Pathophysiology of SARS-CoV-2: the Mount Sinai COVID-19 autopsy experience.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated clinical syndrome COVID-19 are causing overwhelming morbidity and mortality around the globe and disproportionately affected New York City between March and May 2020. Here, we report on the first 100 COVID-19-positive autopsies performed at the Mount Sinai Hospital in New York City. Autopsies revealed large pulmonary emboli in six cases. Diffuse alveolar damage was present in over 90% of cases. We also report microthrombi in multiple organ systems including the brain, as well as hemophagocytosis. We additionally provide electron microscopic evidence of the presence of the virus in our samples. Laboratory results of our COVID-19 cohort disclose elevated inflammatory markers, abnormal coagulation values, and elevated cytokines IL-6, IL-8, and TNFα. Our autopsy series of COVID-19-positive patients reveals that this disease, often conceptualized as a primarily respiratory viral illness, has widespread effects in the body including hypercoagulability, a hyperinflammatory state, and endothelial dysfunction. Targeting of these multisystemic pathways could lead to new treatment avenues as well as combination therapies against SARS-CoV-2 infection.

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines.

OBJECTIVES: Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. METHODS: Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. RESULTS: Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. CONCLUSION: Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc.

NOD2 regulates CXCR3-dependent CD8+ T cell accumulation in intestinal tissues with acute injury.

Polymorphisms in NOD2 confer risk for Crohn's disease, characterized by intestinal inflammation. How NOD2 regulates both inflammatory and regulatory intestinal T cells, which are critical to intestinal immune homeostasis, is not well understood. Anti-CD3 mAb administration is used as therapy in human autoimmune diseases, as well as a model of transient intestinal injury. The stages of T cell activation, intestinal injury, and subsequent T tolerance are dependent on migration of T cells into the small intestinal (SI) lamina propria. Upon anti-CD3 mAb treatment of mice, we found that NOD2 was required for optimal small intestinal IL-10 production, in particular from CD8(+) T cells. This requirement was associated with a critical role for NOD2 in SI CD8(+) T cell accumulation and induction of the CXCR3 ligands CXCL9 and CXCL10, which regulate T cell migration. NOD2 was required in both the hematopoietic and nonhematopoietic compartments for optimal expression of CXCR3 ligands in intestinal tissues. NOD2 synergized with IFN-γ to induce CXCL9 and CXCL10 secretion in dendritic cells, macrophages, and intestinal stromal cells in vitro. Consistent with the in vitro studies, during anti-CD3 mAb treatment in vivo, CXCR3 blockade, CD8(+) T cell depletion, or IFN-γ neutralization each inhibited SI CD8(+) T cell recruitment, and reduced chemokine expression and IL-10 expression. Thus, NOD2 synergizes with IFN-γ to promote CXCL9 and CXCL10 expression, thereby amplifying CXCR3-dependent SI CD8(+) T cell migration during T cell activation, which, in turn, contributes to induction of both inflammatory and regulatory T cell outcomes in the intestinal environment.

Caspase-8 acts as a molecular rheostat to limit RIPK1- and MyD88-mediated dendritic cell activation.

Caspase-8, an executioner enzyme in the death receptor pathway, was shown to initiate apoptosis and suppress necroptosis. In this study, we identify a novel, cell death-independent role for caspase-8 in dendritic cells (DCs): DC-specific expression of caspase-8 prevents the onset of systemic autoimmunity. Failure to express caspase-8 has no effect on the lifespan of DCs but instead leads to an enhanced intrinsic activation and, subsequently, more mature and autoreactive lymphocytes. Uncontrolled TLR activation in a RIPK1-dependent manner is responsible for the enhanced functionality of caspase-8-deficient DCs, because deletion of the TLR-signaling mediator, MyD88, ameliorates systemic autoimmunity induced by caspase-8 deficiency. Taken together, these data demonstrate that caspase-8 functions in a cell type-specific manner and acts uniquely in DCs to maintain tolerance.

ADAM-10 is overexpressed in rheumatoid arthritis synovial tissue and mediates angiogenesis.

OBJECTIVE: To examine the expression of ADAM-10 in rheumatoid arthritis (RA) synovial tissue (ST) and the role it plays in angiogenesis. METHODS: ADAM-10 expression was determined using immunohistology, Western blotting, and quantitative polymerase chain reaction. In order to examine the role of ADAM-10 in angiogenesis, we performed in vitro Matrigel tube formation and chemotaxis assays using human microvascular endothelial cells (HMVECs) transfected with control or ADAM-10 small interfering RNA (siRNA). To determine whether ADAM-10 plays a role in angiogenesis in the context of RA, we performed Matrigel assays using a coculture system of HMVECs and RA synovial fibroblasts. RESULTS: Endothelial cells and lining cells within RA ST expressed high levels of ADAM-10 compared with cells within osteoarthritis ST and normal ST. ADAM-10 expression was significantly elevated at the protein and messenger RNA levels in HMVECs and RA synovial fibroblasts stimulated with proinflammatory mediators compared with unstimulated cells. ADAM-10 siRNA-treated HMVECs had decreased endothelial cell tube formation and migration compared with control siRNA-treated HMVECs. In addition, ADAM-10 siRNA-treated HMVECs from the RA synovial fibroblast coculture system had decreased endothelial cell tube formation compared with control siRNA-treated HMVECs. CONCLUSION: These data show that ADAM-10 is overexpressed in RA and suggest that ADAM-10 may play a role in RA angiogenesis. ADAM-10 may be a potential therapeutic target in inflammatory angiogenic diseases such as RA.

Expression and clinicopathologic significance of HER2 and PD-L1 in high grade urothelial carcinoma of the urinary tract.

BACKGROUND: Urothelial carcinoma (UC) is an aggressive tumor with high recurrence rates and poses a great challenge for clinical management. Programmed death ligand-1 (PD-L1) inhibitors and human epidermal growth factor receptor 2 (HER2) blockers have been approved for the treatment of advanced urothelial carcinoma. PD-L1 and HER2 expression in UC will determine whether patients are likely to respond to these targeted treatments. This study assessed the expressions of HER2 and PD-L1 in UC at our institution and investigated their correlations with gender, tumor location (upper genitourinary (GU) tract vs. lower GU tract), tumor stage, and histologic divergent subtypes. DESIGN: Patients with UC who had PD-L1 or HER2 immunostains performed in the past 3 years at our institution were included in our analysis. A total of 97 cases were identified. PD-L1 and HER2 scores were provided by two experienced GU pathologists. HER2 scores were given according to the criteria used in breast cancer, while PD-L1 scores were reported as the combined positive score. We assessed correlation of the scores with the patients' gender, tumor location, tumor stage, and histologic divergent subtypes. The data for PD-L1 expression were analyzed using the Mann-Whitney U Test for gender and urinary tract location, and one-way analysis of variance (ANOVA) for stage and histology. The data for HER2 expression were analyzed using the chi-square test. For all analyses, significance was set at P<0.05. RESULTS: Of the 97 patients, the average age was 69 years. There were 95 patients who had previously reported HER2 results and 86 patients who had PD-L1 results. PD-L1 expression did not show a significant difference among the histological divergent subtypes (P=0.36). However, HER2 status exhibited a significant difference, with more HER2-positive cases observed in the conventional histology (P=0.008). No correlation between HER2 status and either gender or tumor stage was identified. The median PD-L1 combined positive score was significantly higher in lower urinary tract UC than upper (10 and 2, respectively; P=0.049). No significant differences were observed for gender or pathologic stage. CONCLUSION: Our data suggest that HER2 is more frequently expressed in conventional UC than in divergent subtypes. Additionally, PD-L1 has a higher expression level in lower urinary tract UC compared to upper. However, PD-L1 and HER2 expression are not related to gender or tumor stage in UC.

Applications of artificial intelligence in prostate cancer histopathology.

The diagnosis of prostate cancer (PCa) depends on the evaluation of core needle biopsies by trained pathologists. Artificial intelligence (AI) derived models have been created to address the challenges posed by pathologists' increasing workload, workforce shortages, and variability in histopathology assessment. These models with histopathological parameters integrated into sophisticated neural networks demonstrate remarkable ability to identify, grade, and predict outcomes for PCa. Though the fully autonomous diagnosis of PCa remains elusive, recently published data suggests that AI has begun to serve as an initial screening tool, an assistant in the form of a real-time interactive interface during histological analysis, and as a second read system to detect false negative diagnoses. Our article aims to describe recent advances and future opportunities for AI in PCa histopathology.

Glycoprotein 96 perpetuates the persistent inflammation of rheumatoid arthritis.

OBJECTIVE: The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood. The aim of this study was to determine the contribution of endogenous gp96 in Toll-like receptor (TLR)-mediated macrophage activation in RA. METHODS: RA synovial fluid was used to activate macrophages and HEK-TLR-2 and HEK-TLR-4 cells. Neutralizing antibodies to TLR-2, TLR-4, and gp96 were used to inhibit activation. RA synovial fluid macrophages were isolated by CD14 negative selection. Cell activation was measured by the expression of tumor necrosis factor α (TNFα) or interleukin-8 messenger RNA. Arthritis was induced in mice by K/BxN serum transfer. The expression of gp96 was determined by immunoblot analysis, enzyme-linked immunosorbent assay, and immunohistochemistry. Arthritis was treated with neutralizing anti-gp96 antiserum or control serum. RESULTS: RA synovial fluid induced the activation of macrophages and HEK-TLR-2 and HEK-TLR-4 cells. RA synovial fluid-induced macrophage and HEK-TLR-2 activation was suppressed by neutralizing anti-gp96 antibodies only in the presence of high (>800 ng/ml) rather than low (<400 ng/ml) concentrations of gp96. Neutralization of RA synovial fluid macrophage cell surface gp96 inhibited the constitutive expression of TNFα. Supporting the role of gp96 in RA, joint tissue gp96 expression was induced in mice with the K/BxN serum-induced arthritis, and neutralizing antibodies to gp96 ameliorated joint inflammation, as determined by clinical and histologic examination. CONCLUSION: These observations support the notion that gp96 plays a role as an endogenous TLR-2 ligand in RA and identify the TLR-2 pathway as a therapeutic target.

Requirement of myeloid cell-specific Fas expression for prevention of systemic autoimmunity in mice.

OBJECTIVE: The death receptor Fas is a critical mediator of the extrinsic apoptotic pathway, and its role in mediating lymphoproliferation has been extensively examined. The present study was undertaken to investigate the impact of myeloid cell-specific loss of Fas. METHODS: Mice with Fas flanked by loxP sites (Fas(flox/flox) ) were crossed with mice expressing Cre under control of the murine lysozyme M gene promoter (Cre(LysM) ), which functions in mature lysozyme-expressing cells of the myelomonocytic lineage. The genotype for Cre(LysM) Fas(flox/flox) mice was verified by polymerase chain reaction and flow cytometric analysis. Flow cytometric analysis was also used to characterize myeloid, dendritic, and lymphoid cell distribution and activation in bone marrow, blood, and spleen. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure serum cytokine/chemokine and immunoglobulin levels. Renal damage or dysfunction was examined by immunohistochemical and immunofluorescence analysis. RESULTS: Cre(LysM) Fas(flox/flox) mice exhibited a systemic lupus erythematosus (SLE)-like disease that included leukocytosis, splenomegaly, hypergammaglobulinemia, antinuclear autoantibody and proinflammatory cytokine production, and glomerulonephritis. Loss of Fas in myeloid cells increased levels of both Gr-1(low) and Gr-1(intermediate) blood monocytes and splenic macrophages and, in a paracrine manner, incited activation of conventional dendritic cells and lymphocytes in Cre(LysM) Fas(flox/flox) mice. CONCLUSION: Taken together, these results suggest that loss of Fas in myeloid cells is sufficient to induce inflammatory phenotypes in mice, reminiscent of an SLE-like disease. Thus, Fas in myeloid cells may be considered a suppressor of systemic autoimmunity.

Cyclin-dependent kinase inhibitor p21, via its C-terminal domain, is essential for resolution of murine inflammatory arthritis.

OBJECTIVE: The mechanism responsible for persistent synovial inflammation in rheumatoid arthritis (RA) is unknown. Previously, we demonstrated that expression of the cyclin-dependent kinase inhibitor p21 is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. The present study was undertaken to investigate the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis. METHODS: Experimental arthritis was induced in wild-type or p21-/- (C57BL/6) mice, using the K/BxN serum-transfer model. Mice were administered p21 peptide mimetics as a prophylactic for arthritis development. Lipopolysaccharide-induced cytokine and signal transduction pathways in macrophages that were treated with p21 peptide mimetics were examined by Luminex-based assay, flow cytometry, or enzyme-linked immunosorbent assay. RESULTS: Enhanced and sustained development of experimental inflammatory arthritis, associated with markedly increased numbers of macrophages and severe articular destruction, was observed in p21-/- mice. Administration of a p21 peptide mimetic suppressed activation of macrophages and reduced the severity of experimental arthritis in p21-intact mice only. Mechanistically, treatment with the p21 peptide mimetic led to activation of the serine/threonine kinase Akt and subsequent reduction of the activated isoform of p38 MAPK in macrophages. CONCLUSION: These are the first reported data to reveal that p21 has a key role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.

Gemcitabine and cisplatin plus nivolumab as organ-sparing treatment for muscle-invasive bladder cancer: a phase 2 trial.

Cystectomy is a standard treatment for muscle-invasive bladder cancer (MIBC), but it is life-altering. We initiated a phase 2 study in which patients with MIBC received four cycles of gemcitabine, cisplatin, plus nivolumab followed by clinical restaging. Patients achieving a clinical complete response (cCR) could proceed without cystectomy. The co-primary objectives were to assess the cCR rate and the positive predictive value of cCR for a composite outcome: 2-year metastasis-free survival in patients forgoing immediate cystectomy or

Development of a new humanized mouse model to study acute inflammatory arthritis.

BACKGROUND: Substantial advances have been generated in understanding the pathogenesis of rheumatoid arthritis (RA). Current murine models of RA-like disease have provided great insights into the molecular mechanism of inflammatory arthritis due to the use of genetically deficient or transgenic mice. However, these studies are limited by differences that exist between human and murine immune systems. Thus, the development of an animal model that utilizes human immune cells, will afford the opportunity to study their function in the initiation and propagation of inflammatory arthritis. METHODS: One to two-day old irradiated NOD-scid IL2rγ(null) (NSG) mice were reconstituted with human CD34+ cord blood stem cells. Leukocytes were analyzed by flow cytometry and circulating antibodies were determined by ELISA. Arthritis was induced by injecting complete Freund's adjuvant into knee or ankle joints. Mice were also treated with the TNF inhibitor, Etanercept, or PBS and joints were analyzed histologically. RESULTS: Humanized mice were established with high reconstitution rates and were able to spontaneously produce human immunoglobulins as well as specific IgG in response to immunization. Intraperitoneal injection of thioglycolate or injection of complete Freund's adjuvant into joints resulted in migration of human immune cells to the injected sites. Arthritic humanized mice treated with Etanercept had markedly less inflammation, which was associated with decreased total numbers of human CD45+ cells, including human lymphocytes and neutrophils. CONCLUSIONS: The humanized mouse model is a new model to study inflammatory arthritis disease using human leukocytes without rejection of engrafted tissue. Future studies may adapt this system to incorporate RA patient cord blood and develop a chimeric animal model of inflammatory arthritis using genetically predisposed immune cells.

Long-term adaptation of the human lung tumor cell line A549 to increasing concentrations of hydrogen peroxide.

Previously, we demonstrated that A549, a human lung cancer cell line, could be adapted to the free radical nitric oxide (NO●). NO● is known to be over expressed in human tumors. The original cell line, A549 (parent), and the newly adapted A549-HNO (which has a more aggressive phenotype) serve as a useful model system to study the biology of NO●. To see if tumor cells can similarly be adapted to any free radical with the same outcome, herein we successfully adapted A549 cells to high levels of hydrogen peroxide (HHP). A549-HHP, the resulting cell line, was more resistant and grew better then the parent cell line, and showed the following characteristics: (1) resistance to hydrogen peroxide, (2) resistance to NO●, (3) growth with and without hydrogen peroxide, and (4) resistance to doxorubicin. Gene chip analysis was used to determine the global gene expression changes between A549-parent and A549-HHP and revealed significant changes in the expression of over 1,700 genes. This gene profile was markedly different from that obtained from the A549-HNO cell line. The mitochondrial DNA content of the A549-HHP line determined by quantitative PCR favored a change for a more anaerobic metabolic profile. Our findings suggest that any free radical can induce resistance to other free radicals; this is especially important given that radiation therapy and many chemotherapeutic agents exert their effect via free radicals. Utilizing this model system to better understand the role of free radicals in tumor biology will help to develop new therapeutic approaches to treat lung cancer.

Part I. Development of a model system for studying nitric oxide in tumors: high nitric oxide-adapted head and neck squamous cell carcinoma cell lines.

The free radical nitric oxide (NO) is over-expressed in many tumors, including head and neck squamous cell carcinomas (HNSCC); however, the role NO plays in tumor pathophysiology is still not well understood. We, herein, report the development of an in vitro model system which can be used to probe the role of NO in the carcinogenesis of HNSCC. Five HNSCC cell lines were adapted to a high NO (HNO) environment by gradually introducing increasing concentrations of DETA-NONOate, a nitrogen-based NO donor, to cell media. The adaptation process was carried out until a sufficiently high enough donor concentration was reached which enabled the HNO cells to survive and grow, but which was lethal to the original, unadapted ("parent") cells. The adapted HNO cells exhibited analogous morphology to the parent cells, but grew better than their corresponding parent cells in normal media, on soft agar, and in the presence of hydrogen peroxide, an oxygen-based free radical donor. These results indicate that the HNO cell lines are unique and possess biologically different properties than the parent cell lines from which they originated. The HNO/parent cell lines developed herein may be used as a model system to better understand the role NO plays in HNSCC carcinogenesis.

Part II. Initial molecular and cellular characterization of high nitric oxide-adapted human tongue squamous cell carcinoma cell lines.

It is not understood why some head and neck squamous cell carcinomas, despite having identical morphology, demonstrate different tumor aggressiveness, including radioresistance. High levels of the free radical nitric oxide (NO) and increased expression of the NO-producing enzyme nitric oxide synthase (NOS) have been implicated in tumor progression. We previously adapted three human tongue cancer cell lines to high NO (HNO) levels by gradually exposing them to increasing concentrations of an NO donor; the HNO cells grew faster than their corresponding untreated ("parent") cells, despite being morphologically identical. Herein we initially characterize the HNO cells and compare the biological properties of the HNO and parent cells. HNO/parent cell line pairs were analyzed for cell cycle distribution, DNA damage, X-ray and ultraviolet radiation response, and expression of key cellular enzymes, including NOS, p53, glutathione S-transferase-pi (GST-pi), apurinic/apyrimidinic endonuclease-1 (APE1), and checkpoint kinases (Chk1, Chk2). While some of these properties were cell line-specific, the HNO cells typically exhibited properties associated with a more aggressive behavior profile than the parent cells (greater S-phase percentage, radioresistance, and elevated expression of GST-pi/APE1/Chk1/Chk2). To correlate these findings with conditions in primary tumors, we examined the NOS, GST-pi, and APE1 expression in human tongue squamous cell carcinomas. A majority of the clinical samples exhibited elevated expression levels of these enzymes. Together, the results herein suggest cancer cells exposed to HNO levels can develop resistance to free radicals by upregulating protective mechanisms, such as GST-pi and APE1. These upregulated defense mechanisms may contribute to their aggressive expression profile.

The contribution of accessory toxins of Vibrio cholerae O1 El Tor to the proinflammatory response in a murine pulmonary cholera model.

The contribution of accessory toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera toxin genes (ctxAB) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-toxin (RTX) toxin gene (rtxA) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease (hapA) or hemolysin (hlyA) did not significantly affect virulence in this model. Compound deletion of ctxAB, hlyA, hapA, and rtxA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera toxins.

Long-term adaptation of breast tumor cell lines to high concentrations of nitric oxide.

Nitric oxide (NO), a free radical, has been implicated in the biology of human cancers, including breast cancer, yet it is still unclear how NO affects tumor development and propagation. We herein gradually adapted four human breast adenocarcinoma cell lines (BT-20, Hs578T, T-47D, and MCF-7) to increasing concentrations of the NO donor DETA-NONOate up to 600 muM. The resulting model system consisted of a set of fully adapted high nitric oxide ("HNO") cell lines that are biologically different from the "parent" cell lines from which they originated. Although each of the four parent and HNO cell lines had identical morphologic appearance, the HNO cells grew faster than their corresponding parent cells and were resistant to both nitrogen- and oxygen-based free radicals. These cell lines serve as a novel tool to study the role of NO in breast cancer progression and potentially can be used to predict the therapeutic response leading to more efficient therapeutic regimens.

Nitric oxide: perspectives and emerging studies of a well known cytotoxin.

The free radical nitric oxide (NO(*)) is known to play a dual role in human physiology and pathophysiology. At low levels, NO(*) can protect cells; however, at higher levels, NO(*) is a known cytotoxin, having been implicated in tumor angiogenesis and progression. While the majority of research devoted to understanding the role of NO(*) in cancer has to date been tissue-specific, we herein review underlying commonalities of NO(*) which may well exist among tumors arising from a variety of different sites. We also discuss the role of NO(*) in human physiology and pathophysiology, including the very important relationship between NO(*) and the glutathione-transferases, a class of protective enzymes involved in cellular protection. The emerging role of NO(*) in three main areas of epigenetics-DNA methylation, microRNAs, and histone modifications-is then discussed. Finally, we describe the recent development of a model cell line system in which human tumor cell lines were adapted to high NO(*) (HNO) levels. We anticipate that these HNO cell lines will serve as a useful tool in the ongoing efforts to better understand the role of NO(*) in cancer.

Randomized Double-Blind Phase II Study of Maintenance Pembrolizumab Versus Placebo After First-Line Chemotherapy in Patients With Metastatic Urothelial Cancer.

PURPOSE: Platinum-based chemotherapy for first-line treatment of metastatic urothelial cancer is typically administered for a fixed duration followed by observation until progression. "Switch maintenance" therapy with PD-1 blockade at the time of chemotherapy cessation may be attractive for mechanistic and pragmatic reasons. PATIENTS AND METHODS: Patients with metastatic urothelial cancer achieving at least stable disease on first-line platinum-based chemotherapy were enrolled. Patients were randomly assigned double-blind 1:1 to switch maintenance pembrolizumab 200 mg intravenously once every 3 weeks versus placebo for up to 24 months. Patients with disease progression on placebo could cross over to pembrolizumab. The primary objective was to determine the progression-free survival. Secondary objectives included determining overall survival as well as treatment outcomes according to PD-L1 combined positive score (CPS). RESULTS: Between December 2015 and November 2018, 108 patients were randomly assigned to pembrolizumab (n = 55) or placebo (n = 53). The objective response rate was 23% with pembrolizumab and 10% with placebo. Treatment-emergent grade 3-4 adverse events occurred in 59% receiving pembrolizumab and 38% of patients receiving placebo. Progression-free survival was significantly longer with maintenance pembrolizumab versus placebo (5.4 months [95% CI, 3.1 to 7.3 months] v 3.0 months [95% CI; 2.7 to 5.5 months]; hazard ratio, 0.65; log-rank P = .04; maximum efficiency robust test P = .039). Median overall survival was 22 months (95% CI, 12.9 months to not reached) with pembrolizumab and 18.7 months (95% CI, 11.4 months to not reached) with placebo. There was no significant interaction between PD-L1 CPS ≥ 10 and treatment arm for progression-free survival or overall survival. CONCLUSION: Switch maintenance pembrolizumab leads to additional objective responses in patients achieving at least stable disease with first-line platinum-based chemotherapy and prolongs progression-free survival in patients with metastatic urothelial cancer.