Toll-like receptor 8 agonists improve NK-cell function primarily targeting CD56brightCD16- subset.

BACKGROUND: Toll-like receptors (TLRs) are pattern-recognition sensors mainly expressed in innate immune cells that directly recognize conserved pathogen structures (pathogen-associated molecular patterns-PAMPs). Natural killer (NK) cells have been described to express different endosomal TLRs triggered by RNA and DNA sequences derived from both viruses and bacteria. This study was addressed to establish which endosomal TLR could directly mediate NK activation and function after proper stimuli. It was also important to establish the most suitable TLR agonist to be used as adjuvant in tumor vaccines or in combined cancer immunotherapies. METHODS: We assessed endosomal TLR expression in total NK cells by using RT-qPCR and western blotting technique. In some experiments, we purified CD56brightCD16- and CD56dimCD16+ cells subsets by using NK Cell Isolation Kit Activation marker, cytokine production, CD107a expression and cytotoxicity assay were evaluated by flow cytometry. Cytokine release was quantified by ELISA. NK cells obtained from ovarian ascites underwent the same analyses. RESULTS: Although the four endosomal TLRs (TLR3, TLR7/8, and TLR9) were uniformly expressed on CD56brightCD16- and CD56dimCD16+ cell subsets, the TLR7/8 (R848), TLR3 (polyinosinic-polycytidylic acid, Poly I:C) and TLR9 (ODN2395) ligands promoted NK-cell function only in the presence of suboptimal doses of cytokines, including interleukin (IL)-2, IL-12, IL-15, and IL-18, produced in vivo by other environmental cells. We showed that R848 rather than TLR3 and TLR9 agonists primarily activated CD56brightCD16- NK cells by increasing their proliferation, cytokine production and cytotoxic activity. Moreover, we demonstrated that R848, which usually triggers TLR7 and TLR8 on dendritic cells, macrophages and neutrophils cells, activated CD56brightCD16- NK-cell subset only via TLR8. Indeed, specific TLR8 but not TLR7 agonists increased cytokine production and cytotoxic activity of CD56brightCD16- NK cells. Importantly, these activities were also observed in peritoneal NK cells from patients with metastatic ovarian carcinoma, prevalently belonging to the CD56brightCD16- subset. CONCLUSION: These data highlight the potential value of TLR8 in NK cells as a new target for immunotherapy in patients with cancer.

Celecoxib and Myrtol: A Novel Therapy for Recurrent Appendiceal Mucinous Neoplasms With Extensive Peritoneal Dissemination.

BACKGROUND: Unresectable appendiceal mucinous neoplasms (AMNs) with extensive peritoneal dissemination cause significant morbidity and have limited treatment options. We evaluated a novel combination of Celecoxib and Myrtol in treating such AMNs. METHODS: Patients with recurrent AMNs with extensive peritoneal disease treated with a daily regimen of 200 mg Celecoxib and 1200 mg Myrtol Standardized were included. Progression-free survival (PFS) and overall survival (OS) were calculated, and carcinoembryonic antigen (CEA) trends were compared pretreatment and post-treatment in terms of percentage change. RESULTS: Thirteen patients with extensive, recurrent disease (median peritoneal carcinomatosis index of 36) were included between 2017 and 2020. The median age was 63 years (interquartile range: 55 to 67) and 7 (54%) were male. A total of 85% had undergone prior cytoreductive surgery while 15% underwent cytoreductive surgery >2 times. 54% had received multiple cycles of systemic chemotherapy before starting Celecoxib-Myrtol. After a median follow-up of 8 months, median PFS and OS were 16 months (interquartile range: 5 to 17) and 27 months, respectively. Nine (69.2%) showed improvement in CEA values 3 months after treatment compared with 3-month pretreatment CEA trends. None had adverse events attributable to Celecoxib-Myrtol. CONCLUSIONS: Our feasibility study suggests that a regimen of Celecoxib-Myrtol is well tolerated and may prolong PFS and OS in patients with recurrent AMNs with peritoneal spread.

Single-Cell and Bulk RNA-Sequencing Reveal Differences in Monocyte Susceptibility to Influenza A Virus Infection Between Africans and Europeans.

There is considerable inter-individual and inter-population variability in response to viruses. The potential of monocytes to elicit type-I interferon responses has attracted attention to their role in viral infections. Here, we use single-cell RNA-sequencing to characterize the role of cellular heterogeneity in human variation of monocyte responses to influenza A virus (IAV) exposure. We show widespread inter-individual variability in the percentage of IAV-infected monocytes. Notably, individuals with high cellular susceptibility to IAV are characterized by a lower activation at basal state of an IRF/STAT-induced transcriptional network, which includes antiviral genes such as IFITM3, MX1 and OAS3. Upon IAV challenge, we find that cells escaping viral infection display increased mRNA expression of type-I interferon stimulated genes and decreased expression of ribosomal genes, relative to both infected cells and those never exposed to IAV. We also uncover a stronger resistance of CD16+ monocytes to IAV infection, together with CD16+ -specific mRNA expression of IL6 and TNF in response to IAV. Finally, using flow cytometry and bulk RNA-sequencing across 200 individuals of African and European ancestry, we observe a higher number of CD16+ monocytes and lower susceptibility to IAV infection among monocytes from individuals of African-descent. Based on these data, we hypothesize that higher basal monocyte activation, driven by environmental factors and/or weak-effect genetic variants, underlies the lower cellular susceptibility to IAV infection of individuals of African ancestry relative to those of European ancestry. Further studies are now required to investigate how such cellular differences in IAV susceptibility translate into population differences in clinical outcomes and susceptibility to severe influenza.

Immune related endonucleases and GTPases are not associated with tumor response in patients with advanced non-small cell lung cancer treated with checkpoint inhibitors.

Immune related endonucleases have recently been described as potential therapeutic targets and predictors of response to treatment with immune checkpoint inhibitors (ICI). The aim is to evaluate the association between the expression of 5 biomarkers involved in the immune response (CD73, CD39, VISTA, Arl4d and Cytohesin-3) in parallel with the more common ICI-predictive markers, PD-L1 expression and Tumor Mutation Burden (TMB) with response to ICI therapy in an advanced non-small cell lung cancer (NSCLC) cohort. METHODS: Patients with advanced NSCLC treated with ICI single agent were divided into responders and non-responders according to RECIST v1.1 and duration of response (DOR) criteria. Immunohistochemistry was performed on pretreatment tumor tissue samples for PD-L1, CD73, CD39, VISTA, Arl4d, and Cytohesin-3 expression. TMB was estimated with NEOplus v2 RUO (NEO New Oncology GmbH) hybrid capture next generation sequencing assay. Resistance mutations in STK11/KEAP1 and positive predictive mutations in ARID1A/POLE were also evaluated. RESULTS: Included were 56 patients who were treated with ICI single agent. The median progression-free and overall survival for the whole cohort was 3.0 (95% CI, 2.4-3.6) and 15 (95% CI, 9.7-20.2) months, respectively. The distribution of CD73 in tumor cells and CD39, VISTA, Arl4d and Cytohesin-3 expression in immune cells were not different between responders and non-responders. Also, PD-L1 and TMB were not predictive for response. The frequency of STK11, KEAP1 and ARID1A mutations was low and only observed in the non-responder group. CONCLUSION: Separate and combined expression of 5 biomarkers involved in the immune response (CD73, CD39, VISTA, Arl4d, and Cytohesin-3) was not associated with response in our cohort of advanced NSCLC patients receiving single agent ICI. To confirm our findings the analysis of independent larger cohorts is warranted.

Impact of intratumoural CD73 expression on prognosis and therapeutic response in patients with gastric cancer.

AIM: CD73 overexpression has been reported in several malignancies and is considered to be a novel immune checkpoint. However, the role and significance of CD73 in gastric cancer (GC) still remain obscure. We aim to investigate the role of CD73 expression in predicting prognosis, shaping immune contexture and guiding therapeutic strategy in GC. METHODS: The study enrolled four independent cohorts with a total of 902 patients with GC. CD73 expression and immune contexture were examined by immunohistochemistry, single-sample gene set enrichment analysis and flow cytometry. Clinical outcomes of patient subgroups were evaluated using the Kaplan-Meier curves and Cox proportional hazard analysis. All statistical tests were two-sided. RESULTS: CD73 was identified as an independent adverse prognostic factor for survival in GC. CD73high tumours showed a specific microenvironment with more CD8+ T cell infiltration, but these CD8+ T cells displayed a dysfunctional phenotype. Furthermore, the CD73 (NT5E) mRNA level was associated with the Cancer Genome Atlas molecular subtypes, and NT5E high tumours showed significant fibroblast growth factor receptor 2 activation and vascular endothelial growth factor and receptor enrichment. In addition, CD73high tumours indicated better chemotherapeutic responsiveness to fluorouracil yet a worse objective response rate to pembrolizumab in GC. CONCLUSIONS: High CD73 expression indicated an immunoevasive contexture with CD8+ T cell dysfunction and represented an independent predictor for adverse clinical outcomes. As a potential immunotherapeutic target, CD73 could potentially be a novel biomarker for adjuvant chemotherapy, targeted therapies and immunotherapy. The crucial role of CD73 in the therapeutic landscape of GC needs further validation retrospectively and prospectively.

Adoptive immunotherapy with double-bright (CD56bright /CD16bright ) expanded natural killer cells in patients with relapsed or refractory acute myeloid leukaemia: a proof-of-concept study.

Patients with acute myeloid leukaemia (AML) have a five-year survival rate of 28·7%. Natural killer (NK)-cell have anti-leukaemic activity. Here, we report on a series of 13 patients with high-risk R/R AML, treated with repeated infusions of double-bright (CD56bright /CD16bright ) expanded NK cells at an academic centre in Brazil. NK cells from HLA-haploidentical donors were expanded using K562 feeder cells, modified to express membrane-bound interleukin-21. Patients received FLAG, after which cryopreserved NK cells were thawed and infused thrice weekly for six infusions in three dose cohorts (106 -107 cells/kg/infusion). Primary objectives were safety and feasibility. Secondary endpoints included overall response (OR) and complete response (CR) rates at 28-30 days after the first infusion. Patients received a median of five prior lines of therapy, seven with intermediate or adverse cytogenetics, three with concurrent central nervous system (CNS) leukaemia, and one with concurrent CNS mycetoma. No dose-limiting toxicities, infusion-related fever, or cytokine release syndrome were observed. An OR of 78·6% and CR of 50·0% were observed, including responses in three patients with CNS disease and clearance of a CNS mycetoma. Multiple infusions of expanded, cryopreserved NK cells were safely administered after intensive chemotherapy in high-risk patients with R/R AML and demonstrated encouraging outcomes.

Combined utility of CD177, P53, CD105 and c-kit immunohistochemical stains improves the detection of myelodysplastic syndrome.

The diagnosis of myelodysplastic syndrome (MDS) relies primarily on identifying peripheral blood cytopenia and morphologic dysplasia as well as detecting cytogenetic aberrations in a subset of patients. Accumulating data points to the importance of examining certain immunophenotypic changes characteristic of MDS, most of which are tested by flow cytometry. The role of immunohistochemistry in the diagnostic workup of MDS is less known. In this study, we used immunohistochemistry to survey the expression patterns of CD177, P53, CD105 and c- kit in a cohort of MDS bone marrow specimens (n = 57) and compared the results with a control group of patients who had cytopenia for other benign conditions (n = 49). MDS cases showed significant higher rates of: CD177-loss (13/57, 23% vs 1/49, 2%; P = .0016), P53 overexpression (8/57, 14% vs none; P = .005) and the presence of clusters of CD105-positive cells (6/57, 11% vs none; P = .021). Increased c-kit-positive cells was more common in MDS patients, but not statistically significant (17/57, 30% vs 8/49, 16%; P = .102). On multivariate analysis, only loss of CD177 expression was significantly higher in MDS group (P = .014). These findings suggest that a panel of immunohistochemical stains could serve as an adjunct tool in investigating unexplained cytopenias and warrant further comparative studies with flow cytometry.

Low-affinity immunoglobulin gamma Fc region receptor III-B (FcγRIIIB, CD16B) deficiency in patients with blood and immune system disorders.

Low-affinity immunoglobulin gamma Fc region receptor III-B (FcγRIIIB) deficiency is present in ˜0·05% of the general population. Among our patients, FcγRIIIB deficiency was less frequent in those with immune-system disorders (one of 1815 patients, 0·05%) than in those with blood disorders (nine of 2147 patients, 0·42%, P = 0·023): mainly primary immune thrombocytopenia (4·34%), therapy related myeloid neoplasms (1·16%) and myelodysplastic syndrome with excess blasts (1·28%). Four of the nine (44·4%) patients with blood disorders were diagnosed with or quickly evolved to acute myeloid leukaemia (AML), suggesting that FcγRIIIB deficiency could be an adverse prognostic factor for progression to AML that should be confirmed in large multicentre studies.

Pattern of placental alkaline phosphatase (PLAP) expression in human tumors: a tissue microarray study on 12,381 tumors.

Placental alkaline phosphatase (PLAP) is commonly expressed at high levels in testicular germ cell tumors. PLAP immunohistochemistry (IHC) is thus often used to confirm this diagnosis, especially in cases of putative metastasis. However, other tumors can also express PLAP. To comprehensively determine PLAP expression in normal and tumor tissue, a tissue microarray containing 16,166 samples from 131 different tumor types and subtypes as well as 608 samples from 76 different normal tissue types was analyzed by IHC. Moderate to strong PLAP positivity was found in 27 (21%) of 131 different tumor types including seminoma (96%), embryonal carcinoma (85%), and yolk sac tumors of the testis (56%); endometrioid carcinoma of the endometrium (28%) and the ovary (20%); gastric adenocarcinoma (22%); serous carcinoma (not otherwise specified) of the ovary (17%) and the uterus (11%); adenocarcinoma of the ampulla of Vater (15%); carcinosarcoma of the ovary (11%) and the uterus (8%); esophageal adenocarcinoma (10%); invasive urothelial carcinoma (4%); cholangiocarcinoma (2%); and adenocarcinoma of the lung (1%). Low-level PLAP immunostaining, often involving only a small fraction of tumor cells, was seen in 21 additional tumor entities. The clinical significance of PLAP expression may vary between tumor types as high PLAP expression was linked to advanced pathological tumor stage (p = 0.0086), nodal metastasis (p = 0.0085), and lymphatic (p = 0.0007) and blood vessel invasion (p = 0.0222) in colorectal cancer, but to low pathological tumor stage in endometrial cancer (p = 0.0043). In conclusion, our data identify several tumor entities that can show PLAP expression at comparable levels to testicular germ cell tumors. These tumor entities need to be considered in cases of PLAP-positive metastasis. Low-level PLAP expression can be found in various other tumor entities and should generally not be viewed as a strong argument for germ cell neoplasia.

IL-7 and CCL19-secreting CAR-T cell therapy for tumors with positive glypican-3 or mesothelin.

Although chimeric antigen receptor (CAR)-engineered T cells have shown great success in the treatment of B cell malignancies, this strategy has limited efficacy in patients with solid tumors. In mouse CAR-T cells, IL-7 and CCL19 expression have been demonstrated to improve T cell infiltration and CAR-T cell survival in mouse tumors. Therefore, in the current study, we engineered human CAR-T cells to secrete human IL-7 and CCL19 (7 × 19) and found that these 7 × 19 CAR-T cells showed enhanced capacities of expansion and migration in vitro. Furthermore, 7 × 19 CAR-T cells showed superior tumor suppression ability compared to conventional CAR-T cells in xenografts of hepatocellular carcinoma (HCC) cell lines, primary HCC tissue samples and pancreatic carcinoma (PC) cell lines. We then initiated a phase 1 clinical trial in advanced HCC/PC/ovarian carcinoma (OC) patients with glypican-3 (GPC3) or mesothelin (MSLN) expression. In a patient with advanced HCC, anti-GPC3-7 × 19 CAR-T treatment resulted in complete tumor disappearance 30 days post intratumor injection. In a patient with advanced PC, anti-MSLN-7 × 19 CAR-T treatment resulted in almost complete tumor disappearance 240 days post-intravenous infusion. Our results demonstrated that the incorporation of 7 × 19 into CAR-T cells significantly enhanced the antitumor activity against human solid tumor. Trial registration: NCT03198546. Registered 26 June 2017, https://clinicaltrials.gov/ct2/show/NCT03198546?term=NCT03198546&draw=2&rank=1.

CD73 expression in normal, hyperplastic, and neoplastic thyroid: a systematic evaluation revealing CD73 overexpression as a feature of papillary carcinomas.

CD73 converts AMP to adenosine, an immunosuppressive metabolite that promotes tumorigenesis. This study presents a systematic evaluation of CD73 expression in benign, hyperplastic, and neoplastic thyroid. CD73 expression was assessed by immunohistochemistry in 142 thyroid samples. CD73 was expressed in normal thyroid (3/6) and goiter (5/6), with an apical pattern and mild intensity. Apical and mild CD73 expression was also present in oncocytic cell adenomas/carcinomas (9/10; 5/8) and in follicular adenomas/carcinomas (12/18; 23/27). In contrast, papillary thyroid carcinomas featured extensive and intense CD73 staining (49/50) (vs. normal thyroid/goiter, p < 0.001). Seven of nine anaplastic carcinomas were CD73-positive with heterogeneous extensiveness of staining. Medullary and poorly differentiated carcinomas were mostly CD73-negative (1/6; 2/2). These results were corroborated by NT5E mRNA profiling. Papillary carcinomas feature enhanced CD73 protein and mRNA expression with distinct and intense staining, more pronounced in the invasive fronts of the tumors.

Significance of mesothelin and CA125 expression in endometrial carcinoma: a retrospective analysis.

BACKGROUND: This study aimed to investigate the association between clinicopathologic factors, mesothelin, and cancer antigen (CA) 125 in endometrial carcinoma. METHODS: Between 1989 and 2017, patients with endometrial carcinoma who underwent total hysterectomy and bilateral salpingo-oophorectomy at our hospital were identified. The association between either or both immunochemical expression of mesothelin and CA125 and clinicopathological features were retrospectively examined. RESULTS: Among 485 patients, 171 were positive for mesothelin, 368 were positive for CA125, and 167 were positive for mesothelin and CA125. The expression of mesothelin and CA125 was positively correlated (p < 0.01). More patients with mesothelin expression showed myometrial invasion of more than 50% (p = 0.028) and positive lymphovascular invasion (p = 0.027). Similarly, more patients with co-expression of mesothelin and CA125 had myometrial invasion of more than 50% (p = 0.016) and positive lymphovascular invasion (p = 0.02). Patients with mesothelin expression and co-expression of mesothelin and CA125 demonstrated worse progression-free survival (PFS) and overall survival (OS). In the multivariate analysis, mesothelin expression and co-expression were poor prognostic factors for PFS (mesothelin expression: hazard ratio [HR] = 2.14, p < 0.01; co-expression: HR = 2.19, p < 0.01) and OS (mesothelin expression: HR = 2.18, p < 0.01; co-expression: HR = 2.22, p < 0.01). CONCLUSIONS: Mesothelin expression and co-expression might be associated with tumor aggressiveness and poor prognosis in patients with endometrial carcinoma. Persons with mesothelin-expressing endometrial cancers present a particularly high medical unmet need.

Nonclassical Monocytes (CD14dimCD16+) Are Associated With Carotid Intima-Media Thickness Progression for Men but Not Women: The Multi-Ethnic Study of Atherosclerosis-Brief Report.

[Figure: see text].

Human CD34-negative hematopoietic stem cells: The current understanding of their biological nature.

Hematopoietic stem cell (HSC) heterogeneity and hierarchy are a current topic of interest, having major implications for clinical HSC transplantation and basic research on human HSCs. It was long believed that the most primitive HSCs in mammals, including mice and humans, were CD34 antigen positive (CD34+). However, 2 decades ago, it was reported that murine long-term multilineage reconstituting HSCs were lineage marker negative (Lin-, i.e., c-kit+Sca-1+CD34low/-), known as CD34low/- KSL cells. In contrast, human CD34- HSCs, a counterpart of murine CD34low/- KSL cells, were hard to identify for a long time mainly because of their rarity. We previously identified very primitive human cord blood (CB)-derived CD34- severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection method and proposed the new concept that CD34- SRCs (HSCs) reside at the apex of the human HSC hierarchy. Through a series of studies, we identified two positive/enrichment markers: CD133 and GPI-80. The combination of these two markers enabled the development of an ultrahigh-resolution purification method for CD34- as well as CD34+ HSCs and the successful purification of both HSCs at the single-cell level. Cell population purity is a crucial prerequisite for reliable biological and molecular analyses. Clonal analyses of highly purified human CD34- HSCs have revealed their potent megakaryocyte/erythrocyte differentiation potential. Based on these observations, we propose a revised road map for the commitment of human CB-derived CD34- HSCs. This review updates the current understanding of the stem cell nature of human CB-derived primitive CD34- as well as CD34+ HSCs.

Case Report: In Situ Vaccination by Autologous CD16+ Dendritic Cells and Anti-PD-L 1 Antibody Synergized With Radiotherapy To Boost T Cells-Mediated Antitumor Efficacy In A Psoriatic Patient With Cutaneous Squamous Cell Carcinoma.

The combination of radiotherapy and immunotherapy improves the survival rate of patients with malignancies developed through escape from T-cell-mediated immune surveillance. Immune checkpoint inhibitors, such as anti-programmed cell death protein-ligand 1 (anti-PD-L1) antibody, are used to rescue exhausted T cells. Simultaneously, dendritic cells (DCs) which are antigen-presenting cells that can initiate T-cell activation, are used to induce a tumor-specific immune response. However, the synergistic antitumor efficacy of the aforementioned combinational immunotherapy with intratumoral injection of low-dose DCs has not been reported, and the underlying therapeutic mechanism requires further investigation. Herein, we present the special case of a psoriatic patient with cutaneous squamous cell carcinoma (cSCC) in the right inguinal region, these two diseases characterized by opposing contradiction, further complicating treatments and side-effect management efforts. To treat the intractable SCC without exaggerating psoriasis, we developed the triple-regimen therapy (TRT) with the intratumoral injection of low-dose autologous DCs and anti-PD-L1 combined with radiotherapy. The injected DCs were obtained simply through leukapheresis without prior G-CSF administration for mobilization nor tumor-antigen loading for expansion. The patient received three radiation doses (24, 18, and 18 Gy) combined with three intratumoral injections of anti-PD-L1 antibody (40, 60, and 120 mg) plus autologous DCs (80% of the DC subpopulation being CD16+ myeloid DC with approximate amounts of 7.3 × 104, 2.5 × 106, and 1.7 × 107) within 10 weeks. The efficacy of the TRT was encouraging in shrinking tumor mass with remarkable SUVmax reduction (approximately 42%) on FDG PET-Scan despite relatively low-dose DCs were available. The low-dose intratumoral immunotherapy induced mild cutaneous side effects as expected. The transcriptomes were compared between pre-TRT and post-TRT biopsies to analyze underlying mechanical pathways of the TRT protocol. Over 10 highly significantly enriched T-cell-related pathways (P <0.0001) were identified in post-TRT biopsies. In addition, the activation of both innate and adaptive immunity was significantly enriched in post-TRT peripheral blood samples. We develop the easily accessible TRT which produces both local anti-tumor T-cell responses and systemic antitumor immunity for treating cSCC patients, especially for those with autoimmune disease.

The clinical significance of PNH-phenotype cells accounting for < 0.01% of total granulocytes detected by the Clinical and Laboratory Standards Institute methods in patients with bone marrow failure.

Small populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) cells accounting for up to 0.01% of total granulocytes can be accurately detected by a high-sensitivity flow cytometry (FCM) assay established by the Clinical and Laboratory Standards Institute (CLSI method) and have a prognostic value in bone marrow failure (BMF); however, the significance of GPI(-) granulocytes accounting for 0.001-0.009% of granulocytes remains unclear. To clarify this issue, we examined the peripheral blood of 21 BMF patients in whom minor (around 0.01%) populations of GPI(-) granulocytes had been previously detected by a different high-resolution FCM method (OPTIMA method, which defines ≥ 0.003% GPI(-) granulocytes as an abnormal increase) using both the CLSI and OPTIMA methods simultaneously. These two methods detected an "abnormal increase" in GPI(-) granulocytes in 10 patients (48%) and 17 patients (81%), respectively. CLSI detected 0.002-0.005% (median, 0.004%) GPI(-) granulocytes in 7 patients who were deemed positive for PNH-type cells according to the OPTIMA method, which detected 0.003-0.012% (median 0.006%) GPI(-) granulocytes. The clone sizes of GPI(-) cells detected by each assay were positively correlated (r = 0.994, p < 0.001). Of the seven patients who were judged positive for PNH-type cells by OPTIMA alone, five received immunosuppressive therapy, and all of them achieved a partial or complete response. GPI(-) granulocytes detected in BMF patients by the CLSI method should thus be considered significant, even at percentages of < 0.01%.

Prognostic impact of CD73 expression and its relationship to PD-L1 in patients with radically treated pancreatic cancer.

Immune suppressing molecule CD73 is overexpressed in various cancers and associated with poor survival. Little is so far known about the predictive value of CD73 in pancreatic ductal adenocarcinoma (PDAC). The purpose of this study was to investigate the prognostic significance of CD73 in PDAC. The study material consisted of 110 radically treated patients for PDAC. Tissue microarray blocks were constructed and stained immunohistochemically using CD73 antibody. Staining intensity and numbers of stained tumour cells, inflammatory cells, stroma, and blood vessels were assessed. High-level CD73 expression in tumour cells was positively associated with PD-L1 expression, perineural invasion, and histopathological grade. CD73 positivity in tumour-infiltrating lymphocytes was significantly associated with lymph node metastasis. Lymphocytic CD73 positivity was also associated with staining positivity in both stroma and vascular structures. In addition, CD73 positivity in vascular structures and stroma were associated with each other. There were no significant associations between CD73 positive tumour cells and CD73 positivity in any other cell types. PD-L1 expression was associated with CD73 staining positivity in stroma (p = 0.007) and also with histopathological grade (p = 0.033) and T class (p = 0.016) of the primary tumour. CD73 positivity in tumour cells was significantly associated with poor disease-specific (p = 0.021) and overall survival (p = 0.016). In multivariate analysis, CD73 positivity in tumour cells was an independent negative prognostic factor together with histopathological grade, TNM stage, and low immune cell score. In conclusion, high CD73 expression in tumour cells is associated with poor survival in PDAC independently of the number of tumour-infiltrating lymphocytes or TNM stage.

Matrix metalloproteinase-7, -8, -9, -15, and -25 in minor salivary gland adenoid cystic carcinoma.

Knowledge on the role of matrix metalloproteinases (MMPs) in adenoid cystic carcinoma (ACC) is limited. MMPs are capable of degrading almost all extracellular and pericellular components to promote invasion and metastasis. This study aimed to evaluate the immunohistochemical expression of MMP-7, -8, -9, -15, and -25 in ACC and to relate the results with clinicopathological factors and survival. The study included 68 patients with minor salivary gland ACC treated at the Helsinki University Hospital (Helsinki, Finland) in 1974-2012. Samples from 52 patients were available, consisting of 44 primary tumours and eight recurrent tumours. We scored immunostaining of MMP-7, -8, -9, -15, and -25 and analysed the immunoscore against clinical and pathological parameters using statistical correlation test. MMP-9 immunoexpression in pseudocysts of ACC and in peritumoural inflammatory cells associated with better survival and fewer treatment failures. High tumoural MMP-7 and -25 associated with better survival. High tumoural MMP-15 associated with poorer survival and high tumoural MMP-9 with advanced stage and regional recurrences. Tumour cells did not show MMP-8 immunopositivity. These results suggest that MMP-9 may contribute to ACC carcinogenesis in different roles. MMP-7, -8, and -9 can stimulate signalling pathways that may promote tissue modulation and metastatic potential. MMP-15 and -25 may reflect prognosis.

Numbers and phenotype of non-classical CD14dimCD16+ monocytes are predictors of adverse clinical outcome in patients with coronary artery disease and severe SARS-CoV-2 infection.

AIMS: To elucidate the prognostic role of monocytes in the immune response of patients with coronary artery disease (CAD) at risk for life-threatening heart and lung injury as major complications of SARS-CoV-2 infection. METHODS AND RESULTS: From February to April 2020, we prospectively studied a cohort of 96 participants comprising 47 consecutive patients with CAD and acute SARS-CoV-2 infection (CAD + SARS-CoV-2), 19 CAD patients without infections, and 30 healthy controls. Clinical assessment included blood sampling, echocardiography, and electrocardiography within 12 h of admission. Respiratory failure was stratified by the Horovitz Index (HI) as moderately/severely impaired when HI ≤200 mmHg. The clinical endpoint (EP) was defined as HI ≤200 mmHg with subsequent mechanical ventilation within a follow-up of 30 days. The numbers of CD14dimCD16+ non-classical monocytes in peripheral blood were remarkably low in CAD + SARS-CoV-2 compared with CAD patients without infection and healthy controls (P < 0.0001). Moreover, these CD14dimCD16 monocytes showed decreased expression of established markers of adhesion, migration, and T-cell activation (CD54, CD62L, CX3CR1, CD80, and HLA-DR). Decreased numbers of CD14dimCD16+ monocytes were associated with the occurrence of EP. Kaplan-Meier curves illustrate that CAD + SARS-CoV-2 patients with numbers below the median of CD14dimCD16+ monocytes (median 1443 cells/mL) reached EP significantly more often compared to patients with numbers above the median (log-rank 5.03, P = 0.025). CONCLUSION: Decreased numbers of CD14dimCD16+ monocytes are associated with rapidly progressive respiratory failure in CAD + SARS-CoV-2 patients. Intensified risk assessments comprising monocyte sub- and phenotypes may help to identify patients at risk for respiratory failure.

Expression and localization of the MMP inhibitor RECK in a rat model of COPD: its involvement in the development of COPD.

OBJECTIVE: To unravel the potential function of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) as the matrix metalloproteinase (MMP) inhibitor in the development of chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: Twelve specific pathogen-free Sprague Dawley rats were randomly assigned to the control cohort (n = 6) or the COPD cohort (n = 6). COPD model was developed by tobacco smoke exposure. Functional residual capacity (FRC), static lung compliance (Cchord), ratio of forced expiratory volume in 0.1 s to forced vital capacity (FEV0.1/FVC), and peak expiratory flow (PEF) were detected by respiratory function tests. Immunohistochemistry was performed to determine the pathological changes as well as the expression and localization of RECK in pulmonary tissue. RECK expression was further quantified by real-time polymerase chain reaction (PCR) and Western blot assays. RESULTS: COPD rats had significantly reduced FEV0.1/FVC% and PEF values but increased FRC and Cchord levels, as compared to the control cohort (p < 0.05). Hematoxylin and eosin (HE) staining indicated typical COPD pathological changes, including leukocyte infiltration, airway thickening, alveoli fusion, etc., in the COPD rats. IHC indicated reduced expression of RECK in the COPD cohort, which was mainly expressed on the epithelium and partly expressed on subepithelial cells and inflammatory cells. Real-time PCR and Western blot assays further revealed the significantly lower expression of RECK in lung tissue from the COPD cohort. CONCLUSIONS: RECK is mainly expressed on airway epithelial cells. COPD rats expressed significantly lower RECK levels, indicating that RECK exhibits a protective function in the development of COPD.