The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation.

OBJECTIVES: The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment. METHODS: The Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms. RESULTS: Representative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed. CONCLUSIONS: mIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force

Pitfalls in assessing stromal tumor infiltrating lymphocytes (sTILs) in breast cancer

Abstract: Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls

Report on computational assessment of Tumor Infiltrating Lymphocytes from the International Immuno-Oncology Biomarker Working Group

Funder: U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)Funder: National Center for Research Resources under award number 1 C06 RR12463-01, VA Merit Review Award IBX004121A from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service, the DOD Prostate Cancer Idea Development Award (W81XWH-15-1-0558), the DOD Lung Cancer Investigator-Initiated Translational Research Award (W81XWH-18-1-0440), the DOD Peer Reviewed Cancer Research Program (W81XWH-16-1-0329), the Ohio Third Frontier Technology Validation Fund, the Wallace H. Coulter Foundation Program in the Department of Biomedical Engineering and the Clinical and Translational Science Award Program (CTSA) at Case Western Reserve University.Funder: Susan G Komen Foundation (CCR CCR18547966) and a Young Investigator Grant from the Breast Cancer Alliance.Funder: The Canadian Cancer SocietyFunder: Breast Cancer Research Foundation (BCRF), Grant No. 17-194Abstract: Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring

PD-L1 staining patterns on NSCLC specimens using the 22C3 antibody concentrate on the Dako ASL48 platform (LDT) compared with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard).

<p>(<i>A</i>) The PD-L1 22C3 pharmDx kit on the Dako ASL48 platform; (<i>B</i>) optimised LDT (primary antibody dilution 1:50, 30-minute incubation); (<i>C</i>) LDT using primary antibody dilution 1:100, 30-minute incubation; (<i>D</i>) primary antibody dilution 1:200, 30-minute incubation; (<i>E</i>) primary antibody dilution 1:100, 60-minute incubation; (<i>F</i>) primary antibody dilution 1:100, 120-minute incubation. Original magnification 5×. Inserts, original magnification 40×. PD-L1, programmed death ligand 1; NSCLC, non–small cell lung cancer; ASL48, Autostainer Link 48; LDT, laboratory-developed test; IHC, immunohistochemistry.</p

Use of the 22C3 anti–PD-L1 antibody to determine PD-L1 expression in multiple automated immunohistochemistry platforms

<div><p>Background</p><p>For non–small cell lung cancer (NSCLC), treatment with pembrolizumab is limited to patients with tumours expressing PD-L1 assessed by immunohistochemistry (IHC) using the PD-L1 IHC 22C3 pharmDx (Dako, Inc.) companion diagnostic test, on the Dako Autostainer Link 48 (ASL48) platform. Optimised protocols are urgently needed for use of the 22C3 antibody concentrate to test PD-L1 expression on more widely available IHC autostainers.</p><p>Methods</p><p>We evaluated PD-L1 expression using the 22C3 antibody concentrate in the three main commercially available autostainers Dako ASL48, BenchMark ULTRA (Ventana Medical Systems, Inc.), and Bond-III (Leica Biosystems) and compared the staining results with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Several technical conditions for laboratory-developed tests (LDTs) were evaluated in tonsil specimens and a training set of three NSCLC samples. Optimised protocols were then validated in 120 NSCLC specimens.</p><p>Results</p><p>Optimised protocols were obtained on both the VENTANA BenchMark ULTRA and Dako ASL48 platforms. Significant expression of PD-L1 was obtained on tissue controls with the Leica Bond-III autostainer when high concentrations of the 22C3 antibody were used. It therefore was not tested on the 120 NSCLC specimens. An almost 100% concordance rate for dichotomized tumour proportion score (TPS) results was observed between TPS ratings using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Interpathologist agreement was high on both LDTs and the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform.</p><p>Conclusion</p><p>Availability of standardized protocols for determining PD-L1 expression using the 22C3 antibody concentrate on the widely available Dako ASL48 and VENTANA BenchMark ULTRA IHC platforms will expand the number of laboratories able to determine eligibility of patients with NSCLC for treatment with pembrolizumab in a reliable and concordant manner.</p></div

PD-L1 staining patterns on NSCLC specimens using the 22C3 antibody concentrate on the VENTANA BenchMark ULTRA platform (LDT) compared with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard).

<p>(<i>A</i>) The PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform; (<i>B</i>) optimised LDT (CC1 64 minutes, 22C3 primary antibody dilution 1:50, OptiView amplification 12 minutes); (<i>C</i>) CC1 32 minutes, primary antibody dilution 1:50, OptiView amplification 12 minutes; (<i>D</i>) CC1 64 minutes, primary antibody dilution 1:50, OptiView amplification 4 minutes; (<i>E</i>) CC1 32 minutes, primary antibody dilution 1:100, OptiView amplification 12 minutes; (<i>F</i>) CC1 64 minutes, primary antibody dilution 1:100, OptiView amplification 12 minutes. Original magnification 5×. Inserts, original magnification 40×. PD-L1, programmed death ligand 1; NSCLC, non–small cell lung cancer; ASL48, Autostainer Link 48; LDT, laboratory-developed test; IHC, immunohistochemistry.</p

PPA and NPA for each of the PD-L1 IHC assays using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms (LDTs) relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard).

<p>PPA and NPA for each of the PD-L1 IHC assays using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms (LDTs) relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard).</p

Intraclass correlation coefficient of TPS rating for each of the PD-L1 IHC assays using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms (LDTs) relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard).

<p>Intraclass correlation coefficient of TPS rating for each of the PD-L1 IHC assays using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms (LDTs) relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard).</p