Summary To guide the design of immunotherapy strategies for patients with early stage lung tumors, we developed a multiscale immune profiling strategy to map the immune landscape of early lung adenocarcinoma lesions to search for tumor-driven immune changes. Utilizing a barcoding method that allows a simultaneous single cell analysis of the tumor, non-involved lung and blood cells together with multiplex tissue imaging to assess spatial cell distribution, we provide a detailed immune cell atlas of early lung tumors. We show that stage I lung adenocarcinoma lesions already harbor significantly altered T cell and NK cell compartments. Moreover, we identified changes in tumor infiltrating myeloid cell (TIM) subsets that likely compromise anti-tumor T cell immunity. Paired single cell analyses thus offer valuable knowledge of tumor-driven immune changes, providing a powerful tool for the rational design of immune therapies.

Glioblastoma is the most common primary malignant brain tumor in adults and associated with poor survival. The Ivy Foundation Early Phase Clinical Trials Consortium conducted a randomized, multi-institution clinical trial to evaluate immune responses and survival following neoadjuvant and/or adjuvant therapy with pembrolizumab in 35 patients with recurrent, surgically resectable glioblastoma. Patients who were randomized to receive neoadjuvant pembrolizumab, with continued adjuvant therapy following surgery, had significantly extended overall survival compared to patients that were randomized to receive adjuvant, post-surgical PD-1 blockade alone. Neoadjuvant PD-1 blockade was associated with upregulation of T cell and interferon-γ-related gene expression, but downregulation of cell cycle-related gene expression within the tumor, which was not seen in patients that received adjuvant therapy alone. Focal induction of programmed death-ligand 1 (PD-L1) in the tumor microenvironment, enhanced clonal expansion of T cells, decreased PD-1 expression on peripheral blood T cells, and a decreasing monocytic population was observed more frequently in the neoadjuvant group than patients treated only in the adjuvant setting. These findings suggest that the neoadjuvant administration of PD-1 blockade enhances the local and systemic anti-tumor immune response and may represent a more efficacious approach to the treatment of this uniformly lethal brain tumor.

Therapeutic antibodies blocking programmed death-1 and its ligand (PD-1/PD-L1) induce durable responses in a substantial fraction of melanoma patients. We sought to determine whether the number and/or type of mutations identified using a next generation sequencing (NGS) panel available in the clinic were correlated with response to anti–PD-1 in melanoma. Using archival melanoma samples from anti–PD-1/PD-L1-treated patients, we performed hybrid capture-based NGS on 236–315 genes and T-cell receptor (TCR) sequencing on initial and validation cohorts from two centers. Patients who responded to anti–PD-1/PD-L1 had higher mutational loads in an initial cohort (median 45.6 vs. 3.9 mutations/MB; P = 0.003), and a validation cohort (37.1 vs. 12.8 mutations/MB; P = 0.002) compared to nonresponders. Response rate, progression-free survival, and overall survival was superior in the high, compared to intermediate and low, mutation load groups. Melanomas with NF1 mutations harbored high mutational loads (median 62.7 mutations/MB) and high response rates (74%) whereas BRAF/NRAS/NF1 wild-type melanomas had a lower mutational load. In these archival samples, TCR clonality did not predict response. Mutation numbers in the 315 genes in the NGS platform strongly correlated with those detected by whole exome sequencing in The Cancer Genome Atlas samples, but was not associated with survival. In conclusion, mutational load, as determined by an NGS platform available in the clinic, effectively stratified patients by likelihood of response. This approach may provide a clinically feasible predictor of response to anti–PD-1/PD-L1.

Developing T cells face a series of cell fate choices in the thymus and in the periphery. The role of the individual T cell receptor (TCR) in determining decisions of cell fate remains unresolved. The stochastic/selection model postulates that the initial fate of the cell is independent of TCR specificity, with survival dependent on additional TCR/coreceptor "rescue" signals. The "instructive" model holds that cell fate is initiated by the interaction of the TCR with a cognate peptide-MHC complex. T cells are then segregated on the basis of TCR specificity with the aid of critical coreceptors and signal modulators [Chan S, Correia-Neves M, Benoist C, Mathis (1998) Immunol Rev 165: 195-207]. The former would predict a random representation of individual TCR across divergent T cell lineages whereas the latter would predict minimal overlap between divergent T cell subsets. To address this issue, we have used highthroughput sequencing to evaluate the TCR distribution among key T cell developmental and effector subsets from a single donor. We found numerous examples of individual subsets sharing identical TCR sequence, supporting a model of a stochastic process of cell fate determination coupled with dynamic patterns of clonal expansion of T cells bearing the same TCR sequence among both CD4 + and CD8+ populations.F ollowing production of their T cell receptors (TCRs), T cells experience several developing stages. An encounter with a cognate peptide-MHC complex can induce naïve T (Tn) cells expressing the CD45RA isomer to begin to express CD45RO. Cells expressing both isomers are considered transitional in nature (Tt), thus cells identified on the basis of CD45RA expression alone include Tn and Tt and can thus be referred to as Tn+t. Cells expressing only CD45RO have passed into the memory (Tm) compartment, where they can lay quiescent awaiting repeat stimulation by the same or similar peptide-MHC complexes. Activated T cells (Ta) driven to effector function lose expression of both CD45RA and RO and express CD69. During different developing stages, T cells also face a series of cell fate choices: CD4 + CD8+ cells commit to either the CD4 + helper (Th) or CD8+ cytotoxic (Tc) lineages, a choice closely associated with binding to MHC class II or class I peptide complexes, respectively. Subsequently, CD4 + T cells can develop into regulatory (Tr) CD25+ cells, or into CD25−CD294− Th1 (IFN-γ producing) or CD25−CD294+ Th2 (IL-4 producing) effector subsets. Other choices are also available (1, 2).Although it is generally accepted that the TCR expressed by the developing T lineage cell will determine the response to a specific peptide-MHC complex, the role of the individual TCR in determining decisions of cell fate remains unresolved. To address these issues, we have coupled high-throughput sequencing techniques (3, 4) to high volume antibody covered superparamagnetic polystyrene bead isolation of defined T cell subsets with semiquantitative PCR amplification of the complementarity determining region 3 regions (CDR3) from mR...

Background Immunotherapy with PD-1 or PD-L1 blockade fails to induce a response in about 80% of patients with unselected non-small cell lung cancer (NSCLC), and many of those who do initially respond then develop resistance to treatment. Agonists that target the shared interleukin-2 (IL-2) and IL-15Rβγ pathway have induced complete and durable responses in some cancers, but no studies have been done to assess the safety or efficacy of these agonists in combination with anti-PD-1 immunotherapy. We aimed to define the safety, tolerability, and activity of this drug combination in patients with NSCLC. Methods In this non-randomised, open-label, phase 1b trial, we enrolled patients (aged ≥18 years) with previously treated histologically or cytologically confirmed stage IIIB or IV NSCLC from three academic hospitals in the USA. Key eligibility criteria included measurable disease, eligibility to receive anti-PD-1 immunotherapy, and an Eastern Cooperative Oncology Group performance status of 0 or 1. Patients received the anti-PD-1 monoclonal antibody nivolumab intravenously at 3 mg/kg (then 240 mg when US Food and Drug Administration [FDA]-approved dosing changed) every 14 days (either as new treatment or continued treatment at the time of disease progression) and the IL-15 superagonist ALT-803 subcutaneously once per week on weeks 1–5 of four 6-week cycles for 6 months. ALT-803 was administered at one of four escalating dose concentrations: 6, 10, 15, or 20 μg/kg. The primary endpoint was to define safety and tolerability and to establish a recommended phase 2 dose of ALT-803 in combination with nivolumab. Analyses were per-protocol and included any patients who received at least one dose of study treatment. This trial is registered with ClinicalTrials.gov, number NCT02523469; phase 2 enrolment of patients is ongoing. Findings Between Jan 18, 2016, and June 28, 2017, 23 patients were enrolled and 21 were treated at four dose levels of ALT-803 in combination with nivolumab. Two patients did not receive treatment because of the development of inter-current illness during enrolment, one patient due to leucopenia and one patient due to pulmonary dysfunction. No dose-limiting toxicities were recorded and the maximum tolerated dose was not reached. The most common adverse events were injection-site reactions (in 19 [90%] of 21 patients) and flu-like symptoms (15 [71%]). The most common grade 3 adverse events, occurring in two patients each, were lymphocytopenia and fatigue. A grade 3 myocardial infarction occurred in one patient. No grade 4 or 5 adverse events were recorded. The recommended phase 2 dose of ALT-803 is 20 μg/kg given once per week subcutaneously in combination with 240 mg intravenous nivolumab every 2 weeks. Interpretation ALT-803 in combination with nivolumab can be safely administered in an outpatient setting. The promising clinical activity observed with the addition of ALT-803 to the regimen of patients with PD-1 monoclonal antibody relapsed and refractory disease shows evidence of anti-tumour act...

Fractionated RT modulates the local TCR repertoire 2 Translational Relevance. Radiotherapy (RT) is well documented to be immunogenic; however, systemic anti-tumor immune responses outside of the irradiated tumor field, termed the "abscopal effect", are rare in patients. The lack of abscopal effect is poorly understood, particularly in the context of low-dose daily fractionated RT, the most common regimen used in clinical practice. We demonstrate that 5 daily fractions of 2 Gy induces a polyclonal T-cell response at the irradiation site which is dominated by the expansion of pre-existing T-cell clones. However, Conclusions:These data provide evidence that RT can enhance T-cell trafficking to locally-treated tumor sites and augment pre-existing anti-cancer T-cell responses with the capacity to mediate regression of out-of-field tumor lesions when delivered in combination with αPD-1 mAb therapy.

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Purpose-To use a population-based cancer registry to examine trends in renal cell carcinoma (RCC) incidence and survival among four racial/ethnic groups (White, Black, Hispanic, and Asian/ Pacific Islander (A/PI)) and both genders.Materials and Methods-Race/ethnicity, gender, age, staging, length of survival, and cause of death data were analyzed using 39,434 cases of RCC from 1988 to 2004 from the California Cancer Registry. Annual age-adjusted incidence rates and relative survival rates were calculated for the racial/ethnic and gender groups. These rates and the percent of localized cancer were plotted by year, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. and Microsoft Excel® was used to calculate linear regression equations. Median age was also calculated. Z-tests and X 2 -tests were performed to determine p-values. NIH Public AccessResults-A rise in RCC incidence was found, with localized cancer accounting for most of the increase. Blacks had a significantly higher incidence rate (p<0.0001) and lower survival rate (p<0.0001) than all other races/ethnicities, despite having more localized cancer (p<0.005). Blacks were also diagnosed at a younger age (p<0.0001) than their counterparts. On the other hand, A/PI's had a lower incidence rate (p<0.0001) and higher survival rate (p<0.05) than all other races/ ethnicities. Males had approximately twice the incidence rate of females and a lower survival rate (p<0.005).Conclusions-Higher incidence rates and lower survival rates were identified among Blacks and males when compared to their counterparts, while A/PI's showed the opposite trends. Such racial/ ethnic and gender disparities in RCC incidence and survival may help elucidate biological, behavioral, and environmental factors that can potentially be addressed.