Utilisation of cytological samples for multiplex immunofluorescence assay.

OBJECTIVE: Understanding the immune environment of non-small cell lung cancer (NSCLC) is important for designing effective anticancer immunotherapies. We describe the use of multiplex immunofluorescence (mIF) assays to enable characterisation of the tumour-infiltrating immune cells and their interactions, both across and within immune subtypes. METHODS: Six cytological samples of NSCLC taken by transoesophageal ultrasound-guided fine needle aspiration were tested with an mIF assay designed to detect the expression of key immune cell markers such as CD3, CD8, CD20, CD11b, and CD68. Pan-cytokeratin was used to detect the NSCLC cells. Fluorescence images were acquired on a Vectra-Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences). RESULTS: MIF assay was able to reliably detect and quantify the myeloid cell markers CD11b, CD68, CD3+ and CD8+ T cells, and CD20+ B lymphocytes on cytological samples of NSCLC. Whole-tissue analysis and its correlation with the corresponding H&E stains allowed a better understanding of the tissue morphology and the relationship between tumour and stroma compartments. Additionally, a uniform, specific, and correct staining pattern was seen for every immune marker. CONCLUSION: The implementation of mIF assay on cytological samples taken with minimally invasive methods seems feasible and can be used to explore the immune environment of NSCLC.

Association between adipophilin expression and risk of dyslipidaemia in patients with granuloma annulare.

BACKGROUND: An association between granuloma annulare (GA) and dyslipidaemia has been reported. Adipophilin expression may play a plausible role as a cutaneous biomarker for dyslipidaemia in patients with GA; however, this potential link remains to be explored. METHODS: Patients with GA were identified at our hospital between January 1, 1990, and December 31, 2021, with a thorough review of their clinical and histological characteristics. Adipophilin staining was assessed in biopsies of GA lesions. RESULTS: A total of 107 patients with GA were included. The prevalence of dyslipidaemia in patients with positive adipophilin staining was clearly higher than in those with negative labelling (62.3% vs 13.3%). Relative to the dyslipidaemia risk for patients with negative adipophilin expression, the odds for patients with positive adipophilin expression were increased 10-fold (OR: 10.8; p-value<.01). We identified 23 incident cases of dyslipidaemia over a median follow-up period of 91 months among 54 patients with no history of dyslipidaemia. The patients with positive adipophilin expression showed a higher risk of developing dyslipidaemia (HR: 8.9; p-value<.01). CONCLUSIONS: Patients with positive adipophilin staining in their GA biopsies were found to be associated with a higher risk for both baseline and incident dyslipidaemia.

MHC class I and II-deficient humanized mice are suitable tools to test the long-term antitumor efficacy of immune checkpoint inhibitors and T-cell engagers.

BACKGROUND: Immunodeficient mice engrafted with peripheral blood mononuclear cells (PBMCs) are models to study new cancer immunotherapy agents. However, this approach is associated with xenograft-versus-host disease (xGVHD), which starts early after PBMC transfer and limits the duration and interpretation of experiments. Here, we explore different approaches to overcome xGVHD and better support the development of cancer immunotherapies. METHODS: Immunodeficient NOD-scid IL2Rgnull (NSG) mice were intravenously transferred with human PBMCs and subcutaneously co-engrafted with HT29 human colon carcinoma cells. Diverse strategies to reduce xGVHD while preserving the antitumor activity of human immune cells were evaluated: (1) ex vivo immune graft modification by depleting CD4+ T cells pre-transfer using magnetic beads, (2) post-transplantation cyclophosphamide administration to eliminate proliferating xenoreactive T-cell clones and (3) using major histocompatibility complex (MHC) class I and II-deficient NSG mice: (Kb Db)null (IA)null (MHC-dKO NSG). Body weight and plasma murine alanine aminotransferase levels were measured as indicators of xGVHD and tumor size was measured every 2-3 days to monitor antitumor activity. The antitumor effects and pharmacodynamics of nivolumab plus ipilimumab and an anti-epithelial cell adhesion molecule (EpCAM)/CD3 T-cell engager (αEpCAM/CD3 bispecific antibody (BsAb)) were evaluated in the model. RESULTS: CD4+ T-cell depletion attenuates xGVHD but also abrogates the antitumor activity. Cyclophosphamide limits the antitumor response and does not substantially prevent xGVHD. In contrast, xGVHD was significantly attenuated in MHC-dKO NSG recipients, while the antitumor effect of human PBMCs was preserved. Furthermore, the administration of nivolumab plus ipilimumab caused exacerbated xGVHD in conventional NSG mice, thereby precluding the observation of their antitumor effects. Severe xGVHD did not occur in MHC-dKO NSG mice thus enabling the study of complete and durable tumor rejections. Similarly, NSG mice treated with an αEpCAM/CD3 BsAb showed complete tumor regressions, but died due to xGVHD. In contrast, MHC-dKO NSG mice on treatment with the αEpCAM/CD3 BsAb achieved complete tumor responses without severe xGVHD. A significant proportion of mice rendered tumor-free showed tumor rejection on rechallenge with HT29 cells without further treatment. Finally, tumor-infiltrating CD8+ T-cell number increase, activation and CD137 upregulation were observed on αEpCAM/CD3 BsAb treatment. CONCLUSION: Humanized MHC-dKO immunodeficient mice allow and refine the preclinical testing of immunotherapy agents for which experimentation is precluded in conventional immunodeficient mice due to severe xGVHD.

Osteoclast-rich undifferentiated carcinoma of the bladder and the diagnostic usefulness of immunohistochemistry. A case report.

Osteoclast-rich undifferentiated carcinoma (ORUC) of the urinary tract is a rare variant of urothelial carcinoma, first described in 1985 by Kitazawa et al. It has a worse prognosis compared to other histological variants of invasive urothelial carcinoma and its diagnosis may prove challenging due to the variability in its immunohistochemical profile. We present a case of ORUC in which GATA3 immunostaining was a useful diagnostic tool.

Neutrophil Extracellular Traps, Local IL-8 Expression, and Cytotoxic T-Lymphocyte Response in the Lungs of Patients With Fatal COVID-19.

BACKGROUND: Excessive inflammation is pathogenic in the pneumonitis associated with severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defense mechanism against bacteria, but have also been shown to mediate tissue damage in a number of diseases. RESEARCH QUESTION: Could NETs and their tissue-damaging properties inherent to neutrophil-associated functions play a role in the respiratory failure seen in patients with severe COVID-19, and how does this relate to the SARS-CoV-2 viral loads, IL-8 (CXCL8) chemokine expression, and cytotoxic T-lymphocyte infiltrates? STUDY DESIGN AND METHODS: Sixteen lung biopsy samples obtained immediately after death were analyzed methodically as exploratory and validation cohorts. NETs were analyzed quantitatively by multiplexed immunofluorescence and were correlated with local levels of IL-8 messenger RNA (mRNA) and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by reverse-transcriptase polymerase chain reaction and immunohistochemistry analysis. RESULTS: NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one of eight liver tissues. NET focal presence correlated negatively with CD8+ T-cell infiltration in the lungs. INTERPRETATION: Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, but no correlation was found with viral loads. The strong association between NETs and IL-8 points to this chemokine as a potentially causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered with by the presence of NETs.

Osteoclast-rich undifferentiated carcinoma of the bladder and the diagnostic usefulness of immunohistochemistry. A case report / Carcinoma urotelial indiferenciado con células gigantes tipo osteoclasto: Ayuda inmunohistoquímica para una entidad poco frecuente

Osteoclast-rich undifferentiated carcinoma (ORUC) of the urinary tract is a rare variant of urothelial carcinoma, first described in 1985 by Kitazawa et al. It has a worse prognosis compared to other histological variants of invasive urothelial carcinoma and its diagnosis may prove challenging due to the variability in its immunohistochemical profile. We present a case of ORUC in which GATA3 immunostaining was a useful diagnostic tool.(AU)

El carcinoma urotelial indiferenciado con células gigantes tipo osteoclástico es una variante poco frecuente del carcinoma urotelial, descrito por primera vez en 1985 por Kitazawa et al. que debido al peor pronóstico que asocia y a la variabilidad del perfil inmunohistoquímico que presenta, hace de esta entidad un verdadero reto diagnóstico para los patólogos. Presentamos aquí un caso, exponiendo la utilidad e importancia de GATA3 en el diagnóstico de esta variante infrecuente de carcinoma urotelial.(AU)