. 2020 Oct; 9(10):e1183.
doi: 10.1002/cti2.1183.

Immune contexture analysis in immuno-oncology: applications and challenges of multiplex fluorescent immunohistochemistry

Reshma Shakya 1 Tam Hong Nguyen 2 Nigel Waterhouse 2 Rajiv Khanna 1 
  • PMID: 33072322
  •     159 References
  •     4 citations


The tumor microenvironment is an integral player in cancer initiation, tumor progression, response and resistance to anti-cancer therapy. Understanding the complex interactions of tumor immune architecture (referred to as 'immune contexture') has therefore become increasingly desirable to guide our approach to patient selection, clinical trial design, combination therapies, and patient management. Quantitative image analysis based on multiplexed fluorescence immunohistochemistry and deep learning technologies are rapidly developing to enable researchers to interrogate complex information from the tumor microenvironment and find predictive insights into treatment response. Herein, we discuss current developments in multiplexed fluorescence immunohistochemistry for immune contexture analysis, and their application in immuno-oncology, and discuss challenges to effectively use this technology in clinical settings. We also present a multiplexed image analysis workflow to analyse fluorescence multiplexed stained tumor sections using the Vectra Automated Digital Pathology System together with FCS express flow cytometry software. The benefit of this strategy is that the spectral unmixing accurately generates and analyses complex arrays of multiple biomarkers, which can be helpful for diagnosis, risk stratification, and guiding clinical management of oncology patients.

Keywords: FCS express image cytometry; immune profiling; multiplexed fluorescent immunohistochemistry; quantitative digital pathology; tumor microenvironment; vectra.

Tumor immunosurveillance in human cancers.
Bernhard Mlecnik, Gabriela Bindea, Franck Pagès, Jérôme Galon.
Cancer Metastasis Rev, 2011 Jan 21; 30(1). PMID: 21249426    Free PMC article.
Imaging Mass Cytometry.
Qing Chang, Olga I Ornatsky, +3 authors, David W Hedley.
Cytometry A, 2017 Feb 06; 91(2). PMID: 28160444
Principles for valid histopathologic scoring in research.
K N Gibson-Corley, A K Olivier, D K Meyerholz.
Vet Pathol, 2013 Apr 06; 50(6). PMID: 23558974    Free PMC article.
Highly Cited. Review.
Development of a Companion Diagnostic PD-L1 Immunohistochemistry Assay for Pembrolizumab Therapy in Non-Small-cell Lung Cancer.
Charlotte Roach, Nancy Zhang, +7 authors, Karina Kulangara.
Appl Immunohistochem Mol Morphol, 2016 Jun 23; 24(6). PMID: 27333219    Free PMC article.
Highly Cited.
Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.
H Halse, A J Colebatch, +13 authors, P J Neeson.
Sci Rep, 2018 Jul 26; 8(1). PMID: 30042403    Free PMC article.
Controls for immunohistochemistry: the Histochemical Society's standards of practice for validation of immunohistochemical assays.
Stephen M Hewitt, Denis G Baskin, +2 authors, Eduardo Rosa-Molinar.
J Histochem Cytochem, 2014 Jul 16; 62(10). PMID: 25023613    Free PMC article.
Highly Cited.
Prognostic value of tumor-infiltrating lymphocytes in melanoma: a systematic review and meta-analysis.
Qiaofen Fu, Nan Chen, +9 authors, Gaofeng Li.
Oncoimmunology, 2019 May 31; 8(7). PMID: 31143514    Free PMC article.
Highly Cited. Review.
Spectral imaging in preclinical research and clinical pathology.
Richard Levenson, Joseph Beechem, George McNamara.
Anal Cell Pathol (Amst), 2012 Apr 06; 35(5-6). PMID: 22475632    Free PMC article.
Multiplexed Epitope-Based Tissue Imaging for Discovery and Healthcare Applications.
Bernd Bodenmiller.
Cell Syst, 2016 May 03; 2(4). PMID: 27135535
Telepathology Impacts and Implementation Challenges: A Scoping Review.
Julien Meyer, Guy Paré.
Arch Pathol Lab Med, 2015 Dec 01; 139(12). PMID: 26619028
Effect of neoadjuvant chemotherapy on the immune microenvironment in non-small cell lung carcinomas as determined by multiplex immunofluorescence and image analysis approaches.
Edwin R Parra, Pamela Villalobos, +19 authors, Ignacio I Wistuba.
J Immunother Cancer, 2018 Jun 07; 6(1). PMID: 29871672    Free PMC article.
Automated quantitative multiplex immunofluorescence in situ imaging identifies phospho-S6 and phospho-PRAS40 as predictive protein biomarkers for prostate cancer lethality.
Michail Shipitsin, Clayton Small, +9 authors, Peter Blume-Jensen.
Proteome Sci, 2014 Jul 31; 12. PMID: 25075204    Free PMC article.
Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues.
Edwin R Parra, Naohiro Uraoka, +7 authors, Jaime Rodriguez-Canales.
Sci Rep, 2017 Oct 19; 7(1). PMID: 29042640    Free PMC article.
Highly Cited.
The microenvironment of the tumour-host interface.
L A Liotta, E C Kohn.
Nature, 2001 May 18; 411(6835). PMID: 11357145
Highly Cited. Review.
Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry.
Yun Xing, Qaiser Chaudry, +9 authors, Shuming Nie.
Nat Protoc, 2007 Jun 05; 2(5). PMID: 17546006
Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue.
K Specht, T Richter, +3 authors, H Höfler.
Am J Pathol, 2001 Feb 13; 158(2). PMID: 11159180    Free PMC article.
Highly Cited.
Immune Responses in Bladder Cancer-Role of Immune Cell Populations, Prognostic Factors and Therapeutic Implications.
Magdalene Joseph, Deborah Enting.
Front Oncol, 2019 Dec 12; 9. PMID: 31824850    Free PMC article.
Genetically modified species in research: Opportunities and challenges for the histology core laboratory.
Alicia K Olivier, Paul Naumann, +5 authors, David K Meyerholz.
J Histotechnol, 2012 Aug 21; 35(2). PMID: 22904582    Free PMC article.
Histopathological image analysis: a review.
Metin N Gurcan, Laura E Boucheron, +3 authors, B Yener.
IEEE Rev Biomed Eng, 2009 Jan 01; 2. PMID: 20671804    Free PMC article.
Highly Cited. Review.
Understanding immune phenotypes in human gastric disease tissues by multiplexed immunohistochemistry.
Le Ying, Feng Yan, +10 authors, Yiqun Hu.
J Transl Med, 2017 Oct 14; 15(1). PMID: 29025424    Free PMC article.
Visualization of microscopy-based spectral imaging data from multi-label tissue sections.
James R Mansfield, Clifford Hoyt, Richard M Levenson.
Curr Protoc Mol Biol, 2008 Oct 31; Chapter 14. PMID: 18972383
Quantitative assessment of the spatial heterogeneity of tumor-infiltrating lymphocytes in breast cancer.
Nikita L Mani, Kurt A Schalper, +6 authors, David L Rimm.
Breast Cancer Res, 2016 Jul 31; 18(1). PMID: 27473061    Free PMC article.
Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II.
C R Taylor, R M Levenson.
Histopathology, 2006 Sep 19; 49(4). PMID: 16978205
Highly Cited. Review.
Current Status of Companion and Complementary Diagnostics: Strategic Considerations for Development and Launch.
H Scheerens, A Malong, +14 authors, J A Williams.
Clin Transl Sci, 2017 Jan 26; 10(2). PMID: 28121072    Free PMC article.
Applications of immunohistochemistry.
Jeyapradha Duraiyan, Rajeshwar Govindarajan, Karunakaran Kaliyappan, Murugesan Palanisamy.
J Pharm Bioallied Sci, 2012 Oct 16; 4(Suppl 2). PMID: 23066277    Free PMC article.
Highly multiplexed immunofluorescence imaging of human tissues and tumors using t-CyCIF and conventional optical microscopes.
Jia-Ren Lin, Benjamin Izar, +5 authors, Peter K Sorger.
Elife, 2018 Jul 12; 7. PMID: 29993362    Free PMC article.
Highly Cited.
Novel technologies and emerging biomarkers for personalized cancer immunotherapy.
Jianda Yuan, Priti S Hegde, +16 authors, Bernard A Fox.
J Immunother Cancer, 2016 Jan 21; 4. PMID: 26788324    Free PMC article.
Highly Cited. Review.
Predictive biomarkers for checkpoint inhibitor-based immunotherapy.
Geoffrey T Gibney, Louis M Weiner, Michael B Atkins.
Lancet Oncol, 2016 Dec 08; 17(12). PMID: 27924752    Free PMC article.
Highly Cited. Review.
Close proximity of immune and tumor cells underlies response to anti-PD-1 based therapies in metastatic melanoma patients.
Tuba N Gide, Ines P Silva, +9 authors, James S Wilmott.
Oncoimmunology, 2020 Feb 01; 9(1). PMID: 32002281    Free PMC article.
Quantitative Spatial Profiling of PD-1/PD-L1 Interaction and HLA-DR/IDO-1 Predicts Improved Outcomes of Anti-PD-1 Therapies in Metastatic Melanoma.
Douglas B Johnson, Jennifer Bordeaux, +22 authors, Naveen Dakappagari.
Clin Cancer Res, 2018 Jul 20; 24(21). PMID: 30021908    Free PMC article.
Factors affecting immunoreactivity in long-term storage of formalin-fixed paraffin-embedded tissue sections.
Federica Grillo, Simona Pigozzi, +3 authors, Luca Mastracci.
Histochem Cell Biol, 2015 Mar 12; 144(1). PMID: 25757745
International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study.
Franck Pagès, Bernhard Mlecnik, +96 authors, Jérôme Galon.
Lancet, 2018 May 15; 391(10135). PMID: 29754777
Highly Cited.
Antibody validation.
Jennifer Bordeaux, Allison Welsh, +5 authors, David Rimm.
Biotechniques, 2010 Apr 03; 48(3). PMID: 20359301    Free PMC article.
Highly Cited. Review.
Highly multiplexed imaging of single cells using a high-throughput cyclic immunofluorescence method.
Jia-Ren Lin, Mohammad Fallahi-Sichani, Peter K Sorger.
Nat Commun, 2015 Sep 25; 6. PMID: 26399630    Free PMC article.
Highly Cited.
Spatial computation of intratumoral T cells correlates with survival of patients with pancreatic cancer.
Julienne L Carstens, Pedro Correa de Sampaio, +6 authors, Raghu Kalluri.
Nat Commun, 2017 Apr 28; 8. PMID: 28447602    Free PMC article.
Highly Cited.
Type, density, and location of immune cells within human colorectal tumors predict clinical outcome.
Jérôme Galon, Anne Costes, +13 authors, Franck Pagès.
Science, 2006 Sep 30; 313(5795). PMID: 17008531
Highly Cited.
Differential Expression and Significance of PD-L1, IDO-1, and B7-H4 in Human Lung Cancer.
Kurt A Schalper, Daniel Carvajal-Hausdorf, +7 authors, David L Rimm.
Clin Cancer Res, 2016 Jul 22; 23(2). PMID: 27440266    Free PMC article.
Highly Cited.
Digital pathology and image analysis in tissue biomarker research.
Peter W Hamilton, Peter Bankhead, +5 authors, Manuel Salto-Tellez.
Methods, 2014 Jul 19; 70(1). PMID: 25034370
Autofluorescence contributes to false-positive intracellular Foxp3 staining in macrophages: a lesson learned from flow cytometry.
Fuqiang Li, Mingjie Yang, +5 authors, Behrooz G Sharifi.
J Immunol Methods, 2012 Oct 11; 386(1-2). PMID: 23046996    Free PMC article.
Review of imaging solutions for integrated quantitative immunohistochemistry in the Pathology daily practice.
Marcial García Rojo, Gloria Bueno, Janina Slodkowska.
Folia Histochem Cytobiol, 2010 Feb 19; 47(3). PMID: 20164017
Review of Telemicrobiology.
Daniel D Rhoads, Blaine A Mathison, +2 authors, Liron Pantanowitz.
Arch Pathol Lab Med, 2015 Sep 01; 140(4). PMID: 26317376    Free PMC article.
CellProfiler: free, versatile software for automated biological image analysis.
Michael R Lamprecht, David M Sabatini, Anne E Carpenter.
Biotechniques, 2007 Feb 03; 42(1). PMID: 17269487
Highly Cited.
Diagnostic digital cytopathology: Are we ready yet?
Jarret C House, Evita B Henderson-Jackson, +10 authors, Marilyn M Bui.
J Pathol Inform, 2014 Jan 07; 4. PMID: 24392242    Free PMC article.
Whole-slide imaging and automated image analysis: considerations and opportunities in the practice of pathology.
J D Webster, R W Dunstan.
Vet Pathol, 2013 Oct 05; 51(1). PMID: 24091812
Editorial: Antibody can get it right: confronting problems of antibody specificity and irreproducibility.
Agnes Schonbrunn.
Mol Endocrinol, 2014 Sep 04; 28(9). PMID: 25184858    Free PMC article.
Tim-3 Expression on Tumor-Infiltrating PD-1+CD8+ T Cells Correlates with Poor Clinical Outcome in Renal Cell Carcinoma.
Clémence Granier, Charles Dariane, +17 authors, Eric Tartour.
Cancer Res, 2016 Nov 23; 77(5). PMID: 27872087
Automated image analysis in histopathology: a valuable tool in medical diagnostics.
Laoighse Mulrane, Elton Rexhepaj, +2 authors, William M Gallagher.
Expert Rev Mol Diagn, 2008 Nov 13; 8(6). PMID: 18999923
Web-based oil immersion whole slide imaging increases efficiency and clinical team satisfaction in hematopathology tumor board.
Zhongchuan Will Chen, Jessica Kohan, +2 authors, Mohamed E Salama.
J Pathol Inform, 2014 Nov 08; 5(1). PMID: 25379347    Free PMC article.
Applications and challenges of digital pathology and whole slide imaging.
C Higgins.
Biotech Histochem, 2015 May 16; 90(5). PMID: 25978139
Image analysis and machine learning in digital pathology: Challenges and opportunities.
Anant Madabhushi, George Lee.
Med Image Anal, 2016 Jul 18; 33. PMID: 27423409    Free PMC article.
Highly Cited. Review.
The immune contexture of primary and metastatic human tumours.
Nicolas A Giraldo, Etienne Becht, +3 authors, Wolf H Fridman.
Curr Opin Immunol, 2014 Feb 04; 27. PMID: 24487185
Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue.
A Sally Davis, Anke Richter, +4 authors, Jeffery K Taubenberger.
J Histochem Cytochem, 2014 Apr 12; 62(6). PMID: 24722432    Free PMC article.
Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry.
Charlotte Giesen, Hao A O Wang, +11 authors, Bernd Bodenmiller.
Nat Methods, 2014 Mar 04; 11(4). PMID: 24584193
Highly Cited.
Digital transplantation pathology: combining whole slide imaging, multiplex staining and automated image analysis.
K Isse, A Lesniak, +3 authors, A J Demetris.
Am J Transplant, 2011 Nov 08; 12(1). PMID: 22053785    Free PMC article.
Review of the current state of whole slide imaging in pathology.
Liron Pantanowitz, Paul N Valenstein, +5 authors, Terence J Colgan.
J Pathol Inform, 2011 Sep 03; 2. PMID: 21886892    Free PMC article.
Highly Cited.
Relevance of deep learning to facilitate the diagnosis of HER2 status in breast cancer.
Michel E Vandenberghe, Marietta L J Scott, +3 authors, Craig Barker.
Sci Rep, 2017 Apr 06; 7. PMID: 28378829    Free PMC article.
Altered expression of programmed death-ligand 1 after neo-adjuvant chemotherapy in patients with lung squamous cell carcinoma.
Zhengbo Song, Xinmin Yu, Yiping Zhang.
Lung Cancer, 2016 Aug 28; 99. PMID: 27565935
Multifeature prostate cancer diagnosis and Gleason grading of histological images.
Ali Tabesh, Mikhail Teverovskiy, +4 authors, Olivier Saidi.
IEEE Trans Med Imaging, 2007 Oct 24; 26(10). PMID: 17948727
Multispectral imaging: a review of its technical aspects and applications in anatomic pathology.
J R Mansfield.
Vet Pathol, 2013 Oct 17; 51(1). PMID: 24129898
Orchestration and Prognostic Significance of Immune Checkpoints in the Microenvironment of Primary and Metastatic Renal Cell Cancer.
Nicolas A Giraldo, Etienne Becht, +17 authors, Catherine Sautès-Fridman.
Clin Cancer Res, 2015 Feb 18; 21(13). PMID: 25688160
Highly Cited.
State-of-the-Art of Profiling Immune Contexture in the Era of Multiplexed Staining and Digital Analysis to Study Paraffin Tumor Tissues.
Edwin Roger Parra, Alejandro Francisco-Cruz, Ignacio Ivan Wistuba.
Cancers (Basel), 2019 Feb 23; 11(2). PMID: 30791580    Free PMC article.
Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma.
Zipei Feng, Sachin Puri, +7 authors, Bernard A Fox.
J Immunother Cancer, 2015 Oct 27; 3. PMID: 26500776    Free PMC article.
Immunohistochemical markers in urinary bladder carcinomas from paraffin-embedded archival tissue after storage for 5-70 years.
Jorunn Litlekalsoy, Vendil Vatne, Jens G Hostmark, Ole Didrik Laerum.
BJU Int, 2007 Apr 18; 99(5). PMID: 17437436
Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.
Edward C Stack, Chichung Wang, Kristin A Roman, Clifford C Hoyt.
Methods, 2014 Sep 23; 70(1). PMID: 25242720
Highly Cited. Review.
From the immune contexture to the Immunoscore: the role of prognostic and predictive immune markers in cancer.
Helen Angell, Jérôme Galon.
Curr Opin Immunol, 2013 Apr 13; 25(2). PMID: 23579076
Highly Cited. Review.
Cancer classification using the Immunoscore: a worldwide task force.
Jérôme Galon, Franck Pagès, +61 authors, Bernard A Fox.
J Transl Med, 2012 Oct 05; 10. PMID: 23034130    Free PMC article.
Highly Cited. Review.
Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis.
A C Croce, G Bottiroli.
Eur J Histochem, 2015 Jan 13; 58(4). PMID: 25578980    Free PMC article.
Highly Cited. Review.
Multiplexed tissue biomarker imaging.
Edward C Stack, Periklis G Foukas, Peter P Lee.
J Immunother Cancer, 2016 Feb 18; 4. PMID: 26885371    Free PMC article.
A colorful future of quantitative pathology: validation of Vectra technology using chromogenic multiplexed immunohistochemistry and prostate tissue microarrays.
Wei Huang, Kenneth Hennrick, Sally Drew.
Hum Pathol, 2012 Sep 05; 44(1). PMID: 22944297
Induction of resident memory T cells enhances the efficacy of cancer vaccine.
Mevyn Nizard, Hélène Roussel, +20 authors, Eric Tartour.
Nat Commun, 2017 May 26; 8. PMID: 28537262    Free PMC article.
Highly Cited.
Overview of contemporary guidelines in digital pathology: what is available in 2015 and what still needs to be addressed?
Matthew G Hanna, Liron Pantanowitz, Andrew J Evans.
J Clin Pathol, 2015 May 17; 68(7). PMID: 25979986
Complete Routine Remote Digital Pathology Services.
Aleksandar Vodovnik, Mohammad Reza F Aghdam.
J Pathol Inform, 2018 Dec 07; 9. PMID: 30505622    Free PMC article.
Whole-slide imaging: routine pathologic diagnosis.
Toby C Cornish, Ryan E Swapp, Keith J Kaplan.
Adv Anat Pathol, 2012 Apr 14; 19(3). PMID: 22498580
"Slide less pathology": Fairy tale or reality?
M Indu, R Rathy, M P Binu.
J Oral Maxillofac Pathol, 2016 Sep 08; 20(2). PMID: 27601824    Free PMC article.
Primary histologic diagnosis using automated whole slide imaging: a validation study.
John R Gilbertson, Jonhan Ho, +3 authors, Anil V Parwani.
BMC Clin Pathol, 2006 Apr 29; 6. PMID: 16643664    Free PMC article.
Biomarkers immune monitoring technology primer: Immunoscore® Colon.
Fabienne Hermitte.
J Immunother Cancer, 2016 Sep 24; 4. PMID: 27660711    Free PMC article.
Canadian licensure for the use of digital pathology for routine diagnoses: one more step toward a new era of pathology practice without borders.
Bernard Têtu, Andrew Evans.
Arch Pathol Lab Med, 2013 Jun 28; 138(3). PMID: 23802851
Multiplexed ion beam imaging of human breast tumors.
Michael Angelo, Sean C Bendall, +9 authors, Garry P Nolan.
Nat Med, 2014 Mar 04; 20(4). PMID: 24584119    Free PMC article.
Highly Cited.
Cutoff Finder: a comprehensive and straightforward Web application enabling rapid biomarker cutoff optimization.
Jan Budczies, Frederick Klauschen, +4 authors, Carsten Denkert.
PLoS One, 2012 Dec 20; 7(12). PMID: 23251644    Free PMC article.
Highly Cited.
PD-1 blockade induces responses by inhibiting adaptive immune resistance.
Paul C Tumeh, Christina L Harview, +23 authors, Antoni Ribas.
Nature, 2014 Nov 28; 515(7528). PMID: 25428505    Free PMC article.
Highly Cited.
Multiplexed quantitative analysis of CD3, CD8, and CD20 predicts response to neoadjuvant chemotherapy in breast cancer.
Jason R Brown, Hallie Wimberly, +3 authors, Veerle Bossuyt.
Clin Cancer Res, 2014 Sep 27; 20(23). PMID: 25255793    Free PMC article.
Highly Cited.
The use of digital pathology and image analysis in clinical trials.
Robert Pell, Karin Oien, +6 authors, UK National Cancer Research Institute (NCRI) Cellular-Molecular Pathology (CM-Path) quality assurance working group.
J Pathol Clin Res, 2019 Feb 16; 5(2). PMID: 30767396    Free PMC article.
Digital imaging in pathology: whole-slide imaging and beyond.
Farzad Ghaznavi, Andrew Evans, Anant Madabhushi, Michael Feldman.
Annu Rev Pathol, 2012 Nov 20; 8. PMID: 23157334
Macrophage spatial heterogeneity in gastric cancer defined by multiplex immunohistochemistry.
Yu-Kuan Huang, Minyu Wang, +6 authors, Alex Boussioutas.
Nat Commun, 2019 Sep 04; 10(1). PMID: 31477692    Free PMC article.
QuPath: Open source software for digital pathology image analysis.
Peter Bankhead, Maurice B Loughrey, +10 authors, Peter W Hamilton.
Sci Rep, 2017 Dec 06; 7(1). PMID: 29203879    Free PMC article.
Highly Cited.
Current Status of Whole-Slide Imaging in Education.
Adela Saco, Jose Antoni Bombi, +2 authors, Jaume Ordi.
Pathobiology, 2016 Apr 23; 83(2-3). PMID: 27101397
PD-L1 Expression Correlates with Tumor-Infiltrating Lymphocytes and Response to Neoadjuvant Chemotherapy in Breast Cancer.
Hallie Wimberly, Jason R Brown, +7 authors, David L Rimm.
Cancer Immunol Res, 2014 Dec 21; 3(4). PMID: 25527356    Free PMC article.
Highly Cited.
Quantitative comparison of immunohistochemical staining measured by digital image analysis versus pathologist visual scoring.
Anthony E Rizzardi, Arthur T Johnson, +5 authors, Stephen C Schmechel.
Diagn Pathol, 2012 Apr 21; 7. PMID: 22515559    Free PMC article.
Highly Cited.
World-Wide Immunoscore Task Force: meeting report from the "Melanoma Bridge", Napoli, November 30th-December 3rd, 2016.
Jerome Galon, Alessandro Lugli, +4 authors, Paolo A Ascierto.
J Transl Med, 2017 Oct 24; 15(1). PMID: 29058595    Free PMC article.
Assessment of problems in diagnostic and research immunohistochemistry associated with epitope instability in stored paraffin sections.
L J van den Broek, M J van de Vijver.
Appl Immunohistochem Mol Morphol, 2000 Dec 29; 8(4). PMID: 11127924
Genomics and proteomics approaches for biomarker discovery in sporadic colorectal cancer with metastasis.
María González-González, Jacinto Garcia Garcia, +6 authors, Manuel Fuentes.
Cancer Genomics Proteomics, 2013 Feb 06; 10(1). PMID: 23382583
The use of immunohistochemistry for biomarker assessment--can it compete with other technologies?
Robert W Dunstan, Keith A Wharton, Catherine Quigley, Amanda Lowe.
Toxicol Pathol, 2011 Oct 06; 39(6). PMID: 21971934
Evaluate Cutpoints: Adaptable continuous data distribution system for determining survival in Kaplan-Meier estimator.
Magdalena Ogłuszka, Magdalena Orzechowska, +2 authors, Andrzej K Bednarek.
Comput Methods Programs Biomed, 2019 Jul 20; 177. PMID: 31319941
Emerging Themes in Image Informatics and Molecular Analysis for Digital Pathology.
Rohit Bhargava, Anant Madabhushi.
Annu Rev Biomed Eng, 2016 Jul 16; 18. PMID: 27420575    Free PMC article.
CellProfiler 3.0: Next-generation image processing for biology.
Claire McQuin, Allen Goodman, +12 authors, Anne E Carpenter.
PLoS Biol, 2018 Jul 04; 16(7). PMID: 29969450    Free PMC article.
Highly Cited.
Practical Stereology Applications for the Pathologist.
Danielle L Brown.
Vet Pathol, 2017 Apr 26; 54(3). PMID: 28438109
Neoadjuvant chemotherapy (NACT) increases immune infiltration and programmed death-ligand 1 (PD-L1) expression in epithelial ovarian cancer (EOC).
S J L Mesnage, A Auguste, +13 authors, A F Leary.
Ann Oncol, 2016 Nov 20; 28(3). PMID: 27864219
Telepathology at the Armed Forces Institute of Pathology: A Retrospective Review of Consultations From 1996 to 1997.
Arunima Ghosh, G Thomas Brown, Paul Fontelo.
Arch Pathol Lab Med, 2017 Oct 03; 142(2). PMID: 28967805
Objective measurement and clinical significance of TILs in non-small cell lung cancer.
Kurt A Schalper, Jason Brown, +5 authors, David L Rimm.
J Natl Cancer Inst, 2015 Feb 05; 107(3). PMID: 25650315    Free PMC article.
Highly Cited.
Accurate and reproducible invasive breast cancer detection in whole-slide images: A Deep Learning approach for quantifying tumor extent.
Angel Cruz-Roa, Hannah Gilmore, +6 authors, Anant Madabhushi.
Sci Rep, 2017 Apr 19; 7. PMID: 28418027    Free PMC article.
Highly Cited.
The Gold Standard Paradox in Digital Image Analysis: Manual Versus Automated Scoring as Ground Truth.
Famke Aeffner, Kristin Wilson, +8 authors, G Dave Young.
Arch Pathol Lab Med, 2017 May 31; 141(9). PMID: 28557614
Selecting fluorochrome conjugates for maximum sensitivity.
Holden T Maecker, Tom Frey, Laurel E Nomura, Joe Trotter.
Cytometry A, 2004 Nov 13; 62(2). PMID: 15536642
Histopathologic-based prognostic factors of colorectal cancers are associated with the state of the local immune reaction.
Bernhard Mlecnik, Marie Tosolini, +8 authors, Jérôme Galon.
J Clin Oncol, 2011 Jan 20; 29(6). PMID: 21245428
Highly Cited.
Complete Digital Pathology for Routine Histopathology Diagnosis in a Multicenter Hospital Network.
Juan Antonio Retamero, Jose Aneiros-Fernandez, Raimundo G Del Moral.
Arch Pathol Lab Med, 2019 Jul 12; 144(2). PMID: 31295015
Spatial interaction of tumor cells and regulatory T cells correlates with survival in non-small cell lung cancer.
Souptik Barua, Penny Fang, +4 authors, Steven H Lin.
Lung Cancer, 2018 Feb 08; 117. PMID: 29409671    Free PMC article.
Immunohistochemical evaluation of the effects of paraffin section storage on biomarker stability.
J A Ramos-Vara, J D Webster, D DuSold, M A Miller.
Vet Pathol, 2013 Feb 26; 51(1). PMID: 23435571
The prognostic impact of a combined carbonic anhydrase IX and Ki67 signature in oral squamous cell carcinoma.
A C Klimowicz, P Bose, +3 authors, N T Brockton.
Br J Cancer, 2013 Sep 07; 109(7). PMID: 24008660    Free PMC article.
Towards the introduction of the 'Immunoscore' in the classification of malignant tumours.
Jérôme Galon, Bernhard Mlecnik, +45 authors, Franck Pagès.
J Pathol, 2013 Oct 15; 232(2). PMID: 24122236    Free PMC article.
Highly Cited. Review.
Multiparametric immune profiling in HPV- oral squamous cell cancer.
Zipei Feng, Daniel Bethmann, +14 authors, Bernard A Fox.
JCI Insight, 2017 Jul 21; 2(14). PMID: 28724788    Free PMC article.
Highly Cited.
Multispectral imaging for quantitative and compartment-specific immune infiltrates reveals distinct immune profiles that classify lung cancer patients.
Artur Mezheyeuski, Christian Holst Bergsland, +4 authors, Patrick Micke.
J Pathol, 2017 Dec 29; 244(4). PMID: 29282718
In vivo fluorescence imaging for tissue diagnostics.
S Andersson-Engels, C Klinteberg, K Svanberg, S Svanberg.
Phys Med Biol, 1997 May 01; 42(5). PMID: 9172261
Neoadjuvant immune checkpoint blockade in high-risk resectable melanoma.
Rodabe N Amaria, Sangeetha M Reddy, +42 authors, Jennifer A Wargo.
Nat Med, 2018 Oct 10; 24(11). PMID: 30297909    Free PMC article.
Highly Cited.
Methods for categorizing a prognostic variable in a multivariable setting.
Madhu Mazumdar, Alex Smith, Jennifer Bacik.
Stat Med, 2003 Feb 19; 22(4). PMID: 12590414
Detailed resolution analysis reveals spatial T cell heterogeneity in the invasive margin of colorectal cancer liver metastases associated with improved survival.
Anna Berthel, Inka Zoernig, +6 authors, Niels Halama.
Oncoimmunology, 2017 Apr 14; 6(3). PMID: 28405518    Free PMC article.
Multispectral imaging and pathology: seeing and doing more.
Richard M Levenson, Alessandro Fornari, Massimo Loda.
Expert Opin Med Diagn, 2008 Sep 01; 2(9). PMID: 23495926
The immune contexture in human tumours: impact on clinical outcome.
Wolf Herman Fridman, Franck Pagès, Catherine Sautès-Fridman, Jérôme Galon.
Nat Rev Cancer, 2012 Mar 16; 12(4). PMID: 22419253
Highly Cited. Review.
Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers.
Gillian O'Hurley, Evelina Sjöstedt, +5 authors, Cecilia Lindskog.
Mol Oncol, 2014 Apr 15; 8(4). PMID: 24725481    Free PMC article.
Impact of pre-therapy glioblastoma multiforme microenvironment on clinical response to autologous CMV-specific T-cell therapy.
David G Walker, Reshma Shakya, +5 authors, Rajiv Khanna.
Clin Transl Immunology, 2020 Jan 14; 8(11). PMID: 31929892    Free PMC article.
Epithelial cell shedding and barrier function: a matter of life and death at the small intestinal villus tip.
J M Williams, C A Duckworth, +3 authors, D M Pritchard.
Vet Pathol, 2014 Nov 28; 52(3). PMID: 25428410    Free PMC article.
X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization.
Robert L Camp, Marisa Dolled-Filhart, David L Rimm.
Clin Cancer Res, 2004 Nov 10; 10(21). PMID: 15534099
Highly Cited.
The non-small cell lung cancer immune landscape: emerging complexity, prognostic relevance and prospective significance in the context of immunotherapy.
Andrea Anichini, Elena Tassi, Giulia Grazia, Roberta Mortarini.
Cancer Immunol Immunother, 2018 Mar 09; 67(6). PMID: 29516154
Deep learning for digital pathology image analysis: A comprehensive tutorial with selected use cases.
Andrew Janowczyk, Anant Madabhushi.
J Pathol Inform, 2016 Aug 27; 7. PMID: 27563488    Free PMC article.
Highly Cited.
Multidimensional, quantitative assessment of PD-1/PD-L1 expression in patients with Merkel cell carcinoma and association with response to pembrolizumab.
Nicolas A Giraldo, Peter Nguyen, +24 authors, Janis M Taube.
J Immunother Cancer, 2018 Oct 05; 6(1). PMID: 30285852    Free PMC article.
Immunoprofiling as a predictor of patient's response to cancer therapy-promises and challenges.
Daniel Bethmann, Zipei Feng, Bernard A Fox.
Curr Opin Immunol, 2017 Feb 22; 45. PMID: 28222333
Data integration from pathology slides for quantitative imaging of multiple cell types within the tumor immune cell infiltrate.
Zhaoxuan Ma, Stephen L Shiao, +6 authors, Arkadiusz Gertych.
Diagn Pathol, 2017 Sep 20; 12(1). PMID: 28923066    Free PMC article.
Computer-aided diagnostics in digital pathology.
Ewert Bengtsson, Håvard Danielsen, +3 authors, Béla Molnár.
Cytometry A, 2017 Jun 24; 91(6). PMID: 28640523
Exploring Metabolic Configurations of Single Cells within Complex Tissue Microenvironments.
Anne Miller, Csörsz Nagy, +7 authors, Arvand Haschemi.
Cell Metab, 2017 Sep 12; 26(5). PMID: 28889950
The Pathologist 2.0: An Update on Digital Pathology in Veterinary Medicine.
Christof A Bertram, Robert Klopfleisch.
Vet Pathol, 2017 Jun 06; 54(5). PMID: 28578626
Changes in Expression of Multiple Checkpoint Molecules and Infiltration of Tumor Immune Cells after Neoadjuvant Chemotherapy in Gastric Cancer.
Yue Yu, Xiaopeng Ma, +6 authors, Yixin Zeng.
J Cancer, 2019 Jul 02; 10(12). PMID: 31258783    Free PMC article.
Reprograming the tumor immunologic microenvironment using neoadjuvant chemotherapy in osteosarcoma.
Chuangzhong Deng, Yanyang Xu, +11 authors, Jin Wang.
Cancer Sci, 2020 Apr 02; 111(6). PMID: 32232912    Free PMC article.
Neoadjuvant cisplatin and paclitaxel modulate tumor-infiltrating T cells in patients with cervical cancer.
A Marijne Heeren, Iske F van Luijk, +7 authors, Ekaterina S Jordanova.
Cancer Immunol Immunother, 2019 Oct 17; 68(11). PMID: 31616965    Free PMC article.
The immune score as a new possible approach for the classification of cancer.
Jérôme Galon, Franck Pagès, +5 authors, Paolo A Ascierto.
J Transl Med, 2012 Jan 05; 10. PMID: 22214470    Free PMC article.
Highly Cited.
Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.
Michael J Gerdes, Christopher J Sevinsky, +28 authors, Fiona Ginty.
Proc Natl Acad Sci U S A, 2013 Jul 03; 110(29). PMID: 23818604    Free PMC article.
Highly Cited.
Quantitative image analysis of cellular heterogeneity in breast tumors complements genomic profiling.
Yinyin Yuan, Henrik Failmezger, +14 authors, Florian Markowetz.
Sci Transl Med, 2012 Oct 27; 4(157). PMID: 23100629
Highly Cited.
Aberrant expression of HLA class-I antigens in Burkitt lymphoma cells.
M L Andersson, N J Stam, +2 authors, M G Masucci.
Int J Cancer, 1991 Feb 20; 47(4). PMID: 1899854
An automatic algorithm for the segmentation and morphological analysis of microvessels in immunostained histological tumour sections.
C C Reyes-Aldasoro, L J Williams, +2 authors, G M Tozer.
J Microsc, 2010 Dec 02; 242(3). PMID: 21118252
Paraffin section storage and immunohistochemistry. Effects of time, temperature, fixation, and retrieval protocol with emphasis on p53 protein and MIB1 antigen.
K Wester, E Wahlund, +6 authors, C Busch.
Appl Immunohistochem Mol Morphol, 2000 Aug 11; 8(1). PMID: 10937051
Constant Quest for Quality: Digital Cytopathology.
Simone L Van Es, Janelle Greaves, +3 authors, Tony Badrick.
J Pathol Inform, 2018 May 04; 9. PMID: 29721361    Free PMC article.
Spatial and phenotypic immune profiling of metastatic colon cancer.
Jenny Lazarus, Tomasz Maj, +15 authors, Timothy L Frankel.
JCI Insight, 2018 Nov 16; 3(22). PMID: 30429368    Free PMC article.
Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei.
Carolina Wählby, Fredrik Erlandsson, Ewert Bengtsson, Anders Zetterberg.
Cytometry, 2002 Jan 05; 47(1). PMID: 11774347
The additional facet of immunoscore: immunoprofiling as a possible predictive tool for cancer treatment.
Paolo A Ascierto, Mariaelena Capone, +7 authors, Bernard A Fox.
J Transl Med, 2013 Mar 05; 11. PMID: 23452415    Free PMC article.
Implementation of Whole Slide Imaging for Clinical Purposes: Issues to Consider From the Perspective of Early Adopters.
Andrew J Evans, Mohamed E Salama, Walter H Henricks, Liron Pantanowitz.
Arch Pathol Lab Med, 2017 Apr 26; 141(7). PMID: 28440660
Automated subcellular localization and quantification of protein expression in tissue microarrays.
Robert L Camp, Gina G Chung, David L Rimm.
Nat Med, 2002 Oct 22; 8(11). PMID: 12389040
Highly Cited.
Antigenicity testing by immunohistochemistry after tissue oxidation.
C Blind, A Koepenik, +5 authors, I Petersen.
J Clin Pathol, 2007 Apr 07; 61(1). PMID: 17412873
Accurate quantitation of Ki67-positive proliferating hepatocytes in rabbit liver by a multicolor immunohistochemical (IHC) approach analyzed with automated tissue and cell segmentation software.
Chris M van der Loos, Onno J de Boer, +3 authors, Joanne Verheij.
J Histochem Cytochem, 2012 Sep 04; 61(1). PMID: 22941418    Free PMC article.
NIH Image to ImageJ: 25 years of image analysis.
Caroline A Schneider, Wayne S Rasband, Kevin W Eliceiri.
Nat Methods, 2012 Aug 30; 9(7). PMID: 22930834    Free PMC article.
Highly Cited.
Relationship between magnification and resolution in digital pathology systems.
Tiffany L Sellaro, Robert Filkins, +5 authors, Michael Montalto.
J Pathol Inform, 2013 Oct 02; 4. PMID: 24083056    Free PMC article.
Critical comparison of 31 commercially available digital slide systems in pathology.
Marcial García Rojo, Gloria Bueno García, +2 authors, Manuel Carbajo Vicente.
Int J Surg Pathol, 2006 Oct 17; 14(4). PMID: 17041192
The history of pathology informatics: A global perspective.
Seung Park, Anil V Parwani, +15 authors, Liron Pantanowitz.
J Pathol Inform, 2013 Jul 23; 4. PMID: 23869286    Free PMC article.
Patterns of cancer invasion revealed by QDs-based quantitative multiplexed imaging of tumor microenvironment.
Chun-Wei Peng, Xiu-Li Liu, +5 authors, Yan Li.
Biomaterials, 2011 Jan 26; 32(11). PMID: 21262536
Icy: an open bioimage informatics platform for extended reproducible research.
Fabrice de Chaumont, Stéphane Dallongeville, +10 authors, Jean-Christophe Olivo-Marin.
Nat Methods, 2012 Jun 30; 9(7). PMID: 22743774
Highly Cited.
Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue.
David Robertson, Kay Savage, Jorge S Reis-Filho, Clare M Isacke.
BMC Cell Biol, 2008 Mar 28; 9. PMID: 18366689    Free PMC article.
Commentary: Roles for Pathologists in a High-throughput Image Analysis Team.
Famke Aeffner, Kristin Wilson, +5 authors, G David Young.
Toxicol Pathol, 2016 Jun 28; 44(6). PMID: 27343178
Erratum to: Effect of neoadjuvant chemotherapy on tumor-infiltrating lymphocytes and PD-L1 expression in breast cancer and its clinical significance.
Vasiliki Pelekanou, Daniel E Carvajal-Hausdorf, +7 authors, David L Rimm.
Breast Cancer Res, 2017 Sep 28; 19(1). PMID: 28946899    Free PMC article.
Proteomics in diagnostic pathology: profiling and imaging proteins directly in tissue sections.
Pierre Chaurand, Melinda E Sanders, Roy A Jensen, Richard M Caprioli.
Am J Pathol, 2004 Oct 07; 165(4). PMID: 15466373    Free PMC article.
Neoadjuvant versus adjuvant ipilimumab plus nivolumab in macroscopic stage III melanoma.
Christian U Blank, Elisa A Rozeman, +21 authors, Ton N Schumacher.
Nat Med, 2018 Oct 10; 24(11). PMID: 30297911
Highly Cited.
Categorizing a prognostic variable: review of methods, code for easy implementation and applications to decision-making about cancer treatments.
M Mazumdar, J R Glassman.
Stat Med, 2000 Jan 07; 19(1). PMID: 10623917
Clonal evolution in cancer.
Mel Greaves, Carlo C Maley.
Nature, 2012 Jan 20; 481(7381). PMID: 22258609    Free PMC article.
Highly Cited. Review.
Spatial Mapping of Myeloid Cells and Macrophages by Multiplexed Tissue Staining.
Joshua Saylor, Zhaoxuan Ma, +9 authors, Beatrice S Knudsen.
Front Immunol, 2019 Jan 09; 9. PMID: 30619287    Free PMC article.
Multiplexed Immunohistochemistry and Digital Pathology as the Foundation for Next-Generation Pathology in Melanoma: Methodological Comparison and Future Clinical Applications.
Yannick Van Herck, Asier Antoranz, +4 authors, Francesca Maria Bosisio.
Front Oncol, 2021 Apr 16; 11. PMID: 33854972    Free PMC article.
Next-Generation Digital Histopathology of the Tumor Microenvironment.
Felicitas Mungenast, Achala Fernando, +4 authors, Rupert C Ecker.
Genes (Basel), 2021 May 01; 12(4). PMID: 33917241    Free PMC article.
Challenges and Opportunities in the Statistical Analysis of Multiplex Immunofluorescence Data.
Christopher M Wilson, Oscar E Ospina, +6 authors, Brooke L Fridley.
Cancers (Basel), 2021 Jul 03; 13(12). PMID: 34204319    Free PMC article.
Comparison of liver biopsies before and after direct-acting antiviral therapy for hepatitis C and correlation with clinical outcome.
Omar A Saldarriaga, Bradley Dye, +6 authors, Heather L Stevenson.
Sci Rep, 2021 Jul 17; 11(1). PMID: 34267267    Free PMC article.