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Background: Scoring proliferation through Ki67-immunohistochemistry is an important component in predicting therapy response to chemotherapy in breast cancer patients. Therefore, an accurate and standardized Ki67-scoring is pivotal both in routine diagnostics and larger multi-center studies aiming at improving present or establishing new cut-off values for existing or novel therapy regimens. However, recent studies have cast some doubt on the reliability of "visual" Ki67 scoring by pathologists, especially within the lower - yet clinically important - proliferation range. Here, we present and apply a novel automated image analysis approach for Ki67-quantification in breast cancer tissue. Methods: We perform automated Ki67-scoring in 1219 breast cancer patients from the GeparTrio study cohort using a novel image analysis approach that avoids detection biases due to morphological variability by using a generic minimum-model approach. The method is capable of tumor-stroma-separation and may be used to process large data sets fully unsupervised in batch mode while allowing for efficient visual checks of the results. We compare these results with a different in-house-developed subtiling-based automated quantification method and moreover, gauge our approach with manual scoring performed by pathologists. Results: The results show deviations of 10% (automated method 1 vs. manual), 9% (automated method 2 vs. manual) and 3% (automated method 1 vs. automated method 2) on average. The Ki67 scores show Pearson correlations between automated and manual scoring of r>0.8 (p < 0.001) for both automated methods and r>0.95 (p < 0.001) between the two tested automated methods. Conclusion: Because of the methodological differences of the presented techniques our results suggest a high robustness of the automated methods that at the same time show a good agreement with manual Ki67 scoring. Our approach therefore offers an automated and standardized means of Ki67 quantification applicable in routine diagnostics as well as larger clinical study settings, such as in the GeparTrio cohort shown here. [ABSTRACT FROM AUTHOR]

Abstract Background The virtual microscopy is widely accepted in Pathology for educational purposes and teleconsultation but is far from the routine use in surgical pathology due to the technical requirements and some limitations. A technical problem is the limited bandwidth of a usual network and the delayed transmission rate and presentation time on the screen. Methods In this study the process of secondary diagnostic was evaluated using the "T.Konsult Pathologie" service of the Professional Association of German Pathologists within the German breast cancer screening program. The characteristics of the access to the WSI (Whole Slide Images) have been analyzed to explore the possibilities of prefetching and caching to reduce the presentation and transfer time with the goal to increase user acceptance. The log files of the web server were analyzed to reconstruct the movements of the pathologist on the WSI and to create the observation path. Using a specialized tool the observation paths were extracted automatically from the log files. The attributes linearity, 3-point-linearity, changes per request, and number of consecutive requests were calculated to design, develop and evaluate different caching and prefetching strategies. Results The analysis of the observation paths showed that a complete accordance of two image requests is a very rare event. But more frequently a partial covering of two requested image areas can be found. In total 257 diagnostic paths from 131 WSI have been extracted and analysed. On average a diagnostic path consists of 16 image requests and takes 189 seconds between first and last image request. The mean linearity was 0,41 and the mean 3-point-linearity 0,85. Three different caching algorithms have been compared with respect to hit rate and additional image requests on the WSI server. Tests demonstrated that 95% of the diagnostic paths could be loaded without any deletion of entries in the cache (cache size 12,2 Megapixel). If the image parts are stored after JPEG compression this complies with less than 2 MB. Discussion WSI telepathology is a technology which offers the possibility to break the limitations of conventional static telepathology. The complete histological slide may be investigated instead of sets of images of lesions sampled by the presenting pathologist. The benefit is demonstrated by the high diagnostic security of 95% accordance between first and second diagnosis.

Background: The morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is gaining momentum as evidence strengthens for the clinical relevance of this immunological biomarker. Accumulating evidence suggests that the extent of lymphocytic infiltration in tumor tissue can be assessed as a major parameter by evaluation of hematoxylin and eosin (H&E)-stained tumor sections. TILs have been shown to provide prognostic and potentially predictive value, particularly in triple-negative and human epidermal growth factor receptor 2-overexpressing BC. Design: A standardized methodology for evaluating TILs is now needed as a prerequisite for integrating this parameter in standard histopathological practice, in a research setting as well as in clinical trials. This article reviews current data on the clinical validity and utility of TILs in BC in an effort to foster better knowledge and insight in this rapidly evolving field, and to develop a standardized methodology for visual assessment on H&E sections, acknowledging the future potential of molecular/multiplexed approaches. Conclusions: The methodology provided is sufficiently detailed to offer a uniformly applied, pragmatic starting point and improve consistency and reproducibility in the measurement of TILs for future studies. [ABSTRACT FROM AUTHOR]

Immune cell locomotion is associated with amoeboid migration, a flexible mode of movement, which depends on rapid cycles of actin polymerization and actomyosin contraction 1 . Many immune cells do not necessarily require integrins, the major family of adhesion receptors in mammals, to move productively through three-dimensional tissue spaces 2,3 . Instead, they can use alternative strategies to transmit their actin-driven forces to the substrate, explaining their migratory adaptation to changing external environments 4-6 . However, whether these generalized concepts apply to all immune cells is unclear. Here, we show that the movement of mast cells (immune cells with important roles during allergy and anaphylaxis) differs fundamentally from the widely applied paradigm of interstitial immune cell migration. We identify a crucial role for integrin-dependent adhesion in controlling mast cell movement and localization to anatomical niches rich in KIT ligand, the major mast cell growth and survival factor. Our findings show that substrate-dependent haptokinesis is an important mechanism for the tissue organization of resident immune cells., (© 2023. The Author(s).)

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., Competing Interests: Competing interestsA.E. is on the Roche advisory board and has reported honoraria from Amgen, Novartis and Roche. A.J.L. is a consultant for BMS, Merck, AZ/Medimmune, and Genentech. R.S. reports research funding from Roche, Puma, Merck; advisory board and consultancy for BMS; travel funding from Roche, Merck, and Astra Zeneca. S.G. reports Lab research funding from Lilly, Clinical research funding from Eli Lilly and Novartis and is a Paid advisor to Eli Lilly, Novartis, and G1 Therapeutics. J.v.d.L. is member of the scientific advisory boards of Philips, the Netherlands and ContextVision, Sweden and receives research funding from Philips, the Netherlands and Sectra, Sweden. S.A. reports Research funding to institution from Merck, Genentech, BMS, Novartis, Celgene and Amgen and is an uncompensated consultant /steering committee member for Merck, Genentech and BMS. T.O.N. has consulted for Nanostring and received compensation and has intellectual property rights/ownership interests from Bioclassifier LLC [not related to the subject material under consideration]. S.L. receives research funding to institution from Novartis, Bristol Meyers Squibb, Merck, Roche-Genentech, Puma Biotechnology, Pfizer and Eli Lilly, has acted as consultant (not compensated) to Seattle Genetics, Pfizer, Novartis, BMS, Merck, AstraZeneca and Roche-Genentech and acted as consultant (paid to her institution) to Aduro Biotech. S.R.L. has received travel and educational funding from Roche/Ventana. A.M. is an equity holder in Elucid Bioimaging and in Inspirata Inc., a scientific advisory consultant for Inspirata Inc, has served as a scientific advisory board member for Inspirata Inc, Astrazeneca, Bristol Meyers-Squibb and Merck, has sponsored research agreements with Philips and Inspirata Inc, is involved in a NIH U24 grant with PathCore Inc, and 3 different R01 grants with Inspirata Inc. and his technology has been licensed to Elucid Bioimaging and Inspirata Inc. G.C. is on the advisory boards of Roche, BMS, Pfizer, Seattle Genetics and Ellipsis, and reports personal fees from Roche, BMS, Pfizer, Seattle Genetics, and Ellipsis, outside of the submitted work. J.H. is the director and owner of Vivactiv Ltd. J.H. is the director and owner of Slide Score B.V. F.P.L. reports funding from Astrazeneca, BMS, Roche, MSD, Pfizer, Novartis, Sanofi, Eli Lilly. J.B. reports consultancies from Insight Genetics, BioNTech AG, Biotheranostics, Pfizer, RNA Diagnostics and OncoXchange, research funding from Thermo Fisher Scientific, Genoptix, Agendia, NanoString Technologies, Stratifyer GmbH and Biotheranostics, applied for patents, including Jan 2017: Methods and Devices for Predicting Anthracycline Treatment Efficacy, US utility—15/325,472; EPO—15822898.1; Canada—not yet assigned; Jan 2017: Systems, Devices and Methods for Constructing and Using a Biomarker, US utility—15/328,108; EPO—15824751.0; Canada—not yet assigned; Oct 2016: Histone gene module predicts anthracycline benefit, PCT/CA2016/000247; Dec 2016: 95‐Gene Signature of Residual Risk Following Endocrine Treatment, PCT/CA2016/000304; Dec 2016: Immune Gene Signature Predicts Anthracycline Benefit, PCT/CA2016/000305. M.A.S. reports consulting work for Achilles Therapeutics. C.S. reports receipt of grants/research support from Pfizer, AstraZeneca, BMS and Ventana; receipt of honoraria, consultancy, or SAB Member fees from Pfizer, Novartis, GlaxoSmithKline, MSD, BMS, Celgene, AstraZeneca, Illumina, Sarah Canon Research Institute, Genentech, Roche-Ventana, GRAIL, Medicxi; Advisor for Dynamo Therapeutics; Stock shareholder in Apogen Biotechnologies, Epic Bioscience, GRAIL; Co-Founder & stock options in Achilles Therapeutics. A.H.B. is the co-founder and CEO of PathAI. J.K. is an employee of PathAI. D.D. is on the advisory board for Oncology Analytics, Inc, and a consultant for Novartis. D.L.R. is on the advisory board of Amgen, Astra Zeneca, Cell Signaling Technology, Cepheid, Daiichi Sankyo, GSK, Konica/Minolta, Merck, NanoString, Perkin Elmer, Ventana, Ultivue; receives research support from Astra Zeneca, Cepheid, Navigate BioPharma, NextCure, Lilly, Ultivue; instrument support from Ventana, Akoya/Perkin Elmer, NanoString; paid consultant for Biocept; received travel honoraria from BMS, founder and equity holder for PixelGear and received royalty from Rarecyte. A.T. reports benefits from ICR’s Inventors Scheme associated with patents for one of PARP inhibitors in BRCA1/2 associated cancers, as well as honoraria from Pfizer, Vertex, Prime Oncology, Artios, honoraria and stock in InBioMotion, honoraria and financial support for research from AstraZeneca, Medivation, Myriad Genetics and Merck Serono. This work includes contributions from, and was reviewed by, individuals at the FDA. This work has been approved for publication by the agency, but it does not necessarily reflect official agency policy. Certain commercial materials and equipment are identified in order to adequately specify experimental procedures. In no case does such identification imply recommendation or endorsement by the FDA, nor does it imply that the items identified are necessarily the best available for the purpose. This work includes contributions from, and was reviewed by, individuals who received funding from the National Institutes of Health, the U.S. Department of Veterans Affairs and the Department of Defense. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health, the U.S. Department of Veterans Affairs, the Department of Defense, or the United States Government., (© The Author(s) 2020.)

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., Competing Interests: Competing interestsJ.T. is funded by Visiopharm A/S, Denmark. A.M. is an equity holder in Elucid Bioimaging and in Inspirata Inc. He is also a scientific advisory consultant for Inspirata Inc. In addition he has served as a scientific advisory board member for Inspirata Inc, Astrazeneca, Bristol Meyers-Squibb and Merck. He also has sponsored research agreements with Philips and Inspirata Inc. His technology has been licensed to Elucid Bioimaging and Inspirata Inc. He is also involved in an NIH U24 grant with PathCore Inc, and three different R01 grants with Inspirata Inc. S.R.L. received travel and educational funding from Roche/Ventana. A.J.L. serves as a consultant for BMS, Merck, AZ/Medimmune, and Genentech. He is also provides consulting and advisory work for many other companies not relevant to this work. FPL does consulting for Astrazeneca, BMS, Roche, MSD Pfizer, Novartis, Sanofi, and Lilly. S.Ld.H., A.K., M.K., U.K., and M.B. are employees of Roche. J.M.S.B. is consultant for Insight Genetics, BioNTech AG, Biothernostics, Pfizer, RNA Diagnostics, and OncoXchange. He received funding from Thermo Fisher Scientific, Genoptix, Agendia, NanoString technologies, Stratifyer GmBH, and Biotheranostics. L.F.S.K. is a consultant for Roche and Novartis. J.K.K. and A.H.B. are employees of PathAI. D.L.R. is on the advisory board for Amgen, Astra Xeneca, Cell Signaling Technology, Cepheid, Daiichi Sankyo, GSK, Konica/Minolta, Merck, Nanostring, Perking Elmer, Roche/Ventana, and Ultivue. He has received research support from Astrazeneca, Cepheid, Navigate BioPharma, NextCure, Lilly, Ultivue, Roche/Ventana, Akoya/Perkin Elmer, and Nanostring. He also has financial conflicts of interest with BMS, Biocept, PixelGear, and Rarecyte. S.G. is a consultant for and/or receives funding from Eli Lilly, Novartis, and G1 Therapeutics. J.A.W.M.vdL. is a member of the scientific advisory boards of Philips, the Netherlands and ContextVision, Sweden, and receives research funding from Philips, the Netherlands and Sectra, Sweden. S.A. is a consultant for Merck, Genentech, and BMS, and receives funding from Merck, Genentech, BMS, Novartis, Celgene, and Amgen. T.O.N. has consulted for Nanostring, and has intellectual property rights and ownership interests from Bioclassifier LLC. S.L. receives research funding to her institution from Novartis, Bristol Meyers-Squibb, Merck, Roche-Genentech, Puma Biotechnology, Pfizer and Eli Lilly. She has acted as consultant (not compensated) to Seattle Genetics, Pfizer, Novartis, BMS, Merck, AstraZeneca and Roche-Genentech. She has acted as consultant (paid to her institution) to Aduro Biotech. J.H. is director and owner of Slide Score BV. M.M.S. is a medical advisory board member of OptraScan. R.S. has received research support from Merck, Roche, Puma; and travel/congress support from AstraZeneca, Roche and Merck; and he has served as an advisory board member of BMS and Roche and consults for BMS., (© The Author(s) 2020.)

Background: There are case descriptions of pronounced peri-implant inflammatory reactions and necrosis in non-infectious knee joint replacements with metal-polyethylene pairing., Objectives: Due to the histopathological similarities to the dysfunctional metal-on-metal (MoM) hip joint replacement, MoM-like reactions in knee joint arthroplasty ("ARMD-KEP") are proposed and a histopathological comparison is made., Materials and Methods: This analysis evaluates five cases of "ARMD-KEP" using: (1) the SLIM consensus classification, (2) the particle algorithm, (3) the CD3 focus score and (4) the AVAL score. The comparison groups consist of 11 adverse cases of MoM hip and 20 cases of knee joint arthroplasty without adverse reaction., Results: The ARMD-KEP cases were identified as SLIM type VI. Their median ALVAL score was 10. The CD3 focus score confirmed an adverse reaction. Particle corrosion was found in two of five cases., Conclusions: This data indicates that, in rare cases, an adverse MoM-like reaction may be present in knee replacements, with inflammatory and immunological expression similar to that of the adverse MoM reaction in the hip. The pathomechanisms can be discussed as follows: (1) secondary metal-metal contact, (2) dysfunctional loading of the coupling mechanism and (3) corrosion of the metal components. Much like trunnionosis in the hip, the term "hingiosis" is proposed for corrosion phenomena in dysfunctional conditions of coupled knee endoprosthetic systems.

Conventional dendritic cells (cDCs) are found in all tissues and play a key role in immune surveillance. They comprise two major subsets, cDC1 and cDC2, both derived from circulating precursors of cDCs (pre-cDCs), which exited the bone marrow. We show that, in the steady-state mouse, pre-cDCs entering tissues proliferate to give rise to differentiated cDCs, which themselves have residual proliferative capacity. We use multicolor fate mapping of cDC progenitors to show that this results in clones of sister cDCs, most of which comprise a single cDC1 or cDC2 subtype, suggestive of pre-cDC commitment. Upon infection, a surge in the influx of pre-cDCs into the affected tissue dilutes clones and increases cDC numbers. Our results indicate that tissue cDCs can be organized in a patchwork of closely positioned sister cells of the same subset whose coexistence is perturbed by local infection, when the bone marrow provides additional pre-cDCs to meet increased tissue demand., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The murine epidermis harbors two immune cell lineages, Langerhans cells (LCs) and γδ T cells known as dendritic epidermal T cells (DETCs). LCs develop from both early yolk sac (YS) progenitors and fetal liver monocytes before locally self-renewing in the adult. For DETCs, the mechanisms of homeostatic maintenance and their hematopoietic origin are largely unknown. Here, we exploited multicolor fate mapping systems to reveal that DETCs slowly turn over at steady state. Like for LCs, homeostatic maintenance of DETCs is achieved by clonal expansion of tissue-resident cells assembled in proliferative units. The same mechanism, albeit accelerated, facilitates DETC replenishment upon injury. Hematopoietic lineage tracing uncovered that DETCs are established independently of definitive hematopoietic stem cells and instead originate from YS hematopoiesis, again reminiscent of LCs. DETCs thus resemble LCs concerning their maintenance, replenishment mechanisms, and hematopoietic development, suggesting that the epidermal microenvironment exerts a lineage-independent influence on the initial seeding and homeostatic maintenance of its resident immune cells., (© 2018 Gentek et al.)

Aims: Advanced-stage ovarian high-grade serous carcinoma (HGSC) is a poor-prognosis cancer; however, a small and poorly characterised subset of patients shows long-term survival. We aimed to establish a cohort of HGSC long-term survivors for histopathological and molecular analysis., Methods and Results: Paraffin blocks from 151 patients with primary FIGO III/IV HGSC and progression-free survival (PFS) >5 years were collected within the Tumorbank Ovarian Cancer (TOC) Network; 77 HGSC with a PFS <3 years were used as a control group. A standardised analysis of histological type and morphological features was performed. Ki67 index, tumour-infiltrating lymphocytes (TILs) and major histocompatibility complex expression (MHC1/2) were determined by immunohistochemistry. A total of 117 of 151 tumours (77.5%) in the long-term survivor group fulfilled the World Health Organisation (WHO) criteria of HGSC after review, and of these, 83 patients (70.9%) fulfilled all clinical criteria for inclusion into our cohort. Tumours of long-term survivors had significantly higher CD3 + and CD8 + TILs and were more frequently positive for MHC2 than controls (P = 0.004, P = 0.025, P = 0.048). However, there were also long-term survivors (up to 20%) with low TILs or low MHC expression. TILs and MHC had no impact on survival in long-term survivors. Morphological and Ki67 analysis revealed no differences between long-term survivors and controls., Conclusions: HGSC from long-term survivors have higher-level T cell infiltration and antigen-presentation capacity; however, this is not a prerequisite for an excellent prognosis. Histopathological criteria are not capable to identify these patients. Further extensive clinical and molecular characterisation of this enigmatic subgroup is ongoing to understand the reasons of long-term survival in HGSC., (© 2018 John Wiley & Sons Ltd.)

Morphological evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer is gaining momentum as evidence strengthens the clinical relevance of this immunological biomarker. TILs in the post-neoadjuvant residual disease setting are acquiring increasing importance as a stratifying marker in clinical trials, considering the raising interest on immunotherapeutic strategies after neoadjuvant chemotherapy. TILs in ductal carcinoma in situ, with or without invasive carcinoma, represent an emerging area of clinical breast cancer research. The aim of this report is to update pathologists, clinicians and researchers on TIL assessment in both the post-neoadjuvant residual disease and the ductal carcinoma in situ settings. The International Immuno-Oncology Working Group proposes a method for assessing TILs in these settings, based on the previously published International Guidelines on TIL Assessment in Breast Cancer. In this regard, these recommendations represent a consensus guidance for pathologists, aimed to achieve the highest possible consistency among future studies., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Tumor-infiltrating lymphocytes (TILs) have an established impact on the prognosis of high-grade serous ovarian carcinoma (HGSOC), however, their role in recurrent ovarian cancer is largely unknown. We therefore systematically investigated TIL densities and MHC class I and II (MHC1, 2) expression in the progression of HGSOC., Experimental Design: CD3+, CD4+, CD8+ TILs and MHC1, 2 expression were evaluated by immunohistochemistry on tissue microarrays in 113 paired primary and recurrent HGSOC. TILs were quantified by image analysis. All patients had been included to the EU-funded OCTIPS FP7 project., Results: CD3+, CD4+, CD8+ TILs and MHC1 and MHC2 expression showed significant correlations between primary and recurrent tumor levels (Spearman rho 0.427, 0.533, 0.361, 0.456, 0.526 respectively; P<.0001 each). Paired testing revealed higher CD4+ densities and MHC1 expression in recurrent tumors (Wilcoxon P=.034 and P=.018). There was also a shift towards higher CD3+ TILs levels in recurrent carcinomas when analyzing platinum-sensitive tumors only (Wilcoxon P=.026) and in pairs with recurrent tumor tissue from first relapse only (Wilcoxon P=.031). High MHC2 expression was the only parameter to be significantly linked to prolonged progression-free survival after first relapse (PFS2, log-rank P=.012)., Conclusions: This is the first study that analyzed the development of TILs density and MHC expression in paired primary and recurrent HGSOC. The level of the antitumoral immune response in recurrent tumors was clearly dependent on the one in the primary tumor. Our data contribute to the understanding of temporal heterogeneity of HGSOC immune microenvironment and have implications for selection of samples for biomarker testing in the setting of immune-targeting therapeutics., (Copyright © 2018. Published by Elsevier Inc.)

Assessment of the immune response to tumors is growing in importance as the prognostic implications of this response are increasingly recognized, and as immunotherapies are evaluated and implemented in different tumor types. However, many different approaches can be used to assess and describe the immune response, which limits efforts at implementation as a routine clinical biomarker. In part 1 of this review, we have proposed a standardized methodology to assess tumor-infiltrating lymphocytes (TILs) in solid tumors, based on the International Immuno-Oncology Biomarkers Working Group guidelines for invasive breast carcinoma. In part 2 of this review, we discuss the available evidence for the prognostic and predictive value of TILs in common solid tumors, including carcinomas of the lung, gastrointestinal tract, genitourinary system, gynecologic system, and head and neck, as well as primary brain tumors, mesothelioma and melanoma. The particularities and different emphases in TIL assessment in different tumor types are discussed. The standardized methodology we propose can be adapted to different tumor types and may be used as a standard against which other approaches can be compared. Standardization of TIL assessment will help clinicians, researchers and pathologists to conclusively evaluate the utility of this simple biomarker in the current era of immunotherapy.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

In lymph nodes (LNs), dendritic cells (DCs) are thought to dispose of apoptotic cells, a function pertaining to macrophages in other tissues. We found that a population of CX3CR1 + MERTK + cells located in the T cell zone of LNs, previously identified as DCs, are efferocytic macrophages. Lineage-tracing experiments and shield chimeras indicated that these T zone macrophages (TZM) are long-lived macrophages seeded in utero and slowly replaced by blood monocytes after birth. Imaging the LNs of mice in which TZM and DCs express different fluorescent proteins revealed that TZM-and not DCs-act as the only professional scavengers, clearing apoptotic cells in the LN T cell zone in a CX3CR1-dependent manner. Furthermore, similar to other macrophages, TZM appear inefficient in priming CD4 T cells. Thus, efferocytosis and T cell activation in the LN are uncoupled processes designated to macrophages and DCs, respectively, with implications to the maintenance of immune homeostasis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The histopathological synovitis score evaluates in a graded approach, as is largely usual for diagnostic histopathological scores, the immunological and inflammatory changes caused by synovitis. A synovitis score of between 1 and ≤ 4 is classified as low-grade (osteoarthritis-related synovitis, post-traumatic synovitis, meniscopathy-related synovitis and synovitis in hemochromatosis). Synovitis scores of between ≥ 5 and 9 are classified as high-grade synovitis (rheumatoid arthritis, psoriatic arthritis, Lyme's arthritis, post-infection/reactive arthritis and peripheral arthritis in Bechterew disease); sensitivity is 61.7% and sensitivity 96.1%. According to receiver operating characteristic (ROC) analysis (AUC: 0.8-0.9), diagnostic value is good. National and international acceptance of the synovitis score has grown since the first publication in 2002 and a related follow-up publication in 2006. PubMed data analysis (as of 11.01.2017) yielded the following citation values according to "cited by PubMed Central articles" for two publications relating to the synovitis score: there were 29 cited-by-PubMed articles for DOI: 10.1078/0344-0338-5710261 , and 44 cited-in-PubMed articles for the second publication, DOI: 10.1111/j.1365-2559.2006.02508 . This makes a total of 73 PubMed citations over a period of 15 years, thereby evidencing the score's international acceptance. Immunohistochemical determination of a number of CD antigens relevant to inflammation has been proposed to further specify the synovitis score for the purposes of risk stratification of high-grade synovitis (e.g., risk of progression and sensitivity to biological agents).

The histopathological synovitis score evaluates the immunological and inflammatory changes of synovitis in a graduated manner generally customary for diagnostic histopathological scores. The score results from semiquantitative evaluation of the width of the synovial surface cell layer, the cell density of the stroma and the density of the inflammatory infiltration into 4 semiquantitative levels (normal 0, mild 1, moderate 2, severe 3). The addition of these values results in a final score of 0-9 out of 9. On the basis of this summation the condition is divided into low-grade synovitis and high-grade synovitis: A synovitis score of 1 to≤4 is called low-grade synovitis (arthrosis-associated/OA synovitis, posttraumatic synovitis, meniscopathy-associated synovitis and synovitis with haemochromatosis). A synovitis score of≥5 to 9 is called high-grade synovitis (rheumatoid arthritis, psoriatic arthritis, Lyme arthritis, postinfection/reactive arthritis and peripheral arthritis with Bechterew's disease). By means of the synovitis score it is therefore possible to distinguish between degenerative/posttraumatic diseases (low-grade synovitis) and inflammatory rheumatic diseases (high-grade synovitis) with a sensitivity of 61.7% and a specificity of 96.1%. The diagnostic accuracy according to ROC analysis (AUC: 0.8-0.9) is good. Since the first publication (2002) and an associated subsequent publication (2006), the synovitis score has nationally and internationally been accepted for histopathological assessment of the synovitis. In a PubMed data analysis (status: 14.02.2017), the following citation rates according to Cited by PubMed Central articles resulted for the two synovitis score publications: For DOI: 10.1078/0344-0338-5710261 there were 29 Cited by PubMed Central articles and for the second extended publication DOI:10.1111/j.1365-2559.2006.02508 there were 44 Cited by PubMed Central articles. Therefore a total of 73 PubMed citations are observed over a period of 15 years, which demonstrates an international acceptance of the score. This synovitis score provides for the first time a diagnostic, standardised and reproducible histopathological evaluation method enabling a contribution to the differential diagnosis of chronic inflammatory general joint diseases. This is particularly the case by incorporation into the joint pathology algorithm. To specify the synovitis score an immunohistochemical determination of various inflammation-relevant CD antigens is proposed to enable a risk stratification of high-grade synovitis (e.g.: progression risk and sensitivity for biologicals)., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

There is evidence from studies on fibrotic diseases that a TH2 polarized immune response, characterized by the production of cytokines like IL-4, IL-5, and IL-13 is important for fibrosis development. Data concerning the role of TH2 polarized immune response for fibrogenesis in lymphomas are scarce. Using immunohistochemistry, we investigated paraffin embedded tissue specimens of 58 follicular lymphomas (FL) grade 1/2 with and without fibrosis. In FL with fibrosis, we detected more interfollicular GATA-3 positive lymphocytes and a shift towards a TH2 immune response. In areas of fibrosis, which were enriched with myofibroblasts, we observed a polarization of macrophages towards a M2 phenotype with expression of CD163. Our results hold some evidence that a polarization of the adaptive immune system towards a TH2 immune response in the tumor microenvironment is associated with increased fibrosis, which may indicate its functional relevance for either development or maintenance of fibrosis in FL.

Lymph node (LN) expansion during an immune response relies on the transient remodeling of its vasculature. Although the mechanisms driving LN endothelial cell division are beginning to be understood, a comprehensive view of LN endothelial cell dynamics at the single-cell level is lacking. Here, we used multicolored fluorescent fate-mapping models to track the behavior of blood endothelial cells during LN expansion upon inflammation and subsequent return to homeostasis. We found that expansion of the LN vasculature relied on the sequential assembly of endothelial cell proliferative units. This segmented growth was sustained by the clonal proliferation of high endothelial venule (HEV) cells, which act as local progenitors to create capillaries and HEV neo-vessels at the periphery of the LN. Return to homeostasis was accompanied by the stochastic death of pre-existing and neo-synthesized LN endothelial cells. Thus, our fate-mapping studies unravel-at a single-cell level-the complex dynamics of vascular-tree remodeling during LN expansion and contraction., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Multiple independent studies have shown that tumor-infiltrating lymphocytes (TIL) are prognostic in breast cancer with potential relevance for response to immune-checkpoint inhibitor therapy. Although many groups are currently evaluating TIL, there is no standardized system for diagnostic applications. This study reports the results of two ring studies investigating TIL conducted by the International Working Group on Immuno-oncology Biomarkers. The study aim was to determine the intraclass correlation coefficient (ICC) for evaluation of TIL by different pathologists. A total of 120 slides were evaluated by a large group of pathologists with a web-based system in ring study 1 and a more advanced software-system in ring study 2 that included an integrated feedback with standardized reference images. The predefined aim for successful ring studies 1 and 2 was an ICC above 0.7 (lower limit of 95% confidence interval (CI)). In ring study 1 the prespecified endpoint was not reached (ICC: 0.70; 95% CI: 0.62-0.78). On the basis of an analysis of sources of variation, we developed a more advanced digital image evaluation system for ring study 2, which improved the ICC to 0.89 (95% CI: 0.85-0.92). The Fleiss' kappa value for <60 vs ≥60% TIL improved from 0.45 (ring study 1) to 0.63 in RS2 and the mean concordance improved from 88 to 92%. This large international standardization project shows that reproducible evaluation of TIL is feasible in breast cancer. This opens the way for standardized reporting of tumor immunological parameters in clinical studies and diagnostic practice. The software-guided image evaluation approach used in ring study 2 may be of value as a tool for evaluation of TIL in clinical trials and diagnostic practice. The experience gained from this approach might be applicable to the standardization of other diagnostic parameters in histopathology.

Aims: Antibodies targeting the checkpoint molecules programmed cell death 1 (PD-1) and its ligand PD-L1 are emerging cancer therapeutics. We systematically investigated PD-1 and PD-L1 expression patterns in the poor-prognosis tumor entity high-grade serous ovarian carcinoma., Methods: PD-1 and PD-L1 protein expression was determined by immunohistochemistry on tissue microarrays from 215 primary cancers both in cancer cells and in tumor-infiltrating lymphocytes (TILs). mRNA expression was measured by quantitative reverse transcription PCR. An in silico validation of mRNA data was performed in The Cancer Genome Atlas (TCGA) dataset., Results: PD-1 and PD-L1 expression in cancer cells, CD3+, PD-1+, and PD-L1+ TILs densities as well as PD-1 and PD-L1 mRNA levels were positive prognostic factors for progression-free (PFS) and overall survival (OS), with all factors being significant for PFS (p < 0.035 each), and most being significant for OS. Most factors also had prognostic value that was independent from age, stage, and residual tumor. Moreover, high PD-1+ TILs as well as PD-L1+ TILs densities added prognostic value to CD3+TILs (PD-1+: p = 0.002,; PD-L1+: p = 0.002). The significant positive prognostic impact of PD-1 and PD-L1 mRNA expression could be reproduced in the TCGA gene expression datasets (p = 0.02 and p < 0.0001, respectively)., Conclusions: Despite their reported immune-modulatory function, high PD-1 and PD-L1 levels are indicators of a favorable prognosis in ovarian cancer. Our data indicate that PD-1 and PD-L1 molecules are biologically relevant regulators of the immune response in high-grade serous ovarian carcinoma, which is an argument for the evaluation of immune checkpoint inhibiting drugs in this tumor entity.

Increased proliferation is a hallmark of malignant tumors. The proliferation marker Ki67 has been investigated as a breast cancer biomarker, but despite 32 years of research the best cutpoints and the best methods for determination are still under debate. This review is based on an overview on the efforts to standardize Ki67 and to optimize its performance that was presented at the St. Gallen oncology conference 2015. The clinical validity of Ki67 as a prognostic marker as well as a predictive marker (in the neoadjuvant setting) has been shown in several meta-analyses. Depending on cohort characteristics, molecular subtype and clinical setting, Ki67 is a prognostic marker, a predictive marker, or both. Many different cutpoints for Ki67 have been reported, but it is has not been possible to determine an evidence-based "optimal" cutpoint. This supports the view that Ki67 is continuous marker, reflecting the continuous variation of the proliferation rate in different tumors. We should probably stop looking for an "optimal" cutpoint for Ki67 because it simply does not exist. It is evident from the results of several ring trials that intermediate levels of Ki67 are particularly difficult for standardization. Due to the low analytical validity in the intermediate range as well as intratumoral heterogeneity, the clinical utility of intermediate Ki67 levels is limited. Clinical decisions should not be based on small differences in the intermediate range and additional molecular tests might be necessary for tumors with intermediate Ki67 levels. For the two groups of tumors with a very low or a very high Ki67 a clinical interpretation could be straightforward. Despite these limitations, the assessment of proliferation is a central parameter for tumor characterization and an important element of the pathological assessment., (Copyright © 2015. Published by Elsevier Ltd.)

Purpose: Scoring proliferation through Ki67 immunohistochemistry is an important component in predicting therapy response to chemotherapy in patients with breast cancer. However, recent studies have cast doubt on the reliability of "visual" Ki67 scoring in the multicenter setting, particularly in the lower, yet clinically important, proliferation range. Therefore, an accurate and standardized Ki67 scoring is pivotal both in routine diagnostics and larger multicenter studies., Experimental Design: We validated a novel fully automated Ki67 scoring approach that relies on only minimal a priori knowledge on cell properties and requires no training data for calibration. We applied our approach to 1,082 breast cancer samples from the neoadjuvant GeparTrio trial and compared the performance of automated and manual Ki67 scoring., Results: The three groups of autoKi67 as defined by low (≤ 15%), medium (15.1%-35%), and high (>35%) automated scores showed pCR rates of 5.8%, 16.9%, and 29.5%, respectively. AutoKi67 was significantly linked to prognosis with overall and progression-free survival P values P(OS) < 0.0001 and P(PFS) < 0.0002, compared with P(OS) < 0.0005 and P(PFS) < 0.0001 for manual Ki67 scoring. Moreover, automated Ki67 scoring was an independent prognosticator in the multivariate analysis with P(OS) = 0.002, P(PFS) = 0.009 (autoKi67) versus P(OS) = 0.007, PPFS = 0.004 (manual Ki67)., Conclusions: The computer-assisted Ki67 scoring approach presented here offers a standardized means of tumor cell proliferation assessment in breast cancer that correlated with clinical endpoints and is deployable in routine diagnostics. It may thus help to solve recently reported reliability concerns in Ki67 diagnostics., (©2014 American Association for Cancer Research.)

Purpose: Modulation of immunologic interactions in cancer tissue is a promising therapeutic strategy. To investigate the immunogenicity of human epidermal growth factor receptor 2 (HER2) -positive and triple-negative (TN) breast cancers (BCs), we evaluated tumor-infiltrating lymphocytes (TILs) and immunologically relevant genes in the neoadjuvant GeparSixto trial., Patients and Methods: GeparSixto investigated the effect of adding carboplatin (Cb) to an anthracycline-plus-taxane combination (PM) on pathologic complete response (pCR). A total of 580 tumors were evaluated before random assignment for stromal TILs and lymphocyte-predominant BC (LPBC). mRNA expression of immune-activating (CXCL9, CCL5, CD8A, CD80, CXCL13, IGKC, CD21) as well as immunosuppressive factors (IDO1, PD-1, PD-L1, CTLA4, FOXP3) was measured in 481 tumors., Results: Increased levels of stromal TILs predicted pCR in univariable (P < .001) and multivariable analyses (P < .001). pCR rate was 59.9% in LPBC and 33.8% for non-LPBC (P < .001). pCR rates ≥ 75% were observed in patients with LPBC tumors treated with PMCb, with a significant test for interaction with therapy in the complete (P = .002) and HER2-positive (P = .006), but not the TNBC, cohorts. Hierarchic clustering of mRNA markers revealed three immune subtypes with different pCR rates (P < .001). All 12 immune mRNA markers were predictive for increased pCR. The highest odds ratios (ORs) were observed for PD-L1 (OR, 1.57; 95% CI, 1.34 to 1.86; P < .001) and CCL5 (OR, 1.41; 95% CI, 1.23 to 1.62; P < .001)., Conclusion: Immunologic factors were highly significant predictors of therapy response in the GeparSixto trial, particularly in patients treated with Cb. After further standardization, they could be included in histopathologic assessment of BC., (© 2014 by American Society of Clinical Oncology.)