High Plus Low Dose Radiation Strategy in Combination with TIGIT and PD1 Blockade to Promote Systemic Antitumor Responses.

Tumors deploy various immune-evasion mechanisms that create a suppressive environment and render effector T-cells exhausted and inactive. Therefore, a rational utilization of checkpoint inhibitors may alleviate exhaustion and may partially restore antitumor functions. However, in high-tumor-burden models, the checkpoint blockade fails to maintain optimal efficacy, and other interventions are necessary to overcome the inhibitory tumor stroma. One such strategy is the use of radiotherapy to reset the tumor microenvironment and maximize systemic antitumor outcomes. In this study, we propose the use of anti-PD1 and anti-TIGIT checkpoint inhibitors in conjunction with our novel RadScopal technique to battle highly metastatic lung adenocarcinoma tumors, bilaterally established in 129Sv/Ev mice, to mimic high-tumor-burden settings. The RadScopal approach is comprised of high-dose radiation directed at primary tumors with low-dose radiation delivered to secondary tumors to improve the outcomes of systemic immunotherapy. Indeed, the triple therapy with RadScopal + anti-TIGIT + anti-PD1 was able to prolong the survival of treated mice and halted the growth of both primary and secondary tumors. Lung metastasis counts were also significantly reduced. In addition, the low-dose radiation component reduced TIGIT receptor (PVR) expression by tumor-associated macrophages and dendritic cells in secondary tumors. Finally, low-dose radiation within triple therapy decreased the percentages of TIGIT+ exhausted T-cells and TIGIT+ regulatory T-cells. Together, our translational approach provides a new treatment alternative for cases refractory to other checkpoints and may bring immunotherapy into a new realm of systemic disease control.

Influence of low-dose radiation on abscopal responses in patients receiving high-dose radiation and immunotherapy.

BACKGROUND: Preclinical evidence suggests that low-dose radiation may overcome the inhibitory effects of the tumor stroma and improve a tumor's response to immunotherapy, when combined with high-dose radiation to another tumor. The aim of this study was to evaluate tumor responses to this combination in a clinical setting. METHODS: A post-hoc analysis of 3 ongoing immunoradiation trials was performed. Twenty-six (of 155) patients received low-dose radiation (1-20 Gy total), either as scatter from high-dose radiation or from intentional treatment of a second isocenter with low-dose radiation, were evaluated for response. The low-dose lesions were compared to lesions that received no radiation (< 1 Gy total). Response rates, both defined as complete and partial responses as defined by RECIST criteria were used to compare lesion types. RESULTS: The 26 patients had a total of 83 lesions for comparison (38 receiving low-dose, 45 receiving no-dose). The average dose given to low-dose lesions was 7.3 Gy (1.1-19.4 Gy), and the average time to response was 56 days. Twenty-two out of 38 (58%) low-dose lesions met the PR/CR criteria for RECIST compared with 8 out of 45 (18%) no-dose lesions (P = 0.0001). The median change for longest diameter size for low-dose lesions was - 38.5% compared to 8% in no-dose lesions (P < 0.0001). Among the low-dose lesions that had at least one no-dose lesion within the same patient as a control (33 and 45 lesions respectively), 12 low-dose lesions (36%) responded without a corresponding response in their no-dose lesions; Conversely, two (4%) of the no-dose lesions responded without a corresponding response in their low-dose lesion (P = 0.0004). CONCLUSIONS: Low-dose radiation may increase systemic response rates of metastatic disease treated with high-dose radiation and immunotherapy.

Role of Radiation Therapy in Modulation of the Tumor Stroma and Microenvironment.

In recent decades, there has been substantial growth in our understanding of the immune system and its role in tumor growth and overall survival. A central finding has been the cross-talk between tumor cells and the surrounding environment or stroma. This tumor stroma, comprised of various cells, and extracellular matrix (ECM), has been shown to aid in suppressing host immune responses against tumor cells. Through immunosuppressive cytokine secretion, metabolic alterations, and other mechanisms, the tumor stroma provides a complex network of safeguards for tumor proliferation. With recent advances in more effective, localized treatment, radiation therapy (XRT) has allowed for strategies that can effectively alter and ablate tumor stromal tissue. This includes promoting immunogenic cell death through tumor antigen release to increasing immune cell trafficking, XRT has a unique advantage against the tumoral immune evasion mechanisms that are orchestrated by stromal cells. Current studies are underway to elucidate pathways within the tumor stroma as potential targets for immunotherapy and chemoradiation. This review summarizes the effects of tumor stroma in tumor immune evasion, explains how XRT may help overcome these effects, with potential combinatorial approaches for future treatment modalities.

The HGF/c-MET Pathway Is a Driver and Biomarker of VEGFR-inhibitor Resistance and Vascular Remodeling in Non-Small Cell Lung Cancer.

Purpose: Resistance to VEGFR inhibitors is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). We investigated the cellular mechanisms mediating resistance of NSCLCs to VEGFR tyrosine kinase inhibitors.Experimental Design: We generated murine models of human NSCLC and performed targeted inhibition studies with the VEGFR TKIs cediranib and vandetanib. We used species-specific hybridization of microarrays to compare cancer (human) and stromal (mouse) cell transcriptomes of TKI-sensitive and -resistant tumors. We measured tumor microvascular density and vessel tortuosity to characterize the effects of therapy on the tumor vascular bed. Circulating cytokine and angiogenic factor levels in patients enrolled in VEGFR TKI trials were correlated with clinical outcomes.Results: Murine xenograft models of human lung adenocarcinoma were initially sensitive to VEGFR TKIs, but developed resistance to treatment. Species-specific microarray analysis identified increased expression of stromal-derived hepatocyte growth factor (HGF) as a candidate mediator of TKI resistance and its receptor, c-MET, was activated in cancer cells and tumor-associated stroma. A transient increase in hypoxia-regulated molecules in the initial response phase was followed by adaptive changes resulting in a more tortuous vasculature. Forced HGF expression in cancer cells reduced tumor sensitivity to VEGFR TKIs and produced tumors with tortuous blood vessels. Dual VEGFR/c-MET signaling inhibition delayed the onset of the resistant phenotype and prevented the vascular morphology alterations. In patients with cancer receiving VEGFR TKIs, high pretreatment HGF plasma levels correlated with poorer survival.Conclusions: HGF/c-MET pathway mediates VEGFR inhibitor resistance and vascular remodeling in NSCLC. Clin Cancer Res; 23(18); 5489-501. ©2017 AACR.

Optimizing Radiotherapy with Immunotherapeutic Approaches.

Several factors must be considered to successfully integrate immunotherapy with radiation into clinical practice. One such factor is that concepts arising from preclinical work must be tested in combination with radiation in preclinical models to better understand how combination therapy will work in patients; examples include checkpoint inhibitors, tumor growth factor-beta (TGF-ß) inhibitors, and natural killer (NK) cell therapy. Also, many radiation fields and fractionation schedules typically used in radiation therapy had been standardized before the introduction of advanced techniques for radiation planning and delivery that account for changes in tumor size, location, and motion during treatment, as well as uncertainties introduced by variations in patient setup between treatment fractions. As a result, radiation therapy may involve the use of large treatment volumes, often encompassing nodal regions that may not be irradiated with more conformal techniques. Traditional forms of radiation in particular pose challenges for combination trials with immunotherapy. This chapter explores these issues in more detail and provides insights as to how radiation therapy can be optimized to combine with immunotherapy.

Preclinical Rationale and Clinical Considerations for Radiotherapy Plus Immunotherapy: Going Beyond Local Control.

The use of radiation for cancer therapy has expanded and sparked interest in possible synergistic effects by combining it with current immunotherapies. In this review, we present a case of a patient who responded to programmed cell death 1 (PD1) blockade and radiation therapy and discuss possible mechanisms. We provide background on the blockade of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) and PD1 checkpoints and highlight future immune-based therapies that may synergize with radiation, including cytosine-phosphate-guanine vaccines, OX40 agonists, CD40 agonists, regulatory T-cell depletion, and metabolic "rewiring" of cancer cells. Clinical considerations are noted for combining radiation with immunotherapies to extend the benefit of immunotherapy to more patients. New trials are needed to appropriately investigate the best sequencing and radiation dose to prime an immune response and to identify predictive biomarkers of such responses.

Strategies for combining immunotherapy with radiation for anticancer therapy.

Radiation therapy controls local disease but also prompts the release of tumor-associated antigens and stress-related danger signals that primes T cells to promote tumor regression at unirradiated sites known as the abscopal effect. This may be enhanced by blocking inhibitory immune signals that modulate immune activity through a variety of mechanisms. Indeed, abscopal responses have occurred in patients with lung cancer or melanoma when given anti-CTLA4 antibody and radiation. Other approaches involve expanding and reinfusing T or NK cells or engineered T cells to express receptors that target specific tumor peptides. These approaches may be useful for immunocompromised patients receiving radiation. Preclinical and clinical studies are testing both immune checkpoint-based strategies and adoptive immunotherapies with radiation.

Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma with distinct biology, immune profiles, and therapeutic vulnerabilities.

UNLABELLED: The molecular underpinnings that drive the heterogeneity of KRAS-mutant lung adenocarcinoma are poorly characterized. We performed an integrative analysis of genomic, transcriptomic, and proteomic data from early-stage and chemorefractory lung adenocarcinoma and identified three robust subsets of KRAS-mutant lung adenocarcinoma dominated, respectively, by co-occurring genetic events in STK11/LKB1 (the KL subgroup), TP53 (KP), and CDKN2A/B inactivation coupled with low expression of the NKX2-1 (TTF1) transcription factor (KC). We further revealed biologically and therapeutically relevant differences between the subgroups. KC tumors frequently exhibited mucinous histology and suppressed mTORC1 signaling. KL tumors had high rates of KEAP1 mutational inactivation and expressed lower levels of immune markers, including PD-L1. KP tumors demonstrated higher levels of somatic mutations, inflammatory markers, immune checkpoint effector molecules, and improved relapse-free survival. Differences in drug sensitivity patterns were also observed; notably, KL cells showed increased vulnerability to HSP90-inhibitor therapy. This work provides evidence that co-occurring genomic alterations identify subgroups of KRAS-mutant lung adenocarcinoma with distinct biology and therapeutic vulnerabilities. SIGNIFICANCE: Co-occurring genetic alterations in STK11/LKB1, TP53, and CDKN2A/B-the latter coupled with low TTF1 expression-define three major subgroups of KRAS-mutant lung adenocarcinoma with distinct biology, patterns of immune-system engagement, and therapeutic vulnerabilities.

In vitro effect of glucocorticoids on nasal polyps.

UNLABELLED: Glucocorticoids are considered the main treatment option for nasal polyps, but their effect is only recently being understood. AIM: To evaluate whether fluticasone propionate (FP) inhibits the inflammatory process induced by TNF-alpha in vitro, and to assess if NF-kappaB is associated to this inhibition. STUDY DESIGN: Experimental in vitro study. MATERIALS AND METHODS: Nasal polyp fibroblasts were cultured during 24 hours. Three different concentrations of FP (1, 10 and 100 nM, added to TNF-alpha) were compared to negative (without additive) and positive (TNF-alpha) controls. Gene expression (RTQ-PCR) and protein concentration (ELISA) of VCAM-1, ICAM-1, eotaxin and RANTES were measured, as well as the nuclear translocation of NF-kappaB. RESULTS: TNF-alpha significantly increased protein concentration and RNA expression of all the studied molecules, as well as the nuclear translocation of NF-kappaB, when compared to the negative control. FP decreased these parameters in a dose-dependent manner, statistically different from positive control up to 100nM. CONCLUSIONS: FP extensively inhibited inflammatory recruiters, at both protein and RNA levels, confirming the ability of glucocorticoids to modulate the inflammatory process in nasal polyps. This inhibition was associated to decreased NF-kappaB nuclear translocation, demonstrating that this is an important mechanism of glucocorticoids action for nasal polyps.

In vitro effect of glucocorticoids on nasal polyps / Efeito in vitro de glicocorticosteróides sobre pólipos nasais

Glucocorticoids are considered the main treatment option for nasal polyps, but their effect is only recently being understood. AIM: To evaluate whether fluticasone propionate (FP) inhibits the inflammatory process induced by TNF-alpha in vitro, and to assess if NF-kappaB is associated to this inhibition. STUDY DESIGN: Experimental in vitro study. MATERIALS AND METHODS: Nasal polyp fibroblasts were cultured during 24 hours. Three different concentrations of FP (1, 10 and 100 nM, added to TNF-alpha) were compared to negative (without additive) and positive (TNF-alpha) controls. Gene expression (RTQ-PCR) and protein concentration (ELISA) of VCAM-1, ICAM-1, eotaxin and RANTES were measured, as well as the nuclear translocation of NF-kappaB. RESULTS: TNF-alpha significantly increased protein concentration and RNA expression of all the studied molecules, as well as the nuclear translocation of NF-kappaB, when compared to the negative control. FP decreased these parameters in a dose-dependent manner, statistically different from positive control up to 100nM. CONCLUSIONS: FP extensively inhibited inflammatory recruiters, at both protein and RNA levels, confirming the ability of glucocorticoids to modulate the inflammatory process in nasal polyps. This inhibition was associated to decreased NF-kappaB nuclear translocation, demonstrating that this is an important mechanism of glucocorticoids action for nasal polyps.

Glicocorticoides são considerados a principal opção terapêutica para polipose nasossinusal, mas seus efeitos estão sendo descobertos apenas recentemente. OBJETIVO: Avaliar se proprionato de fluticasona (FP) inibe in vitro o processo inflamatório induzido por TNF-alfa, e se NF-kappaB está associado a esta inibição. FORMA DE ESTUDO: Experimental in vitro. MATERIAIS E MÉTODOS: Fibroblastos de pólipos nasais foram cultivados por 24 horas. Três concentrações diferentes de FP (1, 10 e 100nM, além do TNF-alfa) foram comparados a controles negativo (sem aditivo) e positivo (TNF-alfa). Expressão gênica (RTQ-PCR) concentração proteica (ELISA) de VCAM-1, ICAM-1, eotaxin e RANTES foram medidos, assim como a translocação nuclear de NF-kappaB. RESULTADOS: TNF-alfa aumentou significativamente a concentração proteica e expressão gênica de todas molé¬culas estudadas, assim como a translocação nuclear de NF-kappaB, quando comparado ao controle negativo. O FP diminuiu estes parâmetros numa forma dose-dependente, diferente estatisticamente do controle positivo até 100nM. CONCLUSÕES: O FP extensivamente inibiu os recrutadores inflamatórios, em níveis proteicos e gênicos, confirmando a habilidade dos glicocorticoides em modular o processo inflamatório na polipose nasossinusal. Esta inibição esteve associada à diminuição da translocação nuclear de NF-kappaB, demonstrando que este é um importante mecanismo de ação dos glicocorticoide na polipose nasossinusal.

Low mRNA expression of the apoptosis-related genes CASP3, CASP8, and FAS is associated with low induction treatment response in childhood acute lymphoblastic leukemia (ALL).

BACKGROUND: Defects in apoptosis signaling have been considered to be responsible for treatment failure in many types of cancer, although with controversial results. The objective of the present study was to assess the expression profile of key apoptosis-related genes in terms of clinical and biological variables and of the survival of children with acute lymphoblastic leukemia (ALL). PROCEDURE: The levels of mRNA expression of the apoptosis-related genes CASP3, CASP8, CASP9, FAS, and BCL2 were analyzed by quantitative real-time PCR in consecutive samples from 139 consecutive children with ALL at diagnosis treated by the Brazilian protocol (GBTLI-ALL 99). Gene expression levels and clinical and biological features were compared by the Mann-Whitney test. Event-free survival (EFS) was calculated by Kaplan-Meier plots and log-rank test. RESULTS: A significant correlation was detected between CASP3, CASP8, CASP9, and FAS expression levels (P < 0.01) in ALL samples. Higher levels of BCL2 were significantly associated with white blood cell (WBC) count <50,000/mm(3) at diagnosis (P = 0.01) and low risk group classification (P = 0.008). Lower expression levels of CASP3, CASP8 and FAS gene were associated with a poor response at day 7 according the GBTLI-ALL 99 protocol (P = 0.03, P = 0.02 and P = 0.008, respectively). There was a relationship between FAS gene expression lower than the 75th percentile and lower 5-year EFS (P = 0.02). CONCLUSION: These findings suggest an association between lower expression levels of the pro-apoptotic genes and a poor response to induction therapy at day 7 and prognosis in childhood ALL.

mRNA expression profile of multidrug resistance genes in childhood acute lymphoblastic leukemia. Low expression levels associated with a higher risk of toxic death.

BACKGROUND: Increased activity of multidrug resistance (MDR) genes has been associated with treatment failure in acute leukemias, although with controversial reports. The objective of the present study was to assess the expression profile of the genes related to MDR: ABCB1, ABCC1, ABCC3, ABCG2, and LRP/MVP in terms of the clinical and biological variable and the survival of children with acute lymphoblastic leukemia (ALL). PROCEDURE: The levels of mRNA expression of the drug resistance genes ABCB1, ABCC1, ABCC3, ABCG2, and LRP/MVP were analyzed by quantitative real-time PCR using the median values as cut-off points, in consecutive samples from 140 children with ALL at diagnosis. RESULTS: Expression levels of the ABCG2 gene in the patient group as a whole (P = 0.05) and of the ABCG2 and ABCC1 genes in patients classified as being at high risk were associated with higher rates of 5-year event-free survival (EFS) (P = 0.04 and P = 0.01). Expression levels of the ABCG2 gene below the median were associated with a greater chance of death related to treatment toxicity for the patient group as a whole (P = 0.009) and expression levels below the median of the ABCG2 and ABCC1 genes were associated with a greater chance of death due to treatment toxicity for the high-risk group (P = 0.02 and P = 0.03, respectively). CONCLUSION: The present data suggest a low participation of the drug efflux genes in treatment failure in patients with childhood ALL. However, the low expression of some of these genes may be associated with a higher death risk related to treatment toxicity.

Differential expression of 12 histone deacetylase (HDAC) genes in astrocytomas and normal brain tissue: class II and IV are hypoexpressed in glioblastomas.

BACKGROUND: Glioblastoma is the most lethal primary malignant brain tumor. Although considerable progress has been made in the treatment of this aggressive tumor, the clinical outcome for patients remains poor. Histone deacetylases (HDACs) are recognized as promising targets for cancer treatment. In the past several years, HDAC inhibitors (HDACis) have been used as radiosensitizers in glioblastoma treatment. However, no study has demonstrated the status of global HDAC expression in gliomas and its possible correlation to the use of HDACis. The purpose of this study was to evaluate and compare mRNA and protein levels of class I, II and IV of HDACs in low grade and high grade astrocytomas and normal brain tissue and to correlate the findings with the malignancy in astrocytomas. METHODS: Forty-three microdissected patient tumor samples were evaluated. The histopathologic diagnoses were 20 low-grade gliomas (13 grade I and 7 grade II) and 23 high-grade gliomas (5 grade III and 18 glioblastomas). Eleven normal cerebral tissue samples were also analyzed (54 total samples analyzed). mRNA expression of class I, II, and IV HDACs was studied by quantitative real-time polymerase chain reaction and normalized to the housekeeping gene beta-glucuronidase. Protein levels were evaluated by western blotting. RESULTS: We found that mRNA levels of class II and IV HDACs were downregulated in glioblastomas compared to low-grade astrocytomas and normal brain tissue (7 in 8 genes, p < 0.05). The protein levels of class II HDAC9 were also lower in high-grade astrocytomas than in low-grade astrocytomas and normal brain tissue. Additionally, we found that histone H3 (but not histone H4) was more acetylated in glioblastomas than normal brain tissue. CONCLUSION: Our study establishes a negative correlation between HDAC gene expression and the glioma grade suggesting that class II and IV HDACs might play an important role in glioma malignancy. Evaluation of histone acetylation levels showed that histone H3 is more acetylated in glioblastomas than normal brain tissue confirming the downregulation of HDAC mRNA in glioblastomas.

Gene expression profile analysis of primary glioblastomas and non-neoplastic brain tissue: identification of potential target genes by oligonucleotide microarray and real-time quantitative PCR.

The prognosis of glioblastomas is still extremely poor and the discovery of novel molecular therapeutic targets can be important to optimize treatment strategies. Gene expression analyses comparing normal and neoplastic tissues have been used to identify genes associated with tumorigenesis and potential therapeutic targets. We have used this approach to identify differentially expressed genes between primary glioblastomas and non-neoplastic brain tissues. We selected 20 overexpressed genes related to cell cycle, cellular movement and growth, proliferation and cell-to-cell signaling and analyzed their expression levels by real time quantitative PCR in cDNA obtained from microdissected fresh tumor tissue from 20 patients with primary glioblastomas and from 10 samples of non-neoplastic white matter tissue. The gene expression levels were significantly higher in glioblastomas than in non-neoplastic white matter in 18 out of 20 genes analyzed: P < 0.00001 for CDKN2C, CKS2, EEF1A1, EMP3, PDPN, BNIP2, CA12, CD34, CDC42EP4, PPIE, SNAI2, GDF15 and MMP23b; and NFIA (P: 0.0001), GPS1 (P: 0.0003), LAMA1 (P: 0.002), STIM1 (P: 0.006), and TASP1 (P: 0.01). Five of these genes are located in contiguous loci at 1p31-36 and 2 at 17q24-25 and 8 of them encode surface membrane proteins. PDPN and CD34 protein expression were evaluated by immunohistochemistry and they showed concordance with the PCR results. The present results indicate the presence of 18 overexpressed genes in human primary glioblastomas that may play a significant role in the pathogenesis of these tumors and that deserve further functional investigation as attractive candidates for new therapeutic targets.

Ocular cysticercosis of the anterior segment.

A 6-year-old girl presented with signs of severe anterior uveitis. After initiating treatment, a cyst like lesion was observed in the anterior chamber that led to the diagnosis of ocular cysticercosis that was ultimately confirmed with histopathologic analysis. Ocular cysticercosis usually affects the extraocular muscle. Infection of the anterior chamber has been described, although to a lesser extent. Because of the usually poor visual prognosis and the controversy in treatment, physicians should be aware of this disease and its different variants of presentation.