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5554 Glioblastoma multiforme (GBM) is the most common type of brain tumor and is a leading cause of mortality with mean patient survival 6–12 months following diagnosis. We have recently shown that combined treatment of glioma bearing rats with RAdFlt3L and RAdTK dramatically improved survival. RAdFlt3L encodes human soluble Flt3L and stimulates dendritic cell proliferation and improves antigen presentation. RAdTK expresses herpes simplex type I-thymidine kinase and selectively kills rapidly dividing cells when used in combination with the non-toxic prodrug ganciclovir. We now show that combined treatment with RAdTK and RAdFlt3L improves long-term survival in C57BL/6 mice bearing intracranial GL26 tumors when all individual therapies completely fail (p

UFT. a a combination of tegafur and uracil, is increasingly used in the treatment of gastrointestinal malignancies. We report on a life-threatening toxicity observed in a 74-year-old female while undergoing adjuvant treatment with oral UFT plus leucovorm for adenocarcinoma of the rectum. The patient developed grade 4 thrombocytopenia, grade 4 neutropenia, grade 3 infection, grade 3 neurotoxicity and grade 2 cutaneous toxicity. This oral double-agent regimen should be used with caution in the adjuvant treatment of elderly patients with gastro-intestinal malignancies.

7524 Background: CP-675,206 had a manageable safety profile and antitumor activity in a prior Single-Dose phase I trial (Camacho et al. ProcASCO 2004). The safety, tolerability and recommended phase II dose of Monthly CP-675,206 was assessed in this study. Methods: Eligibility: stage IIIc-IV melanoma, measurable disease, ECOG 0–1, no active brain metastasis. 3 dose levels were sequentially tested. Results: 14 pts (9 M, 5 F) mean age 55 (range 42–67) were enrolled (3 at 3.0 mg/kg, 3 at 6.0 mg/kg, 8 at 10.0 mg/kg). 57% had stage IVc. All pts at 3 and 6 mg/kg received 2 doses, at 10 mg/kg received a median of 4.5 (range 2–8) monthly doses for a total of 51 doses administered. No G4 treatment-related toxicities were observed. G3 diarrhea in 1 pt at 3 mg/kg, none at 6 mg/kg, 2 pts at 10 mg/kg. G2 treatment-related adverse events included 4 dermatitis, and 1 each of diarrhea, pruritus, hypothyroidism, urticaria, and myalgias. Preliminary PK analysis suggests little accumulation of CP-675,206 with multiple dosin...

Top of pageAbstract Dendritic cell (DC) vaccines are conventionally produced ex vivo by culturing DC precursors in the presence of recombinant growth factors. Methods to facilitate the generation of DCs, to prolong their viability, to facilitate their migration to lymphoid tissues and to augment their capacity to establish and maintain antigen-specific immune responses are critical for improving their effective therapeutic potential for immunotherapy. To address these issues, we have genetically engineered purified human CD14+ monocytes with lentiviral vectors (LV) for production of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), which triggered their differentiation into cells with typical characteristics of DCs. We previously demonstrated that these lentivirus programmed DCs (DC/LVs) exhibited more potent immunostimulatory function and prolonged viability in vitro than DCs generated by conventional methods. Using the same strategy, we have now generated murine DC/LVs in order to test the efficacy of this approach in vivo. Third generation self-inactivating LV (5 |[mu]|g/ml p24 equivalent) transduced non-adherent bone marrow (BM) cells harvested from C57BL/6 mice at >90% transduction efficiency. BM cells co-expressing GM-CSF and IL-4 after LV transduction produced these cytokines at concentrations similar to those commonly used for generation of conventionally cultured DCs by addition of exogenous cytokines (> 10ng/ml), resulting in the generation of DC/LVs as evidenced by their morphology and immunophenotype (CD11b+, CD11c high, CD80 high, MHCII high). DC/LVs maintained in culture were metabolically active and viable for 3 weeks, whereas conventional DCs showed a drastic decrease in viability in the first week of culture. In vivo bioluminescence imaging studies were then performed using luciferase as a marking gene to track DCs after s.c. injection into nude syngeneic mice. Persistent and robust bioluminescence signals were detectable from the injected DC/LVs for more than 45 days, whereas signals produced by conv. DCs faded away within the first two weeks. To determine whether the higher viability of DC/LVs correlated with greater potency as vaccines, we next examined their antigen presentation and immunostimulatory functions in vivo. DC/LVs and conv. DCs maintained in culture for 7 days were transduced to express the melanoma associated antigen MART-1 and injected s.c. Splenocytes harvested 2 weeks after vaccination with DC/LVs showed up to 2-fold increase in activated T cells by IFN|[gamma]|- ELISPOT assay (p

Conventional, ex vivo culture of monocytes with recombinant proteins for their differentiation into DCs involves considerable manipulation under “Good Manufacturing Practices” conditions, and is not only more labor intensive but importantly, after ex vivo produced DCs are administered, they lack the stimulatory signals to keep them alive and functional and therefore are short lived. Because of these problems, we have evaluated an one-hit lentiviral transduction approach for genetically modifying monocytes in order to promote autocrine and paracrine production of factors required for their differentiation into immature DCs. High-titer third generation self-inactivating lentiviral vectors expressing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) efficiently achieved simultaneous and persistent co-delivery of the transgenes into purified human CD14+ monocytes. Co-expression of GM-CSF and IL-4 in monocytes was sufficient to induce their differentiation into lentivirus-modified DCs (“DC/LVs”), as evidenced by their morphology, immunophenotype and immune-function*. Mixed lymphocyte reactions showed that the T-cell stimulating activity of DC/LVs was superior to that of DCs grown by conventional methods. DC/LVs displayed efficient antigen-specific, MHC Class-I restricted stimulation of autologous CD8+ T-cells, as shown by IFN-G production and CTL assays. Importantly, DC/LVs could be maintained metabolically active and viable in culture for 2–3 weeks in the absence of exogenously added growth factors, unlike conventional DCs *. We are now evaluating whether DC/LVs can be re-infused immediately after gene transfer to achieve stable and long-lasting differentiation in vivo. Additionally, the genetic engineering of monocytes is anticipated to generate DCs after one hit of lentiviral transduction, instead of the three consecutive steps for development of DCs (differentiation, maturation, gene delivery of tumor antigens). We have thus established a mouse model for testing DC/LVs in vivo for the treatment of melanoma. Bone marrow cells from C57BL/6 mice transduced with lentiviral vectors expressing GM-CSF and IL-4 recapitulated the same DC/LV morphology and immunophenotype obtained in the human system. Mouse DC/LVs were also more viable in vitro and outperformed conventional mouse DCs in pilot immunization assays as followed by CTL assays and IFN-G ELISPOT. We are currently evaluating the immunotherapeutic efficacy of DC/LVs injected into mice developing B16 melanoma tumors. Co-delivery of a gene for DC maturation (CD40L) and of gene encoding a tumor-associated antigens (MART-1) is being performed. Our goal is to evaluate the implications of simultaneous co-expression of GM-CSF/ IL-4/ CD40L/ MART-1 in DC/LV differentiation and migration to lymph nodes in vivo, immunopotency and safety. Once these pre-clinical considerations are addressed, we foresee a broad clinical application of genetically engineered DCs for vaccination purposes against cancer and chronic infectious diseases.

9682 Background: Monitoring tumor antigen-specific immunotherapy requires accurate and reproducible assays to identify pts that elicit an immune response. However, the performance specifications of cellular immunology assays, such as MHC-restricted tetramer assays, have not been well defined. Methods: Following informed consent and screening, 10 healthy subjects and 20 pts with melanoma (all HLA-A2.1) not receiving active therapy underwent 4 blood draws within 2 months. Peripheral blood mononuclear cells were cryopreserved and later subjected to iTAg™ Tetramer assays. Tetramers tested using sequential gating FACS analysis: MART-1 26–35, HIVgag77–85, Flu M1 58–66, CMVpp65, EBV BMLF1, AFP 325–334 and a Negative Tetramer (a non-relevant peptide control, all from Beckman Coulter). Optimal signal was obtained when at least 50,000 CD8+ events were accrued. Results: Primary objectives were to define the performance specifications of the MART-1 tetramer assay, and to identify positive and negative controls. Using...

BACKGROUND Increased proliferation of endogenous bone marrow progenitor cells in response to the administration of hematopoietic growth factors, followed by reduced cell cycling or entrance of the cells into a quiescent state upon withdrawal of the growth factors, may have clinically relevant effects on the tolerance of the hematopoietic system to subsequent myelotoxic treatments. PURPOSE We investigated the ability of granulocyte colony-stimulating factor (G-CSF) to protect progenitor cells in the bone marrow of cancer patients from the toxic effects of subsequent treatments with chemotherapeutic agents. METHODS Thirty-six patients with histologically documented, locally advanced or metastatic breast cancer were randomly assigned to receive doxorubicin once every 3 weeks at a dose of 75 mg/m2 and cyclophosphamide at a dose of 1000 mg/m2, with G-CSF administered either before and after chemotherapy (18 patients) or after chemotherapy only (18 patients). For prechemotherapy administration of G-CSF, recombinant human methionyl (r-met Hu) G-CSF was administered subcutaneously to patients twice per day for 5 days at a dose of 5 micrograms/kg, with the last dose given 48 hours before the start of chemotherapy. For postchemotherapy administration of G-CSF, r-met Hu G-CSF was administered subcutaneously to patients once per day for 7 days at a dose of 5 micrograms/kg, with the first dose given 24 hours after chemotherapy. RESULTS The incidence or the duration of grade 4 neutropenia was not reduced in all patients by the use of prechemotherapy G-CSF; the incidence over all cycles of chemotherapy was 74% for patients treated with prechemotherapy and postchemotherapy G-CSF and 66% for patients treated with postchemotherapy G-CSF only (two-sided P, adjusted for dose = .21) and the median duration in both treatment arms was 3 days (two-sided P = .19). Unexpectedly, the incidence of grades 3 and 4 thrombocytopenia was much greater in patients who received prechemotherapy G-CSF compared with those who did not (54% versus 6%, respectively, over all chemotherapy cycles; two-sided P, adjusted for dose < .001). No difference in the decrease in hemoglobin level (adjusted for red blood cell transfusions) between patients in the two treatment arms was observed. CONCLUSIONS AND IMPLICATIONS No beneficial effects were associated with the administration of G-CSF to cancer patients prior to chemotherapy. The observation of more severe thrombocytopenia in patients treated with prechemotherapy G-CSF led us to conclude that the proliferation of progenitor cells was still increased 48 hours after the last dose of G-CSF and that the administration of chemotherapy at or within this time period actually worsens the toxic effects on bone marrow. This result has important ramifications for the design of clinical cancer treatment protocols, especially those that involve shortened intervals between cycles of chemotherapeutic agent administration.

One of the principal mechanisms by which the immune system fails to reject a tumor is a lack of cytotoxic response activation. In order to circumvent this problem, we have genetically engineered murine colorectal carcinoma cells to actively express the IL-2 gene, introduced using three different retroviral vectors (DCTK-IL2, LNCX-IL2, LXSN-IL2). These three vectors also carried the neoR gene, which confers resistance to in vitro treatment with a neomycin analog (G418). We evaluated the effect of different selection methods on the final gene expression. Previous experiences demonstrated that, out of the three retroviral vectors with the IL-2 gene, one of them (DCTK-IL2) was very effective in stimulating a systemic specific antitumor response, but an inappropriate local response against the immunization dose. In order to control the growth of tumor cells transduced with the DCTK-IL2 vector injected as active immunization, these cells were further transduced with a retroviral vector carrying the neo gene and a “suicidal” gene, which codifies for the HSVTK. For these cells with a double transduction, we designed two selection methods (initially with high doses of G418 or HAT followed by G418) in order to identify the subclones which had the most favorable expression of both the IL-2 and HSVTK genes. IL-2 production increases both with increasing in vitro concentrations, and with duration of G418 selection. Assayed with a commercial IL-2 ELISA, the DCTK-IL2 vector produced the lowest 1L-2 levels in vitro (2 U/10 6 /24 h), whereas the other two retroviral vectors produced similar levels of IL-2 (18 U/10 6 /24 h). Cells with a double transduction were able to successfully tolerate higher levels of G418 in the culture (up to 1.6 mg/ml) due to the presence of two copies of the neoR gene. Both methods of selection of these cells (high dose G418 or HAT) produced cell populations sensible to GCV in vitro . We obtained 55 subclones from different moments of the ongoing cell selection, and the clones with most favorable expression of both genes (IL-2 and HSVTK) were expanded for further in vivo studies. In conclusion, we have shown that both the level and duration of selection of retrovirally transduced cells have an important impact on gene expression, and that it is possible to select cells with a double transduction with two vectors carrying the neoR gene. The most favourable cell clones will be used as active antitumoral immunization (vaccines) in further gene therapy experimental studies in Balb/c mice.

Ifosfamide (IFO) is an oxazophosphorine with a different activity and toxicity profile than cyclophosphamide. Its use together with uroprotective agents has allowed safe administration and dose-escalation. We report the results of a phase II trial of IFO at high doses in heavily pretreated BC and S patients. IFO was administered in a 168 hour CI through a central venous access, for a total dose of 14 g/m2 q3w. MESNA was administered together at equimolar doses. Ondansetron, 8 mg/8 h po was used as antiemetic treatment. No hematopoietic support was used. We included 10 BC and 14 S patients with disease progression during salvage chemotherapy at conventional doses. Mean previous lines of therapy 3 (range 1–5), 20 had received previous treatment with conventional-dose cyclophosphamide or IFO. All had received previous adriamycin. Median age 44 (range 18–62), 12 males and 12 females. Median number of cycle 3 (range 1–8) for a total of 81 cycles of therapy. Worst WHO grade toxic reaction for each patient: grade III–IV leukopenia in 65%, grade III nausea and vomiting in 40%, grade III neurotoxicity in 5%, grade II nephrotoxicity in 5%. Neurologic and renal toxicity were reversible. 9 patients were admitted for neutropenic fever, with two documented septic episodes. Treatment had to be discontinued in 2 patients after 2 cycles (1 renal toxicit); I gastro-intestinal GI-toxicity). 20 patients are evaluable for response (4 did not finish the first cycle of therapy, 3 for early disease progression, 1 for unacceptable GI toxicity). Partial responses in 2/8 (25%) BC, and in 3/12 (25%) S. No complete responses were recorded. 65% had disease stabilization, and 10% had disease progression. 3 with S and 1 BC underwent further high dose chemotherapy with transplantation after assessment of chemotherapy sensitivity with high-dose IFO. Median duration of response was 5 months. Median overall survival was 6 months (range 2–11+). In conclusion, CI of IFO for 168 hours is an active regimen in highly pretreated BC and S. The addition of hematopoietic growth factors and further antiemetic agents could improve the toxicity ofthis regimen. Additionally, this regimen could be combined with non-myelotoxic drugs.

Systemically delivered small interfering RNA (siRNA) therapies for cancer have begun clinical development. The effects of siRNA-mediated knockdown of ribonucleotide reductase subunit-2 (RRM2), a rate-limiting enzyme in cell replication, were investigated in malignant melanoma, a cancer with a paucity of effective treatment options. A panel of human melanoma cell lines was transfected with siRNA to induce the knockdown of RRM2. Sequence-specific, siRNA-mediated inhibition of RRM2 effectively blocked cell proliferation and induced G1/S-phase cell cycle arrest. This effect was independent of the activating oncogenic mutations in the tested cell lines. Synergistic inhibition of melanoma cell proliferation was achieved using the combination of siRNA targeting RRM2 and temozolomide, an analog of the current standard of care for melanoma chemotherapy. In conclusion, siRNA-mediated RRM2 knockdown significantly inhibits proliferation of melanoma cell lines with different oncogenic mutations with synergistic enhancement in combination with temozolomide.

Background. A Phase II trial with a new regimen of methotrexate, carboplatin, and vinblastine (M-CAVI) was conducted for patients with bladder cancer who could not receive cisplatin-based chemotherapy. Methods. Treatment consisted of methotrexate (30 mg/m2) on days 1,15, and 22; carboplatin (300 mg/m2) on day 2; and vinblastine (3 mg/m2) on days 2, 15, and 22, scheduled at 28-day intervals. The dosage of carboplatin was adjusted for creatinine clearance. Twenty-five patients with metastatic (n = 9) or locally advanced or locoregional bladder cancer with a poor prognosis In (= 16) were treated with M-CAW. Fifteen patients had abnormal serum creatinine levels, 4 had a low performance status, 3 had cardiac disease, and 3 were older than 70 years old. Results. Among 23 patients assessable for clinical response, the response rate was 48%. The median duration of response for metastatic disease was 7 months. Among nine patients assessable for pathologic response, there were two complete responses and three partial responses. The toxic effects have been moderate. Conclusions. M-CAVI is an active and well-tolerated regimen that can be used in patients with bladder cancer who are ineligible to receive cisplatin-based chemotherapy. Cancer 1992; 70:1974–1979.

Aims and Background Several reports on prognostic factors for infiltrating bladder cancer have given controversial results. We assessed the prognostic value of p53 nuclear overexpression together with known prognostic factors for survival in patients with invasive T2-4 NO MO bladder cancer treated with neoadjuvant chemotherapy. Study Design Thirty-five paraffi-nized tumor samples from initial transurethral resection of patients with bladder cancer were analyzed immunohistochemi-cally to detect overexpression of p53 protein. Patients were treated with 3 to 4 cycles of neoadjuvant methotrexate, carboplatin, and vinblastine (M-CAVI) and then underwent radical cystectomy. Prechemotherapy, treatment, and postchemotherapy factors were analyzed for correlation with survival by univariate and multivariate analysis. Fifty-seven percent of tumors were positive for p53 protein, 71.5% had grade III-IV tumors, and 72% had organ-confined disease. The median follow-up was 20 months (range 5-71+). Results By univariate analysis, the significant pretreatment factors were initial tumor (T) stage ( P Conclusions Although the median follow-up of the study is still too short, in this group of patients treated with a neoadjuvant carboplatin-based regimen, a classical variable (prechemotherapy T stage) rather than p53 nuclear overexpression was an independent prognostic factor for survival. Further follow-up will be required to assess the value of p53 overexpression as a prognostic factor in invasive bladder cancer patients treated with neoadjuvant carboplatin-based chemotherapy.

Background Malignant melanoma is an aggressive tumor type that often develops drug resistance to targeted therapeutics. The production of colony stimulating factor 1 (CSF-1) in tumors recruits myeloid cells such as M2-polarized macrophages and myeloid derived suppressor cells (MDSC), leading to an immune suppressive tumor milieu. Methods We used the syngeneic mouse model of BRAFV600E-driven melanoma SM1, which secretes CSF-1, to evaluate the ability of the CSF-1 receptor (CSF-1R) inhibitor PLX3397 to improve the antitumor efficacy of the oncogenic BRAF inhibitor vemurafenib. Results Combined BRAF and CSF-1R inhibition resulted in superior antitumor responses compared with either therapy alone. In mice receiving PLX3397 treatment, a dramatic reduction of tumor-infiltrating myeloid cells (TIM) was observed. In this model, we could not detect a direct effect of TIMs or pro-survival cytokines produced by TIMs that could confer resistance to PLX4032 (vemurafenib). However, the macrophage inhibitory effects of PLX3397 treatment in combination with the paradoxical activation of wild type BRAF-expressing immune cells mediated by PLX4032 resulted in more tumor-infiltrating lymphocytes (TIL). Depletion of CD8+ T-cells abrogated the antitumor response to the combination therapy. Furthermore, TILs isolated from SM1 tumors treated with PLX3397 and PLX4032 displayed higher immune potentiating activity. Conclusions The combination of BRAF-targeted therapy with CSF-1R blockade resulted in increased CD8 T-cell responses in the SM1 melanoma model, supporting the ongoing evaluation of this therapeutic combination in patients with BRAFV600 mutant metastatic melanoma. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1377-8) contains supplementary material, which is available to authorized users.

The 27th annual meeting of the Society for Immunotherapy of Cancer (SITC) was held on October 26–28, 2012 in North Bethesda, Maryland and the highlights of the meeting are summarized. The topics covered at this meeting included advances in cancer treatment using adoptive cell therapy (ACT), oncolytic viruses, dendritic cells (DCs), immune check point modulators and combination therapies. Advances in immune editing of cancer, immune modulation by cancer and the tumor microenvironment were also discussed as were advances in single cell analysis and the manufacture and potency testing of tumor infiltrating lymphocytes (TIL).

Meeting abstracts Semaphorin 4D (SEMA4D, CD100) and its receptor plexin-B1 are broadly expressed in cancer and expression correlates with invasive disease in several human tumors. SEMA4D normally functions to regulate the motility and differentiation of multiple cell types, including those of the

2 and 10 mg/kg Q3W, respectively, over control (P

Background Molecular imaging with positron emission tomography (PET) may allow the non-invasive study of the pharmacodynamic effects of agonistic monoclonal antibodies (mAb) to 4-1BB (CD137). 4-1BB is a member of the tumor necrosis factor family expressed on activated T cells and other immune cells, and activating 4-1BB antibodies are being tested for the treatment of patients with advanced cancers. Methods We studied the antitumor activity of 4-1BB mAb therapy using [18 F]-labeled fluoro-2-deoxy-2-D-glucose ([18 F]FDG) microPET scanning in a mouse model of colon cancer. Results of microPET imaging were correlated with morphological changes in tumors, draining lymph nodes as well as cell subset uptake of the metabolic PET tracer in vitro. Results The administration of 4-1BB mAb to Balb/c mice induced reproducible CT26 tumor regressions and improved survival; complete tumor shrinkage was achieved in the majority of mice. There was markedly increased [18 F]FDG signal at the tumor site and draining lymph nodes. In a metabolic probe in vitro uptake assay, there was an 8-fold increase in uptake of [3H]DDG in leukocytes extracted from tumors and draining lymph nodes of mice treated with 4-1BB mAb compared to untreated mice, supporting the in vivo PET data. Conclusion Increased uptake of [18 F]FDG by PET scans visualizes 4-1BB agonistic antibody-induced antitumor immune responses and can be used as a pharmacodynamic readout to guide the development of this class of antibodies in the clinic.

Post-acute sequelae of COVID-19 (PASC) represent an emerging global crisis. However, quantifiable risk-factors for PASC and their biological associations are poorly resolved. We executed a deep multi-omic, longitudinal investigation of 309 COVID-19 patients from initial diagnosis to convalescence (2-3 months later), integrated with clinical data, and patient-reported symptoms. We resolved four PASC-anticipating risk factors at the time of initial COVID-19 diagnosis: type 2 diabetes, SARS-CoV-2 RNAemia, Epstein-Barr virus viremia, and specific autoantibodies. In patients with gastrointestinal PASC, SARS-CoV-2-specific and CMV-specific CD8+ T cells exhibited unique dynamics during recovery from COVID-19. Analysis of symptom-associated immunological signatures revealed coordinated immunity polarization into four endotypes exhibiting divergent acute severity and PASC. We find that immunological associations between PASC factors diminish over time leading to distinct convalescent immune states. Detectability of most PASC factors at COVID-19 diagnosis emphasizes the importance of early disease measurements for understanding emergent chronic conditions and suggests PASC treatment strategies., By correlating patient symptoms with in-depth profiling of blood cells and plasma components throughout COVID-19 infection, this study identifies factors that may predict sustained disease.

Materials and methods Tumor tissues were collected at baseline prior to treatment, on-treatment at cycle 1 day 14 (C1D14) and at disease progression (PD). Modulation of the MAPK and PI3K pathways, cell proliferation, and CD8 T-cells in tumor biopsies were assessed by immunohistochemistry. Changes in transcription profiles upon treatment were measured by qRT-PCR using the Fluidigm/Nanostring platforms.

BackgroundThe clinical use of BRAF inhibitors for treatment of metastatic melanoma is limited by the development of drug resistance. In this study we investigated whether co-targeting the MAPK and the PI3K-AKT pathway can prevent emergence of resistance or provide additional growth inhibitory effects in vitro.MethodsAnti-tumor effects of the combination of the BRAF inhibitor (BRAFi) dabrafenib and GSK2141795B (AKTi) in a panel of 23 BRAF mutated melanoma cell lines were evaluated on growth inhibition by an ATP-based luminescent assay, on cell cycle and apoptosis by flow cytometry and on cell signaling by western blot. Moreover, we investigated the possibilities of delaying or reversing resistance or achieving further growth inhibition by combining AKTi with dabrafenib and/or the MEK inhibitor (MEKi) trametinib by using long term cultures.ResultsMore than 40% of the cell lines, including PTEN-/- and AKT mutants showed sensitivity to AKTi (IC50 5 nM dabrafenib. Combinatorial treatment induced apoptosis only in cell lines sensitive to AKTi. In long term cultures of a PTEN-/- cell line, combinatorial treatment with the MAPK inhibitors, dabrafenib and trametinib, and AKTi markedly delayed the emergence of drug resistance. Moreover, combining AKTi with the MAPK inhibitors from the beginning provided superior growth inhibitory effects compared to addition of AKTi upon development of resistance to MAPK inhibitors in this particular cell line.ConclusionsAKTi combined with BRAFi-based therapy may benefit patients with tumors harboring BRAF mutations and particularly PTEN deletions or AKT mutations.

Background Several single center studies have provided evidence of immune activation and antitumor activity of therapeutic vaccination with dendritic cells (DC) in patients with metastatic melanoma. The efficacy of this approach in patients with favorable prognosis metastatic melanoma limited to the skin, subcutaneous tissues and lung (stages IIIc, M1a, M1b) was tested in a multicenter two stage phase 2 study with centralized DC manufacturing. Methods The vaccine (IDD-3) consisted 8 doses of autologous monocyte-derived matured DC generated in serum-free medium with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-13 (IL-13), pulsed with lysates of three allogeneic melanoma cell lines, and matured with interferon gamma. The primary endpoint was antitumor activity. Results Among 33 patients who received IDD-3 there was one complete response (CR), two partial responses (PR), and six patients had stable disease (SD) lasting more than eight weeks. The overall prospectively defined tumor growth control rate was 27% (90% confidence interval of 13-46%). IDD-3 administration had minimal toxicity and it resulted in a high frequency of immune activation to immunizing melanoma antigens as assessed by in vitro immune monitoring assays. Conclusions The administration of matured DC loaded with tumor lysates has significant immunogenicity and antitumor activity in patients with limited metastatic melanoma. Clinical trial registration NCT00107159.