Background: Tobacco is the main risk factor for developing lung cancer. Yet, some heavy smokers do not develop lung cancer at advanced ages while others develop it at young ages. Here, we assess for the first time the genetic background of these clinically relevant extreme phenotypes using whole exome sequencing (WES). Methods: We performed WES of germline DNA from heavy smokers who either developed lung adenocarcinoma at an early age ( extreme cases, n=50) or did not present lung adenocarcinoma or other tumors at an advanced age (extreme controls, n=50). We selected non-synonymous variants located in exonic regions and consensus splice sites of the genes that showed significantly different allelic frequencies between both cohorts. We validated our results in all the additional extreme cases (i.e., heavy smokers who developed lung adenocarcinoma at an early age) available from The Cancer Genome Atlas (TCGA). Results: The mean age for the extreme cases and controls was respectively 49.7 and 77.5 years. Mean tobacco consumption was 43.6 and 56.8 pack-years. We identified 619 significantly different variants between both cohorts, and we validated 108 of these in extreme cases selected from TCGA. Nine validated variants, located in relevant cancer related genes, such as PARP4, HLA-A or NQO1, among others, achieved statistical significance in the False Discovery Rate test. The most significant validated variant (P=4.48x10(-5)) was located in the tumor-suppressor gene ALPK2. Conclusions: We describe genetic variants associated with extreme phenotypes of high and low risk for the development of tobacco-induced lung adenocarcinoma. Our results and our strategy may help to identify high-risk subjects and to develop new therapeutic strategies.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

Background The immune response to cancer is often conceptualized with the cancer immunity cycle. An essential step in this interpretation is that antigens released by dying tumors are presented by dendritic cells to naive or memory T cells in the tumor-draining lymph nodes. Whether tumor cell death resulting from cytotoxicity, as mediated by T cells or natural killer (NK) lymphocytes, is actually immunogenic currently remains unknown. Methods In this study, tumor cells were killed by antigen-specific T-cell receptor (TCR) transgenic CD8 T cells or activated NK cells. Immunogenic cell death was studied analyzing the membrane exposure of calreticulin and the release of high mobility group box 1 (HMGB1) by the dying tumor cells. Furthermore, the potential immunogenicity of the tumor cell debris was evaluated in immunocompetent mice challenged with an unrelated tumor sharing only one tumor-associated antigen and by class I major histocompatibility complex (MHC)-multimer stainings. Mice deficient in Batf3, Ifnar1 and Sting1 were used to study mechanistic requirements. Results We observe in cocultures of tumor cells and effector cytotoxic cells, the presence of markers of immunogenic cell death such as calreticulin exposure and soluble HMGB1 protein. Ovalbumin (OVA)-transfected MC38 colon cancer cells, exogenously pulsed to present the gp100 epitope are killed in culture by mouse gp100-specific TCR transgenic CD8 T cells. Immunization of mice with the resulting destroyed cells induces epitope spreading as observed by detection of OVA-specific T cells by MHC multimer staining and rejection of OVA(+) EG7 lymphoma cells. Similar results were observed in mice immunized with cell debris generated by NK-cell mediated cytotoxicity. Mice deficient in Batf3-dependent dendritic cells (conventional dendritic cells type 1, cDC1) fail to develop an anti-OVA response when immunized with tumor cells killed by cytotoxic lymphocytes. In line with this, cultured cDC1 dendritic cells uptake and can readily cross-present antigen from cytotoxicity-killed tumor cells to cognate CD8(+) T lymphocytes. Conclusion These results support that an ongoing cytotoxic antitumor immune response can lead to immunogenic tumor cell death.

Checkpoint inhibitors have improved the survival of patients with advanced tumors and show a manageable toxicity profile. However, auto-immune colitis remains a relevant side effect, and combinations of anti-PD1/PDL1 and anti-CTLA-4 increase its incidence and severity. Here, we report the case of a 50-year-old patient diagnosed with stage IV cervical cancer that relapsed following radical surgery, external radiation/brachytherapy and standard chemotherapy. She was subsequently treated with Nivolumab and Ipilimumab combination and developed grade 2 colitis presenting a dissociation between endoscopic and pathological findings. At cycle 10 the patient reported grade 3 diarrhea and abdominal discomfort, without blood or mucus in the stools. Immunotherapy was withheld and a colonoscopy was performed, showing normal mucosa in the entire colon. Puzzlingly, histologic evaluation of randomly sampled mucosal biopsy of the distal colon showed an intense intraepithelial lymphocyte infiltration with crypt loss and some regenerating crypts with a few apoptotic bodies set in a chronically inflamed lamina propria, consistent with the microscopic diagnosis of colitis. Treatment with methylprednisolone 2 mg/kg was initiated which led to a decrease in the number of stools to grade 1. Additional investigations to exclude other causes of diarrhea rendered negative results. The patient experienced a major partial response and, following the resolution of diarrhea, she was re-challenged again with

Intratumoral therapies, especially Toll-like receptor agonists, can trigger both the innate and adaptive immune systems. BO-112 is a nanoplexed form of polyinosinic:polycytidylic acid (poly I:C) that induces local and systemic immunotherapeutic effects in mouse models. In a multicenter phase 1 clinical trial, repeated intratumoral administrations of BO-112 induced an increase in tumor cell necrosis and apoptosis, as well as augmented immune reactivity according to gene expression profiling. The first three cohorts receiving BO-112 as a monotherapy resulted in a recommended dose of 1 mg that could be safely repeated. Two grade 3 to 4 adverse reactions in the form of reversible thrombocytopenia were reported. In a fourth cohort of 28 patients with tumors that had primary resistance to anti-programmed cell death protein-1 (PD-1), the combination of intratumoral BO-112 with nivolumab or pembrolizumab was also well tolerated, and 3 patients (2 with melanoma and 1 with renal cell carcinoma) achieved partial responses, with 10 more patients having stable disease at 8 to 12 weeks. Thus, local BO-112 combined with a systemic anti-PD-1 agent might be a strategy to revert anti-PD-1 resistance.

Caspases are known for their ability to precipitate apoptosis. Our findings indicate that accelerating the terminal inactivation of cells dying in response to radiation therapy limit their immunogenicity as a consequence of reduced type I interferon secretion. Thus, caspase inhibitors stand out as promising combinatorial partners to improve the immunogenicity of radiation therapy in the clinic.

Caspase 3 (CASP3) has a key role in the execution of apoptosis, and many cancer cells are believed to disable CASP3 as a mechanism of resistance to cytotoxic therapeutics. Alongside, CASP3 regulates stress-responsive immunomodulatory pathways, including secretion of type I interferon (IFN). Here, we report that mouse mammary carcinoma TSA cells lacking Casp3 or subjected to chemical caspase inhibition were as sensitive to the cytostatic and cytotoxic effects of radiation therapy (RT) in vitro as their control counterparts, yet secreted increased levels of type I IFN. This effect originated from the accrued accumulation of irradiated cells with cytosolic DNA, likely reflecting the delayed breakdown of cells experiencing mitochondrial permeabilization in the absence of CASP3. Casp3(-/-) TSA cells growing in immunocompetent syngeneic mice were more sensitive to RT than their CASP3-proficient counterparts, and superior at generating bona fide abscopal responses in the presence of an immune checkpoint blocker. Finally, multiple genetic signatures of apoptotic proficiency were unexpectedly found to have robust negative (rather than positive) prognostic significance in a public cohort of breast cancer patients. However, these latter findings were not consistent with genetic signatures of defective type I IFN signaling, which were rather associated with improved prognosis. Differential gene expression analysis on patient subgroups with divergent prognosis (as stratified by independent signatures of apoptotic proficiency) identified SLC7A2 as a new biomarker with independent prognostic value in breast cancer patients. With the caveats associated with the retrospective investigation of heterogeneous, public databases, our data suggest that apoptotic caspases may influence the survival of breast cancer patients (or at least some subsets thereof) via mechanisms not necessarily related to type I IFN signaling as they identify a novel independent prognostic biomarker that awaits prospective validation.

Sunitinib is one of the most widely used targeted therapeutics for renal cell carcinoma (RCC), but acquired resistance against targeted therapies remains a major clinical challenge. To dissect mechanisms of acquired resistance and unravel reliable predictive biomarkers for sunitinib in RCC, we sequenced the exons of 409 tumor-suppressor genes and oncogenes in paired tumor samples from an RCC patient, obtained at baseline and after development of acquired resistance to sunitinib. From newly arising mutations, we selected, using in silico prediction models, six predicted to be deleterious, located in G6PD, LRP1B, SETD2, TET2, SYNE1, and DCC. Consistently, immunoblotting analysis of lysates derived from sunitinib-desensitized RCC cells and their parental counterparts showed marked differences in the levels and expression pattern of the proteins encoded by these genes. Our further analysis demonstrates essential roles for these proteins in mediating sunitinib cytotoxicity and shows that their loss of function renders tumor cells resistant to sunitinib in vitro and in vivo. Finally, sunitinib resistance induced by continuous exposure or by inhibition of the six proteins was overcome by treatment with cabozantinib or a low-dose combination of lenvatinib and everolimus. Collectively, our results unravel novel markers of acquired resistance to sunitinib and clinically relevant approaches for overcoming this resistance in RCC.

Sunitinib is one of the most widely used targeted therapeutics for renal cell-cancer (RCC) but acquired resistance against targeted therapies remains a major clinical challenge. To dissect mechanisms of acquired resistance and unravel reliable predictive biomarkers for sunitinib in renal cell-cancer (RCC), we sequenced the exons of 409 tumor-suppressor genes and oncogenes in paired tumor samples from an RCC patient, obtained at baseline and following development of acquired resistance to sunitinib. From newly arising mutations, we selected, using in-silico prediction models, 6 predicted to be deleterious, located in G6PD, LRP1B, SETD2, TET2, SYNE1 and DCC. Consistently, immunoblotting analysis of lysates derived from sunitinib-desensitized RCC cells and their parental counterparts showed marked differences in the levels and expression pattern of the proteins encoded by these genes. Our further analysis demonstrates essential roles for these proteins in mediating sunitinib cytotoxicity and shows that their loss of function render tumor cells resistant to sunitinib in vitro and in vivo. Finally, sunitinib resistance induced by continuous exposure or by inhibition of the 6 proteins was overcome by treatment with cabozantinib or a low-dose combination of lenvatinib and everolimus. Collectively, our results unravel novel markers of acquired resistance to sunitinib and clinically relevant approaches for overcoming this resistance in RCC.

Poly I:C is a powerful immune adjuvant as a result of its agonist activities on TLR-3, MDA5 and RIG-I. BO-112 is a nanoplexed formulation of Poly I:C complexed with polyethylenimine that causes tumor cell apoptosis showing immunogenic cell death features and which upon intratumoral release results in more prominent tumor infiltration by T lymphocytes. Intratumoral treatment with BO-112 of subcutaneous tumors derived from MC38, 4T1 and B16-F10 leads to remarkable local disease control dependent on type-1 interferon and gamma-interferon. Some degree of control of non-injected tumor lesions following BO-112 intratumoral treatment was found in mice bearing bilateral B16-OVA melanomas, an activity which was enhanced with co-treatment with systemic anti-CD137 and anti-PD-L1 mAbs. More abundant CD8(+) T lymphocytes were found in B16-OVA tumor-draining lymph nodes and in the tumor microenvironment following intratumoral BO-112 treatment, with enhanced numbers of tumor antigen-specific cytotoxic T lymphocytes. Genome-wide transcriptome analyses of injected tumor lesions were consistent with a marked upregulation of the type-I interferon pathway. Inspired by these data, intratumorally delivered BO-112 is being tested in cancer patients (NCT02828098).

Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.

Radiotherapy can be synergistically combined with immunotherapy in mouse models, extending its efficacious effects outside of the irradiated field (abscopal effects). We previously reported that a regimen encompassing local radiotherapy in combination with anti-CD137 plus anti-PD-1 mAbs achieves potent abscopal effects against syngeneic transplanted murine tumors up to a certain tumor size. Knowing that TGF beta expression or activation increases in irradiated tissues, we tested whether TGF beta blockade may further enhance abscopal effects in conjunction with the anti-PD-1 plus anti-CD137 mAb combination. Indeed, TGF beta blockade with 1D11, a TGF beta-neutralizing mAb, markedly enhanced abscopal effects and overall treatment efficacy against subcutaneous tumors of either 4T1 breast cancer cells or large MC38 colorectal tumors. Increases in CD8 T cells infiltrating the nonirradiated lesion were documented upon combined treatment, which intensely expressed Granzyme-B as an indicator of cytotoxic effector capability. Interestingly, tumor tissue but not healthy tissue irradiation results in the presence of higher concentrations of TGF beta in the nonirradiated contralateral tumor that showed smad2/3 phosphorylation increases in infiltrating CD8 T cells. In conclusion, radiotherapy-induced TGF beta hampers abscopal efficacy even upon combination with a potent immunotherapy regimen. Therefore, TGF beta blockade in combination with radioimmunotherapy results in greater efficacy.

Surgically resectable synchronic and metachronic liver metastases of colon cancer have high risk of relapse in spite of standard-of-care neoadjuvant and adjuvant chemotherapy regimens. Dendritic cell vaccines loaded with autologous tumor lysates were tested for their potential to avoid or delay disease relapses (NCT01348256). Patients with surgically amenable liver metastasis of colon adenocarcinoma (n = 19) were included and underwent neoadjuvant chemotherapy, surgery and adjuvant chemotherapy. Fifteen patients with disease-free resection margins were randomized 1: 1 to receive two courses of four daily doses of dendritic cell intradermal vaccinations versus observation. The trial had been originally designed to include 56 patients but was curtailed due to budgetary restrictions. Follow-up of the patients indicates a clear tendency to fewer and later relapses in the vaccine arm (median disease free survival -DFS-) 25.26 months, 95% CI 8. 74-n.r) versus observation arm (median DFS 9.53 months, 95% CI 5.32-18.88).

Background: Combination immunotherapy has the potential to achieve additive or synergistic effects. Combined local injections of dsRNA analogues (mimicking viral RNA) and repeated vaccinations with tumor-lysate loaded dendritic cells shows efficacy against colon cancer mouse models. In the context of immunotherapy, radiotherapy can exert beneficial abscopal effects. Patients and methods: In this two-cohort pilot phase I study, 15 advanced cancer patients received two 4-week cycles of four intradermal daily doses of monocyte-derived dendritic cells preloaded with autologous tumor lysate and matured for 24 h with poly-ICLC (Hiltonol), TNF-alpha and IFN-alpha. On days +8 and +10 of each cycle, patients received intratumoral image-guided 0.25mg injections of the dsRNA-analogue Hiltonol. Cyclophosphamide 600 mg/m(2) was administered 1 week before. Six patients received stereotactic ablative radiotherapy (SABR) on selected tumor lesions, including those injected with Hiltonol. Expression of 25 immune-relevant genes was sequentially monitored by RT-PCR on circulating peripheral blood mononuclear cell (PBMCs) and serum concentrations of a cytokine panel were sequentially determined before and during treatment. Pre-and posttreatment PBMC from patients achieving durable stable disease (SD) were studied by IFNc ELISPOT-assays responding to tumor-lysate loaded DC and by TCR beta sequencing. Results: Combined treatment was, safe and well tolerated. One heavily pretreated castration-resistant prostate cancer patient experienced a remarkable mixed abscopal response to SABR+ immunotherapy. No objective responses were observed, while nine patients presented SD (five of them in the six-patient radiotherapy cohort). Intratumoral Hiltonol increased IFN-beta and IFN-alpha mRNA in circulating PBMC. DC vaccination increased serum IL-12 and IL-1 beta concentrations, especially in patients presenting SD. IFNc-ELISPOT reactivity to tumor lysates was observed in two patients experiencing durable SD. Conclusions: This radio-immunotherapy combination strategy, aimed at resembling viral infection in tumor tissue in combination with a dendritic-cell vaccine and SABR, is safe and shows immune-associated activity and signs of preliminary clinical efficacy.

TRAF2 dependent K63-polyubiquitinations have been recently shown to connect CD137 (4-1BB) stimulation to NF kappa B activation. In a search of deubiquitinase enzymes (DUBs) that could regulate such a signaling route, A20 and CYLD were found to coimmunoprecipitate with CD137 and TRAF2 complexes. Indeed, overexpression of A20 or CYLD downregulated CD137-elicited ubiquitination of TRAF2 and TAK1 upon stimulation with agonist monoclonal antibodies. Moreover, overexpression of A20 or CYLD downregulated CD137-induced NF kappa B activation in cultured cells and in gene-transferred hepatocytes in vivo, while silencing these deubiquitinases enhanced CD137 costimulation of primary human CD8 T cells. Therefore A20 and CYLD directly downregulate the signaling from a T and NK-cell costimulatory receptor under exploitation for cancer immunotherapy in clinical trials.

Single nucleotide polymorphisms (SNPs) may modulate individual susceptibility to carcinogens. We designed a genome-wide association study to characterize individuals presenting extreme phenotypes of high and low risk to develop tobacco-induced non-small cell lung cancer (NSCLC), and we validated our results. We hypothesized that this strategy would enrich the frequencies of the alleles that contribute to the observed traits. We genotyped 2.37 million SNPs in 95 extreme phenotype individuals, that is: heavy smokers that either developed NSCLC at an early age (extreme cases); or did not present NSCLC at an advanced age (extreme controls), selected from a discovery set (n=3631). We validated significant SNPs in 133 additional subjects with extreme phenotypes selected from databases including >39,000 individuals. Two SNPs were validated: rs12660420 (p(combined)=5.66x10(-5); ORcombined=2.80), mapping to a noncoding transcript exon of PDE10A; and rs6835978 (p(combined)=1.02x10(-4); ORcombined=2.57), an intronic variant in ATP10D. We assessed the relevance of both proteins in early-stage NSCLC. PDE10A and ATP10D mRNA expressions correlated with survival in 821 stage I-II NSCLC patients (p=0.01 and p<0.0001). PDE10A protein expression correlated with survival in 149 patients with stage I-II NSCLC (p=0.002). In conclusion, we validated two variants associated with extreme phenotypes of high and low risk of developing tobacco-induced NSCLC. Our findings may allow to identify individuals presenting high and low risk to develop tobacco-induced NSCLC and to characterize molecular mechanisms of carcinogenesis and resistance to develop NSCLC.

Multiple lines of evidence indicate a critical role of antigen cross-presentation by conventional BATF3-dependent type 1 classical dendritic cells (cDC1) in CD8-mediated antitumor immunity. Flt3L and XCL1, respectively, constitute a key growth/differentiation factor and a potent and specific chemoattractant for cDC1. To exploit their antitumor functions in local immunotherapy, we prepared Semliki Forest Virus (SFV)-based vectors encoding XCL1 and soluble Flt3L (sFlt3L). These vectors readily conferred transgene expression to the tumor cells in culture and when engrafted as subcutaneous mouse tumor models. In syngeneic mice, intratumoral injection of SFV-XCL1-sFlt3L (SFV-XF) delayed progression of MC38-and B16-derived tumors. Therapeutic activity was observed and exerted additive effects in combination with anti-PD-1, anti-CD137, or CTLA-4 immunostimulatory mAbs. Therapeutic effects were abolished by CD8 beta T-cell depletion and were enhanced by CD4 T-cell depletion, but not by T regulatory cell predepletion with anti-CD25 mAb. Antitumor effects were also abolished in BATF3- and IFNARdeficient mice. In B16-OVA tumors, SFV-XF increased the number of infiltratingCD8T cells, including those recognizing OVA. Consistently, following the intratumoral SFV-XF treatment courses, we observed increased BATF3-dependent cDC1 among B16-OVA tumor-infiltrating leukocytes. Such an intratumoral increase was not seen in MC38-derived tumors, but both resident and migratory cDC1 were boosted in SFV-XF-treated MC38 tumor-draining lymph nodes. In conclusion, viral gene transfer of sFlt3L and XCL1 is feasible, safe, and biologically active in mice, exerting antitumor effects that can be potentiated by CD4 T-cell depletion. Significance: These findings demonstrate that transgenic expression of sFLT3L and XCL1 in tumor cells mediates crosspriming of, and elicits potent antitumor activity from, CD8 T lymphocytes, particularly in combination with CD4 T-cell depletion. (C) 2018 AACR.

T-cell costimulation and coinhibition can be respectively exploited by blocking and agonist mAbs. Both strategies can be synergistically combined in mouse models. Early clinical results from combinations of anti-PD-1 mAbs in conjunc-tion with agonist anti-CD137 (4-1BB) mAbs show excellent safety and promising efficacy. (C) 2017 AACR.

PURPOSE/OBJECTIVES: Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory monoclonal antibodies (mAb) to act both on irradiated tumor lesions and on tumors at distant, nonirradiated sites. We have recently reported that external beam radiotherapy achieves abscopal effects when combined with antagonist anti-PD1 mAbs and agonist anti-CD137 (4-1BB) mAbs. The goal of this work is to study the abscopal effects of radiotherapy instigated by brachytherapy techniques. METHODS AND MATERIALS: Mice bearing a subcutaneous colorectal carcinoma, MC38 (colorectal cancer), in both flanks were randomly assigned to receive brachytherapy or not (8 Gy × three fractions) to only one of the two grafted tumors, in combination with intraperitoneal immunostimulatory monoclonal antibodies (anti-PD1, anti-CD137, and/or their respective isotype controls). To study the abscopal effects of brachytherapy, we established an experimental set up that permits irradiation of mouse tumors sparing a distant site resembling metastasis. Such second nonirradiated tumor was used as indicator of abscopal effect. Tumor size was monitored every 2 days. RESULTS: Abscopal effects on distant nonirradiated subcutaneous tumor lesions of transplanted MC38-derived tumors only took place when brachytherapy was combined with immunostimulatory anti-PD1 and/or anti-CD137 mAbs. CONCLUSIONS: Our results demonstrate that immunotherapy-potentiated abscopal effects can be attained by brachytherapy. Accordingly, immunotherapy plus brachytherapy combinations are suitable for clinical translation.

Background: Surrogate biomarkers of efficacy are needed for anti-PD1/PD-L1 therapy, given the existence of delayed responses and pseudo-progressions. We evaluated changes in serum IL-8 levels as a biomarker of response to anti-PD-1 blockade in melanoma and non-small-cell lung cancer (NSCLC) patients. Patients and methods: Metastatic melanoma and NSCLC patients treated with nivolumab or pembrolizumab alone or nivolumab plus ipilimumab were studied. Serum was collected at baseline; at 2-4 weeks after the first dose; and at the time-points of response evaluation. Serum IL-8 levels were determined by sandwich ELISA. Changes in serum IL-8 levels were compared with the Wilcoxon test and their strength of association with response was assessed with the Mann-Whitney test. Accuracy of changes in IL-8 levels to predict response was estimated using receiver operation characteristics curves. Results: Twenty-nine melanoma patients treated with nivolumab or pembrolizumab were studied. In responding patients, serum IL-8 levels significantly decreased between baseline and best response (P < 0.001), and significantly increased upon progression (P = 0.004). In non-responders, IL-8 levels significantly increased between baseline and progression (P = 0.013). Early changes in serum IL-8 levels (2-4 weeks after treatment initiation) were strongly associated with response (P < 0.001). These observations were validated in 19 NSCLC patients treated with nivolumab or pembrolizumab (P = 0.001), and in 15 melanoma patients treated with nivolumab plus ipilimumab (P < 0.001). Early decreases in serum IL-8 levels were associated with longer overall survival in melanoma (P = 0.001) and NSCLC (P = 0.015) patients. Serum IL-8 levels also correctly reflected true response in three cancer patients presenting pseudoprogression. Conclusions: Changes in serum IL-8 levels could be used to monitor and predict clinical benefit from immune checkpoint blockade in melanoma and NSCLC patients.

Purpose/Objectives: The goal of this study was to assess the effects of ionizing radiation on the expression of the integrin ligands ICAM-1 and VCAM that control leucocyte transit by lymphatic endothelial cells. Materials/Methods: Confluent monolayers of primary human lymphatic endothelial cells (LEC) were irradiated with single dose of 2, 5, 10 or 20 Gy, with 6 MeV-x-rays using a Linear-Accelerator. ICAM-1 and VCAM expression was determined by flow cytometry. Human tissue specimens received a single dose of 20 Gy with 15 MeV-x-rays. MC38, B16-OVA or B16-VEGF-C tumors grown in C57BL/6 mice were irradiated with single dose of 20Gy using a Linear-Accelerator fitted with a 10mm Radiosurgery collimator. Clinical samples were obtained from patients previous and 4 weeks after complete standard radiotherapy. ICAM-1 and VCAM expression was detected in all tissue specimens by confocal microscopy. To understand the role of TGF beta in this process anti-TGF beta blocking mAb were injected i.p. 30min before radiotherapy. Cell adhesion to irradiated LEC was analyzed in adhesion experiments performed in the presence or in the absence of anti- TGF beta and /or anti-ICAM1 blocking mAb. Results: We demonstrate that lymphatic endothelial cells in tumor samples experience induction of surface ICAM-1 and VCAM when exposed to ionizing radiation in a dose- and time-dependent manner. These effects can be recapitulated in cultured LEC, and are in part mediated by TGF beta. These data are consistent with increases in ICAM-1 and VCAM expression on LYVE-1+ endothelial cells in freshly explanted human tumor tissue and in mouse transplanted tumors after radiotherapy. Finally, ICAM-1 and VCAM expression accounts for enhanced adherence of human T lymphocytes to irradiated LEC. Conclusion: Our results show induction of ICAM-1 and VCAM on LVs in irradiated lesions and offer a starting point for elucidating the biological and therapeutic implications of targeting leukocyte traffic in combination to immunotherapy. (C) 2016 Elsevier Inc. All rights reserved.

Immune mechanisms have evolved to cope with local entry of microbes acting in a confined fashion but eventually inducing systemic immune memory. Indeed, in situ delivery of a number of agents into tumors can mimic in the malignant tissue the phenomena that control intracellular infection leading to the killing of infected cells. Vascular endothelium activation and lymphocyte attraction, together with dendritic cell-mediated cross-priming, are the key elements. Intratumoral therapy with pathogen-associated molecular patterns or recombinant viruses is being tested in the clinic. Cell therapies can be also delivered intratumorally, including infusion of autologous dendritic cells and even tumor-reactive T lymphocytes. Intralesional virotherapy with an HSV vector expressing GM-CSF has been recently approved by the Food and Drug Administration for the treatment of unresectable melanoma. Immunomodulatory monoclonal Abs have also been successfully applied intratumorally in animal models. Local delivery means less systemic toxicity while focusing the immune response on the malignancy and the affected draining lymph nodes.

Preclinical and clinical evidence indicate that the proimmune effects of radiotherapy can be synergistically augmented with immunostimulatory mAbs to act both on irradiated tumor lesions and on distant, nonirradiated tumor sites. The combination of radiotherapy with immunostimulatory anti-PD1 and anti-CD137 mAbs was conducive to favorable effects on distant nonirradiated tumor lesions as observed in transplanted MC38 (colorectal cancer), B16OVA (melanoma), and 4T1 (breast cancer) models. The therapeutic activity was crucially performed by CD8 T cells, as found in selective depletion experiments. Moreover, the integrities of BATF-3-dependent dendritic cells specialized in crosspresentation/crosspriming of antigens to CD8+ T cells and of the type I IFN system were absolute requirements for the antitumor effects to occur. The irradiation regimen induced immune infiltrate changes in the irradiated and nonirradiated lesions featured by reductions in the total content of effector T cells, Tregs, and myeloid-derived suppressor cells, while effector T cells expressed more intracellular IFN¿ in both the irradiated and contralateral tumors. Importantly, 48 hours after irradiation, CD8+ TILs showed brighter expression of CD137 and PD1, thereby displaying more target molecules for the corresponding mAbs. Likewise, PD1 and CD137 were induced on tumor-infiltrating lymphocytes from surgically excised human carcinomas that were irradiated ex vivo These mechanisms involving crosspriming and CD8 T cells advocate clinical development of immunotherapy combinations with anti-PD1 plus anti-CD137 mAbs that can be synergistically accompanied by radiotherapy strategies, even if the disease is left outside the field of irradiation.

D137(4-1BB) costimulation and adoptive T cell therapy strongly synergize in terms of achieving maximal efficacy against experimental cancers. These costimulatory biological functions of CD137 have been exploited by means of introducing the CD137 signaling domain in clinically successful chimeric antigen receptors and to more efficiently expand T cells in culture. In addition, immunomagnetic sorting of CD137-positive T cells among tumor-infiltrating lymphocytes selects for the fittest antitumor T lymphocytes for subsequent cultures. In mouse models, co-infusion of both agonist antibodies and T cells attains marked synergistic effects that result from more focused and intense cytolytic activity visualized under in vivo microscopy and from more efficient entrance of T cells into the tumor through the vasculature. These several levels of dynamic interaction between adoptive T cell therapy and CD137 offer much opportunity to raise the efficacy of current cancer immunotherapies.

Weak and ineffective antitumor cytotoxic T lymphocyte (CTL) responses can be rescued by immunomodulatory mAbs targeting PD-1 or CD137. Using Batf3(-/-) mice, which are defective for cross-presentation of cell-associated antigens, we show that BATF3-dependent dendritic cells (DC) are essential for the response to therapy with anti-CD137 or anti-PD-1 mAbs. Batf3(-/-) mice failed to prime an endogenous CTL-mediated immune response toward tumor-associated antigens, including neoantigens. As a result, the immunomodulatory mAbs could not amplify any therapeutically functional immune response in these mice. Moreover, administration of systemic sFLT3L and local poly-ICLC enhanced DC-mediated cross-priming and synergized with anti-CD137- and anti-PD-1-mediated immunostimulation in tumor therapy against B16-ovalbumin-derived melanomas, whereas this function was lost in Batf3(-/-) mice. These experiments show that cross-priming of tumor antigens by FLT3L- and BATF3-dependent DCs is crucial to the efficacy of immunostimulatory mAbs and represents a very attractive point of intervention to enhance their clinical antitumor effects. Significance: Immunotherapy with immunostimulatory mAbs is currently achieving durable clinical responses in different types of cancer. We show that cross-priming of tumor antigens by BATF3-dependent DCs is a key limiting factor that can be exploited to enhance the antitumor efficacy of anti-PD-1 and anti-CD137 immunostimulatory mAbs.

Immunotherapy strategies against cancer are emerging as powerful weapons for treatment of this disease. The success of checkpoint inhibitors against metastatic melanoma and adoptive T-cell therapy with chimeric antigen receptor T cells against B-cell-derived leukemias and lymphomas are only two examples of developments that are changing the paradigms of clinical cancer management. These changes are a result of many years of intense research into complex and interrelated cellular and molecular mechanisms controling immune responses. Promising advances come from the discovery of cancer mutation-encoded neoantigens, improvements in vaccine development, progress in delivery of cellular therapies, and impressive achievements in biotechnology. As a result, radical transformation of cancer treatment is taking place in which conventional cancer treatments are being integrated with immunotherapeutic agents. Many clinical trials are in progress testing potential synergistic effects of treatments combining immunotherapy with other therapies. Much remains to be learned about the selection, delivery, and off-target effects of immunotherapy used alone or in combination. The existence of numerous escape mechanisms from the host immune system that human tumors have evolved still is a barrier to success. Efforts to understand the rules of immune cell dysfunction and of cancer-associated local and systemic immune suppression are providing new insights and fuel the enthusiasm for new therapeutic strategies. In the future, it might be possible to tailor immune therapy for each cancer patient. The use of new immune biomarkers and the ability to assess responses to therapy by noninvasive monitoring promise to improve early cancer diagnosis and prognosis. Personalized immunotherapy based on individual genetic, molecular, and immune profiling is a potentially achievable future goal. The current excitement for immunotherapy is justified in view of many existing opportunities for harnessing the immune system to treat cancer.

The recent approval by the FDA of the combination of anti-CTLA4 and anti-PD-1 mAbs for the treatment of BRAF-unmutated unresectable or metastatic melanoma is a landmark for the development of cancer immunotherapy. On October 18 to 22, 2015, a symposium was held in Pamplona (Spain) to present and discuss the basic and clinical discoveries that have brought us to this milestone and to explore other targets and immunotherapy strategies aimed at attaining more efficacious oncology practice in the short term.

Surgery remains our strongest treatment pillar against early stages of cancer. In a number of instances, the curative potential of surgery can be enhanced by treatments given before (neoadjuvant) or after (adjuvant) surgical procedures. Immunomodulation has emerged as a powerful tool to fight metastatic disease across cancer histologies and goes now to be tested at earlier surgically amenable stages. The work by Liu and colleagues in this issue provides solid preclinical evidence in support of neoadjuvant immunotherapy over adjuvant approaches.

INTRODUCTION: Anti-programmed cell death 1 (anti-PD-1) and anti-programmed cell death ligand 1 (PD-L1) antagonist monoclonal antibodies (mAbs) against metastatic non-small cell lung cancer with special efficacy in patients with squamous cell lung cancer are being developed in the clinic. However, robust and reliable experimental models to test immunotherapeutic combinations in squamous lung tumors are still lacking. METHODS: We generated a transplantable squamous cell carcinoma cell line (UN-SCC680AJ) from a lung tumor induced by chronic N-nitroso-tris-chloroethylurea mutagenesis in A/J mice. Tumor cells expressed cytokeratins, overexpressed p40, and lacked thyroid transcription factor 1, confirming the squamous lineage reported by histological analysis. More than 200 mutations found in its exome suggested potential for antigenicity. Immunocompetent mice subcutaneously implanted with this syngeneic cell line were treated with anti-CD137 and/or anti-PD-1 mAbs and monitored for tumor growth/progression or assessed for intratumoral leukocyte infiltration using immunohistochemical analysis and flow cytometry. RESULTS: In syngeneic mice, large 12-day-established tumors derived from the transplantable cell line variant UN-SCC680AJ were amenable to curative treatment with anti-PD-1, anti-PD-L1, or anti-CD137 immunostimulatory mAbs. Single-agent therapies lost curative efficacy when treatment was started beyond day +17, whereas a combination of anti-PD-1 plus anti-CD137 achieved complete rejections. Tumor cells expressed weak baseline PD-L1 on the plasma membrane, but this could be readily induced by interferon-¿. Combined treatment efficacy required CD8 T cells and induced a leukocyte infiltrate in which T lymphocytes co-expressing CD137 and PD-1 were prominent. CONCLUSIONS: These promising results advocate the use of combined anti-PD-1/PD-L1 plus anti-CD137 mAb immunotherapy for the treatment of squamous non-small cell lung cancer in the clinical setting.

Mutation analysis of epidermal growth factor receptor (EGFR) gene is essential for treatment selection in non-small cell lung cancer (NSCLC). Analysis is usually performed in tumor samples. We evaluated the clinical utility of EGFR analysis in plasma cell-free DNA (cfDNA) from patients under treatment with EGFR inhibitors. We selected 36 patients with NSCLC and EGFR-activating mutations. Blood samples were collected at baseline and during treatment with EGFR inhibitors. Wild-type EGFR, L858R, delE746-A750, and T790M mutations were quantified in cfDNA by droplet digital PCR. Stage IV patients had higher total circulating EGFR copy levels than stage I (3523 vs. 1003 copies/mL; p < 0.01). There was high agreement for activating mutations between baseline cfDNA and tumor samples, especially for L858R mutation (kappa index = 0.679; p = 0.001). In 34 % of advanced NSCLC patients, we detected mutations in cfDNA not previously detected in tumor samples and double mutations in 17 %. Patients with baseline total EGFR copy levels above the median presented decreased overall survival (OS) (341 vs. 870 days, p < 0.05) and progression-free survival (PFS) (238 vs. 783 days; p < 0.05) compared with those with total EGFR copy levels below the median. Patients with baseline concentrations of activating mutations above the median (94 copies/mL) had lower OS (317 vs. 805 days; p < 0.05) and PFS (195 vs. 724 days; p < 0.05). During follow-up, T790M resistance mutation was detected in 53 % of patients. Total and mutated EGFR analysis in cfDNA seems a relevant tool to characterize the molecular profile and prognosis of NSCLC patients harboring EGFR mutations.

CD137 (4-1BB) is a surface marker discovered on activated T lymphocytes. However, its expression pattern is broader and has also been described on activated NK cells, B-cells and myeloid cells including mature dendritic cells. In this study, we have immunostained for CD137 on paraffin-embedded lymphoid tissues including tonsils, lymph nodes, ectopic tertiary lymphoid tissue in Hashimoto thyroiditis and cancer. Surprisingly, immunostaining mainly decorates intrafollicular lymphocytes in the tissues analyzed, with only scattered staining in interfollicular areas. Moreover, pathologic lymphoid follicles in follicular lymphoma and tertiary lymphoid tissue associated to non-small cell lung cancer showed a similar pattern of immunostaining. Multicolor flow cytometry demonstrated that CD137 expression was restricted to CD4+ CXCR5+ follicular T helper lymphocytes in tonsils and lymph nodes. Short term culture of lymph node cell suspensions in the presence of an agonist anti-CD137 mAb or CD137-ligand results in the functional upregulation of TFH cells, including CD40L surface expression and cytokine production, in three out of six cases. As a consequence, immunostimulatory monoclonal antibodies, anti-CD137 mAb such as urelumab and PF-05082566 should be expected to primarily act on this lymphocyte subset, thus modifying ongoing humoral immune responses.

Hypoxia is a common feature in solid tumors that has been implicated in immune evasion. Previous studies from our group have shown that hypoxia upregulates the co-stimulatory receptor CD137 on activated T lymphocytes and on vascular endothelial cells. In this study, we show that exposure of mouse and human tumor cell lines to hypoxic conditions (1% O-2) promotes CD137 transcription. However, the resulting mRNA is predominantly an alternatively spliced form that encodes for a soluble variant, lacking the transmembrane domain. Accordingly, soluble CD137 (sCD137) is detectable by ELISA in the supernatant of hypoxia-exposed cell lines and in the serum of tumor-bearing mice. sCD137, as secreted by tumor cells, is able to bind to CD137-Ligand (CD137L). Our studies on primed T lymphocytes in co-culture with stable transfectants for CD137L demonstrate that tumor-secreted sCD137 prevents co-stimulation of T lymphocytes. Such an effect results from preventing the interaction of CD137L with the transmembrane forms of CD137 expressed on T lymphocytes undergoing activation. Indeed, silencing CD137 with shRNA renders more immunogenic tumor-cell variants upon inoculation to immunocompetent mice but which readily grafted on immunodeficient or CD8(+) T-cell-depleted mice. These mechanisms are interpreted as a molecular strategy deployed by tumors to repress lymphocyte co-stimulation via CD137/CD137L.

A current pressing need in cancer immunology is the development of preclinical model systems that are immunocompetent for the study of human tumors. Here, we report the development of a humanized murine model that can be used to analyze the pharmacodynamics and antitumor properties of immunostimulatory monoclonal antibodies (mAb) in settings where the receptors targeted by the mAbs are expressed. Human lymphocytes transferred into immunodeficient mice underwent activation and redistribution to murine organs, where they exhibited cell-surface expression of hCD137 and hPD-1. Systemic lymphocyte infiltrations resulted in a lethal CD4(+) T cell-mediated disease (xenograft-versus-host disease), which was aggravated when murine subjects were administered clinical-grade anti-hCD137 (urelumab) and anti-hPD-1 (nivolumab). In mice engrafted with human colorectal HT-29 carcinoma cells and allogeneic human peripheral blood mononuclear cells (PBMC), or with a patient-derived gastric carcinoma and PBMCs from the same patient, we found that coadministration of urelumab and nivolumab was sufficient to significantly slow tumor growth. Correlated with this result were increased numbers of activated human T lymphocytes producing IFN gamma and decreased numbers of human regulatory T lymphocytes in the tumor xenografts, possibly explaining the efficacy of the therapeutic regimen. Our results offer a proof of concept for the use of humanized mouse models for surrogate efficacy and histology investigations of immune checkpoint drugs and their combinations.

Background: Stereotactic ablative body radiation (SABR) is a novel and sophisticated radiation modality that involves the irradiation of extracranial tumors through precise and very high doses in patients with oligometastatic lung disease and primary lung tumors. Case presentation: A 52-year-old female with subclinical idiopathic interstitial lung disease (ILD) and oligometastatic lung disease from squamous urethral cancer who was treated with SABR for a metastatic lesion located in the right lower pulmonary lobe. The patient received a hypo-fractionated course of SABR. A 3D-conformal multifield technique was used with six coplanar and one non-coplanar statics beams. A 48Gy total dose in three fractions over six days was prescribed to the 95% of the PTV. The presence of idiopathic ILD and other identifiable underlying lung conditions were not taken into account as a constraint to prescribe a different than standard total dose or fractionation schedule. Six months after the SABR treatment, a CT-scan showed the presence of a pneumomediastinum with air outside the bronchial tree and within the subcutaneous tissue without co-existing pneumothorax. To our knowledge, this is the first case of pneumomediastinum appearing 6months after SABR treatment for a lung metastasis located in the perihiliar/central tumors region as defined by the RTOG protocols as the proximal bronchial tree. Conclusion: Radiation oncologist should be aware of the potential risk of severe lung toxicity caused by SABR in patients with ILD, especially when chemotherapy-induced pulmonary toxicity is administered in a short time interval.

PURPOSE: To develop a simple clinical model predictive of locoregional failure after complete surgical resection followed by perioperative high-dose-rate brachytherapy (PHDRB) and external beam irradiation (EBRT). PATIENT AND METHODS: Patients (n=166) enrolled in several PHDRB prospective studies conducted at the University of Navarre were analyzed. PHDRB was given to total doses of 16 Gy/4 b.i.d. or 24 Gy/6 b.i.d. treatments for negative or close/positive margins along with 45Gy of EBRT. RESULTS: After a median followup of 7.4 years (range, 3-12+), 50 patients have failed and 116 remain controlled at last followup. Tumor size, with a cutoff point set at 3cm (p=0.041) and margin status (positive and <1mm vs. negative ¿1mm, p=0.0001) were independent predictors of locoregional control. These two parameters were used to develop a four-tiered, hierarchical scoring system that stratified patients into low-risk (negative ¿1mm margins and size ¿3cm), intermediate-risk (negative ¿1mm margins, and size >3cm), high-risk (positive and <1mm margins and size ¿3cm), and very high-risk categories (positive and <1mm margins and size >3cm). This classification yields 5-year locoregional control rates of 92.3%, 78.0%, 65.5%, and 48.0% for low-, intermediate-, high-, and very high-risk categories, respectively. The predictive ability of the model is highly significant (p=0.0001) with an area under the curve of 0.72 (0.64-0.81). CONCLUSIONS: The risk of locoregional failure after combined surgical resection, PHDRB, and EBRT is mainly determined by the number of residual clonogens, which is inversely proportional to the status of the surgical margins and directly related to the size of the resected tumor. These two parameters generate a four-tiered predictive model that seems to be valid for a number of different common tumors and clinical settings.

Inhibitory receptors on immune system cells respond to membrane-bound and soluble ligands to abort or mitigate the intensity of immune responses by raising thresholds of activation, halting proliferation, favoring apoptosis or inhibiting/deviating effector function differentiation. Such evolutionarily selected inhibitory mechanisms are termed check-points and therefore check-point inhibitors empower any ongoing anti-cancer immune response that might have been too weak or exhausted. Monoclonal antibodies (mAb) interfering with CTLA-4-CD80/86, PD-1 - PD-L1, TIM-3-GAL9 and LAG3-MHC-II belong to this category of check-point inhibitors. The anti-CTLA-4 mAb ipilimumab has been approved for metastatic melanoma. Anti-PD-1 and anti-PD-L1 mAbs have shown extremely encouraging clinical activity. The potential of combination strategies with these agents has recently been highlighted by clinical observations on CTLA-4+PD-1 combined blockade in melanoma patients.

PURPOSE: To report the disease-free Grade complication-free survival of a Phase II protocol of reirradiation with high-dose-rate (HDR) interstitial brachytherapy (ITB) in previously irradiated gynecologic cancer. METHODS AND MATERIALS: Fifteen patients with previously irradiated cervical (n = 6), endometrial (n = 6), and vulvovaginal tumors (n = 3) were treated with HDR-ITB alone to a median dose of 38 Gy in 8 b.i.d. fractions over 4 consecutive days. Prior treatments included surgery (n = 12; 80%), external irradiation (n = 15; 100%), and brachytherapy (n = 9; 60%). Average clinical target volume Size was 60.9 cc (range, 14.8-165.3 cc), and median time to reirradiation was 3.9 years (range, 0.4-22.7 years). RESULTS: With a median followup of 2.8 years (range, 1.2-9.2 years), 3 patients (20.0%) developed Grade toxicity consisting of Grade 3 intestinal obstruction (n = 1), Grade 4 rectovesical fistula (n = 1), and Grade 5 intestinal obstruction (n = 1). Six patients remain alive and without evidence of disease at last followup. Two patients are alive with disease progression, and 7 patients have died, 4 of them from disease progression and 3 from other causes. The 2-year disease-free. Grade complication-free survival was 40%. CONCLUSIONS: HDR-ITB alone is a reasonable salvage treatment option in a significant number of patients with previously irradiated gynecologic tumors. (C) 2014 American Brachytherapy Society.

IL8 levels correlate with tumor burden in preclinical models and in patients with cancer. IL8 is a potentially useful biomarker to monitor changes in tumor burden following anticancer therapy, and has prognostic significance.

Background This study aimed to test the safety of using perioperative high-dose-rate brachytherapy (PHDRB) in resected head and neck cancer. Methods From 2000 to 2008, 97 patients received PHDRB after complete macroscopic resection. Group 1 (previously irradiated patients) received 32 to 40 Gray (Gy) of PHDRB in 8 to 10 twice-daily (bid) treatments (R0R1 resections). Group 2 (unirradiated patients) received 16 to 24 Gy of PHDRB in 4 to 6 bid treatments (R0R1 resections) followed by external beam irradiation (EBRT) of 45 Gy/25 daily fractions +/- concomitant chemotherapy. Results The median follow-up was 4.3 years. The cumulative hazard of 2-year grade = 3 complications in group 1 was 45.9%, and the rate of grade = 3 complications in group 2 was 24.6%. Actuarial locoregional control at 2 and 5 years for group 1 was 60.9% and for group 2, 84.1% and 79.4%. Conclusions Complications and locoregional failure rates were similar to those reported in the reference standards despite a much smaller treatment volume. (c) 2012 Wiley Periodicals, Inc. Head Neck, 2012

Background: Stereotactic body radiation therapy (SBRT) is a radiation technique used in patients with oligometastatic lung disease. Lung and chest wall toxicities have been described in the patients but pathological vertebral fracture is an adverse effect no reported in patients treated with SBRT for lung metastases. Case presentation: A 68-year-old woman with the diagnosis of a recurrence of a single lung metastatic nodule of urothelial carcinoma after third line of chemotherapy. The patient received a hypo-fractionated course of SBRT.A 3D-conformal multifield technique was used with six coplanar and one non-coplanar statics beams. A total dose of 48 Gy in three fractions over six days was prescribed to the 95% of the CTV. Ten months after the SBRT procedure, a CT scan showed complete response of the metastatic disease without signs of radiation pneumonitis. However, rib and vertebral bone toxicities were observed with the fracture-collapse of the 7th and 8th vertebral bodies and a fracture of the 7th and 8th left ribs. We report a unique case of pathological vertebral fracture appearing ten months after SBRT for an asymptomatic growing lung metastases of urothelial carcinoma. Conclusion: Though SBRT allows for minimization of normal tissue exposure to high radiation doses SBRT tolerance for vertebral bone tissue has been poorly evaluated in patients with lung tumors. Oncologists should be alert to the potential risk of fatal bone toxicity caused by this novel treatment. We recommend BMD testing in all woman over 65 years old with clinical risk factors that could contribute to low BMD. If low BMD is demonstrated, we should carefully restrict the maximum radiation dose in the vertebral body in order to avoid intermediate or low radiation dose to the whole vertebral body.