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The accumulation of tumor-specific CD4+ and CD8+ effector T cells is key to an effective antitumor response. Locally, CD4+ T cells promote the recruitment and effector function of tumor-specific CD8+ T cells and activate innate killer cells in the tumor. Here, we show that tumor-specific CD4+ T cells were predominantly present in the CD39+ subset of tumor-infiltrating lymphocytes (TIL). The CD39+ CD4+ and CD8+ TILs were detected in three different tumor types, and displayed an activated (PD-1+, HLA-DR+) effector memory phenotype. CD4+CD39+ single-cell RNA-sequenced TILs shared similar well-known activation, tissue residency, and effector cell–associated genes with CD8+CD39+CD103+ TILs. Finally, analysis of directly ex vivo cell-sorted and in vitro expanded pure populations of CD39-positive and negative CD4+ and CD8+ TILs revealed that tumor-specific antigen reactivity was almost exclusively detected among CD39+ cells. Immunotherapy of cancer is based on the activation of tumor-reactive CD4+ and CD8+ T cells. We show that the expression of CD39 can be used to identify, isolate, and expand tumor-reactive T-cell populations in cancers.

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Purpose:The tumor immune microenvironment determines clinical outcome. Whether the original tissue in which a primary tumor develops influences this microenvironment is not well understood.Experimental Design:We applied high-dimensional single-cell mass cytometry [Cytometry by Time-Of-Flight (CyTOF)] analysis and functional studies to analyze immune cell populations in human papillomavirus (HPV)–induced primary tumors of the cervix (cervical carcinoma) and oropharynx (oropharyngeal squamous cell carcinoma, OPSCC).Results:Despite the same etiology of these tumors, the composition and functionality of their lymphocytic infiltrate substantially differed. Cervical carcinoma displayed a 3-fold lower CD4:CD8 ratio and contained more activated CD8+CD103+CD161+ effector T cells and less CD4+CD161+ effector memory T cells than OPSCC. CD161+ effector cells produced the highest cytokine levels among tumor-specific T cells. Differences in CD4+ T-cell infiltration between cervical carcinoma and OPSCC were reflected in the detection rate of intratumoral HPV-specific CD4+ T cells and in their impact on OPSCC and cervical carcinoma survival. The peripheral blood mononuclear cell composition of these patients, however, was similar.Conclusions:The tissue of origin significantly affects the overall shape of the immune infiltrate in primary tumors.

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The accumulation of tumor-specific CD4+ and CD8+ effector T cells is key to an effective antitumor response. Locally, CD4+ T cells promote the recruitment and effector function of tumor-specific CD8+ T cells and activate innate killer cells in the tumor. Here, we show that tumor-specific CD4+ T cells were predominantly present in the CD39+ subset of tumor-infiltrating lymphocytes (TIL). The CD39+ CD4+ and CD8+ TILs were detected in three different tumor types, and displayed an activated (PD-1+, HLA-DR+) effector memory phenotype. CD4+CD39+ single-cell RNA-sequenced TILs shared similar well-known activation, tissue residency, and effector cell–associated genes with CD8+CD39+CD103+ TILs. Finally, analysis of directly ex vivo cell-sorted and in vitro expanded pure populations of CD39-positive and negative CD4+ and CD8+ TILs revealed that tumor-specific antigen reactivity was almost exclusively detected among CD39+ cells. Immunotherapy of cancer is based on the activation of tumor-reactive CD4+ and CD8+ T cells. We show that the expression of CD39 can be used to identify, isolate, and expand tumor-reactive T-cell populations in cancers.

BackgroundA profound insight into the immune landscape of vulvar squamous cell carcinoma (VSCC) is lacking. Here, an in-depth interrogation of T cell infiltration, local immune contexture, signaling pathways and checkpoint molecule expression was performed in early-stage and late-stage VSCC.MethodsThe type, location, and infiltration pattern of T cells were studied in 109 patients with primary VSCC FIGO stage I–III. RNA expression of genes involved in immune oncology and oncogenic signaling pathways was analyzed in 40 VSCC, matched for prognostic clinicopathological variables, analyzed for HPV and p53 status, and selected based on T cell infiltration.ResultsHigh intraepithelial infiltration with CD4 or CD8 T cells was associated with longer overall and recurrence-free survival and formed an independent prognostic factor, outperforming molecular subtype and stage of the disease. Strong T cell infiltrated VSCC displayed a coordinated immune response reflected by a positive association between T cells and different lymphocyte and myeloid cell subsets. The expression of genes involved in the migration of T cells and myeloid cells, T cell activation and costimulation, interferon (IFN)-γ signaling, cytotoxicity and apoptosis was higher than in low infiltrated tumors. An active immune signaling profile was observed in all inflamed, part of the altered-excluded and not in altered-immunosuppressed or deserted VSCC. While several checkpoint molecules were overexpressed, only PD-L1 expression displayed discriminatory ability and clinical usefulness. High PD-L1 expression was detected in all inflamed and ~60% of the altered-excluded VSCC.ConclusionAn active immune signaling profile is present in 35% of primary FIGO I–III VSCCs, suggesting potential responsiveness to neoadjuvant PD-1/PD-L1 immunotherapy.

Objective. There is great need for better risk stratification in vulvar squamous cell carcinoma (VSCC). Our aim was to define the prognostic significance of stratifying VSCC based on p16 and p53 immunohistochemistry (IHC) as surrogate markers for HPV and TP53 mutations.Methods. A large retrospective cohort of surgically treated women with primary VSCC was used. VSCC were classified into three subtypes: HPV-positive (HPVpos), HPV-negative/p53 mutant (HPVneg/p53mut), and HPVnegative/p53 wildtype (HPVneg/p53wt). Overall survival (OS), relative survival (RS), and recurrence-free period (RFP) were depicted using the Kaplan-Meier method and survival curves for relative survival; associations were studied using univariable and multivariable Cox proportional hazard models.Results. Of the 413 VSCCs, 75 (18%) were HPVpos, 63 (15%) HPVneg/p53wt, and 275 (66%) HPVneg/p53mut VSCC. Patients with HPVneg/p53mut VSCC had worse OS and RS (HR 3.43, 95%CI 1.80-6.53, and relative excess risk (RER) of 4.02; 95%CI 1.48-10.90, respectively, and worse RFP (HR 3.76, 95%CI 2.02-7.00). HPVpos VSCC patients showed most favorable outcomes. In univariate analysis, the molecular subtype of VSCC was a prognostic marker for OS, RS and RFP (p = 0.003, p = 0.009, p Conclusions. Stratification of VSCC by p16and p53-IHC has potential to be used routinely in diagnostic pathology. It results in the identification of three clinically distinct subtypes and may be used to guide treatment and follow-up, and in stratifying patients in future clinical trials. (C) 2020 Elsevier Inc. All rights reserved.

The accumulation of tumor-specific CD4

The recent literature has shown that vulvar squamous cell carcinoma (VSCC) can be stratified into two prognostically relevant groups based on human papillomavirus (HPV) status. The prognostic value of p53 for further sub-stratification, particularly in the HPV-independent group, has not been agreed upon. This disagreement is likely due to tremendous variations in p53 immunohistochemical (IHC) interpretation. To address this problem, we sought to compare p53 IHC patterns with TP53 mutation status. We studied 61 VSCC (48 conventional VSCC, 2 VSCC with sarcomatoid features, and 11 verrucous carcinomas) and 42 in situ lesions (30 differentiated vulvar intraepithelial neoplasia [dVIN], 9 differentiated exophytic vulvar intraepithelial lesions [deVIL], and 3 high-grade squamous intraepithelial lesions or usual vulvar intraepithelial neoplasia [HSIL/uVIN]). IHC for p16 and p53, and sequencing of TP53 exons 4-9 were performed. HPV in situ hybridization (ISH) was performed in selected cases. We identified six major p53 IHC patterns, two wild-type patterns: (1) scattered, (2) mid-epithelial expression (with basal sparing), and four mutant patterns: (3) basal overexpression, (4) parabasal/diffuse overexpression, (5) absent, and (6) cytoplasmic expression. These IHC patterns were consistent with TP53 mutation status in 58/61 (95%) VSCC and 39/42 (93%) in situ lesions. Cases that exhibited strong scattered staining and those with a weak basal overexpression pattern could be easily confused. The mid-epithelial pattern was exclusively observed in p16-positive lesions; the basal and parabasal layers that had absent p53 staining, appeared to correlate with the cells that were positive for HPV-ISH. This study describes a pattern-based p53 IHC interpretation framework, which can be utilized as a surrogate marker for TP53 mutational status in both VSCC and vulvar in situ lesions.

INTRODUCTION:The most commonly mutated gene in vulvar squamous cell carcinoma (VSCC) is TP53 and its prognostic value, particularly in HPV-independent VSCC, is uncertain. In other tumors, p53 immunohistochemistry (IHC) is an excellent surrogate marker for TP53 mutations. In order to study this in VSCC, we assigned six p53-IHC patterns into two final classes: 'wildtype' or 'mutant'. We determined the performance and interobserver variability of this pattern-based p53-IHC approach.METHODS:Two experienced gynecologic pathologists scored the predefined p53-IHC patterns of 59 VSCC, independently and blinded for molecular data. Agreement was calculated by Cohen's kappa. All disagreements regarding p53-IHC patterns were resolved by a consensus meeting. After DNA isolation, the presence of pathogenic TP53 variants were determined by next-generation sequencing (NGS). Sensitivity, specificity, and accuracy of p53-IHC as a surrogate marker for TP53 mutation status were calculated.RESULTS:Initial p53-IHC pattern interpretation showed substantial agreement between both observers (k=0.71, pCONCLUSIONS:Pattern-based p53-IHC classification is highly reproducible amongst experienced gynecologic pathologists and accurately reflects TP53 mutations in VSCC. This approach to p53-IHC interpretation offers guidance and provides necessary clarity for resolving the proposed prognostic relevance of final p53-IHC class within HPV-independent VSCC.

Introduction/Background Vulvar squamous cell carcinoma (VSCC) consists of three subtypes; HPV-related, HPV-negative TP53 wildtype and HPV-negative mutated TP53 (HPVposVSCC, HPVnegVSCC/p53wt, and HPVnegVSCC/p53abn, respectively) which are all treated by mutilating radical surgery and/or (chemo)radiotherapy. Despite the fact that the immune system plays a key role in cancer, the knowledge on its effect in VSCC is limited at best. A study, elucidating the clinical impact of tumor-immunity in VSCC was, therefore, performed with the aim to foster the development of immunotherapeutic approaches. Methodology Sixty-five patients with early-stage VSCC were categorized based on HPV and p53 status. Archived tissues were analyzed for expression of CD3, CD8, FoxP3, PD-1, and pan-keratin in randomly selected areas using immunofluorescence. Additional phenotyping of T cells was performed ex-vivo on VSCC and blood samples by flow cytometry. Healthy vulvar tissue and blood served as controls. Results T-cell infiltration of VSCC was highly variable between patients, ranging from completely absent to very high numbers, and differed per VSCC subtype. Approximately 80% of the HPVposVSCC showed high T-cell infiltration, followed by 60% of the HPVnegVSCC/p53wt, and 40% of the HPVnegVSCC/p53abn. Importantly, high T-cell infiltration was associated with longer recurrence-free period and overall survival, irrespective of the HPV and p53 status. In-depth analysis of tumor-infiltrating T cells with flow cytometry confirmed the tumor-specific presence of activated effector memory T cells in VSCC and revealed that most of the CD4+ and CD8+ T cells expressed PD-1. Conclusion This study is the first to show a strong correlation between T-cell infiltration and clinical outcome. Our data suggest the application of two immunotherapeutic strategies depending on immune phenotype. The high expression of PD-1 in T-cell infiltrated tumors alludes to anti-PD1 blockade, whereas VSCC tumors with low numbers of intratumoral T cells should be stimulated with inflammatory reagents to stimulate local immune responses. Disclosure Nothing to disclose.

Supplemental Digital Content is available in the text., Surgical resection with free surgical margins is the cornerstone of successful primary treatment of vulvar squamous cell carcinoma (VSCC). In general reexcision is recommended when the minimum peripheral surgical margin (MPSM) is

Purpose: The tumor immune microenvironment determines clinical outcome. Whether the original tissue in which a primary tumor develops influences this microenvironment is not well understood. Experimental Design: We applied high-dimensional single-cell mass cytometry [Cytometry by Time-Of-Flight (CyTOF)] analysis and functional studies to analyze immune cell populations in human papillomavirus (HPV)–induced primary tumors of the cervix (cervical carcinoma) and oropharynx (oropharyngeal squamous cell carcinoma, OPSCC). Results: Despite the same etiology of these tumors, the composition and functionality of their lymphocytic infiltrate substantially differed. Cervical carcinoma displayed a 3-fold lower CD4:CD8 ratio and contained more activated CD8+CD103+CD161+ effector T cells and less CD4+CD161+ effector memory T cells than OPSCC. CD161+ effector cells produced the highest cytokine levels among tumor-specific T cells. Differences in CD4+ T-cell infiltration between cervical carcinoma and OPSCC were reflected in the detection rate of intratumoral HPV-specific CD4+ T cells and in their impact on OPSCC and cervical carcinoma survival. The peripheral blood mononuclear cell composition of these patients, however, was similar. Conclusions: The tissue of origin significantly affects the overall shape of the immune infiltrate in primary tumors.

Vulvar squamous cell carcinoma (VSCC) develops via two different pathways: TP53 mutations in a background of lichen sclerosus or a persistent infection with high-risk human papilloma virus (HPV). The latter group of tumor responds better to treatment than the non-virally induced VSCC. This may be explained by a difference in the tumor immune microenvironment (TME).This review summarizes literature on TME of VSCC and its precursors, and extrapolates this to foster the development of new therapeutic strategies.Both types of VSCC and their precursors are infiltrated with variable numbers of M2 macrophages, regulatory T cells and CD8+ T cells, indicating that they express targetable tumor antigens. Type 1 T cell immunity in precursor lesions is associated with fewer recurrences and better clinical responses to immunotherapy. Escape of these lesions and progression toward VSCC is associated with the downregulation of HLA Class I, increased expression of co-inhibitory molecules, infiltration with immunosuppressive cells and the local production of immunosuppressive enzymes and cytokines. More in-depth studies of the VSCC TME are required to fully comprehend the impact of the immune system on VSCC, and subsequently to identify patients who will benefit from immunotherapeutic strategies.

Introduction Medical students must be able to read and critically appraise scientific papers. Therefore, they have to be taught specific skills. There is evidence from a range of studies that research-tutored learning is an effective way to achieve this. We examined the learning gain and opinion of students exposed to research-tutored education.

Raptor populations in Sudano-Sahelian West Africa are being severely affected by widespread habitat alteration which depletes prey populations, potentially aggravated by changing rainfall patterns. We studied Grasshopper Buzzards Butastur rufipennis at nests in natural and transformed habitats in the Sudano-Sahelian region of northern Cameroon to assess the effects of habitat transformation and rainfall on nestling diet and growth. Grasshoppers and small mammals were more frequently taken in natural habitat, whereas lizards were most frequently taken in transformed habitats. These dietary differences reflected differences in prey availability around nests in natural and transformed habitats. Land use was a significant predictor of asymptotic weight: nestlings in natural habitat attained a higher mass than those in transformed habitats, when potentially confounding variables such as hatch order, gender, hatch date, rainfall or the presence of siblings were taken into consideration. These results suggest that body condition at fledging was habitat-dependent, with potential consequences for subsequent survival. However, we recorded no differences in caloric content of food delivered to nests in natural and transformed habitat, which was possibly related to prey caught during twilight hours. There was a positive relationship between precipitation levels during the nestling phase and nestling growth rate. We predict unfavourable future conditions for nestling growth of raptors in Sudano-Sahelian savannas as a consequence of continued widespread habitat transformation and diminished rainfall.

Background Independent of their blood pressure lowering effect, ACE inhibitors are thought to reduce vascular inflammation. The clinical relevance of this effect is unclear with the current knowledge. Abdominal aortic aneurysms (AAA) are characterized by a broad, non-specific inflammatory response, and thus provide a clinical platform to evaluate the anti-inflammatory potential of ACE inhibitors. Methods and results Eleven patients scheduled for open AAA repair received ramipril (5 mg/day) during 2-4 weeks preceding surgery. Aortic wall samples were collected during surgery, and compared to matched samples obtained from a biobank. An anti-inflammatory potential was evaluated in a comprehensive analysis that included immunohistochemistry, mRNA and protein analysis. A putative effect of ACE inhibitors on AAA growth was tested separately by comparing 18-month growth rate of patients on ACE inhibitors (n = 82) and those not taking ACE inhibitors (n = 204). Ramipril reduces mRNA expression of multiple pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, TNF -α, Interferon-[Formula: see text], and MCP-1, as well as aortic wall IL-8 and MCP-1 (P = 0.017 and 0.008, respectively) protein content. The is followed by clear effects on cell activation that included a shift towards anti-inflammatory macrophage (M2) subtype. Evaluation of data from the PHAST cohort did not indicate an effect of ACE inhibitors on 18-month aneurysm progression (mean difference at 18 months: -0.24 mm (95% CI: -0.90-0.45, P = NS). Conclusions ACE inhibition quenches multiple aspects of vascular inflammation in AAA. However, this does not translate into reduced aneurysm growth. Trial registration Nederlands Trial Register 1345.

Contains fulltext : 118070.pdf (Publisher’s version ) (Open Access) Statins are thought to reduce vascular inflammation through lipid independent mechanisms. Evaluation of such an effect in atherosclerotic disease is complicated by simultaneous effects on lipid metabolism. Abdominal aortic aneurysms (AAA) are part of the atherosclerotic spectrum of diseases. Unlike atherosclerotic occlusive disease, AAA is not lipid driven, thus allowing direct evaluation of putative anti-inflammatory effects. The anti-inflammatory potency of increasing doses (0, 20 or 40 mg/day) simvastatin or atorvastatin was evaluated in 63 patients that were at least 6 weeks on statin therapy and who underwent open AAA repair. A comprehensive analysis using immunohistochemistry, mRNA and protein analyses was applied on aortic wall samples collected during surgery. The effect of statins on AAA growth was analyzed in a separate prospective study in incorporating 142 patients. Both statins equally effectively and dose-dependently reduced aortic wall expression of NFkappaB regulated mediators (i.e. IL-6 (P

Objective— Genomewide association studies have implicated allelic variation at 9p21.3 in multiple forms of vascular disease, including atherosclerotic coronary heart disease and abdominal aortic aneurysm. As for other genes at 9p21.3, human expression quantitative trait locus studies have associated expression of the tumor suppressor gene CDKN2B with the risk haplotype, but its potential role in vascular pathobiology remains unclear. Methods and Results— Here we used vascular injury models and found that Cdkn2b knockout mice displayed the expected increase in proliferation after injury, but developed reduced neointimal lesions and larger aortic aneurysms. In situ and in vitro studies suggested that these effects were attributable to increased smooth muscle cell apoptosis. Adoptive bone marrow transplant studies confirmed that the observed effects of Cdkn2b were mediated through intrinsic vascular cells and were not dependent on bone marrow–derived inflammatory cells. Mechanistic studies suggested that the observed increase in apoptosis was attributable to a reduction in MDM2 and an increase in p53 signaling, possibly due in part to compensation by other genes at the 9p21.3 locus. Dual inhibition of both Cdkn2b and p53 led to a reversal of the vascular phenotype in each model. Conclusion— These results suggest that reduced CDKN2B expression and increased smooth muscle cell apoptosis may be one mechanism underlying the 9p21.3 association with aneurysmal disease.

Research into the effect of environmental variables on reproductive success of tropical raptors is often constrained by the lack of information on breeding biology. We provide the first detailed information of the breeding biology and nestling development of the Grasshopper Buzzard Butastur rufipennis, an Afrotropical migratory raptor threatened by extensive land transformation in its breeding range. Breeding coincided with the transition from the dry to the wet season. The mean incubation period was 30 d and mean fledging period 36 d. The breeding period was characterised by rapid establishment of territories and short post-fledging dependency periods related to the onset of migration shortly after breeding. Growth rate of body mass, tarsus, wing and primary feathers were similar between sexes, which showed significant but moderate body mass dimorphism at fledging (♂85–90% of ♀). Second hatchlings were smaller in body mass and structural dimensions compared to similarly-aged first hatchlings and singles of the same sex. Survival of second hatchlings was related to body mass ratio with first hatchlings, whereas brood reduction occurred through food competition and siblicide. We provide ageing formulae and photographic records to facilitate further studies of Grasshopper Buzzard nestling development and reproductive success in the region.Keywords: ageing, Butastur rufipennis, hatch order, reproduction, siblicideOSTRICH 2012, 83(3): 137–146

Few studies have examined raptor reproduction in response to land-use change in sub-Saharan Africa, hampering conservation efforts to address regional declines. To further our understanding of mechanisms underlying the dramatic declines of West African raptors, we examined the relationship between environmental conditions, nest density, and measures of reproduction in the Grasshopper Buzzard (Butastur rufipennis). Analyses were based on 244 nest sites divided between transformed and natural habitat in northern Cameroon. At the landscape scale, nest density increased with the density of preferred nest trees. Nests were more widely spaced in transformed than in natural habitat. Dispersion was adjusted to differences in availability of small mammals, which was negatively associated with distance to nearest neighbor, and in the area under cultivation, which was positively associated with distance to nearest neighbor. Productivity was positively associated with rainfall, canopy shielding the nest, availability of grasshoppers, and the nest's visibility from ground level; canopy shielding, grass cover, rainfall, and distance to nearest neighbor were positively associated with nest success. In natural habitat, losses of eggs and nestlings to natural predators were greater than in transformed habitats, while losses through human predation were small. Productivity and nest success were unaffected by land use because of the opposing effects of greater predation pressure, closer spacing of nests, and more food in natural habitat than in transformed habitat. Thus transformed habitat may provide adequate breeding habitat for the Grasshopper Buzzard, but declining rainfall and intensifying anthropogenic land use are likely to affect future reproductive output.