Yoshinobu Koguchi

Serum Immunoregulatory Proteins as Predictors of Overall Survival of Metastatic Melanoma Patients Treated with Ipilimumab.

Treatment with ipilimumab improves overall survival (OS) in patients with metastatic melanoma. Because ipilimumab targets T lymphocytes and not the tumor itself, efficacy may be uniquely sensitive to immunomodulatory factors present at the time of treatment. We analyzed serum from patients with metastatic melanoma (247 of 273, 90.4%) randomly assigned to receive ipilimumab or gp100 peptide vaccine. We quantified candidate biomarkers at baseline and assessed the association of each using multivariate analyses. Results were confirmed in an independent cohort of similar patients (48 of 52, 92.3%) treated with ipilimumab. After controlling for baseline covariates, elevated chemokine (C-X-C motif) ligand 11 (CXCL11) and soluble MHC class I polypeptide-related chain A (sMICA) were associated with poor OS in ipilimumab-treated patients [log10 CXCL11: HR, 1.88; 95% confidence interval (CI), 1.14-3.12; P = 0.014; and log10 sMICA quadratic effect P = 0.066; sMICA (≥ 247 vs. 247): HR, 1.75; 95% CI, 1.02-3.01]. Multivariate analysis of an independent ipilimumab-treated cohort confirmed the association between log10 CXCL11 and OS (HR, 3.18; 95% CI, 1.13-8.95; P = 0.029), whereas sMICA was less strongly associated with OS [log10 sMICA quadratic effect P = 0.16; sMICA (≥ 247 vs. 247): HR, 1.48; 95% CI, 0.67-3.27]. High baseline CXCL11 and sMICA were associated with poor OS in patients with metastatic melanoma after ipilimumab treatment but not vaccine treatment. Thus, pretreatment CXCL11 and sMICA may represent predictors of survival benefit after ipilimumab treatment as well as therapeutic targets.

Preliminary analysis of immune responses in patients enrolled in a Phase II trial of Cyclophosphamide with Allogenic DRibble Vaccine Alone (DPV-001) or with GM-CSF or Imiquimod for adjuvant treatment of Stage IIIA or IIIB NSCLC

Preliminary analysis of immune responses in patients enrolled in a Phase II trial of cyclophosphamide with allogenic dribble vaccine alone (DPV-001) or with GM-CSF or imiquimod for adjuvant treatment of stage IIIa or IIIb NSCLC Rachel Sanborn, Brian Boulmay, Rui Li, Bradley Spieler, Kyle Happel, Christopher Paustian, Tarsem Mougdil, Zipei Feng, Christopher Dubay, Brenda Fisher, Yoshinobu Koguchi, Sandra Aung, Eileen Mederos, Carlo Bifulco, Michael McNamara, Keith S Bahjat, William Redmond, Augusto C Ochoa, Hong Ming Hu, Bernard Fox, Walter Urba, Traci Hilton

A phase I/II study of pembrolizumab (P) with gemcitabine (G) in patients (Pts) with previously-treated advanced non-small cell lung cancer (NSCLC): Phase I safety results.

e20605Background: P, an anti-PD1 antibody active in advanced NSCLC, has optimal benefit in tumors with high PD-L1 expression. G may increase antigen cross-presentation and prime CD8 T cell responses via increased tumor cell apoptosis. The combination of antigen cross-presentation and T cell activation may have a synergistic effect with concurrent anti-PD1 immunotherapy. This study is evaluating P + G in pts with previously-treated advanced NSCLC with goal to establish feasible dosing prior to phase II evaluation. Methods: The phase I portion enrolled sequential cohorts of 2 pts each with advanced NSCLC (1-3 prior lines of therapy), regardless of PD-L1 status. Treatment consisted of G, 1250 mg/m2, Days (D) 1 & 8 + P 200 mg D 1; 21-day cycles. Pts received G to max 6 cycles, P to max 2 years. Each cohort closed until pts completed 6 weeks therapy (2 cycles). Dose limiting toxicity (DLT) was defined as Gr 4 hematologic or Gr 3/4 non-hematologic toxicity at least possibly treatment-related within the first 6 ...

Abstract 37: Anti-OX40 (MEDI6469) prior to definitive surgical resection in patients with head and neck squamous cell carcinoma

Background: Head and neck squamous cell carcinomas (HNSCC) produce suppressive factors that impair the immune system, thus limiting effective antitumor immunity. OX40 is a member of the tumor necrosis factor (TNF) receptor family and its biologic activity leads to potent co-stimulation, which can enhance T-cell memory, proliferation and antitumor activity in patients with metastatic cancer. However, its effect on wound healing and the optimal timing of administration in relation to surgery to induce immune changes within the tumor microenvironment (TME) is not known. Objectives: To determine the safety and peak immunologic activity of neoadjuvant anti-OX40 treatment administered prior to definitive surgical resection in patients with locoregionally advanced HNSCC. Methods: Between January 2016 and July 2016, 10 patients with locoregionally advanced HNSCC were enrolled into this phase Ib neoadjuvant time course trial testing a murine antibody to OX40 (MEDI6469) administered 2 days, 1 week and 2 weeks prior to definitive surgical resection. In order to assess changes in the tumor microenvironment (TME), a tissue biopsy and peripheral blood samples were obtained prior to MEDI6469 infusion and tissue was also harvested at the time of surgical resection from the primary tumor site, metastatic and draining lymph nodes along with peripheral blood. Assessments of tumor infiltrating lymphocyte (TIL) populations were performed based on flow cytometry and fluorescent multiplex immunohistochemistry (mIHC); other circulating immunologic parameters that correlate with changes induced by MEDI6469 administration were also measured. These immune changes were assessed and compiled in a “cumulative suppression index,” which incorporates immunosuppressive elements within the tumor, such as FoxP3+ and PD-L1+ cells, to be correlated with clinical variables and outcome. Surgical complications were described using the Clavien-Dindo grading scale. Clinical trial information: NCT02274155. Results: MEDI6469 administration was well tolerated and there were no grade 3 or 4 adverse events (AEs) attributable to anti-OX40 treatment. The toxicity profile was mild, most commonly consisting of low-grade fever prior to surgery, which was performed in all patients without delay. Postoperative grade 3 and 4 complications per Clavien-Dindo scale were observed in two patients. Immunologic changes were observed at all time courses with significant activation and proliferation of CD4+ and CD8+ central and effector memory T-cell populations in both the TME and circulation occurring between 12 and 19 days following MEDI6469 infusion. Ki67 was specifically induced in the TME and on peripheral blood PBMCs after MEDI6469 administration, returning to baseline at Day 55. Up-regulation of PD-L1 was also seen in the tumor post treatment in the majority of specimens. In the tumor, expression of CD39, ICOS and PD-1 is increased on CD4+ T cells in almost all patients and a recently identified tumor-reactive T-cell subset of CD39+CD103+CD8+ T cells, with resident memory phenotype, was increased in some patients. Conclusion: Preoperative MEDI6469 administration prior to surgery is feasible and safe in patients with HNSCC and results in activation and proliferation of T cell populations and up-regulation of PD-L1 in tumor cells occurring between 12 and 19 days following infusion. Citation Format: R. Bryan Bell, Rom S. Leidner, Rebekka A. Duhen, Carmen Ballesteros-Merino, Zipei Feng, Yoshinobu Koguchi, Carlo B. Bifulco, Brendan D. Curti, Walter J. Urba, Bernard A. Fox, Andrew D. Weinberg. Anti-OX40 (MEDI6469) prior to definitive surgical resection in patients with head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 37.

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability. Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay. A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Adoptive TIL in HPV-negative oral scca

Meeting abstracts A 22 year old male was diagnosed with clinical T2 squamous cell carcinoma of the lateral oral tongue. He underwent partial glossectomy with elective neck dissection of ipsilateral cervical nodes levels I-IV. Pathology showed a 2.1cm primary tumor with clear margins to 5mm, 1.6cm

A scalable platform for clinical immunophenotyping: assay design and quality control for high complexity flow cytometry

In the early 1980s it was demonstrated that density gradient isolation of peripheral blood mononuclear cells led to an unpredictable loss of lymphocyte populations and unacceptable levels of error for even routine CD4+ and CD8+ T cell enumeration. The error associated with this manipulation was further amplified by errors associated with cryopreservation. Based on these results, clinical flow cytometry laboratories have uniformly adopted whole blood lysis techniques for enumeration of leukocytes in peripheral blood. We have developed an extended menu of standardized immunophenotyping assays performed using anticoagulated whole blood. Based on the recommendations of the Human Immunology Project, the assays offer results that can be correlated with other clinical sites where these recommendations are followed, while offering flexibility for characterization of additional antigens and cell populations. The “Core” assays are cocktails of 8-10 antibodies that identify the cell populations of interest. To each core, an unlimited number of additional antigens of interest can be quantified. This is accomplished by replicating the core cocktail as needed. Added to this core cocktail are antibodies for detection of inducible antigens. These inducible antigens are detected using fluorochromes and detectors optimized for sensitivity and reproducibility. Included with each assay is a process control. This control uses a stabilized whole blood preparation that allows the entire staining procedure to be evaluated, from pipetting of the blood through RBC lysis and analysis. Target values are determined internally for each lot of control material and each run is verified to fall within the acceptable range for each parameter. The laboratory also performs a standardization calibration protocol to adjust photomultiplier tube voltages based on a fluorescent calibration bead. This calibration allows for quantitative fluorescence (Median Fluorescence Intensity, or MFI) to be compared over the duration of a study. Through the use of process controls, instrument standardization, and reagent validation, day-to-day and user-to-user variability is minimized. This approach allows for the complexity of clinical research while minimizing the error typically associated with such assays in the research setting. Adoption of similar practices can improve data quality and thus, the opportunity to identify changes in peripheral immune composition related to disease state and treatment.

Phase I study of alpha-tocopherlyoxyacetic acid in patients with advanced cancer.

Alpha-tocopheryloxyacetic acid (α-TEA) is a pro-apoptotic agent that demonstrates in vivo anti-tumor activity in pre-clinical models in a T cell-dependent fashion. In IND-enabling studies, orally administered α-TEA lysine salt demonstrated a safety profile in mice and dogs providing rationale for initiating a Phase I clinical trial of α-TEA in patients with advanced cancer (NCT02192346). α-TEA lysine salt was administered to patients in gelatin capsules daily for 28 days. The main clinical objectives of the trial are to characterize α-TEA related toxicity, and determine the maximum tolerated dose, and pharmacokinetics of a-TEA in humans. Secondary objectives are to characterize the phenotype of T-cell subsets in the peripheral blood by flow cytometry. The diagnoses of patients in the ongoing trial include renal cancer, esophageal cancer, thyroid cancer, duodenal cancer, squamous cell oropharyngeal cancer and advanced squamous cell carcinoma of the head and neck. Six patients have been treated at the 2.4mg/kg dose level, and five patients at the 4.8mg/kg dose level. Two patients had atrial fibrillation possibly associated with α-TEA at the 4.8 mg/kg dose level. Grade 2/3 toxicities (24 hour diarrhea) were observed in Subject 1 that in retrospect was not α-TEA related. Subject 3 had grade 4 elevations of hepatocellular enzymes on day 28 from disease progression and grade 3 transient lymphopenia, possibly related to α-TEA. Three patients have stable disease, one lasting 5 months and another lasting 2+ months, which is ongoing. Of interest is that the two patients with stable disease demonstrated an expansion of the CD8+ T cell subset and another patient demonstrated increased CD8 effector memory and substantial increase in expression of the activation markers, CD38 and HLA-DR at day 8 post-treatment. The trial is still accruing patients and the pharmacokinetics, pharmacodynamics, and identification and characterization of α-TEA metabolites in human plasma are ongoing.