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Genetic instability is known to drive colorectal carcinogenesis. Generally, a distinction is made between two types of genetic instability: chromosomal instability (CIN) and microsatellite instability (MIN or MSI). Most CIN tumours are aneuploid, whereas MSI tumours are considered near-diploid. However, for MUTYH-associated polyposis (MAP) the genetic instability involved in the carcinogenesis remains unclear, as near-diploid adenomas, aneuploid adenomas and near-diploid carcinomas have been reported. Remarkably, our analysis of 26 MAP carcinomas, using SNP arrays and flow sorting, showed that these tumours are often near-diploid (52%) and mainly contain chromosomal regions of copy-neutral loss of heterozygosity (LOH) (71%). This is in contrast to sporadic colon cancer, where physical loss is the main characteristic. The percentage of chromosomal gains (24%) is comparable to sporadic colorectal cancers with CIN. Furthermore, we verified our scoring of copy-neutral LOH versus physical loss in MAP carcinomas by two methods: fluorescence in situ hybridization, and LOH analysis using polymorphic markers on carcinoma fractions purified by flow sorting. The results presented in this study suggest that copy-neutral LOH is an important mechanism in the tumorigenesis of MAP. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Objectives: To provide an outline of the existing data on penile intraepithelial neoplasia (PeIN), as well as a narrative review on imiquimod (IQ; a toll-like receptor 7 agonist) treatment and immune microenvironment markers that may predict response to treatment., Methods: A narrative review of the literature from 2000 to the present was conducted on PubMed, and we describe the most relevant data and cross references., Results: The incidence of PeIN is increasing. Local therapy with IQ may offer an easy applicable treatment with complete response rates of up to 63% but can be associated with considerable side-effects. There is no conclusive data on the optimal treatment schedule for PeIN, but evaluation of treatment results for other human papillomavirus-related pre-malignancies suggest three times a week for a duration up to 16 weeks. There are no published studies concerning the PeIN immune microenvironment. However, findings from the few studies on penile cancer and pre-cancerous vulvar and cervical lesions imply that specific immune cell subpopulations can serve as future predictors for successful immunomodulation treatments such as IQ., Conclusions: Overall, limited data are available on IQ treatment for PeIN and no published data exists on the PeIN immune microenvironment. Further translational studies are warranted to gain more understanding on the pathophysiology of PeIN and potential predictors of progression and of response to topical treatments., (© 2024 BJU International.)

Purpose: Patients with advanced penile squamous cell carcinoma have a poor prognosis (21% 2-year overall survival [OS] from diagnosis). We assessed the activity of atezolizumab (anti-PD-L1) in patients with advanced penile cancer, with or without radiotherapy (RT)., Patients and Methods: A single-center, nonrandomized phase II study with two treatment arms was conducted in 32 patients with histologically confirmed advanced penile cancer. All patients received atezolizumab (1,200 mg) once every 3 weeks. Twenty patients, who were expected to benefit from RT for locoregional disease control, received additional irradiation. The primary end point was 1-year progression-free survival (PFS) for the complete cohort and was reached if the actual 1-year PFS was at least 35%. Secondary end points included OS, objective response rate (ORR), and tolerability. Exploratory biomarker analyses were conducted in pretreatment specimens., Results: Median follow-up was 29.1 months (IQR, 18.1-33.5). Grade 3-4 adverse events related to atezolizumab or RT were observed in 3/32 (9.4%) and 13/20 (65%) patients, respectively. One-year PFS was 12.5% (95% CI, 5.0 to 31.3), which did not meet the study's primary end point. Median OS was 11.3 months (95% CI, 5.5 to 18.7). In the objective response-evaluable population (n = 30; 93.8%), the ORR was 16.7% (95% CI, 6 to 35), including 2 (6.7%) complete responders and 3 (10%) partial responders. Improved PFS was observed in patients with high-risk human papillomavirus (hrHPV)-positive tumors ( P = .003) and those with high infiltration of intratumoral CD3 + CD8 + T cells ( P = .037)., Conclusion: Although the primary end point of 1-year PFS was not met, durable antitumor activity to atezolizumab was observed in a subset of patients. Biomarkers, such as hrHPV and intratumoral CD3 + CD8 + T-cell infiltration, may help to better select responders.

Purpose: Ongoing angiogenesis renders the tumor endothelium unresponsive to inflammatory cytokines and interferes with adhesion of leukocytes, resulting in escape from immunity. This process is referred to as tumor endothelial cell anergy. We aimed to investigate whether anti-angiogenic agents can overcome endothelial cell anergy and provide pro-inflammatory conditions., Experimental Design: Tissues of renal cell carcinoma (RCC) patients treated with VEGF pathway-targeted drugs and control tissues were subject to RNAseq and immunohistochemical profiling of the leukocyte infiltrate. Analysis of adhesion molecule regulation in cultured endothelial cells, in a preclinical model and in human tissues was performed and correlated to leukocyte infiltration., Results: It is shown that treatment of RCC patients with the drugs sunitinib or bevacizumab overcomes tumor endothelial cell anergy. This treatment resulted in an augmented inflammatory state of the tumor, characterized by enhanced infiltration of all major leukocyte subsets, including T cells, regulatory T cells, macrophages of both M1- and M2-like phenotypes and activated dendritic cells. In vitro, exposure of angiogenic endothelial cells to anti-angiogenic drugs normalized ICAM-1 expression. In addition, a panel of tyrosine kinase inhibitors was shown to increase transendothelial migration of both non-adherent and monocytic leukocytes. In primary tumors of RCC patients, ICAM-1 expression was found to be significantly increased in both the sunitinib and bevacizumab-treated groups. Genomic analysis confirmed the correlation between increased immune cell infiltration and ICAM-1 expression upon VEGF-targeted treatment., Conclusion: The results support the emerging concept that anti-angiogenic therapy can boost immunity and show how immunotherapy approaches can benefit from combination with anti-angiogenic compounds., (© 2022. The Author(s).)

Introduction: CD4+ T cells are thought to have a central role in the pathogenesis of autoimmune hepatitis (AIH). Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) directs homing of CD4+ T cells in the alimentary tract and is a therapeutic target in inflammatory bowel diseases. Here we assessed MAdCAM-1 expression in AIH and viral hepatitis and related its expression with immune infiltrate analysis and histopathological key features., Methods: Hepatic portal areas of pretreatment biopsies (n=10) and follow-up biopsies (n=9) of patients with a confirmed diagnosis of AIH were assessed for MAdCAM-1 expression and infiltrate composition using immunohistochemistry and multispectral imaging (Vectra® Polaris™). Controls consisted of biopsies of patients with untreated chronic viral hepatitis B or C (n=22)., Results: MAdCAM-1 expression on endothelium was sparsely present in portal fields of two treatment-naïve AIH patients. Three patients showed MAdCAM-1 expression within lymphoid aggregates. No expression of significance (including single-cell expression) was observed in the remaining 6 patients. In contrast, viral hepatitis C biopsies showed endothelial MAdCAM-1 expression in 8 of 13 untreated patients. Densities of both B-cells (CD20+) and CD4+ T-cells (CD3+ CD8-) were increased in AIH and viral hepatitis patients with MAdCAM-1 expression., Conclusion: MAdCAM-1 was detected in liver biopsies in a minority of patients with AIH at the time of diagnosis suggesting no central role in its pathophysiology. Lymphoid or reticular MAdCAM-1 pattern expression was associated with more dense infiltrates of both B-cells and CD4 + T-cells, and may be related to the formation of secondary lymphoid follicles., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Although various neurodegenerative disorders have been associated with coeliac disease (CD), the underlying neuropathological link between these brain and gut diseases remains unclear. We postulated that the neuronal damage sporadically observed in CD patients is immune-mediated. Our aim was to determine if the loss of neurons, especially Purkinje cells, coincides with microglia activation and T- and B-cell infiltration in the cerebellum of patients with CD and a concomitant idiopathic neurological disease affecting the cerebellum (NeuroCD). Post-mortem cerebellar tissue was collected of validated NeuroCD cases. Gender- and age-matched genetic spinocerebellar ataxia (SCA) controls and non-neurological controls (NNC) were selected based on clinical reports and pathological findings. Cerebellar tissue of seventeen patients was included (6 NeuroCD, 5 SCA, 6 NNC). In SCA cases we found that the Purkinje cell layer was 58.6% reduced in comparison with NNC. In NeuroCD cases this reduction was even more prominent with a median reduction of 81.3% compared to NNC. Marked increased numbers of both CD3+ and CD8+ cells were observed in the NeuroCD but not in SCA patients. This coincided with significantly more microglial reactivity in NeuroCD patients. These findings demonstrate that the massive loss of Purkinje cells in the cerebellum of neuro CD patients is accompanied by local innate and T-cell mediated immune responses., (© 2023. The Author(s).)

There is evidence for increased angiogenesis in the (ectopic) endometrium of adenomyosis patients under the influence of vascular endothelial growth factor (VEGF). VEGF stimulates both angiogenesis and lymph-angiogenesis. However, information on lymph vessels in the (ectopic) endometrium of adenomyosis patients is lacking. In this retrospective matched case-control study, multiplex immunohistochemistry was performed on thirty-eight paraffin embedded specimens from premenopausal women who had undergone a hysterectomy at the Amsterdam UMC between 2001 and 2018 to investigate the evidence for (lymph) angiogenesis in the (ectopic) endometrium or myometrium of patients with adenomyosis versus controls with unrelated pathologies. Baseline characteristics of both groups were comparable. In the proliferative phase, the blood and lymph vessel densities were, respectively, higher in the ectopic and eutopic endometrium of patients with adenomyosis than in the endometrium of controls. The relative number of blood vessels without α-smooth muscle actinin (α SMA) was higher in the eutopic and ectopic endometrium of adenomyosis patients versus controls. The level of VEGF staining intensity was highest in the myometrium but did not differ between patients with adenomyosis or controls. The results indicate increased angiogenesis and lymphangiogenesis in the (ectopic) endometrium affected by adenomyosis. The clinical relevance of our findings should be confirmed in prospective clinical studies.

According to recent studies and observations in clinical practice, uterine fibroids increase the risk of preterm birth. There are several theories on the pathogenesis of preterm birth in the presence of fibroids. One theory proclaims that fibroid necrosis leads to preterm birth, though pathophysiological mechanisms have not been described. Necrotic tissue secretes specific cytokines and proteins and we suggest these to be comparable to the inflammatory response leading to spontaneous preterm birth. We hypothesize that fibroid necrosis could induce preterm parturition through a similar inflammatory response. This new hypothesis generates novel perspectives for future research and the development of preventative strategies for preterm birth. Moreover, we emphasize the importance of the recognition of fibroids and especially fibroid necrosis by clinicians during pregnancy.

Cervical cancer is the fourth most common cancer in women worldwide. Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are the most common histological types, with AC patients having worse prognosis. Over the last two decades, incidence rates of AC have increased, highlighting the importance of further understanding AC tumorigenesis, and the need to investigate new treatment options. The cytokine TGF-β functions as a tumour suppressor in healthy tissue. However, in tumour cells this suppressive function can be overcome. Therefore there is an increasing interest in using TGF-β inhibitors in the treatment of cancer. Here, we hypothesize that TGF-β plays a different role in SCC and AC. Analysis of RNA-seq data from the TCGA, using a TGF-β response signature, resulted in separate clustering of the two subtypes. We further investigated the expression of TGF-β-signalling related proteins (TβR1/2, SMAD4, pSMAD2, PAI-1, αvβ6 and MMP2/9) in a cohort of 62 AC patients. Low TβR2 and SMAD4 expression was associated with worse survival in AC patients and interestingly, high PAI-1 and αvβ6 expression was also correlated with worse survival. Similar correlations of TβR2, PAI-1 and αvβ6 with clinical parameters were found in previously reported SCC analyses. However, when comparing expression levels between SCC and AC patient samples, pSMAD2, SMAD4, PAI-1 and αvβ6 showed lower expression in AC compared to SCC. Because of the low expression of core TβR1/2, (p-)SMAD2 and SMAD4 proteins and the correlation with worse prognosis, TGF-β pathway most likely leads to tumour inhibitory effects in AC and therefore the use of TGF-β inhibitors would not be recommended. However, given the correlation of PAI-1 and αvβ6 with poor prognosis, the use of TGF- β inhibitors might be of interest in SCC and in the subsets of AC patients with high expression of these TGF-β associated proteins., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Marvin, Spaans, de Kroon, Slieker, Khelil, ten Dijke, Ritsma and Jordanova.)

Competing Interests: We declare no competing interests.

Cervical cancer remains a public health concern despite all the efforts to implement vaccination and screening programs. Conventional treatment for locally advanced cervical cancer consists of surgery, radiotherapy (with concurrent brachytherapy), combined with chemotherapy, or hyperthermia. The response rate to combination approaches involving immunomodulatory agents and conventional treatment modalities have been explored but remain dismal in patients with locally advanced disease. Studies exploring the immunological effects exerted by combination treatment modalities at the different levels of the immune system (peripheral blood (PB), tumor-draining lymph nodes (TDLN), and the local tumor microenvironment (TME)) are scarce. In this systemic review, we aim to define immunomodulatory and immunosuppressive effects induced by conventional treatment in cervical cancer patients to identify the optimal time point for immunotherapy administration. Radiotherapy (RT) and chemoradiation (CRT) induce an immunosuppressive state characterized by a long-lasting reduction in peripheral CD3, CD4, CD8 T cells and NK cells. At the TDLN level, CRT induced a reduction in Nrp1+Treg stability and number, naïve CD4 and CD8 T cell numbers, and an accompanying increase in IFNγ-producing CD4 helper T cells, CD8 T cells, and NK cells. Potentiation of the T-cell anti-tumor response was particularly observed in patients receiving low irradiation dosage. At the level of the TME, CRT induced a rebound effect characterized by a reduction of the T-cell anti-tumor response followed by stable radioresistant OX40 and FoxP3 Treg cell numbers. However, the effects induced by CRT were very heterogeneous across studies. Neoadjuvant chemotherapy (NACT) containing both paclitaxel and cisplatin induced a reduction in stromal FoxP3 Treg numbers and an increase in stromal and intratumoral CD8 T cells. Both CRT and NACT induced an increase in PD-L1 expression. Although there was no association between pre-treatment PD-L1 expression and treatment outcome, the data hint at an association with pro-inflammatory immune signatures, overall and disease-specific survival (OS, DSS). When considering NACT, we propose that posterior immunotherapy might further reduce immunosuppression and chemoresistance. This review points at differential effects induced by conventional treatment modalities at different immune compartments, thus, the compartmentalization of the immune responses as well as individual patient's treatment plans should be carefully considered when designing immunotherapy treatment regimens.

Rationale: von Willebrand factor (vWF) mediates platelet adhesion during thrombosis. While chronic thromboembolic pulmonary hypertension (CTEPH) is associated with increased plasma levels of vWF, the role of this protein in CTEPH has remained enigmatic. Objectives: To identify the role of vWF in CTEPH. Methods: CTEPH-specific patient plasma and pulmonary endarterectomy material from patients with CTEPH were used to study the relationship between inflammation, vWF expression, and pulmonary thrombosis. Cell culture findings were validated in human tissue, and proteomics and chromatin immunoprecipitation were used to investigate the underlying mechanism of CTEPH. Measurements and Main Results: vWF is increased in plasma and the pulmonary endothelium of CTEPH patients. In vitro , the increase in vWF gene expression and the higher release of vWF protein upon endothelial activation resulted in elevated platelet adhesion to CTEPH endothelium. Proteomic analysis revealed that nuclear factor (NF)-κB2 was significantly increased in CTEPH. We demonstrate reduced histone tri-methylation and increased histone acetylation of the vWF promoter in CTEPH endothelium, facilitating binding of NF-κB2 to the vWF promoter and driving vWF transcription. Genetic interference of NFκB2 normalized the high vWF RNA expression levels and reversed the prothrombotic phenotype observed in CTEPH-pulmonary artery endothelial cells. Conclusions: Epigenetic regulation of the vWF promoter contributes to the creation of a local environment that favors in situ thrombosis in the pulmonary arteries. It reveals a direct molecular link between inflammatory pathways and platelet adhesion in the pulmonary vascular wall, emphasizing a possible role of in situ thrombosis in the development or progression of CTEPH.

Human papillomavirus (HPV) infection drives tumorigenesis in almost all cervical cancers and a fraction of vulvar and penile cancers. Due to increasing incidence and low vaccination rates, many will still have to face HPV-related morbidity and mortality in the upcoming years. Current treatment options (i.e., surgery and/or chemoradiation) for urogenital (pre-)malignancies can have profound psychosocial and psychosexual effects on patients. Moreover, in the setting of advanced disease, responses to current therapies remain poor and nondurable, highlighting the unmet need for novel therapies that prevent recurrent disease and improve clinical outcome. Immunotherapy can be a useful addition to the current therapeutic strategies in various settings of disease, offering relatively fewer adverse effects and potential improvement in survival. This review discusses immune evasion mechanisms accompanying HPV infection and HPV-related tumorigenesis and summarizes current immunotherapeutic approaches for the treatment of HPV-related (pre-)malignant lesions of the uterine cervix, vulva, and penis.

Purpose: Almost all cervical cancers are caused by human papillomavirus (HPV) and patients with advanced stage are at high risk for relapse. Circulating HPV DNA (HPV ctDNA) may serve as a residual tumor marker at the end of chemoradiation or to predict relapse during the follow-up period., Experimental Design: We analyzed serum samples from 94 HPV16- or HPV18-related CCs from the BioRAIDs prospective cohort. Samples were collected before and after treatment and during an 18-month follow-up period. Using digital droplet PCR (ddPCR), we assessed the relevance of circulating HPV E7 gene as a marker for residual disease compared to HPV integration site and PIK3CA mutations. Finally, the prognostic impact of circulating HPV E7 gene was assessed with its prediction value of relapse., Results: HPV E7 gene was the most sensitive tumor marker, superior to both HPV integration sites and PIK3CA mutations in serum. Circulating HPV DNA (HPV ctDNA) was detected in 63% (59/94) of patients, before treatment. HPV ctDNA detection in serum sample was associated with high FIGO stage ( P = 0.02) and para-aortic lymph node involvement ( P = 0.01). The level of HPV ctDNA was positively correlated with HPV copy number in the tumor ( R = 0.39, P < 0.001). Complete clearance of HPV ctDNA by the end of treatment was significantly associated with a longer PFS ( P < 0.0001). Patients with persistent HPV ctDNA in serum relapsed with a median time of 10 months (range, 2-15) from HPV ctDNA detection., Conclusions: HPV ctDNA detection is a useful marker to predict relapse in cervical cancer. See related commentary by Wentzensen and Clarke, p. 5733 ., (©2021 The Authors; Published by the American Association for Cancer Research.)

Esophageal adenocarcinoma (EAC) is a disease with dismal treatment outcomes. Response to neoadjuvant chemoradiation (CRT) varies greatly. Although the underlying mechanisms of CRT resistance are not identified, accumulating evidence indicates an important role for local antitumor immunity. To explore the immune microenvironment in relation to response to CRT we performed an in-depth analysis using multiplex immunohistochemistry, flow cytometry and mRNA expression analysis (NanoString) to generate a detailed map of the immunological landscape of pretreatment biopsies as well as peripheral blood mononuclear cells (PBMCs) of EAC patients. Response to CRT was assessed by Mandard's tumor regression grade (TRG), disease-free- and overall survival. Tumors with a complete pathological response (TRG 1) to neoadjuvant CRT had significantly higher tumor-infiltrating T cell levels compared to all other response groups (TRG 2-5). These T cells were also in closer proximity to tumor cells in complete responders compared to other response groups. Notably, immune profiles of near-complete responders (TRG 2) showed more resemblance to non-responders (TRG 3-5) than to complete responders. A high CD8:CD163 ratio in the tumor was associated with an improved disease-free survival. Gene expression analyses revealed that T cells in non-responders were Th2-skewed, while complete responders were enriched in cytotoxic immune cells. Finally, complete responders were enriched in circulating memory T cells. preexisting immune activation enhances the chance for a complete pathological response to neoadjuvant CRT. This information can potentially be used for future patient selection, but also fuels the development of immunomodulatory strategies to enhance CRT efficacy., Competing Interests: Adam Bass has research support from Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, U.S.A, which has also provided financial support for the study. Tanja D. de Gruijl has a consultant or advisory role in DCprime, Macrophage Pharma, Partner Tx, is a co-founder of LAVA Therapeutics, and receives research funding from Idera Pharmaceuticals. Mark I. van Berge Henegouwen has a consultant role for Medtronic, Johnson and Johnson, Alesi Surgical and Mylan. Unrestricted research funds from Olympus and Stryker. All fees are paid to the institution. Hanneke W.M. van Laarhoven has consultant or advisory role in BMS, Lilly, MSD, Nordic Pharma, Servier and receives research funding/medication: Bayer, BMS, Celgene, Janssen, Lilly, Merck, Nordic Pharma, Philips, Roche, Servier. Johannes J. van der Vliet is CSO at Lava Therapeutics., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Usual vulvar intraepithelial neoplasia (uVIN) is a premalignancy caused by persistent infection with high-risk types of human papillomavirus (HPV), mainly type 16. Even though different treatment modalities are available (eg, surgical excision, laser evaporation or topical application of imiquimod), these treatments can be mutilating, patients often have recurrences and 2%-8% of patients develop vulvar carcinoma. Therefore, immunotherapeutic strategies targeting the pivotal oncogenic HPV proteins E6 and E7 are being explored to repress carcinogenesis., Method: In this phase I/II clinical trial, 14 patients with HPV16+ uVIN were treated with a genetically enhanced DNA vaccine targeting E6 and E7. Safety, clinical responses and immunogenicity were assessed. Patients received four intradermal HPV-16 E6/E7 DNA tattoo vaccinations, with a 2-week interval, alternating between both upper legs. Biopsies of the uVIN lesions were taken at screening and +3 months after last vaccination. Digital photography of the vulva was performed at every check-up until 12 months of follow-up for measurement of the lesions. HPV16-specific T-cell responses were measured in blood over time in ex vivo reactivity assays., Results: Vaccinations were well tolerated, although one grade 3 suspected unexpected serious adverse reaction was observed. Clinical responses were observed in 6/14 (43%) patients, with 2 complete responses and 4 partial responses (PR). 5/14 patients showed HPV-specific T-cell responses in blood, measured in ex vivo reactivity assays. Notably, all five patients with HPV-specific T-cell responses had a clinical response., Conclusions: Our results indicate that HPV-16 E6/E7 DNA tattoo vaccination is a biologically active and safe treatment strategy in patients with uVIN, and suggest that T-cell reactivity against the HPV oncogenes is associated with clinical benefit., Trial Registration Number: NTR4607., Competing Interests: Competing interests: JB is (part time) employee of Modra Pharmaceuticals and stockholder in Modra Pharmaceuticals. (not related to the manuscript). TS is advisor for Adaptive Biotechnologies, Allogene Therapeutics, Merus, Neogene Therapeutics, and Scenic Biotech; is a recipient of research support from Merck KgaA; is a stockholder in Allogene Therapeutics, Merus, Neogene Therapeutics and Scenic Biotech; and is venture partner at Third Rock Ventures, all not related to the current work. JH is advisor to Achilles Therapeutics, Bristol-Myers Squibb, BioNTech USA, Ipsen, Gadeta, Immunocore, MSD, Merck Serono, Molecular Partners, Neogene Therapeutics, Novartis, Pfizer, Roche/Genentech, Sanofi, Seattle Genetics, Third Rock Ventures, is stock holder in Neogene Therapeutics, and is a recipient of grant or research support from Bristol-Myers Squibb, MSD, Novartis and BioNTech USA. KDV reports research funding from Roche and is consultant for Third Rock Ventures, all outside the scope of this work. TDdG has served as advisor to TILT Biotherapeutics, LAVA Therapeutics, Macrophage Pharma and DCPrime, is a recipient of a grant from Idera Pharmaceuticals, and is co-founder and shareholder of LAVA Therapeutics. JHvdB is a recipient of grant or research support from BioNTech USA and Astra Zeneca, and is currently employed at CellPoint B.V., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Comprehensive analysis of tumor infiltrating myeloid cells in the tumor microenvironment of penile squamous cell carcinoma (PSCC) is lacking. In this retrospective study, for the first time, PSCC resection specimens (N = 103) were annotated into the following compartments: intratumoral tumor (IT Tumor), intratumoral stroma (IT Stroma), peritumoral tumor (PT Tumor) and peritumoral stroma (PT Stroma) compartments. We then quantified CD14+, CD68+ and CD163+ myeloid cells within these compartments using an image analysis software and assessed their association with various clinical parameters, including high-risk human papillomavirus (hrHPV) status. In the total cohort, hrHPV status, grade of differentiation, age and tumor size were associated with myeloid cell densities. hrHPV + tumors had higher infiltration rates of CD14+, CD68+ and CD163+ myeloid cells in the IT tumor compartment (p < 0.001, for all) compared to hrHPV - tumors. Furthermore, when examining the association between compartment-specific infiltration and differentiation grade, increased myeloid cell densities in the IT tumor compartment were associated with a more advanced histological grade (p < 0.001, for all). This association remained significant when the hrHPV - cohort (N = 60) was analyzed (CD14+ p = 0.001; CD68+ p < 0.001; CD163+ p = 0.004). Subgroup analysis in the hrHPV + group (N = 43) showed that high infiltration rates of CD68+ and CD163+ cells in the PT tumor compartment were associated with lymph node (LN) metastasis (p = 0.031 and p = 0.026, respectively). Regarding the association between myeloid cell densities and disease-specific survival, the risk of death was found to decrease slightly as the number of myeloid cells in the IT tumor compartment increased (CD14+ p = 0.04; CD68+ p = 0.05; CD163+ p = 0.02). However, after adjusting for hrHPV, no independent association between myeloid densities and disease-specific survival were found. Altogether, these findings demonstrate the importance of assessing myeloid cell densities within the spatial context of the tumor. Further studies are needed to unravel the specific phenotype of myeloid cells residing in the different compartments, their effect on clinical parameters and the impact of hrHPV on the recruitment of myeloid cell populations in PSCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rafael, de Vries, Ottenhof, Hofland, Broeks, de Jong, Bekers, Horenblas, de Menezes, Jordanova and Brouwer.)

Human papillomavirus (HPV) drives high-grade intraepithelial neoplasia and cancer; for unknown reasons, this occurs most often in the cervical transformation zone. Either mutation or HPV E6-driven inhibition of Notch1 can drive neoplastic development in stratified squamous epithelia. However, the contribution of Notch1 and its Delta-like ligands (DLL) to site susceptibility remains poorly understood. Here, we map DLL1/DLL4 expression in cell populations present in normal cervical biopsies by immunofluorescence. In vitro keratinocyte 2D monolayer models, growth assays, and organotypic raft cultures were used to assess the functional role of DLL-Notch signaling in uninfected cells and its modulation by HPV16 in neoplasia. An RNA sequencing-based gene signature was used to suggest the cell of origin of 279 HPV-positive cervical carcinomas from The Cancer Genome Atlas and to relate this to disease prognosis. Finally, the prognostic impact of DLL4 expression was investigated in three independent cervical cancer patient cohorts. Three molecular cervical carcinoma subtypes were identified, with reserve cell tumors the most common and linked to relatively good prognosis. Reserve cells were characterized as DLL1 - /DLL4 + , a proliferative phenotype that is temporarily observed during squamous metaplasia and wound healing but appears to be sustained by HPV16 E6 in raft models of low-grade and, more prominently, high-grade neoplasia. High expression of DLL4 was associated with an increased likelihood of cervical cancer-associated death and recurrence. Taken together, DLL4-Notch1 signaling reflects a proliferative cellular state transiently present during physiologic processes but inherent to cervical reserve cells, making them strongly resemble neoplastic tissue even before HPV infection has occurred. SIGNIFICANCE: This study investigates cervical cancer cell-of-origin populations and describes a DLL-Notch1 phenotype that is associated with disease prognosis and that might help identify cells that are susceptible to HPV-induced carcinogenesis., (©2021 American Association for Cancer Research.)

Background: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality with infection by human papilloma virus (HPV) being the most important risk factor. We analysed the association between different viral integration signatures, clinical parameters and outcome in pre-treated CCs., Methods: Different integration signatures were identified using HPV double capture followed by next-generation sequencing (NGS) in 272 CC patients from the BioRAIDs study [NCT02428842]. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed., Results: Episomal HPV was much less frequent in CC as compared to anal carcinoma (p < 0.0001). We identified >300 different HPV-chromosomal junctions (inter- or intra-genic). The most frequent integration site in CC was in MACROD2 gene followed by MIPOL1/TTC6 and TP63. HPV integration signatures were not associated with histological subtype, FIGO staging, treatment or PFS. HPVs were more frequently episomal in PIK3CA mutated tumours (p = 0.023). Viral integration type was dependent on HPV genotype (p < 0.0001); HPV18 and HPV45 being always integrated. High HPV copy number was associated with longer PFS (p = 0.011)., Conclusions: This is to our knowledge the first study assessing the prognostic value of HPV integration in a prospectively annotated CC cohort, which detects a hotspot of HPV integration at MACROD2; involved in impaired PARP1 activity and chromosome instability.

PD-1/PD-L1 immune checkpoint inhibitors show potential for cervical cancer treatment. However, low response rates suggest that patient selection based on PD-L1 protein expression is not optimal. Here, we evaluated different PD-L1 detection methods and studied transcriptional regulation of PD-L1/PD-L2 expression by The Cancer Genome Atlas (TCGA) mRNAseq analysis. First, we determined the copy number of the PD-L1/PD-L2 locus by fluorescence in situ hybridization (FISH), PD-L1 mRNA expression by RNA in situ hybridization (RNAish), and PD-L1/PD-L2 protein expression by immunohistochemistry (IHC) on tissue microarrays containing a cohort of 60 patients. Additionally, distribution of PD-L1/PD-L2 was visualized based on flow cytometry analysis of single-cell suspensions (n = 10). PD-L1/PD-L2 locus amplification was rare (2%). PD-L1 mRNA expression in tumor cells was detected in 56% of cases, while 41% expressed PD-L1 protein. Discordant scores for PD-L1 protein expression on tumor cells between cores from one patient were observed in 27% of cases. Interestingly, with RNAish, PD-L1 heterogeneity was observed in only 11% of the cases. PD-L2 protein expression was found in 53%. PD-L1 mRNA and protein expression on tumor cells were strongly correlated (p < 0.001). PD-L1 and PD-L2 protein expression showed no correlation on tumor cells (p = 0.837), but a strong correlation on cells in stromal fields (p < 0.001). Co-expression of PD-L1 and PD-L2 on macrophage-like populations was also observed with flow cytometry analysis. Both PD-L1 and PD-L2 TCGA transcript levels strongly correlated in the TCGA data, and both PD-L1 and PD-L2 strongly correlated with interferon gamma (IFNG) expression/transcript levels (p < 0.0001). Importantly, patients with high PD-L1/PD-L2/IFNG transcript levels had a survival advantage over patients with high PD-L1/PD-L2 and low IFNG expression. Based on these findings, we conclude that PD-L1/PD-L2 expression in cervical cancer is mainly associated with interferon induction and not gene amplification, which makes FISH unsuitable as biomarker. The heterogeneous PD-L1 and PD-L2 expression patterns suggest IHC unreliable for patient selection. RNAish, in conjunction with interferon signaling evaluation, seems a promising technique for immune checkpoint detection. These results warrant further investigation into their prognostic and predictive potential., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Rotman, Otter, Bleeker, Samuels, Heeren, Roemer, Kenter, Zijlmans, van Trommel, de Gruijl and Jordanova.)

Preclinical assessment of novel therapies to fight cancer requires models that reflect the human physiology and immune response. Here, we established an in vitro three-dimensional (3D) reconstructed organotypic human melanoma-in-skin (Mel-RhS) model to investigate cellular and molecular features of tumor formation over a period of 6 weeks. Tumor nests developed over time at the epidermal-dermal junction and spread towards the dermis, in places disrupting the basement membrane. This coincided with secretion of matrix metalloproteinase 9 (MMP-9) by melanoma cells. These features resemble the initial stages of invasive melanoma. Interestingly, while the SK-MEL-28 cell line did not secrete detectable levels of interleukin-10 (IL-10) in traditional two-dimensional monolayers, it did express IL-10 in the 3D Mel-RhS, as did the surrounding keratinocytes and fibroblasts. This cellular cross-talk-induced secretion of IL-10 in the Mel-RhS indicated the generation of an immune suppressive microenvironment. Culture supernatants from Mel-RhS interfered with monocyte-to-dendritic-cell differentiation, leading to the development of M2-like macrophages, which was in part prevented by antibody-mediated IL-10 blockade. Indeed, high-dimensional single-cell analysis revealed a shift within the monocyte population away from a CD163 + PD-L1 + M2-like phenotype upon IL-10 blockade. Thus, the 3D configuration of the Mel-RhS model revealed a role for IL-10 in immune escape through misdirected myeloid differentiation, which would have been missed in classical monolayer cultures.

Peritoneal metastases of high-grade serous ovarian cancer (HGSOC) are small-sized deposits with superficial growth toward the peritoneal cavity. It is unknown whether integrity of the peritoneal elastic lamina (PEL) correlates with the peritoneal tumor microenvironment (pTME) and whether neoadjuvant chemotherapy (NACT) affects the pTME. We explored integrity of PEL, composition of pTME, effects of NACT, and the prognostic implications in patients with extensive peritoneal metastases of HGSOC. Peritoneal samples (n = 69) were collected during cytoreductive surgery between 2003 and 2016. Clinical data were collected from medical charts. Integrity of PEL was evaluated with elastic stains. T cell (CD3, CD8) and M2-macrophage markers (CD163) were scored using algorithms created in definiens tissue studio. Patients with a disrupted PEL (n = 39; 57%), more often had residual disease after surgery (p = 0.050), compared to intact PEL. An intact PEL was associated with increased intraepithelial (ie) CD8+ cells (p = 0.032), but was not correlated with improved survival. After NACT, increased ieCD3+ cells were shown, compared to no-NACT (p = 0.044). Abundance of total CD3+ and CD8+ cells were associated with PFS (multivariate HR 0.40; 95%CI 0.23-0.69 and HR 0.49; 95%CI 0.29-0.83) and OS (HR 0.33; 95%CI 0.18-0.62 and HR 0.36; 95%CI 0.20-0.64). M2-macrophage infiltration was not correlated with survival. NACT increases abundance of ieCD3+ cells in peritoneal metastases of HGSOC. Increase of CD3+ and CD8+ cells is associated with improved PFS and OS. This suggests that CD3+ and CD8+ cells may function as prognostic biomarkers. Their role as predictive biomarker for chemotherapy or immunotherapy response in HGSOC warrants further research.

Purpose: Adenocarcinoma of the uterine cervix is the second most common type of cervical cancer after squamous cell carcinoma (SCC). Although both subtypes are treated similarly, patients with adenocarcinoma have a worse prognosis. In this study, immunologic features of the tumor microenvironment in these two subsets were pursued with potential therapeutic implications., Experimental Design: The immune microenvironment of primary tumors and nonmetastatic tumor-draining lymph nodes (TDLN) was compared between patients with cervical adenocarcinoma ( n = 16) and SCC ( n = 20) by polychromatic flow cytometry and by transcriptional profiling of the primary tumors ( n = 299) using publicly available data from The Cancer Genome Atlas (TCGA)., Results: Flow cytometric analyses revealed intact T-cell differentiation in TDLNs, but hampered effector T-cell trafficking to the primary tumors in adenocarcinoma, as compared with SCC. TCGA analysis demonstrated higher expression of chemokines involved in effector T-cell homing (CXCL9/10/11) in SCC primary tumors as compared with adenocarcinoma primary tumors, which was highly correlated to a transcriptional signature for type I conventional dendritic cells (cDC1). This was consistent with elevated frequencies of CD141/BDCA3 + cDC1 in primary tumor SCC samples relative to adenocarcinoma and correspondingly elevated levels of CXCL9 and CXCL10 in 24-hour ex vivo cultures. Hampered cDC1 recruitment in adenocarcinoma was in turn related to lower transcript levels of cDC1-recruiting chemokines and an elevated β-catenin activation score and was associated with poor overall survival., Conclusions: Our data have identified an opportunity for the investigation of potentially novel therapeutic interventions in adenocarcinoma of the cervix, that is, β-catenin inhibition and cDC1 mobilization., (©2020 American Association for Cancer Research.)

Background: Little is known about immune cell infiltrate type in the kidney allograft of patients with chronic-active antibody-mediated rejection (c-aABMR). Methods: In this study, multiplex immunofluorescent staining was performed on 20 cases of biopsy-proven c-aABMR. T-cell subsets (CD3, CD8, Foxp3, and granzyme B), macrophages (CD68 and CD163), B cells (CD20), and natural killer cells (CD57) were identified and counted in the glomeruli (cells/glomerulus) and the tubulointerstitial (TI) compartment [cells/high-power field (HPF)]. Results: In the glomerulus, T cells and macrophages were the dominant cell types with a mean of 5.5 CD3 + cells/glomerulus and 4 CD68 + cells/glomerulus. The majority of T cells was CD8 + (62%), and most macrophages were CD68 + CD163 + (68%). The TI compartment showed a mean of 116 CD3 + cells/HPF, of which 54% were CD8 + . Macrophage count was 21.5 cells/HPF with 39% CD68 + CD163 + . CD20 + cells were sporadically present in glomeruli, whereas B-cell aggregates in the TI compartment were frequently observed. Natural killer cells were rarely identified. Remarkably, increased numbers of CD3 + FoxP3 + cells in the TI compartment were associated with decreased graft survival ( p = 0.004). Conclusions: Renal allograft biopsies showing c-aABMR show a predominance of infiltrating CD8 + T cells, and increased numbers of interstitial FoxP3 + T cells are associated with inferior allograft survival., (Copyright © 2020 Sablik, Jordanova, Pocorni, Clahsen-van Groningen and Betjes.)

Purpose: MYC rearrangement ( MYC -R) occurs in approximately 10% of diffuse large B-cell lymphomas (DLBCLs) and has been associated with poor prognosis in many studies. The impact of MYC- R on prognosis may be influenced by the MYC partner gene (immunoglobulin [IG] or a non-IG gene). We evaluated a large cohort of patients through the Lunenburg Lymphoma Biomarker Consortium to validate the prognostic significance of MYC-R (single-, double-, and triple-hit status) in DLBCL within the context of the MYC partner gene., Methods: The study cohort included patients with histologically confirmed DLBCL morphology derived from large prospective trials and patient registries in Europe and North America who were uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone therapy or the like. Fluorescence in situ hybridization for the MYC , BCL2 , BCL6 , and IG heavy and light chain loci was used, and results were correlated with clinical outcomes., Results: A total of 5,117 patients were identified of whom 2,383 (47%) had biopsy material available to assess for MYC -R. MYC -R was present in 264 (11%) of 2,383 patients and was associated with a significantly shorter progression-free and overall survival, with a strong time-dependent effect within the first 24 months after diagnosis. The adverse prognostic impact of MYC- R was only evident in patients with a concurrent rearrangement of BCL2 and/or BCL6 and an IG partner (hazard ratio, 2.4; 95% CI, 1.6 to 3.6; P < .001)., Conclusion: The negative prognostic impact of MYC -R in DLBCL is largely observed in patients with MYC double hit/triple-hit disease in which MYC is translocated to an IG partner, and this effect is restricted to the first 2 years after diagnosis. Our results suggest that diagnostic strategies should be adopted to identify this high-risk cohort, and risk-adjusted therapeutic approaches should be refined further.

Resistance to chemotherapy is widely recognized as one of the major factors limiting therapeutic efficacy and influences clinical outcomes in patients with cancer. Many studies on various tumor types have focused on combining standard-of-care chemotherapy with immunotherapy. However, for cervical cancer, the role of neoadjuvant chemotherapy (NACT) on the local immune microenvironment is largely unexplored. We performed a pilot study on 13 primary cervical tumor samples, before and after NACT, to phenotype and enumerate tumor-infiltrating T-cell subpopulations using multiplex immunohistochemistry (CD3, CD8, FoxP3, Ki67, and Tbet) and automated co-expression analysis software. A significant decrease in proliferating (Ki67 + ) CD3 + CD8 - T cells and FoxP3 + (CD3 + CD8 - ) regulatory T cells was observed in the tumor stroma after cisplatin and paclitaxel treatment, with increased rates of cytotoxic CD8 + T cells, including activated and CD8 + Tbet + T cells. No effect was observed on the number of tumor-infiltrating T cells in the cervical tumor microenvironment after treatment with cisplatin only. Therefore, we conclude that patients treated with cisplatin and paclitaxel had more tumor-infiltrating T-cell modulation than patients treated with cisplatin monotherapy. These findings enhance our understanding of the immune-modulating effect of chemotherapy and warrant future combination of the standard-of-care therapy with immunotherapy to improve clinical outcome in patients with cervical cancer.

Lymph nodes draining the primary tumor are essential for the initiation of an effective anti-tumor T-cell immune response. However, cancer-derived immune suppressive factors render the tumor-draining lymph nodes (TDLN) immune compromised, enabling tumors to invade and metastasize. Unraveling the different mechanisms underlying this immune escape will inform therapeutic intervention strategies to halt tumor spread in early clinical stages. Here, we review our findings from translational studies in melanoma, breast, and cervical cancer and discuss clinical opportunities for local immune modulation of TDLN in each of these indications.

Current chemotherapeutic treatment for advanced penile squamous cell carcinoma has substantial side effects and no randomized data to support an overall survival benefit. Immunotherapy with checkpoint blockade is currently being tested in penile cancer patients in clinical trials. The high PD-L1 expression and CD8 + T-cell infiltration in penile cancer represent a promising prospect for immunotherapy response in the treatment of locally advanced disease. For efficacious immunotherapy treatment, a better understanding of the tumor microenvironment (TME) is critical. Here we use the structure revealed by cancer immunograms to define current knowledge of the penile cancer TME as a backbone for future research. PATIENT SUMMARY: Advanced penile cancer has a poor prognosis with a need for more effective therapy. In this manuscript we describe the potential of immunotherapy as a new treatment modality in penile cancer., (Copyright © 2019 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Background: Cervical cancer (CxCa) is mainly a locally invading disease that metastasizes to loco-regional lymph node basins before involving distant organs in more advanced stages. Local immune potentiation of tumor-draining lymph nodes (TDLN) may thus protect against tumor progression., Methods: To identify therapeutic targets for local immune modulation, multi-parameter flow cytometric T-cell profiling of primary cervical tumors (PT) and TDLN (n = 37) was performed. The in-vitro effect of PD-1 blockade on T-cell reactivity to HPV16 E6 oncoproteins was determined in cultures of TDLN and PT single cell suspensions (n = 19). Also, intracellular cytokine staining (ICS) upon anti-CD3 stimulation was performed in metastatic TDLN (LN+) and PT (n = 7), as well as multiplexed immunofluorescence histochemistry staining (n = 8)., Results: Our data revealed elevated rates of activated regulatory T cells (aTregs) and of central or effector memory CD8 + T cells in metastatic TDLN (LN+) as compared to tumor-free TDLN (LN-), and equally high or even higher rates of these subsets in PT. Both memory subsets co-expressed multiple immune checkpoints. PD-1 blockade significantly enhanced detectable E6-specific T-cell responses in 4/5 HPV16+ LN+ and in 1/5 HPV16+ PT. Whereas aTreg rates were higher in anti-PD-1 non-responders, in responders elevated levels of CD8 + FoxP3 + CD25 + T cells were observed, which correlated with the efficacy of PD-1 blockade (P = 0.018). This subset was characterized by an early effector memory phenotype with particularly high levels of co-expressed PD-1, CTLA-4, TIM-3 and LAG-3 checkpoints, but, rather than exhausted, was shown upon polyclonal activation to produce higher levels of Granzyme-B and effector cytokines as compared to its CD8 + FoxP3 - counterparts., Conclusion: These observations support local PD-(L)1 blockade to interrupt loco-regional immune suppression in CxCa and control metastatic spread to TDLN. Furthermore, our data identify CD8 + FoxP3 + CD25 + T cells as therapeutic targets, which may also serve as predictive biomarker for PD-(L)1 checkpoint blockade.

Background: Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is a highly immunogenic tumor and differences in tumor microenvironment might contribute to the improved survival of HPV-positive OPSCC patient., Methods: A comprehensive multivariate analysis with clinical and immune variables (human leukocyte antigen [HLA] I/II, programmed death ligand 1 (PD-L1), programmed death receptor 1 (PD1), T cells, and macrophages) was performed in 142 OPSCC patients., Results: We found an inverse correlation between the expression of HLA class II molecules on tumor cells and CD68+ CD163+ tumor-associated macrophages (TAMs). High HLA-DP/DQ/DR expression and low number of TAMs were associated with longer disease-specific survival and disease-free survival (DFS). Furthermore, a new population of CD8+ FoxP3+ T cells was correlated with shorter DFS in multivariate analysis., Conclusions: \We identified new prognostic markers for patients with oropharyngeal cancer, which can be used for selecting patients that can benefit from immunotherapy., (© 2018 The Authors. Head & Neck published by Wiley Periodicals, Inc.)

Cervical cancer is the fourth most common cancer type in women worldwide and is characterized by a highly immune-suppressive microenvironment. Here, we describe aberrant glycosylation as a factor mediating this immunosuppressive microenvironment. Expression of a specific carbohydrate ligand for the immune-regulatory C-type lectin MGL was correlated to poor disease-specific survival and distant recurrences in squamous cell carcinoma (SCC) and adenosquamous carcinoma (ASC), the most common histological subtypes of cervical cancer. MGL ligand expression was also associated with lymph node metastasis, the absence of CD14 + myeloid cells and the presence of CD14 - CD163 + myeloid cells. Indeed, expression of the MGL receptor itself could be detected on CD163 + cells, suggesting that MGL + myeloid cells are able to interact locally with MGL ligand + tumor cells. Additionally, MGL ligand expression correlated to the occurrence of PIK3CA mutations, the most frequently observed oncogenic alteration in cervical cancer. In conclusion, we present prognostic value for MGL ligand expression in SCC/ASC patients, which further supports an immune evasive role for the C-type lectin MGL in the tumor immune compartment.

Regulatory T cells (Tregs) may comprise different subsets allowing them to efficiently suppress different types of effector T cells. In this study, we show that high numbers of both conventional and Tbet co-expressing Foxp3 hi Tregs accumulate in human papilloma virus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC). The infiltration of Tbet+ Foxp3+ Tregs was strongly correlated with a concomitant tumor-specific and conventional type 1-oriented intratumoral T cell infiltrate. Both conventional CD4+CD25+CD127-Foxp3 hi Tregs and their Tbet hi counterparts exhibited an activated phenotype, co-expressed high levels of CTLA4 and Helios and exhibited a maximally demethylated Foxp3 gene locus TSDR, indicating their full capacity to impede a type 1 effector T cell response. Interestingly, while the prognostic value of conventional Tregs was neutral, a high intratumoral frequency of Tbet+ Tregs was associated with prolonged disease-specific survival, most likely because their presence reflected high numbers of effector T cells. The presence of these Tbet+ Tregs may in part explain why a dense type 1-oriented immune infiltrate in OPSCC is not enough to fully control tumor growth.

Checkpoint blockade immunotherapies have revolutionised cancer treatment in the last decade. Nevertheless, these are only beneficial for a small proportion of cancer patients. Important prognosticators for response to immunotherapy are the mutation burden of tumours as well as the quality and quantity of tumour-infiltrating immune cells. High-throughput multiplex immunophenotyping technologies have a central role in deciphering the complexity of anti-tumour immune responses. Current techniques for the immunophenotyping of solid tumours are held back by the lack of spatial context, limitations in the number of targets that can be visualised simultaneously, and/or cumbersome protocols. We developed a tyramide signal amplification-free method for the simultaneous detection of seven cellular targets by immunofluorescence. This method overcomes limitations posed by most widespread techniques and provides a unique tool for extensive phenotyping by multispectral fluorescence microscopy. Furthermore, it can be easily implemented as a high-throughput technology for validation of discovery sets generated by RNA sequencing or mass cytometry and may serve in the future as a complementary diagnostic tool., (© 2018 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Uveal melanoma progression can be predicted by gene expression profiles enabling a clear subdivision between tumors with a good (class I) and a poor (class II) prognosis. Poor prognosis uveal melanoma can be subdivided by expression of immune-related genes; however, it is unclear whether this subclassification is justified; therefore, T cells in uveal melanoma specimens were quantified using a digital PCR approach. Absolute T-cell quantification revealed that T-cell influx is present in all uveal melanomas associated with a poor prognosis. However, this infiltrate is only accompanied by differential immune-related gene expression profiles in uveal melanoma with the highest T-cell infiltrate. Molecular deconvolution of the immune profile revealed that a large proportion of the T-cell-related gene expression signature does not originate from lymphocytes but is derived from other immune cells, especially macrophages. Expression of the lymphocyte-homing chemokine CXCL10 by activated macrophages correlated with T-cell infiltration and thereby explains the correlation of T-cell numbers and macrophages. This was validated by in situ analysis of CXCL10 in uveal melanoma tissue with high T-cell counts. Surprisingly, CXCL10 or any of the other genes in the activated macrophage-cluster was correlated with reduced survival due to uveal melanoma metastasis. This effect was independent of the T-cell infiltrate, which reveals a role for activated macrophages in metastasis formation independent of their role in tumor inflammation. IMPLICATIONS: The current report uses an innovative digital PCR method to study the immune environment and demonstrates that absolute T-cell quantification and expression profiles can dissect disparate immune components., (©2018 American Association for Cancer Research.)