Immune Cell Staining Research Articles

The effects of combustible cigarettes and electronic nicotine delivery systems on immune cell-driven inflammation and mucosal healing in ulcerative colitis.

The effects of combustible cigarettes (CCs) and electronic nicotine delivery systems (ENDS) on immune cell-driven colon inflammation and intestinal healing of patients with ulcerative colitis (UC) are still unknown and, therefore, were examined in this study. Intracellular staining and flow cytometry analysis of immune cells isolated from UC patients who used ENDS (UCENDS), CCs (UCCC) and who were non-smokers (UCAIR) were performed to elucidate cellular mechanisms which were responsible for CCs and ENDS-dependent modulation of immune response during UC progression. Additionally, dextran sulfate sodium (DSS)-colitis was induced in ENDS/CC/air-exposed mice (DSSENDS/ DSSCC/DSSAIR groups) to support clinical findings. Significantly increased number of immunosuppressive, IL-10, TGF-β and IL-35-producing, FoxP3-expressing CD3+CD4+T regulatory cells (Tregs) was observed in the blood of UCENDS patients while reduced presence of inflammatory, TNF-α and IFN-γ-producing, Tbx21-expressing CD3+CD4+ Th1, IL-4-producing Gata3-expresing Th2 and IL-17, IL-22-producing, RORγT, IL-23R-expressing Th17 cells were noticed in the blood of UCCC patients. Exposure to either CCs or ENDS was associated with enhanced mucosal healing, ameliorated spontaneous recovery and improved survival of DSS-treated mice. An expansion of immunosuppressive cells (IL-10-producing tolerogenic CD11c+ dendritic cells, alternatively activated CD206, Arginase 1-expressing, IL-10-producing F4/80+macrophages, IL-10-producing FoxP3-expressing Tregs) was noticed in the colons of DSSENDS-treated mice, while reduced number of inflammatory, IL-17- and IL-4-producing T lymphocytes was observed in the colons of DSSCC-compared to DSSAIR-treated mice. Despite different mechanisms of action, both ENDS and CCs attenuated on-going colon inflammation, enhanced healing and ameliorated recovery of injured intestines of DSS-treated mice and UC patients. This is the first study that compared the effects of CCs and ENDS on immune cells of patients suffering from ulcerative colitis, providing new information about molecular and cellular mechanisms which were responsible for ENDS and CCs-dependent modulation of immune cell-driven colon injury and inflammation. Obtained results showed that both ENDS and CCs had capacity to attenuate detrimental immune response, enhanced healing and ameliorated recovery of injured intestines.

Effects of combustible cigarettes and heated tobacco products on immune cell-driven inflammation in chronic obstructive respiratory diseases.

Since long-term effects of heated tobacco products (HTP) on the progression of chronic obstructive pulmonary disease (COPD) are unknown, we used COPD mice model to compare immune cell-dependent pathological changes in the lungs of animals which were exposed to HTP or combustible cigarettes (CCs). We also performed intracellular staining and flow cytometry analysis of immune cells which were present in the blood of CCs and HTP users who suffered from immune cell-driven chronic obstructive respiratory diseases. CCs enhanced NLRP3 inflammasome-dependent production of inflammatory cytokines in lung-infiltrated neutrophils and macrophages and increased influx of cytotoxic Th1, Th2, and Th17 lymphocytes in the lungs of COPD mice. Similarly, CCs promoted generation of inflammatory phenotype in circulating leukocytes of COPD patients. Opposite to CCs, HTP favored expansion of immunosuppressive, IL-10-producing, FoxP3-expressing T, NK, and NKT cells in inflamed lungs of COPD mice. Compared with CCs, HTP had weaker capacity to promote synthesis of inflammatory cytokines in lung-infiltrated immune cells. Significantly lower number of inflammatory neutrophils, monocytes, Th1, Th2, and Th17 lymphocytes were observed in the blood of patients who consumed HTP than in the blood of CCs users, indicating different effects of CCs and HTP on immune cells' phenotype and function.

Alteration of PD-L1 (SP142) status after neoadjuvant chemotherapy and its clinical significance in triple-negative breast cancer

PurposeThe tumor immune microenvironment can change after neoadjuvant chemotherapy (NAC) for triple-negative breast cancer (TNBC). We aimed to investigate the effects of NAC on PD-L1 (SP142) status and its clinical significance in TNBC.MethodsPaired samples of biopsy and resection specimens were collected from 182 patients with TNBC before and after NAC. PD-L1 (SP142) expression in immune cells in pre- and post-NAC breast cancer samples and the changes between them were analyzed, along with their relationships with the clinicopathological features and clinical outcomes of the patients.ResultsOf the 182 patients, 61 (33.5%) achieved pathologic complete response (pCR) after NAC. PD-L1 (SP142) positivity, defined as immune cell staining in ≥ 1% of tumor area, was a predictor for pCR. PD-L1-positive immune cells significantly increased after NAC (2.8% to 5.2% on average) in 109 patients with measurable residual disease. Alteration of PD-L1 status was observed in 24 (22.0%) of the 109 patients with measurable residual tumors after NAC, and all PD-L1 status-converted patients, except one, revealed negative-to-positive conversion. Regarding chemotherapeutic agents, the use of platinum agents was associated with a significant increase in PD-L1-positive immune cells after NAC. In survival analyses, a positive PD-L1 status after NAC and increase of PD-L1-positive immune cells after NAC were associated with better recurrence-free survival of the patients.ConclusionPD-L1 (SP142) status changes after NAC, mostly as a positive conversion. As PD-L1 (SP142) status can convey prognostic and predictive information, it needs to be tested before and after NAC.

Predictive value of dynamic changes in ctDNA and baseline biomarkers with neoadjuvant atezolizumab in operable urothelial carcinoma in the ABACUS trial.

534 Background: Circulating tumour DNA (ctDNA) is being explored in the neoadjuvant setting in multiple prospective trials. Dynamic changes may be a surrogate marker of pathological complete response. ABACUS was a multi-centre, single-arm phase II trial investigating two cycles of atezolizumab before cystectomy in patients with muscle-invasive urothelial cancer who were ineligible for or refused neoadjuvant cisplatin-based chemotherapy (NCT02662309). Results of primary and secondary endpoints have been previously published. Here we perform exploratory biomarker analysis using different definitions of ctDNA response and assess correlation with tissue response at time of cystectomy in the ABACUS trial. We also assess how these definitions of response correlate with baseline biomarker expression. Methods: Patients with baseline PDL-1, CD8, TMB and sequential ctDNA measurements (baseline and at cystectomy) were included. ctDNA analysis was performed using the Signatera assay, PD-L1 positivity was defined as ≥5% of immune cell staining, TMB was assessed using the FoundationOne CDx assay and CD8 measurement was performed via immunohistochemistry analyses. Two definitions of ctDNA response were used—ctDNA clearance and a 50% reduction (or greater) in ctDNA variant allele frequency (VAF). Results were correlated with pathological complete response (pCR) rate at time of cystectomy and relapse-free survival. Results: The 2-yr DFS and OS rates in ABACUS were 68% and 77%, respectively (N=95). 40 patients had sequential DNA analysis. 43% (17/40) had pathological complete response and 20% (8/40) experienced relapse. 63% (25/40) patients were ctDNA+ at baseline, with 40% (10/25) having ctDNA response of 50% VAF reduction and 8% (3/40) achieving ctDNA clearance. 30% (3/10) of patients who had VAF reduction experienced relapse and 40% (4/10) achieved pCR. All patients with ctDNA clearance achieved pCR none relapsed. There was no association between VAF reduction of 50% and tissue response (pCR) (p=0.24). Additional analysis showed no association between a ctDNA reduction of 75% (rather than 50%) and pCR (p=0.24). Baseline PDL-1, TMB and not predictive of pathological complete response (p=0.18, p=0.77, p=0.10, respectively) or ctDNA response (p=0.54, p=0.77, p=0.74) to neoadjuvant atezolizumab. There was no difference in relapse rates between ctDNA/PD-L1+ve vs ctDNA+ve/PD-L1-ve patients (16% vs 16%, respectively). Conclusions: ctDNA clearance is rare but appears more accurate than 50% reduction in VAF to predict response/relapse. This is relevant for ongoing neoadjuvant trials planning to use this as an endpoint. Combining immune tissue based biomarkers with ctDNA does not appear to improve biomarker accuracy.

Key Considerations when Evaluating Whether to Establish a PD-L1 Laboratory Developed Test (LDT)

Abstract Introduction/Objective Programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) testing is often used to identify patients with cancer who may be eligible for immune checkpoint inhibitor therapy. The complexity around using different assays and platforms has caused confusion and reluctance to perform in-house PD-L1 testing. Some labs have established a laboratory developed test (LDT) that may be used across multiple types of tumors. To evaluate the role of PD-L1 LDTs, ASCP formed an ad-hoc working group to identify key considerations. Methods/Case Report In early 2023, 40 pathologists and laboratory professionals joined the ASCP PD-L1 Learning Collaborative to explore ways to improve PD-L1 testing processes. An ad-hoc working group focused on LDTs and reviewed the literature, spoke with labs using LDTs, and developed guidance questions. Results (if a Case Study enter NA) The working group identified these key topics and questions: Testing volume and types of cancers: How many tests are performed each month to justify an in-house PD-L1 test? Which types of cancers are tested? Do we have (or do we plan to develop) reflex PD-L1 testing protocols for certain types of tumors? Buy-in from oncologists: Do our medical oncologists feel that a PD-L1 LDT provides the results they need to make treatment decisions across different types of cancers? Assay selection and testing platforms: Which IHC platform (eg, Ventana, Dako, Leica, etc.) do we currently use and which assay (eg, 22C3, 28-8, SP263, E1L3N, etc.) should we use? How well do these assays stain tumor cells vs. immune cells? IHC testing processes: What is our volume of IHC testing and how would adding PD-L1 impact our workflow for all IHC tests? How often do we encounter technical issues? How much staff time will it take to add PD-L1? What are the maintenance costs? Interpretation and scoring: Which pathologists are trained to interpret PD-L1 tests? Are they trained to interpret tumor cells, immune cells, or both? Do we train residents? Which scoring systems will we use and how will we report results? Validation: How many cases are needed for validation? Should validation samples include tumor cell and immune cell staining? Reimbursement: How will the lab be reimbursed for performing PD-L1 testing and interpretation? Conclusion Before establishing a PD-L1 LDT, the medical laboratory must review these questions against the backdrop of an evolving PD-L1 testing landscape. An LDT may be an appropriate approach for labs that aim to simplify PD-L1 testing based on the IHC platform(s) they are using.

Effects of combustible cigarettes and electronic nicotine delivery systems on the development and progression of chronic lung inflammation in mice.

Although detrimental effects of combustible cigarettes (CCs) on the progression of lung inflammatory diseases are well known, changes in electronic nicotine delivery systems (ENDS)-exposed lung-infiltrated immune cells are still unrevealed. The analysis of blood gas parameters, descriptive and quantitative histology of lung tissues, determination of serum cytokines, intracellular staining and flow cytometry analysis of lung-infiltrated immune cells were used to determine the differences in the extent of lung injury and inflammation between mice from experimental (CC and ENDS-exposed animals) and control group (Air-exposed mice). Continuous exposition to either CCs or ENDS induced severe systemic inflammatory response, increased activation of NLRP3 inflammasome in neutrophils and macrophages and enhanced dendritic cell-dependent activation of Th1 and Th17 cells in the lungs. ENDS induced less severe immune response than CCs. Serum concentrations of inflammatory cytokines were significantly lower in the samples of ENDS-exposed mice. Compared to CCs, ENDS recruited lower number of circulating leukocytes in injured lungs and had less capacity to induce CD14/TLR-2-dependent activation of NLRP3 inflammasome in lung-infiltrated neutrophils and macrophages. ENDS-primed dendritic cells had reduced capacity for the generation of Th1 and Th17 cell-driven lung inflammation. Accordingly, extensive immune cell-driven lung injury resulted in severe respiratory dysfunction in CCs-exposed mice, while ENDS caused moderate respiratory dysfunction in experimental animals. Continuous exposition to either CCs or ENDS induced immune cell-driven lung damage in mice. ENDS triggered immune response which was less potent than inflammatory response elicited by CCs and, therefore, caused less severe lung injury and inflammation. This is the first study that compared the effects of CCs and ENDS on lung-infiltrated immune cells. Although both CCs and ENDS elicited systemic inflammatory response, immune cell-driven lung injury and inflammation were less severe in ENDS-exposed than in CC-exposed animals. Continuous exposition to ENDS-sourced aerosols was less harmful for respiratory function of experimental animals than CC-derived smoke.

Immune profiling and RNA-seq uncover the cause of partial unexplained recurrent implantation failure.

Detailed knowledge of the changes in endometrial immune cells during the window of implantation in unexplained recurrent implantation failure (RIF) patients, the functions performed by immune cells, and the interactions between them is largely lacking. This study aimed to classify RIF patients and explore the mechanism through endometrial immune profiling and RNA-seq analysis. This study enrolled a total of 172 patients, comprising 144 women with unexplained RIF and 28 fertile women. Endometrial samples were collected using endometrial scratching at the mid-luteal phase before in vitro fertilization treatment or pregnancy. Transcriptome sequencing and immunohistochemical staining of endometrial immune cells including natural killer (NK) cells, macrophages, T cells, and B cells were performed. Comparison of the percentage of endometrial immune cells and the RNA-seq information between RIF patients and fertile control patients. The proportions of uterine CD56+ uNK cells, CD57+ NKT cells, CD68+ macrophages, and CD19+ B cells were significantly elevated in RIF patients. In addition, the number of positive CD68 glandular lumens was significantly higher in RIF patients than in the fertile group. In addition, based on this result, we classified RIF patients into three categories. Hyperactivation of endometrial immune cells may be associated with reduced endometrial tolerance and recurrent implantation failure, affecting pregnancy outcomes in RIF patients.

HPV-positive murine oral squamous cell carcinoma: development and characterization of a new mouse tumor model for immunological studies

BackgroundInfection with high-risk human papillomavirus (HPV) strains is one of the risk factors for the development of oral squamous cell carcinoma (OSCC). Some patients with HPV-positive OSCC have a better prognosis and respond better to various treatment modalities, including radiotherapy or immunotherapy. However, since HPV can only infect human cells, there are only a few immunocompetent mouse models available that enable immunological studies. Therefore, the aim of our study was to develop a transplantable immunocompetent mouse model of HPV-positive OSCC and characterize it in vitro and in vivo.MethodsTwo monoclonal HPV-positive OSCC mouse cell lines were established by inducing the expression of HPV-16 oncogenes E6 and E7 in the MOC1 OSCC cell line using retroviral transduction. After confirming stable expression of HPV-16 E6 and E7 with quantitative real-time PCR and immunofluorescence staining, the cell lines were further characterized in vitro using proliferation assay, wound healing assay, clonogenic assay and RNA sequencing. In addition, tumor models were characterized in vivo in C57Bl/6NCrl mice in terms of their histological properties, tumor growth kinetics, and radiosensitivity. Furthermore, immunofluorescence staining of blood vessels, hypoxic areas, proliferating cells and immune cells was performed to characterize the tumor microenvironment of all three tumor models.ResultsCharacterization of the resulting MOC1-HPV cell lines and tumor models confirmed stable expression of HPV-16 oncogenes and differences in cell morphology, in vitro migration capacity, and tumor microenvironment characteristics. Although the cell lines did not differ in their intrinsic radiosensitivity, one of the HPV-positive tumor models, MOC1-HPV K1, showed a significantly longer growth delay after irradiation with a single dose of 15 Gy compared to parental MOC1 tumors. Consistent with this, MOC1-HPV K1 tumors had a lower percentage of hypoxic tumor area and a higher percentage of proliferating cells. Characteristics of the newly developed HPV-positive OSCC tumor models correlate with the transcriptomic profile of MOC1-HPV cell lines.ConclusionsIn conclusion, we developed and characterized a novel immunocompetent mouse model of HPV-positive OSCC that exhibits increased radiosensitivity and enables studies of immune-based treatment approaches in HPV-positive OSCC.

Translational analysis of combination of 5-fluorouracil (FU), interferon (IFN)-alpha2, and nivolumab in unresectable fibrolamellar liver cancer.

e16198 Background: Fibrolamellar liver cancer (FLC) is a rare malignancy and involves multidisciplinary approaches including surgery, liver-directed, and systemic treatment. Combination of 5-fluorouracil (FU) and interferon (IFN)-a2 has long been used for FLC. We had a hypothesis that addition of anti-PD1 to 5FU+IFN-a2 may further improve clinical outcome and modulation of FLC tumor microenvironment. Methods: We treated 3 patients with 5FU+IFN-a2+nivolumab (N): 5FU 200 mg/m2 7-day-on/7-day-off and IFN-a2 (4 million U/m2 3 times/week while on 5-FU) for 8 weeks, followed by on-treatment biopsy and addition of N 480 mg every 4 weeks to 5FU+IFN-a2. Those who had conversion of unresectable disease to resectable one had the resected tumors analyzed. Gene expression profiling and immunohistochemistry (IHC) staining of immune cells were performed to investigate the evolution of tumor microenvironment on treatment. Results: 26 and 29 year-old patients had unresectable FLC with a 6.1 cm tumor in seg IV liver invading the common hepatic duct, and a 19.5 cm tumor, metastatic peritoneal nodules, and abdominal lymphadenopathy, respectively. After 9 and 5 months of therapy, unresectable tumors were converted to resectable ones with partial response (PR): the former had left hepatectomy and bile duct resection; the latter, left hepatectomy, resection of the omentum, spleen, left diaphragm, and lymph node dissection. A 20 year-old patient had multifocal liver tumors with peritoneal carcinomatosis and lymph nodes. He received treatment for 5 months and achieved stable disease, but hepatic tumor bleeding led to treatment discontinuation. 2 patients with PR had increased expression of genes related to T cells, CD8+T cells, Th1 cells, NK cells, cytotoxic cells, macrophages, dendritic cells, neutrophils, and B cells, subsequently on tumors before-treatment, on-treatment (5FU+IFN-a2), post-surgery (5FU+IFN-a2+N). IHC staining confirmed an increase in CD8+T cell infiltration after treatment with 5FU+IFN-a2 and 5FU+IFN-a2+N. The third patient whose disease was not converted to a resectable disease showed a decrease in genes related to Th1 cells, cytotoxic cells, dendritic cells, and neutrophils on-treatment biopsy (5FU+IFN-a2). This patient had a marginal increase in CD8+T cell infiltration on exposure to 5FU+IFN-a2. Conclusions: This clinical outcome of converting unresectable disease to resectable one suggests synergy of 5FU+IFN-a2 combined with N. The robust increased expression of genes related to immune system activation and increased infiltration of CD8+T cells on 5FU+IFN-a2+N may explain this clinical efficacy. Pro-inflammatory evolution of tumors on treatment could be used as predictive biomarkers. More prospective data are indicated to further confirm our hypothesis. Clinical trial information: NCT04380545 .

Programmed death‑ligand 1 expression in tumor cells and tumor‑infiltrating lymphocytes are associated with depth of tumor invasion in penile cancer.

The present study aimed to demonstrate the proportion of the programmed death-ligand 1 (PD-L1) expression in penile cancer patients and the association with clinicopathological parameters. Formalin-fixed paraffin-embedded specimens were obtained from 43 patients with primary penile squamous cell carcinoma treated at Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, between 2008 and 2018. PD-L1 expression was evaluated by the immunohistochemistry using an SP263 monoclonal antibody. PD-L1 positivity was defined as >25% tumor cell staining or >25% tumor-associated immune cell staining. The correlation between PD-L1 expression and clinicopathological parameters was analyzed. A total of eight of 43 patients (18.6%) were identified as positive for PD-L1 expression in tumor cells and tumor-infiltrating lymphocytes. In the PD-L1 positive group, there was a significant association with pathological T stage (P=0.014) with a higher percentage of PD-L1 positive tumors in T1 stage compared with T2-T4 stage. In this cohort, there was a trend towards longer survival in patients with positive PD-L1 expression (5-year OS: 75% vs. 61.2%, P=0.19). Lymph node involvement and the location of tumor at the shaft of penis were two independent prognostic factors for survival. In conclusion, the PD-L1 expression was detected in 18% of penile cancer patients and high expression of PD-L1 was associated with the early T stage.

The effectiveness of calcium electroporation combined with gene electrotransfer of a plasmid encoding IL-12 is tumor type-dependent.

In calcium electroporation (CaEP), electroporation enables the cellular uptake of supraphysiological concentrations of Ca2+, causing the induction of cell death. The effectiveness of CaEP has already been evaluated in clinical trials; however, confirmatory preclinical studies are still needed to further elucidate its effectiveness and underlying mechanisms. Here, we tested and compared its efficiency on two different tumor models to electrochemotherapy (ECT) and in combination with gene electrotransfer (GET) of a plasmid encoding interleukin-12 (IL-12). We hypothesized that IL-12 potentiates the antitumor effect of local ablative therapies as CaEP and ECT. The effect of CaEP was tested in vitro as well as in vivo in murine melanoma B16-F10 and murine mammary carcinoma 4T1 in comparison to ECT with bleomycin. Specifically, the treatment efficacy of CaEP with increasing calcium concentrations alone or in combination with IL-12 GET in different treatment protocols was investigated. We closely examined the tumor microenvironment by immunofluorescence staining of immune cells, as well as blood vessels and proliferating cells. In vitro, CaEP and ECT with bleomycin reduced cell viability in a dose-dependent manner. We observed no differences in sensitivity between the two cell lines. A dose-dependent response was also observed in vivo; however, the efficacy was better in 4T1 tumors than in B16-F10 tumors. In 4T1 tumors, CaEP with 250 mM Ca resulted in more than 30 days of growth delay, which was comparable to ECT with bleomycin. In contrast, adjuvant peritumoral application of IL-12 GET after CaEP prolonged the survival of B16-F10, but not 4T1-bearing mice. Moreover, CaEP with peritumoral IL-12 GET modified tumor immune cell populations and tumor vasculature. Mice bearing 4T1 tumors responded better to CaEP in vivo than mice bearing B16-F10 tumors, even though a similar response was observed in vitro. Namely, one of the most important factors might be involvement of the immune system. This was confirmed by the combination of CaEP or ECT with IL-12 GET, which further enhanced antitumor effectiveness. However, the potentiation of CaEP effectiveness was also highly dependent on tumor type; it was more pronounced in poorly immunogenic B16-F10 tumors compared to moderately immunogenic 4T1 tumors.

Development of swine immune reagents for swine immunity and biomedical research

Abstract The USDA-NIFA Swine Immune Toolkit Initiative has been involved in generating priority immune reagents for swine immunity and biomedical research and pipelining them for marketing. With the help of commercial partners we expressed each target immune molecule as a soluble protein using yeast expression systems and then produced panels of monoclonal antibodies (mAbs) against each target. Most recently we generated panels of mAbs reactive with porcine IL-5, IL-6, IL-13, IL-21, IL-28B, CXCL10, and BAFF and screened their reactivity in multiple immune assays. We determined mAb reactivity with orthologous proteins for most panels of mAbs. A sensitive sandwich ELISA is now available for IL-13 and CXCL10; other targets are being screened for best mAb pairs. Reactivity tests of labeled α-CXCL10, α-IL-6 and α-IL-13 mAbs for intracellular staining of porcine immune cells using flow cytometry assays are underway using different cell stimulation conditions. Immunohistochemistry analyses for binding of α-CXCL10 and other mAbs on formalin fixed pig lymph nodes and spleen tissues are in progress. Further, analysis of a large panel of commercial α-human CD antigen mAbs for cross-reactivity with porcine cells is ongoing. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized assay techniques for their research efforts. Finally, efforts are proceeding to develop SLA tetramers for antigen presentation studies. Tools and reagents generated by this project will undoubtedly advance our understanding of swine immune responses to disease and vaccine and use for biomedical research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815 and USDA ARS project 8042-32000-117.

Primary results of the ICONIC phase 2 trial of perioperative FLOT plus avelumab (FLOT-A) in operable oesophagogastric adenocarcinoma (OGA).

446 Background: ICONIC evaluated 4+4 cycles of FLOT-A (2-weekly standard FLOT with 10mg/kg of the anti-PDL1 antibody avelumab) for perioperative treatment of early-stage OGA. We report R0 resection rates, pathologic complete response rates (pCR), pathologic tumour regression grades (TRG) according to Mandard classification and progression free survival (PFS) data in the modified intention to treat population (mITT), and translational analyses. Methods: ICONIC is a single-arm phase II trial of FLOT-A in patients (pts) with ≥cT2-4 or N+ OGA. The pCR rate in surgical specimens was the primary endpoint. Response evaluation according to Mandard, R0 rates and PFS were among secondary endpoints. PD-L1 expression was assessed according to the combined positive score (CPS) with the 22C3 pharmDx kit. Results: The trial closed early after the pCR rate was 15% (95% CI: 49%-83%) once 34 pts in the mITT population (defined as pts who had 1-4 cycles FLOT-A and surgery) as the pre-specified aim to demonstrate a pCR rate of 25% in 40 pts was unlikely to be met. Baseline characteristics of the mITT population were: median age 64y, 79% male, 79% OG junction, 6% oesophageal, 15% stomach, 65% poorly differentiated, 79% T3/4, 59% N+. 91% of pts received all 4 pre-operative cycles and 97% of pts achieved R0 resections. PDL1 CPS ≥1 and CPS ≥5 were nevertheless associated with increased TRG3 and decreased TRG4/5 rates. 3/34 pts (9%) had hypermutated/MMRd tumours and after their exclusion, the association of higher CPS with better TRG3 and decreased TRG4/5 remained. With a median follow up duration of 15.8 months the 12-month PFS was 93.1% (95% CI: 75.1%-98.2%) which is promising compared to historic results with peri-operative FLOT. Conclusions: Although FLOT-A failed to increase pCR rates to 25%, there is a trend towards higher TRG3 and lower TRG4/5 in pts with PDL1 CPS≥1 and CPS≥5 and promising PFS indicate activity of immunotherapy in combination with FLOT chemotherapy. Translational and biomarker analyses by exome- and RNA-sequencing, and multiplex immune cell staining are ongoing and will be reported. Clinical trial information: NCT03399071 . [Table: see text]

Investigation of the Status of Immune Checkpoint Molecules in Meningiomas by Immunohistochemistry.

To investigate the status of immune checkpoint molecules (CTLA-4 and TIM-3) in meningiomas and thus contribute to the development of new personalized treatment strategies. We utilized 402 cases of meningioma for this study. New blocks were prepared using the tissue microarray method, and sections obtained from these blocks were immunohistochemically stained with CTLA-4 and TIM-3 antibodies. Subsequently, statistical analysis were performed. Our findings revealed that CTLA-4 expression were observed in 25.1% of meningiomas. CTLA-4 expression and the number of expressing lymphocytes were found to be significantly higher in high-grade tumors and in those with brain invasion. Meningiomas with staining of immune cells with TIM-3 are 3.5%, and the tumor grade was correlated with the number of immune cells expressing TIM-3. Immune checkpoint molecules (CTLA-4 and TIM-3) with varying levels of expression can serve as prognostic and predictive biomarkers, as well as important targets for therapy. Drugs developed for CTLA-4 and TIM-3 molecules may prove to be more effective in treating meningiomas with high-grade, brain-invading, spontaneous necrosis, and macronucleolus.

Results of PD-L1 Analysis of Women Treated with Durvalumab in Advanced Endometrial Carcinoma (PHAEDRA).

Women with advanced endometrial carcinoma (EC) with mismatch repair (MMR) deficiency have improved outcomes when treated with immune checkpoint inhibitors; however, additional biomarkers are needed to identify women most likely to respond. Scores for programmed death ligand 1 (PD-L1), immunohistochemical staining of tumor (TC+), immune cells (IC+) and presence of tumor-associated immune cells (ICP) on MMR deficient (n = 34) and proficient (n = 33) EC from women treated with durvalumab in the PHAEDRA trial (ANZGOG1601/CTC0144) (trial registration number ACTRN12617000106336, prospectively registered 19 January 2017) are reported and correlated with outcome. Receiver operating characteristic (ROC) analyses and area under the ROC curve were used to determine optimal cutpoints. Performance was compared with median cutpoints and two algorithms; a novel algorithm derived from optimal cutpoints (TC+ ≥ 1 or ICP ≥ 10 or IC+ ≥ 35) and the Ventana urothelial carcinoma (UC) algorithm (either TC+ ≥ 25, ICP > 1 and IC+ ≥ 25 or ICP = 1 and IC+ = 100). The cutpoint ICP ≥ 10 had highest sensitivity (53%) and specificity (82%), being prognostic for progression-free survival (PFS) (p = 0.01), while the optimal cutpoints algorithm was associated with overall survival (p = 0.02); these results were not significant after adjusting for MMR status. The optimal cutpoints algorithm identified non-responders (p = 0.02) with high sensitivity (88%) and negative predictive value (92%), remaining significant after adjustment for MMR. Although MMR status had the strongest association with response, further work to determine the significance of ICP ≥ 10 and the novel optimal cutpoint algorithm is needed.

The Ring Study: an international comparison of PD-L1 diagnostic assays and their interpretation in non-small cell lung cancer, head and neck squamous cell cancer and urothelial cancer

PD-L1 immunohistochemistry has been approved as a diagnostic assay for immunotherapy. However, an international comparison across multiple cancers is lacking. This study aimed to assess the performance of PD-L1 diagnostic assays in non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC) and urothelial cancer (UC). The excisional specimens of NSCLC, HNSCC and UC were assayed by Ventana SP263 and scored at three sites in each country, including Australia, Brazil, Korea, Mexico, Russia and Taiwan. All slides were rotated to two other sites for interobserver scoring. The same cohort of NSCLC was assessed with Dako 22C3 pharmDx PD-L1 for comparison. The PD-L1 immunopositivity was scored according to the approved PD-L1 scoring algorithms which were the percentage of PD-L1-expressing tumour cell (TC) and tumour proportion score (TPS) by Ventana SP263 and Dako 22C3 staining, respectively. In NSCLC, the comparison demonstrated the comparability of the SP263 and 22C3 assays (cut-off of 1%, κ=0.71; 25%, κ=0.75; 50%, κ=0.81). The interobserver comparisons showed moderate to almost perfect agreement for SP263 in TC staining at 25% cut-off (NSCLC, κ=0.72 to 0.86; HNSCC, κ=0.60 to 0.82; UC, κ=0.68 to 0.91) and at 50% cut-off for NSCLC (κ=0.64 to 0.90). Regarding the immune cell (IC) scoring in UC, there was a lower correlation (concordance correlation coefficient=0.10 to 0.68) and poor to substantial agreements at the 1%, 5%, 10% and 25% cut-offs (κ= -0.04 to 0.76). The interchangeability of SP263 and 22C3 in NSCLC might be acceptable, especially at the 50% cut-off. In HNSCC, the performance of SP263 is comparable across five countries. In UC, there was low concordance of IC staining, which may affect treatment decisions. Overall, the study showed the reliability and reproducibility of SP263 in NSCLC, HNSCC and UC.

High interobserver and intraobserver reproducibility among pathologists assessing PD-L1 CPS across multiple indications.

A common concern among pathologists scoring PD-L1 immunohistochemical staining is interobserver and intraobserver variability. We assessed the interobserver and intraobserver reproducibility of PD-L1 scoring among trained pathologists using a combined positive score (CPS; tumour cell and tumour-associated immune cell staining). Data were collected for 2 years (2017-2019) from 456 pathologists worldwide. Digital training encompassed unique, tumour-specific training, and test sets. Samples were stained using PD-L1 IHC 22C3 pharmDx and evaluated at specific CPS cutoffs for gastric cancer (GC), cervical cancer (CC), urothelial cancer (UC), oesophageal cancer (OC), and head and neck squamous cell carcinoma (HNSCC). Pathologists underwent expert-to-peer training and scored 20 blinded samples on day 1 and 25 blinded samples on day 2 (including 15 of the day 1 samples). Interobserver and intraobserver reproducibility were assessed. For GC (120 observers) and CC (32 observers) samples assessed at CPS ≥1, average interobserver agreement was 91.5% and 91.0%, respectively, and average intraobserver agreement was 90.2% and 96.6%, respectively. For UC (139 observers) and OC (52 observers) samples measured at CPS ≥10, average interobserver agreement was 93.4% and 93.7%, respectively, and average intraobserver agreement was 92.0% and 92.5%, respectively. For HNSCC samples (113 observers), average interobserver agreement was 94.1% at CPS ≥1 and 86.5% at CPS ≥20; intraobserver agreement was 94.7% at CPS ≥1 and 90.5% at CPS ≥20. The consistently high interobserver and intraobserver concordance rates support the effectiveness of face-to-face training of many global pathologists for scoring PD-L1 CPS across multiple indications at several specific cutoffs.

A retrospective study of PD-L1 immunohistochemistry for hepatocellular carcinoma

Programmed cell death ligand 1 (PD-L1) expression plays an important role in selecting hepatocellular carcinoma (HCC) patients who will benefit from anti-programmed cell death 1 (PD-1)/PD-L1 monotherapy and directing those with lower levels to alternative treatments. In total, 156 HCC tumors were stained with three PD-L1 immunohistochemistry assays (SP142, 28-8, and E1L3N). Two junior pathologists and one senior pathologist evaluated the pathological slides and recorded the percentages of tumor and immune cells stained at any intensity. Analytical comparisons demonstrated that the anti-PD-L1 assay SP142 is a significantly stronger reagent that stains a higher percent of both tumor and immune cells, while 28-8 and E1L3N behave similarly to each other. The correlation coefficients of the three assays ranged from 0.77 to 0.92 for tumor cells and from 0.66 to 0.75 for immune cells. Next, 1% and 5% tumor cell staining cutoffs were designated using various combinations of assays and cutoffs. Across all three reagents, 14 samples showed concordance above the 5% threshold, eight showed concordance within the 1% to 5% interval, and 93 showed concordance below the 1% threshold. The remaining 41 samples showed a combination of discordant results across all three reagents. High PD-L1 expression in tumor and immune cells tended to not be recurrent. The anti-PD-L1 assays 28-8 and E1L3N were closely aligned with regards to staining tumor and immune cells, whereas SP142 showed higher percentages of staining for both cell types. All three assays indicated greater variability in immune cell staining than in tumor cells.

In Vivo Generation of SSA/Ro Antigen-Specific Regulatory T Cells Improves Experimental Sjögren's Syndrome in Mice.

Sjögren's syndrome (SS) is a systemic autoimmune disease, and T cells play an important role in the initiation and perpetuation of the disease. In this study, we developed an immunotherapy for NOD/LtJ mice with SS-like symptoms by combining a transient depletion of CD4+ T cells with the administration of autoantigen-specific peptide Ro480. NOD/LtJ mice were treated with single anti-CD4 monoclonal antibody (mAb) followed 2 days later by a series of 6 intraperitoneal injections of Ro480-494 every other day. Salivary flow rates were determined pre- and posttreatment once a week. Mice were euthanized 6 weeks after the initial anti-CD4 mAb treatment, salivary glands (SGs) were collected for analyses of histologic disease scores and inflammatory cell infiltration, polymerase chain reaction determination of genes was conducted, and flow cytometry analysis including major histocompatibility complex class II tetramer staining of immune cells was performed. In addition, adoptive transfer of Treg cells was administrated to investigate the function of the newly generating Treg cells in vivo. The combination of anti-CD4 mAb with autoantigen-specific peptide Ro480 generated SSA/Ro antigen-specific Treg cells in vivo, which can suppress interferon-γ production of CD4+ T cells and inflammation infiltration in SGs and maintain the function of SGs. Our findings provide a new approach to generating antigen-specific Treg cells in vivo for SS treatment, which may have implications for potential therapy for patients with SS.

Vascular endothelial growth factor receptor 2 expression and immunotherapy efficacy in non-small cell lung cancer.

It is unclear whether tumor vascular endothelial growth factor receptor 2 expression affects the therapeutic efficacy of immune‐checkpoint inhibitors and antiangiogenic agents. This retrospective, multicenter study included patients with advanced non–small cell lung cancer who were treated with immune‐checkpoint inhibitors. We constructed tissue microarrays and performed immunohistochemistry with an anti‐vascular endothelial growth factor receptor 2 antibody. We analyzed immune and tumor cell staining separately in order to determine their correlation with the objective response rate, progression‐free survival, and overall survival in patients receiving immune‐checkpoint inhibitors. Of 364 patients, 37 (10%) expressed vascular endothelial growth factor receptor 2 in immune cells and 165 (45%) in tumor cells. The objective response rate, progression‐free survival, and overall survival were significantly worse in patients treated with immune checkpoint inhibitor monotherapy who expressed vascular endothelial growth factor receptor 2 in immune cells than those who did not (10% vs 30%, p = 0.028; median = 2.2 vs 3.6 months, p = 0.012; median = 7.9 vs 17.0 months, p = 0.049, respectively), while there was no significant difference based on tumor cell expression (24% vs 30%, p = 0.33; median = 3.1 vs 3.5 months, p = 0.55; median = 13.6 vs 16.8 months, p = 0.31). There was no significant difference in overall survival between patients treated with and without antiangiogenic agents in any treatment period based on vascular endothelial growth factor receptor 2 expression. Immune checkpoint inhibitor efficacy was limited in patients expressing vascular endothelial growth factor receptor 2 in immune cells.