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Date Title Author Journal
2015 Optimizing HER2 assessment in breast cancer: application of automated image analysis.

Oncotopix Clinical, Publicly Sharable

In breast cancer, analysis of HER2 expression is pivotal for treatment decision. This study aimed at comparing digital, automated image analysis with manual reading using the HER2-CONNECT algorithm (Visiopharm) in order to minimize the number of equivocal 2+ scores and the need for reflex fluorescence in situ hybridization (FISH) analysis. Consecutive samples from 462 patients were included. Tissue micro arrays (TMAs) were routinely manufactured including two 2 mm cores from each patient, and each core was assessed in order to ensure the presence of invasive carcinoma. Immunohistochemical staining (IHC) was performed with Roche/Ventanaś HER2 ready-to-use kit. TMAs were scanned in a Zeiss Axio Z1 scanner, and one batch analysis of the HER2-CONNECT algorithm including all core samples was run using Visiopharmś cloud-based software. The automated reading was compared to conventional manual assessment of HER2 protein expression, together with FISH analysis of HER2 gene amplification for borderline (2+) protein expression samples. Compared to FISH analysis, manual assessment of the HER2 protein expression demonstrated a sensitivity of 85.8% and a specificity of 86.0% with 14.0% equivocal samples. With HER2-CONNECT, sensitivity increased to 100 % and specificity to 95.5% with less than 4.5% equivocal. Total agreement when comparing HER2-CONNECT with manual IHC assessment supplemented by FISH for borderline (2+) cases was 93.6%. Application of automated image analysis for HER2 protein expression instead of manual assessment decreases the need for supplementary FISH testing by 68%. In the routine diagnostic setting, this would have significant impact on cost reduction and turn-around time.

Henrik Holten-Rossing, Maj-Lis Møller Talman, Martin Kristensson, Ben Vainer

Henrik Holten-Rossing, Maj-Lis Møller Talman, Martin Kristensson... et al. Breast Cancer Res Treat
2017 Application of automated image analysis reduces the workload of manual screening of sentinel Lymph node biopsies in breast cancer

Oncotopix Clinical, Publicly Sharable

Introduction Breast cancer is one of the most common cancer diseases in women with more than 1.67 million cases diagnosed worldwide each year. In breast cancer, the sentinel lymph node (SLN) pinpoints the first lymph node(s) into which the tumor spreads and it is usually located in the ipsilateral axilla. In patients with no clinical signs of metastatic disease in the axilla, a SLN biopsy (SLNB) is performed. Assessment of metastases in the SLNB is done in a conventional microscope by manually observing a metastasis and measuring its size and/or counting the number of tumor cells. This is done essentially to categorize the type of metastases as macrometastases, micrometastases or isolated tumor cells, which is used to determine which treatment the breast cancer patient will benefit mostly from. The aim of this study was to evaluate whether digital image analysis can be applied as a screening tool for SNLB assessment without compromising the diagnostic accuracy. Materials and methods Consecutive SLNB from 135 patients with localized breast cancer receiving surgery in the period of February to August 2015 were collected and included in this study. Of the 135 patients, 35 were received at Dept. of Pathology, Rigshospitalet, Copenhagen University Hospital, 50 at Dept. of Pathology, Zealand University Hospital, and 50 at Dept. of Pathology, Odense University Hospital. Formalin-fixed and paraffin-embedded tissue sections were analyzed by immunohistochemistry (IHC) using the BenchMark ULTRA Ventana platform. Rigshospitalet used a mixture of cytokeratin CK7 and CK19, Zealand University Hospital used pancytokeratin AE1/AE3 and Odense used pancytokeratin CAM5.2 for detection of epithelial tumor cells. Slides were stained locally. SLNB sections were assessed in a conventional microscope according to national guidelines for SLNB in breast cancer patients. The IHC stained sections were scanned by a Hamamatsu NanoZoomer-XR digital whole slide scanner and the images were analyzed by Visiopharm's software using a custommade algorithm for SLNB in breast cancer. The algorithm was optimized to the cytokeratin antibodies and the local laboratory conditions, based on staining intensity and background staining. Results Conventional microscopy was used as golden standard for assessment of positive tumor cells and compared with digital image analysis (DIA). The algorithm demonstrated a sensitivity of 100% (i.e. no false negative slides were observed), including 67.2%, 19.2% and 56.1% of the slides from the three pathology departments being negative, respectively. This means that on average, the workload could have been decreased by 58.2% by using the digital SLNB algorithm as a screening tool. Discussion and conclusion The SLNB algorithm demonstrated a sensitivity of 100% regardless of the antibody used for IHC and the staining protocol. No false negative slides were observed, which proves that the SLNB algorithm is an ideal screening tool for selecting those slides not necessary for a pathologist to see. Implementation of automated digital image analysis of SLNB in breast cancer would decrease the workload in this context for examining pathologists by almost 60%. This article is protected by copyright. All rights reserved.

Henrik Holten-Rossing, Maj-Lis Møller Talman, Anne Marie Bak Jylling, Anne-Vibeke Laenkholm, Martin Kristensson, Ben Vainer

Henrik Holten-Rossing, Maj-Lis Møller Talman, Anne Marie Bak Jylling... et al. Histopathology
2017 Digital image analysis of her2 immunostained gastric and gastroesophageal junction adenocarcinomas

Oncotopix, Oncotopix Discovery, Publicly Sharable

Manual assessment of human epidermal growth factor receptor 2 (HER2) protein expression by immunohistochemistry (IHC) in gastric and gastroesophageal junction (GGEJ) adenocarcinomas is prone to interobserver variability and hampered by tumor heterogeneity and different scoring criteria. Equivocal cases are frequent, requiring additional in situ hybridization analysis. This study aimed to evaluate the accuracy of digital image analysis for the assessment of HER2 protein expression. In total, 110 GGEJ adenocarcinomas were included in tissue microarrays with 3 tissue cores per case. Two immunoassays, PATHWAY and HercepTest, and fluorescent in situ hybridization analysis were performed. The Visiopharm HER2-CONNECT Analysis Protocol Package was applied through the ONCOtopix digital image analysis software platform. HER2 membrane connectivity, calculated by the Analysis Protocol Package, was converted to standard IHC scores applying predetermined cutoff values for breast carcinoma as well as novel cutoff values. Cases with excessive cytoplasmic staining as well as HER2 amplified IHC negative cases were excluded from analysis. Applying HER2-CONNECT with connectivity cutoff values established for breast carcinoma resulted in 72.7% sensitivity and 100% specificity for the identification of HER2 positive gene amplified cases. By application of new cutoff values, the sensitivity increased to 100% without decreased specificity. With the new cutoff values, a 36% to 50% reduction of IHC equivocal cases was obtained. In conclusion, HER2-CONNECT with adjusted cutoff values seem to be an effective tool for standardized assessment of HER2 protein expression in GGEJ adenocarcinomas, decreasing the need for in situ hybridization analyzes.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Sofie L. Nielsen, Soren Nielsen, Mogens Vyberg

Sofie L. Nielsen, Soren Nielsen, Mogens Vyberg Applied Immunohistochemistry and Molecular Morphology
2018 PD1hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

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Background: Abnormally sustained immune reactions drive B-cell proliferation in some cases of marginal zone lymphoma but the CD4+ T-cell subsets, which are likely to contribute to the B-cell responses in the tumour microenvironment, are not well characterised and neither has the spatial distribution of the different subsets in involved lymph nodes been investigated. Methods: Employing a workflow of multiplex semi-automated immunohistochemistry combined with image processing we investigated association between infiltrating T-cells and proliferating lymphoma B-cells. Results: Both total numbers of activating follicular helper (Tfh) cells (defined by high expression of PD1) and suppressive regulatory (Treg) T-cells (defined by FOXP3+ expression) and the Tfh:Treg ratio, assessed over relatively large areas of tissue, varied among cases of marginal zone lymphoma. We determined spatial distribution and demonstrated that PD1hi cells showed significantly more clustering than did FOXP3+. To investigate the association of infiltrating T-cells with lymphoma B-cells we employed Pearson correlation and Morisita-Horn index, statistical measures of interaction. We demonstrated that PD1hi cells were associated with proliferating B-cells and confirmed this by nearest neighbour analysis. Conclusions: The unexpected architectural complexity of T-cell infiltration in marginal zone lymphoma, revealed in this study, further supports a key role for Tfh cells in driving proliferation of lymphoma B-cells. We demonstrate the feasibility of digital analysis of spatial architecture of T-cells within marginal zone lymphoma and future studies will be needed to determine the clinical importance of these observations.

Katherine Wickenden, Nadia Nawaz, Sami Mamand, Deevia Kotecha, Amy L. Wilson, Simon D. Wagner, Matthew J. Ahearne

Katherine Wickenden, Nadia Nawaz, Sami Mamand... et al. Diagnostic Pathology
2018 Clinical significance of CD73 in triple-negative breast cancer: multiplex analysis of a phase III clinical trial

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Background: CD73 is an ecto-enzyme that promotes tumor immune escape through the production of immunosuppressive extracellular adenosine in the tumor microenvironment. Several CD73 inhibitors and adenosine receptor antagonists are being evaluated in phase I clinical trials. Patients and methods: Full-face sections from formalin-fixed paraffin-embedded primary breast tumors from 122 samples of triple-negative breast cancer (TNBC) from the BIG 02-98 adjuvant phase III clinical trial were included in our analysis. Using multiplex immunofluorescence and image analysis, we assessed CD73 protein expression on tumor cells, tumor-infiltrating leukocytes and stromal cells. We investigated the associations between CD73 protein expression with disease-free survival (DFS), overall survival (OS) and the extent of tumor immune infiltration. Results: Our results demonstrated that high levels of CD73 expression on epithelial tumor cells were significantly associated with reduced DFS, OS and negatively correlated with tumor immune infiltration (Spearman's R=-0.50, P < 0.0001). Patients with high levels of CD73 and low levels of tumor-infiltrating leukocytes had the worse clinical outcome. Conclusions: Taken together, our study provides further support that CD73 expression is associated with a poor prognosis and reduced anti-tumor immunity in human TNBC and that targeting CD73 could be a promising strategy to reprogram the tumor microenvironment in this BC subtype.

L. Buisseret, S. Pommey, B. Allard, S. Garaud, M. Bergeron, I. Cousineau, L. Ameye, Y. Bareche, M. Paesmans, J. P.A. Crown, A. Di Leo, S. Loi, M. Piccart-Gebhart, K. Willard-Gallo, C. Sotiriou, John Stagg

L. Buisseret, S. Pommey, B. Allard... et al. Annals of Oncology
2019 Early Treatment with Empagliflozin and GABA Improves β -Cell Mass and Glucose Tolerance in Streptozotocin-Treated Mice

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While the autoimmune character of T1D (type 1 diabetes) is being challenged, it is currently recognized that inflammation plays a key role in its development. We hypothesized that glucotoxicity could contribute to β-cell mass destruction through participation in islet inflammation. We evaluated the potential of empagliflozin (EMPA) and GABA (gamma-aminobutyric acid) to protect β-cell mass against glucotoxicity and to increase β-cell mass after diagnosis of T1D. Empagliflozin is a SGLT2 (sodium-dependent glucose cotransporter) inhibitor which thereby blocks glucose recapture by the kidney and promotes glucose excretion in urine. GABA is an inhibitory neurotransmitter, which stimulates α-to-β cell transdifferentiation. In streptozotocin-treated mice, empagliflozin and/or GABA were delivered for a period of five days or three weeks. As compared to untreated T1D mice, EMPA-treated T1D mice had decreased FFA (free fatty acid) levels and improved glucose homeostasis. EMPA-treated T1D mice had higher islet density, with preserved architecture, compared to T1D mice, and EMPA-treated T1D mice also differed from T1D mice by the total absence of immune cell infiltration within islets. Islets from EMPA-treated mice were also less subjected to ER (endoplasmic reticulum) stress and inflammation, as shown by qPCR analysis. Glucose homeostasis parameters and islet area/pancreas area ratio improved, as compared to diabetic controls, when T1D mice were treated for three weeks with GABA and EMPA. T1D EMPA+GABA mice had higher glucagon levels than T1D mice, without modifications of glucagon area/islet area ratios. In conclusion, empagliflozin and GABA, used in monotherapy in streptozotocin-induced diabetic mice, have positive effects on β-cell mass preservation or proliferation through an indirect effect on islet cell inflammation and ER stress. Further research is mandatory to evaluate whether empagliflozin and GABA may be a potential therapeutic target for the protection of β-cell mass after new-onset T1D.

Caroline Daems, Sophie Welsch, Hasnae Boughaleb, Juliette Vanderroost, Annie Robert, Etienne Sokal, Philippe A. Lysy

Caroline Daems, Sophie Welsch, Hasnae Boughaleb... et al. Journal of Diabetes Research
2019 Immune-enrichment of non-small cell lung cancer baseline biopsies for multiplex profiling define prognostic immune checkpoint combinations for patient stratification

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Background Permanence of front-line management of lung cancer by immunotherapies requires predictive companion diagnostics identifying immune-checkpoints at baseline, challenged by the size and heterogeneity of biopsy specimens. Methods An innovative, tumor heterogeneity reducing, immune-enriched tissue microarray was constructed from baseline biopsies, and multiplex immunofluorescence was used to profile 25 immune-checkpoints and immune-antigens. Results Multiple immune-checkpoints were ranked, correlated with antigen presenting and cytotoxic effector lymphocyte activity, and were reduced with advancing disease. Immune-checkpoint combinations on TILs were associated with a marked survival advantage. Conserved combinations validated on more than 11,000 lung, breast, gastric and ovarian cancer patients demonstrate the feasibility of pan-cancer companion diagnostics. Conclusions In this hypothesis-generating study, deepening our understanding of immune-checkpoint biology, comprehensive protein-protein interaction and pathway mapping revealed that redundant immune-checkpoint interactors associate with positive outcomes, providing new avenues for the deciphering of molecular mechanisms behind effects of immunotherapeutic agents targeting immune-checkpoints analyzed. Derek Bergeron and Amira Ben Amor contributed equally to this work. * Abbreviations: ACT : Adoptive cell transfer ADC : Adenocarcinoma APC : Antigen presenting cells CD3-ICP : ICP expressed on CD3+ TIL CDx : Companion diagnostics CTLA-4 : Cytotoxic T lymphocyte-associated antigen 4 EGA : European Genome-phenome Archive GEO : Gene Expression Omnibus GZMB : Granzyme B HEV : High endothelial venules HLA-DR : Human leukocyte antigen-DR ICP : Immune checkpoint IF : Immunofluorescence IFN-γ : Interferon-gamma IHC : Immunohistochemistry IIC : Infiltrating immune cells IID : Integrated Interaction Database IM : Immunoscore K-M : Kaplan-Meier survival analysis LUAD : Lung adenocarcinoma LUSC : Lung squamous cell carcinoma MFI : Mean fluorescence intensity MP-IF : Multiplex immunofluorescence NAViGaTOR : Network Analysis, Visualization and Graphing, TORonto NK cells : Natural killer cells NSCLC : Non-small cell lung carcinoma OS : Overall survival pathDIP : Pathway Data Integration Portal PD-1 : Programmed death-1 PD-L1 and PD-L2 : Programmed death-1 ligands 1 and 2 PNAd : Peripheral node addressin SCC : Squamous-cell carcinoma TAM : Tumor-associated macrophages TCGA : The Cancer Genome Atlas TCR : T cell receptor TIL : Tumor infiltrating lymphocytes TMA : Tissue microarray TNM : Tumor, node, metastases

Anne Monette, Derek Bergeron, Amira Ben Amor, Liliane Meunier, Christine Caron, Anne Marie Mes-Masson, Nidhameddine Kchir, Kamel Hamzaoui, Igor Jurisica, Réjean Lapointe

Anne Monette, Derek Bergeron, Amira Ben Amor... et al. Journal for ImmunoTherapy of Cancer
2020 Validation of an Automated Quantitative Digital Pathology Approach for Scoring TMEM, a Prognostic Biomarker for Metastasis

Oncotopix Discovery, Publicly Sharable

Metastasis causes ~90% of breast cancer mortality. However, standard prognostic tests based mostly on proliferation genes do not measure metastatic potential. Tumor MicroEnvironment of Metastasis (TMEM), an immunohistochemical biomarker for doorways on blood vessels that support tumor cell dissemination is prognostic for metastatic outcome in breast cancer patients. Studies quantifying TMEM doorways have involved manual scoring by pathologists utilizing static digital microscopy: a labor-intensive process unsuitable for use in clinical practice. We report here a validation study evaluating a new quantitative digital pathology (QDP) tool (TMEM-DP) for identification and quantification of TMEM doorways that closely mimics pathologists’ workflow and reduces pathologists’ variability to levels suitable for use in a clinical setting. Blinded to outcome, QDP was applied to a nested case-control study consisting of 259 matched case-control pairs. Sixty subjects of these were manually scored by five pathologists, digitally recorded using whole slide imaging (WSI), and then used for algorithm development and optimization. Validation was performed on the remainder of the cohort. TMEM-DP shows excellent reproducibility and concordance and reduces pathologist time from ~60 min to ~5 min per case. Concordance between manual scoring and TMEM-DP was found to be >0.79. These results show that TMEM-DP is capable of accurately identifying and scoring TMEM doorways (also known as MetaSite score) equivalent to pathologists.

David Entenberg, Maja H. Oktay, Timothy D’alfonso, Paula S. Ginter, Brian D. Robinson, Xiaonan Xue, Thomas E. Rohan, Joseph A. Sparano, Joan G. Jones, John S. Condeelis

David Entenberg, Maja H. Oktay, Timothy D’alfonso... et al. Cancers
2020 PUMA and NOXA Expression in Tumor-Associated Benign Prostatic Epithelial Cells Are Predictive of Prostate Cancer Biochemical Recurrence

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Background: Given that treatment decisions in prostate cancer (PC) are often based on risk, there remains a need to find clinically relevant prognostic biomarkers to stratify PC patients. We evaluated PUMA and NOXA expression in benign and tumor regions of the prostate using immunofluorescence techniques and determined their prognostic significance in PC. Methods: PUMA and NOXA expression levels were quantified on six tissue microarrays (TMAs) generated from radical prostatectomy samples (n = 285). TMAs were constructed using two cores of benign tissue and two cores of tumor tissue from each patient. Association between biomarker expression and biochemical recurrence (BCR) at 3 years was established using log-rank (LR) and multivariate Cox regression analyses. Results: Kaplan–Meier analysis showed a significant association between BCR and extreme levels (low or high) of PUMA expression in benign epithelial cells (LR = 8.831, p = 0.003). Further analysis revealed a significant association between high NOXA expression in benign epithelial cells and BCR (LR = 14.854, p < 0.001). The combination of extreme PUMA and high NOXA expression identified patients with the highest risk of BCR (LR = 16.778, p < 0.001) in Kaplan–Meier and in a multivariate Cox regression analyses (HR: 2.935 (1.645–5.236), p < 0.001). Conclusions: The combination of PUMA and NOXA protein expression in benign epithelial cells was predictive of recurrence following radical prostatectomy and was independent of PSA at diagnosis, Gleason score and pathologic stage.

Sylvie Clairefond, Benjamin Péant, Véronique Ouellet, Véronique Barrès, Zhe Tian, Dominique Trudel, Pierre I. Karakiewicz, Anne Marie Mes-Masson, Fred Saad

Sylvie Clairefond, Benjamin Péant, Véronique Ouellet... et al. Cancers 2020, Vol. 12, Page 3187
2020 Detailed Molecular and Immune Marker Profiling of Archival Prostate Cancer Samples Reveals an Inverse Association between TMPRSS2:ERG Fusion Status and Immune Cell Infiltration

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Prostate cancer is a significant global health issue, and limitations to current patient management pathways often result in overtreatment or undertreatment. New ways to stratify patients are urgently needed. We conducted a feasibility study of such novel assessments, looking for associations between genomic changes and lymphocyte infiltration. An innovative workflow using an in-house targeted sequencing panel, immune cell profiling using an image analysis pipeline, RNA sequencing, and exome sequencing in select cases was tested. Gene fusions were profiled by RNA sequencing in 27 of 27 cases, and a significantly higher tumor-infiltrating lymphocyte (TIL) count was noted in tumors without a TMPRSS2:ERG fusion compared with those with the fusion (P = 0.01). Although this finding was not replicated in a larger validation set (n = 436) of The Cancer Genome Atlas images, there was a trend in the same direction. Differential expression analysis of TIL-high and TIL-low tumors revealed the enrichment of both innate and adaptive immune response pathways. Mutations in mismatch repair genes (MLH1 and MSH6 mutations in 1 of 27 cases) were identified. We describe a potential immune escape mechanism in TMPRSS2:ERG fusion-positive tumors. Detailed profiling, as shown herein, can provide novel insights into tumor biology. Likely differences with findings with other cohorts are related to methods used to define region of interest, but this warrants further study in a larger cohort.

Srinivasa R. Rao, Nasullah K. Alham, Elysia Upton, Stacey McIntyre, Richard J. Bryant, Lucia Cerundolo, Emma Bowes, Stephanie Jones, Molly Browne, Ian Mills, Alastair Lamb, Ian Tomlinson, David Wedge, Lisa Browning, Korsuk Sirinukunwattana, Claire Palles, Freddie C. Hamdy, Jens Rittscher, Clare Verrill

Srinivasa R. Rao, Nasullah K. Alham, Elysia Upton... et al. The Journal of Molecular Diagnostics
2020 A Novel Approach for Quantifying Cancer Cells Showing Hybrid Epithelial/Mesenchymal States in Large Series of Tissue Samples: Towards a New Prognostic Marker

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In cancer biology, epithelial-to-mesenchymal transition (EMT) is associated with tumorigenesis, stemness, invasion, metastasis, and resistance to therapy. Evidence of co-expression of epithelial and mesenchymal markers suggests that EMT should be a stepwise process with distinct intermediate states rather than a binary switch. In the present study, we propose a morphological approach that enables the detection and quantification of cancer cells with hybrid E/M states, i.e., which combine partially epithelial (E) and partially mesenchymal (M) states. This approach is based on a sequential immunohistochemistry technique performed on the same tissue section, the digitization of whole slides, and image processing. The aim is to extract quantitative indicators able to quantify the presence of hybrid E/M states in large series of human cancer samples and to analyze their relationship with cancer aggressiveness. As a proof of concept, we applied our methodology to a series of about a hundred urothelial carcinomas and demonstrated that the presence of cancer cells with hybrid E/M phenotypes at the time of diagnosis is strongly associated with a poor prognostic value, independently of standard clinicopathological features. Although validation on a larger case series and other cancer types is required, our data support the hybrid E/M score as a promising prognostic biomarker for carcinoma patients.

Louis Godin, Cédric Balsat, Yves Rémi Van Eycke, Justine Allard, Claire Royer, Myriam Remmelink, Ievgenia Pastushenko, Nicky D’Haene, Cédric Blanpain, Isabelle Salmon, Sandrine Rorive, Christine Decaestecker

Louis Godin, Cédric Balsat, Yves Rémi Van Eycke... et al. Cancers 2020, Vol. 12, Page 906
2020 Prevalence of PD-L1 expression is associated with EMAST, density of peritumoral T-cells and recurrence-free survival in operable non-metastatic colorectal cancer

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Introduction: Microsatellite instability (MSI) predict response to anti-PD1 immunotherapy in colorectal cancer (CRC). CRCs with MSI have higher infiltration of immune cells related to a better survival. Elevated Microsatellite Alterations at Tetranucleotides (EMAST) is a form of MSI but its association with PD-L1 expression and immune-cell infiltration is not known. Methods: A consecutive, observational cohort of patients undergoing surgery for CRC. EMAST and clinicopathological characteristics were investigated against PD-L1, as well as CD3 and CD8 expression in the invasive margin or tumour centre (Immunoscore). Difference in survival between groups was assessed by log rank test. Results: A total of 149 stage I–III CRCs patients, with a median follow up of 60.1 months. Patients with PD-L1+ tumours (7%) were older (median 79 vs 71 years, p = 0.045) and had EMAST+ cancers (OR 10.7, 95% CI 2.2–51.4, p = 0.001). Recurrence-free survival was longer in cancers with PD-L1+ immune cells (HR 0.35, 95% CI 0.16–0.76, p = 0.008, independent of EMAST) and high Immunoscore (HR 0.10, 95% CI 0.01–0.72, p = 0.022). Patients expressing PD-L1 in immune cells had longer disease-specific survival (HR 0.28, 95% CI 0.10–0.77, p = 0.014). Conclusions: Higher Immunoscore (CD3/CD8 cells) and expression of tumour PD-L1 is found in CRCs with EMAST. Lymphocytic infiltrate and peritumoral PD-L1 expression have prognostic value in CRC.

Martin M. Watson, Dordi Lea, Einar Gudlaugsson, Ivar Skaland, Hanne R. Hagland, Kjetil Søreide

Martin M. Watson, Dordi Lea, Einar Gudlaugsson... et al. Cancer Immunology, Immunotherapy
2020 Dual Targeting of Mesothelin and CD19 with Chimeric Antigen Receptor-Modified T Cells in Patients with Metastatic Pancreatic Cancer

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B cells infiltrate pancreatic ductal adenocarcinoma (PDAC) and in preclinical cancer models, can suppress T cell immunosurveillance in cancer. Here, we conducted a pilot study to assess the safety and feasibility of administering lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous T cells redirected against mesothelin to target tumor cells along with CART cells redirected against CD19 to deplete B cells. Both CARs contained 4-1BB and CD3ζ signaling domains. Three patients with chemotherapy-refractory PDAC received 1.5 g/m2 cyclophosphamide prior to separate infusions of lentiviral-transduced T cells engineered to express chimeric anti-mesothelin immunoreceptor SS1 (CART-Meso, 3 × 107/m2) and chimeric anti-CD19 immunoreceptor (CART-19, 3 × 107/m2). Treatment was well tolerated without dose-limiting toxicities. Best response was stable disease (1 of 3 patients). CART-19 (compared to CART-Meso) cells showed the greatest expansion in the blood, although persistence was transient. B cells were successfully depleted in all subjects, became undetectable by 7–10 days post-infusion, and remained undetectable for at least 28 days. Together, concomitant delivery of CART-Meso and CART-19 cells in patients with PDAC is safe. CART-19 cells deplete normal B cells but at the dose tested in these 3 subjects did not improve CART-Meso cell persistence.

Andrew H. Ko, Alexander C. Jordan, Evan Tooker, Simon F. Lacey, Renee B. Chang, Yan Li, Alan P. Venook, Margaret Tempero, Lloyd Damon, Lawrence Fong, Mark H. O'Hara, Bruce L. Levine, J. Joseph Melenhorst, Gabriela Plesa, Carl H. June, Gregory L. Beatty

Andrew H. Ko, Alexander C. Jordan, Evan Tooker... et al. Molecular Therapy
2020 Reduced Protumorigenic Tumor-Associated Macrophages With Statin Use in Premalignant Human Lung Adenocarcinoma

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Background: Statins have anticancer properties by acting as competitive inhibitors of the mevalonate pathway. They also have anti-inflammatory activity, but their role in suppressing inflammation in a cancer context has not been investigated to date. Methods: We have analyzed the relationship between statin use and tumor-Associated macrophages (TAMs) in a cohort of 262 surgically resected primary human lung adenocarcinomas. TAMs were evaluated by multiplex immunostaining for the CD68 pan-TAM marker and the CD163 protumorigenic TAM marker followed by digital slide scanning and partially automated quantitation. Links between statin use and tumor stage, virulence, and cancer-specific survival were also investigated in a wider cohort of 958 lung adenocarcinoma cases. All statistical tests were two-sided. Results: We found a statin dose-dependent reduction in protumorigenic TAMs (CD68 CD163 ) in both stromal (P .021) and parenchymal (P .003) compartments within regions of in situ tumor growth, but this association was lost in invasive regions. No statistically significant relationship between statin use and tumor stage was observed, but there was a statin dose-dependent shift towards lower histological grade as assessed by growth pattern (P .028). However, statin use was a predictor of slightly worse cancer-specific survival (P .032), even after accounting for prognostic variables in a multivariable Cox proportional hazards survival model (hazard ratio 1.38, 95% confidence interval 1.04 to 1.84). Conclusions: Statin use is associated with reduced numbers of protumorigenic TAMs within preinvasive lung adenocarcinoma and is related to reduced tumor invasiveness, suggesting a chemo-preventive effect in early tumor development. However, invasive disease is resistant to these effects, and no beneficial relationship between statin use and patient outcome is observed.

Esraa Al Dujaily, Juvenal Baena, Madhumita Das, Marco Sereno, Claire Smith, Tamihiro Kamata, Leah Officer, Catrin Pritchard, John Le Quesne

Esraa Al Dujaily, Juvenal Baena, Madhumita Das... et al. JNCI Cancer Spectrum
2020 Molecular correlates of cisplatin-based chemotherapy response in muscle invasive bladder cancer by integrated multi-omics analysis

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Overtreatment with cisplatin-based chemotherapy is a major issue in the management of muscle-invasive bladder cancer (MIBC), and currently none of the reported biomarkers for predicting response have been implemented in the clinic. Here we perform a comprehensive multi-omics analysis (genomics, transcriptomics, epigenomics and proteomics) of 300 MIBC patients treated with chemotherapy (neoadjuvant or first-line) to identify molecular changes associated with treatment response. DNA-based associations with response converge on genomic instability driven by a high number of chromosomal alterations, indels, signature 5 mutations and/or BRCA2 mutations. Expression data identifies the basal/squamous gene expression subtype to be associated with poor response. Immune cell infiltration and high PD-1 protein expression are associated with treatment response. Through integration of genomic and transcriptomic data, we demonstrate patient stratification to groups of low and high likelihood of cisplatin-based response. This could pave the way for future patient selection following validation in prospective clinical trials. There are currently only a few biomarkers to predict the response of muscle invasive bladder cancer to therapy. Here, the authors analyse 300 tumors using exome and RNA sequencing and find that tumors with a high degree of genomic instability and a non-basal/squamous gene expression subtype are most likely to respond to treatment.

Ann Taber, Emil Christensen, Philippe Lamy, Iver Nordentoft, Frederik Prip, Sia Viborg Lindskrog, Karin Birkenkamp-Demtröder, Trine Line Hauge Okholm, Michael Knudsen, Jakob Skou Pedersen, Torben Steiniche, Mads Agerbæk, Jørgen Bjerggaard Jensen, Lars Dyrskjøt

Ann Taber, Emil Christensen, Philippe Lamy... et al. Nature Communications 2020 11:1
2020 Analysis of spatial relationships between CD8 and FoxP3 cells using digital imaging in head and neck squamous cell carcinoma

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Purpose/Objective(s): T cell-mediated anti-tumor immune responses are gated in part by the relative abundance of cytotoxic T cells (Teffs) and regulatory (Tregs) in the tumor microenvironment (TME) whereby Treg may impair the efficacy of Teffs. Tregs can affect Teff function by direct cell-to-cell contact or by secretion of soluble mediators, consistent with the hypothesis that proximity of Tregs to Teff within tumor tissues may have prognostic significance. Here we used a novel chromogenic multiplex assay to investigate the spatial relationship of these functionally diverse T cell subsets in HPV positive and HPV negative head and neck squamous cell carcinomas (HNSCCs). Materials/Methods: Twenty head and neck cancer patients with primary tumors of the oropharynx or oral cavity were included in this study of which 10 were HPV positive and 10 HPV negative. Formalin-fixed tissues were stained with antibodies detecting CD8 (Teffs) and FoxP3 (Tregs). At least three different fields at the leading edge of tumor and adjacent stroma were evaluated. Stained tissue sections were digitally scanned at 40x magnification utilizing an iScan HT (Roche, Switzerland) whole-slide imaging scanner. Visiopharm (Visiopharm, Denmark) image analysis software was utilized by a pathologist to analyze the digitized slide images. Result(s): HPV positive tumor tissues contained >3-fold higher numbers of both CD8 and FoxP3 expressing cells when compared to HPV negative tumors whereas the ratios between these cell subsets (CD8/FoxP3) were comparable. The differential density of T-cells in the sampled areas was reflected in a significantly shorter mean distance between CD8+ and FoxP3+ cells in HPV positive (29.92 mum) tumors compared to that of HPV negative (52.56 mum) tumors (p=0.0045, 95% CI: 8.17-37.11). The mean frequency of distances <30mum measured in HPV positive tumors was 98.46 contrasted by 39.44 in HPV negative tumors; this difference was statistically different (p=0.0194). The mean frequency of distances >30mum measured in HPV positive tumors was 37.02 and in HPV negative tumors 36.06. Conclusion(s): Consistent with earlier reports, HPV positive lesions contained more CD8 and FoxP3 expressing T cells than HPV negative lesions. This difference was reflected in statistically significantly closer proximity of Teffs and Tregs in HPV positive lesions, potentially enhancing functional interaction of these T cell subsets in the tumor microenvironment of HPV positive lesions. The implications of these observations for prognosis and response to immunotherapeutic intervention remain to be investigated.Copyright © 2020

M. Stewart, R. Stapp, D. Amin, R. Ganti, U. Nwagu, T. Richa, M. Crippen, R. Zinner, A. Luginbuhl, J. Johnson, V. Bar-Ad, U. Martinez-Outschoorn, C. Solomides, U. Rodeck, J.M. Curry

M. Stewart, R. Stapp, D. Amin... et al. International Journal of Radiation Oncology*Biology*Physics
2020 Visualization, quantification, and mapping of immune cell populations in the tumor microenvironment

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The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Manuel Flores Molina, Thomas Fabre, Aurélie Cleret-Buhot, Geneviève Soucy, Liliane Meunier, Mohamed N. Abdelnabi, Nicolas Belforte, Simon Turcotte, Naglaa H. Shoukry

Manuel Flores Molina, Thomas Fabre, Aurélie Cleret-Buhot... et al. Journal of Visualized Experiments
2020 Multispectral Imaging Enables Characterization of Intrahepatic Macrophages in Patients With Chronic Liver Disease

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Intrahepatic macrophages influence the composition of the microenvironment, host immune response to liver injury, and development of fibrosis. Compared with stellate cells, the role of macrophages in the development of fibrosis remains unclear. Multispectral imaging allows detection of multiple markers in situ in human formalin-fixed, paraffin-embedded tissue. This cutting-edge technology is ideal for analyzing human liver tissues, as it allows spectral unmixing of fluorophore signals, subtraction of auto-fluorescence, and preservation of hepatic architecture. We analyzed five different antibodies commonly observed on macrophage populations (CD68, MAC387, CD163, CD14, and CD16). After optimization of the monoplex stains and development of a Spectral Library, we combined all of the antibodies into a multiplex protocol and used them to stain biopsies collected from representative patients with chronic liver diseases, including chronic hepatitis C, nonalcoholic steatohepatitis, and autoimmune hepatitis. Various imaging modalities were tested, including cell phenotyping, tissue segmentation, t-distributed stochastic neighbor embedding plots, and phenotype matrices that facilitated comparison and visualization of the identified macrophage and other cellular profiles. We then tested the feasibility of this platform to analyze numerous regions of interest from liver biopsies with multiple patients per group, using batch analysis algorithms. Five populations showed significant differences between patients positive for hepatitis C virus with advanced fibrosis when compared with controls. Three of these were significantly increased in patients with advanced fibrosis when compared to controls, and these included CD163+CD16+, CD68+, and CD68+MAC387+. Conclusion: Spectral imaging microscopy is a powerful tool that enables in situ analysis of macrophages and other cells in human liver biopsies and may lead to more personalized therapeutic approaches in the future.

Omar A. Saldarriaga, Benjamin Freiberg, Santhoshi Krishnan, Arvind Rao, Jared Burks, Adam L. Booth, Bradley Dye, Netanya Utay, Monique Ferguson, Abdellah Akil, Minkyung Yi, Laura Beretta, Heather L. Stevenson

Omar A. Saldarriaga, Benjamin Freiberg, Santhoshi Krishnan... et al. Hepatology Communications
2020 A validation study of human epidermal growth factor receptor 2 immunohistochemistry digital imaging analysis and its correlation with human epidermal growth factor receptor 2 fluorescence in situ hybridization results in breast carcinoma

Oncotopix Clinical, Publicly Sharable

Background: The Visiopharm human epidermal growth factor receptor 2 (HER2) digital imaging analysis (DIA) algorithm assesses digitized HER2 immunohistochemistry (IHC) by measuring cell membrane connectivity. We aimed to validate this algorithm for clinical use by comparing with pathologists' scoring and correlating with HER2 fluorescence in situ hybridization (FISH) results. Materials and Methods: The study cohort consisted of 612 consecutive invasive breast carcinoma specimens including 395 biopsies and 217 resections. HER2 IHC slides were scanned using Philips IntelliSite Scanners, and the digital images were analyzed using Visiopharm HER2-CONNECT App to obtain the connectivity values (0-1) and scores (0, 1+, 2+, and 3+). HER2 DIA scores were compared with Pathologists' manual scores, and HER2 connectivity values were correlated with HER2 FISH results. Results: The concordance between HER2 DIA scores and pathologists' scores was 87.3% (534/612). All discordant cases (n = 78) were only one-step discordant (negative to equivocal, equivocal to positive, or vice versa). Five cases (0.8%) showed discordant HER2 IHC DIA and HER2 FISH results, but all these cases had relatively low HER2 copy numbers (between 4 and 6). HER2 IHC connectivity showed significantly better correlation with HER2 copy number than HER2/CEP17 ratio. Conclusions: HER2 IHC DIA demonstrates excellent concordance with pathologists' scores and accurately discriminates between HER2 FISH positive and negative cases. HER2 IHC connectivity has better correlation with HER2 copy number than HER2/CEP17 ratio, suggesting HER2 copy number may be more important in predicting HER2 protein expression, and response to anti-HER2-targeted therapy.

Ramon Hartage, Aidan Li, Scott Hammond, Anil Parwani

Ramon Hartage, Aidan Li, Scott Hammond... et al. Journal of Pathology Informatics
2020 Quantitative digital imaging analysis of HER2 immunohistochemistry predicts the response to anti-HER2 neoadjuvant chemotherapy in HER2-positive breast carcinoma

Oncotopix Clinical, Publicly Sharable

Purpose: Patients with HER2-positive breast cancer commonly receive anti-HER2 neoadjuvant chemotherapy and pathologic complete response (pCR) can be achieved in up to half of the patients. HER2 protein expression detected by immunohistochemistry (IHC) can be quantified using digital imaging analysis (DIA) as a value of membranous connectivity. We aimed to investigate the association HER2 IHC DIA quantitative results with response to anti-HER2 neoadjuvant chemotherapy. Methods: Digitized HER2 IHC whole slide images were analyzed using Visiopharm HER2-CONNECT to obtain quantitative HER2 membranous connectivity from a cohort of 153 HER2+ invasive breast carcinoma cases treated with anti-HER2 neoadjuvant chemotherapy (NAC). HER2 connectivity and other factors including age, histologic grade, ER, PR, and HER2 fluorescence in situ hybridization (FISH) were analyzed for association with the response to anti-HER2 NAC. Results: Eighty-three cases (54.2%) had pCR, while 70 (45.8%) showed residual tumor. Younger age, negative ER/PR, higher HER2 DIA connectivity, higher HER2 FISH ratio and copy number were significantly associated with pCR in univariate analysis. Multivariate analysis demonstrated only age, HER2 DIA connectivity, PR negativity, and HER2 copy number was significantly associated with pCR, whereas HER2 DIA connectivity had the strongest association. Conclusions: HER2 IHC DIA connectivity is the most important factor predicting pCR to anti-HER2 neoadjuvant chemotherapy in patients with HER2-positive breast cancer.

Aidan C. Li, Jing Zhao, Chao Zhao, Zhongliang Ma, Ramon Hartage, Yunxiang Zhang, Xiaoxian Li, Anil V. Parwani

Aidan C. Li, Jing Zhao, Chao Zhao... et al. Breast Cancer Research and Treatment
2021 Optimized Culture Conditions for Improved Growth and Functional Differentiation of Mouse and Human Colon Organoids

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Background & Aims: Diligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids. Methods: Mouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors. Results: In contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures. Conclusion: Our study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.

Sarah S. Wilson, Martha Mayo, Terry Melim, Heather Knight, Lori Patnaude, Xiaoming Wu, Lucy Phillips, Susan Westmoreland, Robert Dunstan, Edda Fiebiger, Sonia Terrillon

Sarah S. Wilson, Martha Mayo, Terry Melim... et al. Frontiers in Immunology
2021 Increased angiotensin-converting enzyme 2 and loss of alveolar type II cells in COVID-19–related acute respiratory distress syndrome

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Rationale: ACE2 (angiotensin-converting enzyme 2), the entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is expressed in type 2 alveolar epithelial cells (AT2) that may play key roles in postinjury repair. An imbalance between ACE2 and ACE has also been hypothesized to contribute to lung injury. Objectives: To characterize the expression and distribution of ACE2 and ACE and to compare AT2 with endothelial cell expression in coronavirus disease (COVID-19)–related or –unrelated acute respiratory distress syndrome (ARDS) and controls. Methods: Lung tissue stainings (using multiplex immunofluorescence) and serum concentrations of ACEs were determined retrospectively in two different cohorts of patients. AT2 and endothelial cells were stained in lung tissue for ProSPC (pro-surfactant protein C) and CD31, respectively. Measurements and Main Results: Pulmonary ACE2 expression was increased in patients with COVID-19–related and –unrelated ARDS (0.06% of tissue area and 0.12% vs. 0.006% for control subjects; P = 0.013 and P, 0.0001, respectively). ACE2 was upregulated in endothelial cells (0.32% and 0.53% vs. 0.01%; P = 0.009 and P, 0.0001) but not in AT2 cells (0.13% and 0.08% vs. 0.03%; P = 0.94 and P = 0.44). Pulmonary expression of ACE was decreased in both COVID-19–related and –unrelated ARDS (P = 0.057 and P = 0.032). Similar increases in ACE2 and decreases in ACE were observed in sera of COVID-19 (P = 0.0054 and P, 0.0001) and non–COVID-19 ARDS (P, 0.0001 and P = 0.016). In addition, AT2 cells were decreased in patients with COVID-19–related ARDS compared with COVID-19–unrelated ARDS (1.395% vs. 2.94%, P = 0.0033). Conclusions: ACE2 is upregulated in lung tissue and serum of both COVID-19–related and –unrelated ARDS, whereas a loss of AT2 cells is selectively observed in COVID-19–related ARDS.

Ludovic Gerard, Marylene Lecocq, Caroline Bouzin, Delphine Hoton, Gregory Schmit, Joao Pinto Pereira, Virginie Montiel, Thomas Plante-Bordeneuve, Pierre François Laterre, Charles Pilette

Ludovic Gerard, Marylene Lecocq, Caroline Bouzin... et al. American Journal of Respiratory and Critical Care Medicine
2021 Signalling of multiple interleukin (IL)-17 family cytokines via IL-17 receptor A drives psoriasis-related inflammatory pathways

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Background: The interleukin (IL)-23/IL-17 immune axis is of central importance in psoriasis. However, the impact of IL-17 family cytokines other than IL-17A in psoriasis has not been fully established. Objectives: To elucidate the contribution of IL-17 family cytokines in psoriasis. Methods: To address the expression and localization of IL-17 family cytokines, lesional and nonlesional skin samples from patients with psoriasis were analysed by several complementary methods, including quantitative polymerase chain reaction, immunoassays, in situ hybridization and immunohistochemistry. Mechanistic studies assessing the functional activity of IL-17 family cytokines were performed using ex vivo cultured human skin biopsies and primary human keratinocytes. Results: We demonstrated that IL-17A, IL-17F, IL-17A/F and IL-17C are expressed at increased levels in psoriasis lesional skin and induce overlapping gene expression responses in ex vivo cultured human skin that correlate with the transcriptomic signature of psoriasis skin. Furthermore, we showed that brodalumab, in contrast to ixekizumab, normalizes gene expression responses induced by the combination of IL-17A, IL-17F, IL-17A/F and IL-17C in human keratinocytes. Conclusions: Several IL-17 ligands signalling through IL-17RA are overexpressed in psoriasis skin and induce similar psoriasis-related inflammatory pathways demonstrating their relevance in relation to therapeutic intervention in psoriasis.

M. A.X. Tollenaere, J. Hebsgaard, D. A. Ewald, P. Lovato, S. Garcet, X. Li, S. D. Pilger, M. L. Tiirikainen, M. Bertelsen, J. G. Krueger, H. Norsgaard

M. A.X. Tollenaere, J. Hebsgaard, D. A. Ewald... et al. British Journal of Dermatology
2021 Mechanisms and regulation of IL-22-mediated intestinal epithelial homeostasis and repair

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Aims: Ulcerative colitis and Crohn's disease, collectively known as inflammatory bowel disease (IBD), are chronic inflammatory disorders of the intestine for which key elements in disease initiation and perpetuation are defects in epithelial barrier integrity. Achieving mucosal healing is essential to ameliorate disease outcome and so new therapies leading to epithelial homeostasis and repair are under investigation. This study was designed to determine the mechanisms by which IL-22 regulates intestinal epithelial cell function. Main methods: Human intestinal organoids and resections, as well as mice were used to evaluate the effect of IL-22 on stem cell expansion, proliferation and expression of mucus components. IL-22 effect on barrier function was assessed in polarized T-84 cell monolayers. Butyrate co-treatments and organoid co-cultures with immune cells were performed to monitor the impact of microbial-derived metabolites and inflammatory environments on IL-22 responses. Key findings: IL-22 led to epithelial stem cell expansion, proliferation, barrier dysfunction and anti-microbial peptide production in human and mouse models evaluated. IL-22 also altered the mucus layer by inducing an increase in membrane mucus but a decrease in secreted mucus and goblet cell content. IL-22 had the same effect on anti-microbial peptides and membrane mucus in both healthy and IBD human samples. In contrast, this IL-22-associated epithelial phenotype was different when treatments were performed in presence of butyrate and organoids co-cultured with immune cells. Significance: Our data indicate that IL-22 promotes epithelial regeneration, innate defense and membrane mucus production, strongly supporting the potential clinical utility of IL-22 as a mucosal healing therapy in IBD.

Lori Patnaude, Martha Mayo, Regina Mario, Xiaoming Wu, Heather Knight, Kelly Creamer, Sarah Wilson, Valerie Pivorunas, Jozsef Karman, Lucy Phillips, Robert Dunstan, Rajesh V. Kamath, Bradford McRae, Sonia Terrillon

Lori Patnaude, Martha Mayo, Regina Mario... et al. Life Sciences
2021 A phase Ib/II study of pepinemab in combination with avelumab in advanced non–small cell lung cancer

Oncotopix Discovery, Publicly Sharable, Tissuealign

Purpose: The CLASSICAL-Lung clinical trial tested the combination of pepinemab, an IgG4 humanized mAb targeting semaphorin 4D, with the PD-L1 inhibitor avelumab to assess the effects of coupling increased T-cell infiltration and reversal of immune suppression via pepinemab with sustained T-cell activation via checkpoint inhibition. Patients and Methods: This phase Ib/II, single-arm study was designed to evaluate the safety, tolerability, and efficacy of pepinemab in combination with avelumab in 62 patients with advanced non–small cell lung cancer (NSCLC), including immunotherapy-naïve (ION) patients and patients whose tumors progressed following anti-PD-1/L1 monotherapy (IOF). The main objectives were to evaluate safety/tolerability, establish a recommended phase 2 dose (RP2D), obtain a preliminary evaluation of antitumor activity, and investigate candidate biomarker activity. Results: The combination was well tolerated with no major safety signals identified. Pepinemab, 10 mg/kg with avelumab, 10 mg/kg, every 2 weeks, was selected as the RP2D. Among 21 evaluable ION patients, 5 patients experienced partial responses (PR), 4 patients evidenced clinical benefit ≥1 year, and the disease control rate (DCR) was 81%. Notably, overall response rate with the combination therapy was higher than previously reported for single-agent avelumab in the PD-L1-negative/low population. Among 29 evaluable IOF patients, the combination resulted in a DCR of 59%, including 2 PR and 7 patients with durable clinical benefit of ≥23 weeks. Biomarker analysis of biopsies demonstrated increased CD8 T-cell density correlating with RECIST response criteria. Conclusions: The combination of pepinemab with avelumab was well tolerated in NSCLC and showed signs of antitumor activity in immunotherapy-resistant and PD-L1-negative/low tumors.

Michael R. Shafique, Terrence L. Fisher, Elizabeth E. Evans, John E. Leonard, Desa Rae E. Pastore, Crystal L. Mallow, Ernest Smith, Vikas Mishra, Andreas Schröder, Kevin M. Chin, Joseph T. Beck, Megan A. Baumgart, Ramaswamy Govindan, Nashat Y. Gabrail, Alexander I. Spira, Nagashree Seetharamu, Yanyan Lou, Aaron S. Mansfield, Rachel E. Sanborn, Jonathan W. Goldman, Maurice Zauderer

Michael R. Shafique, Terrence L. Fisher, Elizabeth E. Evans... et al. Clinical Cancer Research
2021 A template to quantify the location and density of CD3 + and CD8 + tumor-infiltrating lymphocytes in colon cancer by digital pathology on whole slides for an objective, standardized immune score assessment

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Background: In colon cancer, the location and density of tumor-infiltrating lymphocytes (TILs) can classify patients into low and high-risk groups for prognostication. While a commercially available ‘Immunoscore®’ exists, the incurred expenses and copyrights may prevent universal use. The aim of this study was to develop a robust and objective quantification method of TILs in colon cancer. Methods: A consecutive, unselected series of specimens from patients with colon cancer were available for immunohistochemistry and assessment of TILs by automated digital pathology. CD3 + and CD8 + cells at the invasive margin and in tumor center were assessed on consecutive sections using automated digital pathology and image analysis software (Visiopharm®). An algorithm template for whole slide assessment, generated cell counts per square millimeters (cells/mm2), from which the immune score was calculated using distribution volumes. Furthermore, immune score was compared with clinical and histopathological characteristics to confirm its relevance. Results: Based on the quantified TILs numbers by digital image analyses, patients were classified into low (n = 83, 69.7%), intermediate (n = 14, 11.8%) and high (n = 22, 18.5%) immune score groups. High immune score was associated with stage I–II tumors (p = 0.017) and a higher prevalence of microsatellite instable (MSI) tumors (p = 0.030). MSI tumors had a significantly higher numbers of CD3 + TILs in the invasive margin and CD8 + TILs in both tumor center and invasive margin, compared to microsatellite stable (MSS) tumors. Conclusion: A digital template to quantify an easy-to-use immune score corresponds with clinicopathological features and MSI in colon cancer.

Dordi Lea, Martin Watson, Ivar Skaland, Hanne R. Hagland, Melinda Lillesand, Einar Gudlaugsson, Kjetil Søreide

Dordi Lea, Martin Watson, Ivar Skaland... et al. Cancer Immunology, Immunotherapy
2021 Changes in Immune Cell Types with Age in Breast are Consistent with a Decline in Immune Surveillance and Increased Immunosuppression

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A majority of breast cancers (BC) are age-related and we seek to determine what cellular and molecular changes occur in breast tissue with age that make women more susceptible to cancer initiation. Immune-epithelial cell interactions are important during mammary gland development and the immune system plays an important role in BC progression. The composition of human immune cell populations is known to change in peripheral blood with age and in breast tissue during BC progression. Less is known about changes in immune populations in normal breast tissue and how their interactions with mammary epithelia change with age. We quantified densities of T cells, B cells, and macrophage subsets in pathologically normal breast tissue from 122 different women who ranged in age from 24 to 74 years old. Donor-matched peripheral blood from a subset of 20 donors was analyzed by flow cytometry. Tissue immune cell densities and localizations relative to the epithelium were quantified in situ with machine learning-based image analyses of multiplex immunohistochemistry-stained tissue sections. In situ results were corroborated with flow cytometry analyses of peri-epithelial immune cells from primary breast tissue preparations and transcriptome analyses of public data from bulk tissue reduction mammoplasties. Proportions of immune cell subsets in breast tissue and donor-matched peripheral blood were not correlated. Density (cells/mm2) of T and B lymphocytes in situ decreased with age. T cells and macrophages preferentially localized near or within epithelial bilayers, rather than the intralobular stroma. M2 macrophage density was higher than M1 macrophage density and this difference was due to higher density of M2 in the intralobular stroma. Transcriptional signature analyses suggested age-dependent decline in adaptive immune cell populations and functions and increased innate immune cell activity. T cells and macrophages are so intimately associated with the epithelia that they are embedded within the bilayer, suggesting an important role for immune-epithelial cell interactions. Age-associated decreased T cell density in peri-epithelial regions, and increased M2 macrophage density in intralobular stroma suggests the emergence of a tissue microenvironment that is simultaneously immune-senescent and immunosuppressive with age.

Arrianna Zirbes, Jesuchristopher Joseph, Jennifer C. Lopez, Rosalyn W. Sayaman, Mudaser Basam, Victoria L. Seewaldt, Mark A. LaBarge

Arrianna Zirbes, Jesuchristopher Joseph, Jennifer C. Lopez... et al. Journal of Mammary Gland Biology and Neoplasia
2021 Stabilized epithelial phenotype of cancer cells in primary tumors leads to increased colonization of liver metastasis in pancreatic cancer

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Pancreatic ductal adenocarcinoma (PDAC) is therapeutically recalcitrant and metastatic. Partial epithelial to mesenchymal transition (EMT) is associated with metastasis; however, a causal connection needs further unraveling. Here, we use single-cell RNA sequencing and genetic mouse models to identify the functional roles of partial EMT and epithelial stabilization in PDAC growth and metastasis. A global EMT expression signature identifies ∼50 cancer cell clusters spanning the epithelial-mesenchymal continuum in both human and murine PDACs. The combined genetic suppression of Snail and Twist results in PDAC epithelial stabilization and increased liver metastasis. Genetic deletion of Zeb1 in PDAC cells also leads to liver metastasis associated with cancer cell epithelial stabilization. We demonstrate that epithelial stabilization leads to the enhanced collective migration of cancer cells and modulation of the immune microenvironment, which likely contribute to efficient liver colonization. Our study provides insights into the diverse mechanisms of metastasis in pancreatic cancer and potential therapeutic targets.

Julienne L. Carstens, Sujuan Yang, Pedro Correa de Sampaio, Xiaofeng Zheng, Souptik Barua, Kathleen M. McAndrews, Arvind Rao, Jared K. Burks, Andrew D. Rhim, Raghu Kalluri

Julienne L. Carstens, Sujuan Yang, Pedro Correa de Sampaio... et al. Cell Reports
2021 Immunological tumor heterogeneity and diagnostic profiling for advanced and immune therapies

Phenoplex, Publicly Sharable, Ultivue

Immunotherapies have changed the way how we treat cancer at all stages. The understanding of the immune system in individual tumor specimens guides the selection of immune-modulating agents such as immune checkpoint inhibitors alone or in combination with other therapeutic agents that target, modulate or unleash the patient's immune system. Despite the similar histopathological diagnosis, each tumor is unique at its primary site and site of metastasis, also depending on previous treatment regimens or genetic alterations, such as chromosomal instability or acquired mutations. The clinically well-established use of PD-1/PD-L1 inhibitors already requires the assessment of its target molecules in different cells (viable tumor cells alone or in combination with immune cells or immune cells alone) with different thresholds in various indications. Anyhow, checkpoint inhibitors show the best overall response rate when immune effec-tor cells like tumor-infiltrating lymphocytes are in close spatial proximity without being suppressed by other humoral or cellular regulatory mechanisms. Therefore, immune cell-rich tumors ("hot tumors") are usually quite reactive to immune-modulating agents, whereas other immune-depleted or immune-excluded tumor areas are less responsive and require alternative treatment regimens such as modified immune effectors cells or immune-stimulating agents, for example, oncolytic viruses. Here, we summarize the relevance to understand the entire tumor heterogeneity and its environment, the contextual relationship and spatial quantification of all immune and tumor cells along with the genetic background of the individual cancer through the application of multiplex in-situ technologies and the application of machine learning tools. K E Y W O R D S cell therapy, immunotherapy 1 | BACKGROUND Immunology discoveries and advancements come in waves. More than 100 years ago, different immune cells and their separate role in infectious and neoplastic diseases became obvious and some improvement in light microscopy contributed to the development of cancer immunology as a separate subject. With the advancement of analytical methods like immunohistochemistry (IHC), molecular tools, and computational solutions, immunotherapies make a greater impact in our clinical practice. 1 Today, we have advanced diagnostic tools at hand such as digital imaging for the objective and reproducible assessment of multiple markers at a time or on a single tissue slide precisely quantifying the absolute numbers of functionally distinct immune cells as well as their spatial distribution and contextual

Ralf Huss, Christoph Schmid, | Mael Manesse, | Jeppe Thagaard, Bruno Maerkl

Ralf Huss, Christoph Schmid, | Mael Manesse... et al. Advances in Cell and Gene Therapy
2021 Immune microenvironment characterisation and dynamics during anti-HER2-based neoadjuvant treatment in HER2-positive breast cancer

Oncotopix Discovery, Publicly Sharable

Despite their recognised role in HER2-positive (HER2+) breast cancer (BC), the composition, localisation and functional orientation of immune cells within tumour microenvironment, as well as its dynamics during anti-HER2 treatment, is largely unknown. We here investigate changes in tumour-immune contexture, as assessed by stromal tumour-infiltrating lymphocytes (sTILs) and by multiplexed spatial cellular phenotyping, during treatment with lapatinib-trastuzumab in HER2+ BC patients (PAMELA trial). Moreover, we evaluate the relationship of tumour-immune contexture with hormone receptor status, intrinsic subtype and immune-related gene expression. sTIL levels increase after 2 weeks of HER2 blockade in HR-negative disease and HER2-enriched subtype. This is linked to a concomitant increase in cell density of all four immune subpopulations (CD3+, CD4+, CD8+, Foxp3+). Moreover, immune contexture analysis showed that immune cells spatially interacting with tumour cells have the strongest association with response to anti-HER2 treatment. Subsequently, sTILs consistently decrease at the surgery in patients achieving pathologic complete response, whereas most residual tumours at surgery remain inflamed, possibly reflecting a progressive loss of function of T cells. Understanding the features of the resulting tumour immunosuppressive microenvironment has crucial implications for the design of new strategies to de-escalate or escalate systemic therapy in early-stage HER2+ BC.

G. Griguolo, G. Serna, T. Pascual, R. Fasani, X. Guardia, N. Chic, L. Paré, S. Pernas, M. Muñoz, M. Oliveira, M. Vidal, A. Llombart-Cussac, J. Cortés, P. Galván, B. Bermejo, N. Martínez, R. López, S. Morales, I. Garau, L. Manso, J. Alarcón, E. Martínez, P. Villagrasa, A. Prat, P. Nuciforo

G. Griguolo, G. Serna, T. Pascual... et al. npj Precision Oncology 2021 5:1
2021 Impaired Dendritic Cell Homing in COVID-19

Phenoplex, Publicly Sharable

The high mortality of COVID-19 is mostly attributed to acute respiratory distress syndrome (ARDS), whose histopathological correlate is diffuse alveolar damage (DAD). Furthermore, severe COVID-19 is often accompanied by a cytokine storm and a disrupted response of the adaptive immune system. Studies aiming to depict this dysregulation have mostly investigated the peripheral cell count as well as the functionality of immune cells. We investigated the impact of SARS-CoV-2 on antigen-presenting cells using multiplexed immunofluorescence. Similar to MERS-CoV and SARS-CoV, SARS-CoV-2 appears to be impairing the maturation of dendritic cells (DCs). DC maturation involves a switch in surface antigen expression, which enables the cells' homing to lymph nodes and the subsequent activation of T-cells. As quantitative descriptions of the local inflammatory infiltrate are still scarce, we compared the cell population of professional antigen-presenting cells (APC) in the lungs of COVID-19 autopsy cases in different stages of DAD. We found an increased count of myeloid dendritic cells (mDCs) in later stages. Interestingly, mDCs also showed no significant upregulation of maturation markers in DAD-specimens with high viral load. Accumulation of immature mDCs, which are unable to home to lymph nodes, ultimately results in an inadequate T-cell response.

Lukas Borcherding, Alime Sema Teksen, Bianca Grosser, Tina Schaller, Klaus Hirschbühl, Rainer Claus, Oliver Spring, Michael Wittmann, Christoph Römmele, Éva Sipos, Bruno Märkl

Lukas Borcherding, Alime Sema Teksen, Bianca Grosser... et al. Frontiers in Medicine
2021 A Preliminary Study of Deep-Learning Algorithm for Analyzing Multiplex Immunofluorescence Biomarkers in Body Fluid Cytology Specimens

Phenoplex, Publicly Sharable

Introduction: Multiplex biomarker analysis of cytological body fluid specimens is often used to assist cytologists in distiguishing metastatic cancer cells from reactive meso-thelial cells. However, evaluating biomarker expression visually may be challenging, especially when the cells of interest are scant. Deep-learning algorithms (DLAs) may be able to assist cytologists in analyzing multiple biomarker expression at the single cell level in the multiplex fluores-cence imaging (MFI) setting. This preliminary study was performed to test the feasibility of using DLAs to identify immunofluorescence-stained metastatic adenocarcinoma cells in body fluid cytology samples. Methods: A DLA was developed to analyze MFI-stained cells in body fluid cyto-logical samples. A total of 41 pleural fluid samples, comprising of 20 positives and 21 negatives, were retrospectively collected. Multiplex immunofluorescence labeling for MOC31, BerEP4, and calretinin, were performed on cell block sections, and results were analyzed by manual analysis (manual MFI) and DLA analysis (MFI-DLA) independently. Results: All cases with positive original cytological diagnoses showed positive results either by manual MFI or MFI-DLA, but 2 of the 14 (14.3%) original cytologically negative cases had rare cells with positive MOC31 and/or BerEP4 staining in addition to calretinin. Manual MFI analysis and MFI-DLA showed 100% concordance. Conclusion: MFI combined with DLA provides a potential tool to assist in cytological diagnosis of metastatic malignancy in body fluid samples. Larger studies are warranted to test the clinical validity of the approach.

Weibo Yu, Elizabeth Rao, Curtis D Chin, Josephine S Aguilar-Jakthong, Yunfeng Li, Christine Chow, Shu Yu, Grace Wang, Jianyu Rao

Weibo Yu, Elizabeth Rao, Curtis D Chin... et al. Acta Cytologica
2022 Artificial Intelligence Applications in Human Pathology

General, Publicly Sharable

Ralf Huss, Michael Grunkin

Ralf Huss, Michael Grunkin Artificial Intelligence Applications in Human Pathology
2022 Combining TMEM Doorway Score and MenaCalc Score Improves the Prediction of Distant Recurrence Risk in HR+/HER2− Breast Cancer Patients

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Purpose: to develop several digital pathology‐based machine vision algorithms for combining TMEM and MenaCalc scores and determine if a combination of these biomarkers improves the ability to predict development of distant metastasis over and above that of either biomarker alone. Methods: This retrospective study included a subset of 130 patients (65 patients with no recurrence and 65 patients with a recurrence at 5 years) from the Calgary Tamoxifen cohort of breast cancer patients. Patients had confirmed invasive breast cancer and received adjuvant tamoxifen therapy. Of the 130 patients, 86 cases were suitable for analysis in this study. Sequential sections of formalin‐fixed paraffin‐embedded patient samples were stained for TMEM doorways (immunohistochemistry triple staining) and MenaCalc (immunofluorescence staining). Stained sections were imaged, aligned, and then scored for TMEM doorways and MenaCalc. Different ways of combining TMEM doorway and MenaCalc scores were evaluated and compared to identify the best performing combined marker by using the restricted mean survival time (RMST) difference method. Results: the best performing combined marker gave an RMST difference of 5.27 years (95% CI: 1.71–8.37), compared to 3.56 years (95% CI: 0.95–6.1) for the associated standalone TMEM doorway analysis and 2.94 years (95% CI: 0.25–5.87) for the associated standalone MenaCalc analysis. Conclusions: combining TMEM doorway and MenaCalc scores as a new biomarker improves prognostication over that observed with TMEM doorway or MenaCalc Score alone in this cohort of 86 patients.

Xianjun Ye, Maja H. Oktay, Xiaonan Xue, Thomas E. Rohan, Paula S. Ginter, Timothy D’alfonso, Elizabeth N. Kornaga, Don G. Morris, David Entenberg, John S. Condeelis

Xianjun Ye, Maja H. Oktay, Xiaonan Xue... et al. Cancers
2022 Semi-automated validation and quantification of CTLA-4 in 90 different tumor entities using multiple antibodies and artificial intelligence

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CTLA-4 is an inhibitory immune checkpoint receptor and a negative regulator of anti-tumor T-cell function. This study is aimed for a comparative analysis of CTLA-4+ cells between different tumor entities. To quantify CTLA-4+ cells, 4582 tumor samples from 90 different tumor entities as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Two different antibody clones (MSVA-152R and CAL49) were validated and quantified using a deep learning framework for automated exclusion of unspecific immunostaining. Comparing both CTLA-4 antibodies revealed a clone dependent unspecific staining pattern in adrenal cortical adenoma (63%) for MSVA-152R and in pheochromocytoma (67%) as well as hepatocellular carcinoma (36%) for CAL49. After automated exclusion of non-specific staining reaction (3.6%), a strong correlation was observed for the densities of CTLA-4+ lymphocytes obtained by both antibodies (r = 0.87; p < 0.0001). A high CTLA-4+ cell density was linked to low pT category (p < 0.0001), absent lymph node metastases (p = 0.0354), and PD-L1 expression in tumor cells or inflammatory cells (p < 0.0001 each). A high CTLA-4/CD3-ratio was linked to absent lymph node metastases (p = 0.0295) and to PD-L1 positivity on immune cells (p = 0.0026). Marked differences exist in the number of CTLA-4+ lymphocytes between tumors. Analyzing two independent antibodies by a deep learning framework can facilitate automated quantification of immunohistochemically analyzed target proteins such as CTLA-4. A convolutional neural network (U-Net) for the assessment of aberrant CTLA-4 antibody staining using two independent antibody clones (MSVA-152R and CAL49) was trained and validated on 4582 tumor samples in this study. The deep learning-based framework facilitated automated CTLA-4 quantification in more than 90 different tumor entities via compensating for individual antibody shortcomings.

David Dum, Tjark L.C. Henke, Tim Mandelkow, Cheng Yang, Elena Bady, Jonas B. Raedler, Ronald Simon, Guido Sauter, Maximilian Lennartz, Franziska Büscheck, Andreas M. Luebke, Anne Menz, Andrea Hinsch, Doris Höflmayer, Sören Weidemann, Christoph Fraune, Katharina Möller, Patrick Lebok, Ria Uhlig, Christian Bernreuther, Frank Jacobsen, Till S. Clauditz, Waldemar Wilczak, Sarah Minner, Eike Burandt, Stefan Steurer, Niclas C. Blessin

David Dum, Tjark L.C. Henke, Tim Mandelkow... et al. Laboratory Investigation 2022 102:6
2022 Sex-Dependent Hepatoprotective Role of IL-22 Receptor Signaling in Non-Alcoholic Fatty Liver Disease-Related Fibrosis

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Background & Aims: Nonalcoholic fatty liver disease (NAFLD) is a major health problem with complex pathogenesis. Although sex differences in NAFLD pathogenesis have been reported, the mechanisms underlying such differences remain understudied. Interleukin (IL)22 is a pleiotropic cytokine with both protective and/or pathogenic effects during liver injury. IL22 was shown to be hepatoprotective in NAFLD-related liver injury. However, these studies relied primarily on exogenous administration of IL22 and did not examine the sex-dependent effect of IL22. Here, we sought to characterize the role of endogenous IL22-receptor signaling during NAFLD-induced liver injury in males and females. Methods: We used immunofluorescence, flow cytometry, histopathologic assessment, and gene expression analysis to examine IL22 production and characterize the intrahepatic immune landscape in human subjects with NAFLD (n = 20; 11 men and 9 women) and in an in vivo Western high-fat diet–induced NAFLD model in IL22RA knock out mice and their wild-type littermates. Results: Examination of publicly available data sets from 2 cohorts with NAFLD showed increased hepatic IL22 gene expression in females compared with males. Furthermore, our immunofluorescence analysis of liver sections from NAFLD subjects (n = 20) showed increased infiltration of IL22-producing cells in females. Similarly, IL22-producing cells were increased in wild-type female mice with NAFLD and the hepatic IL22/IL22 binding protein messenger RNA ratio correlated with expression of anti-apoptosis genes. The lack of endogenous IL22-receptor signaling (IL22RA knockout) led to exacerbated liver damage, inflammation, apoptosis, and liver fibrosis in female, but not male, mice with NAFLD. Conclusions: Our data suggest a sex-dependent hepatoprotective antiapoptotic effect of IL22-receptor signaling during NAFLD-related liver injury in females.

Mohamed N. Abdelnabi, Manuel Flores Molina, Geneviève Soucy, Vincent Quoc-Huy Trinh, Nathalie Bédard, Sabrina Mazouz, Nathalie Jouvet, Jessica Dion, Sarah Tran, Marc Bilodeau, Jennifer L. Estall, Naglaa H. Shoukry

Mohamed N. Abdelnabi, Manuel Flores Molina, Geneviève Soucy... et al. Cellular and Molecular Gastroenterology and Hepatology
2022 α Cell dysfunction in islets from nondiabetic, glutamic acid decarboxylase autoantibody–positive individuals

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BACKGROUND. Multiple islet autoantibodies (AAbs) predict the development of type 1 diabetes (T1D) and hyperglycemia within 10 years. By contrast, T1D develops in only approximately 15% of individuals who are positive for single AAbs (generally against glutamic acid decarboxylase [GADA]); hence, the single GADA+ state may represent an early stage of T1D. METHODS. Here, we functionally, histologically, and molecularly phenotyped human islets from nondiabetic GADA+ and T1D donors. RESULTS. Similar to the few remaining β cells in the T1D islets, GADA+ donor islets demonstrated a preserved insulin secretory response. By contrast, α cell glucagon secretion was dysregulated in both GADA+ and T1D islets, with impaired glucose suppression of glucagon secretion. Single-cell RNA-Seq of GADA+ α cells revealed distinct abnormalities in glycolysis and oxidative phosphorylation pathways and a marked downregulation of cAMP-dependent protein kinase inhibitor β (PKIB), providing a molecular basis for the loss of glucose suppression and the increased effect of 3-isobutyl-1-methylxanthine (IBMX) observed in GADA+ donor islets. CONCLUSION. We found that α cell dysfunction was present during the early stages of islet autoimmunity at a time when β cell mass was still normal, raising important questions about the role of early α cell dysfunction in the progression of T1D.

Nicolai M. Doliba, Andrea V. Rozo, Jeffrey Roman, Wei Qin, Daniel Traum, Long Gao, Jinping Liu, Elisabetta Manduchi, Chengyang Liu, Maria L. Golson, Golnaz Vahedi, Ali Naji, Franz M. Matschinsky, Mark A. Atkinson, Alvin C. Powers, Marcela Brissova, Klaus H. Kaestner, Doris A. Stoffers

Nicolai M. Doliba, Andrea V. Rozo, Jeffrey Roman... et al. The Journal of Clinical Investigation
2022 Neutrophil-mediated fibroblast-tumocell il-6/stat-3 signaling underlies the association between neutrophil-to-lymphocyte ratio dynamics and chemotherapy response in localized pancreatic cancer: A hybrid clinical-preclinical study

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Background: Partial/complete pathologic response following neoadjuvant chemotherapy (NAC) in pancreatic cancer (PDAC) patients undergoing pancreatectomy is associated with improved survival. We sought to determine whether neutrophil-to-lymphocyte ratio (NLR) dynamics predict pathologic response following chemotherapy in PDAC, and if manipulating NLR impacts chemosensitivity in preclinical models and uncovers potential mechanistic underpinnings underlying these effects. Methods: Pathologic response in PDAC patients (n=94) undergoing NAC and pancreatectomy (7/2015-12/2019) was dichotomized as partial/complete or poor/absent. Bootstrap-validated multi-variable models assessed associations between pre-chemotherapy NLR (%neutrophils÷%lympho-cytes) or NLR dynamics during chemotherapy (ΔNLR = pre-surgery—pre-chemotherapy NLR) and pathologic response, disease-free survival (DFS), and overall survival (OS). To preclinically model effects of NLR attenuation on chemosensitivity, Ptf1aCre/+; KrasLSL-G12D/+;Tgfbr2flox/flox (PKT) mice and C57BL/6 mice orthotopically injected with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1Cre(KPC) cells were randomized to vehicle, gemcitabine/paclitaxel alone, and NLR-attenuating anti-Ly6G with/without gemcitabine/paclitaxel treatment. Results: In 94 PDAC patients undergoing NAC (median:4 months), pre-chemotherapy NLR (p<0.001) and ΔNLR attenuation during NAC (p=0.002) were independently associated with partial/ complete pathologic response. An NLR score = pre-chemotherapy NLR+ΔNLR correlated with DFS (p=0.006) and OS (p=0.002). Upon preclinical modeling, combining NLR-attenuating anti-Ly6G treatment with gemcitabine/paclitaxel—compared with gemcitabine/paclitaxel or anti-Ly6G alone—not only significantly reduced tumor burden and metastatic outgrowth, but also augmented tumor-infiltrating CD107a+-degranulating CD8+ T-cells (p<0.01) while dampening inflammatory cancer-associated fibroblast (CAF) polarization (p=0.006) and chemoresistant IL-6/STAT-3 signaling in vivo. Neutrophil-derived IL-1β emerged as a novel mediator of stromal inflammation, inducing inflammatory CAF polarization and CAF-tumor cell IL-6/STAT-3 signaling in ex vivo co-cultures. Conclusions: Therapeutic strategies to mitigate neutrophil-CAF-tumor cell IL-1β/IL-6/STAT-3 signaling during NAC may improve pathologic responses and/or survival in PDAC.

Iago de Castro Silva, Anna Bianchi, Nilesh U. Deshpande, Prateek Sharma, Siddharth Mehra, Vanessa Tonin Garrido, Shannon Jacqueline Saigh, Jonathan England, Peter Joel Hosein, Deukwoo Kwon, Nipun B. Merchant, Jashodeep Datta

Iago de Castro Silva, Anna Bianchi, Nilesh U. Deshpande... et al. eLife
2022 Original research: Necroptosis-driving genes RIPK1, RIPK3 and MLKL-p are associated with intratumoral CD3+ and CD8+ T cell density and predict prognosis in hepatocellular carcinoma

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Background Hepatocellular carcinoma (HCC) is a highly lethal cancer and the second leading cause of cancer-related deaths worldwide. As demonstrated in other solid neoplasms and HCC, infiltrating CD8 + T cells seem to be related to a better prognosis, but the mechanisms affecting the immune landscape in HCC are still mostly unknown. Necroptosis is a programmed, caspase-independent cell death that, unlike apoptosis, evokes immune response by releasing damage-associated molecular factors. However, in HCC, the relationship between the necroptotic machinery and the tumor-infiltrating lymphocytes has not been fully investigated so far. Methods We investigated the association between the main necroptosis-related genes, that is, RIPK1, RIPK3, MLKL-p, and CD3 + /CD8 + tumor-infiltrating T cell by RNA-seq data analysis in 371 patients with primary HCC from The Cancer Genome Atlas and then by immunohistochemistry in two independent cohorts of HCC patients from Italy (82) and Japan (86). Results Our findings highlighted the immunogenetic role of necroptosis and its potential prognostic role in HCC: RIPK1, RIPK3 and MLKL-p were found significantly associated with intratumoral CD3 + and CD8 + T cells. In addition, multivariate survival analysis showed that the expression of RIPK1, RIPK3 and MLKL-p was associated with better overall survival in the two independent cohorts. Conclusions Our results confirmed the immunogenetic properties of necroptosis (NCP) in human HCC, showing that tumor-infiltrating lymphocytes (TILs) and, specifically, CD8+ T cells accumulate in tumors with higher expression of the necroptosis-related genes. These results suggest the importance of further studies to better assess the specific composition, as well as the functional features of the immune environment associated with a necroptotic signature in order to explore new possible diagnostic and immunotherapeutic scenarios.

Lorenzo Nicolè, Tiziana Sanavia, Rocco Cappellesso, Valeria Maffeis, Jun Akiba, Akihiko Kawahara, Yoshiki Naito, Claudia Maria Radu, Paolo Simioni, Davide Serafin, Giuliana Cortese, Maria Guido, Giacomo Zanus, Hirohisa Yano, Ambrogio Fassina

Lorenzo Nicolè, Tiziana Sanavia, Rocco Cappellesso... et al. Journal for Immunotherapy of Cancer
2022 Multi-parametric analysis of human livers reveals variation in intrahepatic inflammation across phases of chronic hepatitis B infection

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Background & Aims: Chronic HBV is clinically categorized into 4 phases by a combination of serum HBV DNA levels, HBeAg status and alanine aminotransferase (ALT): immunotolerant (IT), immune-active (IA), inactive carrier (IC) and HBeAg-negative hepatitis (ENEG). Immune and virological measurements in the blood have proven useful but are insufficient to explain the interrelation between the immune system and the virus since immune dynamics differ in the blood and liver. Furthermore, the inflammatory response in the liver and parenchymal cells cannot be fully captured in blood. Methods: Immunological composition and transcriptional profiles of core needle liver-biopsies in chronic HBV phases were compared to those of healthy controls by multiplex immunofluorescence and RNA-sequencing (n = 37 and 78, respectively) analyses. Results: Irrespective of the phase-specific serological profiles, increased immune-gene expression and frequency was observed in chronic HBV compared to healthy livers. Greater transcriptomic deregulation was seen in IA and ENEG (172 vs. 243 DEGs) than in IT and IC (13 vs. 35 DEGs) livers. Interferon-stimulated genes, immune-activation and exhaustion genes (ICOS, CTLA4, PDCD1) together with chemokine genes (CXCL10, CXCL9) were significantly induced in IA and ENEG livers. Moreover, distinct immune profiles associated with ALT elevation and a more accentuated immune-exhaustion profile (CTLA4, TOX, SLAMF6, FOXP3) were observed in ENEG, which set it apart from the IA phase (LGALS9, PDCD1). Interestingly, all HBV phases showed downregulation of metabolic pathways vs. healthy livers (fatty and bile acid metabolism). Finally, increased leukocyte infiltrate correlated with serum ALT, but not with HBV DNA or viral proteins. Conclusion: Our comprehensive multi-parametric analysis of human livers revealed distinct inflammatory profiles and pronounced differences in intrahepatic gene profiles across all chronic HBV phases in comparison to healthy liver. Lay summary: Immunological studies on chronic HBV remain largely restricted to assessment of peripheral responses due to the limited access to the site of infection, the liver. In this study, we comprehensively analyzed livers from a well-defined cohort of patients with chronic HBV and uninfected controls with state-of-the-art techniques, and evaluated the differences in gene expression profiles and inflammation characteristics across distinct disease phases in patients with chronic HBV.

Noe Rico Montanari, Ricardo Ramírez, Abhishek Aggarwal, Nick van Buuren, Michael Doukas, Christina Moon, Scott Turner, Lauri Diehl, Li Li, Jose D. Debes, Becket Feierbach, Andre Boonstra

Noe Rico Montanari, Ricardo Ramírez, Abhishek Aggarwal... et al. Journal of Hepatology
2022 Immune Contexture and Differentiation Features Predict Outcome in Bladder Cancer

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BACKGROUND: An improved risk assessment of patients with bladder cancer (BC) is important to optimize clinical management. OBJECTIVE: To identify whether immune cell subpopulations and cancer cell-intrinsic features are associated with outcome and response to first-line chemotherapy in BC. DESIGN, SETTING, AND PARTICIPANTS: Primary tumor tissue from 785 patients with BC (stage Ta-T4b) were stained using multiplex immunofluorescence (CD3, CD8, FOXP3, CD20, CD68, CD163, and MHC-I) and immunohistochemistry (pancytokeratin, CK5/6, GATA3, programmed death 1 [PD-1], and programmed death ligand 1 [PD-L1]). A digital image analysis quantified staining results within the carcinoma cell and stromal part of the tumor. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Primary endpoints were progression-free survival, recurrence-free survival, and response to first-line chemotherapy. Optimal cutoff values for investigated markers were estimated using maximally selected rank statistics and receiver operating characteristic for each primary endpoint. Time-to-event analyses were performed using Cox regression analyses. RESULTS AND LIMITATIONS: Several immune subpopulations were independently associated with clinical outcomes. Especially, high PD-1 and PD-L1 expression was independently associated with an increased risk of recurrence and progression in non-muscle-invasive tumors, but with a lower risk of recurrence in muscle-invasive tumors. Furthermore, we observed a lower likelihood of response to first-line chemotherapy in patients with basal differentiation features. Finally, a model combining clinical risk factors with our most evident prognosticator improved prediction accuracy compared with clinical risk factors alone for progression in non-muscle-invasive BC and recurrence in muscle-invasive BC. The use of tissue microarrays and a long inclusion period are limitations to this study. CONCLUSIONS: Immune cell subpopulations and cancer cell-intrinsic features are associated with different clinical outcomes in BC. PATIENT SUMMARY: Immune cells play an important role in cancer development and treatment outcomes. Infiltration with specific immune cells and the presence of markers associated with immune evasion in the tumor predict clinical outcomes in bladder cancer.

Ann Taber, Frederik Prip, Philippe Lamy, Mads Agerbæk, Jørgen Bjerggaard Jensen, Torben Steiniche, Lars Dyrskjøt

Ann Taber, Frederik Prip, Philippe Lamy... et al. European Urology Oncology
2022 Immunologic Features in de Novo and Recurrent Glioblastoma Are Associated with Survival Outcomes

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Glioblastoma (GBM) is an immunologically "cold"tumor characterized by poor responsiveness to immunotherapy. Standard of care for GBM is surgical resection followed by chemoradiotherapy and maintenance chemotherapy. However, tumor recurrence is the norm, and recurring tumors are found frequently to have acquired molecular changes (e.g., mutations) that may influence their immunobiology. Here, we compared the immune contexture of de novo GBM and recurrent GBM (rGBM) using high-dimensional cytometry and multiplex IHC. Although myeloid and T cells were similarly abundant in de novo and rGBM, their spatial organization within tumors differed and was linked to outcomes. In rGBM, T cells were enriched and activated in perivascular regions and clustered with activated macrophages and fewer regulatory T cells. Moreover, a higher expression of phosphorylated STAT1 by T cells in these regions at recurrence was associated with a favorable prognosis. Together, our data identify differences in the immunobiology of de novo GBM and rGBM and identify perivascular T cells as potential therapeutic targets.

Cécile Alanio, Zev A. Binder, Renee B. Chang, MacLean P. Nasrallah, Devora Delman, Joey H. Li, Oliver Y. Tang, Logan Y. Zhang, Jiasi Vicky Zhang, E. John Wherry, Donald M. O'Rourke, Gregory L. Beatty

Cécile Alanio, Zev A. Binder, Renee B. Chang... et al. Cancer Immunology Research
2022 Identification of Similarities Between Skin Lesions in Patients With Antisynthetase Syndrome and Skin Lesions in Patients With Dermatomyositis by Highly Multiplexed Imaging Mass Cytometry

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Objective: Antisynthetase syndrome (ASyS) and dermatomyositis (DM) are autoimmune disorders that overlap clinically. Given the presence of DM-like skin lesions in ASyS patients, there is debate about whether ASyS is a distinct disease or a subclassification of DM. Recent studies identified differences in type I interferon (IFN) expression between ASyS and DM muscle and finger eruptions. This study was undertaken to elucidate similarities and differences in the pathogenesis of cutaneous disease in ASyS and DM at the single-cell level. Methods: Five ASyS patients and 7 DM patients were recruited from a prospectively collected database of well-characterized DM patients. ASyS patients were clinically confirmed as having ASyS according to the Connors et al criteria and the Solomon et al criteria and the presence of aminoacyl–transfer RNA synthetase antibodies. Immunophenotyping was conducted using immunofluorescence (IF) and imaging mass cytometry (IMC). Results: IF staining for MxA and IFNβ expression revealed up-regulation of type I IFN in ASyS and DM samples compared to healthy control samples (P < 0.05). IMC showed similar numbers of macrophages, T cells, B cells, and dendritic cells in ASyS and DM samples, with no differences in counts (P > 0.05), but an increase in myeloid dendritic cell percentage in DM samples (P < 0.05). Key type I IFN, cytokine, and JAK/STAT pathways were similarly expressed in both ASyS and DM (P > 0.05). At the single-cell level, macrophages positive for phosphorylated stimulator of IFN genes in ASyS samples expressed increased levels of tumor necrosis factor, interluekin-17 (IL-17), and IFNβ (P < 0.001). Conclusion: IMC is a powerful tool that identifies a role for the type I IFN system in DM-like skin lesions in ASyS and DM with some differences at the cellular level, but overall significant overlap, supporting similar therapeutic decision making.

Jay Patel, Adarsh Ravishankar, Spandana Maddukuri, Thomas Vazquez, Madison Grinnell, Victoria P. Werth

Jay Patel, Adarsh Ravishankar, Spandana Maddukuri... et al. Arthritis & Rheumatology
2022 Central nervous system immune interactome is a function of cancer lineage, tumor microenvironment, and STAT3 expression

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BACKGROUND. Immune cell profiling of primary and metastatic CNS tumors has been focused on the tumor, not the tumor microenvironment (TME), or has been analyzed via biopsies. METHODS. En bloc resections of gliomas (n = 10) and lung metastases (n = 10) were analyzed via tissue segmentation and high-dimension Opal 7-color multiplex imaging. Single-cell RNA analyses were used to infer immune cell functionality. RESULTS. Within gliomas, T cells were localized in the infiltrating edge and perivascular space of tumors, while residing mostly in the stroma of metastatic tumors. CD163+ macrophages were evident throughout the TME of metastatic tumors, whereas in gliomas, CD68+, CD11c+CD68+, and CD11c+CD68+CD163+ cell subtypes were commonly observed. In lung metastases, T cells interacted with CD163+ macrophages as dyads and clusters at the brain-tumor interface and within the tumor itself and as clusters within the necrotic core. In contrast, gliomas typically lacked dyad and cluster interactions, except for T cell CD68+ cell dyads within the tumor. Analysis of transcriptomic data in glioblastomas revealed that innate immune cells expressed both proinflammatory and immunosuppressive gene signatures. CONCLUSION. Our results show that immunosuppressive macrophages are abundant within the TME and that the immune cell interactome between cancer lineages is distinct. Further, these data provide information for evaluating the role of different immune cell populations in brain tumor growth and therapeutic responses.

Hinda Najem, Martina Ott, Cynthia Kassab, Arvind Rao, Ganesh Rao, Anantha Marisetty, Adam M. Sonabend, Craig Horbinski, Roel Verhaak, Anand Shankar, Santhoshi N. Krishnan, Frederick S. Varn, Víctor A. Arrieta, Pravesh Gupta, Sherise D. Ferguson, Jason T. Huse, Gregory N. Fuller, James P. Long, Daniel E. Winkowski, Ben A. Freiberg, Charles David James, Leonidas C. Platanias, Maciej S. Lesniak, Jared K. Burks, Amy B. Heimberger

Hinda Najem, Martina Ott, Cynthia Kassab... et al. JCI Insight
2023 Artificial Intelligence-Aided Diagnosis of Breast Cancer Lymph Node Metastasis on Histologic Slides in a Digital Workflow

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Identifying lymph node (LN) metastasis in invasive breast carcinoma (IBC) can be tedious and time consuming. We investigated an artificial intelligence (AI) algorithm to detect LN metastasis by screening H&E slides in a clinical digital workflow. The study included two sentinel LN (SLN) cohorts (validation cohort with 234 SLNs and consensus cohort with 102 SLNs) and one non-sentinel LN (NSLN) cohort (258 LNs enriched with lobular carcinoma and post-neoadjuvant therapy cases). All H&E slides were scanned into whole slide images (WSI) in clinical digital workflow and WSIs were automatically batch analyzed using Visiopharm (VIS) metastasis AI algorithm. For SLN validation cohort, VIS metastasis AI algorithm detected all 46 metastases including 19 macrometastases, 26 micrometastases, 1 with isolated tumor cells (ITC) with a sensitivity of 100%, specificity of 41.5%, positive predictive value (PPV) of 29.5% and negative predictive value (NPV) of 100%. The false positivity was caused by histiocytes (52.7%), crushed lymphocytes (18.2%), and others, which were readily recognized during pathologists' reviews. For SLN consensus cohort, three pathologists examined all VIS AI annotated H&E slides and cytokeratin IHC slides with similar average concordance rates (99% for both modalities). However, the average time consumed by pathologists using VIS AI annotated slides was significantly less than the time using IHC slides (0.6 vs 1.0 minute, p=0.0377). For NSLN cohort, AI algorithm detected all 81 metastases, including 23 from lobular carcinoma and 31 from post-neoadjuvant chemotherapy cases with sensitivity of 100%, specificity of 78.5%, PPV of 68.1%, and NPV of 100%. VIS AI algorithm showed perfect sensitivity and NPV in detecting LN metastasis and less time consumed, suggesting its potential utility as a screening modality in routine clinical digital pathology workflow to improve efficiency.

Bindu Challa, Maryam Tahir, Yan Hu, David Kellough, Giovani Lujan, Shaoli Sun, Anil V. Parwani, Zaibo Li

Bindu Challa, Maryam Tahir, Yan Hu... et al. Modern Pathology
2023 Multiplex Immunochromogenic Tissue Staining Employing Primary Antibodies from the Same Species

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Chromogenic immunohistochemistry (IHC) serves as an essential assay for the diagnoses of many diseases including cancer. Single-marker IHC detection is the standard used for clinical diagnostic assays. A technology to stain multiple biomarkers chromogenically on a single tissue will also yield contextual biomarker information. Methods to chromogenically stain multiple biomarkers simultaneously employing antibodies from the same species are limited and require complex protocols. Here we describe both manual and automated protocols using the UltraPlex™ mxIHC technology that allows simultaneous detection of up to three biomarkers on a single tissue using a single heat-induced antigen retrieval step in formaldehyde-fixed paraffin-embedded (FFPE) tissue and using primary antibodies from any species.

Matthew Levin, Zohreh AkhavanAghdam, David Schwartz

Matthew Levin, Zohreh AkhavanAghdam, David Schwartz Methods in Molecular Biology
2023 Expansion of Phenotypically Altered Dendritic Cell Populations in the Small Airways and Alveolar Parenchyma in Patients with Chronic Obstructive Pulmonary Disease

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Contrasting the antigen-presenting dendritic cells (DCs) in the conducting airways, the alveolar DC populations in human lungs have remained poorly investigated. Consequently , little is known about how alveolar DCs are altered in diseases such as chronic obstructive pulmonary disease (COPD). This study maps multiple tissue DC categories in the distal lung across COPD severities. Specifically, single-multiplex immunohistochemistry was applied to quantify langerin/ CD207 + , CD1a + , BDCA2 + , and CD11c + subsets in distal lung compartments from patients with COPD (GOLD stage I-IV) and never-smoking and smoking controls. In the alveolar pa-renchyma, increased numbers of CD1a + langerin − (p < 0.05) and BDCA-2 + DCs (p < 0.001) were observed in advanced COPD compared with controls. Alveolar CD11c + DCs also increased in advanced COPD (p < 0.01). In small airways, lan-gerin + and BDCA-2 + DCs were also significantly increased. Contrasting the small airway DCs, most alveolar DC subsets frequently extended luminal protrusions. Importantly, alve-olar and small airway langerin + DCs in COPD lungs displayed site-specific marker profiles. Further, multiplex immunohis-tochemistry with single-cell quantification was used to specifically profile langerin DCs and reveal site-specific expression patterns of the maturation and activation markers S100, fascin, MHC2, and B7. Taken together, our results show that clinically advanced COPD is associated with increased levels of multiple alveolar DC populations exhibiting features of both adaptive and innate immunity phenotypes. This expansion is likely to contribute to the distal lung immunopathol-ogy in COPD patients.

Michiko Mori, Carl-Magnus Clausson, Caroline Sanden, Jimmie Jönsson, Cecilia K Andersson, Premkumar Siddhuraj, Medya Shikhagaie, Karolina Åkesson, Anders Bergqvist, Claes-Göran Löfdahl, Jonas S Erjefält

Michiko Mori, Carl-Magnus Clausson, Caroline Sanden... et al. Research Article J Innate Immun
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