SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion.

Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.

Single-cell protein activity analysis identifies recurrence-associated renal tumor macrophages.

Clear cell renal carcinoma (ccRCC) is a heterogeneous disease with a variable post-surgical course. To assemble a comprehensive ccRCC tumor microenvironment (TME) atlas, we performed single-cell RNA sequencing (scRNA-seq) of hematopoietic and non-hematopoietic subpopulations from tumor and tumor-adjacent tissue of treatment-naive ccRCC resections. We leveraged the VIPER algorithm to quantitate single-cell protein activity and validated this approach by comparison to flow cytometry. The analysis identified key TME subpopulations, as well as their master regulators and candidate cell-cell interactions, revealing clinically relevant populations, undetectable by gene-expression analysis. Specifically, we uncovered a tumor-specific macrophage subpopulation characterized by upregulation of TREM2/APOE/C1Q, validated by spatially resolved, quantitative multispectral immunofluorescence. In a large clinical validation cohort, these markers were significantly enriched in tumors from patients who recurred following surgery. The study thus identifies TREM2/APOE/C1Q-positive macrophage infiltration as a potential prognostic biomarker for ccRCC recurrence, as well as a candidate therapeutic target.

Perioperative nivolumab versus observation in patients with renal cell carcinoma undergoing nephrectomy (PROSPER ECOG-ACRIN EA8143): an open-label, randomised, phase 3 study.

BACKGROUND: The standard of care for patients with intermediate-to-high risk renal cell carcinoma is partial or radical nephrectomy followed by surveillance. We aimed to investigate use of nivolumab before nephrectomy followed by adjuvant nivolumab in patients with high-risk renal cell carcinoma to determine recurrence-free survival compared with surgery only. METHODS: In this open-label, randomised, phase 3 trial (PROSPER EA8143), patients were recruited from 183 community and academic sites across the USA and Canada. Eligible patients were aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0-1, with previously untreated clinical stage T2 or greater or Tany N+ renal cell carcinoma of clear cell or non-clear cell histology planned for partial or radical nephrectomy. Selected patients with oligometastatic disease, who were disease free at other disease sites within 12 weeks of surgery, were eligible for inclusion. We randomly assigned (1:1) patients using permuted blocks (block size of 4) within stratum (clinical TNM stage) to either nivolumab plus surgery, or surgery only followed by surveillance. In the nivolumab group, nivolumab 480 mg was administered before surgery, followed by nine adjuvant doses. The primary endpoint was investigator-reviewed recurrence-free survival in patients with renal cell carcinoma assessed in all randomly assigned patients regardless of histology. Safety was assessed in all randomly assigned patients who started the assigned protocol treatment. This trial is registered with ClinicalTrials.gov, NCT03055013, and is closed to accrual. FINDINGS: Between Feb 2, 2017, and June 2, 2021, 819 patients were randomly assigned to nivolumab plus surgery (404 [49%]) or surgery only (415 [51%]). 366 (91%) of 404 patients assigned to nivolumab plus surgery and 387 (93%) of 415 patients assigned to surgery only group started treatment. Median age was 61 years (IQR 53-69), 248 (30%) of 819 patients were female, 571 (70%) were male, 672 (88%) were White, and 77 (10%) were Hispanic or Latino. The Data and Safety Monitoring Committee stopped the trial at a planned interim analysis (March 25, 2022) because of futility. Median follow-up was 30·4 months (IQR 21·5-42·4) in the nivolumab group and 30·1 months (21·9-41·8) in the surgery only group. 381 (94%) of 404 patients in the nivolumab plus surgery group and 399 (96%) of 415 in the surgery only group had renal cell carcinoma and were included in the recurrence-free survival analysis. As of data cutoff (May 24, 2023), recurrence-free survival was not significantly different between nivolumab (125 [33%] of 381 had recurrence-free survival events) versus surgery only (133 [33%] of 399; hazard ratio 0·94 [95% CI 0·74-1·21]; one-sided p=0·32). The most common treatment-related grade 3-4 adverse events were elevated lipase (17 [5%] of 366 patients in the nivolumab plus surgery group vs none in the surgery only group), anaemia (seven [2%] vs nine [2%]), increased alanine aminotransferase (ten [3%] vs one [<1%]), abdominal pain (four [1%] vs six [2%]), and increased serum amylase (nine [2%] vs none). 177 (48%) patients in the nivolumab plus surgery group and 93 (24%) in the surgery only group had grade 3-5 adverse events due to any cause, the most common of which were anaemia (23 [6%] vs 19 [5%]), hypertension (27 [7%] vs nine [2%]), and elevated lipase (18 [5%] vs six [2%]). 48 (12%) of 404 patients in the nivolumab group and 40 (10%) of 415 in the surgery only group died, of which eight (2%) and three (1%), respectively, were determined to be treatment-related. INTERPRETATION: Perioperative nivolumab before nephrectomy followed by adjuvant nivolumab did not improve recurrence-free survival versus surgery only followed by surveillance in patients with high-risk renal cell carcinoma. FUNDING: US National Institutes of Health National Cancer Institute and Bristol Myers Squibb.

The composition and signaling of the IL-35 receptor are unconventional.

Interleukin 35 (IL-35) belongs to the IL-12 family of heterodimeric cytokines but has a distinct functional profile. IL-35 suppresses T cell proliferation and converts naive T cells into IL-35-producing induced regulatory T cells (iTr35 cells). Here we found that IL-35 signaled through a unique heterodimer of receptor chains IL-12Rß2 and gp130 or homodimers of each chain. Conventional T cells were sensitive to IL-35-mediated suppression in the absence of one receptor chain but not both receptor chains, whereas signaling through both chains was required for IL-35 expression and conversion into iTr35 cells. Signaling through the IL-35 receptor required the transcription factors STAT1 and STAT4, which formed a unique heterodimer that bound to distinct sites in the promoters of the genes encoding the IL-12 subunits p35 and Ebi3. This unconventional mode of signaling, distinct from that of other members of the IL-12 family, may broaden the spectrum and specificity of IL-35-mediated suppression.

BCG invokes superior STING-mediated innate immune response over radiotherapy in a carcinogen murine model of urothelial cancer.

Radiation and bacillus Calmette-Guérin (BCG) instillations are used clinically for treatment of urothelial carcinoma, but the precise mechanisms by which they activate an immune response remain elusive. The role of the cGAS-STING pathway has been implicated in both BCG and radiation-induced immune response; however, comparison of STING pathway molecules and the immune landscape following treatment in urothelial carcinoma has not been performed. We therefore comprehensively analyzed the local immune response in the bladder tumor microenvironment following radiotherapy and BCG instillations in a well-established spontaneous murine model of urothelial carcinoma to provide insight into activation of STING-mediated immune response. Mice were exposed to the oral carcinogen, BBN, for 12 weeks prior to treatment with a single 15 Gy dose of radiation or three intravesical instillations of BCG (1 × 108 CFU). At sacrifice, tumors were staged by a urologic pathologist and effects of therapy on the immune microenvironment were measured using the NanoString Myeloid Innate Immunity Panel and immunohistochemistry. Clinical relevance was established by measuring immune biomarker expression of cGAS and STING on a human tissue microarray consisting of BCG-treated non-muscle-invasive urothelial carcinomas. BCG instillations in the murine model elevated STING and downstream STING-induced interferon and pro-inflammatory molecules, intratumoral M1 macrophage and T-cell accumulation, and complete tumor eradication. In contrast, radiotherapy caused no changes in STING pathway or innate immune gene expression; rather, it induced M2 macrophage accumulation and elevated FoxP3 expression characteristic of immunosuppression. In human non-muscle-invasive bladder cancer, STING protein expression was elevated at baseline in patients who responded to BCG therapy and increased further after BCG therapy. Overall, these results show that STING pathway activation plays a key role in effective BCG-induced immune response and strongly indicate that the effects of BCG on the bladder cancer immune microenvironment are more beneficial than those induced by radiation. © 2021 The Pathological Society of Great Britain and Ireland.

PD-1 Signaling Promotes Tumor-Infiltrating Myeloid-Derived Suppressor Cells and Gastric Tumorigenesis in Mice.

BACKGROUND & AIMS: Immune checkpoint inhibitors have limited efficacy in many tumors. We investigated mechanisms of tumor resistance to inhibitors of programmed cell death-1 (PDCD1, also called PD-1) in mice with gastric cancer, and the role of its ligand, PD-L1. METHODS: Gastrin-deficient mice were given N-methyl-N-nitrosourea (MNU) in drinking water along with Helicobacter felis to induce gastric tumor formation; we also performed studies with H/K-ATPase-hIL1B mice, which develop spontaneous gastric tumors at the antral-corpus junction and have parietal cells that constitutively secrete interleukin 1B. Mice were given injections of an antibody against PD-1 or an isotype control before tumors developed, or anti-PD-1 and 5-fluorouracil and oxaliplatin, or an antibody against lymphocyte antigen 6 complex locus G (also called Gr-1), which depletes myeloid-derived suppressor cells [MDSCs]), after tumors developed. We generated knock-in mice that express PD-L1 specifically in the gastric epithelium or myeloid lineage. RESULTS: When given to gastrin-deficient mice before tumors grew, anti-PD-1 significantly reduced tumor size and increased tumor infiltration by T cells. However, anti-PD-1 alone did not have significant effects on established tumors in these mice. Neither early nor late anti-PD-1 administration reduced tumor growth in the presence of MDSCs in H/K-ATPase-hIL-1ß mice. The combination of 5-fluorouracil and oxaliplatin reduced MDSCs, increased numbers of intra-tumor CD8+ T cells, and increased the response of tumors to anti-PD-1; however, this resulted in increased tumor expression of PD-L1. Expression of PD-L1 by tumor or immune cells increased gastric tumorigenesis in mice given MNU. Mice with gastric epithelial cells that expressed PD-L1 did not develop spontaneous tumors, but they developed more and larger tumors after administration of MNU and H felis, with accumulation of MDSCs. CONCLUSIONS: In mouse models of gastric cancer, 5-fluorouracil and oxaliplatin reduce numbers of MDSCs to increase the effects of anti-PD-1, which promotes tumor infiltration by CD8+ T cells. However, these chemotherapeutic agents also induce expression of PD-L1 by tumor cells. Expression of PD-L1 by gastric epithelial cells increases tumorigenesis in response to MNU and H felis, and accumulation of MDSCs, which promote tumor progression. The timing and site of PD-L1 expression is therefore important in gastric tumorigenesis and should be considered in design of therapeutic regimens.

Nivolumab plus ipilimumab, with or without enzalutamide, in AR-V7-expressing metastatic castration-resistant prostate cancer: A phase-2 nonrandomized clinical trial.

BACKGROUND: AR-V7-positive metastatic prostate cancer is a lethal phenotype with few treatment options and poor survival. METHODS: The two-cohort nonrandomized Phase 2 study of combined immune checkpoint blockade for AR-V7-expressing metastatic castration-resistant prostate cancer (STARVE-PC) evaluated nivolumab (3 mg/kg) plus ipilimumab (1 mg/kg), without (Cohort 1) or with (Cohort 2) the anti-androgen enzalutamide. Co-primary endpoints were safety and prostate-specific antigen (PSA) response rate. Secondary endpoints included time-to-PSA-progression-free survival (PSA-PFS), time-to-clinical/radiographic-PFS, objective response rate (ORR), PFS lasting greater than 24 weeks, and overall survival (OS). RESULTS: Thirty patients were treated with ipilimumab plus nivolumab (N = 15, Cohort 1, previously reported), or ipilimumab plus nivolumab and enzalutamide (N = 15, Cohort 2) in patients previously progressing on enzalutamide monotherapy. PSA response rate was 2/15 (13%) in cohort 1 and 0/15 in cohort 2, ORR was 2/8 (25%) in Cohort 1 and 0/9 in Cohort 2 in those with measureable disease, median PSA-PFS was 3.0 (95% confidence interval [CI]: 2.1-NR) in cohort 1 and 2.7 (95% CI: 2.1-5.9) months in cohort 2, and median PFS was 3.7 (95% CI: 2.8-7.5) in cohort 1 and 2.9 (95% CI: 1.3-5.8) months in cohort 2. Three of 15 patients in cohort 1 (20%, 95% CI: 7.1%-45.2%) and 4/15 patients (26.7%, 95% CI: 10.5%-52.4%) in cohort 2 achieved a durable PFS lasting greater than 24 weeks. Median OS was 8.2 (95% CI: 5.5-10.4) in cohort 1 and 14.2 (95% CI: 8.5-NA) months in cohort 2. Efficacy results were not statistically different between cohorts. Grade-3/4 adverse events occurred in 7/15 cohort 1 patients (46%) and 8/15 cohort 2 patients (53%). Combined cohort (N = 30) baseline alkaline phosphatase and cytokine analysis suggested improved OS for patients with lower alkaline phosphatase (hazards ratio [HR], 0.30; 95% CI: 0.11-0.82), lower circulating interleukin-7 (IL-7) (HR, 0.24; 95% Cl: 0.06-0.93) and IL-6 (HR, 0.13; 95% Cl: 0.03-0.52) levels, and higher circulating IL-17 (HR, 4.53; 95% CI: 1.47-13.93) levels. There was a trend towards improved outcomes in men with low sPD-L1 serum levels. CONCLUSION: Nivolumab plus ipilimumab demonstrated only modest activity in patients with AR-V7-expressing prostate cancer, and was not sufficient to justify further exploration in unselected patients. Stratification by baseline alkaline phosphatase and cytokines (IL-6, -7, and -17) may be prognostic for outcomes to immunotherapy.

LAG3 (CD223) as a cancer immunotherapy target.

Despite the impressive impact of CTLA4 and PD1-PDL1-targeted cancer immunotherapy, a large proportion of patients with many tumor types fail to respond. Consequently, the focus has shifted to targeting alternative inhibitory receptors (IRs) and suppressive mechanisms within the tumor microenvironment. Lymphocyte activation gene-3 (LAG3) (CD223) is the third IR to be targeted in the clinic, consequently garnering considerable interest and scrutiny. LAG3 upregulation is required to control overt activation and prevent the onset of autoimmunity. However, persistent antigen exposure in the tumor microenvironment results in sustained LAG3 expression, contributing to a state of exhaustion manifest in impaired proliferation and cytokine production. The exact signaling mechanisms downstream of LAG3 and interplay with other IRs remain largely unknown. However, the striking synergy between LAG3 and PD1 observed in multiple settings, coupled with the contrasting intracellular cytoplasmic domain of LAG3 as compared with other IRs, highlights the potential uniqueness of LAG3. There are now four LAG3-targeted therapies in the clinic with many more in preclinical development, emphasizing the broad interest in this IR. Given the translational relevance of LAG3 and the heightened interest in the impact of dual LAG3/PD1 targeting in the clinic, the outcome of these trials could serve as a nexus; significantly increasing or dampening enthusiasm for subsequent targets in the cancer immunotherapeutic pipeline.

Impact of baseline serum IL-8 on metastatic hormone-sensitive prostate cancer outcomes in the Phase 3 CHAARTED trial (E3805).

BACKGROUND: The immunosuppressive cytokine interleukin- 8 (IL-8), produced by tumor cells and some myeloid cells, promotes inflammation, angiogenesis, and metastasis. In our discovery work, elevated serum IL-8 at androgen deprivation therapy (ADT) initiation portended worse overall survival (OS). Leveraging serum samples from the phase 3 CHAARTED trial of patients treated with ADT +/- docetaxel for metastatic hormone-sensitive prostate cancer (mHSPC), we validated these findings. METHODS: Two hundred and thirty-three patients had serum samples drawn within 28 days of ADT initiation. The samples were assayed using the same Mesoscale Multiplex ELISA platform employed in the discovery cohort. After adjusting for performance status, disease volume, and de novo/metachronous metastases, multivariable Cox proportional hazards models assessed associations between IL-8 as continuous and binary variables on OS and time to castration-resistant prostate cancer (CRPC). The median IL-8 level (9.3 pg/ml) was the a priori binary cutpoint. Fixed-effects meta-analyses of the discovery and validation sets were performed. RESULTS: Higher IL-8 levels were prognostic for shorter OS (continuous: hazard ratio [HR] 2.2, 95% confidence interval [CI]: 1.4-3.6, p = .001; binary >9.3: HR 1.7, 95% CI: 1.2-2.4, p = .007) and time to CRPC (continuous: HR 2.3, 95% CI: 1.6-3.3, p < .001; binary: HR 1.8, 95% CI: 1.3-2.5, p < .001) and independent of docetaxel use, disease burden, and time of metastases. Meta-analysis including the discovery cohort, also showed that binary IL-8 levels >9.3 pg/ml from patients treated with ADT alone was prognostic for poorer OS (HR 1.8, 95% CI: 1.2-2.7, p = .007) and shorter time to CRPC (HR 1.4, 95% CI: 0.99-1.9, p = .057). CONCLUSIONS: In the phase 3 CHAARTED study of men with mHSPC at ADT initiation, elevated IL-8 portended worse survival and shorter time to castration-resistant prostate cancer independent of docetaxel administration, metastatic burden, and metachronous versus de novo metastatic presentation. These findings support targeting IL-8 as a strategy to improve mHSPC outcomes.

Association between immunosuppressive cytokines and PSA progression in biochemically recurrent prostate cancer treated with intermittent hormonal therapy.

BACKGROUND: Immunosuppressive cytokines have the potential to promote prostate cancer progression. Assessing their longitudinal changes may implicate mechanisms of progression, treatment resistance, and suggest new therapeutic targets. METHODS: Thirty-seven men with biochemically recurrent (BCR) prostate cancer who received 6 months of androgen deprivation therapy (ADT) and were monitored until the time to prostate-specific antigen progression (TTPP) were identified from a completed phase III trial (NCT00020085). Serum samples were archived at baseline, 3 months after ADT, and at TTPP. Cytokine concentrations were quantified using a 36-parameter electrochemiluminescence assay. The Wilcoxon signed-rank sum test was used to compare observations between time points. Kaplan-Meier analysis was used to calculate TTPP dichotomized by cytokine values above or below the median. Pearson's rank correlation coefficient was used to compare continuous variables. RESULTS: Median TTPP was 399 days (range, 114-1641). Median prostate-specific antigen (PSA) at baseline and progression were 8.5 and 5.3 ng/mL, respectively. Twenty-three patients (62%) achieved undetectable PSA with ADT. Castrate levels of testosterone (<50 ng/dL) after 3 months of ADT occurred in 35 patients (95%). TNF-α (P = .002), IL-23 (P = .002), and CXCL10 (P = .001) significantly increased from baseline to post ADT. Certain cytokines correlated longitudinally: TNF-α correlated with IL-23 (r = .72; P < .001) and IL-8 (r = .59; P < .001) from baseline to post ADT and to PSA progression. Neutrophil-to-lymphocyte ratio correlated with IL-27 (r = .57; P < .001) and MIP-3α (r = .56; P < .001). Patients with a detectable PSA after ADT had elevated levels of IL-6 (P = .049) and IL-8 (P = .013) at PSA progression as compared with those with an undetectable PSA. There was a trend toward shorter TTPP in patients with TNF-α levels above the median (P = .042). CONCLUSIONS: Several innate cytokines were associated with biochemically recurrent prostate cancer.

Optimizing the management of castration-resistant prostate cancer patients: A practical guide for clinicians.

BACKGROUND: Advanced prostate cancer (PC) patients, especially those with metastatic prostate cancer (mPC), often require complex management pathways. Despite the publication of clinical practice guidelines by leading urological and oncological organizations that provide a substantial and comprehensive framework, there are numerous clinical scenarios that are not always addressed, especially as new treatments become available, new imaging modalities are developed, and advances in genetic testing continue. METHODS: A 14-member expert review panel comprised of urologists and medical oncologists were chosen to provide guidance on addressing specific topics and issues regarding metastatic castration-resistant prostate cancer (mCRPC) patients. Panel members were chosen based upon their experience and expertise in the management of PC patients. Four academic members (two urologists and two medical oncologists) of the panel served as group leaders; the remaining eight panel members were from Large Urology Group Practice Association (LUGPA) practices with proven experience in leading their advanced PC clinics. The panel members were assigned to four separate working groups, each assigned a specific mCRPC topic to review and discuss with the entire panel. RESULTS: This article describes the practical recommendations of an expert panel on the management of mCRPC patients. The target reading audience for this publication is all providers (urologists, medical oncologists, radiation oncologists, or advanced practice providers) who evaluate and manage advanced PC patients, regardless of their practice setting. CONCLUSION: The panel has provided recommendations for managing mCRPC with regard to specific issues: (a) biomarker monitoring and the role of genetic and molecular testing; (b) rationale, current strategies, and optimal sequencing of the various approved therapies, including hormonal therapy, cytotoxic chemotherapy, radiopharmaceuticals and immunotherapy; (c) adverse event management and monitoring; and (d) imaging advanced PC patients. These recommendations seek to complement national guidelines, not replace them, and a discussion of where the panel agreed or disagreed with national guidelines is included.

A Phase I Trial of Intravesical Cabazitaxel, Gemcitabine and Cisplatin for the Treatment of Nonmuscle Invasive bacillus Calmette-Guérin Unresponsive or Recurrent/Relapsing Urothelial Carcinoma of the Bladder.

PURPOSE: For patients with bacillus Calmette-Guérin unresponsive or recurrent/relapsing nonmuscle invasive bladder cancer, multi-agent intravesical trials have been limited. In this study we investigate the safety of intravesical cabazitaxel, gemcitabine and cisplatin in the salvage setting. MATERIALS AND METHODS: This was a dose escalation, drug escalation trial for patients with bacillus Calmette-Guérin unresponsive or recurrent/relapsing nonmuscle invasive bladder cancer who declined or were ineligible for radical cystectomy. All patients underwent a 6-week induction regimen of sequentially administered cabazitaxel, gemcitabine and cisplatin. Complete response was defined as no cancer on post-induction transurethral bladder tumor resection and negative urine cytology, while partial response allowed for positive cytology. Responders continued with maintenance cabazitaxel and gemcitabine monthly for the first year and bimonthly for the second year. RESULTS: A total of 18 patients were enrolled. Mean age was 71 years, median followup was 27.8 months (range 16.3 to 46.9) and mean number of previous rounds of intravesical therapies before trial enrollment was 3.7. Nine patients (50%) had received intravesical chemotherapy after bacillus Calmette-Guérin and 7 (39%) were previously treated in a phase I clinical trial setting. At enrollment 6 (33%) subjects had T1 disease and 13 (72%) had carcinoma in situ. There were no dose limiting toxicities. Initial partial and complete response rates were 94% and 89%, respectively. At 1 year recurrence-free survival was 0.83 (range 0.57 to 0.94) and at 2 years estimated recurrence-free survival was 0.64 (0.32 to 0.84). CONCLUSIONS: In this high risk and highly pretreated cohort of bacillus Calmette-Guérin unresponsive or recurrent/relapsing nonmuscle invasive bladder cancer cases combination intravesical cabazitaxel, gemcitabine and cisplatin was a well tolerated and potentially effective regimen.

Inhibitors of the PD-1 Pathway in Tumor Therapy.

The programmed death 1 (PD-1) pathway delivers inhibitory signals that function as a brake for immune responses. This pathway limits the initiation and duration of immune responses, thereby protecting tissues from immune-mediated damage and autoimmune diseases. However, the PD-1 pathway also inhibits immune responses to tumors. The critical role of PD-1 in preventing antitumor immunity is demonstrated by the transformative effects of PD-1 pathway blockade in a broad range of cancers with the hallmark of durability of response. Despite this success, most patients do not respond to PD-1 monotherapy, and some patients experience adverse events. In this review, we discuss the functions of the PD-1 pathway and its translation to cancer immunotherapy. We also consider current challenges and opportunities for PD-1 cancer immunotherapy, including mechanisms of response and resistance, identification of biomarkers of response to PD-1 therapy, characterization and treatment of PD-1 therapy-related adverse events, and development of safe and effective combination therapies.

A role for paracrine interleukin-6 signaling in the tumor microenvironment in prostate tumor growth.

BACKGROUND: Interleukin-6 (IL-6) is a mediator of inflammation that can facilitate prostate cancer progression. We previously demonstrated that IL-6 is present in the prostate tumor microenvironment and is restricted almost exclusively to the stromal compartment. The present study examined the influence of paracrine IL-6 signaling on prostate tumor growth using allograft models of mouse prostate cancer (TRAMP-C2), colon cancer (MC38), and melanoma (B16) cell lines in wildtype (WT) and IL-6 knockout (IL-6-/- ) mice. METHODS: Cells were implanted into WT or IL-6-/- mice and tumor sizes were measured at a 3 to 4 day interval. Serum, tumors, and other organs were collected for IL-6 analysis by ELISA and RNA in situ hybridization (RISH). RESULTS: There was a significant reduction in TRAMP-C2 and B16 tumor size grown in IL-6-/- mice versus WT mice (P = 0.0006 and P = 0.02, respectively). This trend was not observed for the MC38 cell line. RISH analysis of TRAMP-C2 tumors grown in WT mice showed that cells present in the tumor microenvironment were the primary source of IL-6 mRNA, not the TRAMP-C2 cells. Serum IL-6 ELISA analyses showed an increase in the circulating levels of IL-6 in WT mice bearing TRAMP-C2 tumors. Similar phospho-STAT3 expression and tumor vascularization were observed in TRAMP-C2 tumors grown in WT and IL-6-/- mice. CONCLUSIONS: Our results are consistent with previous studies in prostate cancer patients demonstrating that paracrine IL-6 production in the tumor microenvironment may influence tumor growth. Additionally, these data provide evidence that elevated systemic IL-6 levels may be involved in tumor growth regulation in prostate cancer, and are not simply caused by or indicative of tumor burden.

Consequences of interleukin 1ß-triggered chronic inflammation in the mouse prostate gland: Altered architecture associated with prolonged CD4+ infiltration mimics human proliferative inflammatory atrophy.

BACKGROUND: Elevated expression of the proinflammatory cytokine interleukin 1ß (IL-1ß) has been observed in expressed prostatic secretions of patients with chronic prostatitis/chronic pelvic pain syndrome, and genetic polymorphisms associated with the IL1B gene are linked to increased risk for aggressive prostate cancer. METHODS: To study the role of IL-1ß expression in prostate inflammation, we examined IL1B expression in human prostatic proliferative inflammatory atrophy (PIA) lesions and developed a tetracycline-regulated human IL1B transgene in the mouse prostate. RESULTS: Here, we demonstrate that IL1B expression is a common finding in human PIA lesions, which harbored focal IL1B expression in epithelial and stromal compartments. Human IL1B expression in the mouse prostate elicited acute and chronic inflammation. Penetrance and expressivity were variable and tunable by altering transgene dosage and the presence of an exogenous inducible marker antigen (green fluorescent protein). Inflammation was characterized by infiltration of CD4+ T cells, demonstrating an adaptive immune response. Chronic inflammation persisted after doxycycline (Dox) withdrawal. Reactive epithelia increased expression of downstream cytokines, and altered glandular architecture was observed upon sustained induction of IL1B. Immunohistochemical analyses revealed a higher proliferative index and decreased Nkx3.1 expression in inflamed mouse prostates. CONCLUSIONS: These data implicate IL-1ß in human prostate pathology and this model provides a versatile platform to interrogate molecular mechanisms of inflammation-associated prostate pathologies associated with episodic or sustained IL-1ß expression.

Comprehensive Evaluation of Programmed Death-Ligand 1 Expression in Primary and Metastatic Prostate Cancer.

Antibodies targeting the programmed cell death protein 1/programmed death-ligand 1 (PD-L1) interaction have shown clinical activity in multiple cancer types. PD-L1 protein expression is a clinically validated predictive biomarker of response for such therapies. Prior studies evaluating the expression of PD-L1 in primary prostate cancers have reported highly variable rates of PD-L1 positivity. In addition, limited data exist on PD-L1 expression in metastatic castrate-resistant prostate cancer (mCRPC). Here, we determined PD-L1 protein expression by immunohistochemistry using a validated PD-L1-specific antibody (SP263) in a large and representative cohort of primary prostate cancers and prostate cancer metastases. The study included 539 primary prostate cancers comprising 508 acinar adenocarcinomas, 24 prostatic duct adenocarcinomas, 7 small-cell carcinomas, and a total of 57 cases of mCRPC. PD-L1 positivity was low in primary acinar adenocarcinoma, with only 7.7% of cases showing detectable PD-L1 staining. Increased levels of PD-L1 expression were noted in 42.9% of small-cell carcinomas. In mCRPC, 31.6% of cases showed PD-L1-specific immunoreactivity. In conclusion, in this comprehensive evaluation of PD-L1 expression in prostate cancer, PD-L1 expression is rare in primary prostate cancers, but increased rates of PD-L1 positivity were observed in mCRPC. These results will be important for the future clinical development of programmed cell death protein 1/PD-L1-targeting therapies in prostate cancer.

The future of perioperative therapy in advanced renal cell carcinoma: how can we PROSPER?

Patients with high-risk renal cell carcinoma (RCC) experience high rates of recurrence despite definitive surgical resection. Recent trials of adjuvant tyrosine kinase inhibitor therapy have provided conflicting efficacy results at the cost of significant adverse events. PD-1 blockade via monoclonal antibodies has emerged as an effective disease-modifying treatment for metastatic RCC. There is emerging data across other solid tumors of the potential efficacy of neoadjuvant PD-1 blockade, and preclinical evidence supporting a neoadjuvant over adjuvant approach. PROSPER RCC is a Phase III, randomized trial evaluating whether perioperative nivolumab increases recurrence-free survival in patients with high-risk RCC undergoing nephrectomy. The neoadjuvant component, intended to prime the immune system for enhanced efficacy, distinguishes PROSPER from other purely adjuvant studies and permits highly clinically relevant translational studies.

Association of PD-1/PD-L axis expression with cytolytic activity, mutational load, and prognosis in melanoma and other solid tumors.

Programmed cell death protein-1 (PD-1)/programmed death ligand-1 (PD-L1) checkpoint blockade has led to remarkable and durable objective responses in a number of different tumor types. A better understanding of factors associated with the PD-1/PD-L axis expression is desirable, as it informs their potential role as prognostic and predictive biomarkers and may suggest rational treatment combinations. In the current study, we analyzed PD-L1, PD-L2, PD-1, and cytolytic activity (CYT) expression, as well as mutational density from melanoma and eight other solid tumor types using The Cancer Genome Atlas database. We found that in some tumor types, PD-L2 expression is more closely linked to Th1/IFNG expression and PD-1 and CD8 signaling than PD-L1 In contrast, mutational load was not correlated with a Th1/IFNG gene signature in any tumor type. PD-L1, PD-L2, PD-1, CYT expression, and mutational density are all positive prognostic features in melanoma, and conditional inference modeling revealed PD-1/CYT expression (i.e., an inflamed tumor microenvironment) as the most impactful feature, followed by mutational density. This study elucidates the highly interdependent nature of these parameters, and also indicates that future biomarkers for anti-PD-1/PD-L1 will benefit from tumor-type-specific, integrated, mRNA, protein, and genomic approaches.

Bipolar androgen therapy in men with metastatic castration-resistant prostate cancer after progression on enzalutamide: an open-label, phase 2, multicohort study.

BACKGROUND: Prostate cancer that progresses after enzalutamide treatment is poorly responsive to further antiandrogen therapy, and paradoxically, rapid cycling between high and low serum testosterone concentrations (bipolar androgen therapy [BAT]) in this setting might induce tumour responses. We aimed to evaluate BAT in patients with metastatic castration-resistant prostate cancer that progressed after enzalutamide. METHODS: We did this single-centre, open-label, phase 2, multicohort study in the USA. We included patients aged 18 years or older who had histologically confirmed and radiographically documented metastatic castration-resistant prostate cancer, with no more than two previous second-line hormonal therapies, and a castrate concentration of testosterone. Patients were asymptomatic, with Eastern Cooperative Oncology Group performance status of 0-2, and did not have high-risk lesions for tumour flare (eg, >5 sites of visceral disease or bone lesions with impending fracture). For the cohort reported here, we required patients to have had progression on enzalutamide with a continued prostate-specific antigen (PSA) rise after enzalutamide treatment discontinuation. Patients received BAT, which consisted of intramuscular testosterone cipionate 400 mg every 28 days until progression and continued luteinising hormone-releasing hormone agonist therapy. Upon progression after BAT, men were rechallenged with oral enzalutamide 160 mg daily. The co-primary endpoints were investigator-assessed 50% decline in PSA concentration from baseline (PSA50) for BAT (for all patients who received at least one dose) and for enzalutamide rechallenge (based on intention-to-treat analysis). These data represent the final analysis for the post-enzalutamide cohort, while two additional cohorts (post-abiraterone and newly castration-resistant prostate cancer) are ongoing. The trial is registered with ClinicalTrials.gov, number NCT02090114. FINDINGS: Between Aug 28, 2014, and May 18, 2016, we accrued 30 eligible patients and treated them with BAT. Nine (30%; 95% CI 15-49; p<0·0001) of 30 patients achieved a PSA50 to BAT. 29 patients completed BAT and 21 proceeded to enzalutamide rechallenge, of whom 15 (52%; 95% CI 33-71; p<0·0001) achieved a PSA50 response. During BAT, the only grade 3-4 adverse event occurring in more than one patient was hypertension (three [10%] patients). Other grade 3 or worse adverse events occurring during BAT in one [3%] patient each were pulmonary embolism, myocardial infarction, urinary obstruction, gallstone, and sepsis. During enzalutamide retreatment, no grade 3-4 toxicities occurred in more than one patient. No treatment-related deaths were reported during either BAT or enzalutamide retreatment. INTERPRETATION: BAT is a safe therapy that resulted in responses in asymptomatic men with metastatic castration-resistant prostate cancer and also resensitisation to enzalutamide in most patients undergoing rechallenge. Further studies with BAT are needed to define the potential clinical role for BAT in the management of metastatic castration-resistant prostate cancer and the optimal strategy for sequencing between androgen and antiandrogen therapies in metastatic castration-resistant prostate cancer to maximise therapeutic benefit to patients. FUNDING: National Institutes of Health and National Cancer Institute.

Expression of LAG-3 and efficacy of combination treatment with anti-LAG-3 and anti-PD-1 monoclonal antibodies in glioblastoma.

Like in many tumor types, immunotherapy is currently under investigation to assess its potential efficacy in glioblastoma patients. Trials are under way to assess the efficacy of new immune checkpoint inhibitors including anti-PD-1 or CTLA4. We here investigate the expression and efficacy of a novel immune-checkpoint inhibitor, called LAG-3. We show that LAG-3 is expressed in human glioblastoma samples and in a mouse glioblastoma model we show that knock out or LAG-3 inhibition with a blocking antibody is efficacious against glioblastoma and can be used in combination with other immune checkpoint inhibitors toward complete eradication of the model glioblastoma tumors. From a mechanistic standpoint we show that LAG-3 expression is an early marker of T cell exhaustion and therefore early treatment with LAG-3 blocking antibody is more efficacious than later treatment. These data provide insight and support the design of trials that incorporate LAG-3 in the treatment of glioblastoma.