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Background: Low-dose CT (LDCT) screening trials have shown that lung cancer early detection saves lives. However, a better stratification of the screening population is still needed. In this respect, we generated and prospectively validated a plasma miRNA signature classifier (MSC) able to categorize screening participants according to lung cancer risk. Here, we aimed to deeply characterize the peripheral immune profile and develop a diagnostic immune signature classifier to further implement blood testing in lung cancer screening. Methods: Peripheral blood mononuclear cell (PBMC) samples collected from 20 patients with LDCT-detected lung cancer and 20 matched cancer-free screening volunteers were analyzed by flow cytometry using multiplex panels characterizing both lymphoid and myeloid immune subsets. Data were validated in PBMC from 40 patients with lung cancer and 40 matched controls and in a lung cancer specificity set including 27 subjects with suspicious lung nodules. A qPCR-based gene expression signature was generated resembling selected immune subsets. Results: Monocytic myeloid-derived suppressor cell (MDSC), polymorphonuclear MDSC, intermediate monocytes and CD8+PD-1+ T cells distinguished patients with lung cancer from controls with AUCs values of 0.94/0.72/0.88 in the training, validation, and lung cancer specificity set, respectively. AUCs raised up to 1.00/0.84/0.92 in subgroup analysis considering only MSC-negative subjects. A 14-immune genes expression signature distinguished patients from controls with AUC values of 0.76 in the validation set and 0.83 in MSC-negative subjects. Conclusions: An immune-based classifier can enhance the accuracy of blood testing, thus supporting the contribution of systemic immunity to lung carcinogenesis. Impact: Implementing LDCT screening trials with minimally invasive blood tests could help reduce unnecessary procedures and optimize cost-effectiveness.

Supplementary Methods

Supplementary Tables S1-S6_S17-S18

Supplementary Figures

In tumor-bearing mice, cyclic fasting or fasting-mimicking diets (FMD) enhance the activity of antineoplastic treatments by modulating systemic metabolism and boosting antitumor immunity. Here we conducted a clinical trial to investigate the safety and biological effects of cyclic, five-day FMD in combination with standard antitumor therapies. In 101 patients, the FMD was safe, feasible, and resulted in a consistent decrease of blood glucose and growth factor concentration, thus recapitulating metabolic changes that mediate fasting/FMD anticancer effects in preclinical experiments. Integrated transcriptomic and deep-phenotyping analyses revealed that FMD profoundly reshapes anticancer immunity by inducing the contraction of peripheral blood immunosuppressive myeloid and regulatory T-cell compartments, paralleled by enhanced intratumor Th1/cytotoxic responses and an enrichment of IFNγ and other immune signatures associated with better clinical outcomes in patients with cancer. Our findings lay the foundations for phase II/III clinical trials aimed at investigating FMD antitumor efficacy in combination with standard antineoplastic treatments.Significance:Cyclic FMD is well tolerated and causes remarkable systemic metabolic changes in patients with different tumor types and treated with concomitant antitumor therapies. In addition, the FMD reshapes systemic and intratumor immunity, finally activating several antitumor immune programs. Phase II/III clinical trials are needed to investigate FMD antitumor activity/efficacy.This article is highlighted in the In This Issue feature, p. 1

Supplementary Table S7-S16

PDF file - 31K, Polyfunctional analysis of vaccine-induced T-cell cultures

PDF file - 64K, Analysis of chemokine receptor expression

Supplementary Figure S4. Pharmacokinetics and biodistribution of i.v.-injected TRAIL exosomes. Detection and quantification of TRAIL exosomes in plasma by Near-Infrared imaging (A) and TRAIL ELISA (B) after 1, 5, 60 and 180 minutes post-injection. Imaging and quantification of Near-Infrared-labeled TRAIL exosomes in organs (spleen, kidneys, liver and lungs) 1, 3 and 24 h after injection.

PDF file - 90K

PDF file - 66K, Correlations between multiple immunological parameters and DFS

Homing of TRAIL exosomes to tumor site. Labeling of exosomes with PKH26, treatment schedule and evaluation of lesions by confocal microscopy and flow cytometry. Pharmacokinetics and biodistribution of TRAIL exosomes. Near-Infrared labeling of exosomes, injection schedule and evaluation methods in plasma (Near-Infrared Imaging and ELISA) and organs (Near-Infrared Imaging).

PDF file - 44K, Patient characteristics

PDF file - 54K, Flow diagram for phase II randomized trial of adjuvanted multipeptide vaccination

PDF file - 100K, Assessment of peptide-specific recognition by in vitro short-term peptide sensitization assay (IVS)

Legends to Supplementary Figures S1-S4

PDF file - 56K, Number of IFNγ-forming spots/2 � 105 CD8+ T cells in APL-vaccinated patients

Purpose: Exosomes deliver signals to target cells and could thus be exploited as an innovative therapeutic tool. We investigated the ability of membrane TRAIL-armed exosomes to deliver proapoptotic signals to cancer cells and mediate growth inhibition in different tumor models.Experimental Methods and Results: K562 cells, transduced with lentiviral human membrane TRAIL, were used for the production of TRAIL+ exosomes, which were studied by nanoparticle tracking analysis, cytofluorimetry, immunoelectronmicroscopy, Western blot, and ELISA. In vitro, TRAIL+ exosomes induced more pronounced apoptosis (detected by Annexin V/propidium iodide and activated caspase-3) in TRAIL-death receptor (DR)5+ cells (SUDHL4 lymphoma and INT12 melanoma), with respect to the DR5−DR4+KMS11 multiple myeloma. Intratumor injection of TRAIL+ exosomes, but not mock exosomes, induced growth inhibition of SUDHL4 (68%) and INT12 (51%), and necrosis in KMS11 tumors. After rapid blood clearance, systemically administered TRAIL+ exosomes accumulated in the liver, lungs, and spleen and homed to the tumor site, leading to a significant reduction of tumor growth (58%) in SUDHL4-bearing mice. The treatment of INT12-bearing animals promoted tumor necrosis and a not statistically significant tumor volume reduction. In KMS11-bearing mice, despite massive perivascular necrosis, no significant tumor growth inhibition was detected.Conclusions: TRAIL-armed exosomes can induce apoptosis in cancer cells and control tumor progression in vivo. Therapeutic efficacy was particularly evident in intratumor setting, while depended on tumor model upon systemic administration. Thanks to their ability to deliver multiple signals, exosomes thus represent a promising therapeutic tool in cancer. Clin Cancer Res; 22(14); 3499–512. ©2016 AACR.

Purpose: The progressive immune dysfunctions that occur in patients with advanced melanoma make them unlikely to efficiently respond to cancer vaccines. A multicenter randomized phase II trial was conducted to test whether immunization with modified HLA class I tumor peptides in the context of adjuvant therapy results in better immunologic responses and improved clinical outcomes in patients with early melanoma (stages IIB/C-III).Experimental Design: Forty-three patients were enrolled to undergo vaccination (n = 22) or observation (n = 21). The vaccine included four HLA-A*0201–restricted modified peptides (Melan-A/MART-1[27L], gp100[210M], NY-ESO-1[165V], and Survivin[97M]) emulsified in Montanide ISA51 and injected subcutaneously in combination with cyclophosphamide (300 mg/m2) and low-dose IL-2 (3 × 106 IU). The immune responses were monitored using ex vivo IFN-γ–ELISpot, HLA/multimer staining, and in vitro short-term peptide sensitization assays.Results: Vaccination induced a rapid and persistent increase in specific effector memory CD8+ T cells in 75% of the patients. However, this immunization was not associated with any significant increase in disease-free or overall survival as compared with the observation group. An extensive immunologic analysis revealed a significantly reduced cross-recognition of the corresponding native peptides and, most importantly, a limited ability to react to melanoma cells.Conclusions: Adjuvant setting is an appealing approach for testing cancer vaccines because specific CD8+ T cells can be efficiently induced in most vaccinated patients. However, the marginal antitumor activity of the T cells induced by modified peptides in this study largely accounts for the observed lack of benefit of vaccination. These findings suggest reconsidering this immunization strategy, particularly in early disease. Clin Cancer Res; 18(23); 6485–96. ©2012 AACR.

712 Background: Nivolumab, an immune checkpoint inhibitor of PD-1, demonstrated a significant OS benefit in patients with metastatic renal cell carcinoma (mRCC), in progression after a previous line of therapy with anti-VEGFR agents. However, the features of effective immune response and predictive biomarkers of clinical benefit to PD-1 blockade have not yet been recognized. Methods: I-RENE is a prospective translational multicenter Italian study of a real-life mRCC patients treated with nivolumab or cabozantinib after failure of therapy with anti-VEGFR agents. 82 patients were enrolled from December 2018 to August 2022 (nivo 60, cabo 22), with blood samples obtained at baseline and at different time points in both treatment groups. An extended concept of "immune liquid biopsy" is being applied to the study, consisting in the phenotypic and transcriptional profile of lymphoid and myeloid subsets, immune-related miRNA quantification, cyto/chemo-kinome and RNAseq of extracellular vesicle. Results: Multiparametric flow cytometry, performed to monitor the blood frequency and different myeloid and lymphoid cells, show that monocyte subsets (classical, intermediate and non-classical CD14+ cells), monocytic myeloid derived suppressor cells (MDSC, such as CD14+HLA-DRneg and CD14+PD-L1+) and polymorphonucleate (PMN)-MDSC, remain either stable or increase during treatment; concomitantly, CD8+PD-1+ T cells (detected by anti-nivolumab IgG4) increment frequency, acquire the effector CD45RA-CCR7+ phenotype and express the proliferating marker Ki67. Patients receiving cabozantinib display instead a remarkable decrease of all myeloid cell subsets, paired by the boost of cytotoxic CD3-CD16+CD56dim NK cells and more marginally of CD8+PD1+ cells, Preliminary correlations indicate that clinical benefit of nivolumab seems to cluster with the lack of CD14+ cells and M-MDSC increase and blood frequency of total, and the boost in CD8+CD45RA-CCR7+Ki67+ effector T cells. In contrast, the cabozantinib-induced immune modulation occurring in patients treated with does not associate with clinical response Conclusions: This first set of data indicate blood as promising source of dynamic biomarkers for the development of algorithms predicting response to PD-1 blockade. Furthermore, the results so far collected suggest that the potent immunomodulation induced by cabozantinib on the immunosuppressive myeloid compartment may not lead to any tumor control in the absence of specific immune stimulation by checkpoint inhibitors. This study was supported by the Italian Ministry of health (RF-2016_02363001). Clinical trials.gov: NCT04891055 Clinical trial information: NCT04891055 .

In tumor-bearing mice, cyclic fasting or fasting-mimicking diets (FMD) enhance the activity of antineoplastic treatments by modulating systemic metabolism and boosting antitumor immunity. Here we conducted a clinical trial to investigate the safety and biological effects of cyclic, five-day FMD in combination with standard antitumor therapies. In 101 patients, the FMD was safe, feasible, and resulted in a consistent decrease of blood glucose and growth factor concentration, thus recapitulating metabolic changes that mediate fasting/FMD anticancer effects in preclinical experiments. Integrated transcriptomic and deep-phenotyping analyses revealed that FMD profoundly reshapes anticancer immunity by inducing the contraction of peripheral blood immunosuppressive myeloid and regulatory T-cell compartments, paralleled by enhanced intratumor Th1/cytotoxic responses and an enrichment of IFNγ and other immune signatures associated with better clinical outcomes in patients with cancer. Our findings lay the foundations for phase II/III clinical trials aimed at investigating FMD antitumor efficacy in combination with standard antineoplastic treatments. Significance: Cyclic FMD is well tolerated and causes remarkable systemic metabolic changes in patients with different tumor types and treated with concomitant antitumor therapies. In addition, the FMD reshapes systemic and intratumor immunity, finally activating several antitumor immune programs. Phase II/III clinical trials are needed to investigate FMD antitumor activity/efficacy. This article is highlighted in the In This Issue feature, p. 1

The recent introduction of immune checkpoint inhibitors has been forcing the oncologists to face an unprecedented paradigm shift in cancer therapy. Unusual rationale and mechanisms of action, unexpected types of clinical response, and totally novel side effects, requested clinicians to rapidly gain familiarity with the immune system and its complex rules. A tight collaboration with researchers has become a common practice, to allow a bench-to bedside-and back approach and the fastest implementation of preclinical knowledge into clinical setting. This is particularly true for melanoma, one of the immunogenic—if not the most immunogenic—human malignancies, from which the vast majority of what we now know about cancer immunity has been learned. The present review is the authors’ view on the most recent and relevant information emerged in literature that should be taken into account when facing immunotherapy of melanoma patients. Having a general understanding of the pathways driving melanoma antigenicity and immune escape would help in applying these unique therapeutics under optimized conditions, tailoring treatment on individual patient immune profiles.

Natural killer (NK) cells contribute to immunosurveillance and first-line defense in the control of tumor growth and metastasis diffusion. NK-cell-derived extracellular vesicles (NKEVs) are constitutively secreted and biologically active. They reflect the protein and genetic repertoire of originating cells, and exert antitumor activity in vitro and in vivo. Cancer can compromise NK cell functions, a status potentially reflected by their extracellular vesicles. Hence, NKEVs could, on the one hand, contribute to improve cancer therapy by interacting with tumor and/or immune cells and on the other hand, sense the actual NK cell status in cancer patients. Here, we investigated the composition of healthy donors' NKEVs, including NK microvesicles and exosomes, and their interaction with uncompromised cells of the immune system. To sense the systemic NK cell status in cancer patients, we developed an immune enzymatic test (NKExoELISA) that measures plasma NK-cell-derived exosomes, captured as tsg101+CD56+ nanovesicles. NKEV mass spectrometry and cytokine analysis showed the expression of NK cell markers, i.e., NKG2D and CD94, perforin, granzymes, CD40L, and other molecules involved in cytotoxicity, homing, cell adhesion, and immune activation, together with EV markers tsg101, CD81, CD63, and CD9 in both NK-derived exosomes and microvesicles. Data are available via Proteome Xchange with identifier PXD014894. Immunomodulation studies revealed that NKEVs displayed main stimulatory functions in peripheral blood mononuclear cells (PBMCs), inducing the expression of human leukocyte antigen DR isotype (HLA-DR) and costimulatory molecules on monocytes and CD25 expression on T cells, which was maintained in the presence of lipopolysaccharide (LPS) and interleukin (IL)-10/transforming growth factor beta (TGFβ), respectively. Furthermore, NKEVs increased the CD56+ NK cell fraction, suggesting that effects mediated by NKEVs might be potentially exploited in support of cancer therapy. The measurement of circulating NK exosomes in the plasma of melanoma patients and healthy donors evidenced lower levels of tsg101+CD56+ exosomes in patients with respect to donors. Likewise, we detected lower frequencies of NK cells in PBMCs of these patients. These data highlight the potential of NKExoELISA to sense alterations of the NK cell immune status.

719 Background: Patients (pts) displaying a full-fledged immune response show high levels of circulating extracellular vesicles (EVs) containing immune transcripts that might be transferred to neighboring cells to amplify immune responses, as in autoimmunity and transplantation. We hypothesized EV immune transcripts could reflect immune activation in cancer pts receiving immunotherapy with immune checkpoint inhibitors (ICI). These RNA-containing EVs may surrogate immune functionality and represent a plasma transcriptional signature of adaptive immunity that can be readily detected by liquid biopsy. Methods: Blood samples from metastatic renal cell cancer pts,treated with ICI nivolumab (n=8) or tyrosine kinase inhibitor cabozantinib (n=9) were collected at baseline, week 4 and 12. EV-RNA was isolated from plasma with membrane affinity spin columns and evaluated for CD274(PD-L1), IFNG, PDCD1 (PD-1), CD3 and GZMB (granzyme B) transcripts by qRT-PCR. Results: All pts showed a statistically significant increase of CD3, IFNG, GZMB, PD-1 and PD-L1 immune transcripts in plasma EVs, evidenced at 4 and 12 weeks of therapy. Treatment subgroup analysis revealed an upregulation of CD3, IFNG, GZMB and PD-1 transcripts only in plasma EVs of patients receiving nivolumab that was more evident in those showing clinical benefit (4/8), while PD-L1 increased significantly during cabozantinib treatment. Upon comparing transcripts with soluble proteins, PD-L1 displayed similar kinetics, while PD-1 increased only as transcript but not as protein, given its potential sequestration by the anti-PD-1 antibody. This effect was detectable only in nivolumab but not in cabozantinib treated pts. Conclusions: Monitoring immune transcripts carried by plasma EVs holds promise as potential tool to assess the entity of the ongoing immune activation during immunotherapy. Plasma EVs may be exploited as indicators of immune response and help to predict clinical efficacy at early on-treatment time points. This approach based on plasma EVs might represent a starting point for the development of a novel strategy to study immune responses in clinical setting.

Purpose: Exosomes deliver signals to target cells and could thus be exploited as an innovative therapeutic tool. We investigated the ability of membrane TRAIL-armed exosomes to deliver proapoptotic signals to cancer cells and mediate growth inhibition in different tumor models. Experimental Methods and Results: K562 cells, transduced with lentiviral human membrane TRAIL, were used for the production of TRAIL+ exosomes, which were studied by nanoparticle tracking analysis, cytofluorimetry, immunoelectronmicroscopy, Western blot, and ELISA. In vitro, TRAIL+ exosomes induced more pronounced apoptosis (detected by Annexin V/propidium iodide and activated caspase-3) in TRAIL-death receptor (DR)5+ cells (SUDHL4 lymphoma and INT12 melanoma), with respect to the DR5−DR4+KMS11 multiple myeloma. Intratumor injection of TRAIL+ exosomes, but not mock exosomes, induced growth inhibition of SUDHL4 (68%) and INT12 (51%), and necrosis in KMS11 tumors. After rapid blood clearance, systemically administered TRAIL+ exosomes accumulated in the liver, lungs, and spleen and homed to the tumor site, leading to a significant reduction of tumor growth (58%) in SUDHL4-bearing mice. The treatment of INT12-bearing animals promoted tumor necrosis and a not statistically significant tumor volume reduction. In KMS11-bearing mice, despite massive perivascular necrosis, no significant tumor growth inhibition was detected. Conclusions: TRAIL-armed exosomes can induce apoptosis in cancer cells and control tumor progression in vivo. Therapeutic efficacy was particularly evident in intratumor setting, while depended on tumor model upon systemic administration. Thanks to their ability to deliver multiple signals, exosomes thus represent a promising therapeutic tool in cancer. Clin Cancer Res; 22(14); 3499–512. ©2016 AACR.

Purpose: The progressive immune dysfunctions that occur in patients with advanced melanoma make them unlikely to efficiently respond to cancer vaccines. A multicenter randomized phase II trial was conducted to test whether immunization with modified HLA class I tumor peptides in the context of adjuvant therapy results in better immunologic responses and improved clinical outcomes in patients with early melanoma (stages IIB/C-III). Experimental Design: Forty-three patients were enrolled to undergo vaccination (n = 22) or observation (n = 21). The vaccine included four HLA-A*0201–restricted modified peptides (Melan-A/MART-1[27L], gp100[210M], NY-ESO-1[165V], and Survivin[97M]) emulsified in Montanide ISA51 and injected subcutaneously in combination with cyclophosphamide (300 mg/m2) and low-dose IL-2 (3 × 106 IU). The immune responses were monitored using ex vivo IFN-γ–ELISpot, HLA/multimer staining, and in vitro short-term peptide sensitization assays. Results: Vaccination induced a rapid and persistent increase in specific effector memory CD8+ T cells in 75% of the patients. However, this immunization was not associated with any significant increase in disease-free or overall survival as compared with the observation group. An extensive immunologic analysis revealed a significantly reduced cross-recognition of the corresponding native peptides and, most importantly, a limited ability to react to melanoma cells. Conclusions: Adjuvant setting is an appealing approach for testing cancer vaccines because specific CD8+ T cells can be efficiently induced in most vaccinated patients. However, the marginal antitumor activity of the T cells induced by modified peptides in this study largely accounts for the observed lack of benefit of vaccination. These findings suggest reconsidering this immunization strategy, particularly in early disease. Clin Cancer Res; 18(23); 6485–96. ©2012 AACR.

Background: The fasting mimicking diet (FMD), a plant-based, low-calorie, low-protein, low-carbohydrate diet, modulates systemic metabolism and anticancer immunity, and displays anticancer activity in many preclinical tumor models. However, safety, metabolic, and activity data in the clinical setting are lacking. Methods: At Fondazione IRCCS Istituto Nazionale dei Tumori, a prospective study is ongoing to assess feasibility, tolerability, and metabolic effects of the FMD in cancer patients (pts) following a 5-day FMD (700 Kcal on day 1 and 300 kCal on days 2-5) every 21-28 days. This study is enrolling pts with any cancer type (except for small cell lung cancer), stage, and concomitantly administered anticancer treatment. Main exclusion criteria are: insulin-dependent diabetes, severe comorbidities, BMI 5%. We are regularly collecting blood and urine samples before FMD initiation and at diet completion to measure diet-induced changes of metabolites and growth factors. Here we report data on metabolic modifications detected in 38 pts after the first FMD cycle, and preliminary peripheral blood immunomonitoring in selected cases. Results: 38 pts were enrolled from January 2017 to July 2017. 29 had advanced disease, and 34 were receiving concomitant anticancer therapy at study enrollment. The most represented tumor types were breast (14), prostate (4), pancreatic (4), and lung (5) cancer. All pts completed at least one cycle of FMD, and no G3-G4 adverse event (AE) were reported. After the first FMD cycle, median glycemia was reduced by 15% (p Citation Format: Filippo de Braud, Claudio Vernieri, Veronica Huber, Agata Cova, Paola Squarcina, Monica Milano, Giovanni Fucà, Valter Longo, Licia Rivoltini. Metabolic and immunologic effects of the fasting mimicking diet in cancer patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B022.

The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of antitumor immune responses, through a mechanism known as ‘Fas tumor counterattack,’ has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in the cytoplasm of melanoma cells and its expression is confined to multivesicular bodies that contain melanosomes. In these structures FasL colocalizes with both melanosomal (i.e., gp100) and lysosomal (i.e., CD63) antigens. Isolated melanosomes express FasL, as detected by Western blot and cytofluorimetry, and they can exert Fas-mediated apoptosis in Jurkat cells. We additionally show that melanosome-containing multivesicular bodies degranulate extracellularly and release FasL-bearing microvesicles, that coexpress both gp100 and CD63 and retain their functional activity in triggering Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their antitumor activity.

CTL reactivity to the epitope MART-1(27-35), of the melanoma (self) antigen MART-1/melan A is frequently observed in tumor-infiltrating lymphocytes and may be readily elicited from the peripheral blood of melanoma patients that express HLA-A*0201. Available data suggest that these observations contrast with those made for other HLA-A*0201-presented melanoma self antigens regarding the regularity of observed CTL responses. Based on preliminary findings, we hypothesized that the CTL response to MART-1 might be augmented in part by T cell encounters with peptides derived from sources other than MART-1, which show sequence similarity to MART-1(27-35). To test this idea, a protein database search for potential MART-1 epitope mimics was done using criteria developed from analyses of effector recognition of singly-substituted peptide analogues of MART-1(27-35). Synthetic peptides were made for a portion of the sequences retrieved; 12/40 peptides tested were able to sensitize target cells for lysis by one or more anti-MART-1 effectors. The peptides recognized correspond to sequences occurring in a variety of proteins of viral, bacterial, and human (self) origin. One peptide derives from glycoprotein C of the common pathogen HSV-1; cells infected with recombinant vaccinia virus encoding native glycoprotein C were lysed by anti-MART-1 effectors. Our results overall indicate that sequences conforming to the A2.1 binding motif and possessing features essential to recognition by anti-MART-1 CTL occur frequently in proteins. These findings further suggest that T cells might encounter a variety of such sequences in vivo, and that epitope mimicry may play a role in modulating the CTL response to MART-1(27-35).

Background & Aims: Normal and neoplastic cells release microvesicles, whose effects on the immune system still need to be elucidated. Because human colorectal cancer cells are hypothesized to escape immune recognition by expressing proapoptotic molecules, we investigated whether microvesicles bearing Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand and inducing apoptosis of activated T cells are secreted by colorectal cancer cells both in vitro and in affected patients. Methods: Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand expression were analyzed in colorectal cancer cells and purified microvesicles by flow cytometry, Western blotting, and immunoelectron microscopy. Microvesicle tumor origin was assessed through simultaneous detection of lysosomal (CD63) and adenocarcinoma (carcinoembryonic antigen) markers. Proapoptotic activity of microvesicles was evaluated by annexin V/propidium iodide staining and caspase activation in T cells, including CD8 + T lymphocytes from colorectal cancer patients. Results: Colorectal cancer cells showed a granular pattern of tumor necrosis factor-related apoptosis-inducing ligand and Fas ligand expression, suggesting a secretory behavior. These proapoptotic molecules were detected on isolated microvesicles, together with class I HLA, CD63, and carcinoembryonic antigen. Microvesicles induced Fas ligand-mediated and tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis of activated CD8 + T cells generated from colorectal cancer patients. Microvesicles with comparable phenotypes and functions were found in plasma from patients with advanced disease, whereas vesicular structures expressing Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand were also detected in colorectal cancer specimens. Conclusions: These data show that colorectal cancer induces T-cell apoptosis through the release of Fas ligand-bearing and tumor necrosis factor-related apoptosis-inducing ligand-bearing microvesicles both in vitro and in vivo. This mechanism of immune escape has potential implications as a prognostic factor and could be targeted for the development of new antitumor therapies in colorectal cancer patients.

Heat shock proteins (HSPs) are involved in the activation of both adaptive and innate immune systems. Here, we report that vaccination with autologous tumor-derived HSP96 of colorectal cancer patients, radically resected for liver metastases, induced a significant boost of natural killer (NK) activity detected as cytokine secretion and cytotoxicity in the presence of NK-sensitive targets. Increased NK activity was associated with a raise in CD3−CD56+ NK and/or CD3+CD56+ NK-like T cells, displaying enhanced expression of NKG2D and/or NKp46 receptors. Up-regulated expression of CD83 and CD40 and increased interleukin-12 release on stimulation were observed in CD14+ cells from post-HSP96 peripheral blood mononuclear cells, suggesting an indirect pathway of NK stimulation by HSP96-activated monocytes. Additionally, CD3−CD56+ and CD3+CD56+ lymphocytes were found to undergo functional and phenotypic activation on in vitro exposure to HSP96 even in the absence of monocytes, supporting a potential direct activity of HSP96 on these cell subsets. This evidence was confirmed by the specific binding of FITC-conjugated HSP96 to a subset of both CD3−CD56+ and CD3+CD56+ cells in peripheral blood mononuclear cells from colorectal cancer patients. Altogether, these findings identify the activation of the NK compartment as an additional immunologic effect of autologous tumor-derived HSP96 administration in cancer patients.

Suboptimal activation of T lymphocytes by tumor cells may contribute to the failure of the immune system to control tumor growth. We recently demonstrated that Melan-A/MART-1-reactive CTLs can be anergized by peptide analogues with partial agonist/antagonist functions, which selectively impair interleukin (IL)-2 release. Here we analyze the potential expression of partial agonist/antagonist peptides by tumor cells and their role in suboptimal T-cell activation. HLA-bound peptide fractions were eluted from HLA-A*0201/Melan-A/MART-1(+) melanoma cells and analyzed for reconstitution of the MART-1-specific T-cell epitope. Among the peptide fractions able to induce IFN-gamma release by MART-1-specific T cells, only fraction 43-44 activated IL-2 production by anti-MART-1 T cells, whereas the remaining two fractions acted as peptide antagonists by inhibiting IL-2 release in response to the native epitope. A comparable down-modulation of IL-2 release could also be induced by the MART-1-derived peptide 32-40, previously identified in one of the two anergizing fractions. A substantial deficit in IL-2 release was additionally detected in tumor-specific CD8(+) T cells infiltrating melanoma lesions. To overcome IL-2 impairment by peptide antagonists, anti-MART-1 T cells were generated by in vitro sensitization with the two optimized analogues Melan-A/MART-1(27-35) 1L (with superagonist features) and Melan-A/MART-1(26-35) 2L (with improved HLA-A*0201 binding). T cells raised with the superagonist Melan-A/MART-1(27-35) 1L showed resistance to the inhibition of IL-2 release mediated by melanoma-derived peptide fractions, whereas Melan-A/MART-1(26-35) 2L-specific T cells appeared to be as sensitive as T cells raised with the parental epitope. This resistance was associated with the enhanced ability of Melan-A/MART-1(27-35) 1L-specific T cells to release IL-2. Taken together, these data indicate that melanoma cells can process and present on their surface peptides inhibiting optimal T-cell activation against immunodominant epitopes and that the usage of optimized peptide analogues could represent a promising approach for overcoming tumor-induced immunosuppression and possibly designing more successful vaccines for cancer patients.

The identification of appropriate mouse models could be useful in carefully evaluating the actual role of the in vivo development of antigen-loss variants during antigen-specific vaccine therapy of human tumors. In this study we investigated the level of efficacy of a MART-1/Melan-A-specific CD8+ T cell clone against its autologous melanoma in a severe combined immunodeficiency (SCID) mouse model, in which the tumor cells expressed in vivo heterogeneous and suboptimal levels of MART-1. The subcutaneous co-injection of the MART-1/Melan-A-reactive T cell clone A42 with MART-1/Melan-A+ autologous human melanoma cells into SCID mice caused a total inhibition of tumor growth. However, the systemic treatment with A42 clone lymphocytes resulted in only 50-60% inhibition of tumor growth, although the T cell clone targeted the tumors and the MART-1+ cells virtually disappeared from the tumors. This study suggests that an immunotherapy based on the expansion of an antigen-specific T cell clone generated in vitro is highly efficient in abolishing tumor growth when the target antigen is fully expressed, but leads to in vivo immunoselection of antigen-loss variants in the presence of suboptimal levels of antigen expression. Furthermore, this work shows that human tumors/SCID mouse models may be useful in evaluating the in vivo efficacy of adoptive immunotherapies.

e16042 Background: A significant percentage of patients (pts) progress after first line treatment of prostate cancer (PCa). We present preliminary results of pilot study using a multiple peptide-based anti-tumor vaccine. Methods: A phase I-II trial of vaccination (vax) with HLA-A*0201-restricted peptides from PSMA and Survivin was carried out in 20 pts with b-failure after surgery or radiotherapy (mean pre-vax PSA: 1.83 ng/ml). Vax consisted of two peptides from PSMA (PSMA4–12 and PSMA711–719) and one from Survivin (SVV96–104/97M) given by 4 fortnightly (priming) and 4 monthly administrations (boosting). To selectively eliminate regulatory T cells (Treg) and possibly enhance immunization, peptides were preceded by low dose cyclophosphamide (CTX, 300 mg/mq, i.v.). Antigen (Ag) and tumor-specific T cell responses were extensively monitored in peripheral blood together with CD4+CD25+Foxp3+ Treg frequency. PSA trend was also registered. Results: Vax was well tolerated. Most pts (19/20) showed a significant increase of SVV96–104/97M-specific T cells (mean 14 in pre-vax vs 170 in post-vax PBMC), while response to PSMA was achieved in about half of the pts and only to PSMA711–719 peptide (mean 12 in pre-vax vs 86 in post-vax PBMC). Increments of HLA-A*0201/SVV96–104/97M or PSMA711–719 multimer+ CD8+ T cells were induced in 50 and 35% pts, respectively. Vax-induced Ag-specific T cells displayed however limited cross-reactivity with HLA-A*0201+ PCa cells. No effect of CTX on Treg frequency was observed. 6 pts had no biochemical response to vax and switched to hormonal therapy, while 14/20 exhibited a significant although transient PSA decrease during vax (11 in the priming phase and 3 in boosting). Conclusions: Peptide vax could rapidly enhance Ag-specific immune responses in most treated pts. However, the reduced ability of vax-induced Ag-specific CD8+ T cells to cross-recognize PCa cells, together with their low frequency in PBMC, could explain why PSA control was achieved only transiently and in strict dependence with vax administration. Anti- tumor vax represent a useful tool for controlling PCa biochemical recurrence in the absence of major side effects, but immunization protocols inducing efficient tumor cell killing still need to be identified. No significant financial relationships to disclose.

13502 Background: Despite effective therapeutic strategies for the treatment of prostate cancer (PC), patients still recur and progress in a significant percentage. Here we describe the preliminary clinical and immunological data of a peptide-based strategy of vaccination in biochemical relapsed PC patients. Methods: We planned to enroll 30 HLA-A*0201 patients with biochemical failure after surgery, radiation or androgen deprivation therapy (ADT), according to the ASTRO criteria. On entering the study, patients discontinue ADT. No selections were made based on PSA levels at diagnosis, Gleason pattern score or other pathological features. Vaccination includes three peptides derived from proteins highly expressed in PC, PSMA (PSMA-P14–12 and PSMA-P2711–719) and Survivin (SVV96- 104/97M). Peptides, emulsified in Montanide ISA 51, are administered subcutaneously (8 injections) at two-weeks intervals. Vaccine is preceded by systemic administration of low dose cyclophosphamide (300 mg/m2/four weeks), aimed at selectively down- modulating regulatory T cells (Treg). Immunological monitoring includes evaluation of peptide and tumor-specific CD8+T cells in peripheral blood (IFN?-Elispot and HLA/multimer staining) and analysis of the frequency and suppressive activity of CD4+CD25+Foxp3+Treg. Results: So far, ten patients have been enrolled. Treatment is well tolerated, with mild cutaneous toxicity due to Montanide injection. A significant increase in the frequency of antigen-specific CD8+ T cells was observed in 8/10 patients. Treg, only marginally expanded in these patients, do not seem to decrease by cyclophosphamide treatment, although studies on the potential modulation of their suppressive activity are in progress. After a follow-up of 6–12 months, PSA stabilization was observed in 7 patients, 2 of which entered the trial after maximal androgen blockade discontinuation. Interestingly, PSA stabilization appears to directly correlate with the induction of antigen-specific T cell responses. Conclusions: Our vaccination strategy proved to be safe, non toxic and well tolerated. Initial monitoring data show an expansion of antigen specific T cells, with an apparent correlation between immunological and biochemical responses. No significant financial relationships to disclose.

Peripheral blood lymphocytes (PBL) of a patient with metastatic melanoma were cultured with autologous melanoma cells (Auto-Me) and recombinant interleukin 2 (IL-2) (MLTC-PBL). Thirty-five days later, when no cytotoxicity against Auto-Me or K562 was detectable, MLTC-PBL were cloned in the presence of Auto-Me, IL-2 (25 U/ml) and Daudi cells as feeder. Eighty-one growing clones were simultaneously screened for proliferative and cytotoxic activity to Auto-Me. Twenty-two clones proliferated in the presence of Auto-Me only, 29 in the presence of IL-2 only and 41 in the presence of Auto-Me plus IL-2; 12 clones showed cytotoxic activity against Auto-Me. Six clones expressed both cytotoxic and proliferative activity to Auto-Me. The phenotype of 6 proliferative clones tested was CD3+, CD4+, WT31+, CD8-, CD16-, Leu19-, whereas that of 2 cytotoxic-proliferative clones tested was CD3+, CD8+, Leu19+, WT31+, CD4-, CD16-. Specificity analysis of proliferative response of 6 clones and of cytotoxicity of 7 clones, tested on a panel of 14 different target cells, revealed a complex pattern of reactivity, each clone expressing a peculiar specificity. Our results suggest the possibility of isolating, from melanoma patients' PBL, T-cell clones with proliferative activity to Auto-Me and Auto-Me plus IL-2, and T-cell clones which apparently express both proliferative and cytotoxic activity to Auto-Me.

9092 Background: To select melanoma patients for treatment with BRAF inhibitors, the BRAF mutational status is determined in the most recent tumor biopsy. However, tumor specimens are not always available for the analysis, and relying on a single biopsy specimen can potentially exclude from treatment patients with heterogeneity among metastatic tumors due to polyclonality of BRAF mutation, which appears as a rather common condition. As an alternative approach we explored a blood-based mutation detection assay. Methods: We developed a method including enrichment for the BRAV600E variant by selective elimination of the wild type allele by TspRI digestion and BRAFV600E detection by TaqMan Mutation Detection Assay (BRAF_476_mu, Life Technologies). Sensitivity testing showed that BRAFV600E variant was detected starting from 6.25X10-5 ng of DNA, and specificity testing showed that the variant can be detected when diluted in 8X105 copies of wild-type alleles. Results: Mutational analysis performed by Sanger sequencing of exon15 in 114 melanoma biopsies showed that 4 (3%) harbored the c.1798_1799GT>AA (V600K) mutation, 1 (1%) the c.1799_1800TG>AA mutation (V600E), and 56 (49%) the most common c.1799T>A mutation (V600E), while by the novel method the latter mutation was detected in 9 additional specimens (8% increment) and confirmed by sequencing the PCR product after TspRI digestion. Pre-surgery plasma was available for 50/114 patients at advanced stages, 26/50 (52%) showing a mutated specimen, including 4 with a double nucleotide substitution (V600K and V600E). The matched plasma samples resulted mutated in 25/26 cases with mutated biopsy and in further 14/25 samples with biopsy resulting wild type; in contrast, plasma of 50 healthy controls tested negative. Taken together, these results indicate that V600E circulating variant was detectable in 39/50 (78%) plasma samples and in 22/50 (44%) tumor specimens. Conclusions: Detection of BRAF V600E variant in circulating free DNA may represent a more sensitive approach for patient selection and decision making process during the treatment with BRAF inhibitors.

Recent studies increasingly point to a pivotal role of CD4+ T cells in human anti-tumor immune response. Here we show that lymphocytes purified from a tumor-infiltrated lymph node of a melanoma patient that had remained disease free for 10 years after surgical resection of a lymph node metastasis comprised oligoclonal class II HLA-restricted CD4+ T cells recognizing the autologous tumor cells in vitro. In fact, the CD4+ T cell clones isolated from these lymphocytes displayed a tumor-specific, cytotoxic activity in addition to a Th1-like cytokine profile. By a genetic approach, a peptide derived from a mutated receptor-like protein tyrosine phosphatase κ was identified as a novel HLA-DR10-restricted epitope for all the melanoma-specific CD4+ T cell clones. The immunogenic peptide was shown to contain the mutated residue that was crucial for T cell recognition and activation. Moreover, a systemic immunity against the mutated peptide was detectable in the patient’s peripheral blood T lymphocytes obtained during the disease-free period of follow-up. These findings further support the relevance of CD4+ T cells directed against mutated epitopes in tumor immunity and provide the rationale for a possible usage of mutated, tumor-specific Ags for immunotherapy of human cancer.

Several T cell-recognized epitopes presented by melanoma cells have been identified recently. Despite the large array of epitopes potentially available for clinical use, it is still unclear which of these antigens could be effective in mediating anti-tumor responses when used as a vaccine. Preliminary studies showed that immunization of melanoma patients with epitopes derived from proteins of the MAGE family may result in significant clinical regressions. However, no sign of systemic immunization could be observed in peripheral blood of treated patients. Conversely, significant immunization (detected as increased antigen-specific CTL activity in peripheral blood) was obtained by vaccinating HLA-A2.1+ melanoma patients with the immunodominant epitope (residues 27-35) of the differentiation antigen MART-1, but this immunization was not accompanied by a significant clinical response. To implement immunotherapeuties capable of significantly impacting disease outcome, it is necessary to identify the potential mechanisms responsible for the failure of some antigens to mediate significant anti-tumor responses in vivo. In the case of the MART-1(27-35) epitope, we hypothesize that one of these mechanisms may be related to the existence of natural analogs of this peptide in other human normal proteins.

HLA-A2-restricted, CD3+, CD8+, alpha/beta+ cytotoxic T cell (CTL) clones were isolated from peripheral blood (PBL) or tumor infiltrating lymphocytes (TIL) of two HLA-A2+ melanoma patients (9742 and 5810), to evaluate the possible recognition of autologous melanoma and of allogeneic HLA-A2-matched normal melanocytes. These CTL clones lysed not only fresh and cultured autologous melanoma cells, but also allogeneic HLA-A2+, but not HLA-A2-, normal melanocytes. The lysis of autologous neoplastic cells and of melanocytes could be inhibited by an anti-HLA-A2 monoclonal antibody (mAb). Lysis of the normal melanocytes was not dependent on the presence of human or fetal calf serum in the culture medium. HLA-A2-restricted CTL clones recognized not only proliferating melanocytes cultured in complete melanocyte medium, but also melanocytes made quiescent by culture for up to 6 d in a basal medium devoid of exogenous factors such as phorbol ester (O-tetradecanoyl phorbol 13-acetate [TPA]), epidermal growth factor, insulin, and pituitary extracts. Analysis of specificity of four CTL clones (A75, A83, A94, and 119) from patient 9742, performed on a panel of 39 targets, indicated that the three HLA-A2-restricted CTL (A75, A83, and A94) lysed all but one of nine allogeneic melanomas expressing the HLA-A2 molecule with no reactivity on nine HLA-A2- allogeneic melanomas. Only a few instances of borderline reactivity were seen by the same effectors on 21 targets of nonmelanocyte lineage, including 12 carcinomas of different histology, four Epstein-Barr virus-transformed B cells (lymphoblastoid cell lines [LCL]), including the autologous LCL, four lines of normal fibroblasts, and normal kidney cells. Lack of reactivity on allogeneic targets of nonmelanocyte lineage occurred in spite of expression of HLA-A2 on 14 of these targets as determined by conventional tissue typing and cytofluorimetric analysis with four different anti-HLA-A2 mAb. These data indicate that tissue-related antigens can be expressed on normal and neoplastic cells of the melanocyte lineage and can be recognized in association with HLA-A2 by CTL clones from melanoma patients.

Heat shock proteins (hsp) 96 play an essential role in protein metabolism and exert stimulatory activities on innate and adaptive immunity. Vaccination with tumor-derived hsp96 induces CD8+ T cell-mediated tumor regressions in different animal models. In this study, we show that hsp96 purified from human melanoma or colon carcinoma activate tumor- and Ag-specific T cells in vitro and expand them in vivo. HLA-A*0201-restricted CD8+ T cells recognizing Ags expressed in human melanoma (melanoma Ag recognized by T cell-1 (MART-1)/melanoma Ag A (Melan-A)) or colon carcinoma (carcinoembryonic Ag (CEA)/epithelial cell adhesion molecule (EpCAM)) were triggered to release IFN-γ and to mediate cytotoxic activity by HLA-A*0201-matched APCs pulsed with hsp96 purified from tumor cells expressing the relevant Ag. Such activation occurred in class I HLA-restricted fashion and appeared to be significantly higher than that achieved by direct peptide loading. Immunization with autologous tumor-derived hsp96 induced a significant increase in the recognition of MART-1/Melan-A27–35 in three of five HLA-A*0201 melanoma patients, and of CEA571–579 and EpCAM263–271 in two of five HLA-A*0201 colon carcinoma patients, respectively, as detected by ELISPOT and HLA/tetramer staining. These increments in Ag-specific T cell responses were associated with a favorable disease course after hsp96 vaccination. Altogether, these data provide evidence that hsp96 derived from human tumors can present antigenic peptides to CD8+ T cells and activate them both in vitro and in vivo, thus representing an important tool for vaccination in cancer patients.