bluuebunny/crossref_metadata_2025 huggingface dataset:
Final report of a pilot trial combining ipilimumab and adoptive cell therapy.
Authors: Mullinax John , Weber Jeffrey S. , Khushalani Nikhil I. , Eroglu Zeynep , Brohl Andrew Scott , Markowitz Joseph , Royster Erica , Richards Allison , Zager Jonathan S. , Sondak Vernon K. , Mule' James J , Pilon-Thomas Shari , Sarnaik Amod
Published: November 2019

147

Background: We previously confirmed the feasibility of adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) for patients with advanced melanoma. Patients successfully treated had a 38% response rate, but patient attrition due to progression before ACT (21%) resulted in a response rate of only 26% based on intent to treat (IIT) analysis. We hypothesized that combination immunotherapy would decrease attrition due to progression. Here we present the final report of a pilot trial combining ipilimumab (IPI) and ACT. Methods: Thirteen patients with metastatic melanoma were accrued to an IRB-approved trial. All had >1 cm3 of soft tissue/nodal metastases amenable to resection leaving residual disease. Patients received 3 mg/kg of IPI two weeks prior to tumor resection for TIL generation. One week following resection, a second dose of IPI was administered. ACT included pre-conditioning chemotherapy and TIL infusion followed by IL-2. Two additional doses of IPI were given 2 and 5 weeks after ACT. Feasibility was a primary endpoint and considered achieved if ≥ 2 doses of IPI and TIL were infused to at least 60% of all patients. The clinical responses were assessed by RECIST 1.1 criteria at 12 weeks following TIL transfer. Results: All patients received at least 2 doses of IPI, and 12 of the 13 patients (92%) received TIL. One patient (7%) dropped out due to progression before TIL infusion. TIL were expanded from 47.2% of tumor fragments and 58.7% of these fragments generated TIL reactive to autologous tumor. Median number of infused TIL was 6.5e10 (2.29e10-1.04e11) and median 85% were CD8+ (27-99). At 12 weeks following infusion, of the 13 patients enrolled, there were 6 responders (46%). Median progression-free survival was 7.4 months (1.4-42.2). Grade ≥3 immune-related adverse events included colitis (1), uveitis (1), and hypothyroidism (1). Conclusions: IPI combined with ACT with TILs for patients with advanced melanoma is feasible with decreased attrition due to progression and is associated with promising clinical results based upon ITT analysis. This combination approach of TIL cell therapy and co-inhibitory blockade serves as a model for future efforts to improve the efficacy of immunotherapy for patients with cancer. Clinical trial information: NCT01701674.
Antigen-dependent costimulation to improve T-cell therapy for cancer.
Authors: DeRenzo Christopher C. , Nguyen Phuong , Gottschalk Stephen
Published: November 2019

151

Background: T-cell therapy for cancer faces several challenges, including limited T-cell expansion at tumor sites, and lack of unique tumor antigens that are not expressed in normal tissues. To overcome the first obstacle, we developed Engager (ENG) T cells, which secrete bispecific molecules consisting of single chain variable fragments specific for CD3 and a tumor antigen. ENG T cells have the unique ability to redirect bystander T cells to tumors, amplifying antitumor effects. Costimulatory chimeric antigen receptors (CoCARs) are one potential strategy to restrict full T-cell activation to tumor sites that express a unique "antigen address." The goal of this project was now to generate T cells that express engager molecules and CoCARs (ENG/CoCAR T cells), which recognize distinct tumor antigens, and evaluate their effector function. Methods: We focused on two tumor antigens, EphA2 and HER2, which are expressed in a broad range of solid tumors. RD114-pseudotyped retroviral particles encoding an EphA2-ENG or a HER2-CoCAR were used to transduce CD3/CD28-activated human T cells. Transduced T cells were cocultured with EphA2+/HER2- or EphA2+/HER2+ tumor cells. Results: Both EphA2-ENG and EphA2-ENG/HER2-CoCAR T cells were activated by EphA2+ targets, as judged by IFNγ secretion. EphA2-ENG T cells secreted little IL-2 and died after one stimulation with EphA2+/HER2- or EphA2+/HER2+ tumor cells. In contrast, EphA2-ENG/HER2-CoCAR T cells secreted high levels of IL-2 and proliferated when stimulated with EphA2+/HER2+ cells. Little IL-2 secretion and no proliferation was observed after stimulation of the same T cells with EphA2+/HER2- cells, indicating these T cells are only fully activated in the presence of both target antigens. Upon repeated stimulation with EphA2+/HER2+ tumor cells, EphA2-ENG/HER2-CoCAR T cells continued to secrete IL-2 and proliferate without the addition of external cytokines for at least 10 weeks. Conclusions: EphA2-ENG/HER2-CoCAR T cells demonstrated robust dual antigen dependent IL-2 secretion, and continued proliferation upon repeat stimulation with EphA2+/HER2+ cells. Thus, providing antigen-specific costimulation is a potential strategy to improve the safety and efficacy of T-cell therapy for cancer.
Phase I study with pegylated human IL-10 (AM0010) alone or in combination with anti-PD-1 or FOLFOX-immune biomarker update.
Authors: Naing Aung , Infante Jeffrey R. , Hecht J. Randolph , Papadopoulos Kyriakos P. , Wong Deborah Jean Lee , Autio Karen A. , Falchook Gerald Steven , Patel Manish R. , Ott Patrick Alexander , Patnaik Amita , Bendell Johanna C. , Van Vlasselaer Peter , Hong David S. , Tannir Nizar M. , Oft Martin
Published: November 2019

157

Background: Persistence of activated CD8 T cells is essential for the durability of tumor immune responses. IL-10 is an anti-inflammatory cytokine, PEGylated IL-10 (AM0010) enhances tumor specific CD8 T cell expansion and cytotoxicity. We evaluated tolerability and efficacy of AM0010 alone, with chemotherapy or anti-PD-1 in a phase 1 study. AM0010 alone induces objective anti-tumor responses without auto-immune toxicities. AM0010 with anti-PD-1 was evaluated in RCC (n=8) or NSCLC (n=5) and showed improved response rates (ORR 50 and 40% respectively) compared to published ORR with anti-PD-1 monotherapy. AM0010 and FOLFOX for 2nd LOT pancreatic cancer increased the ORR compared to FOLFOX. Expansion cohorts for AM0010 and FOLFOX in pancreatic cancer (n=20) or with anti-PD-1 in RCC (n=30) or NSCLC (n=30) were evaluated. Methods: Observed tumor responses were monitored using irRC criteria. Immune responses were analysed for 96 serum cytokines. Activation of PBMC derived CD4 and CD8 T cells were analyzed by FACS. The expansion of the T cell repertoire was measured by deep sequencing of the TCR. Results: AM0010 induced systemic, sustained elevation of Th1 and Th2 type cytokines and cytotoxic products of CD8+ T cells. Th17 related cytokines were reduced. In-vitro, AM0010 inhibits activation induced cell death. Phosphorylated STAT3 (p-STAT3) was induced in activated CD8+ T cells in vitro and in tumors. In addition, PD1+ Lag-3+ KI-67+ CD8+ T cells expanded in the blood, remained Tim-3- CTLA-4-. Expansion of PD-1+ Lag-3+ CD8+ T cells correlate with objective tumor response. AM0010 treatment also resulted in the progressive systemic expansion of novel T cell clones, which were not detectable in the patient prior to treatment. This de-novo expansion coincided with tumor responses in several patients. The magnitude of this expansion correlated with the magnitude of objective tumor responses. The immune activation was similar in monotherapy and in the combination arms, suggesting a AM0010 specific and non-redundant mechanism of action. Conclusions: AM0010 has predominantly immune stimulatory activity in cancer patients, alone and in combination with other immune oncology therapies. Clinical trial information: NCT02009449.
Use of CMV gB/pp65 eVLPs to harness the immunogenicity of foreign viral antigens to target solid tumors.
Authors: Anderson David E , Ahmed Tanvir , Soare Catalina , Fluckiger Anne-Catherine , Ontsouka Barthelemy , Diress Abebaw , Bozic Jasminka , Yorke Matthew , Waziri Allen , Kirchmeier Marc , Diaz-Mitoma Francisco
Published: November 2019

165

Background: Human cytomegalovirus (CMV) is a ubiquitous, generally asymptomatic virus that is present in over 90% of GBM and breast cancer tumors which have comparatively low mutational burdens and predicted neoantigens relative to melanoma or lung cancer. Memory CD4+ and CD8+T cells are most frequently directed against the gB and pp65 antigens, respectively. Thus, CMV gB and pp65 represent highly immunogenic “foreign” antigen components of a vaccine against GBM and breast cancer. Methods: Enveloped virus-like particles (eVLPs) are produced after transfection of HEK 293 cells with a plasmid encoding murine leukemia virus Gag plasmid fused in-frame with CMV pp65 antigen, which gives rise to particles. Co-transfected CMV gB plasmid enables particles budding from the cell surface to incorporate the gB protein into the lipid bilayer. Surface expression of gB and internal expression of pp65 have been confirmed by CryoEM and immunogold labelling. Production and purification of multiple batches at a GMP-compliant manufacturer has demonstrated consistent purity and yields that meet regulatory requirements. Results: eVLPs restimulate IFN-g secreting CD4+ and CD8+ T cells in ex vivo PBMCs from healthy subjects (n=8) at mean frequencies of 0.27% and 1.28%, respectively. eVLP formulation with GM-CSF augments IFN-g and CCL3 secretion in stimulated PBMCs from healthy subjects (n=5), GBM patients (n=5), and breast cancer (n=1) patients to comparable levels. This vaccine candidate also induces de novo CD4+ and CD8+responses in mice. The vaccine induces high titer antibody responses against CMV gB, and the potential for ADCC activity against gB-expressing GBM tumor cells is currently being investigated. Conclusions: CMV gB/pp65 eVLPs address several shortcomings of past theraeputic vaccines, including: 1) the poor immunogenicity of "self" tumor-associated antigens, 2) failure to induce robust CD4+ responses that support CD8+ tumor-specific responses, 3) inclusion of only one or a few epitopes that rapidly encourage tumor cell immunoselection of variants that no longer express the target(s). An IND submission to FDA for a phase I/IIa trial in GBM patients is planned for H1 2017.
Trials in progress: A phase II study of in situ therapeutic vaccination against refractory solid cancers with intratumoral poly-ICLC.
Authors: Kyi Chrisann , Sabado Rachel Lubong , Saenger Yvonne M. , Loging William , Donovan Michael J. , Lehrer Deborah , Mandeli John P , Salazar Andres M. , Friedlander Philip Adam , Bhardwaj Nina
Published: November 2019

166

Background: Poly-ICLC, a RNA complex, can activate dendritic cells and trigger NK cells to kill tumor cells. It can be given intramuscularly (IM) to induce systemic inflammation and intratumorally (IT) to induce immune infiltration of tumors. Methods: In this phase II study, eligible subjects are head and neck, and skin cancer patients with refractory or metastatic disease. In each treatment cycle, one accessible tumor site was targeted for IT injection of 1 mg of Poly-ICLC 3 times a week for 2 weeks followed by IM boosters biweekly for 6 weeks and then a 2 week rest period. Tumor biopsies were performed at baseline, week 3, and week 26. Pre- and post-vaccination tumors were evaluated by quantitative immunohistochemistry (IHC) and RNA sequencing. Blood samples were collected at baseline and prior to each cycle for immune response evaluations. Results: A phase I pilot portion of this study showed 1 patient who achieved clinical benefit with stable disease (progression-free survival of 6 months). Poly-ICLC was well tolerated with principal side effects of fatigue and inflammation at injection site (< grade 2). One case of grade 3 tumor necrosis was observed. In the patient with clinical benefit, IHC analysis of tumor showed increased CD4(60x), CD8(10x), PD-1(20x), and PD-L1(3x) compared to patients with progressive disease whose tumor biopsies showed unchanged/decreased CD4, CD8, PD-1, and PD-L1 levels over treatment period. Furthermore, RNASeq analysis of the same patient’s tumor and peripheral blood mononuclear cells (PBMC) showed dramatic changes such as upregulation of interferon-stimulated genes, chemokines, and genes associated with T cell activation and antigen presentation indicative of local and systemic immune activation in response to Poly-ICLC treatment. Conclusions: Preliminary findings show that Poly-ICLC is well tolerated in advanced solid cancer patients, and generates local immune response in tumor microenvironment and systemic immune response as evident in the patient achieving clinical benefit. These results warrant further investigation, and are currently being explored in this ongoing larger multicenter adaptive phase II clinical trial. Clinical trial information: NCT01984892.
IgG response against prostate-related antigen as potential biomarker for personalized peptide vaccination in metastatic recurrent breast cancer.
Authors: Saku Shuko , Toh Uhi , Okabe Mina , Iwakuma Nobutaka , Shichijo Shigeki , Yamada Akira , Itoh Kyogo , Akagi Yoshito
Published: November 2019

167

Background: The aim of this study is to identify the prognostic role of serum value of IgG antibody against prostate related-antigen (PRA), including prostate-specific antigen (PSA), prostate specific membrane antigen (PSMA), and prostate acid phosphatase (PAP) in mrBC patients. Methods: Peripheral blood samples of 77 patients with mrBC were analyzed for serum anti-PRA IgG levels before and after 6th and 12th PPV therapy prospectively. Most of the peptides used for PPV are derived from cancer associated antigens expressing in various types of advanced cancers, but the peptides derived from PRAs were not used in this study. Results: After PPV therapy, total serum levels of anti-PRA IgG were significantly increased in 31 mrBC patients (Group 1) compared with remaining 46 rmBC patients (Group 2). Either serum anti-PSA, anti-PAP, and/or anti-PMSA IgGs showed a significant increase in patients of Group 1 after 6th (p=0.045) and 12th PPV treatment (p < 0.001), irrespective of their intrinsic subtypes. The median PFS and median OS of Group 1 patients were 8.1 and 14.3 months, while those of Group 2 patients were 5.1 and 10.8 months, respectively. Anti-PRA IgG levels were significantly associated with PFS (p=0.0073; HR: 0.37) and OS (p=0.025; HR: 0.43) between these two groups, whereas no significant relation was found with age, clinical response rate, and recurrent metastatic status. Conclusions: The Group 1 patients with elevated anti-PRA IgG may have better prognosis compared to Group 2 patients who showed no IgG elevation after PPV treatment for rmBC. These results indicated a clinical significance between pre-and post- treatment measurement of serum anti-PRA IgGs in mrBC patients receiving PPV therapy, and may be a useful prognostic marker for monitoring the outcome to PPV treatment of breast cancer.
Immune profiling of circulating T cells and TILs following neoadjuvant ipilimumab and chemotherapy in non-small cell lung cancer (NSCLC).
Authors: Yi John , Clarke Jeffrey Melson , Healy Patrick , Wang Xiaofei F. , Shoemaker Debra , Onaitis Mark W. , Dumbauld Chelsae , Osborne Robyn , Crawford Jeffrey , Harpole David , D'Amico Thomas A. , Dunphy Frank , Christensen Jared , Ready Neal , Weinhold Kent
Published: November 2019

26

Background: Ipilimumab (Ipi) is a humanized CTLA-4 antibody that blocks binding of CTLA-4 to B7, permitting T cell activation through CD28. Phased in Ipi added to chemotherapy (C) may enhance efficacy in NSCLC. Methods: Patients with stage 2 or 3A NSCLC received neoadjuvant carboplatin AUC6 plus paclitaxel 200 mg/m2 every 21 days for 3 cycles and Ipi (10 mg/kg) was given on day 1 for cycles 2 and 3. Blood for immune profiling of circulating T cells was collected at baseline, after chemotherapy alone, and after chemotherapy plus Ipi. Tumor infiltrating lymphocytes (TIL) were derived from 7 available tumors. Polychromatic flow cytometry (PFC) analyses were performed on peripheral blood mononuclear cells (PBMC) and TIL. Objective response rates were assessed according to RECIST 1.1 criteria. Results: Of the 24 patients enrolled on this study, objective responses after 3 cycles of neoadjuvant C plus ipi included 2 PD, 8 SD, and 14 PR. Phenotypic analyses revealed that PBMC from all 24 patients were highly activated following two cycles of Ipi (cycle 3) as evidenced by significantly increased frequencies of CD28, ICOS, HLA-DR, PD-1, and CTLA-4 expressing CD4+ cells; and ICOS, HLA-DR, and CTLA-4 expressing CD8+ cells. The frequencies of Tregs were highly variable among the 24 participants. Two of the 24 participants had levels of MDSC cells above 15%. TIL contained far greater frequencies of activated CD4+ and CD8+ cells than found in the PBMC at cycle 3. Tumor associated antigen (TAA)-specific CD4+ or CD8+ cells were detected at baseline in 4 patients (24%), but their relative frequencies remained unaltered by Ipi therapy. No patients developed detectable de novo TAA reactivities while on Ipi therapy. Conclusions: Combined neoadjuvant Ipi plus chemotherapy produced significantly increased frequencies of highly activated CD4+ and CD8+ populations in the peripheral blood and the tumor microenvironment. TAA-specific CD4+ or CD8+ cells were detected in PBMC at baseline in a subset of patients. No TAA-reactive T cells were detected among the 7 TIL samples analyzed. Analysis for predictive or pharmacodynamic biomarkers is ongoing. Clinical trial information: NCT01820754.
Clinical and immunologic variables associated with outcomes following combination ipilimumab and nivolumab immunotherapy in melanoma.
Authors: Rosner Samuel , Kwong Erica , Panageas Katherine , Postow Michael Andrew
Published: November 2019

55

Background: Prior studies have shown routine laboratory parameters such as lymphocytes (lymphs) and eosinophils (eos) are associated with outcome in patients (pts) treated with ipilimumab (ipi) or programmed death-1 (PD-1) agents as single drugs. Less is known about these parameters as potential biomarkers for pts treated with ipi + nivolumab (nivo) in combination. Methods: After IRB approval, a single institution, retrospective review was performed of pts with melanoma who received ipi + nivo on phase I-III clinical trials (n=122) as well as via commercial use (n=87). Prior to treatment initiation, routine laboratory parameters were investigated and correlated with overall survival (OS) and disease response by RECIST 1.1 (partial or complete response vs. stable/progressive disease). Absolute as well as relative frequencies of cell counts were examined. Kaplan-Meier estimators and Cox regression were performed. Results: 209 pts, 85 (41%) women and 124 (59%) men, with a median age of 60.5 years were analyzed. Median OS was 44.4 months, 95% CI (32.9, not reached). 23 pts had a complete response (CR) and 87 pts had a partial response (PR), while 39 pts had stable disease (SD) and 49 pts had progressive disease (PD). By univariate analysis, significant favorable factors for OS included: low absolute monocytes, low absolute/relative neutrophils, high relative eos, and high relative lymphs. Low LDH and the neutrophil to lymph ratio were also associated with better OS. None of these factors were significantly associated with response by RECIST 1.1. Conclusions: Routinely assessed laboratory parameters were associated with OS but not disease response among pts receiving ipi + nivo. Some of these factors were similar to those which previously correlated with OS after ipi or nivo as single-agents, suggesting they may be prognostic. Given the ongoing randomized phase III study of ipi + nivo and nivo vs. ipi, there is an opportunity to examine whether these factors are relevant in refining which pts obtain the greatest benefit from ipi + nivo vs. single agent therapy.
Prognostic value of PD-L1+CD4+ T cells in non-small cell lung cancer (NSCLC) patients treated with a PD-1 inhibitor.
Authors: Vetsika Kiriaki E. , Aggouraki Despoina , Lyristi Zacharoula , Koinis Filippos , Georgoulias Vassilis , Kotsakis Athanasios
Published: November 2019

58

Background: One of the tumour escape mechanisms is the expression of programmed cell death-1 (PD-1) receptor on immune cells. Clinical trials have demonstrated that antibodies against PD-L1 or PD-1 molecules induce objective clinical responses in pre-treated patients with NSCLC. In this study, we evaluated the levels of PD-1+ and PD-L1+expressing- immune cells and their association with the clinical outcome in NSCLC patients. Methods: Peripheral blood was collected from 32 advanced/metastatic pre-treated NSCLC patients before anti-PD-1 therapy (Nivolumab). Flow cytometry was performed to identify and quantify PD-1 and PD-L1 expression on circulating immune cells with anti-tumor function (CD4+ and CD8+ T cells, B cells, Dendritic cells) , as well as on immunosuppressivecells [(CD4+ and CD8+ Treg cells, Bregs, Myeloid-derived Suppressor cells (MDSC)]. Correlation between the levels of PD-1+ and PD-L1+-expressing immune cells, as well as their association with overall (OS) and progression-free survival (PFS) was evaluated; high expression of immune cells was defined as the percentage of the cells above the mean value. Results: PD-1 and PD-L1 expression was detected on all tested immune cells with anti-tumor activity. PD-1 expression was found on all immunosuppressive cells, whereas only CD4+ and CD8+ Treg cells and Granulocytic -MDSCs expressed PD-L1. No PD-L1 expression was observed on Bregs and monocytic-MDSCs. There was no correlation of PD-1 or PD-L1-expressing cells with response to anti-PD1 treatment. A significantly decreased PFS was observed in the low CD4+ PD-L1+ T cells group compared with the high CD4+ PD-L1+ group (3.4 months vs. 5.2 months, p=0.03). Univariate analysis for PFS revealed that the low percentages of CD4+ PDL-1+ T cells were prognostic for shorter PFS (HR= 3.1, p=0.04). On the multivariate analysis, low PD-L1+ CD4+T cells were independently associated with decreased PFS (HR: 3.3, p=0.03). Conclusions: These data provide evidence that PD-L1+ expression on peripheral CD4+ T cells was a significant predictor of better PFS in NSCLC patients. Therefore, PD-L1 expression could be used for the selection of patients who might benefit from PD-1/PD-L1 inhibitors.
Correlation of consistent blood-based gene expression with change in CTLA4 in two large independent clinical studies of patients with advanced melanoma treated with tremelimumab.
Authors: Friedlander Philip Adam , Wassman Karl , Kyi Chrisann , Oh William K. , Bhardwaj Nina
Published: November 2019

6

Background: We previously reported 16-gene pre- and 8-gene post-treatment response predictive gene signatures in whole blood that were trained in one cohort (N=210) of advanced melanoma patients treated with tremelimumab and validated in an independent test dataset (N=150) for both response and survival.(SITC 2016 Annual Meeting Poster Submission ID: 214348.) Methods: A correlation matrix was calculated for the change between pre- and post-treatment gene expression for all 169 genes in two independent clinical trials. The genes were ranked by their relationship with CTLA4. Comparison was made between the training and test datasets. Reliable biological signals exceeded technical variance of +/- 0.5 fold change. Results: Correlation of the change in gene expression relative to CTLA4 between the 169 genes in two independent datasets was consistent as to fold change and ranking. The literature describes the genes in the Table as playing a role in anti-CTLA4 and PD-1 immunotherapy. We found only ICOS to be predictive of gene response in our 16-gene pre- and 8-gene post-treatment algorithms. Conclusions: Several genes, such as CTLA4, CD28, CXCR3, IL32, FOXP3, CD25, CCR5, and IFNG, are consistently upregulated one to three fold from anti-CTLA4 immunotherapy but are not predictive of response. ClinicalTrials.gov NCT00257205. [Table: see text]
Effect of paclitaxel and carboplatin on tumor-reactive T cells and the efficacy of PD-1 blockade.
Authors: Yan Yiyi , Dronca Roxana Stefania , Liu Xin , Markovic Svetomir , Dong Haidong
Published: November 2019

65

Background: The addition of chemotherapy to immune checkpoint inhibitors has been explored clinically in solid tumors in order to overcome one of the challenges of immunotherapy: clinical outcomes are variable, with only a subset of patients achieving durable responses. Further understanding of the immunoregulatory mechanisms of chemotherapy in the setting of immunotherapy is crucial for the development of optimal combinatorial chemo-immunotherapeutic strategies to improve clinical outcomes in patients with advanced cancer. Methods: Metastatic melanoma (MM) patients who failed anti-PD-1 single therapy were subsequently treated with paclitaxel and carboplatin in addition to PD-1 blockade. Using peripheral blood (PB) from MM patients and healthy donor, the phenotypic and functional changes in T cells induced by chemotherapy, in the setting of PD-1 blockade, were examined. Results: Paclitaxel and carboplatin, in combination with PD-1 blockade, were able to induce significant clinical responses in a subset of MM patients who were resistant to anti-PD-1 therapy alone. In patients who responded to this combination, a subset of tumor-reactive effector T cells (CD8+CD11a+PD1+GranzymeB+) survived the treatment, with increased frequency after the addition of chemotherapy. This T cell population preserved its effector phenotype, although ongoing PD-1 blockade was needed to improve its anti-tumor activity. In vitro experiments, using CD8+CD11a+PD1+GranzymeB+isolated from healthy donor PB, also demonstrated its ability to survive chemotherapy treatment. In addition, after cultured with chemotherapy, this T cell subset demonstrated higher cytotoxic T cell functions as well as increased chemotherapy efflux. Conclusions: Chemotherapy regulates a subset of tumor-reactive effector T cells (CD8+CD11a+PD1+GranzymeB+) and increases clinical efficacy of PD-1 blockade. This novel effector T cell population underlies the key cellular and molecular immunoregulatory mechanisms of chemotherapy. It serves as a meaningful marker to measure these collaborative effects and to develop the optimal chemo-immunotherapy strategy to improve clinical responses to current immune checkpoint blocking agents.
Analytical validation of an immune response assay for classifying solid tumors.
Authors: Conroy Jeff , Glenn Sean , Burgher Blake , Papanicolau-Sengos Antonios , Andreas Jonathan , Giamo Vincent , Odunsi Kunle , Ernstoff Marc S. , Eng Kevin H. , He Ji , Gardner Mark , Morrison Carl
Published: November 2019

68

Background: An advanced diagnostic laboratory test (Immune Advance) was developed that analyzes multiple DNA and RNA biomarkers to predict the likelihood of response to checkpoint inhibitors in patients with solid tumors. Using RNA-seq and DNA-seq, the NGS test measures gene expression of immune response genes and overall mutational burden. The studies described here were designed to validate the analytical performance of the test on the Ion Torrent System in our CLIA lab. Methods: Studies were designed to characterize the analytical performance of an immune response NGS assay using total nucleic acids from >100 FFPE tissues representing NSCLC, melanoma, renal cell carcinoma, and bladder cancer. Performance variables with respect to gene-specific amplicon specificity, linearity, and limits of detection were estimated with various sample dilutions and input nucleic acids. The effects of the tumor micro-environment (adjacent benign tissue, necrosis) was evaluated by including these potential interferents in the assay. Analytical precision including intra-assay, inter-assay, and inter-operator reproducibility was measured by testing replicate FFPE tissue sections. Accuracy was determined by comparing select transcript and DNA level variants with those from established IHC, RT-PCR, and NGS assays. Transcript stability in FFPE specimens was evaluated in serial sections from blocks with routine storage and compared to originating matched fresh frozen specimens. Results: RNA stability was demonstrated by high degree of correlation between matched frozen and FFPE samples. Analytic precision was demonstrated by high correlation between RNA-Seq and TaqMan results for genes evaluated. As compared to IHC the results for RNA-Seq were continuous rather than bimodal and allowed for a much more detailed analysis of the immune response. Immune signatures were maintained with variable RNA/DNA input amounts, altered tumor micro-environments, and potential interferents demonstrating tolerance to typical sample types tested. Reproducibility results show little variation between runs and operators. Conclusions: The analytical performance of the Immune Advance assay has been validated for clinical use using FFPE tumor specimens.
Immunotoxin and bcl-2 inhibitor combination therapy targeting chondroitin sulfate proteoglycan 4.
Authors: Yu Xin , Keir Stephen T. , Szafranski Scott , Clayton Steven , Pastan Ira , Bigner Darell D. , Chandramohan Vidyalakshmi
Published: November 2019

74

Background: Immunotoxins (ITs) are a class of bifunctional chimeric proteins composed of an antibody fragment linked to a toxin. When ITs internalize into target cells, they induce protein synthesis inhibition and apoptosis. While ITs are highly specific and potent, the efficacy of IT-based therapies in some tumor cells is limited by hyperactive anti-apoptotic pathways and inefficient translocation of ITs from the endoplasmic reticulum to the cytosol. Therefore, to improve the efficacy of IT-based therapies, we evaluated a dual-pathway therapy that combines an IT with the ABT-737, ABT-263, or ABT-199 small molecule Bcl-2 inhibitor. Methods: The immunotoxin 9.2.27-PE38KDEL (9.2.27-IT) was generated by fusing a truncated mutant form of Pseudomonas exotoxin A to a single-chain variable fragment antibody. It targets human chondroitin sulfate proteoglycan 4 (CSPG4), an antigen highly expressed in a variety of cancer cells. We screened and identified 3 human glioblastoma xenografts, 3 human melanoma cell lines, and 5 human breast cancer cell lines resistant to the 9.2.27-IT despite their high levels of cell surface expression of CSPG4 (IC50 of IT alone was >100 ng/ml in all cell lines except for one melanoma cell line). In vitro cytotoxicity of the 9.2.27-IT —alone or in combination with the individual Bcl-2 inhibitors ABT-737, ABT-263, or ABT199—was assessed. Concentrations of ABT analogues were chosen so that ABT alone did not induce cytotoxicity. Results: The treatment groups that responded to the combination therapy yielded IC50 values ranging from 0.04 – 9 ng/ml for glioblastoma xenografts, 0.21-15 ng/ml for melanoma cell lines, and 4.5-50 ng/ml for breast cancer cell lines. The most potent combination group showed >1000 fold improvement of IC50 compared to using the immunotoxin alone. ABT-737 produced the strongest synergistic effects among the ABT analogues. Preliminary results from in vivo studies further demonstrated that this approach engendered a synergistic response and delayed tumor growth in immunotoxin-resistant mouse tumor models. Conclusions: This new combinatorial approach will potentially help to overcome immunotoxin resistance in cancer patients and provide better therapeutic outcomes.
Immune monitoring outcomes of patients with stage III/IV melanoma treated with a combination of pembrolizumab and intratumoral plasmid interleukin 12 (pIL-12).
Authors: Algazi Alain Patrick , Tsai Katy K. , Rosenblum Michael , Fox Bernard A. , Andtbacka Robert Hans Ingemar , Li Amy , Takamura Kathryn Toshimi , Dwyer Mary , Browning Erica , Talia Reneta , Twitty Chris , Le Mai H. , Gargosky Sharron , Campbell Jean S. , Ballesteros-Merino Carmen , Bifulco Carlo Bruno , Pierce Robert , Daud Adil
Published: November 2019

78

Background: Recent publications support the emergence of predictive biomarkers for pembrolizomab in melanoma based on the expression of PD-L1 in the tumor microenvironment (Daud 2016a) or the frequency of PD-1hiCTLA-4hi on CD8+ TIL (Daud 2016b) whereby immunologically inactive tumors show poor response to immune checkpoint inhibition alone. Since intratumoral pIL-12 with electroporation (IT-pIL12-EP) increases TIL in both treated and untreated lesions, we hypothesize that non-response can be rescued with the combination of IT-pIL12-EP and anti-PD-1. Phase II clinical and immunological data are shown. Methods: Melanoma stage III/IV patients were enrolled with CD8+ TIL < 25% PD-1hiCTLA-4+measured by flow cytometry (NCT02493361). PD-L1 IHC (22C3 Ab) was also evaluated. Patients were treated with pembrolizumab (200mg every 3 weeks) and IT-pIL12-EP. Patients were evaluated for ORR every 12 weeks (RECISTv1.1). Pre- and post-treatment blood and tumor specimens were collected, and analyzed for immune phenotyping, gene expression, T cell receptor diversity, and changes in the tumor microenvironment by IHC. Results: TIL assessment for evaluable enrolled patients were < 22% PD-1hiCTLA-4+, a value associated with anti-PD-1 non-response (Daud 2016b). Remarkably, although predicted to be an unresponsive population, the ORR was 40% (4CR/ 2PR) by RECISTv1.1. In patients with CR/PR, analysis of the tumor microenvironment revealed many significant immunological changes not seen in non-responders, including number and ratios of CD8+:PD-L1+by IHC, increased expression of NK, CD8 and ‘adaptive resistance’ markers by NanoString, as well as increased clonality and T cells by TCR sequencing. Conclusions: The excellent safety profile and striking 40% clinical response rate is encouraging as treated patients were predicted to be non-responsive to pembrolizumab. The correlative data shows an immune-directed mechanism that is differentiated between responders and non-responders suggesting the combination can effectively alter the tumor microenviroment to benefit patients otherwise unlikely to respond to anti-PD-1 monotherapy. Clinical trial information: NCT02493361.
Extended survival analysis of ipilimumab for the treatment of advanced malignant melanoma in pretreated patients: Five-year long-term follow-up of the South African expanded access program.
Authors: Rapoport Bernardo Leon , Vorobiof Daniel A. , Dreosti Lydia Mary , Nosworthy Adam L. , McAdam Georgina Laird , Jordaan Johann Petrus , Miller-Janson Helen , de Beer Janetta Catharina , de Necker Margreet
Published: November 2019

84

Background: Ipilimumab is a human monoclonal IgG1 antibody against CTLA-4 that has been shown to prolong the overall survival of patients with advanced pretreated melanoma. In 2015, a retrospective, multi-centre, non-interventional analysis was performed on data collected from the ipilimumab expanded access programme in South Africa, with last follow-up date (or death) in December 2014. The current study extends this analysis by follow-up on the long-term survival of pre-treated metastatic patients up to September 2016. Methods: Follow-up questions were sent to participating investigators, who had patients who were still alive (29) or for whom it was not known whether they were still alive (11) following the last ipilimumab infusion. Investigators had to confirm whether patients were still alive, the date of death or last contact, clinical response at last contact, and whether the patient was still responding to ipilimumab. Results: Of the 108 patients, 84 (78%) had cutaneous melanoma and 24 patients (22%) had non-cutaneous melanoma, including uveal, mucosal, and melanoma of unknown primary. Twenty patients previously received two or more lines of treatment for metastatic melanoma. The median age was 59 years (range 27 – 86) and there were 73 (68%) males and 35 (32%) females. Baseline ECOG PS was 0 in 33%, PS 1 in 58% and PS 2 in 6% of patients. The longest follow-up time available was 5.4 years. The median OS was 9.36 months (95% CI 7.48 – 11.84). One-year survival was 39% (95% CI 29% - 48%), 2-year survival was 22% (95% CI 15% - 30%), 3-year survival was 19% (95% CI 12% - 27%), 4- and 5-year survival was 15% (95% CI 8% - 21%). In the group of cutaneous melanoma patients, the 4- and 5-year survival was 17% (95% CI 9% - 25%) while in the non-cutaneous group the 4- and 5-year survival was 6% (95% CI 0% - 16%). Conclusions: Ipilimumab at a dose of 3mg/kg is an effective treatment for patients with pre-treated advanced (unresectable or metastatic) melanoma and is associated with durable remissions and long-term survival.
Perspectives from the community setting: A retrospective chart review of nivolumab for previously treated non-small cell lung cancer.
Authors: Nhan Brian Richard , Kulkarni Swati
Published: November 2019

86

Background: Lung cancer is among the most commonly diagnosed cancers in Canada and is the leading cause of cancer deaths. Non-small cell lung cancer (NSCLC) is one of the most common types representing 85-90% of cases. Compared to other cancers, the five-year survival rate for lung cancer in Canada is among the lowest at 17%. Nivolumab has been in use in the setting of progression of previously treated NSCLC at the Windsor Regional Cancer Center since June 2015. This retrospective chart review abstracts data from 26 patients at WRCC with NSCLC who were treated with nivolumab. Methods: A retrospective chart review of 26 patients treated with nivolumab in the setting of progressive NSCLC. Results: 26 patients were reviewed (1 stage 2a, 2 stage 3a, 3 stage 3b, 20 stage 4 with 12 M1b and 8 M1a). Grade 1-2 side effects were documented in 4 patients treated with monitoring and no discontinuation or dose modification of nivolumab. A grade 3 side effect was documented in 1 patient leading to discontinuation of treatment. 3 living patients discontinued treatment due to progression. 6 patients died while on treatment secondary to disease progression. The remaining patients are on nivolumab with no side effects documented. Conclusions: As the use of immuno-oncology agents use expand in community clinics, opportunities arise from community data to further learn which patients benefit from these treatments, what side effects can occur and how to manage them, and how response to treatment may look. Out of the 26 patients receiving nivolumab, 5 patients had documented side effects resulting in discontinuation of treatment in 1 patient. Overall nivolumab has been well tolerated in patients with NSCLC in this community setting.
Preliminary results from a phase II study of nivolumab for patients with metastatic adrenocortical carcinoma (ACC).
Authors: Cavalcante Ludimila , Carneiro Benedito A. , Costa Ricardo Lima Barros , Chae Young Kwang , Rademaker Alfred , Giles Francis J.
Published: November 2019

96

Background: ACC is an exceedingly rare malignancy with an estimated incidence of 0.7 cases per million per year. Patients presenting with metastatic disease have an estimated 5-year survival rate of < 15%, without effective standard treatment options. Immunotherapy approaches such as checkpoint inhibitors have emerged as effective therapies for multiple malignancies. Nivolumab, a monoclonal IgG4 antibody against the programmed death-1 (PD-1) receptor on T-cells, acts by inhibiting the interaction with PD-L1 and PD-L2 and thus eliciting an increased anti-tumor immune response. This phase II study will assess the efficacy of nivolumab monotherapy according to objective response rate (ORR) in patients with advanced ACC – the study is currently in the patient enrollment phase. Methods: Patients with metastatic or locally advanced ACC with disease progression after treatment with ≥ 1 line of therapy (including mitotane and/or chemotherapy), or not eligible for first-line chemotherapy are currently being enrolled on this phase II study. Nivolumab is given at a fixed dose of 240mg IV q2 weeks until confirmed disease progression, unacceptable toxicity, or withdrawal. Planned accrual of up to 33 patients will follow a Simon two stage design with interim analysis for efficacy after 10 evaluable patients are enrolled. Results: This is an initial report of the first 7 evaluable patients enrolled on study. Median age 57 years; female (4/7); endocrine hyperfunction (2/7); prior chemotherapy with EDP +/- mitotane (5/7). Patients were treated with a median of 4 doses of nivolumab. Median time to progression of 8 weeks. Best overall response rate thus far has been progression of disease in 5 pts, with 2 pts pending evaluation. Grade 3 or 4 adverse events (AEs) at least possibly related to nivolumab: tremor, hypokalemia (1 pt each). Reported grade 1 and 2 AEs: LFT elevation (3 pts), lower extremity edema (2 pts), lymphopenia, urinary frequency and fatigue (1 pt each). Conclusions: Nivolumab has been well tolerated in this population of patients with metastatic ACC. Clinical trial information: NCT02720484.
Effect of exogenous interferon-gamma (IFN-gamma) on peripheral blood immune markers as part of a phase I clinical trial of combined IFN-gamma with nivolumab (Nivo) in patients (pts) with select solid tumors.
Authors: Zibelman Matthew R. , Macfarlane Alexander , Alpaugh R. Katherine , Dulaimi Essel , Costello Kim , O'Neill John , Borghaei Hossein , Denlinger Crystal Shereen , Geynisman Daniel M. , Dotan Efrat , Jain Angela , Martin Lainie P. , Obeid Elias , Nieves Jeffery A. , Crouteau David , Grahn Amy Y. , Campbell Kerry , Plimack Elizabeth R.
Published: November 2019

97

Background: IFN-gamma is a known activator of PD-L1 expression and plays a key role in immune activation. We present initial correlative peripheral blood findings after the first 2 cohorts of pts enrolled in a phase I trial of combined IFN-gamma/Nivo in pts with various solid tumors. Methods: Pts with advanced solid tumors who had progressed after at least 1 prior therapy were recruited. An induction phase of IFN-gamma (50 or 75 mcg/m2 subq every other day for 1 week) preceded the combination of Nivo (3 mg/kg IV every 2 weeks) with continuation of IFN-gamma at same dose and schedule for 3 months. Pts with clinical benefit could remain on Nivo alone for up to 1 year. Peripheral blood was analyzed via flow cytometry at baseline, after IFN-gamma induction (prior to Nivo start), and after 6 weeks of combo treatment. Results: 13 pts have been accrued. Pre and post induction samples were collected on all pts. Nine pts had available on-treatment samples at time of data analysis. Comparing blood collections from baseline to post-IFN-gamma induction revealed a substantial expansion of the CD14high, CD16+ monocyte population (p=0.001) and a statistically significant increase in MHC class II expression on all monocyte populations (p≤ 0.002 for all types). Natural killer (NK) cells demonstrated greater numbers of cells per ml of blood (p=0.03) and increased NKp30 expression, an important receptor mediating tumor cell lysis (p= 0.005 for CD56dim; p= 0.03 for CD56bright). With the addition of Nivo, evidence for activation of CD56dim NK cells was detected as increased CD69 expression compared to post-induction (p=0.02). PD-1 expression on T-cells showed reduced expression in most comparators after initiation of Nivo (p=0.055 CD8+; p=0.008 CD4+). Conclusions: Early correlative peripheral blood data demonstrate expected systemic IFN-gamma effect as measured by monocyte activation and increased MHC II expression. Evidence of NK cell activation and proliferation was also observed. Decreased PD-1 expression on T-cells may reflect down-regulation in response to PD-1 blockade. Correlations with biopsy specimens and clinical response are planned. Clinical trial information: NCT02614456.
Mutations in LAMB2 Are Associated With Albuminuria and Optic Nerve Hypoplasia With Hypopituitarism
Authors: Tahoun Mona , Chandler Jennifer C , Ashton Emma , Haston Scott , Hannan Athia , Kim Ji Soo , D’Arco Felipe , Bockenhauer D , Anderson G , Lin Meei-Hua , Marzouk Salah , Saied Marwa H , Miner Jeffrey H , Dattani Mehul T , Waters Aoife M
Published: November 2019

Context

Mutations in LAMB2, encoding the basement membrane protein, laminin β2, are associated with an autosomal recessive disorder characterized by congenital nephrotic syndrome, ocular abnormalities, and neurodevelopmental delay (Pierson syndrome).

Case description

This report describes a 12-year-old boy with short stature, visual impairment, and developmental delay who presented with macroscopic hematuria and albuminuria. He had isolated growth hormone deficiency, optic nerve hypoplasia, and a small anterior pituitary with corpus callosum dysgenesis on his cranial magnetic resonance imaging, thereby supporting a diagnosis of optic nerve hypoplasia syndrome. Renal histopathology revealed focal segmental glomerulosclerosis. Using next-generation sequencing on a targeted gene panel for steroid-resistant nephrotic syndrome, compound heterozygous missense mutations were identified in LAMB2 (c.737G>A p.Arg246Gln, c.3982G>C p.Gly1328Arg). Immunohistochemical analysis revealed reduced glomerular laminin β2 expression compared to control kidney and a thin basement membrane on electron microscopy. Laminin β2 is expressed during pituitary development and Lamb2–/– mice exhibit stunted growth, abnormal neural retinae, and here we show, abnormal parenchyma of the anterior pituitary gland.

Conclusion

We propose that patients with genetically undefined optic nerve hypoplasia syndrome should be screened for albuminuria and, if present, screened for mutations in LAMB2.
Implementation of a Preoperative Anemia Clinic Utilizing a Minimal Staffing Model
Authors: Mabry Christian , Perelman Seth , Kim Jung T. , Blitz Jeanna D.
Published: November 2019

We present a process map for the implementation of a program to treat preoperative anemia utilizing 1 existing anesthesiologist in the preoperative evaluation clinic. In the first 7 months postimplementation, 342 patients were screened for anemia, 166 were diagnosed, and 107 were treated. The mean increase in hemoglobin in treated patients was ~2 g/dL (range 0–4.9 g/dL). Two patients’ surgeries were delayed in favor of treatment and 3 surgical patients, who had received 2 complete iron infusions, received an intraoperative transfusion. The total revenue generated for the institution was enough to subsidize the cost of an additional anesthesiologist.
Intraoperative Multipoint Acupuncture for Reducing Postoperative Nausea and Vomiting in High-Risk Children: A Case Series
Authors: Ralston-Wilson Jaime A. , Karlik Joelle B.
Published: November 2019

Acupuncture studies have demonstrated varying effects on pediatric postoperative nausea and vomiting (PONV). Unanswered questions include whether the type of therapy, number of points used, or timing of treatments vary the effect of acupuncture. We present a case series of intraoperative multipoint acupuncture treatments for high-risk pediatric patients. Fourteen patients were included, and 19 treatments were provided. Twelve patients (85.7%) had previous PONV. Patients who received intraoperative acupunctur e reported no early phase PONV, and 3 patients (15.8%) reported late-phase PONV. One patient required postoperative antiemetics. Intraoperative multipoint acupuncture may be a safe and efficacious adjunct for PONV in high-risk pediatric patients.
Ventilation Failure due to Endotracheal Tube Migration Into a Tracheal Pouch in a Toddler With a Repaired Tracheoesophageal Fistula: A Case Report
Authors: Mathew Preethy J. , Ashok Vighnesh , Malik Muneer Abas
Published: November 2019

A tracheal pouch is a rare complication of successful repair of a congenital tracheoesophageal fistula (TEF). An 18-month-old child with a repaired congenital TEF was scheduled for esophageal dilation to treat his esophageal stricture. Migration of the distal end of the endotracheal tube into a previously undetected tracheal pouch caused an abrupt failure to ventilate at the end of surgery. Given our experience, we recommend to screen the trachea of every patient with corrected TEF for a tracheal pouch when they are scheduled for another procedure requiring general anesthesia.