bluuebunny/crossref_metadata_2025 huggingface dataset:
Survival of neuroblastoma patients treated by long-term infusion of anti-GD2 antibody ch14.18/CHO and killer-cell Ig-like receptor (KIR) genotypes and Fcγ-receptor polymorphisms.
Authors: Lode Holger N. , Loibner Hans , Troschke-Meurer Sascha , Valteau Couanet Dominique , Garaventa Alberto , Gray Juliet , Castel Victoria , Yaniv Isaac , Siebert Nikolai , Eger Christin , Juettner Madlen , Kietz Silke , Ehlert Karoline , Janzek Evelyne , Mueller Ina , Ladenstein Ruth
Published: November 2019

111

Background: Receptors expressed on natural killer (NK) cells and their ligands on target cells involved in antibody-dependent cell-mediated cytotoxicity (ADCC) may influence outcome in patients (pts) with high-risk relapsed/refractory neuroblastoma (NB) treated by long-term infusion (LTI) of anti-GD2 antibody ch14.18/CHO Methods: 53 pts received 6x106 IU/m2 sc IL-2 (d1-5; 8-12), LTI of 100 mg/m2 ch14.18/CHO (d8-17) and 160 mg/m2 oral 13-cis-RA (d19-32) in a closed single center program (53 pts) (APN311-303). Polymorphisms in Fcγ-receptor genes 2A (H131R), -3A (V158F) and -3B (NA1/NA2), and KIR- as well as KIR ligand expression (HLA-C1; HLA-C2; HLA-Bw4+) were determined by real-time PCR. Results: The survival update of the APN311-303 cohort revealed a 5-y OS of 56.4±7.1% (mean OS 4.35y [0.3-6.2y]) and a 5-y PFS of 29.1±6.3% (mean PFS 2.4y [0.1 - 5.9y]). Median TTP/PFS was 1.35 y (95% CI: 0.21-2.48 y). This result is clearly superior to historical controls not treated with ch14.18/CHO with a 5-y OS of 14.8±6.8% (p < 0.005), indicating clinical efficacy of the treatment. In this cohort, we identified 26/53 pts with the NK- stimulatory KIR haplotype (2DS2+) who had superior OS- and PFS-rates compared to 18/53 pts with an inhibitory haplotype and KIR/KIR-ligand match (2DS2-, 3DL1+, Bw4+) (p < 0.02). Similarly, we found 33/53 pts with low affinity FCGR alleles (FCGR2A-H131R/R and/or FCGRA3A-V158 F/F). These patients showed lower PFS rates compared to 20/53 patients with high affinity FCGR polymorphisms (p < 0.01). ADCC analysis on day 15 of cycle 1 showed higher levels in the pt group with NK- stimulatory versus NK- inhibitory KIR/KIR ligand haplotypes (23±6 % vs. 6±2%, p < 0.01). A similar effect was observed in pts with high affinity FCGR polymorphisms with an ADCC increase of 20±6% compared to 11±2% in the low affinity FCGR control. Conclusions: KIR-haplotypes and FCGR- polymorphisms correlated with the functional immune parameter ADCC and clinical outcome, and thus support the relevance of ADCC for clinical efficacy of ch14.18/CHO in neuroblastoma.
Genome-wide, CRISPR-Cas9-based genetic screening for regulators of interferon-induced PD-L1 expression in AML cells.
Authors: Tse William T. , Bland Joshua B.
Published: November 2019

116

Background: Expression of PD-L1 in AML cells is induced by pro-inflammatory signals, but how the induction is regulated is incompletely known. Genome-wide screening using the CRISPR-Cas9 technology could provide a strategy to interrogate the regulators of PD-L1 expression. Methods: A lentiviral CRISPR-Cas9 library, containing 3 sgRNA targeting each of 19,050 human genes (Nat Methods 2014, 11:783), was used to transduce THP-1 AML cells. Transduced cells were treated with IFN-γ and stained with an anti-PD-L1 antibody. Two subsets of cells were isolated by cell sorting according to their levels of PD-L1 expression. One subset consisted of cells that showed the median 5% of PD-L1 expression and the other subset, the lowest 5%. sgRNA integrated in the sorted subsets were amplified and their frequencies quantified by deep sequencing. Results: As expected, sgRNA targeting the PD-L1 gene itself were highly enriched in the subset that expressed a low level of PD-L1. sgRNA that targeted IFNGR2, β-chain of the IFN-γ receptor, were also enriched in this subset, whereas those for IFNGR1 were not. sgRNA targeting both JAK1 and JAK2 were enriched. By contrast, those for JAK3 and TYK2 were not. Enrichment was seen for sgRNA that targeted STAT1, a transcriptional activator downstream of JAK1/2, whereas sgRNA targeting the other STAT activators (STAT-2, 3, 4, 5A, 5B and 6) showed no enrichment. sgRNA targeting several serine kinases that regulate STAT1 phosphorylation (PKCδ, PKCε, PKCα, CAMKII, ERK, JNK, and CDK8) were not enriched. Nor were sgRNA targeting several phosphatases that modulate STAT1 activity (TC-PTP, PTPN1, SHP1 and SHP2). sgRNA targeting IRF1 (IFN regulatory transcription factor 1) were highly enriched, whereas sgRNA for IRF-2 to IRF-9 were not. In each of the affected genes, at least 2 out of the 3 sgRNA targeting the gene were enriched, indicating a generality of the findings. Conclusions: A CRISPR-Cas9-based screening strategy provided an unbiased interrogation of genes essential for PD-L1 expression on AML cells. It confirmed the importance of the IFNGR2-JAK1/2-STAT1-IRF1 axis, which could serve as targets for the development of drugs to suppress the ability of AML cells to evade host immunity.
The utility of molecular profiling in clinical trial enrollment and monitoring.
Authors: Hardison Matthew Thomas , Stover James
Published: November 2019

12

Background: With the growing number of therapies that are directed against specific biomarkers, or discrete subsets of cancers, it is increasingly important to prospectively identify the most appropriate subjects for enrollment in a randomized, controlled clinical trial. Historically, clinical trial enrollment was strictly guided phenotypically. Subjects were included in a study simply based on presentation and disease progression, regardless of any underlying molecular mechanisms. Methods: Aegis involves the use of multiple molecular genetic procedures to investigate clinical trial subjects, whether for enrollment or disease monitoring. Fluorescent in situ hybridization (FISH) to detect chromosomal aberrations and single-gene Sanger sequencing are utilized to identify subjects with abnormalities associated with cancers such as chronic lymphocytic leukemia (CLL) prior to enrollment. Gene expression analysis using Nanostring digital RNA counting to stratify diffuse large B-cell lymphoma subjects, B-cell receptor clonal assessment through IgVH analyses, and next generation sequencing are also used to provide vital data throughout the clinical trial process. Results: The use of modern molecular genetic techniques increases and improves the data available to companies for subject enrollment, stratification, and monitoring throughout a clinical trial. Genetic analysis can indicate disease response through reduced mutation burden, changes in clonality of tumor cells, or a resolution of the underlying genetic cause. This data, in conjunction with clinical presentation is employed by pharmaceutical companies to design subsequent trial stages or identify additional potential disease states as targets for a particular therapeutic. Specifically, TP53 sequencing and FISH analysis in CLL are vital to assigning risk stratification to potential subjects. Conclusions: With the advent of immunotherapy and the precision nature of such therapeutic development, molecular profiling will continue to be a vital tool in the identification of optimal clinical trial subjects. This could ultimately lead to smaller, more definitive clinical trials, which would be helpful to clinicians and patients in terms of cost and time.
Preclinical assessment of PM21-particle expanded natural killer cells for ovarian cancer treatment.
Authors: Oyer Jeremiah , Gitto Sarah B. , Altomare Deborah , Lee Dean A. , Copik Alicja
Published: November 2019

127

Background: Ovarian cancer has high recurrence rate and could benefit from immunotherapy with NK cells. A necessity for NK cell therapy is an efficient way to generate high doses of NK cells. The best method currently used in clinical trials is ex vivo NK cell expansion by co-culture with K562 CML cells, modified to express 41-BBL and membrane bound IL21 (K562.mb21). However feeder cell based methods are limited to ex vivo co-culture, difficult to disseminate, and not allowed in many jurisdictions. To overcome these limitations and to further improve NK cell therapy, we developed a feeder cell free particle based method for NK cell stimulation. These particles (PM21) are nano-scale, made from cell membranes of K562.mb21 cells, and efficiently stimulate NK cell expansion (mean 825 fold in 14 days, range 163–2216, n = 13). Methods: PM21 particles were prepared from K562.mb21 cells with a procedure developed by our group. NK cells were expanded by culturing CD3 depleted PBMCs with PM21 particles or by co-culture with K562.mb21 cells for 14 days as previously described. NSG mice ( ≥ 8 per group) were implanted ip with 1 x 106 SKOV3 ovarian tumor cells, seeded 8 days, and then treated with vehicle or NK cells expanded with PM21 or K562.mb21 cells (two doses of 10 x 106, injected 6 days apart), with or without in vivo administration of PM21 particles (600 µg, 3x weekly), and IL2 (25 KU, 3x weekly), all delivered ip. Survival analysis was performed with log rank (Mantel-Cox) test. Results: Treatment of SKOV3 engrafted NSG mice with NK cells, expanded with K562.mb21 cells or with PM21 particles, allowed significant ( < 0.0001) 10 day increase in survival compared to untreated animals that succumbed on average 21 days after start of treatment. Administration of ip PM21 particles enhanced survival by 5 days (p = 0.056) over no in vivo PM21 groups. Conclusions: NK cells prepared with PM21 particles or with K562.mb21 cells are equivalent in anti-SKOV3 efficacy and in vivo application of PM21 particles provides further benefit. Clinical translation is underway and clinical trials are being planned. PM21 particles can be the next step in development of NK cell therapy for enhancing both efficacy and dissemination of NK cell therapeutics for ovarian cancer.
Induction of tumor cell-based ADCC resistance to create new tumor cell survival and therapy resistance mechanisms.
Authors: Aldeghaither Dalal S. , Murray Joseph C. , Roth Sarah Martinez , Fertig Elana J. , Tang Shaojun , Jablonski Sandra A , Weiner Louis M.
Published: November 2019

130

Background: EGFR overexpression & amplification is seen in many types of cancer. Cetuximab is an EGFR targeting monoclonal antibody that acts by signal perturbation, complement activation & inducing antibody dependent cellular cytotoxicity(ADCC) by recruiting effector cells. Cetuximab resistance has been extensively investigated, but it has proven difficult to study the molecular mechanisms underlying ADCC resistance because such models are not readily available. Methods: We established an EGFR-targeted ADCC resistance model system using NK92-CD16V effector cells, Cetuximab, and the A431 EGFR-expressing squamous cell carcinoma cell line. Continuous ADCC challenge of A431 (26+ passages) yielded an ADCC-resistant phenotype (ADCCr) that exhibits a stable phenotype in the absence of continued ADCC selection. To further understand the evolution of resistance to ADCC, we explored the ADCCr cell surface signature, gene expression profile & ability to activate NK cells. Results: We find that ADCCr cells(when compared to its ADCC-sensitive(ADCCs) counterpart) have a modest reduction of EGFR expression, a distinctive transcriptional profile highlighted by overexpression of histone & interferon related genes, altered cell surface expression of important cell adhesion molecules, reduced in vitro proliferation and in vivo growth & resistance that is specific to the formation and function of immune synapses. ADCCr can be partially reversed by the exposure to an inhibitor of the histone acetyl transferase p300 subunit. Interestingly, the ADCCr cell line has reduced sensitivity (compared with ADCCs) to antimetabolite, DNA-intercalating and ABC transporter-regulated cytotoxic agents.The PD-L1 immune checkpoint does not modulate ADCC in this model system. Conclusions: These findings suggest that the induction of resistance to EGFR-targeted immune attack in this model system creates broadly useful survival and potential therapy resistance mechanisms.
Assessment of antitumor function of NK cells expanded with exosomes from K562.mb21 cells.
Authors: Oyer Jeremiah , Gitto Sarah B. , Khederzadeh Sara , Shaver Kari , Lee Dean A. , Altomare Deborah , Copik Alicja
Published: November 2019

132

Background: NK cells can kill malignant cells to provide innate immunity against tumors. Due to their low abundance in blood, a focus is to expand NK cells ex vivo having enhanced anti-tumor cytotoxicity to be used as a treatment. Our group has pioneered a cell-free method using plasma membrane (PM) particles derived from K562 cells expressing 41BBL and membrane-bound IL-21 (K562.mb21) which were developed for NK cell expansion. Compared to feeder cell based methods for NK cell expansion, PM21-particles improve safety and allow for potential wide-spread dissemination, and also allows direct in vivo use. Exosomes, vesicles naturally secreted by cells, may yet be another novel feeder cell free way for NK cell expansion and may have further advantageous therapeutic dimensions. Methods: EX21-exosomes and PM21-particles were prepared from K562.mb21 cells and characterized by Nanosight and Western blot analysis. CD3-depleted PBMCs were cultured with EX21 for 14 days, NK cell amounts were monitored and media changed every 2-3 days. In vitro cytotoxicity against K562 cells were comparatively assessed for EX21-NK cells and PM21-NK cells. In vivo anti-tumor efficacy of EX21- and PM21-NK cells was assessed in NSG mice implanted ip with SKOV3_luc ovarian tumor cells (1 x 106 cells seeded for 4 days). SKOV3-bearing mice were treated with vehicle, or two doses of EX21-NK cells or PM21-NK cells (1 x 107, in 5 day intervals), and with or without in vivo administration of EX21 (10 µg, 3x/week) or PM21-particles (600 µg, 3x/week). All groups were injected ip with IL-2 (10 KU, 3x/week). Survival analysis was performed with a Log-rank (Mantel-Cox) test. Results: NK cells cultured with EX21 expanded 530 fold (344-710) over 14 days compared to 735 fold (667-802) in presence of PM21-particles. Treatment of SKOV3 engrafted NSG mice with NK cells, expanded with either EX21 or with PM21, allowed significant ( < 0.0001) increase in survival compared to untreated animals (41-44 vs 29 days post treatment). Ip delivery of EX21 to SKOV3 bearing mice had no effect on survival in either untreated control or EX21-NK cell treated groups. Conclusions: EX21 efficiently expands NK cells and EX21-NK cells have equal anti-tumor effect as PM21-NK cells, both in vitro and in vivo.
Suppression of EBV-induced LCLs using CAR T cells redirected against HLA-DR.
Authors: Han Chungyong , Singh Rohit , Kim Seon-Hee , Choi Beom K. , Kwon Byoung S.
Published: November 2019

146

Background: Recent studies demonstrated a therapeutic potential of T cells with chimeric antigen receptor (CAR) targeting CD19 in refractory B cell malignancies. However, CD19-CAR T cells frequently caused on-target off-tumor side effect, i.e. B cell aplasia, and led to the recurrence of CD19-negative leukemic cells. Alternative target antigen for B cell malignancies has to be excavated. Methods: We developed antibody clone, MVR, which specifically bound to HLA-DR that is highly expressed on malignant B cells. In particular, MVR recognized polymorphic region of HLA-DR, and indicated different binding affinity against various HLA-DR alleles. Based on MVR binding strength, PBMCs from high binder (MVRHigh) and low binder (MVRLow) were tested to generate MVR-CAR T cells. To evaluate the anti-tumor efficacy on B cell malignancies, MVR-CAR T cells were assessed for immune responses against Epstein-Barr virus (EBV)-induced lymphoblastoid cell line (LCL) in vitro and in vivo. Results: Final yield of MVR-CAR T cells generated from MVRHigh PBMCs was 10-fold lower than that of CD19-CAR T cells, presumably caused by "fratricide" among HLA-DR-upregulated MVR-CAR T cells. In contrast, fratricidal effect was ameliorated in MVR-CAR T cells generated from MVRLow PBMCs indicating that the interaction between MVR-CAR and MVRLow-HLA-DR was weak enough to achieve tolerance to fratricide. Of note, in spite of such low binding, MVRLow-LCLs were killed efficiently by the CAR T cells. Further quantitative analysis revealed that HLA-DR was far more upregulated on LCLs compared with normal T and B cells which did not undergo EBV-transformation. In accordance with this observation, MVR-CAR T cells successfully induced LCL-specific cytotoxicity without causing normal B cell damage in vitro and efficiently suppressed the outgrowth of metastasized tumors in LCL-xenografted immune-deficient mice. Conclusions: MVR-CAR T cells redirected against HLA-DR for B cell malignancies were evaluated for the cytotoxic efficacy in vitro and in vivo. Considering the alleviated on-target off-tumor side effect and the feasibility of targeting HLA-DR for CD19-deficient malignant B cells, MVR-CAR T cells can be an alternative option for B cell malignancies.
Therapeutic effect of local Interleukin-15 with radiotherapy in breast cancer.
Authors: Garcia Martinez Elena , Pilones Karsten A , Aryankalayil Joseph , Formenti Silvia , Demaria Sandra
Published: November 2019

158

Background: Interleukin (IL)-15 is a key regulator of T cell homeostasis with activity in cancer and a favorable toxicity profile compared to IL-2. IL-15 stimulates the proliferation and effector differentiation of CD8+T cells, and the proliferation and activation of natural killer (NK) cells. We observed IL-15 upregulation by gene arrays in radiotherapy (RT)-treated TSA mouse breast cancer, suggesting that it may play a role in RT-induced anti-tumor immunity. However, the upregulation was modest prompting us to test the hypothesis that administration of IL-15 may enhance in situ vaccination by RT. Methods: BALB/c mice with established poorly immunogenic TSA tumors were sham-treated, treated with tumor-targeted RT (8GyX3 days), IL-15 given peri-tumorally (2 ug/mouse/day for 10 days) starting on the first day of RT, and RT+IL-15, and monitored for tumor growth and survival. Tumor infiltrating lymphocytes (TIL) were analyzed by flow cytometry and immunostaining. In some experiments, Batf3-/-mice were used as tumor recipient. Results: IL-15 by itself was ineffective, but it significantly increased tumor control by RT (p=0.0007, RT versus RT+IL-15) leading to complete responses in 50% of the mice, most of them durable. Analysis of TILs showed significantly increased NK cells (CD45+ CD3- DX5+) in tumors treated with RT+IL-15 (p<0.0004 versus sham-treated; p<0.02 versus RT). NK cells were also more activated as indicated by expression of CD122 and CD137. Depletion of NK cells completely abrogated the therapeutic effect of the combination, while CD8 T cell depletion reduced tumor control and rate of complete regression. Interestingly, Batf3-/- mice, which lack CD103+ DCs, showed reduced response to RT+IL-15 compared to WT mice. Conclusions: Data suggest that local IL-15 with RT is an effective strategy to induce anti-tumor immunity to poorly immunogenic breast cancer. NK cells are critical mediators of the response, and may act by both killing tumor cells and promoting priming of CD8 T cells. Experiments are ongoing to determine the mechanisms of durable complete responses. Acknowledgments: IL-15 was provided by NCI BRB. Garcia-Martinez E was supported by GEICAM grant.
An analysis of the National Cancer Database (NCDB): Immunotherapy and survival in stage III melanoma.
Authors: Krishnan Mridula , Ahmed Aabra , Khanal Nabin , Silberstein Peter T.
Published: November 2019

159

Background: High dose Interferon (IFN) was the standard adjuvant treatment used for stage III melanoma between 2004-2010. To our knowledge, this is the largest study using the NCDB to determine the impact of immunotherapy used prior to 2011 in stage III melanoma. Methods: We identified 19,864 patients with stage III melanoma between 2004-2010. Among these, 5,406 of them received immunotherapy. Chi-square analysis was used to determine demographic differences between those with versus without immunotherapy. Between-therapy survival differences were estimated by the Kaplan-Meier method and associated log-rank tests; Tukey-Kramer adjusted p < 0.05 indicated statistical significance. Results: Patients who received immunotherapy had a mean survival of 89.8 months while those who did not had a mean survival of 71 months. The percentage of patients alive at 5 and 10 years was 62% and 52% (among those who received immunotherapy) compared to 46% and 32% (among those who did not receive immunotherapy) respectively. A much higher percentage of these patients were privately insured (73% vs. 47.1%, p<0.001). Those who received immunotherapy were more likely to be younger, have a higher income (36.2% vs. 34.8%, p<0.001), and a greater percentage of females received immunotherapy compared to males. (See Table 1.) Conclusions: Previously, Kirkwood et al. demonstrated a modest improvement in overall survival with IFN by 12 months (2.8 to 3.8 years). In our study, patients who received immunotherapy had a significant improvement in mean survival by 19 months. It was observed that patients who received immunotherapy were substantially younger, had private insurance and fewer comorbidities. [Table: see text]
Cancer epigenetics and effector T cells in immunotherapy and chemotherapy.
Authors: Zou Weiping
Published: November 2019

17

Background: The tumor microenvironment is comprised of immune cells that have been reprogrammed by active tumor-mediated processes to defeat tumor-specific immunity and promote tumor growth in a highly effective manner. Previous studies have helped define the nature of immune responses in the tumor microenvironment, and provide new insights into designing novel immune therapies to target the immune suppressive mechanisms including Tregs and inhibitory B7 family members (e.g. PD-L1/PD-1) and treat patients with cancer. However, the mechanisms controlling tumor immune phenotype and biological phenotype, and their relevance in cancer therapy are not well understood. Methods: Using human colon and ovarian cancer as our models, we have studied the cross-talk between immune cell subsets and tumor/stem cells, and between immunogenic and oncogenic pathways in the tumor microenvironment, and its impact on tumor immunity and therapy. Results: In patients with colon and ovarian cancer as our models, we demonstrate that Th22 cells and myeloid derived suppressor cells (MDSCs) regulate tumor stemness via epigenetic mechanisms. Furthermore, we found that histone modification and DNA methylation repress the tumor production of Th1-type chemokines, and subsequently determine effector T cell trafficking to the tumor microenvironment, affect tumor progression, and therapeutic efficacy of checkpoint blockade and adoptive T cell transfusion, chemotherapy and patient outcome. Conclusions: Epigenetic silencing of Th1-type chemokine is a novel tumor immune evasion mechanism. Selective epigenetic reprogramming alters T cell landscape in cancer and may enhance clinical efficacy of cancer therapy.
Correlation of T-cell infiltration and clonality with PD-L1 expression in soft tissue sarcomas.
Authors: Pollack Seth , He Qianchuan , Yearley Jennifer , Emerson Ryan O , Vignali Marissa , Zhang Yuzheng , Redman Mary , Loggers Elizabeth Trice , Cranmer Lee D. , Pillarisetty Venu Gopal , Ricciotti Robert , Hoch Benjamin L , Murphy Erin , McClanahan Terrill K , Blumenschein Wendy , Townson Steven Matthew , Benzeno Sharon , Riddell Stanley R. , Jones Robin Lewis
Published: November 2019

23

Background: The success of immunotherapy has raised new issues regarding the selection of patients, design of combination strategies, and sequencing of various regimens. Sarcomas have poor outcomes in the metastatic setting but may be amenable to immune therapies. However, we currently have limited knowledge of the immunologic profiles of different soft tissue sarcoma (STS) subtypes. Methods: We identified patients with the relatively common STS subtypes: leiomyosarcoma (LMS), undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS) and liposarcoma. Formalin fixed paraffin embedded (FFPE) tumor samples from 81 patients underwent gene expression analysis, immunohistochemistry for PD-1 and PD-L1, and sequencing of the T cell receptor Vβ region. Differences in liposarcoma subsets were also evaluated. Results: UPS and LMS had high expression levels of genes related to antigen presentation and T cell infiltration. UPS had higher levels of PD-L1 (p ≤ 0.001) and PD-1 (p ≤ 0.05) on IHC. UPS also had the highest T cell infiltration based on TCR sequencing, significantly more than SS, which had the lowest (p ≤ 0.05). UPS and LMS both had higher clonality compared with SS and liposarcoma (p ≤ 0.05). A model adjusted for STS histologic subtype found that for all sarcoma T cell infiltration and clonality were highly correlated with PD-1 and PD-L1 staining levels (p ≤ 0.01). Conclusions: In a model adjusted for sarcoma histologic subtypes, T cell infiltration and clonality were highly correlated with PD-1 and PD-L1 expression, consistent with the emerging view of tumor immunity that highly inflamed tumors acquire inhibitory ligands to evade tumor-specific T cells. UPS, which is a more highly mutated STS subtype, provokes a strong immune response evidenced by multiple inflammatory features suggesting that it may be well-suited to checkpoint inhibitor based approaches. SS and liposarcoma subsets are less highly mutated but do express immunogenic self-antigens therefore strategies to improve antigen presentation and T cell infiltration may be valuable for allowing immunotherapeutic success in these tumor types.
A new approach for immuno-oncology biomarker discovery: High-plex, spatial protein profiling based on NanoString digital quantification.
Authors: Lee David , Liang Yan , Merritt Chris , Pakiam Fiona , Ong Giang , Weng Shaobu , Dunaway Dwayne , Jung Jaemyeong , Warren Sarah , Crowder Scott , Beechem Joseph M , Winckler Wendy
Published: November 2019

27

Background: The immune response to cancer is shaped by the abundance and localization of immune cell populations, their activation status, and expression of immunomodulatory factors. To detect proteins at high multiplex with spatial resolution, NanoString optical barcoding technology was used to digitally profile protein expression in formalin fixed paraffin embedded (FFPE) samples and compared to traditional IHC. Methods: NanoString has enabled digital profiling of immuno-oncology protein targets, including immune cell markers and checkpoint proteins. Protein detection is enabled via primary antibodies (Abs) which are attached via a UV cleavable linker to DNA indexing oligos. FFPE samples are stained with a multiplex cocktail of labeled Abs, then DNA oligos are released by UV light exposure. Liberated oligos are then hybridized to optical barcodes for quantitation on a NanoString instrument. This technique enables quantitative, multiplex protein detection over 5 logs of dynamic range. IHC and NanoString spatial protein profiling were performed on alternating sections from blocks of tonsil, melanoma, and colorectal cancer. Sections were fluorescently labeled with panCK, Ki67, and Syto-83 to visualize morphology, and regions of interest (ROI) were selected for profiling using up to 30 oligo-tagged Abs. Results: NanoString counts strongly correlated with quantification of CD3, CD4, CD8, PanCK, Ki67, PD-1, and PD-L1 derived from IHC. To explore limits of detection, ROIs of 1, 2, 4, or 8 cells were profiled. Digital counts were detected above background at the single cell level and linearly correlated with cell count. Furthermore, quantification with two cell lineage markers and double positive cells were demonstrated using a 30-plex Ab cocktail. Conclusions: Multiplex, digital protein profiling with spatial resolution will enable deep characterization of immune responses in tumors. Additionally, single cell profiling enables inter-cellular characterization of variations in immune response. The strong correlation of NanoString data to IHC indicates the feasibility to spatially profile multiple key proteins with minimal consumption of patient tissue.
Distinct dominant T-cell receptors with a tissue resident memory phenotype in individual melanoma metastases.
Authors: Dhodapkar Kavita M. , Boddupalli Chandra S , Bar Noffar , Kadaveru Krishna , Krauthammer Michael , Pornputtapong Natapol , Mai Zifeng , Ariyan Stephan , Narayan Deepak , Kluger Harriet M. , Deng Yanhong , Verma Rakesh , Das Rituparna , Bacchiocchi Antonella , Halaban Ruth , Sznol Mario , Dhodapkar Madhav V.
Published: November 2019

3

Background: Expression of inhibitory immune checkpoints (ICPs) within tumors has emerged as an important barrier for effective anti-tumor immunity. Antibody-mediated blockade of ICPs can lead to durable responses in patients. Interestingly, only a small subset of tumor infiltrating lymphocytes (TILs) express these checkpoints and there is a need to better understand the characteristics of this subset. We undertook this study to understand characteristics of TILs within melanoma. Methods: We used single cell mass cytometry, gene expression profiling of purified T cell subsets, T cell receptor (TCR) sequencing as well as functional studies to understand the characteristics of TILs in melanoma patients (n=50). We also performed exome sequencing of tumor cells in some patients. Results: We find that TILs are functionally and phenotypically distinct from circulating T cells. They express higher levels of inhibitory ICPs (PD-1, TIM-3) and secrete less IL2, IFNg and TNFa than T cells in circulation. Expression of vascular endothelial growth factor within tumors correlated with reduced T cell infiltration. Expression of ICPs (PD-1, TIM-3, PD-L1) were enriched in T cells with a phenotype and expression profile of tissue resident memory T (TRM) cells with most cells expressing multiple checkpoints. Within the myeloid compartment, ICPs were predominantly expressed on CD14+CD16+ subset. TCR sequencing revealed that individual melanoma metastases revealed that the top clones within each of the lesions have distinct TCRs. Concurrent TCR and tumor exome sequencing of individual metastases in the same patient revealed that inter-lesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. Conclusions: Our findings suggest that TRM cells and CD16+ myeloid cells may be the major target of ICP blockade within tumors. The ability to activate, and retain TRM cells may be an important determinant of the T cell content of the tumor microenvironment and should be a goal for future vaccines. Importantly, our study illustrates inter-lesional diversity of TCRs within individual metastases which may differentially impact the outcome of immune therapy at each site.
Transcripts of immune cell surface proteins from tumor samples to predict patient clinical outcome.
Authors: He Aiwu Ruth , Gusev Yuriy , Bhuvaneshwar Krithika , Smith Coleman , Kroemer Alexander H , Prins Petra
Published: November 2019

32

Background: Hepatocellular carcinoma (HCC) is the second most common cause of cancer death worldwide, and its incidence and mortality are rapidly increasing in the US. Treatments for advanced HCC are limited despite best efforts to develop therapeutics that target the deregulated pathways of HCC. Recent studies reveal a direct causal relationship between cancer and immune dysfunction, whereby tumor cells and their microenvironment can evade immune attack by exploiting various immunoregulatory mechanisms in a process termed cancer immune editing. This study objective is to determine the relationship between immune cell surface protein transcripts from tumor samples and the clinical outcome of pts by performing exploratory analyses of liver cancer data from the cancer genome atlas (TCGA). Methods: We analyzed RNAseq data from a cohort of 75 HCC samples in the TCGA collection. We explored the association of expression of a group of specific markers for infiltrating lymphocytes (several subtypes) with overall survival (OS) and tumor immune infiltrates (IHC data). Results: Among the 75 HCC cases analyzed, 25 pts had Hepatitis C (HCV), 25 had Hepatitis B (HBV), and 25 had HCV and HBV co-infection. Fourteen of the 75 cases were found to express the CD8B isoform. The LOG RANK survival test showed a significant association between CD8B level and OS (ChiSq = 5.2, 1df; p= 0.0222), as did the Cox proportional hazard model for survival. The latter test showed that pts who’s HCC did not express the CD8B were at higher risk of death compared with those who did. Additional immune cell subtype specific transcripts are being tested. Based on analysis of differentially expressed genes between immune cell subtype "Low" and vs. "High" groups, pathways highly relevant to lymphocyte signaling and immune response and infiltration are identified. Conclusions: Immune cell specific transcripts in tumor samples may serve as a predictor of response to treatments that target immune evasion in cancer pts.
A multi-institutional study of immunosuppressed and immunocompetent patients with cutaneous squamous cell carcinoma of the head and neck treated with definitive surgery and radiotherapy: Outcomes after disease recurrence.
Authors: Koyfman Shlomo A. , Manyam Bindu V. , Garsa Adam Arebi , Chin Re-I , Reddy Chandana A. , Gastman Brian , Thorstad Wade , Yom Sue S. , Nussenbaum Brian , Wang Steven , Vidimos Allison T.
Published: November 2019

63

Background: Our multi-institutional group previously demonstrated that immunosuppressed (IS) patients with cutaneous squamous cell cancer of the head and neck (cSCC-HN) treated with surgery and adjuvant radiotherapy had inferior survival compared to immunocompetent (IC) patients. This study further examines those patients who experienced disease recurrence and compares the impact of immune status on overall survival after recurrence. Methods: Pts who received surgical resection and postoperative RT for primary or recurrent, stage I-IV (non-metastatic) cSCC-HN between 1995-2015 at Cleveland Clinic, Washington University St. Louis, and University of California San Francisco were included in this IRB approved study. Pts were categorized as IS if they were diagnosed with chronic hematologic malignancy, HIV/AIDS, or were treated with immunosuppressive therapy for organ transplantation ≥ 6 months prior to diagnosis. Pts with recurrence included those who experienced local, regional, or distant failure after completion of definitive surgery and radiation. Overall survival (OS) and progression free survival (PFS) were calculated using the Kaplan Meier method. Results: In the initial study of 205 pts, 138 (67.3%) were immunocompetent (IC) and 67 (32.7%) were IS, and were followed for a median of 25 months. PFS was significantly lower in IS pts compared to IC at 2 years (38.7% vs. 71.6%; p = 0.002), while 2yr OS demonstrated a similar but non-significant trend (60.9% vs. 78.1%; p = 0.135). A total of 72 patients (35%) recurred, including 31 with distant failure. 1yr post-recurrence OS was 42% with a median survival of 8.4 months. Median survival did not significantly differ between the IS and IC groups (8.0 vs. 12.9 months; p = 0.9). Conclusions: Pts with cSCC-HN who experience disease recurrence after definitive treatment with surgery and radiation have poor survival, irrespective of immune status. Clinical trials testing immunotherapies are needed for both IC and IS patients with this understudied disease.
Auto-antibody detection of ovarian cancer with paraneoplastic antigens.
Authors: Hurley Laura C.
Published: November 2019

70

Background: The high mortality rate of ovarian cancer (OVCA) is due to late detection and high incidence of recurrence. Autoantibody diagnostic tests for early detection and prediction of recurrence have the potential to improve patient outcome. Paraneoplastic neurological syndromes (PNSs) are rare disorders due to remote effects of the immune response against a tumor, targeting both the tumor and healthy tissues such as muscle, brain, and nervous system. Symptoms of PNSs generally precede detection of a tumor; therefore paraneoplastic antibodies are a logical resource for the early detection of cancer. Two PNSs associated with OVCA are myositis and paraneoplastic cerebellar degeneration (PCD), which target muscle and purkinje cells, respectively. Our laboratory has detected the presence of several myositis and PCD associated paraneoplastic antibodies in the sera of patients with OVCA. Methods: Incubation of OVCA patient sera with commercially available paraneoplastic diagnostic line blots indicated presence of paraneoplastic antibodies. OVCA specific epitopes discovered previously in our laboratory by phage-display biopanning were evaluated for both homology to paraneoplastic antigens and for reactivity with PNS patient sera. Selected paraneoplastic antigens reactive with OVCA sera and OVCA specific epitopes homologous with paraneoplastic antigens or reactive with PNS patient sera were evaluated on western blot with OVCA sera, healthy control sera, and sera from patients with benign gynecological conditions. The sera screening will be replicated on ELISA. Recently identified paraneoplastic antigens associated with myositis and PCD that are expressed in OVCA tissue are being evaluated for reactivity with OVCA sera on western blot. Results: We have established a panel of four paraneoplastic antigens that in combination are able to discriminate OVCA sera from healthy patient sera and sera from patients with benign gynecological conditions. Conclusions: Although paraneoplastic syndromes are rare, paraneoplastic antibodies can be present in patients with ovarian cancer without PNS. A panel of paraneoplastic antigens for the autoantibody detection of ovarian cancer has the potential for development of a clinical test.
Clinical utility of clonality testing by next generation sequencing in the monitoring of B-cell and T-cell malignancies.
Authors: Syed Mustafa H. , Ho Caleb , Petrova-Drus Kseniya , Yao JinJuan , Yu Wayne , Zehir Ahmet , Nafa Khedoudja , Arcila Maria E.
Published: November 2019

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Background: Clonality testing is an integral part of the initial assessment of B and T cell malignancies. The further use of conventional clonality assays for monitoring of disease post treatment is limited by low assay sensitivity and inability to track clones based on their unique sequences. Clonality assessment by next generation sequencing (NGS) provides increased diagnostic capabilities, allowing the tracking of disease specific clones as well as the full spectrum of clonal sequences that arise in response to therapy. Here we describe our initial experience using an NGS based assay for monitoring and our comparison to conventional clonality assays and flow cytometry. Methods: DNA was extracted from hematologic samples received for routine clonality assessment including diagnostic (DS) and follow-up post therapy (PT) samples. Clonality testing was performed by conventional PCR-based assays using biomed II primers and by NGS utilizing Lymphotrack IGH FR1 and TRG kits (Invivoscribe). The amplified products were sequenced on Illumina MiSeq. For MRD assessment, we created dedicated laboratory protocols as well as in-house developed software, MSK-LymphoClone (LC), containing a data analysis pipeline, analytical tools for clonality assessment and a signout portal for easy search and retrieval of pertinent clones. Results: Samples from 48 patients were included (48 DS and 60 PT) encompassing 12 plasma cell neoplasms, 11 acute lymphoblastic leukemias, 16 mature B-cell and 9 T-cell lymphomas. All diagnostic samples showed clonal rearrangement with 100% concordance between conventional and NGS assays. Residual disease was detected in 27/60 (45%) PT samples using conventional fragment size based assays, 27/57 (47%) using flow and 38/60 (63%) using NGS. Diagnostic clonal sequences were detected in as low as 0.0019% of total reads in PT samples tested by NGS. 18/60 (30.0%) PT samples (17 patients) were disease negative by all assays; 16 patients remained disease-free (median follow-up - 2.4 months). Conclusions: NGS clonality testing is a valuable tool for monitoring patients with B and T cell neoplasms showing higher sensitivity and specificity than conventional assays.
CD38 as a novel immune checkpoint and a mechanism of resistance to the blockade of the PD-1/PD-L1 axis.
Authors: Chen Limo , Byers Lauren Averett , Ullrich Stephen , Wistuba Ignacio Ivan , Qin Xiao-Feng , Gibbons Don Lynn
Published: November 2019

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Background: Although immune checkpoint inhibitors including PD-L1 blockade provide significant clinical benefit for patients with lung cancer, barriers to immunotherapy clinical successes have been due to a high rate of resistance. The therapeutic improvement requires a thorough understanding of the biological process of resistance. Until recently, there have been only a few studies reporting the mechanisms of resistance to PD-L1 blockade. The mechanistic basis remains poorly defined. Methods: In multiple immunocompetent syngeneic and K-rasLA1/+p53R172H?g/+ spontaneous animal models of lung cancer, we have explored the resistance mechanisms using pharmacological and genetic approaches (monoclonal antibody treatment and CRISPR/Cas9-mediated editing). The molecular and immune profiles of the tumor microenvironment were evaluated. More importantly, to determine the applicability to patients with lung cancer, we analyzed 259 patients specimens with IHC staining and mined many immune markers in TCGA adeno and squamous datasets. Results: We identified the up-regulation of CD38 on tumor cells as well as enrichment of CD38highTregs and CD38highMDSCs in tumor as the markers of treatment resistance. We observed the same resistance mechanism caused by CD38 in PD-L1 KO mice bearing PD-L1 KO Lewis lung tumors edited with the CRISPR/Cas9 system. Furthermore, by manipulating CD38 on a panel of lung cancer cell lines, in vitro and in vivo data demonstrates that CD38 inhibits CD8+ T cell proliferation, antitumor cytokine secretion, and tumor cell killing capability. To test whether CD38 blockade might be therapeutically efficacious to anti-PD-L1 resistance, we applied the combination therapy of anti-CD38 and anti-PD-L1 and demonstrated dramatic therapeutic benefit on primary tumor growth and metastasis. Additionally, in 259 lung patients, 18.5% of cases exhibited positive staining for CD38 on tumor cells, showing a great potential benefit for treating lung patients. Conclusions: CD38 is defined as a novel immune checkpoint and acts as a mechanism of resistance in the context of PD-L1 therapy. Targeting this novel immune checkpoint may broaden the benefit of PD-L1/PD-1 axis blockade for lung cancer treatment.
Characterization of a humanized PD-L1 blocking antibody with pH-dependent antigen binding in a humanized syngenic tumor model.
Authors: Qian Xueming , Teng Fei , Guo Huanhuan , Li Zhen , Qin Xiao-Feng
Published: November 2019

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Background: Checkpoint inhibition is a promising approach for cancer treatment with durable responses and survival benefits. Currently, immune-checkpoint inhibitors are delivered at maximally tolerated doses, resulting in significant toxicities and cost burden. There is significant need to develop more efficacious and safer immune checkpoint inhibitor. Towards this goal, we have developed a next-generation humanized PD-L1 antibody with pH-dependent antigen binding and evaluated its biological activities in vitro and in vivo. Methods: Hybridoma approach was employed to generate anti-PD-L1 antibodies. A cell-based receptor-ligand blocking assay was employed to identify neutralizing antibodies. Antibodies with pH-dependent antigen binding property were identified and characterized further with those without using PD-L1/PD-1 binding assay, mixed lymphocyte reaction assay, and in vivo tumor growth inhibition assay in syngeneic MC38 tumor model expressing human PD-L1 protein. Results: A panel of neutralizing antibodies blocking PD-L1/PD-L1 binding was isolated. Among them we identified antibodies with or without pH-dependent binding property. While the in vitro activities of these antibodies are similar, their tumor growth inhibition activities differ significantly. The in vivo activity of 23F11, an antibody with ph-dependent binding, is maximized at 1mg/kg in MC38/hPD-L1 model. At 1 mg/kg, 23F11 induced complete tumor regression in 7/12 of the mice treated, more potent than antibodies without pH-dependent binding. Imaging study revealed that significantly higher drug concentration in the tumor for 23F11 and correlated with extended period of target occupancy. Furthermore 23F11 treatment is correlated with sustained up-regulation of plasma mIL-18, a potential pharmacodynamics response marker. Conclusions: We have identified antibodies with pH-dependent PD-L1 binding property. These antibodies can maintain high antibody level in PD-L1 expressing tumor and have potent tumor inhibition activity at low dose. Current work will evaluate whether humanized 23F11 can be at least as efficacious as benchmark antibodies yet with improved safety profile in patients.
Retrospective study comparing survival when ipilimimab used with and without radiotherapy in patients with advanced melanoma.
Authors: Parslow Dominique Siobhan , Talbot Toby Laurence James
Published: November 2019

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Background: Ipilimumab, a monoclonal antibody targeting CTLA-4, has increased median survival in patients with advanced melanoma from 6.4 to 10 months, with 20% of patients showing a sustained response. Preclinical and clinical studies have shown possible synergistic and abscopal effects between ipilimumab and radiotherapy. The aim of this study was to compare survival curves of patients who have received ipilimumab with and without radiotherapy, and also look at other potential prognostic indicators for patients with advanced melanoma. Methods: Data was collected retrospectively from patients with advanced melanoma treated with ipilimumab at Royal Cornwall Hospital (RCH), United Kingdom. The data collection proforma included: demographic details, BRAF status, treatments prior to and since ipilimumab, whether and when radiotherapy given, response, autoimmune phenomena documented, overall survival, and survival time since ipilimumab. Results: 43 patients (20 men and 23 women) have received ipilimumab for metastatic melanoma at RCH between 2011 and 2015. Age range 27-88 years, with a median age of 60 years. 13 were BRAF mutated, 28 were BRAF wild-type and 2 were unknown. 33% of patients who received ipilimumab had some response to it, with 21% achieving a complete response. 25% of patient who displayed autoimmune phenomena clinically responded to ipilimumab; only 15% of those who did not display autoimmune phenomena clinically responded. 20 patients received radiotherapy and 23 did not; there was no significant survival difference related to radiotherapy usage. Patients with the BRAF mutation had a shorter median survival than those who were BRAF wild-type. Conclusions: Although this study was retrospective and not randomised, it appears to show an increased likelihood of a response to ipilimumab in patients who also had autoimmune side effects from treatment. Patients with a BRAF mutation had a shorter median survival than those who were BRAF wild type. The addition of radiotherapy to ipilimumab did not appear to impact on survival or response, although, as the patients were not randomised as to whether they received radiotherapy it is difficult to draw firm conclusions from this.