptive, non-comparative phase 2 trial, 67 patients with metastatic TNBC were randomized to nivolumab (1) without induction or with 2-week low-dose induction, or with (2) irradiation (3 × 8 Gy), (3) cyclophosphamide, (4) cisplatin or (5) doxorubicin, all followed by nivolumab. In the overall cohort, the objective response rate (ORR; iRECIST) was 20%. The majority of responses were observed in the cisplatin (ORR 23%) and doxorubicin (ORR 35%) cohorts. After doxorubicin and cisplatin induction, we detected an upregulation of immune-related genes involved in PD-1-PD-L1 (programmed death ligand 1) and T cell cytotoxicity pathways. This was further supported by enrichment among upregulated genes related to inflammation, JAK-STAT and TNF-α signaling after doxorubicin. Together, the clinical and translational data of this study indicate that short-term doxorubicin and cisplatin may induce a more favorable tumor microenvironment and increase the likelihood of response to PD-1 blockade in TNBC. These data warrant confirmation in TNBC and exploration of induction treatments prior to PD-1 blockade in other cancer types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-019-0432-4DOI Listing
June 2019

Multiplexed Cas9 targeting reveals genomic location effects and gRNA-based staggered breaks influencing mutation efficiency.

Nat Commun 2019 04 8;10(1):1598. Epub 2019 Apr 8.

Division of Molecular Genetics, Oncode and The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, 1066 CX, The Netherlands.

Understanding the impact of guide RNA (gRNA) and genomic locus on CRISPR-Cas9 activity is crucial to design effective gene editing assays. However, it is challenging to profile Cas9 activity in the endogenous cellular environment. Here we leverage our TRIP technology to integrate ~ 1k barcoded reporter genes in the genomes of mouse embryonic stem cells. We target the integrated reporters (IRs) using RNA-guided Cas9 and characterize induced mutations by sequencing. We report that gRNA-sequence and IR locus explain most variation in mutation efficiency. Predominant insertions of a gRNA-specific nucleotide are consistent with template-dependent repair of staggered DNA ends with 1-bp 5' overhangs. We confirm that such staggered ends are induced by Cas9 in mouse pre-B cells. To explain observed insertions, we propose a model generating primarily blunt and occasionally staggered DNA ends. Mutation patterns indicate that gRNA-sequence controls the fraction of staggered ends, which could be used to optimize Cas9-based insertion efficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-09551-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453899PMC
April 2019

Genetic Factors Associated with a Poor Outcome in Head and Neck Cancer Patients Receiving Definitive Chemoradiotherapy.

Cancers (Basel) 2019 Mar 29;11(4). Epub 2019 Mar 29.

Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

About half of advanced stage head and neck squamous cell carcinoma (HNSCC) patients can be cured by chemoradiotherapy. Patient outcome may be partially determined by the genetic alterations in HNSCC, rendering these alterations promising candidate prognostic factors and/or therapeutic targets. However, their relevance in patient outcome prognosis remains to be assessed in patients that receive standard-of-care chemoradiotherapy. We therefore tested whether frequent genetic alterations were associated with progression free survival (PFS) in advanced stage HNSCC patients who were uniformly treated with definitive platinum-based chemoradiotherapy. To this end, we performed targeted DNA sequencing on frozen pre-treatment tumor biopsy material from 77 patients with advanced stage oro- and hypopharyngeal carcinoma. This provided somatic point mutation and copy number aberration data of 556 genes. The most frequently mutated genes, (62%), (51%), (30%) and (21%), were not associated with PFS. However, co-occurring and mutations were associated with short PFS (HR 2.24, = 0.028) in HPV-negative tumors. Furthermore, tumor mutational burden (sum of somatic point mutations) showed a trend towards decreased PFS (HR 1.9, = 0.089), and chromosomal instability (CIN) was associated with shorter PFS (HR 2.3, = 0.023), independent of HPV status. Our results show that tumor mutational burden, CIN markers, and co-occurring and mutations are associated with chemoradiotherapy outcomes in advanced stage oro- and hypopharyngeal HNSCC patients, thereby highlighting their prognostic potential. Given their poor prognosis association and link to biological targets, they may also identify patients for novel targeted therapies and immunotherapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers11040445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521057PMC
March 2019

Inhibition of the Replication Stress Response Is a Synthetic Vulnerability in SCLC That Acts Synergistically in Combination with Cisplatin.

Mol Cancer Ther 2019 04 14;18(4):762-770. Epub 2019 Mar 14.

Oncode Institute, Amsterdam, the Netherlands.

Small cell lung cancer (SCLC) is generally regarded as very difficult to treat, mostly due to the development of metastases early in the disease and a quick relapse with resistant disease. SCLC patients initially show a good response to treatment with the DNA damaging agents cisplatin and etoposide. This is, however, quickly followed by the development of resistant disease, which urges the development of novel therapies for this type of cancer. In this study, we set out to compile a comprehensive overview of the vulnerabilities of SCLC. A functional genome-wide screen where all individual genes were knocked out was performed to identify novel vulnerabilities of SCLC. By analysis of the knockouts that were lethal to these cancer cells, we identified several processes to be synthetic vulnerabilities in SCLC. We were able to validate the vulnerability to inhibition of the replication stress response machinery by use of Chk1 and ATR inhibitors. Strikingly, SCLC cells were more sensitive to these inhibitors than nontransformed cells. In addition, these inhibitors work synergistically with either etoposide and cisplatin, where the interaction is largest with the latter. ATR inhibition by VE-822 treatment in combination with cisplatin also outperforms the combination of cisplatin with etoposide Altogether, our study uncovered a critical dependence of SCLC on the replication stress response and urges the validation of ATR inhibitors in combination with cisplatin in a clinical setting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-18-0972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451635PMC
April 2019

Comparative oncogenomics identifies combinations of driver genes and drug targets in BRCA1-mutated breast cancer.

Nat Commun 2019 01 23;10(1):397. Epub 2019 Jan 23.

Division of Molecular Pathology, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands.

BRCA1-mutated breast cancer is primarily driven by DNA copy-number alterations (CNAs) containing large numbers of candidate driver genes. Validation of these candidates requires novel approaches for high-throughput in vivo perturbation of gene function. Here we develop genetically engineered mouse models (GEMMs) of BRCA1-deficient breast cancer that permit rapid introduction of putative drivers by either retargeting of GEMM-derived embryonic stem cells, lentivirus-mediated somatic overexpression or in situ CRISPR/Cas9-mediated gene disruption. We use these approaches to validate Myc, Met, Pten and Rb1 as bona fide drivers in BRCA1-associated mammary tumorigenesis. Iterative mouse modeling and comparative oncogenomics analysis show that MYC-overexpression strongly reshapes the CNA landscape of BRCA1-deficient mammary tumors and identify MCL1 as a collaborating driver in these tumors. Moreover, MCL1 inhibition potentiates the in vivo efficacy of PARP inhibition (PARPi), underscoring the therapeutic potential of this combination for treatment of BRCA1-mutated cancer patients with poor response to PARPi monotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-08301-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344487PMC
January 2019