Host Antitumor Response Research Articles

Isolation and activation of porcine natural killer cells

Background & Aim Natural killer (NK) cells are major effector cells that are critical to the innate immune system and are typically considered to play a fundamental role, particularly in anti-viral and anti-tumor host responses. NK cells provide rapid responses to virus-infected cells, acting at around 3 days after infection, and respond to tumor formation. Typically, immune cells detect the major histocompatibility complex presented on infected cell surfaces, triggering cytokine release, causing lysis or apoptosis. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter into the circulation. In this study, we isolated, characterized and activated NK cells from porcine peripheral blood mononuclear cells (PBMCs). Methods, Results & Conclusion We firstly peripheral blood mononuclear cells (PBMCs) were isolated from pigs (10 weeks old, n=3). Primary porcine NK cells were isolated from PBMCs by negative MACS depletion of CD3 positive cells followed by FACS purification using antibodies against porcine CD3 (IgG1, clone PPT3) and co-cultured with feeder cells in specific culture condition supplemented with IL-2 (1000 IU/mL), IL-12 (20 ng/mL), IL-15 (20 ng/mL), IL-18 (100 ng/mL) and IL-21 (5 ng/mL) or without supplement for 7 and 14 days. Gamma ray (100 Gy) irradiated K562 cells were used as feeder cells for specific activation of porcine NK cells. As results, porcine CD3 negative cells expressed specific markers of activated NK cells. Therefore, gene levels of activated NK cell cytotoxic receptors (NKp30, NKp44, NKG2D, CD244, perforin, and granzyme B) were significantly higher in culture condition with supplement. These results revealed that porcine NK cells are activated in CD3 negative population when culture condition is supplemented with IL-2, IL-12, IL-15, IL-18 and IL-21.

Isolation and characterization of canine natural killer cell-derived exosomes

Background & Aim Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are naturally released from a cell and cannot replicate. Exosomes are membrane bound EVs that are produced in the endosomal compartment of most eukaryotic cells. Compared with EVs in general, it is unclear whether exosomes have unique characteristics or functions or can be separated or distinguished effectively from other EVs. Exosomes have been the focus of great interest, as they appear to be involved in numerous important cellular processes. They deliver bioactive macromolecules such as proteins, lipids, and nucleic acids, allowing intercellular communication in multicellular organisms. Exosomes are secreted by all cell types, including immune cells such as natural killer (NK) cells, and they are innate immune effector cells that contain lytic granules that are released to induce cellular cytotoxicity upon stimulation. NK cells are major effect or cells that are critical to the innate immune system and are typically considered to play a fundamental role, particularly in anti-viral and anti-tumor host responses. Methods, Results & Conclusion We peripheral blood mononuclear cells (PBMCs) were isolated from beagle dog. Subsequently, CD5 negative cells were isolated by immunomagnetic separation and cultured in specific culture condition supplemented with IL-2, IL-15 and IL-21. After 21 days, the culture medium with exosome-depleted fetal bovine serum was replaced, and the culture supernatant was harvested. The supernatant was centrifuged to remove cells and debris and then filtered using a 0.22 μm filter. The filtrate centrifuged at 100,000 g for 90 min at 4°C using ultracentrifugation. We also observed that the exosomes were isolated from culture medium of canine NK cells. As results, canine NK cells contained amounts of protein determined bicinochoninic acid assay with absorbance at 562 nm. Using western blotting, exosome markers (Flotillin-1, CD63, CD81, Alix, HSP70, and TSG101) and activated NK cell markers (perforin and granzyme B) were identified in the samples. The isolated exosomes typically possess a diameter of 40-200 nm, and are characterized by nanoparticle tracking analysis and electron microscope.

Intratumor injection of CCL21-coupled vault nanoparticles is associated with reduction in tumor volume in an in vivo model of glioma.

Glioblastoma (GBM) is the most common and malignant primary adult brain tumor. Current care includes surgical resection, radiation, and chemotherapy. Recent clinical trials for GBM have demonstrated extended survival using interventions such as tumor vaccines or tumor-treating fields. However, prognosis generally remains poor, with expected survival of 20months after randomization. Chemokine-based immunotherapy utilizing CCL21 locally recruits lymphocytes and dendritic cells to enhance host antitumor response. Here, we report a preliminary study utilizing CPZ-vault nanoparticles as a vehicle to package, protect, and steadily deliver therapy to optimize CCL21 therapy in a murine flank model of GBM. GL261 cells were subcutaneously injected into the left flank of eight-week-old female C57BL/6 mice. Mice were treated with intratumoral injections of either: (1) CCL21-packaged vault nanoparticles (CPZ-CCL21), (2) free recombinant CCL21 chemokine empty vault nanoparticles, (3) empty vault nanoparticles, or 4) PBS. The results of this study showed that CCL21-packaged vault nanoparticle injections can decrease the tumor volume in vivo. Additionally, this study showed mice injected with CCL21-packaged vault nanoparticle had the smallest average tumor volume and remained the only treatment group with a negative percent change in tumor volume. This preliminary study establishes vault nanoparticles as a feasible vehicle to increase drug delivery and immune response in a flank murine model of GBM. Future animal studies involving an intracranial orthotopic tumor model are required to fully evaluate the potential for CCL21-packaged vault nanoparticles as a strategy to bypass the blood brain barrier, enhance intracranial immune activity, and improve intracranial tumor control and survival.

Versatile cationic liposomes for RIP3 overexpression in colon cancer therapy and RIP3 downregulation in acute pancreatitis therapy

Because the induction of strong host antitumor responses plays a very important role in antitumor therapy, identifying effective approaches to elicit immunogenic cell death could have important implications. RIP3-dependent necroptotic cancer cells have been reported to release damage-associated molecular patterns and enhance antitumor immunity. In this study, hyaluronic acid-conjugated cationic liposomes (DOTAP/DOPE/PEG-DSPE/CHOL) (HA-P-LP) were prepared as a vector for mRIP3-pDNA overexpression in tumours. Compared with standard cationic liposomes, this vector markedly increased cellular gene internalisation in vitro, enhanced the tumour-targeting effect in vivo and exhibited a significant antitumor effect in combination with adjuvant chloroquine. Considering the dramatic increase in RIP3 under the pathological condition of pancreatitis and the correlation between pancreatitis and necroptosis, non-HA-conjugated liposomes with the same formulation loaded with shRNA mRIP3-pDNA effectively controlled the disease by decreasing the serum amylase concentration and inflammatory cell infiltration. The versatile cationic liposomes loaded with plasmids with opposing functions in this study provide a new concept and method for both tumour therapy and pancreatitis therapy.

Photodynamic Therapy Combined with Antihypoxic Signaling and CpG Adjuvant as an In Situ Tumor Vaccine Based on Metal-Organic Framework Nanoparticles to Boost Cancer Immunotherapy.

Photodynamic therapy (PDT) usually aggravates tumor hypoxia, which promotes the survival and metastasis of residue cancer cells; furthermore, although PDT-induced immunogenic death of cancer cells can induce host antitumor responses, such responses are generally weak and not enough to eliminate the residue cancer cells. Here, metal-organic framework (MOF)-based nanoparticles to combine PDT, antihypoxic signaling, and CpG adjuvant as an in situ tumor vaccine to boost host anticancer responses after PDT are designed. The MOF-based nanoparticles are self-assembled from H2 TCPP and zirconium ions with hypoxia inducible factor (HIF) signaling inhibitor (ACF) and immunologic adjuvant (CpG) loading, and hyaluronic acid (HA) coating on the surface. The final nanoparticles (PCN-ACF-CpG@HA) can specifically target cancer cells overexpressing CD44 receptor though HA; the aggravated hypoxic survival signaling after PDT can be blocked by ACF to inhibit the HIF-1α induced survival and metastasis. With the help of CpG adjuvant, the tumor associated antigens generated from PDT-based cancer cell destruction can initiate strong antitumor immune responses to eliminate residue cancer cells. Taken together, a novel in situ immunostimulatory strategy is designed to synergistically enhance therapeutic effects of PDT by activating host antitumor immune-responses both in vitro and in vivo, which may have great potential for clinical translation in future.

The prognostic value of the neutrophil-to-lymphocyte ratio in patients with muscle-invasive bladder cancer treated with neoadjuvant chemotherapy and radical cystectomy

IntroductionThe neutrophil-to-lymphocyte ratio (NLR) is an attractive marker because it is derived from routine bloodwork. NLR has shown promise as a prognostic factor in muscle invasive bladder cancer (MIBC) but its value in patients receiving neoadjuvant chemotherapy (NAC) before radical cystectomy (RC) is not yet established. Since NLR is related to an oncogenic environment and poor antitumor host response, we hypothesized that a high NLR would be associated with a poor response to NAC and would remain a poor prognostic indicator in patients receiving NAC. MethodsA retrospective analysis was performed on patients with nonmetastatic MIBC (cT2-4aN0M0) who received NAC prior to RC between 2000 and 2013 at 1 of 19 centers across Europe and North America. The pre-NAC NLR was used to split patients into a low (NLR ≤ 3) and high (NLR > 3) group. Demographic and clinical parameters were compared between the groups using Student's t test, chi-squared, or Fisher's exact test. Putative risk factors for disease-specific and overall survival were analyzed using Cox regression, while predictors of response to NAC (defined as absence of MIBC in RC specimen) were investigated using logistic regression. ResultsData were available for 340 patients (199 NLR ≤ 3, 141 NLR > 3). Other than age and rate of lymphovascular invasion, demographic and pretreatment characteristics did not differ significantly. More patients in the NLR > 3 group had residual MIBC after NAC than the NLR ≤ 3 group (70.8% vs. 58.3%, P = 0.049). NLR was the only significant predictor of response (odds ratio: 0.36, P = 0.003) in logistic regression. NLR was a significant risk factor for both disease-specific (hazard ratio (HR): 2.4, P = 0.006) and overall survival (HR:1.8, P = 0.02). ConclusionNLR > 3 was associated with a decreased response to NAC and shorter disease-specific and overall survival. This suggests that NLR is a simple tool that can aid in MIBC risk stratification in clinical practice.

The immune microenvironment in malignant lymphoma

Recent advances in immunotherapy have highlighted the importance of the tumor microenvironment. Lymphomas are a heterogeneous group of malignancies that arise from lymphocytes and typically develop in lymphoid tissues. It has been recognized that the elements of the lymphoma microenvironment are not mere bystanders to a host antitumor inflammatory response but are important components of the tumor that support proliferation, survival, and chemoresistance of lymphoma cells. Lymphoma cells have individual expression patterns of chemokine receptors and adhesion molecules, according to their histological subtypes, and the patterns of surface molecules determine the sites of tumor involvement. Lymphoma cells often depend on the signals provided by non-tumor cells, such as stromal cells and macrophages, through direct cell contact and paracrine factors. On the other hand, there are genetic and non-genetic mechanisms allowing lymphoma cells to escape from anti-tumor immunity, such as downregulation of HLA molecules, B2M, CD58, CD70, and/or upregulation of PD-L1 and PD-L2. Further understanding of the interactions of lymphoma cells and the tumor microenvironment gives an insight into the pathogenesis of lymphomas and supports new approaches to their treatment.

Is radiotherapy the missing link to enhancing the outcomes in non-small cell lung cancer patients treated with immunotherapy?

Significant improvements in outcomes for locally advanced and metastatic non-small cell lung cancer (NSCLC) have been seen with increased use of immune checkpoint inhibitors (ICIs), and their role in the treatment of early-stage NSCLC is a subject of active investigation. Mounting evidence from preclinical and clinical trials involving immunotherapy for NSCLC has demonstrated the importance of understanding the tumor microenvironment (TME) and the host anti-tumor response. Incorporation of radiation, specifically in hypofractionated regimens, may potentiate the anti-tumor response stimulated by ICIs and thereby improve the efficacy of immunotherapy for these patients. Additional studies are in progress to determine the optimal sequence, dose, fractionation, and duration of combination therapy.

Quantification and phenotypic characterization of peripheral blood Vδ1 + T cells in chronic lymphocytic leukemia and monoclonal B cell lymphocytosis.

Vδ1+ T cells, a subset of γδ T cells, are responsible for innate-like immune responses. Recently, an anti-tumor function mediated by MHC-unrestricted recognition of lipid and stress molecules, has also been described in these cells. This study aimed to quantify and phenotypically characterize circulating Vδ1+ T cells in B cell Chronic Lymphocytic Leukemia (CLL) and Monoclonal B cell lymphocytosis (MBL). This study enrolled 58 individuals distributed in five groups: Binet B and C CLL (n = 9), Binet A CLL (n = 26), High count-MBL (n = 10), Low count-MBL (n = 5), and a control group (n = 8). The phenotypic characterization of Vδ1+ T cells, as well as the other T cell subpopulations (CD4+ , CD8+ , CD4+ /CD8+ , and Vδ1- γδT cells), were assessed by flow cytometry, evaluating the frequency of each subset expressing CD27, CD69, and cytoplasmic granzyme B. We observed an increasing percentage of Vδ1+ T cells belonging to CD27- compartment from controls to advanced stages of the disease, which was accompanied by an increasing percentage of these cells expressing granzyme B, a phenotypic pattern that was also observed in the other T cell subpopulations under study since earlier stages of the disease. Moreover, a striking expansion of Vδ1+ T cells in Binet B and C CLL was observed. These experiment findings point to an expansion of CD27- Vδ1+ T cells with a cytotoxic profile, from controls to advanced stages of the disease, which points to a role of Vδ1+ T cells in the host's anti-tumor responses against clonal B-cells in MBL and CLL. © 2018 Clinical Cytometry Society.

Abstract 5071: The functional relevance of Atypical Chemokine Receptor 1 (ACKR1/DARC) genetic isoforms in breast cancer

Abstract Chemokines and their receptors can interact to affect the host's anti-tumor response by influencing the migration of host immune cells indirectly via a chemokine gradient. The regulation and function of these receptors is paramount in ensuring an appropriate immune response to an inflammatory stimulus, especially in the case of cancer. One such receptor, Atypical Chemokine Receptor 1 (ACKR1/DARC), is a genetically diverse transmembrane GPCR that can buffer pro-inflammatory CXC- and CC- chemokine activity in circulation and within tissues, influencing leukocyte migration. While the basic immunological function of this receptor is known, the specific function of its two genetic isoforms, A and B, is not. These isoforms are produced by two alternative promoters and distinct mRNA splicing events. Isoform A is not split by introns, even in the untranslated regions, and offers homology to CXCL8. The alternative splice variant, Isoform B, does contain an intron, and follows the GT-AG splicing rule, including a novel exon coding for an additional 7 amino acids. This localizes to the N-terminus, making up part of the extracellular binding region of the receptor. We aim to determine if the two major ACKR1 isoforms have distinct functional abilities to bind or sequester pro-inflammatory chemokines in epithelial breast cell lines. By creating isoform specific overexpression and knockout constructs, and transiently transfecting the DNA into our subtype specific cell lines, we can use an ELISA assay to determine chemokine concentration in the media, and measure differences based on isoform specificity. Using immunofluorescence, we first show that ACKR1 and our two proinflammatory chemokines, CCL2 and CXCL8, are expressed on the surface of epithelial cells and colocalize. We then indicate that there is an isoform specific difference in chemokine concentration between different breast cancer subtypes, in addition to differences seen in overexpression and knockout cell lines. Determining the functionality of these two isoforms can help us better understand ACKR1's role in the immune system, and its potential role as a prognostic marker in breast cancer. Citation Format: Brittany D. Jenkins, Rachel N. Martini, Inasia Brown, Melissa B. Davis. The functional relevance of Atypical Chemokine Receptor 1 (ACKR1/DARC) genetic isoforms in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5071.

Expression of programmed death ligand 1 is associated with poor prognosis in myeloid sarcoma patients.

Myeloid sarcoma (MS) is a rare condition and is an extramedullary tumour of immature myeloid cells. It is now known that the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway suppresses the host antitumor responses and that these products are expressed on both tumour cells and tumour-infiltrating cells in various malignancies. However, little is known about the significance of PD-1/PD-L1 expression on tumour cells and tumour microenvironmental cells in MS. To investigate the clinicopathological significance of PD-1/PD-L1 expression in MS, we analyzed 98 patients by immunohistochemistry. Of these, 10.2% of cases had neoplastic tumour cells positive for PD-L1 (nPD-L1+ ). However, the rate of nPD-L1+ was <5% (range: 0.27 to 2.97%). On the other hand, PD-L1 expression on 1 or more of stromal cells in the tumour microenvironment (miPD-L1+ ) was observed in 37.8% of cases. Because all nPD-L1+ cases expressed PD-1 on less than 5% of tumour cells, we compared the miPD-L1+ and miPD-L1- groups. There was a correlation between miPD-L1+ status and the number of PD-1-expressing tumour -infiltrating lymphocytes (PD-1+ TILs; P=.0229). miPD-L1+ was found to be associated with poorer overall survival and progression-free survival (P=.00392, P=.00261, respectively). Multivariate analysis also confirmed miPD-L1+ to be an independent poor prognostic factor. In conclusion, our study indicated that the immunotherapy blocking the PD-1/PD-L1 pathway may improve the clinical outcome of MS.

Immunotherapeutic Approaches to Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is an immunosuppressive disease with multiple mechanisms to impair immune-mediated recognition and control of tumor cell proliferation and metastasis. Based on successes experienced with cancer immunotherapy in the treatment of other solid tumors, considerable efforts are underway to develop immunotherapeutics that can enhance the host antitumor response to HNSCC. Promising results in preclinical studies and early clinical trials have been reported, prompting the FDA to approve the use of the immune checkpoint PD-1 receptor antagonist pembrolizumab for the treatment of platinum-refractory recurrent or metastatic HNSCC in 2016. Ongoing clinical trials are investigating the use of various immunotherapies, such as vaccines, adoptive T-cell transfer, and immune modulating antibodies alone or in conjunction with traditional treatment modalities, to achieve more efficacious, more durable, and less toxic treatments. In this article, we provide a basic review of the role of the immune system in developing malignancy, and we discuss some notable mechanisms of immune evasion in HNSCC. We highlight current approaches and published clinical results of immunotherapy in the treatment of HNSCC.

Crk adaptor protein promotes PD-L1 expression, EMT and immune evasion in a murine model of triple-negative breast cancer

ABSTRACTThe tumor infiltration of immune cells in solid cancers can profoundly influence host antitumor responses. In recent years, immunotherapeutic regimens, that target immune checkpoints, demonstrated significant antitumor response by increasing intra-tumoral immune cell populations, including CD8+ effector T cells. However, administration of such immune checkpoint inhibitors is largely inefficacious in inducing immunogenicity and treating breast cancer. Currently, there is a great need to better understand cell autonomous mechanisms of immune evasion in breast cancer to identify upstream therapeutic targets that increase the efficacy of immunotherapy. Here we show that Crk, an SH2 and SH3 domain-containing adaptor protein implicated in focal adhesion signaling, cell migration, and invasion, and frequently up-regulated in human cancers, has an important role in regulating the tumor immune microenvironment. Using a murine 4T1 breast adenocarcinoma model of spontaneous metastasis in immune-competent BALB/C mice, we show that genetic ablation of Crk by CRISPR-Cas9 leads to enhanced anti-tumor immune cell populations, cytotoxic effector and immune surveillance cytokines in primary tumor. Pathologically, this leads to a significant reduction in tumor growth and lung metastasis. Mechanistically, Crk KO suppresses EMT and PD-L1 expression on tumor cells and acts additively with anti-PD1 therapy to suppress tumor growth and metastasis outcomes. Taken together, these data reveal a previously un-described function of Crk adaptor protein expression in tumor cells for cell autonomous regulation of tumor immune microenvironment.

Melanoma subtypes demonstrate distinct PD-L1 expression profiles

PD-L1 expression in the tumor immune microenvironment is recognized as both a prognostic and predictive biomarker in patients with cutaneous melanoma, a finding closely related to its adaptive (IFN-γ-mediated) mechanism of expression. Approximately 35% of cutaneous melanomas express PD-L1, however, the expression patterns, levels, and prevalence in rarer melanoma subtypes are not well described. We performed immunohistochemistry for PD-L1 and CD8 on 200 formalin-fixed paraffin-embedded specimens from patients with acral (n=16), mucosal (n=36), uveal (n=103), and chronic sun-damaged (CSD) (n=45) melanomas (24 lentigo maligna, 13 ‘mixed’ desmoplastic, and 8 ‘pure’ desmoplastic melanomas). CD8+ tumor-infiltrating lymphocyte (TIL) densities were characterized as mild, moderate, or severe, and their geographic association with PD-L1 expression was evaluated. Discrete lymphoid aggregates, the presence of a spindle cell morphology, and the relationship of these features with PD-L1 expression were assessed. PD-L1 expression was observed in 31% of acral melanomas, 44% of mucosal melanomas, 10% of uveal melanomas, and 62% of CSD melanomas (P<0.0001). Compared to our previously characterized cohort of cutaneous melanomas, the proportion of PD-L1(+) tumors was lower in uveal (P=0.0002) and higher in CSD (P=0.0073) melanomas, while PD-L1 expression in the acral and mucosal subtypes was on par. PD-L1 expression in all subtypes correlated with a moderate–severe grade of CD8+ TIL (all, P<0.003), supporting an adaptive mechanism of expression induced during the host antitumor response. The tumor microenvironments observed in CSD melanomas segregated by whether they were the pure desmoplastic subtype, which showed lower levels of PD-L1 expression when compared to other CSD melanomas (P=0.047). The presence of lymphoid aggregates was not associated with the level of PD-L1 expression, while PD-L1(+) cases with spindle cell morphology demonstrated higher levels of PD-L1 than those with a nested phenotype (P<0.0001). Our findings may underpin the reported clinical response rates for anti-PD-1 monotherapy, which vary by subtype.

Abstract 1819: Reprogramming the tumor microenvironment by targeting endothelial DNA repair

Abstract The tumor microenvironment (TME) plays a critical role in orchestrating immune infiltration, tumor progression and response to therapeutics. Therefore, strategies to manipulate the TME including cytokine gene therapy, antibodies to reverse macrophage polarization etc are under active investigation. We have identified an alternative approach to target the TME by disrupting DNA repair in the tumor endothelial cells (ECs). We discovered a seven-microRNA (miR) signature induced in ECs in in vitro and in vivo by oxidative stress and DNA damage. The top miR candidate in this signature, miR-103 altered the TME by inducing DNA damage in ECs, eliciting type I interferons, upregulating immune costimulatory receptors and decreasing PD-L1 expressing tumor associated macrophages and granulocytes. Moreover, miR-103 treatment had a paracrine effect on the tumors and upregulated Fas and TRAIL receptors. Mechanistically, these functions of miR-103 were largely due to its downregulation of the three prime exonuclease TREX1. Local, systemic or vascular targeted delivery of miR-103 decreased both angiogenesis and tumor burden in multiple mouse tumor models. Complementary to the role of miR-103, two additional miRs in the miR signature, miRs 494 and 99b each induced senescence in the vasculature by downregulating the Mre11a-Rad50-NBN (MRN) complex. Ectopic expression of miRs 494 or 99b decreased telomerase activity, increase p21 levels with a concomitant decrease in pRb levels. Vascular targeted delivery of miR-494 decreased angiogenesis in vivo whereas systemic delivery decreased tumor growth. Interestingly, both miR mimics and the MRN knockdowns induced the transcription of a number of senescence associated genes including CD44. Taken together these data suggest that miRs 494 and 99b targeting of the MRN complex induces senescence. The MRN complex interacts with the ATM kinase, histone H2AX and TREX1 suggesting that the miRs we identified disrupt critical nodes of a DNA Damage Response (DDR) network. Our findings reveal a complex, miR mediated cross talk between EC DNA damage pathways, the TME and tumor cells. These interactions can be exploited for designing therapies that synergize with tumor cell killing to enhance host anti-tumor responses. Citation Format: Cristina Espinosa-Diez, RaeAnna Wilson, Rebecca Ruhl, Nathan Kanner, Namita Chatterjee, Clay Hudson, Sudarshan Anand, Shushan Rana. Reprogramming the tumor microenvironment by targeting endothelial DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1819. doi:10.1158/1538-7445.AM2017-1819

Current Immunotherapeutic Strategies to Enhance Oncolytic Virotherapy.

Oncolytic viruses (OV) represent a promising strategy to augment the spectrum of cancer therapeutics. For efficacy, they rely on two general mechanisms: tumor-specific infection/cell-killing, followed by subsequent activation of the host’s adaptive immune response. Numerous OV genera have been utilized in clinical trials, ultimately culminating in the 2015 Food and Drug Administration approval of a genetically engineered herpes virus, Talminogene laherparepvec (T-VEC). It is generally accepted that OV as monotherapy have only modest clinical efficacy. However, due to their ability to elicit specific antitumor immune responses, they are prime candidates to be paired with other immune-modulating strategies in order to optimize therapeutic efficacy. Synergistic strategies to enhance the efficacy of OV include augmenting the host antitumor response through the insertion of therapeutic transgenes such as GM-CSF, utilization of the prime-boost strategy, and combining OV with immune-modulatory drugs such as cyclophosphamide, sunitinib, and immune checkpoint inhibitors. This review provides an overview of these immune-based strategies to improve the clinical efficacy of oncolytic virotherapy.

CheckMate 436: A phase 1-2 study to evaluate safety and efficacy of nivolumab plus brentuximab vedotin in patients with CD30-expressing relapsed/refractory non-Hodgkin lymphomas.

TPS7577 Background: Nivolumab (nivo) is a PD-1 immune checkpoint inhibitor that augments T-cell activation and host anti-tumor responses. PD-1 blockade has shown promise in B- and T-cell non-Hodgkin lymphoma (NHL),1 but many patients (pts) with NHL do not respond or progress after response. Combination therapy using anti-tumor agents with complementary mechanisms of action and low immunosuppressive impact may result in more frequent and durable responses. Brentuximab vedotin (BV) is an anti-CD30 antibody–drug conjugate that induces cell cycle arrest and apoptosis, with activity in a range of NHL tumors.2,3 Tumor cells undergoing BV-induced apoptosis have shown subsequent immune-mediated anti-tumor cytotoxicity.4 Therefore, nivo and BV may synergize if combined for relapsed/refractory (RR) NHL. Methods: CheckMate 436 (NCT02581631) is a phase 1–2, open-label, international, single-arm study evaluating nivo + BV for CD30-expressing RR NHL (study start: Dec 2015) in pts with RR diffuse large B-cell lymphoma, peripheral T-cell lymphoma (excluding anaplastic large cell lymphoma), and cutaneous T-cell lymphoma (mycosis fungoides/Sézary syndrome); cohorts with primary mediastinal B-cell lymphoma (PMBL) and mediastinal gray zone lymphoma were made eligible in Sept 2016. Pts with PMBL must be aged ≥15 y (≥18 y for other histologies). All pts must have CD30-expressing disease, defined by CD30 on ≥1% of tumor cells or tumor-infiltrating lymphocytes by immunohistochemistry. In the phase 1 component, 6 pts will receive nivo and BV until disease progression or unacceptable toxicity. In the phase 2 component, ~130 more pts across the 5 histologies will be enrolled and treated at the recommended dose. Primary endpoints: safety, tolerability, and investigator-assessed objective response rate; secondary endpoints: duration of response and complete response (CR), CR rate, and progression-free and overall survival. Accrual is ongoing.

The association between neutrophil to lymphocyte ratio and overall survival in patients with triple-negative breast cancer.

e12591 Background: Recent reports have suggested that an elevated neutrophil to lymphocyte ratio (NLR) is associated with poor outcomes in various malignancies. Furthermore, radiation therapy (RT) alone or in combination with chemotherapy often results in reduced absolute lymphocyte counts (ALC), which has been postulated to impair host immune surveillance and anti-tumor responses. We sought to better understand whether an elevated NLR results in impaired OS in patients with triple-negative breast cancer. Methods: Our retrospective analysis was performed on a database of patients with biopsy proven triple-negative breast cancer treated at Washington University in St. Louis between 2002 and 2010. Patients who received radiation therapy and had adequate follow up in terms of clinical documentations and laboratory data were included. NLR and ALC were collected at baseline, post-adjuvant chemotherapy, prior to RT, end of RT, 2 months post RT, 1 year post RT, and completion of all planned treatments. Results: Total of 155 patients were included. We used a cutoff value of 3.0 to classify high NLR. Wilcoxon rank-sum test was used to compare NLR at each time point between deceased and alive patients. High NLR post chemotherapy is associated with worse OS (HR 1.127, 95% CI = 1.016-1.249; p&lt;0.05). In addition, NLRs from end of RT, 2 months post and 1 year post RT are associated with worse OS: (HR 1.109, 95% CI = 1.059–1.161; p &lt; 0.001); (HR 1.074, 95% CI = 1.041–1.109; p &lt; 0.001); (HR 1.069, 95% CI = 1.048–1.091; p &lt; 0.001) respectively. Conclusions: High NLR post-chemotherapy and RT are associated with worse OS in patients with triple-negative breast cancer.Further studies are warranted to establish NLR as a prognostic marker and an addition to risk prediction models. [Table: see text]

Glioblastoma usurps host extracellular adenosine to circumvent host anti-tumor Responses

Abstract Glioblastoma (GB) is the most aggressive, fatal, and least successfully treated of all solid brain tumors due to its high capacity to proliferate and extreme chemoresistance. Treatment for GB patients is managed with multimodal therapies including surgical resection (if possible), radiotherapy and chemotherapy with temozolomide (one of the few drugs shown to have some effect). Despite this, patient survival ranges between 6–18 months. Because GB is highly infiltrative, it often invades normal brain tissue, sometimes preventing surgical resection, which inevitably leads to recurrence. Adenosine is an immune suppressive signaling molecule that is generated by the enzymatic action of CD73. I tested the hypothesis that GB usurps host extracellular adenosine to circumvent host anti-tumor mechanisms by down regulating macrophage/microglial cell immune responses and promote M2 instead of M1 macrophage/microglial phenotype. To test this, I isolated macrophage/microglial cells from the tumor microenvironment of mice lacking or expressing CD73. Compared to WT mice, the frequency of M1 verses M2 phenotypes showed that eighty percent of the macrophage/microglial cells present in the tumor microenvironment of CD73−/− mice exhibited an M1 phenotype, while WT mice had about 30 percent M1 phenotype.

Role of necroptotic cancer cells in anti-cancer immunity

Abstract Successful immunogenic apoptosis in experimental cancer therapy depends on the induction of strong host anti-tumor responses. Given that tumors are often resistant to apoptosis, it is important to identify alternative molecular mechanisms that elicit immunogenic cell death. Necroptosis is one form of regulated necrosis and is mediated by RIPK1, RIPK3, and its substrate mixed lineage kinase domain-like and has been reported to contribute to inflammation under pathological conditions. Necroptosis is commonly induced by ligand-dependent activation of certain members of the TNFR and TLR families. To circumvent the use of cell death-inducing ligands or other immune active molecules that would not allow us to evaluate the immune response resulting uniquely from necroptosis induction, we opted for the use of inducible Tet-On systems for the expression of the downstream necroptotic effector proteins, allowing us to induce necroptosis independently of any receptor activation. In our genetic model a direct dimerization of FADD combined with inducible expression of RIPK3 promotes necroptosis. We report that necroptotic cancer cells release DAMPs and promote maturation of dendritic cells, the cross-priming of cytotoxic T cells, and the production of IFN-γ in response to tumor antigen stimulation. Using both FADD-dependent and FADD-independent RIPK3 induction systems, we demonstrate the efficient vaccination potential of immunogenic necroptotic cells. Our study broadens the current concept of immunogenic cell death and opens doors for the development of new strategies in cancer therapy.