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Purpose: This study aims to develop radiomics models and a nomogram based on machine learning techniques, preoperative dual-energy computed tomography (DECT) images, clinical and pathological characteristics, to explore the tumor microenvironment (TME) of clear cell renal cell carcinoma (ccRCC)., Methods: We retrospectively recruited of 87 patients diagnosed with ccRCC through pathological confirmation from Center I (training set, n = 69; validation set, n = 18), and collected their DECT images and clinical information. Feature selection was conducted using variance threshold, SelectKBest, and the least absolute shrinkage and selection operator (LASSO). Radiomics models were then established using 14 classifiers to predict TME cells. Subsequently, we selected the most predictive radiomics features to calculate the radiomics score (Radscore). A combined model was constructed through multivariate logistic regression analysis combining the Radscore and relevant clinical characteristics, and presented in the form of a nomogram. Additionally, 17 patients were recruited from Center II as an external validation cohort for the nomogram. The performance of the models was assessed using methods such as the area under the receiver operating characteristic curve (AUC), calibration curve, and decision curve analysis (DCA)., Results: The validation set AUC values for the radiomics models assessing CD8 + , CD163 + , and αSMA + cells were 0.875, 0.889, and 0.864, respectively. Additionally, the external validation cohort AUC value for the nomogram reaches 0.849 and shows good calibration., Conclusion: Radiomics models could allow for non-invasive assessment of TME cells from DECT images in ccRCC patients, promising to enhance our understanding and management of the tumor., Competing Interests: Declarations. Conflict of interest: The data from this study was analyzed and controlled by authors who are from Central Hospital Affiliated to Shandong First Medical University. The authors declare that they have no conflict of interest. Ethical approval: This study protocol was reviewed and approved by the Medical Ethics Committee of Central Hospital Affiliated to Shandong First Medical University, approval number 2023-012-01. The study has been granted an exemption from requiring written informed consent., (© 2024. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

Purpose: Radiotherapy (RT) with concomitant cisplatin (CRT) or cetuximab (ERT) are accepted treatment options for locally advanced squamous cell carcinoma of the head and neck (LA-SCCHN). Long-term adverse events (AEs) have a vast impact on patients' quality of life. This study explored tissue biomarkers which could help predict late toxicity., Methods/patients: Single-institution prospective study including patients aged ≥ 18 with histologically confirmed newly diagnosed LA-SCCHN treated with RT and either concomitant cisplatin q3w or weekly cetuximab, according to institutional protocols. All patients underwent pre- and post-treatment skin biopsies of neck regions included in the clinical target volume. Angiogenesis, macrophages, and extracellular matrix (ECM) markers were evaluated by immunohistochemistry (IHC)., Results: From April 15, 2016, to December 11, 2017; 31 patients were evaluated [CRT = 12 (38.7%) and ERT = 19 (61.3%)]. 27 patients (87%) had received induction chemotherapy. All patients finished RT as planned. IHC expression of vasculature (CD34) and collagen (Masson's Trichrome) did not differ significantly between and within CRT and ERT arms. Conversely, an increased CD68 and CD163 macrophage infiltration expression was observed after treatment, without significant impact of treatment modality. Patients with higher late toxicity showed lower expression of macrophage markers in pre-treatment samples compared with those with lower late toxicity, with statistically significant differences for CD68., Conclusions: Angiogenesis and ECM biomarkers did not differ significantly between CRT and ERT. Macrophage markers increased after both treatments and deserve further investigation as predictors of late toxicity in LA-SCCHN patients. [Protocol code: TOX-TTCC-2015-01/Spanish registry of clinical studies (REec): 2015-003012-21/Date of registration: 27/01/2016]., Competing Interests: Declarations Competing interests The authors declare the following finantial interests/personal relationships which may be considered as potential competing interests. MP has received advisory honoraria from BMS and speaker honoraria from MSD. MO has received advisory honoraria from Merck, MSD and Transgene; research support from Merck and Roche; and declares clinical trial support from Abbvie, Ayala Pharmaceutical, MSD, ALX Oncology, Debiopharm International, Merck, ISA Pharmaceuticals, Roche Pharmaceuticals, Boehringer Ingelheim, Seagen and Gilead. RM has received advisory honoraria from Merck, Nanobiotix, MSD, BMS, Roche, Seatle Genetics, AZ, Pfizer, Boehringer and Seagen: speaker honoraria from Merck, MSD and Boehringer; and travel support from BMS, MSD and Merck. The authors AJ, JAM-J, AL, OB, NR, IL, MT, and XP have no relevant financial or non-financial interests to disclose. Ethics approval and consent This study was performed in line with the principles of the Declaration of Helsinki. The entire study was approved by Hospital Universitari de Bellvitge Ethics Committee under the approval number AC112/15. All the participants provided written informed consent prior to their inclusion in the study., (© 2024. The Author(s).)

Introduction: Skewed immune response plays a pivotal role in tumor progression. Systemic inflammatory responses represented by combined peripheral leukocyte fractions are prognostic predictors of multiple cancers, including thyroid cancer. We previously reported the prognostic significance of lymphocyte-to-monocyte ratio (LMR) in curatively resected papillary thyroid cancer (PTC). Therefore, this study aimed to analyze immune cell profiles in the tumor microenvironment and their association with LMR in curatively resected PTC., Materials and Methods: The immune cell profiles of primary tumors in 162 patients with curatively resected PTC were analyzed clinicopathologically. Immunohistochemistry of tumor-associated macrophages (TAMs), myeloid-derived suppressor cells, and lymphocytes was performed using CD163, CD33, and CD3 antibodies, respectively. Prognostic analysis and correlation assays were performed using the immunocyte profiles. The gene expression of tumor-derived chemokines was assessed using a The Cancer Genome Atlas database., Results: Patients with a higher density of CD163 + TAMs exhibited a significantly worse prognosis than their counterparts (10-y recurrence-free survival: 80.9% versus 91.2%, P = 0.011). Multivariate prognostic analyses revealed that high CD163 + cell density (P = 0.011), low preoperative LMR (P = 0.003), pN1b (P = 0.005), and high thyroglobulin level (P = 0.038) were independent predictors of recurrence. High CD163 + cell density had a prognostic impact on stage II and III PTC. Interestingly, high CD163 + cell density correlated with low LMR and high monocyte fraction in peripheral blood. Indeed, the expression of TAM-inducible, tumor-derived chemokines is increased in the The Cancer Genome Atlas database., Conclusions: A high density of infiltrated CD163 + TAMs predicts recurrence in correlation with low LMR and circulating monocyte accumulation. Thus, TAMs should be considered when assessing advanced PTC., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: Carcinoma ex-pleomorphic adenoma (CEXPA) represents a malignant transformation from a recurrent or primary pleomorphic adenoma (PA), and the immune response may be essential in this process. Therefore, in this study, we aimed to identify and quantify subpopulations of dendritic cells (DCs) in CEXPA, residual PA in CEXPA (rPA), and PA., Materials and Methods: A multicenter study was performed collecting salivary gland tumor (SGT) samples from three Oral and Maxillofacial Pathology Centers. A tissue microarray containing 41 samples of CEXPA and 22 samples of PA was included in this study and submitted to immunohistochemical reactions against CD1a, CD83, CD207, and Ki67 antibodies., Results: Both PA and rPA showed a higher quantification of CD207+ and CD83+ cells when compared to CEXPA (p < 0.001 and p < 0.01, respectively). There was also a difference when comparing the cell proliferation index between PA/rPA and CEXPA using the Ki-67 marker (p = 0.043). However, there was no difference in the DC population regarding clinical parameters such as sex, anatomical location, size, and metastases (p > 0.06)., Conclusions: Immunohistochemical profile of DC subpopulations and cell proliferation biomarkers in SGTs can contribute as an important tool in the differentiation of benign and malignant tumors or detection of initial areas with malignant transformation., (© 2024 Wiley Periodicals LLC.)

Allergic fungal rhinosinusitis (AFRS) shares similarities with eosinophilic chronic rhinosinusitis (ECRS), both characterized by intractable nasal polyps. The key distinction lies in the presence of fungal infection within the nasal cavity. While ECRS nasal polyps are known for significant infiltration of M2 macrophages and eosinophils, as well as an increase in high endothelial venule (HEV)-like vessels, these features are less commonly reported in AFRS. This study compared clinicopathological findings between AFRS ( n =10), ECRS ( n =12), and non-ECRS ( n =10) patients' nasal polyps using immunohistochemical analysis for CD163 and CD68 to assess the M2/M1 macrophage ratio, and peripheral lymph node addressin (PNAd) and CD34 to evaluate the proportion of HEV-like vessels. AFRS showed a significantly higher number of CD163-positive M2 macrophages and an increased M2/M1 ratio compared with ECRS. However, the percentage of HEV-like vessels and the number of eosinophils infiltrating the nasal polyps were similar in both AFRS and ECRS. The observed increase in M2 macrophages in AFRS nasal polyps is presumed to be induced by fungal infection in the nasal cavity, in comparison with ECRS. These results highlight the distinctive immunological profiles of AFRS and ECRS, emphasizing the role of macrophage polarization in their pathogenesis.

Objective To investigate the phenotypic and functional characteristics of normal human peripheral blood neutrophils. Methods Normal human peripheral blood neutrophils were isolated through density gradient centrifugation followed by red blood cell lysis, and their surface markers and distribution characteristics in different neutrophil subsets were detected using multi-color immunofluorescence staining and flow cytometry. The phagocytic activity of neutrophils was evaluated with pHrodo TM Green E.coli BioParticles TM . The morphological characteristics of neutrophils undergoing the formation of neutrophil extracellular traps (NET) in vitro were examined via Wright-Giemsa staining and immunofluorescence staining, and were observed using standard light microscopy and confocal microscopy, respectively. Results CD66b, CD11b and CD16 were all highly expressed on normal human peripheral blood neutrophils and the percentage of CD16 positive neutrophils was higher compared to CD11b positive neutrophils. Neutrophils could be classified into three subsets based on the density of CD16 expression: namely, CD16 hi neutrophil, CD16 int neutrophil and CD16 lo neutrophil. The proportion of the CD16 hi neutrophil was the highest among the three subsets. The inhibitory molecule programmed cell death ligand 1 (PD-L1) was expressed on all neutrophil subsets without significant differences; however, the expression level of another inhibitory molecule, CD300LD, was markedly higher in the CD16 hi neutrophil compared to CD16 int or CD16 lo neutrophil. Each neutrophil subset demonstrated high levels of adhesion molecule CD62L without notable differences. Notably, the mean fluorescence intensity (MFI) value for pHrodo TM Green E.coli BioParticles TM in the CD16 int neutrophil was the highest among three subsets; and that in CD16 hi neutrophil was higher compared to CD16 lo neutrophil. Results of Wright-Giemsa staining showed that normal peripheral blood neutrophils typically possessed lobulated nuclei; upon stimulation with phorbol 12-myristate 13-acetate (PMA), the nuclei of these neutrophils formed a network structure while losing their lobulated nuclear morphology. Laser confocal microscopy results showed an increased release of neutrophil elastase (NE) with network formation in neutrophils after PMA stimulation. Conclusions Normal human peripheral blood neutrophils are heterogenous population with different expression of surface marker molecules, which might exert different immune functions. NET formation can be induced by PMA stimulation, and the differences of NET formation in different neutrophil subsets need to be further studied.

Objective: The current study aimed to investigate the characteristics of tumor-associated macrophages (TAMs) and their association with microvascular density (MVD) in tumor progression in different grades of orofacial squamous cell carcinoma (OSCC) in the Pakistani population., Study Design: This prospective study included 234 patients with oral cancer reported at different hospitals in Pakistan diagnosed with OSCC. Tumors were graded on the Anneroth grading system and the association between the frequency of TAMs and MVD was examined in vivo. The macrophages visible through immunohistochemistry for CD68 and the microvessels observed through immunohistochemistry for CD34 were manually counted in 3 high-power fields., Results: The CD68 and CD34 counts were significantly lower in well-differentiated squamous cell carcinoma compared to poorly differentiated squamous cell carcinoma. Linear regression analysis revealed a positive correlation between the area percentage of CD68 immunoreactivity and the grade of the tumor (r = 0.776). Vice versa, a positive correlation also existed between the area percentage of CD34 immunoreactivity and the grade of the tumor (r = 0.690). Pearson correlation revealed a positive association between the TAMs and MVD (r = 0.680; P < .001)., Conclusions: There was an increased population of tumor-associated macrophages and tumor angiogenesis with the increasing grade of orofacial squamous cell carcinoma. (Oral Surg Oral Med Oral Pathol Oral Radiol YEAR;VOL:page range)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Invasive lobular carcinoma of the breast is the most common special type breast cancer. It has been defined using morphological features, has a characteristic immunophenotype associated with the loss of E-cadherin mediated intercellular adhesion, and the background of this immunohistochemistry and morphology is generally a biallelic genetic alteration of the CDH-1 gene coding E-cadherin. However, the morphology may often deviate from the classical, and immunohistochemistry may also deviate from the typical, and then the diagnosis of invasive lobular carcinoma becomes less straight forward. Eventually, the definitions of this histological type, although similar, are not identical and this may also give ground to occasional different interpretations. This review summarizes different approaches to invasive lobular carcinomas and the deviations from what is considered normal., (Copyright © 2024 Società Italiana di Anatomia Patologica e Citopatologia Diagnostica, Divisione Italiana della International Academy of Pathology.)

Background: Oral lichen planus is a well-known chronic inflammatory mucocutaneous disorder, which has clinical and histological presentation that mimics oral lichenoid reaction. According to the fifth edition of WHO, both conditions are considered as oral potentially malignant disorders. Recent studies on oral potential disorders documented deregulation of some signaling molecules related to the Wnt/β-catenin pathway. Therefore this study aimed to compare the immune expression of β-catenin & CD163 in dysplastic /non-dysplastic cases of Oral lichen planus & oral lichenoid lesion. In addition, a statistical correlation between both immune markers was done regardless of the type of the study group., Methods: Four study groups were designated as 2 groups of Oral lichen planus (one dysplastic & one non -dysplastic) and the other 2 groups were oral lichenoid lesions (one dysplastic & one non -dysplastic). Ten cases in each group were collected and investigated by immunohistochemistry. The area percent of beta catenin and also counting of m2 macrophages expressing + CD163 marker was calculated in the study groups., Results: The Statistical analysis highlighted a statistically significant difference between the studied groups. Moreover, Pearson correlation test reported a significant moderate positive correlation between beta catenin & CD163 expression in the studied cases., Conclusion: Our findings supported new perceptions of the mechanism by which tumor associated macrophage specific β-catenin signaling promotes the aggressive behavior of oral potential malignant disorders., Clinical Relevance: Evidence of the relationship between beta catenin and M2 macrophages (+ CD163) may enhance the development of macrophage-based strategies for treatment and improve the prognosis of such cases., (© 2024. The Author(s).)

Aims: Microbial flora of dental plaque trigger innate and adaptive immune responses. The function of antigen-presenting cells (APCs) is to bridge the innate and adaptive immune systems. The human immune system contains three main types of APCs: dendritic cells (DC) (Langerhans cells (LCs) and interstitial DCs, IDCs), macrophages and B lymphocytes. In this study, the distribution and density of all APCs in healthy and inflamed human gingival tissue were comparatively analysed., Methods: Research was conducted on gingival biopsy specimens obtained from 55 patients and classified in three groups: healthy gingiva (control group, n=10), moderate periodontal disease (PD) (n=21) and severe PD (n=24). For APCs' identification antibodies raised against CD 1a (for LCs), S 100 protein (for iDCs), CD 68 (for macrophages) and CD 20 (for B lymphocytes) were used., Results: Increased density of IDCs, macrophages and B lymphocytes in lamina propria and reduced density of LCs in the gingival epithelium were found in patients with periodontitis. Simultaneously, it was noticed an increased concentration of macrophages and B cells in the gingival epithelium in patients with PD. No statistically significant difference in the distribution and density of APC was found among patients with moderate and advanced periodontitis., Conclusions: It was hypothesised that in the periodontitis the role of antigen presentation was largely taken from LCs by the DCs, macrophages and B cells. These APCs are thought to have less protective and tolerogenic potential than LCs and this is a significant reason for alveolar bone destruction in periodontitis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Langerhans cell histiocytosis (LCH) is a myeloid neoplasm characterized by clonal neoplastic proliferation of Langerhans-type dendritic cells associated with an inflammatory infiltrate predominantly composed of lymphocytes and eosinophils. In this article, we present an unusual case of LCH with significant swelling in the left lacrimal sac region in a 3-year-old child, clinically mimicking acute dacryocystitis. Microscopically, it showed intense inflammatory infiltrate and histiocytes with irregular nuclei. The tumor cells were positive for S-100 protein, CD1a, and CD207 (langerin). Molecular study was positive for the V600E/E2/D mutation (EXON 15). This case emphasizes the importance of careful clinical, radiographic, and microscopic evaluation, as some neoplasms may mimic common benign lesions., (Copyright © 2024 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.)

Purpose: Stereotactic body radiation therapy (SBRT) versus surgery for operable early-stage non-small cell lung cancer (ES-NSCLC) remains highly debated. Herein, we used spatial proteomics to identify whether any molecular biomarker(s) associate with the efficacy of either modality, in efforts to optimize treatment selection between surgery and SBRT for this population., Methods and Materials: We evaluated biopsy tissue samples from 44 patients with ES-NSCLC treated with first-line SBRT (cohort 1) by GeoMx Digital Spatial Profiling (DSP) with a panel of 70 proteins in 5 spatial molecular compartments: tumor (panCK+), leukocyte (CD45+), lymphocyte (CD3+), macrophage (CD68+), and stroma (α-SMA+). To validate the findings in cohort 1, biopsy samples from 52 patients with ES-NSCLC who received SBRT (cohort 2) and 62 patients with ES-NSCLC who underwent surgery (cohort 3) were collected and analyzed by multiplex immunofluorescence (mIF)., Results: In cohort 1, higher CD44 expression in the lymphocyte compartment was associated with poorer recurrence-free survival (RFS) (DSP: P < .001; mIF: P < .001) and higher recurrence rate (DSP: P = .001; mIF: P = .004). mIF data from cohort 2 validated these findings (P < .05 for all). From cohort 3, higher lymphocyte CD44 predicted higher RFS after surgery (P = .003). Intermodality comparisons demonstrated that SBRT was associated with significantly higher RFS over surgery in CD44-low patients (P < .001), but surgery was superior to SBRT in CD44-high cases (P = .016)., Conclusions: Lymphocyte CD44 may not only be a predictor of SBRT efficacy in this population but also an important biomarker (pending validation by large prospective data) that could better sharpen selection for SBRT versus surgery in ES-NSCLC., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background and Objectives: This study presents a retrospective analysis of 26 autopsy cases from a single centre, primarily focusing on forensic cases, with a majority of male individuals. Materials and Methods: We systematically analysed autopsy reports and cardiac tissue slides using haematoxylin-eosin stain and immunohistochemistry for CD3, CD163, and IL-6. The histological assessment evaluated key variables such as inflammation severity, necrosis, and background changes using a standardised grading system. Quantitative analysis of immunohistochemical markers was performed, calculating the percentage of positively stained cells within the inflammatory infiltrate. Results: The average age was 51.6 years, slightly skewed towards older males. The fatalities varied widely, with sudden death and drug abuse being the most common conditions linked to myocarditis findings on histological examination. A strong correlation was found between the severity of inflammation (measured by size within a myocardium section) and the scoring system based on the number of inflammatory foci per section ( p ≤ 0.001). Most cases showed mild to minimal fibrosis, with some exhibiting moderate to severe fibrosis, arteriosclerosis, and myocyte hypertrophy. The presence of protein CD3 in the inflammatory infiltrate revealed a moderate inverse correlation between the CD3 values and the severity of inflammation and necrosis, and a strong inverse correlation with neutrophil levels. CD3 levels were higher in sudden death cases and lower in cases with numerous inflammatory foci, highlighting the discreet nature of lymphocytic myocarditis. Macrophage presence, assessed using CD163, showed a moderate inverse correlation with neutrophil levels and significant differences between sudden death and non-sudden death cases. Macrophage-rich inflammation was observed in cases with pneumonia/bronchopneumonia-associated lesions. IL-6 expression showed a moderate direct correlation with inflammation severity ( p = 0.028), severity of necrosis ( p = 0.005), and the number of inflammatory foci per section ( p = 0.047). A moderate inverse correlation was found between CD3 and IL-6 expression ( p = 0.005). Conclusions : These findings highlight the need for a unique immunohistochemical approach in forensic cases of myocarditis, differing from guidelines for endomyocardial biopsies due to diverse inflammatory cells. The study suggests exploring inflammatory chemokines within myocarditis foci for their significance in clinical scenarios. Specifically, IL-6, a crucial pro-inflammatory interleukin, correlated significantly with the severity of inflammation and necrosis ( p < 0.05). This study provides novel and valuable insights into the histopathological and immunological markers of myocarditis in autopsy cases.

Introduction: Despite extensive research, comprehensive characterization of leukaemic stem cells (LSC) and information on their immunophenotypic differences from normal haematopoietic stem cells (HSC) is lacking. Herein, we attempted to unravel the immunophenotypic (IPT) characteristics and heterogeneity of LSC using multiparametric flow cytometry (MFC) and single-cell sequencing., Materials and Methods: Bone marrow aspirate samples from patients with acute myeloid leukaemia (AML) were evaluated using MFC at diagnostic and post induction time points using a single tube-10-colour-panel containing LSC-associated antibodies CD123, CD45RA, CD44, CD33 and COMPOSITE (CLL-1, TIM-3, CD25, CD11b, CD22, CD7, CD56) with backbone markers that is, CD45, CD34, CD38, CD117, sCD3. Single-cell sequencing of the whole transcriptome was also done in a bone marrow sample., Results: LSCs and HSCs were identified in 225/255 (88.2%) and 183/255 (71.6%) samples, respectively. Significantly higher expression was noted for COMPOSITE, CD45RA, CD123, CD33, and CD44 in LSCs than HSCs (p < 0.0001). On comparing the LSC specific antigen expressions between CD34+ (n = 184) and CD34- LSCs (n = 41), no difference was observed between the groups. More than one sub-population of LSC was demonstrated in 4.4% of cases, which further revealed high concordance between MFC and single cell transcriptomic analysis in one of the cases displaying three LSC subpopulations by both methods., Conclusion: A single tube-10-colour MFC panel is proposed as an easy and reproducible tool to identify and discriminate LSCs from HSCs. LSCs display both inter- and intra-sample heterogeneity in terms of antigen expressions, which opens the facets for single cell molecular analysis to elucidate the role of subpopulations of LSCs in AML progression., (© 2024 John Wiley & Sons Ltd.)

Abstract: Histiocytic disorders mostly occur as de-novo nodal or extranodal benign masses with rare secondary malignant transformation. A 10-year-old female presented with 10-cm cervical swelling since 9 months associated with fever. Computed tomography revealed left cervical lymphadenopathy and bilateral lung nodules. Lymph node excision biopsy showed effacement of architecture by atypical histiocytes with marked nuclear pleomorphism and frequent mitosis. Focal areas showed mature histiocytes with emperipolesis. The cells were immunopositive for CD68, CD163, and S100 (focal), whereas they were negative for Langerin and CD1a. The Ki67 proliferative index was 30%. A diagnosis of histiocytic sarcoma in a background of Rosai-Dorfman disease was made., (Copyright © 2023 Copyright: © 2023 Indian Journal of Pathology and Microbiology.)

Background: This study was designed to assess the influence of non-surgical periodontal therapy (NSPT) on gingival crevicular fluid (GCF) levels of semaphorin-4D (SEMA-4D), peptidylarginine deiminase-2 (PAD-2), and matrix metalloproteinase-8 (MMP-8) levels in periodontally healthy, Stage III periodontitis non-smoker and smoker patients., Methods: Sixty patients were equally divided into three groups, Group I: Periodontally healthy, Group II: Non-smokers with Stage III periodontitis, and Group III: Smokers with Stage III periodontitis. The patients underwent NSPT with clinical and biochemical parameters examined at baseline and 3 months post therapy. GCF was collected for levels of SEMA-4D, PAD-2, and MMP-8 through enzyme-linked immunosorbent assay (ELISA)., Results: Greater values of PPD (8.06 ± 0.19 mm), CAL (8.94 ± 0.19 mm), PI (2.58 ± 0.19) while lower PBI (1.39 ± 0.19%) and GI (1.72 ± 0.19) scores were seen in Group III as compared to Group II, which reduced significantly from baseline to 3 months in both the groups after NSPT. Minimum values of SEMA-4D, PAD-2, and MMP-8 levels in GCF were seen for Group I, which increased incrementally to Group II and III. Also, among Group II and III the SEMA-4D, PAD-2, and MMP-8 levels in GCF reduced from baseline to 3 months indicating a favorable response within the tissues., Conclusion: Greater levels in GCF of Levels of SEMA-4D, PAD-2, and MMP-8 in Group II and III, which reduced significantly post NSPT, implied that these biomarkers play a pivotal role in the inflammatory process and can be utilized for early diagnosis., (© 2023 American Academy of Periodontology.)

Introduction: Lymphocyte subset enumeration by flow cytometry is important for the therapeutic monitoring of a range of conditions. However, current bead-based methodologies do not produce metrologically traceable results. Here we compare an established bead-based methodology with a volumetric-based system traceable to an internationally recognised reference method., Method: A total of 118 samples received for lymphocyte subset analysis were tested using an established bead-based technique (BD Multitest™ 6-colour TBNK assay using Trucount™ tubes on a BD FACSLyric flow cytometer), followed by a volumetric method on the Sysmex XF-1600 flow cytometer using Exbio Kombitest 6-colour TBNK reagent. All samples were tested in accordance with the manufacturer's instructions., Results: Absolute count values from both methodologies for CD3+, CD3 + CD4+, CD3 + CD8+, CD19+ and CD3-CD16+/CD56+ lymphocyte populations were compared using linear regression (R 2 for all parameters >0.95) and Bland-Altman analysis. There was no significant bias (where p < 0.05) for absolute CD3 + CD4+ lymphocytes in the defined therapeutic range of 0-250 cells/μL (mean bias: 0.27 cells/μL). Although positive biases were seen for CD3 + CD4+ lymphocytes (over the entire range tested: 14-1798 cells/μL) and CD3-CD16+/CD56+ lymphocytes (mean bias: 10.83 cells/μL and 6.79 cells/μL, respectively). Negative biases were seen for CD3 + CD8+ and CD19+ lymphocytes (mean bias: -29.17 cells/μL and - 18.76 cells/μL, respectively)., Conclusion: A high degree of correlation was found for results from both methodologies and observed bias was within the limits of clinical acceptability for all populations. This shows that the metrologically traceable lymphocyte subset absolute counts produced by the Sysmex XF-1600 are robust within clinically required limits., (© 2023 The Authors. International Journal of Laboratory Hematology published by John Wiley & Sons Ltd.)

Endometriosis's pathophysiology remains incompletely understood, with evidence pointing towards a dysregulated immune response. Regulatory T (Treg) cells, pivotal in maintaining self-tolerance, may facilitate the survival of ectopic endometrial cells within the abdominal cavity, thereby contributing to endometriosis development. This study aimed to assess the prevalence of CD39+CD73+ suppressor Treg cell subsets in the peripheral blood of endometriosis patients. This research focuses on the pivotal role of regulatory T-cells (Tregs), which are essential for maintaining immune tolerance and preventing autoimmune diseases. A case-control study was conducted, including 32 women diagnosed with endometriosis and 22 control subjects. The frequency of peripheral blood CD39+CD73+ suppressor Treg cells was quantified using flow cytometry. No significant differences were observed in the frequency of CD3+CD4+CD25High cells (Median [M]: 10.1; Interquartile Range [IQR]: 6.32‒18.3 vs. M: 9.72; IQR: 6.22-19.8) or CD3+CD4+CD25HighCD39+Foxp3+ cells (M: 31.1; IQR: 19.7-44.0 vs. M: 30.55; IQR: 18.5-45.5) between controls and patients. However, a significantly lower frequency of CD3+CD4+CD25HighCD39+CD73+ cells was observed in the endometriosis group compared to controls (M: 1.98; IQR: 0.0377-3.17 vs. M: 2.25; IQR: 0.50-4.08; p = 0.0483), suggesting a reduction in systemic immune tolerance among these patients. This finding highlights the potential role of CD39 and CD73 expression on Treg cells as biomarkers for assessing disease severity and progression. Furthermore, elucidating the mechanisms driving these alterations may unveil new therapeutic strategies to restore immune equilibrium and mitigate endometriosis symptoms., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier España, S.L.U.)

Non muscle invasive bladder cancer (NMIBC) has unpredictable outcomes with a variable risk of recurrence and progression. Many clinic-pathological prognostic factors have been identified but remain insufficient, raising the need to investigate new biomarkers. The aim of our study was to assess the prognostic value of the immunohistochemical (IHC) markers E-Cadherin and B-Catenin in NMIBC. All cases of NMIBC were collected between 2008 and 2013. IHC analysis was performed using E-Cadherin and B-Catenin. Reduced or loss of E-Cadherin expression was assessed as abnormal. Only cases with B-Catenin intense membranous staining were considered normal. A correlation was found between abnormal E-Cadherin expression and stage ( p  = 0.001), grade ( p  = 0.0000000), recurrence ( p  = 0.0000000), progression ( p  = 0.01), recurrence-free survival ( p  = 0.00000001), and progression-free survival ( p  = 0.01). A statistically significant association was found between B-Catenin and stage ( p  = 0. 05), grade ( p  = 0.02), and recurrence ( p  = 0.02). The abnormal expression of these markers could help to identify a high-risk subgroup of NMIBC that might benefit from either more accurate follow-up or more aggressive treatment.

A dysfunctional immune tumor microenvironment facilitates disease progression in multiple myeloma (MM). Using multiplex immunohistochemistry (mIHC), we describe the quantitative and qualitative changes in CD3+CD8+ cytotoxic T cells and assess their proximity to malignant plasma cells (PC) in patients with monoclonal gammopathy of undetermined significance (MGUS), and newly diagnosed (ND) and relapsed and/or refractory (RR) MM. Formalin-fixed, paraffin-embedded trephine sections from patients with MGUS (N=32), NDMM (N=65), and RRMM (N=59) were sequentially stained for CD138, CD3, CD8, and checkpoint receptors (CPR) Tim-3, Lag-3, and PD-1. The Halo® image analysis platform was used for cell segmentation and phenotyping, facilitating enumeration of cytotoxic T cells and analysis of proximity to PC. The percentage of CD8+ cytotoxic T cells in proximity to PC is greater in patients with NDMM than patients with RRMM (at 50 μm distance, 90.8% vs. 81.5%; P=0.038). There is a trend for more CD3+ T cells in MGUS (P=0.08) but no difference was observed in the prevalence of CD8+ cytotoxic T cells (P=0.48). Lag-3 is the most common CPR expressed on cytotoxic T cells in myeloma (P<0.0001), while PD-1 is the most common CPR on CD8- T cells of patients with MGUS and RRMM. Our study is the first to report on the spatial relationship between T cells and PC using mIHC on FFPE bone marrow trephine sections from patients with PC dyscrasia. The proximity of T cells to PC during early stages of MM, and overexpression of Lag-3, validate the move of immune therapeutic strategies, including T-cell engagers and checkpoint inhibitors, to upfront treatment or in early-line treatment of MM.

Assessing minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is essential for adjusting therapeutic strategies and predicting relapse. Quantitative polymerase chain reaction (qPCR) is the gold standard for MRD. Alternatively, flow cytometry is a quicker and cost-effective method that typically uses leukaemia-associated immunophenotype (LAIP) or different-from-normal (DFN) approaches for MRD assessment. This study describes an optimized 12-colour flow cytometry antibody panel designed for BCP-ALL diagnosis and MRD monitoring in a single tube. This method robustly differentiated hematogones and BCP-ALL cells using two specific markers: CD43 and CD81. These and other markers (e.g. CD73, CD66c and CD49f) enhanced the specificity of BCP-ALL cell detection. This innovative approach, based on a dual DFN/LAIP strategy with a principal component analysis method, can be used for all patients and enables MRD analysis even in the absence of a diagnostic sample. The robustness of our method for MRD monitoring was confirmed by the strong correlation (r = 0.87) with the qPCR results. Moreover, it simplifies and accelerates the preanalytical process through the use of a stain/lysis/wash method within a single tube (<2 h). Our flow cytometry-based methodology improves the BCP-ALL diagnosis efficiency and MRD management, offering a complementary method with considerable benefits for clinical laboratories., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Objectives: Early identification of vocal fold leukoplakia (VFL), which has a risk of progressing to malignant transformation, remains a controversial topic. The identification of biological markers for diagnosing these lesions would lead to a more effective treatment. We aimed to analyze the immunoexpression of cathepsin B and E-cadherin in VFL and correlate it with clinical and epidemiological data and disease prognosis., Methods: Thirty-two patients with VFL treated with microsurgery were retrospectively evaluated. The patients were distributed according to the histological results into Group A (low grade) and Group B (high grade). The expression of markers was quantitatively determined as per their staining intensity and tissue distribution using ImageLab. The index of expression (IE) of each marker was correlated with tobacco and alcohol consumption, signs of laryngopharyngeal reflux, and local recurrence of the lesion., Results: The correlation between the IE of markers and variables within the two groups (A and B) demonstrated that patients in Group B with local recurrence had a higher IE of cathepsin B. When all patients (A + B) were included, the same analysis demonstrated that the IE of cathepsin B was higher among smokers and patients who did not show signs of reflux and that the IE of E-cadherin was higher only in patients with recurrence., Conclusion: Patients with moderate to severe dysplasia and carcinoma in situ who smoked as well as had a high IE of cathepsin B were more prone to local recurrence. Regardless of the type of histological lesion, patients with signs of laryngopharyngeal reflux had a lower IE of cathepsin B. The IE of E-cadherin was higher among patients with VFL who relapsed after initial treatment., (Copyright © 2021 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Contrast enhancement of intracranial aneurysm wall during MRI with targeted visualization of vascular wall correlates with previous aneurysm rupture and, according to some data, may be a predictor of further rupture of unruptured aneurysms., Objective: To analyze possible causes of aneurysm contrast enhancement considering morphological data of aneurysm walls., Material and Methods: The study included 44 patients with intracranial aneurysms who underwent preoperative MRI between November 2020 and September 2022. Each aneurysm was assessed regarding contrast enhancement pattern. Microsurgical treatment of aneurysm was accompanied by resection of its wall for subsequent histological and immunohistochemical analysis regarding thrombosis, inflammation and neovascularization. Specimens were subjected to histological and immunochemical analysis. Immunohistochemical analysis was valuable to estimate inflammatory markers CD68 and CD3, as well as neurovascularization marker SD31., Results: Aneurysms with contrast-enhanced walls were characterized by higher number of CD3+, CD68+, CD31+ cells and parietal clots. Intensity of contrast enhancement correlated with aneurysm wall abnormalities., Conclusion: Contrast enhancement of aneurysm wall can characterize various morphological abnormalities.

Placental foetal vascular malperfusion (FVM) may be responsible for complicated foetal or neonatal condition. By highlighting endothelial fragmentation, the double E-cadherin/CD34 immunostain highlights distal villous endothelial fragmentation of recent FVM not seen on haematoxylin-eosin stained sections. We routinely perform the stain on a grossly unremarkable placental sections of placentas predominantly from pregnancies with mass-forming foetal anomalies and umbilical cord complications. The stain can upgrade the FVM and/or reveal its temporal heterogeneity, both useful in establishing the cause of foetal death or poor neonatal condition. It also highlights the basement membranes of syncytiotrophoblastic cells which, in conjunction with endothelial staining, is helpful in the diagnosis of widening thereof in distal villous hypomaturity. It can distinguish mineralised stem occluding thrombi from mineralised trophoblastic pseudo inclusions - the former outlined by CD34, the latter by E-cadherin - thus helping to differentiate FVM from placental aneuploidies. The E-cadherin component helps in the diagnosis of trophoblastic lesions of shallow placental implantation featuring an increased number of extravillous trophoblasts in placental membranes and chorionic disc. Therefore, the double immunostain is helpful in histological diagnosis of placental lesions and patterns of injury.

Objectives: To identify the effect of distribution characteristic of macrophages on placental function and angiogenesis in pregnancies with preeclampsia (PE) in presence of fetal growth restriction (FGR) or preeclampsia without FGR., Material and Methods: The study tested the hypothesis that there was association between distribution characteristic of macrophage subsets (marked by CD68, CD163, respectively) and placental capillary development, leading to placental dysfunction in PE pregnancies with FGR (n = 36). Changes in placental parameters related with efficiency and angiogenesis and macrophage phenotypes (CD68 and CD163) were evaluated by immunohistochemistry. Pearson correlation analysis was performed to analysis the association between macrophage phenotype and placental function as well the CD34 staining, respectively. Additionally, the localization of CD68 and CD163 was assessed by using immunoflurorescence staining., Results: Pearson correlation analysis had shown the positive association between CD68 expression and microvessel formation and the reverse linear relationship between CD163 staining and placental sufficiency in PE + FGR placenta. The co-localization of CD163 and CD34 may pointed to the compensatory role of CD163 distribution involved in prompting neovascularization., Conclusions: The association between disturbed distribution of macrophages and placental efficiency and angiogenesis were only found in PE with FGR not in PE pregnancies without FGR, underlying the discrepancy role of macrophage subsets depending on the clinical phenotype of PE pregnancies.

Introduction: Pulmonary Langerhans cell histiocytosis (PLCH) is a rare interstitial lung disease characterized by the accumulation of Langerhans cells within the lung tissue. The diagnosis of PLCH traditionally involves clinical, radiological, and lung biopsy histopathological evaluations., Case Presentation: We present 2 cases where the diagnosis of PLCH was confirmed through the analysis of bronchoalveolar lavage (BAL) fluid cytology using immunoperoxidase technique, highlighting the significance of this minimally invasive technique in the diagnostic process. Clinical and radiological examination suggested advanced interstitial lung disease characterized by a fibrocystic pattern in both cases. The cytologic analysis of the BAL fluid revealed typical histiocytes with longitudinal grooves and eosinophils, which was better seen on liquid-based cytology (LBC) smears. ICC with CD1a, Langerin, and S-100 confirmed the diagnosis of PLCH., Conclusion: Detecting PLCH through the examination of BAL cytology poses challenges, yet it is achievable, particularly with the assistance of LBC and ICC., (© 2024 S. Karger AG, Basel.)

Objectives: Epstein-Barr virus-positive large B-cell lymphoma (EBV+ LBCL) is a heterogeneous group of diseases that may resemble classic Hodgkin lymphoma (CHL) both morphologically and immunophenotypically. However, these diseases are treated with different therapies and carry distinct prognoses. We examined CD200 expression by immunohistochemistry in EBV+ LBCL and evaluated its diagnostic utility in the differential diagnosis with CHL., Methods: CD200 immunohistochemistry was performed on archival material from 20 cases of CHL (11 EBV+, 9 EBV-), 11 cases of EBV+ LBCL, and 10 cases of diffuse large B-cell lymphoma, not otherwise specified (DLBCL NOS). Staining pattern and intensity (0-3+ scale) were recorded., Results: CD200 positivity was seen in Reed-Sternberg cells in 19 (95%) of 20 cases of CHL, predominantly in a strong (3+, 15/19) and diffuse (>50% of cells, 17/19) pattern. In contrast, CD200 was negative in 8 (73%) of 11 cases of EBV+ LBCL; the 3 positive cases showed 1 to 2+ staining in less than 50% of lesional cells. All cases of DLBCL NOS were negative for CD200., Conclusions: CD200 may be a useful immunophenotypic marker in differentiating EBV+ LBCL from CHL, with negative to partial/weak staining favoring a diagnosis of EBV+ LBCL and strong diffuse staining favoring a diagnosis of CHL., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Aberrant phenotypes in acute myeloid leukemia have variable frequencies and their prognostic value with adverse hematological and other biological prognostic factors is still controversial, despite several reports of clinical significance. To date, no study has been reported evaluating the incidence of these phenotypic aberrations in the Moroccan population. The aim is to evaluate the incidence of aberrant phenotype expressions in acute myeloid leukemia and correlate their presence with the different AML subtypes, and clinical and biological characteristics in Moroccan patients., Methods: Fifty-four AML patients were diagnosed according to WHO classification 2016 criteria. Immunopheno-typing by flow cytometric analysis was performed to evaluate aberrant phenotypes on myeloblasts., Results: The occurrence of lymphoid antigens in AML was higher (51.8%), which was closer to that reported in the literature. CD7 has been revealed to be the most commonly expressed lymphoid antigen. Besides, CD19 was expressed in all 3.7% of M2 AML subtype. However, we could find no statistically significant differences between these aberrant phenotypes regarding FAB subtypes or clinical and biological outcomes. The great majority of AML cases showed asynchronous expression (57%) with significant differences regarding FAB subtypes., Conclusions: Aberrant phenotypes might be associated with different leukemia subtypes that should be studied for a better understanding of their biological significance and adding important information for prognosis and, at the same time, could be of help when looking for minimal residual disease during morphologic remission.

Currently, there are no curative treatment options for mycosis fungoides (MF) and Sézary syndrome (SS) other than stem cell transplant. Understanding the interplay between tumor cells and tumor microenvironment could aid in the development of new therapies. Tumor-associated macrophages (TAMs) mostly have M2 phenotype that promotes tumor progression. This study investigated CD68+ and CD163+ TAMs as well as CD163/CD68 ratio in skin lesions from different stages of MF, large-plaque parapsoriasis, and SS. Moreover, we analyzed serum levels of sCD163 and CCL22 in correlation with TAMs count and CD163/CD68 ratio. CD68+ and CD163+ TAMs count significantly increased as the disease progressed. CD163/CD68 ratio was highest at MF tumor stage and SS indicating M2 polarization with disease progression. Significant positive correlations were detected between serum levels of sCD163 and CCL22 and CD68+ and CD163+ TAMs count and CD163/CD68 ratio. We concluded that TAMs play an important role in MF progression. High CD163/CD68 ratio in tumor stage MF and SS indicates M2 polarization of TAMs with tumor progression. CD163/CD68 ratio should be considered in assessing TAMs rather than total TAMs count. Also, sCD163 and CCL22 serum levels reflect M2 load and thus could be used as markers to assess disease progression., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The tight junction membrane protein claudin 1 and the adherens junction protein E-cadherin play critical roles in cell-cell communication and in cell signaling. As a result, their protein levels and distribution in cancer have been a focus of cancer researchers in recent years. The loss of sensitivity to contact inhibition and the establishment of invasive properties in cancer are thought to be a result of the mislocalization of these membrane proteins to the cytoplasm. However, reports on their distribution and levels have been inconsistent. It is therefore critical that the techniques used to determine the cellular localization of these proteins be both consistent and reliable. This study was undertaken to determine the optimal fixation method, methanol or formalin, for the detection of claudin 1 and E-cadherin by immunofluorescence in five different human breast cancer cell lines. Both methods exhibited staining of the cell membrane and cytoplasm, but the strongest and most distinct signals were obtained using methanol fixation. Interestingly, cell-specific differences were also observed that appeared to be associated with levels of claudin 1 and E-cadherin as seen by Western blotting. Therefore, when evaluating cellular localization of the junction proteins claudin 1 and E-cadherin, expression level and cell type differences must be considered.

Disease Overview: Hairy cell leukemia (HCL) and HCL-like disorders, including HCL variant (HCL-V) and splenic diffuse red pulp lymphoma (SDRPL), are a very heterogeneous group of mature lymphoid B-cell disorders characterized by the identification of hairy cells, a specific genetic profile, a different clinical course, and the need for appropriate treatment., Diagnosis: Diagnosis of HCL is based on morphological evidence of hairy cells, an HCL immunologic score of 3 or 4 based on the CD11C, CD103, CD123, and CD25 expression, the trephine biopsy which makes it possible to specify the degree of tumoral medullary infiltration and the presence of BRAF V600E somatic mutation., Risk Stratification: Progression of patients with HCL is based on a large splenomegaly, leukocytosis, a high number of hairy cells in the peripheral blood, and the immunoglobulin heavy chain variable region gene mutational status. VH4-34-positive HCL cases are associated with a poor prognosis., Treatment: Patients should be treated only if HCL is symptomatic. Chemotherapy with risk adapted therapy purine analogs (PNAs) are indicated in first-line HCL patients. The use of chemo-immunotherapy combining PNAs and rituximab (R) represents an increasingly used therapeutic approach. Management of relapsed/refractory disease is based on the use of BRAF inhibitors (BRAFi) plus rituximab or MEK inhibitors (MEKi), recombinant immunoconjugates targeting CD22 or Bruton Tyrosine Kinase inhibitors (BTKi). However, the optimal sequence of the different treatments remains to be determined. The Bcl2-inhibitors (Bcl-2i) can play a major role in the future., (© 2021 Wiley Periodicals LLC.)

Invariant NK T (iNKT) cells are implicated in viral clearance; however, their role in hepatitis C virus (HCV) infection remains controversial. Here, iNKT cells were studied during different stages of HCV infection. iNKT cells from patients with acute HCV infection and people who inject drugs (PWID) with chronic or spontaneously resolved HCV infection were characterized by flow cytometry. In a longitudinal analysis during acute HCV infection, frequencies of activated CD38+ iNKT cells reproducibly declined in spontaneously resolving patients, whereas they were persistently elevated in patients progressing to chronic infection. During the first year of infection, the frequency of activated CD38+ or CD69+ iNKT cells strongly correlated with alanine transaminase levels with particularly pronounced correlations in spontaneously resolving patients. Increased frequencies of activated iNKT cells in chronic HCV infection were confirmed in cross-sectional analyses of PWID with chronic or spontaneously resolved HCV infection; however, no apparent functional differences were observed with various stimulation protocols. Our data suggest that iNKT cells are activated during acute hepatitis C and that activation is sustained in chronic infection. The correlation between the frequency of activated iNKT cells and alanine transaminase may point toward a role of iNKT cells in liver damage.

Nivolumab, an immune checkpoint blocker, has been approved for advanced gastric cancer (GC), but predictive factors of nivolumab's efficacy in patients with GC, especially immune cells such as tissue-resident memory T cells or those forming tertiary lymphoid structures (TLS), remain unclear. Tissue samples were obtained from surgically resected specimens of patients with GC who were treated with nivolumab as third-line or later treatment. Immunohistochemical staining was performed to detect the presence of TLS and CD103+ T cells and assess the association between TLSs and response to nivolumab treatment. A total of 19 patients were analyzed. In patients with partial response (PR) to nivolumab, numerous TLS were observed, and CD103+ T cells were found in and around TLS. Patients with many TLS experienced immune-related adverse events more often than those with few TLS (p = 0.018). The prognosis of patients with TLS high was better than those with TLS low. Patients with a combination of TLS high and CD103 high tended to have a better prognosis than other groups. Our results suggested that TLS status might be a predictor of nivolumab effectiveness., Competing Interests: The authors have declared that no competing interests exist.

Cancer and atherosclerosis are chronic diseases that share common characteristics at both early and advanced stages and can arise from multiple factors. Both diseases are characterized by uncontrolled cell proliferation, inflammation, angiogenesis and apoptosis. Herein we investigated the ability of a peptide (CTHRSSVVC), that was previously reported to bind atherosclerotic lesions to home in the tumor microenvironment. The CTHRSSVVC peptide was synthesized on solid phase and N-terminally labeled with a sulfo-Cy5 dye. The specific binding to macrophage was evaluated in vitro with flow cytometry and immunofluorescence and in vivo for tumor targeting in BALB/c mice bearing a 4T1 tumor using optical imaging. The sulfo-Cy5-CTHRSSVVC peptide was synthesized in greater than 99% purity. No selective binding of the sulfo-Cy5-CTHRSSVVC peptide to macrophages in vitro was observed, however in vivo the sulfo-Cy5-CTHRSSVVC peptide accumulated in the 4T1 tumor, with a tumor-to-normal tissue ratio of 7.21 ± 1.44 at 2 h post injection. Ex vivo analysis of tumor tissue by confocal microscopy suggested that the sulfo-Cy5-CTHRSSVVC peptide had accumulated in the stroma of the tumor specifically, in regions of spindle shaped cells. In conclusion, although the target for the sulfo-Cy5-CTHRSSVVC peptide remains to be identified, the Cy5-CTHRSSVVC peptide warrants further investigation as a tumor imaging agent., (Copyright © 2021. Published by Elsevier Masson SAS.)

Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. Despite advances in therapy, prognosis remains poor, with a 5-year overall survival of 30%. The immunophenotype of Sézary cells is diverse, which hampers efficient diagnosis, sensitive disease monitoring, and accurate assessment of treatment response. Comprehensive immunophenotypic profiling of Sézary cells with an in-depth analysis of maturation and functional subsets has not been performed thus far. We immunophenotypically profiled 24 patients with SS using standardized and sensitive EuroFlow-based multiparameter flow cytometry. We accurately identified and quantified Sézary cells in blood and performed an in-depth assessment of their phenotypic characteristics in comparison with their normal counterparts in the blood CD4+ T-cell compartment. We observed inter- and intrapatient heterogeneity and phenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and nonclassical T helper subsets with different maturation phenotypes. We combined multiparameter flow cytometry analyses with fluorescence-activated cell sorting and performed RNA sequencing studies on purified subsets of malignant Sézary cells and normal CD4+ T cells of the same patients. We confirmed pure monoclonality in Sézary subsets, compared transcriptomes of phenotypically distinct Sézary subsets, and identified novel downregulated genes, most remarkably THEMIS and LAIR1, which discriminate Sézary cells from normal residual CD4+ T cells. Together, these findings further unravel the heterogeneity of Sézary cell subpopulations within and between patients. These new data will support improved blood staging and more accurate disease monitoring., (© 2021 by The American Society of Hematology.)

Minimal residual disease (MRD)-negative status in multiple myeloma (MM) is associated with favorable outcomes. Although EuroFlow next-generation flow (NGF) is a global standard for MRD detection, its operating cost is high. Therefore, it is desirable to develop a less expensive method with equivalent sensitivity to that of EuroFlow-NGF. In this study, we compared the analytical ability of our BML 10-color multiparameter flow cytometry (MFC) to that of EuroFlow-NGF. Bone marrow samples collected from 51 patients with MM were subjected to MRD detection using BML 10-color-MFC and EuroFlow-NGF. Our antibody panel consisted of CD38 multiepitope, CD138, CD45, CD56, CD19, CD27, CD81, CD117, cytoplasmic immunoglobulin (cIg) κ, and cIgλ in a single tube. The median percentages of total plasma cells, as per 10-color-MFC and EuroFlow-NGF, were 0.2148% and 0.2200%, respectively, with a good correlation between the methods (r = 0.950). The median percentages of myeloma cells determined via 10-color-MFC and EuroFlow-NGF were 0.0012% and 0.0007%, respectively, with a strong correlation (r = 0.954). Our 10-color-MFC demonstrated high sensitivity to detect MRD; the results showed a good correlation with those obtained using EuroFlow-NGF. Therefore, our cost-effective single-tube MFC (approximately 100 USD/sample) is a promising alternative method for the detection of MRD in patients with MM., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The mouse hematopoietic system has served as a paradigm for analysis of developmental fate decisions in tissue homeostasis and regeneration. However, multiple immunophenotypic definitions of, and sometimes divergent nomenclatures used to classify, murine multipotent progenitors (MPPs) have emerged in the field over time. This has created significant confusion and inconsistency in the hematology field. To facilitate easier comparison of murine MPP phenotypes between research laboratories, a working group of four International Society for Experimental Hematology (ISEH) members with extensive experience studying the functional activities associated with different MPP phenotypic definitions reviewed the current state of the field with the goal of developing a position statement toward a simplified and unified immunophenotypic definition of MPP populations. In November of 2020, this position statement was presented as a webinar to the ISEH community for discussion and feedback. Hence, the Simplified MPP Identification Scheme presented here is the result of curation of existing literature, consultation with leaders in the field, and crowdsourcing from the wider experimental hematology community. Adoption of a unified definition and nomenclature, while still leaving room for individual investigator customization, will benefit scientists at all levels trying to compare these populations between experimental settings., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Objectives: This study aims to investigate if addition of fibroblast-stromal cell markers to a classification of synovial pathotypes improves their predictive value on clinical outcomes in rheumatoid arthritis (RA)., Methods: Active RA patients with a knee needle synovial biopsy at baseline and finished 1-year follow-up were recruited from a real-world prospective cohort. Positive staining for CD20, CD38, CD3, CD68, CD31, and CD90 were scored semiquantitatively (0-4). The primary outcome was radiographic progression defined as a minimum increase of 0.5 units of the modified total Sharp score from baseline to 1 year., Results: Among 150 recruited RA patients, 123 (82%) had qualified synovial tissue. Higher scores of CD20+ B cells, sublining CD68+ macrophages, CD31+ endothelial cells, and CD90+ fibroblasts were associated with less decrease in disease activity and greater increase in radiographic progression. A new fibroblast-based classification of synovial pathotypes giving more priority to myeloid and stromal cells classified samples as myeloid-stromal (57.7%, 71/123), lymphoid (31.7%, 39/123), and paucicellular pathotypes (10.6%, 13/123). RA patients with myeloid-stromal pathotype showed the highest rate of radiographic progression (43.7% vs. 23.1% vs. 7.7%, p = 0.011), together with the lowest rate of Boolean remission at 3, 6, and 12 months. Baseline synovial myeloid-stromal pathotype independently predicted radiographic progression at 1 year (adjusted OR: 3.199, 95% confidence interval (95% CI): 1.278, 8.010). Similar results were obtained in a subgroup analysis of treatment-naive RA., Conclusions: This novel fibroblast-based myeloid-stromal pathotype could predict radiographic progression at 1 year in active RA patients which may contribute to the shift of therapeutic decision in RA., 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 Zhang, Ma, Mo, Jing, Zheng, Chen, Wu, Chen, Zhang, Zou, Lin, Xu, Zou, Yang, Ling, Miossec and Dai.)

Purpose: Dendritic cells (DCs) are the most potent antigen-presenting cells that play a major role in initiating the antitumor immune response in different types of cancer. However, the prognostic significance of the accumulation of these cells in human early breast tumors is not totally clear. The aim of this study is to evaluate the prognostic relevance of CD1a( +) and CD83( +) dendritic cells in early breast cancer patients., Methods: We conducted immunohistochemical assays to determine the number of stromal CD1a( +) and CD83( +) DCs in primary tumors from early invasive ductal breast cancer patients, and analyzed their association with clinico-pathological characteristics., Results: Patients with high CD1a( +) DC number had lower risk of bone metastatic occurrence, as well as, longer disease-free survival (DFS), bone metastasis-free survival (BMFS) and overall survival (OS). Moreover, CD1a( +) DC number was an independent prognostic factor for BMFS and OS. In contrast, we found that patients with high number of CD83( +) DCs had lower risk of mix (bone and visceral)-metastatic occurrence. Likewise, these patients presented better prognosis with longer DFS, mix-MFS and OS. Furthermore, CD83( +) DC number was an independent prognostic factor for DFS and OS., Conclusion: The quantification of the stromal infiltration of DCs expressing CD1a or CD83 in early invasive breast cancer patients serves to indicate the prognostic risk of developing metastasis in a specific site., (© 2021. The Japanese Breast Cancer Society.)

The coronavirus disease 2019(COVID-19) is recognized as systemic inflammatory response syndrome. It was demonstrated that a rapid increase of cytokines in the serum of COVID-19 patients is associated with the severity of disease. However, the mechanisms of the cytokine release are not clear. By using immunofluorescence staining we found that the number of CD11b positive immune cells including macrophages in the spleens of died COVID-19 patients, was significantly higher than that of the control patients. The incidence of apoptosis as measured by two apoptotic markers, TUNEL and cleaved caspase-3, in COVID-19 patients' spleen cells is higher than that in control patients. By double immunostaining CD11b or CD68 and SARS-CoV-2 spike protein, it was found that up to 67% of these immune cells were positive for spike protein, suggesting that viral infection might be associated with apoptosis in these cells. Besides, we also stained the autophagy-related molecules (p-Akt、p62 and BCL-2) in spleen tissues, the results showed that the number of positive cells was significantly higher in COVID-19 group. And compared with non-COVID-19 patients, autophagy may be inhibited in COVID-19 patients. Our research suggest that SARS-CoV-2 may result in a higher rate of apoptosis and a lower rate of autophagy of immune cells in the spleen of COVID-19 patients. These discoveries may increase our understanding of the pathogenesis of COVID-19., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The receptor-interacting protein kinase 4 (RIPK4) plays an important role in the development and maintenance of various tissues including skin, but its role in melanoma has not been reported. Using patient-derived cell lines and clinical samples, we show that RIPK4 is expressed in melanomas at different levels. This heterogenous expression, together with very low level of RIPK4 in melanocytes, indicates that the role of this kinase in melanoma is context-dependent. While the analysis of microarray data has revealed no straightforward correlation between the stage of melanoma progression and RIPK4 expression in vivo, relatively high levels of RIPK4 are in metastatic melanoma cell lines. RIPK4 down-regulation by siRNA resulted in the attenuation of invasive potential as assessed by time-lapse video microscopy, wound-healing and transmigration assays. These effects were accompanied by reduced level of pro-invasive proteins such as MMP9, MMP2, and N-cadherin. Incubation of melanoma cells with phorbol ester (PMA) increased PKC-1β level and hyperphosphorylation of RIPK4 resulting in degradation of RIPK4. Interestingly, incubation of cells with PMA for short and long durations revealed that cell migration is controlled by the NF-κB signaling in a RIPK4-dependent (RIPK4 high ) or independent (RIPK4 low ) manner depending on cell origin (distant or lymph node metastasis) or phenotype (mesenchymal or epithelial).

Neuroinflammation is a process related to the onset of neurodegenerative diseases; one of the hallmarks of this process is microglial reactivation and the secretion by these cells of proinflammatory cytokines such as TNF α . Numerous studies report the relationship between neuroinflammatory processes and exposure to anthropogenic air pollutants, but few refer to natural pollutants. Volcanoes are highly inhabited natural sources of environmental pollution that induce changes in the nervous system, such as reactive astrogliosis or the blood-brain barrier breakdown in exposed individuals; however, no neuroinflammatory event has been yet defined. To this purpose, we studied resting microglia, reactive microglia, and TNF α production in the brains of mice chronically exposed to an active volcanic environment on the island of São Miguel (Azores, Portugal). For the first time, we demonstrate a proliferation of microglial cells and an increase in reactive microglia, as well an increase in TNF α secretion, in the central nervous system of individuals exposed to volcanogenic pollutants., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 A. Navarro-Sempere et al.)

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8 + and CD4 + tumor-specific reactive TILs in vitro , using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8 + TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4 + and CD8 + tumor-specific reactive TILs. In situ , the combined detection of TNFRSF9 , TNF , and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro , which can be rapidly adopted in most cancer immunology laboratories., Competing Interests: MD has received honoraria for lectures from Roche and Novartis (past two years). IMS has received honoraria for consultancies and lectures from Novartis, Roche, Merck, and Bristol-Myers Squibb; a restricted research grant from Novartis; and financial support for attending symposia from Bristol-Myers Squibb, Merck, Novartis, Pfizer, and Roche. HDR is currently an employee at Novo Nordisk. The remaining 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 Draghi, Chamberlain, Khan, Papp, Lauss, Soraggi, Radic, Presti, Harbst, Gokuldass, Kverneland, Nielsen, Westergaard, Andersen, Csabai, Jönsson, Szallasi, Svane and Donia.)

Leptomeningeal disease (LMD) in melanoma patients is associated with significant neurological sequela and has a dismal outcome, with survival measured typically in weeks. Despite the therapeutic benefit of targeted therapies and immunotherapies for Stage IV melanoma, patients with LMD do not typically benefit. A deeper understanding of the tumor microenvironment (TME) of LMD may provide more appropriate therapeutic selection. A retrospective analysis of subjects who underwent surgical resection with LMD (n=8) were profiled with seven color multiplex staining to evaluate the expression of the global immune suppressive hub - the signal transducer and activator of transcription 3 (STAT3) and for the presence of CD3+ T cells, CD68+ monocyte-derived cells, CD163+ immune suppressive macrophages, and CD11c+ cells [potential dendritic cells (DCs)] in association with the melanoma tumor marker S100B and DAPI for cellular nuclear identification. High-resolution cellular imaging and quantification was conducted using the Akoya Vectra Polaris. CD11c+ cells predominate in the TME (10% of total cells), along with immunosuppressive macrophages (2%). Another potential subset of DCs co-expressing CD11c+ and the CD163+ immunosuppressive marker is frequently present (8/8 of specimens, 8%). Occasional CD3+ T cells are identified, especially in the stroma of the tumor (p=0.039). pSTAT3 nuclear expression is heterogeneous in the various immune cell populations. Occasional immune cluster interactions can be seen in the stroma and on the edge. In conclusion, the TME of LMD is largely devoid of CD3+ T cells but is enriched in immune suppression and innate immunity., Competing Interests: AH serves on the advisory boards of Caris Life Sciences and WCG Oncology, receives royalties on licensed intellectual property from Celldex Therapeutics and DNAtrix, and received research support from Codiak BioSciences Inc. The remaining 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 Najem, Marisetty, Horbinski, Long, Huse, Glitza Oliva, Ferguson, Kumthekar, Wainwright, Chen, Lesniak, Burks and Heimberger.)