Davide Bedognetti

The Continuum of Cancer Immunosurveillance: Prognostic, Predictive, and Mechanistic Signatures

Numerous analyses of large patient cohorts identified specific patterns of immune activation associated with patient survival. We established these as the immune contexture, encompassing the type, functional orientation, density, and location of adaptive immune cells within distinct tumor regions. Based on the immune contexture, a standardized, powerful immune stratification system, the Immunoscore, was delineated. The immune contexture is characterized by immune signatures also observed in association with the broader phenomenon of immune-mediated, tissue-specific destruction. We defined these as the immunologic constant of rejection. Predictive, prognostic, and mechanistic immune signatures overlap, and a continuum of intratumor immune reactions exists. The balance between tumor cell growth and elimination may be tipped upon a crescendo induced by immune manipulations aimed at enhancing naturally occurring immunosurveillance. Here, we propose a broader immunological interpretation of these three concepts--immune contexture, Immunoscore, and immunologic constant of rejection--that segregates oncogenic processes independently of their tissue origin.

Functionalized multiwalled carbon nanotubes as ultrasound contrast agents

Ultrasonography is a fundamental diagnostic imaging tool in everyday clinical practice. Here, we are unique in describing the use of functionalized multiwalled carbon nanotubes (MWCNTs) as hyperechogenic material, suggesting their potential application as ultrasound contrast agents. Initially, we carried out a thorough investigation to assess the echogenic property of the nanotubes in vitro. We demonstrated their long-lasting ultrasound contrast properties. We also showed that ultrasound signal of functionalized MWCNTs is higher than graphene oxide, pristine MWCNTs, and functionalized single-walled CNTs. Qualitatively, the ultrasound signal of CNTs was equal to that of sulfur hexafluoride (SonoVue), a commercially available contrast agent. Then, we found that MWCNTs were highly echogenic in liver and heart through ex vivo experiments using pig as an animal model. In contrast to the majority of ultrasound contrast agents, we observed in a phantom bladder that the tubes can be visualized within a wide variety of frequencies (i.e., 5.5–10 MHz) and 12.5 MHz using tissue harmonic imaging modality. Finally, we demonstrated in vivo in the pig bladder that MWCNTs can be observed at low frequencies, which are appropriate for abdominal organs. Importantly, we did not report any toxicity of CNTs after 7 d from the injection by animal autopsy, organ histology and immunostaining, blood count, and chemical profile. Our results reveal the enormous potential of CNTs as ultrasound contrast agents, giving support for their future applications as theranostic nanoparticles, combining diagnostic and therapeutic modalities.

Functionalized carbon nanotubes as immunomodulator systems

In view of the broad potential biomedical applications of carbon nanotubes (CNTs) different studies were performed to assess their effect on the immune system. However, the work performed to date was able to give a restricted view looking only at some activation markers and cytokine expression. The immune system is rarely limited to few molecule interactions being instead always a balance of switching several genes on and off. Whole genome expression (microarray) is a technology able to give the full picture on genome expression. Here we describe a microarray genome-wide study on Jurkat cells, a T lymphocyte cell line, and THP1, a monocytic cell line, representative of both types of immune response, the adaptive and innate, respectively. Since any structure or molecule modification may lead to very different immune reactions, we treated the two cell lines with four types of functionalized multi-walled CNTs that differ in terms of functionalization and diameter. After having assessed the internalization and the lack of toxicity of CNTs in both cell types, we used the Affymetrix technology to analyze the expression of about 32,000 transcripts. Three of the tested nanotubes (i.e., ox-MWCNT-1, ox-MWCNT-NH3(+)-1, and ox-MWCNT-NH3(+)-2) activated immune-related pathways in monocytes but not in T cells. In view of these charateristics they were named as monocyte activating CNTs (MA-CNTs). Molecular pathways upregulated by MA-CNTs included IL6, CD40, dendritic cell maturation, tumor necrosis factor-(TNF)-α/TNFR1-2, NFKB signaling and T helper 1 chemokine pathways (CXCR3 and CCR5 ligand pathways). These pathways are commonly activated during acute inflammatory processes as those associated with immune-mediated tumor rejection and pathogen clearance. One of them (i.e., ox-MWCNT-2) downregulated genes associated with ribosomal proteins in both monocytes and T cells. We validated our findings at gene expression level by performing real-time PCR assessing the most highly modulated genes in monocytes. To confirm the results at protein level, the secretion of IL1β, TNFα, IL6 and IL10 by THP1 and primary monocytes was assessed by ELISA, corroborating gene-expression data. Our results provide new insights into the whole gene expression modulation by different CNTs on immune cells. Considering the well known drug carrier ability of CNTs, our findings demonstrate that MA-CNTs here behave as cell specific immunostimulatory systems, giving very interesting future perspectives for their application also as immunotherapeutic agents and/or vaccine adjuvants.

An immunologic portrait of cancer

The advent of high-throughput technology challenges the traditional histopathological classification of cancer, and proposes new taxonomies derived from global transcriptional patterns. Although most of these molecular re-classifications did not endure the test of time, they provided bulk of new information that can reframe our understanding of human cancer biology. Here, we focus on an immunologic interpretation of cancer that segregates oncogenic processes independent from their tissue derivation into at least two categories of which one bears the footprints of immune activation. Several observations describe a cancer phenotype where the expression of interferon stimulated genes and immune effector mechanisms reflect patterns commonly observed during the inflammatory response against pathogens, which leads to elimination of infected cells. As these signatures are observed in growing cancers, they are not sufficient to entirely clear the organism of neoplastic cells but they sustain, as in chronic infections, a self-perpetuating inflammatory process. Yet, several studies determined an association between this inflammatory status and a favorable natural history of the disease or a better responsiveness to cancer immune therapy. Moreover, these signatures overlap with those observed during immune-mediated cancer rejection and, more broadly, immune-mediated tissue-specific destruction in other immune pathologies. Thus, a discussion concerning this cancer phenotype is warranted as it remains unknown why it occurs in immune competent hosts. It also remains uncertain whether a genetically determined response of the host to its own cancer, the genetic makeup of the neoplastic process or a combination of both drives the inflammatory process. Here we reflect on commonalities and discrepancies among studies and on the genetic or somatic conditions that may cause this schism in cancer behavior.

Gene-expression profiling in vaccine therapy and immunotherapy for cancer

The identification of tumor antigens recognized by T cells led to the design of therapeutic strategies aimed at eliciting adaptive immune responses. The last decade of experience has shown that, although active immunization can induce enhancement of anticancer T-cell precursors (easily detectable in standard assays), most often they are unable to induce tumor regression and, consequently, have scarcely any impact on overall survival. Moreover, in the few occasions when tumor rejection occurs, the mechanisms determining this phenomenon remain poorly understood, and data derived from in vivo human observations are rare. The advent of high-throughput gene-expression analysis (microarrays) has cast new light on unrecognized mechanisms that are now deemed to be central for the development of efficient immune-mediated tumor rejection. The aim of this article is to review the data on the molecular signature associated with this process. We believe that the description of how the mechanism of immune-mediated tissue destruction occurs would contribute to our understanding of why it happens, thereby allowing us to develop more effective immune therapeutic strategies.

Impact of carbon nanotubes and graphene on immune cells

It has been recently proposed that nanomaterials, alone or in concert with their specific biomolecular conjugates, can be used to directly modulate the immune system, therefore offering a new tool for the enhancement of immune-based therapies against infectious disease and cancer. Here, we revised the publications on the impact of functionalized carbon nanotubes (f-CNTs), graphene and carbon nanohorns on immune cells. Whereas f-CNTs are the nanomaterial most widely investigated, we noticed a progressive increase of studies focusing on graphene in the last couple of years. The majority of the works (56%) have been carried out on macrophages, following by lymphocytes (30% of the studies). In the case of lymphocytes, T cells were the most investigated (22%) followed by monocytes and dendritic cells (7%), mixed cell populations (peripheral blood mononuclear cells, 6%), and B and natural killer (NK) cells (1%). Most of the studies focused on toxicity and biocompatibility, while mechanistic insights on the effect of carbon nanotubes on immune cells are generally lacking. Only very recently high-throughput gene-expression analyses have shed new lights on unrecognized effects of carbon nanomaterials on the immune system. These investigations have demonstrated that some f-CNTs can directly elicitate specific inflammatory pathways. The interaction of graphene with the immune system is still at a very early stage of investigation. This comprehensive state of the art on biocompatible f-CNTs and graphene on immune cells provides a useful compass to guide future researches on immunological applications of carbon nanomaterials in medicine.

Impaired Response to Influenza Vaccine Associated with Persistent Memory B Cell Depletion in Non-Hodgkin’s Lymphoma Patients Treated with Rituximab-Containing Regimens

Influenza vaccination is generally recommended for non-Hodgkin’s lymphoma (NHL) patients, but no data are available about the activity of this vaccine after treatment with rituximab-containing regimens. We evaluated the humoral response to the trivalent seasonal influenza vaccine in a group of NHL patients in complete remission for ≥6 mo (median, 29 mo) after treatment with rituximab-containing regimens (n = 31) compared with age-matched healthy subjects (n = 34). B cell populations and incidence of influenza-like illness were also evaluated. For each viral strain, the response was significantly lower in patients compared with controls and was particularly poor in patients treated with fludarabine-based regimens. In the patient group, the response to vaccination did not fulfill the immunogenic criteria based on the European Committee for Medicinal Products for Human Use requirements. Among the patients, CD27+ memory B cells were significantly reduced, and their reduction correlated with serum IgM levels and vaccine response. Episodes of influenza-like illness were recorded only in patients. These results showed that NHL patients treated with rituximab-containing regimens have persisting perturbations of B cell compartments and Ig synthesis and may be at particular risk for infection, even in long-standing complete remission.

Interaction of a traditional Chinese Medicine (PHY906) and CPT-11 on the inflammatory process in the tumor microenvironment

Background -Traditional Chinese Medicine (TCM) has been used for thousands of years to treat or prevent diseases, including cancer. Good manufacturing practices (GMP) and sophisticated product analysis (PhytomicsQC) to ensure consistency are now available allowing the assessment of its utility. Polychemical Medicines, like TCM, include chemicals with distinct tissue-dependent pharmacodynamic properties that result in tissue-specific bioactivity. Determining the mode of action of these mixtures was previously unsatisfactory; however, information rich RNA microarray technologies now allow for thorough mechanistic studies of the effects complex mixtures. PHY906 is a long used four herb TCM formula employed as an adjuvant to relieve the side effects associated with chemotherapy. Animal studies documented a decrease in global toxicity and an increase in therapeutic effectiveness of chemotherapy when combined with PHY906.Methods -Using a systems biology approach, we studied tumor tissue to identify reasons for the enhancement of the antitumor effect of CPT-11 (CPT-11) by PHY906 in a well-characterized pre-clinical model; the administration of PHY906 and CPT-11 to female BDF-1 mice bearing subcutaneous Colon 38 tumors.Results -We observed that 1) individually PHY906 and CPT-11 induce distinct alterations in tumor, liver and spleen; 2) PHY906 alone predominantly induces repression of transcription and immune-suppression in tumors; 3) these effects are reverted in the presence of CPT-11, with prevalent induction of pro-apoptotic and pro-inflammatory pathways that may favor tumor rejection.Conclusions -PHY906 together with CPT-11 triggers unique changes not activated by each one alone suggesting that the combination creates a unique tissue-specific response.

Potent synergistic interaction between the Nampt inhibitor APO866 and the apoptosis activator TRAIL in human leukemia cells

OBJECTIVE The nicotinamide phosphoribosyltransferase (Nampt) inhibitor APO866 depletes intracellular nicotinamide adenine dinucleotide (NAD(+)) and shows promising anticancer activity in preclinical studies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to plasma membrane receptors DR4 and DR5 and induces apoptosis via caspase-8 and -10. Here we have explored the interaction between APO866 and TRAIL in leukemia cell lines and in primary B-cell chronic lymphocytic leukemia cells. MATERIALS AND METHODS Cells were treated with APO866, TRAIL, or their combination. Viability and mitochondrial transmembrane potential (ΔΨ(m)) were determined by cell staining with propidium iodide and tetramethylrhodamine ethyl ester, respectively, and flow cytometry. Nampt and γ-tubulin levels, as well as caspase-3 cleavage were detected by immunoblotting. DR4 and DR5 expression were assessed by immunostaining and flow cytometry. Caspases were inhibited with zVAD-FMK and zDEVD-FMK; autophagy with 3-methyladenine, LY294002, and wortmannin. Intracellular NAD(+) and adenosine triphosphate (ATP) were measured by cycling assays and high-performance liquid chromatography (HPLC), respectively. RESULTS APO866 induced NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage in leukemia cells, thereby leading to autophagic cell death. TRAIL induced caspase-dependent apoptosis. TRAIL addition to APO866 synergistically increased its activity in leukemia cells by enhancing NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage. No DR5 upregulation at the cell surface in response to APO866 was observed. Remarkably, in healthy leukocytes APO866 and TRAIL were poorly active and failed to show any cooperation. CONCLUSIONS Activation of the extrinsic apoptotic cascade with TRAIL selectively amplifies the sequelae of Nampt inhibition in leukemia cells, and appears as a promising strategy to enhance APO866 activity in hematological malignancies.

Prediction of Response to Anticancer Immunotherapy Using Gene Signatures

Accompanying this article are three reports involving clinical outcomes and correlative parameters associated with the administration of adjuvant melanoma-associated antigen 3 (MAGE-A3) antigen-specific cancer vaccine for the treatment of resected, stage IB-II, non–small-cell lung cancer (NSCLC) and metastatic melanoma. The first study, directed at patients with stage III or IV M1a melanoma (N 75), compared the earlier immune stimulant AS02B to a newer one (AS15) in a randomized, multicenter, phase II trial. This trial showed improved clinical outcomes with AS15, administration of which resulted in a 44% reduction in the risk of death (overall survival [OS]: 33 months v 20 months in the AS15 and AS02B groups, respectively). This observation corroborates the concept that, beyond antigen specificity, the therapeutic activity of cancer vaccines can be significantly enhanced by the coadministration of an appropriate pro-inflammatory molecule. AS15, which contains a TLR9 agonist in addition to a TLR4 agonist and the QS21 saponin (already present in the AS02B) was selected for subsequent phase III trials. 1,2 A second randomized, multicenter, phase II trial performed in patients with NSCLC (N 182) did not observe any significant difference in terms of disease-free survival and OS between the experimental and placebo groups. MAGE-A3 administration was confirmed to be well tolerated. The major limitation of this (proof-of-concept) trial is represented by the small sample size. With an estimated power of 50% to detect a difference of 10% in absolute recurrence after 30 months (for 0.2), the study was unlikely to achieve conclusive results in term of efficacy. The trial, moreover, used as adjuvant in combination with the vaccine a relatively inert immune stimulant (AS02B). Despite these limitations, an encouraging reduction in the risk of relapse of 25% in the treatment goup compared with the placebo group (P .25) was observed. In line with the majority of the studies performed so far, no significant correlation between cellular and/or humoral immune response against the administered antigen and clinical outcome was detected in either the melanoma or the NSCLC trial. The third report, which is the focus of this editorial, summarizes the biologic insights obtained through the systematic application of global transcript analysis (microarray) for the phenotyping of tumor deposits obtained before initiation of treatment. By comparing patients with melanoma who achieved clinical benefit with those who did not, Ulloa-Montoya et al identified an 84-gene expression signature associated with favorable outcome. The signature was internally cross-validated and found to be predictive of prolonged OS. The expressions of 61 out of 84 genes was assessed by qualitative real-time polymerase chain reaction (qRT-PCR), with superimposable results. The predictive role of the qRT-PCR classifier (gene signature; GS) was further confirmed in the NSCLC adjuvant study. In addition, when survival analysis in the NSCLC trial was restricted to patients bearing the favorable gene signature (GS ), the investigators detected a reduction in the risk of relapse of 58% (P .06) associated with vaccine administration, whereas no such effect was found in GSpatients. Similar trends were observed in the OS analysis. These observations indicate that immune manipulations could modify the postoperative course of (some) patients with NSCLC, as suggested earlier by the positive results of a randomized phase trial assessing the impact of adoptive therapy and human recombinant interlekin-2 (hrIL-2) in this setting. The report of Ulloa-Montoya et al does not present isolated findings but substantiates previous observations from several groups that point to the existence of a cancer immune phenotype conducive to immune responsiveness. A decade ago, serial analysis of melanoma metastases using minimally invasive biopsies was applied to patients undergoing melanoma antigen-specific vaccination in conjunction with the systemic administration hrIL-2. Biopsies of the same lesion were obtained before and after at least one cycle of treatment. This strategy allowed prospective assessment within the same lesion of the weight that the pretreatment tumor phenotype had on responsiveness to treatment. The results suggested that melanoma metastases likely to undergo complete regression following immunotherapy display an immunologically active microenvironment before treatment. Those early efforts were, however, curtailed by the limited number of transcripts represented by prototype platforms and failed to provide a global view of the program associated with immune responsiveness. More recently, application of the same serial biopsy strategy to a small cohort of patients undergoing treatment with systemic administration of hrIL-2 using a new generation genome-wide array platform allowed a more comprehensive characterization. Serial sampling of tissues before and during treatment identified in lesions about to undergo clinical regression a signature predictive of immune responsiveness in pretreatmentbiopsies thatwasqualitativelysimilar, thoughquantitatively less, to the one observable during treatment. Moreover, this signature largely overlapped with the one described by Ulloa-Montoya et aland others. This observation suggests that the cancer phenotype associated JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 19 JULY 1 2013