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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 24h with poly-ICLC (Hiltonol), TNF-α and IFN-α. On days+8 and+10 of each cycle, patients received intratumoral image-guided 0.25mg injections of the dsRNA-analogue Hiltonol. Cyclophosphamide 600mg/m2 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 post-treatment PBMC from patients achieving durable stable disease (SD) were studied by IFNγ ELISPOT-assays responding to tumor-lysate loaded DC and by TCRβ 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-β and IFN-α mRNA in circulating PBMC. DC vaccination increased serum IL-12 and IL-1β concentrations, especially in patients presenting SD. IFNγ-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.

Treatment of metastatic colorectal cancer is based upon the assumption that metastases are homogeneous within a patient. We quantified immune cell types of 603 whole-slide metastases and primary colorectal tumors from 222 patients. Primary lesions, and synchronous and metachronous metastases, had a heterogeneous immune infiltrate and mutational diversity. Small metastases had frequently a low Immunoscore and T and B cell score, while a high Immunoscore was associated with a lower number of metastases. Anti-epidermal growth factor receptor treatment modified immune gene expression and significantly increased T cell densities in the metastasis core. The predictive accuracy of the Immunoscore from a single biopsy was superior to the one of programmed cell death ligand 1 (PD-L1). The immune phenotype of the least-infiltrated metastasis had a stronger association with patient outcome than other metastases.

The recent success of checkpoint blockers to treat cancer has demonstrated that the immune system is a critical player in the war against cancer. Historically, anticancer therapeutics have been tested in syngeneic mouse models (with a fully murine immune system) or in immunodeficient mice that allow the engraftment of human xenografts. Animal models with functioning human immune systems are critically needed to more accurately recapitulate the complexity of the human tumor microenvironment. Such models are integral to better predict tumor responses to both immunomodulatory agents and directly antineoplastic therapies. In this regard, the development of humanized models is a promising, novel strategy that offers the possibility of testing checkpoint blockers' capacity and their combination with other antitumor drugs. In this review, we discuss the strengths and weaknesses of the available animal models regarding their capacity to evaluate checkpoint blockers and checkpoint blocker-based combination immunotherapy.

Retroviral gene transfer of interleukin-12 (IL-12) into T cells markedly enhances antitumor efficacy upon adoptive transfer but has clinically shown unacceptable severe side effects. To overcome the toxicity, we engineered tumor-specific CD8+ T cells to transiently express IL-12. Engineered T cells injected intratumorally, but not intravenously, led to complete rejections not only of the injected lesion but also of distant concomitant tumors. Efficacy was further enhanced by co-injection with agonist anti-CD137 mAb or by transient co-expression of CD137 ligand. This treatment induced epitope spreading of the endogenous CD8+ T cell immune response in a manner dependent on cDC1 dendritic cells. Mouse and human tumor-infiltrating T lymphocyte cultures can be transiently IL-12 engineered to attain marked immunotherapeutic effects.

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.

BRAF V600 mutation has been reported in more than 50% of melanoma cases and its presence predicts clinical activity of BRAF inhibitors (iBRAF). We evaluated the role of MIA, S100 and LDH to monitor iBRAF efficiency in advanced melanoma patients presenting BRAF V600 mutations. This was a prospective study of melanoma patients harboring the BRAF V600 mutation and treated with iBRAF within a clinical trial (dabrafenib) or as part of an expanded access program (vemurafenib). MIA, S100 and LDH were analyzed in serum at baseline, and every 4–6 weeks during treatment. Eighteen patients with melanoma stages IIIc–IV were enrolled with 88.8% of response rate to iBRAF. Baseline concentrations of all the tumor markers correlated with tumor burden. MIA and S100 concentrations decreased significantly one month after the beginning of treatment and, upon progression, their concentrations increased significantly above the minimum levels previously achieved. MIA levels lower than 9 μg/L one month after the beginning of treatment and S100 concentrations lower than 0.1 μg/L at the moment of best response were associated with improved progression-free survival. In conclusion, MIA and S100 are useful to monitor response in melanoma patients treated with iBRAF.

The discovery of reliable biomarkers to predict efficacy and toxicity of anticancer drugs remains one of the key challenges in cancer research. Despite its relevance, no efficient study designs to identify promising candidate biomarkers have been established. This has led to the proliferation of a myriad of exploratory studies using dissimilar strategies, most of which fail to identify any promising targets and are seldom validated. The lack of a proper methodology also determines that many anti-cancer drugs are developed below their potential, due to failure to identify predictive biomarkers. While some drugs will be systematically administered to many patients who will not benefit from them, leading to unnecessary toxicities and costs, others will never reach registration due to our inability to identify the specific patient population in which they are active. Despite these drawbacks, a limited number of outstanding predictive biomarkers have been successfully identified and validated, and have changed the standard practice of oncology. In this manuscript, a multidisciplinary panel reviews how those key biomarkers were identified and, based on those experiences, proposes a methodological framework—the DESIGN guidelines—to standardize the clinical design of biomarker identification studies and to develop future research in this pivotal field.

Purpose The present study was undertaken to determine factors predictive of toxicity, patterns of failure, and survival in 60 adult patients with soft tissue sarcomas of the extremity and superficial trunk treated with combined perioperative high-dose-rate brachytherapy and external beam radiotherapy. Methods and Materials The patients were treated with surgical resection and perioperative high-dose-rate brachytherapy (16 or 24 Gy) for negative and close/microscopically positive resection margins, respectively. External beam radiotherapy (45 Gy) was added postoperatively to reach a 2-Gy equivalent dose of 62.9 and 72.3 Gy, respectively. Adjuvant chemotherapy with ifosfamide and doxorubicin was given to patients with advanced high-grade tumors. Results Grade 3 toxic events were observed in 18 patients (30%) and Grade 4 events in 6 patients (10%). No Grade 5 events were observed. A location in the lower limb was significant for Grade 3 or greater toxic events on multivariate analysis ( p = .013), and the tissue volume encompassed by the 150% isodose line showed a trend toward statistical significance ( p = .086). The local control, locoregional control, and distant control rate at 9 years was 77.4%, 69.5%, and 63.8%, respectively. On multivariate analysis, microscopically involved margins correlated with local control ( p = .036) and locoregional control ( p = .007) and tumor size correlated with distant metastases ( p = .004). The 9-year disease-free survival and overall survival rate was 47.0% and 61.5%, respectively. Multivariate analysis showed poorer disease-free survival rates for patients with tumors >6 cm ( p = .005) and microscopically involved margins ( p = .043), and overall survival rates decreased with increasing tumor size ( p = .011). Conclusions Grade 3 or greater wound complications can probably be decreased using meticulous treatment planning to decrease the tissue volume encompassed by the 150% isodose line, especially in lower limb locations. Microscopically involved margins remain a predictor of local and locoregional failure, despite radiation doses >70 Gy. Patients with tumors ≥6 cm and microscopically involved margins are at high risk of treatment failure and death from the development of distant metastases.

Background Vemurafenib is approved for the first-line treatment of BRAF-V600E+ metastatic melanoma (MM). Also, preliminary clinical experience with imatinib in MM patients with gene amplification or activating mutations of c-KIT, have shown high response rates and prolonged progression free survival. These results emphasise the importance of molecular information, even in those cases in which the diagnosis is made on small samples obtained through minimally invasive approaches. Cytology is an accurate and cost-effective tool for the diagnosis of MM and cytological smears are occasionally the only samples available from these patients. Methods We have studied BRAF and c-KIT mutations in 62 cytological samples from MM patients. Preliminary results of 56 cases (32 men (58%) and 23 women (42%); median age 52 years) are included in this abstract. Diagnosis was made on fine needle aspiration in 41 cases, imprints in 10, direct eye touch in 1, a smear from cellular culture in 1, and pleural and peritoneal fluid in 3. To assure tissue quality, DNA was extracted directly from Papanicolau stained smears on which cytological diagnosis was made. BRAF and c-KIT mutations were analysed by PCR and direct sequencing using an ABI PRISM TM 310XL. Results BRAF mutations was found in 30 cases (53.5%): V600E mutation in 26 (86,7%) and V600K in 4 (13,3%). c-KIT mutations were studied in 18 cases. L576P mutation was present in 1 patient with liver metastasis from acral melanoma (5.56%). BRAF status did not correlated with primary melanoma histology, age at diagnosis, disease free survival, brain metastases rate and overall survival. Conclusions The frequency of activating mutations in BRAF is 53.5% in this series. V600E is the most commonly observed mutations, but V600K appears in 13.3% of the cases. c-KIT mutations incidence is low (5.56%). Mutational analysis of BRAF and c-KIT using cytological samples from patients with metastatic melanoma is feasible and can be used to extend the benefits of targeted therapy to those patients from which biopsies are not available. Updated results will be presented. Disclosure S. Martin Algarra: Participation in Advisory Boards of Roche. All other authors have declared no conflicts of interest.

Aim: IL-8 is produced by multiple tumors and its functions include regulation of tumor angiogenesis and immune response. We evaluated the correlation of IL-8 with tumor burden and its potential role as a cancer biomarker by studying sequential levels of serum IL-8 in preclinical tumor models and in patients with multiple tumor types, at baseline and following anticancer treatment. Methods: IL-8 levels were monitored sequentially by sandwich ELISAs in three different models: a) cultured tumor cells supernatant; b) serum of tumor-xenografted mice and; c) serum and urine samples from 119 cancer patients, at baseline and following diverse anticancer treatments. We correlated IL-8 levels with tumor burden, treatment induced response and cancer prognosis. Results: IL-8 levels showed a strong correlation with tumor burden in all the models explored: a) in tumor cultures, IL-8 levels correlated accurately with the number of cancer cells; b) in tumor-xenografted mice, IL-8 serum levels correlated with tumor burden and rapidly dropped following surgical excision; and c) in patients with melanoma, renal-cell carcinoma, non-small cell lung cancer and hepatocellular carcinoma, serum IL-8 levels correlated with tumor burden and stage, survival and objective responses to therapy, including BRAF inhibitors and immunomodulatory monoclonal-antibodies. IL-8 concentrations in urine were mainly elevated in tumors with direct contact with the urinary tract. Conclusions: IL-8 levels correlate with tumor burden in preclinical models and in cancer patients and are a potentially useful biomarker to monitor response to cancer treatment. This might be particularly relevant to assess the therapeutic effects of drugs that may induce “pseudo-progressions”, such as immunomodulatory monoclonal antibodies. Disclosure: All authors have declared no conflicts of interest.

Aim: We analyzed the genome of individuals presenting extreme phenotypes of sensitivity and resistance to develop tobacco induced NSCLC to identify SNPs associated with these phenotypes. For this purpose, we used one of the most powerful GWAS platforms available. We hypothesized that SNPs may modulate individual susceptibility to carcinogens and that selection of extreme phenotypes would enrich the frequencies of alleles that contribute to the trait, thus increasing the power to identify them. Methods: From an identification cohort (n=3631) we selected caucasian heavy smokers that either developed NSCLC at an early age (cancer-cohort) or that did not present NSCLC at an advanced age (cancer-free cohort). We analyzed their genomic DNA using the array Illumina HumanOmni 2.5 Quad that includes over 2 million powerful markers selected from the 1000 Genomes Project, targeting genetic variation down to 1% minor allele frequency. Statistical significance of SNPs was calculated using logistic regression and Fisher's test. Results: 96 patients (48 per cohort) were selected. Mean age for the cancer and cancer-free cohorts was 49 years (range 38-55) and 76 years (72-84). Mean tobacco consumption was 41 pack-years (range 18-99) and 68 pack-years (40-120). GWAS identified 8 SNPs differentially expressed by logistic regression and 54 SNP by Fisher's test (p Function Gene Oncogenes MSX2 SOX11 Tumor supressors CSMD1 FOXF1 Tobacco induced NSCLC ABCB5 Regulators of transcription DROSHA HDAC9 KIAA0947 Regulators of inflammation PellinoE3 TRIM9 Related with cancer ABHD6 GRIK1 RAB40B SCN1A SLC24A2 SLC25A26 ZFYVE26 Conclusions: We identified candidate SNPs associated with the risk of developing tobacco-induced NSCLC in individuals with extreme phenotypes. Several identified SNPs were located within or near genes that constitute potentially relevant targets for modulation of cancer risk. Validation of the most significant SNPs in an independent set of individuals with similar phenotypes, selected from the EPIC-Spain project (www.epic-spain.com, n=40,000) is ongoing. Disclosure: All authors have declared no conflicts of interest.

Purpose BMS-936558 is a monoclonal antibody that blocks the PD-1 co-inhibitory receptor expressed by activated T cells. This study describes its activity and safety in pts with previously treated advanced MEL. Methods BMS-936558 was administered IV q2wk to pts with various tumors at 0.1 - 10 mg/kg during dose-escalation and/or cohort expansion. Pts received up to 12 cycles (4 doses/cycle) of treatment or until unacceptable toxicity, confirmed progressive disease, or complete response. Clinical activity was assessed by RECIST v1.0. Results As of Feb 24, 2012, 104 MEL pts had received BMS-936558 at 0.1 (n = 17), 0.3 (n = 19), 1 (n = 31), 3 (n = 17), or 10 mg/kg (n = 20). ECOG performance status was 0/1/2 in 63/38/3 pts, respectively. Most pts (67/104) had received prior immunotherapy (IT); prior anti-CTLA-4, -PD-1, or -PD-L1 was not permitted. The number of prior therapies was 1 (39%), 2 (35%), or ≥3 (26%). Median therapy duration was 20 wks (range 2.0 - 121.7 wks). The incidence of grade 3 - 4 related AEs was 20% and included gastrointestinal (4%), endocrine (2%), and hepatobiliary disorders (1%). There were no drug-related deaths in MEL pts. Clinical activity (responses or prolonged stable disease) was observed at all doses (Table). Of the 26/94 (28%) evaluable responders, 19 (73%) are ongoing ranging from 1.9+ to 24.9+ months. For the 23 responders followed ≥6 months from first dose on study, 16 (70%) are progression free. ORs occurred in pts with visceral or bone metastases. Six pts (6%; 95% CI 2 - 13%) had prolonged SD (≥24 wk); 3 pts had a persistent decrease in target lesion tumor burden in the presence of new lesions and were not categorized as responders. Conclusions BMS-936558 had durable clinical benefit in pts with advanced MEL, including those who had received prior IT. Additional long-term follow-up data will be reported. Dose, (mg/kg) No. ptsa ORR, No. pts (%) [95% CI] PFSR at 24 wk (%) [95% CI] 0.1 14 4 (29) [8 - 58] 40 [13 - 66] 0.3 16 3 (19) [4 - 46] 31 [9 - 54] 1 27 8 (30) [14 - 50] 45 [26 - 65] 3 17 7 (41) [18 - 67] * 55 [30 - 80] 10 20 4 (20) [6 - 44] 30 [9 - 51] * 1 CR aResponse-evaluable pts dosed by 7/01/2011 ORR = objective response rate ([{CR + PR} ÷ n] × 100); PFSR = progression-free survival rate. Disclosure J. Sosman: Research Funding: Bristol-Myers Squibb (myself). M. Sznol: Consultant or Advisory Role: Bristol-Myers Squibb (myself, compensated). Research Funding: Bristol-Myers Squibb (myself, clinical trials funding). D.F. McDermott: Advisory Board Role: Bristol-Myers Squibb (myself). R. Carvajal: Research Funding: Bristol-Myers Squibb (myself). S.L. Topalian: Consultant or Advisory Role: Bristol-Myers Squibb (myself, immediate family member, uncompensated). Research Funding: Bristol-Myers Squibb (myself). J.M. Wigginton: Employment or Leadership Position: Bristol-Myers Squibb (myself, employment, compensated). Stock Ownership: Bristol-Myers Squibb (myself). G. Kollia: Employment or Leadership Role: Bristol-Myers Squibb (employment, myself, compensated). Stock Ownership: Bristol-Myers Squibb/BMY stocks (myself). A. Gupta: Employment or Leadership Role: Bristol-Myers Squibb (employment, myself, compensated). Stock Ownership: Bristol-Myers Squibb (myself). F.S. Hodi: Consultant or Advisory Role: Bristol-Myers Squibb (myself, uncompensated). Research Funding: Bristol-Myers Squibb (myself). All other authors have declared no conflicts of interest.