Allele-Specific HLA Loss and Immune Escape in Lung Cancer Evolution
Nicholas McGranahan, Rachel Rosenthal, Crispin T Hiley, Andrew J Rowan, Thomas B K Watkins, Gareth A Wilson, Nicolai J Birkbak, Selvaraju Veeriah, Peter Van Loo, Javier Herrero, Charles Swanton, TRACERx Consortium, Charles Swanton, Mariam Jamal-Hanjani, Selvaraju Veeriah, Seema Shafi, Justyna Czyzewska-Khan, Diana Johnson, Joanne Laycock, Leticia Bosshard-Carter, Rachel Rosenthal, Pat Gorman, Robert E Hynds, Gareth Wilson, Nicolai J Birkbak, Thomas B K Watkins, Nicholas McGranahan, Stuart Horswell, Richard Mitter, Mickael Escudero, Aengus Stewart, Peter Van Loo, Andrew Rowan, Hang Xu, Samra Turajlic, Crispin Hiley, Christopher Abbosh, Jacki Goldman, Richard Kevin Stone, Tamara Denner, Nik Matthews, Greg Elgar, Sophia Ward, Marta Costa, Sharmin Begum, Ben Phillimore, Tim Chambers, Emma Nye, Sofia Graca, Maise Al Bakir, Kroopa Joshi, Andrew Furness, Assma Ben Aissa, Yien Ning Sophia Wong, Andy Georgiou, Sergio Quezada, John A Hartley, Helen L Lowe, Javier Herrero, David Lawrence, Martin Hayward, Nikolaos Panagiotopoulos, Shyam Kolvekar, Mary Falzon, Elaine Borg, Teresa Marafioti, Celia Simeon, Gemma Hector, Amy Smith, Marie Aranda, Marco Novelli, Dahmane Oukrif, Sam M Janes, Ricky Thakrar, Martin Forster, Tanya Ahmad, Siow Ming Lee, Dionysis Papadatos-Pastos, Dawn Carnell, Ruheena Mendes, Jeremy George, Neal Navani, Asia Ahmed, Magali Taylor, Junaid Choudhary, Yvonne Summers, Raffaele Califano, Paul Taylor, Rajesh Shah, Piotr Krysiak, Kendadai Rammohan, Eustace Fontaine, Richard Booton, Matthew Evison, Phil Crosbie, Stuart Moss, Faiza Idries, Leena Joseph, Paul Bishop, Anshuman Chaturved, Anne Marie Quinn, Helen Doran, Angela Leek, Phil Harrison, Katrina Moore, Rachael Waddington, Juliette Novasio, Fiona Blackhall, Jane Rogan, Elaine Smith, Caroline Dive, Jonathan Tugwood, Ged Brady, Dominic G Rothwell, Francesca Chemi, Jackie Pierce, Sakshi Gulati, Babu Naidu, Gerald Langman, Simon Trotter, Mary Bellamy, Hollie Bancroft, Amy Kerr, Salma Kadiri, Joanne Webb, Gary Middleton, Madava Djearaman, Dean Fennell, Jacqui A Shaw, John Le Quesne, David Moore, Apostolos Nakas, Sridhar Rathinam, William Monteiro, Hilary Marshall, Louise Nelson, Jonathan Bennett, Joan Riley, Lindsay Primrose, Luke Martinson, Girija Anand, Sajid Khan, Anita Amadi, Marianne Nicolson, Keith Kerr, Shirley Palmer, Hardy Remmen, Joy Miller, Keith Buchan, Mahendran Chetty, Lesley Gomersall, Jason Lester, Alison Edwards, Fiona Morgan, Haydn Adams, Helen Davies, Malgorzata Kornaszewska, Richard Attanoos, Sara Lock, Azmina Verjee, Mairead MacKenzie, Maggie Wilcox, Harriet Bell, Allan Hackshaw, Yenting Ngai, Sean Smith, Nicole Gower, Christian Ottensmeier, Serena Chee, Benjamin Johnson, Aiman Alzetani, Emily Shaw, Eric Lim, Paulo De Sousa, Monica Tavares Barbosa, Alex Bowman, Simon Jordan, Alexandra Rice, Hilgardt Raubenheimer, Chiara Proli, Maria Elena Cufari, John Carlo Ronquillo, Angela Kwayie, Harshil Bhayani, Morag Hamilton, Yusura Bakar, Natalie Mensah, Lyn Ambrose, Anand Devaraj, Silviu Buderi, Jonathan Finch, Leire Azcarate, Hema Chavan, Sophie Green, Hillaria Mashinga, Andrew G Nicholson, Kelvin Lau, Michael Sheaff, Peter Schmid, John Conibear, Veni Ezhil, Babikir Ismail, Melanie Irvin-Sellers, Vineet Prakash, Peter Russell, Teresa Light, Tracey Horey, Sarah Danson, Jonathan Bury, John Edwards, Jennifer Hill, Sue Matthews, Yota Kitsanta, Kim Suvarna, Patricia Fisher, Allah Dino Keerio, Michael Shackcloth, John Gosney, Pieter Postmus, Sarah Feeney, Julius Asante-Siaw, Hugo J W L Aerts, Stefan Dentro, Christophe Dessimoz, Nicholas McGranahan, Rachel Rosenthal, Crispin T Hiley, Andrew J Rowan, Thomas B K Watkins, Gareth A Wilson, Nicolai J Birkbak, Selvaraju Veeriah, Peter Van Loo, Javier Herrero, Charles Swanton, TRACERx Consortium, Charles Swanton, Mariam Jamal-Hanjani, Selvaraju Veeriah, Seema Shafi, Justyna Czyzewska-Khan, Diana Johnson, Joanne Laycock, Leticia Bosshard-Carter, Rachel Rosenthal, Pat Gorman, Robert E Hynds, Gareth Wilson, Nicolai J Birkbak, Thomas B K Watkins, Nicholas McGranahan, Stuart Horswell, Richard Mitter, Mickael Escudero, Aengus Stewart, Peter Van Loo, Andrew Rowan, Hang Xu, Samra Turajlic, Crispin Hiley, Christopher Abbosh, Jacki Goldman, Richard Kevin Stone, Tamara Denner, Nik Matthews, Greg Elgar, Sophia Ward, Marta Costa, Sharmin Begum, Ben Phillimore, Tim Chambers, Emma Nye, Sofia Graca, Maise Al Bakir, Kroopa Joshi, Andrew Furness, Assma Ben Aissa, Yien Ning Sophia Wong, Andy Georgiou, Sergio Quezada, John A Hartley, Helen L Lowe, Javier Herrero, David Lawrence, Martin Hayward, Nikolaos Panagiotopoulos, Shyam Kolvekar, Mary Falzon, Elaine Borg, Teresa Marafioti, Celia Simeon, Gemma Hector, Amy Smith, Marie Aranda, Marco Novelli, Dahmane Oukrif, Sam M Janes, Ricky Thakrar, Martin Forster, Tanya Ahmad, Siow Ming Lee, Dionysis Papadatos-Pastos, Dawn Carnell, Ruheena Mendes, Jeremy George, Neal Navani, Asia Ahmed, Magali Taylor, Junaid Choudhary, Yvonne Summers, Raffaele Califano, Paul Taylor, Rajesh Shah, Piotr Krysiak, Kendadai Rammohan, Eustace Fontaine, Richard Booton, Matthew Evison, Phil Crosbie, Stuart Moss, Faiza Idries, Leena Joseph, Paul Bishop, Anshuman Chaturved, Anne Marie Quinn, Helen Doran, Angela Leek, Phil Harrison, Katrina Moore, Rachael Waddington, Juliette Novasio, Fiona Blackhall, Jane Rogan, Elaine Smith, Caroline Dive, Jonathan Tugwood, Ged Brady, Dominic G Rothwell, Francesca Chemi, Jackie Pierce, Sakshi Gulati, Babu Naidu, Gerald Langman, Simon Trotter, Mary Bellamy, Hollie Bancroft, Amy Kerr, Salma Kadiri, Joanne Webb, Gary Middleton, Madava Djearaman, Dean Fennell, Jacqui A Shaw, John Le Quesne, David Moore, Apostolos Nakas, Sridhar Rathinam, William Monteiro, Hilary Marshall, Louise Nelson, Jonathan Bennett, Joan Riley, Lindsay Primrose, Luke Martinson, Girija Anand, Sajid Khan, Anita Amadi, Marianne Nicolson, Keith Kerr, Shirley Palmer, Hardy Remmen, Joy Miller, Keith Buchan, Mahendran Chetty, Lesley Gomersall, Jason Lester, Alison Edwards, Fiona Morgan, Haydn Adams, Helen Davies, Malgorzata Kornaszewska, Richard Attanoos, Sara Lock, Azmina Verjee, Mairead MacKenzie, Maggie Wilcox, Harriet Bell, Allan Hackshaw, Yenting Ngai, Sean Smith, Nicole Gower, Christian Ottensmeier, Serena Chee, Benjamin Johnson, Aiman Alzetani, Emily Shaw, Eric Lim, Paulo De Sousa, Monica Tavares Barbosa, Alex Bowman, Simon Jordan, Alexandra Rice, Hilgardt Raubenheimer, Chiara Proli, Maria Elena Cufari, John Carlo Ronquillo, Angela Kwayie, Harshil Bhayani, Morag Hamilton, Yusura Bakar, Natalie Mensah, Lyn Ambrose, Anand Devaraj, Silviu Buderi, Jonathan Finch, Leire Azcarate, Hema Chavan, Sophie Green, Hillaria Mashinga, Andrew G Nicholson, Kelvin Lau, Michael Sheaff, Peter Schmid, John Conibear, Veni Ezhil, Babikir Ismail, Melanie Irvin-Sellers, Vineet Prakash, Peter Russell, Teresa Light, Tracey Horey, Sarah Danson, Jonathan Bury, John Edwards, Jennifer Hill, Sue Matthews, Yota Kitsanta, Kim Suvarna, Patricia Fisher, Allah Dino Keerio, Michael Shackcloth, John Gosney, Pieter Postmus, Sarah Feeney, Julius Asante-Siaw, Hugo J W L Aerts, Stefan Dentro, Christophe Dessimoz
Abstract
Immune evasion is a hallmark of cancer. Losing the ability to present neoantigens through human leukocyte antigen (HLA) loss may facilitate immune evasion. However, the polymorphic nature of the locus has precluded accurate HLA copy-number analysis. Here, we present loss of heterozygosity in human leukocyte antigen (LOHHLA), a computational tool to determine HLA allele-specific copy number from sequencing data. Using LOHHLA, we find that HLA LOH occurs in 40% of non-small-cell lung cancers (NSCLCs) and is associated with a high subclonal neoantigen burden, APOBEC-mediated mutagenesis, upregulation of cytolytic activity, and PD-L1 positivity. The focal nature of HLA LOH alterations, their subclonal frequencies, enrichment in metastatic sites, and occurrence as parallel events suggests that HLA LOH is an immune escape mechanism that is subject to strong microenvironmental selection pressures later in tumor evolution. Characterizing HLA LOH with LOHHLA refines neoantigen prediction and may have implications for our understanding of resistance mechanisms and immunotherapeutic approaches targeting neoantigens. VIDEO ABSTRACT.
Keywords: bioinformatics; cancer evolution; chromosomal instability; copy number; heterogeneity; immune-editing; immune-escape; loss of heterozygosity; lung cancer; neoantigen.
Copyright © 2017 The Francis Crick Institute. Published by Elsevier Inc. All rights reserved.
Figures
References
- Alexandrov L.B., Nik-Zainal S., Wedge D.C., Aparicio S.A., Behjati S., Biankin A.V., Bignell G.R., Bolli N., Borg A., Børresen-Dale A.L., Australian Pancreatic Cancer Genome Initiative. ICGC Breast Cancer Consortium. ICGC MMML-Seq Consortium. ICGC PedBrain Signatures of mutational processes in human cancer. Nature. 2013;500:415–421.
- Andreatta M., Nielsen M. Gapped sequence alignment using artificial neural networks: application to the MHC class I system. Bioinformatics. 2016;32:511–517.
- Brastianos P.K., Carter S.L., Santagata S., Cahill D.P., Taylor-Weiner A., Jones R.T., Van Allen E.M., Lawrence M.S., Horowitz P.M., Cibulskis K. Genomic characterization of brain metastases reveals branched evolution and potential therapeutic targets. Cancer Discov. 2015;5:1164–1177.
- Brown S.D., Warren R.L., Gibb E.A., Martin S.D., Spinelli J.J., Nelson B.H., Holt R.A. Neo-antigens predicted by tumor genome meta-analysis correlate with increased patient survival. Genome Res. 2014;24:743–750.
- Campbell J.D., Alexandrov A., Kim J., Wala J., Berger A.H., Pedamallu C.S., Shukla S.A., Guo G., Brooks A.N., Murray B.A., Cancer Genome Atlas Research Network Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. Nat. Genet. 2016;48:607–616.
- Campoli M., Ferrone S. HLA antigen changes in malignant cells: epigenetic mechanisms and biologic significance. Oncogene. 2008;27:5869–5885.
- Carter S.L., Cibulskis K., Helman E., McKenna A., Shen H., Zack T., Laird P.W., Onofrio R.C., Winckler W., Weir B.A. Absolute quantification of somatic DNA alterations in human cancer. Nat. Biotechnol. 2012;30:413–421.
- Cibulskis K., Lawrence M.S., Carter S.L., Sivachenko A., Jaffe D., Sougnez C., Gabriel S., Meyerson M., Lander E.S., Getz G. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat. Biotechnol. 2013;31:213–219.
- Davoli T., Uno H., Wooten E.C., Elledge S.J. Tumor aneuploidy correlates with markers of immune evasion and with reduced response to immunotherapy. Science. 2017;355:eaaf8399.
- del Campo A.B., Kyte J.A., Carretero J., Zinchencko S., Méndez R., González-Aseguinolaza G., Ruiz-Cabello F., Aamdal S., Gaudernack G., Garrido F., Aptsiauri N. Immune escape of cancer cells with beta2-microglobulin loss over the course of metastatic melanoma. Int. J. Cancer. 2014;134:102–113.
- Favero F., Joshi T., Marquard A.M., Birkbak N.J., Krzystanek M., Li Q., Szallasi Z., Eklund A.C. Sequenza: allele-specific copy number and mutation profiles from tumor sequencing data. Ann. Oncol. 2015;26:64–70.
- Ha G., Roth A., Khattra J., Ho J., Yap D., Prentice L.M., Melnyk N., McPherson A., Bashashati A., Laks E. TITAN: inference of copy number architectures in clonal cell populations from tumor whole-genome sequence data. Genome Res. 2014;24:1881–1893.
- Hanahan D., Weinberg R.A. Hallmarks of cancer: the next generation. Cell. 2011;144:646–674.
- Herbst R.S., Soria J.C., Kowanetz M., Fine G.D., Hamid O., Gordon M.S., Sosman J.A., McDermott D.F., Powderly J.D., Gettinger S.N. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature. 2014;515:563–567.
- Hicklin D.J., Marincola F.M., Ferrone S. HLA class I antigen downregulation in human cancers: T-cell immunotherapy revives an old story. Mol. Med. Today. 1999;5:178–186.
- Hiraki A., Fujii N., Murakami T., Kiura K., Aoe K., Yamane H., Masuda K., Maeda T., Sugi K., Darzynkiewicz Z. High frequency of allele-specific down-regulation of HLA class I expression in lung cancer cell lines. Anticancer Res. 2004;24(3a):1525–1528.
- Hoof I., Peters B., Sidney J., Pedersen L.E., Sette A., Lund O., Buus S., Nielsen M. NetMHCpan, a method for MHC class I binding prediction beyond humans. Immunogenetics. 2009;61:1–13.
- Jamal-Hanjani M., Wilson G.A., McGranahan N., Birkbak N.J., Watkins T.B.K., Veeriah S., Shafi S., Johnson D.H., Mitter R., Rosenthal R., TRACERx Consortium Tracking the Evolution of Non-Small-Cell Lung Cancer. N. Engl. J. Med. 2017;376:2109–2121.
- Koboldt D.C., Zhang Q., Larson D.E., Shen D., McLellan M.D., Lin L., Miller C.A., Mardis E.R., Ding L., Wilson R.K. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res. 2012;22:568–576.
- Lawrence M.S., Stojanov P., Mermel C.H., Robinson J.T., Garraway L.A., Golub T.R., Meyerson M., Gabriel S.B., Lander E.S., Getz G. Discovery and saturation analysis of cancer genes across 21 tumour types. Nature. 2014;505:495–501.
- Li H., Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25:1754–1760.
- Li B., Severson E., Pignon J.C., Zhao H., Li T., Novak J., Jiang P., Shen H., Aster J.C., Rodig S. Comprehensive analyses of tumor immunity: implications for cancer immunotherapy. Genome Biol. 2016;17:174.
- Martin M.P., Carrington M. Immunogenetics of HIV disease. Immunol. Rev. 2013;254:245–264.
- McGranahan N., Furness A.J., Rosenthal R., Ramskov S., Lyngaa R., Saini S.K., Jamal-Hanjani M., Wilson G.A., Birkbak N.J., Hiley C.T. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science. 2016;351:1463–1469.
- McKenna A., Hanna M., Banks E., Sivachenko A., Cibulskis K., Kernytsky A., Garimella K., Altshuler D., Gabriel S., Daly M., DePristo M.A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20:1297–1303.
- Mehta A.M., Jordanova E.S., Kenter G.G., Ferrone S., Fleuren G.J. Association of antigen processing machinery and HLA class I defects with clinicopathological outcome in cervical carcinoma. Cancer Immunol. Immunother. 2008;57:197–206.
- Moretta L., Montaldo E., Vacca P., Del Zotto G., Moretta F., Merli P., Locatelli F., Mingari M.C. Human natural killer cells: origin, receptors, function, and clinical applications. Int. Arch. Allergy Immunol. 2014;164:253–264.
- Nielsen M., Lundegaard C., Worning P., Lauemøller S.L., Lamberth K., Buus S., Brunak S., Lund O. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations. Protein Sci. 2003;12:1007–1017.
- Ott P.A., Hu Z., Keskin D.B., Shukla S.A., Sun J., Bozym D.J., Zhang W., Luoma A., Giobbie-Hurder A., Peter L. An immunogenic personal neoantigen vaccine for patients with melanoma. Nature. 2017;547:217–221.
- Piha-Paul S.A., Bennouna J., Albright A., Nebozhyn M., McClanahan T., Ayers M., Lunceford J.K., Ott P.A. T-cell inflamed phenotype gene expression signatures to predict clinical benefit from pembrolizumab across multiple tumor types. J. Clin. Oncol. 2016;34:1536.
- Ribas A., Robert C., Hodi F.S., Wolchok J.D., Joshua A.M., Hwu W.-J., Weber J.S., Zarour H.M., Kefford R., Loboda A. Association of response to programmed death receptor 1 (PD-1) blockade with pembrolizumab (MK-3475) with an interferon-inflammatory immune gene signature. J. Clin. Oncol. 2015;33:3001.
- Rimmer A., Phan H., Mathieson I., Iqbal Z., Twigg S.R.F., Wilkie A.O.M., McVean G., Lunter G., Lunter G., WGS500 Consortium Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications. Nat. Genet. 2014;46:912–918.
- Rizvi N.A., Hellmann M.D., Snyder A., Kvistborg P., Makarov V., Havel J.J., Lee W., Yuan J., Wong P., Ho T.S. Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. Science. 2015;348:124–128.
- Rooney M.S., Shukla S.A., Wu C.J., Getz G., Hacohen N. Molecular and genetic properties of tumors associated with local immune cytolytic activity. Cell. 2015;160:48–61.
- Rosenthal R., McGranahan N., Herrero J., Taylor B.S., Swanton C. DeconstructSigs: delineating mutational processes in single tumors distinguishes DNA repair deficiencies and patterns of carcinoma evolution. Genome Biol. 2016;17:31.
- Roth A., Khattra J., Yap D., Wan A., Laks E., Biele J., Ha G., Aparicio S., Bouchard-Côté A., Shah S.P. PyClone: statistical inference of clonal population structure in cancer. Nat. Methods. 2014;11:396–398.
- Sahin U., Derhovanessian E., Miller M., Kloke B.P., Simon P., Löwer M., Bukur V., Tadmor A.D., Luxemburger U., Schrörs B. Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature. 2017;547:222–226.
- Schumacher T.N., Schreiber R.D. Neoantigens in cancer immunotherapy. Science. 2015;348:69–74.
- Shen R., Seshan V.E. FACETS: allele-specific copy number and clonal heterogeneity analysis tool for high-throughput DNA sequencing. Nucleic Acids Res. 2016;44:e131.
- Shukla S.A., Rooney M.S., Rajasagi M., Tiao G., Dixon P.M., Lawrence M.S., Stevens J., Lane W.J., Dellagatta J.L., Steelman S. Comprehensive analysis of cancer-associated somatic mutations in class I HLA genes. Nat. Biotechnol. 2015;33:1152–1158.
- Snyder A., Makarov V., Merghoub T., Yuan J., Zaretsky J.M., Desrichard A., Walsh L.A., Postow M.A., Wong P., Ho T.S. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N. Engl. J. Med. 2014;371:2189–2199.
- Spranger S., Spaapen R.M., Zha Y., Williams J., Meng Y., Ha T.T., Gajewski T.F. Up-regulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvironment is driven by CD8(+) T cells. Sci Transl Med. 2013;5:200ra116.
- Szolek A., Schubert B., Mohr C., Sturm M., Feldhahn M., Kohlbacher O. OptiType: precision HLA typing from next-generation sequencing data. Bioinformatics. 2014;30:3310–3316.
- Tran E., Robbins P.F., Lu Y.C., Prickett T.D., Gartner J.J., Jia L., Pasetto A., Zheng Z., Ray S., Groh E.M. T-cell transfer therapy targeting mutant KRAS in cancer. N. Engl. J. Med. 2016;375:2255–2262.
- Tumeh P.C., Harview C.L., Yearley J.H., Shintaku I.P., Taylor E.J., Robert L., Chmielowski B., Spasic M., Henry G., Ciobanu V. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515:568–571.
- Van Allen E.M., Miao D., Schilling B., Shukla S.A., Blank C., Zimmer L., Sucker A., Hillen U., Foppen M.H., Goldinger S.M. Genomic correlates of response to CTLA-4 blockade in metastatic melanoma. Science. 2015;350:207–211.
- Van Loo P., Nordgard S.H., Lingjærde O.C., Russnes H.G., Rye I.H., Sun W., Weigman V.J., Marynen P., Zetterberg A., Naume B. Allele-specific copy number analysis of tumors. Proc. Natl. Acad. Sci. USA. 2010;107:16910–16915.
- Yoshihama S., Roszik J., Downs I., Meissner T.B., Vijayan S., Chapuy B., Sidiq T., Shipp M.A., Lizee G.A., Kobayashi K.S. NLRC5/MHC class I transactivator is a target for immune evasion in cancer. Proc. Natl. Acad. Sci. USA. 2016;113:5999–6004.
- Zaretsky J.M., Garcia-Diaz A., Shin D.S., Escuin-Ordinas H., Hugo W., Hu-Lieskovan S., Torrejon D.Y., Abril-Rodriguez G., Sandoval S., Barthly L. Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N. Engl J. Med. 2016;375:819–829.
- Zhao F., Sucker A., Horn S., Heeke C., Bielefeld N., Schrors B., Bicker A., Lindemann M., Roesch A., Gaudernack G. Melanoma Lesions Independently Acquire T-cell Resistance during Metastatic Latency. Cancer Res. 2016;76:4347–4358.
Source: PubMed