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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 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 Methods

Supplementary Table S7-S16

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).

Supplementary Figure S2. Homing of PKH26-labeled TRAIL exosomes. Schedule of injections (A), detection of fluorescent TRAIL exosomes by confocal microscopy in lesions (B) and of apoptotic cells in suspensions of tumors (C).

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

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

PDF file - 64K, Analysis of chemokine receptor expression

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

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.

The use of IFN-α in clinical oncology has generally been based on the rationale of exploiting its antiproliferative and antiangiogenic activities. However, IFN-α also exhibits enhancing effects on T-cell and dendritic cell functions, which may suggest a novel use as a vaccine adjuvant. We have carried out a pilot phase I-II trial to determine the effects of IFN-α, administered as an adjuvant of Melan-A/MART-1:26-35(27L) and gp100:209-217(210M) peptides, on immune responses in stage IV melanoma patients. In five of the seven evaluable patients, a consistent enhancement of CD8+ T cells recognizing modified and native MART-1 and gp100 peptides and MART-1+gp100+ melanoma cells was observed. Moreover, vaccination induced an increase in CD8+ T-cell binding to HLA tetramers containing the relevant peptides and an increased frequency of CD45RA+CCR7− (terminally differentiated effectors) and CD45RA−CCR7− (effector memory) cells. In all patients, treatment augmented significantly the percentage of CD14+ monocytes and particularly of the CD14+CD16+ cell fraction. An increased expression of CD40 and CD86 costimulatory molecules in monocytes was also observed. Notably, postvaccination monocytes from two of the three patients showing stable disease or long disease-free survival showed an enhanced antigen-presenting cell function and capability to secrete IP10/CXCL10 when tested in mixed leukocyte reaction assays, associated to a boost of antigen and melanoma-specific CD8+ T cells. Although further clinical studies are needed to show the adjuvant activity of IFN-α, the present data represent an important starting point for considering a new clinical use of IFN-α and new immunologic end points, potentially predictive of clinical response. (Cancer Res 2006; 66(9): 4943-51)

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Supplementary Figure 1 from Immunization of Stage IV Melanoma Patients with Melan-A/MART-1 and gp100 Peptides plus IFN-α Results in the Activation of Specific CD8+ T Cells and Monocyte/Dendritic Cell Precursors

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.

BackgroundRescue of exhausted T cell immunity through the inhibition of PD-1/PD-L1 interaction is a pillar of immunotherapeutic anti-PD-1 monoclonal antibodies (mAbs), as Nivolumab and Pembrolizumab. Despite the IgG4 subclass, anti-PD-1 mAbs can bind different FcγRs and trigger immunosuppressive activity in FcR-expressing myeloid cells. This effect is evident when anti-PD-1 mAbs engage soluble PD-1 (sPD-1) forming a stable sPD-1-antiPD-1mAb immune complex (PD-1 IC). In the present study we dissect the process by investigating which of the FcγRs displays the highest affinity for monomeric or complexed Nivolumab. In addition, methods for detecting and quantifying PD-1 ICs in plasma of patients treated with PD-1 blockers mAbs are under development for clinical application.MethodsBy surface plasmon resonance (SRP) (BiacoreTM T200, Cytiva), the interaction of FcγRI/CD64, FcγRIIa/CD32a, FcγRIIb/CD32b, FcγRIIIa/CD16a and FcγRIIIb/CD16b with anti-PD1 Nivolumab and the corresponding PD-1-IC has been evaluated, and their cellular localization has been assessed by confocal microscopy. Further, sPD-1 and PD-1 IC has been determined by customized ELISAs and western blot approaches in plasma of anti-PD-1 mAb-treated patients.ResultsThe binding of anti-PD1 and PD-1-IC occurred with all the FcγRs tested, albeit sensorgrams revealed diverse degrees of affinity. No major difference in the overall affinity of the monomeric anti-PD-1 versus PD-1 IC is observed for FcγRI/CD64, FcγRIIa/CD32a, FcγRIIb/CD32b. Instead, the dissociation phase was clearly slowest for PD-1 IC with respect to the monomeric mAb in FcγRIIIa/CD16a and FcγRIIIb/CD16b binding studies. Nevertheless, only PD-1 IC undergoes internalization when interacting with human FcγR+ myeloid cells in vitro, through pathways that does not appear to lead to lysosomal co-localization. Customized ELISA for PD-1 IC quantification has been developed to detect the complex after enrichment of plasma IgG4 by specific matrix. Data concerning the evaluation of PD-1 IC concentration in plasma of cancer patients treated with Nivolumab, will be presented.ConclusionsDespite the IgG4 subclass is expected to display the lowest binding affinity to Fcγs, we report here that anti-PD-1 therapeutic antibodies bind significantly to CD16, CD32 and CD64, particularly if stabilized in the IC form by engaging soluble PD-1. This evidence, if occurring in vivo, could introduce novel functional properties to these therapeutic agents, with potential detrimental effects on their clinical efficacy. Our findings imply that tools to antagonize PD-1-related exhaustion by Fc-null mAbs or non-mAb-based strategies could be preferred to engage full-fledged antitumor immune responses without unwanted effects related to FcR triggering.

miRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. Little is known on the origin of circulating miRNAs and their relationship with the tumor microenvironment in lung cancer. Here, we focused on the cellular source and relative contribution of different cell types to circulating miRNAs composing our risk classifier of lung cancer using in vitro/in vivo models and clinical samples. A cell‐type specific expression pattern and topography of several miRNAs such as mir‐145 in fibroblasts, mir‐126 in endothelial cells, mir‐133a in skeletal muscle cells was observed in normal and lung cancer tissues. Granulocytes and platelets are the major contributors of miRNAs release in blood. miRNAs modulation observed in plasma of lung cancer subjects was consistent with de‐regulation of the same miRNAs observed during immunosuppressive conversion of immune cells. In particular, activated neutrophils showed a miRNA profile mirroring that observed in plasma of lung cancer subjects. Interestingly mir‐320a secreted by neutrophils of high‐risk heavy‐smokers promoted an M2‐like protumorigenic phenotype through downregulation of STAT4 when shuttled into macrophages. These findings suggest a multifactorial and nonepithelial cell‐autonomous origin of circulating miRNAs associated with risk of lung cancer and that circulating miRNAs may act in paracrine signaling with causative role in lung carcinogenesis and immunosuppression., What's new? microRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. However, little is known on the origin of circulating miRNAs and their mechanisms of action. This study found a multifactorial and non‐epithelial cell‐autonomous origin of circulating miRNAs associated with lung cancer risk. The findings also suggest a link between an immunosuppressive and pro‐tumorigenic microenvironment and modulation of circulating miRNAs associated with lung cancer risk. The authors propose a novel mechanism whereby miRNA released by neutrophils induce macrophage polarization to support lung cancer growth, highlighting the potential for reprogramming macrophages toward an anti‐tumor polarization.

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.

The accrual of myeloid-derived suppressor cells (MDSCs) represents a major obstacle to effective immunotherapy in cancer patients, but the mechanisms underlying this process in the human setting remain elusive. Here, we describe a set of microRNAs (miR-146a, miR-155, miR-125b, miR-100, let-7e, miR-125a, miR-146b, miR-99b) that are associated with MDSCs and resistance to treatment with immune checkpoint inhibitors in melanoma patients. The miRs were identified by transcriptional analyses as being responsible for the conversion of monocytes into MDSCs (CD14(+)HLA-DR(neg) cells) mediated by melanoma extracellular vesicles (EVs) and were shown to recreate MDSC features upon transfection. In melanoma patients, these miRs were increased in circulating CD14(+) monocytes, plasma, and tumor samples, where they correlated with the myeloid cell infiltrate. In plasma, their baseline levels clustered with the clinical efficacy of CTLA-4 or programmed cell death protein 1 (PD-1) blockade. Hence, MDSC-related miRs represent an indicator of MDSC activity in cancer patients and a potential blood marker of a poor immunotherapy outcome.

TPS685 Background: First-line treatment landscape of metastatic renal cell carcinoma (mRCC) is evolving with strong evidence in favour of PD-1/PD-L1 combinations over tyrosine kinase inhibitors (TKIs). No prospective data about efficacy of TKIs post immune-checkpoint inhibitor (CPI) combinations are available. Among TKIs, cabozantinib has demonstrated progression-free survival (PFS) and overall survival (OS) benefit over everolimus in pre-treated mRCC patients (pts). Methods: Overall 49 mRCC pts who received a previous CPI (anti PD-1/PD-L1) will be treated with cabozantinib. Pts will be stratified according to Heng prognostic group, duration of first-line and type of previous therapy received (CPI+CPI or CPI+TKI or CPI+anti-VEGF or CPI monotherapy). Key inclusion criteria include: one previous treatment with a PD-1/PD-L1 inhibitor in first-line and histological diagnosis of clear-cell RCC. The primary endpoint is to assess the efficacy of cabozantinib based on PFS. Secondary endpoints include evaluation of OS, objective response rate and safety profile of the drug. Exploratory endpoints include evaluation of PD-L1 levels by immunohistochemistry in tumor samples; the analysis of the immunological signature/profile of tumor cells; the state of circulating immune cells, as well as the modulating activity of cabozantinib on systemic tumor immunity; the evaluation of bone formation and reabsorption markers in pts with or without bone involvement. Cabozantinib will be administered orally at a dose of 60 mg/day continuously until evidence of disease progression or onset of unacceptable toxicity. Statistical design: By the methodology of Brookmeyer and Crowley, assuming an accrual period of 18 months and a minimum follow-up of 10 months (mos), 49 pts are necessary to detect an increment of the median PFS time from 3.8 mos to 7.4 mos with a power of 90% and one-sided alpha of 5%. The large sample critical value detecting the increment of the PFS median survival time will be 5.54 mos. To date, 2 pts have been enrolled. Clinical trial information: NCT03463681.

Purpose We assessed the presence of salivary cytokines, their modulation during chemoradiation therapy (CTRT), and their association with oral mucositis severity in patients with head and neck cancer (HNC). Methods and Materials The present prospective observational study enrolled 55 patients with locally advanced HNC requiring CTRT. We also studied 10 healthy volunteers and 10 patients with other cancers. The salivary levels of 13 cytokines were analyzed. We constructed a cytokine predictive score of oral mucositis severity. Results The baseline salivary cytokine levels were not associated with the severity of treatment-induced oral mucositis. The cytokine levels overall increased during treatment, especially in patients with worse mucositis. In particular, on univariable analysis, an increase of interleukin (IL)-1β (area under the curve [AUC] 0.733; P =.009), IL-6 (AUC 0.746; P =.005), and tumor necrosis factor-α (AUC 0.710; P =.005) at the third week of treatment was significantly associated with the development of severe oral mucositis. On multivariable analysis, the predictive score based on the IL-1β and IL-6 changes from baseline to week 3 was an early strong predictor of higher grade oral mucositis. Conclusions The treatment of HNC patients with concurrent CTRT induces a significant increase in the salivary levels of IL-1β, IL-6, and tumor necrosis factor-α, all positively associated with the severity of mucosal toxicity. A greater increase of IL-1β and IL-6 3 weeks after treatment initiation is predictive of worse oral mucositis, representing a potential tool for the early identification of patients at risk.

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.

Stimulating the effector functions of tumor-infiltrating T lymphocytes (TIL) in primary and metastatic tumors could improve active and adoptive T-cell therapies for cancer. Abnormal glycolysis, high lactic acid production, proton accumulation, and a reversed intra–extracellular pH gradient are thought to help render tumor microenvironments hostile to roving immune cells. However, there is little knowledge about how acidic microenvironments affect T-cell immunity. Here, we report that lowering the environmental pH to values that characterize tumor masses (pH 6–6.5) was sufficient to establish an anergic state in human and mouse tumor-specific CD8+ T lymphocytes. This state was characterized by impairment of cytolytic activity and cytokine secretion, reduced expression of IL-2Rα (CD25) and T-cell receptors (TCR), and diminished activation of STAT5 and extracellular signal–regulated kinase (ERK) after TCR activation. In contrast, buffering pH at physiologic values completely restored all these metrics of T-cell function. Systemic treatment of B16-OVA–bearing mice with proton pump inhibitors (PPI) significantly increased the therapeutic efficacy of both active and adoptive immunotherapy. Our findings show that acidification of the tumor microenvironment acts as mechanism of immune escape. Furthermore, they illustrate the potential of PPIs to safely correct T-cell dysfunction and improve the efficacy of T-cell–based cancer treatments. Cancer Res; 72(11); 2746–56. ©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.

To present virus and tumor Ags, HLA class I molecules undergo a complex multistep assembly involving discrete but transient folding intermediates. The most extensive folding abnormalities occur in cells lacking the class I L chain subunit, called β2-microglobulin (β2m). Herein, this issue was investigated taking advantage of eight conformational murine mAbs (including the prototypic W6/32 mAb) to mapped H chain epitopes of class I molecules, four human mAbs to class I alloantigens, as well as radioimmunoprecipitation, in vitro assembly, pulse-chase, flow cytometry, and peptide-pulse/ELISPOT experiments. We show that endogenous (HLA-A1, -A66, and -B58) as well as transfected (HLA-A2) heavy chains in β2m-defective Burkitt lymphoma Daudi cells are capable of being expressed on the cell surface, although at low levels, and exclusively as immature glycoforms. In addition, HLA-A2 is: 1) partially folded at crucial interfaces with β2m, peptide Ag, and CD8; 2) receptive to exogenous peptide; and 3) capable of presenting exogenous peptide epitopes (from virus and tumor Ags) to cytotoxic T lymphocytes (bulk populations as well as clones) educated in a β2m-positive environment. These experiments demonstrate a precursor-product relationship between novel HLA class I folding intermediates, and define a stepwise mechanism whereby distinct interfaces of the class I H chain undergo successive, ligand-induced folding adjustments in vitro as well as in vivo. Due to this unprecedented class I plasticity, Daudi is the first human cell line in which folding and function of class I HLA molecules are observed in the absence of β2m. These findings bear potential implications for tumor immunotherapy.

The frequency and function of regulatory T cells (Tregs) were studied in stage II–III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4+ T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8+ T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8+ T cell effectors when used in the inflammatory environment of vaccination. Electronic supplementary material The online version of this article (doi:10.1007/s00262-013-1397-7) contains supplementary material, which is available to authorized users.

Exosomes are endosome-derived nanovesicles involved in intercellular cross-talk through the transfer of proteins and genetic material. In cancer, exosomes can contribute to mould host micro- and macroenvironment, conditioning tumour immunity both at local and systemic level. We have been collecting evidence that these nanovesicles exert a broad array of detrimental effects on the immune system, ranging from apoptosis in activated antitumour T cells to impairment of monocyte differentiation into dendritic cells and induction of suppressive effectors. More recently, we observed that cells with phenotypic and functional properties overlapping with those displayed by myeloid-derived suppressor cells (MDSC) can be generated in vitro by culturing normal monocytes with exosomes released by melanoma cells (Exo-MDSC). Compared to untreated monocytes, Exo-MDSC show increased mRNA level and release of protumourigenic and immunosuppressive cyto/chemokines, down-modulate the expression of HLA-DR and TLRs, and acquire the ability to inhibit T cell proliferation. A defined gene-expression and miRNA signature, involving HIF1alfa, IL6 and several immune-related genes, has been identified in Exo-MDSC as triggered by exosomal TGFb, CCL2 and selected miRNA. Given their ability to efficiently recirculate in body fluids, tumour exosomes could spread systemically and directly condition myelopoiesis. This hypothesis is supported by the evidence that peripheral MDSC from melanoma patients share defined traits of Exo-MDSC gene-expression and miRNA signature, in addition with phenotypic and functional features of these cells. MDSC accumulate in peripheral blood of melanoma patients as an early event and in association with disease progression. High MDSC frequency is an acknowledged negative prognostic factor in several tumour histologies and predicts poor response to immunotherapy, including checkpoint blockade. The identification of exosomes as a potential pathway responsible for MDSC mobilisation and accrual paves the way to the development of novel immune-based therapeutic strategies and prognostic biomarkers in cancer patients. Supported by Italian Association For Cancer Research – AIRC Grant code 12162.