Andrea Anichini

Interleukin-12: Biological properties and clinical application

Interleukin-12 (IL-12) is a heterodimeric protein, first recovered from EBV-transformed B cell lines. It is a multifunctional cytokine, the properties of which bridge innate and adaptive immunity, acting as a key regulator of cell-mediated immune responses through the induction of T helper 1 differentiation. By promoting IFN-γ production, proliferation, and cytolytic activity of natural killer and T cells, IL-12 induces cellular immunity. In addition, IL-12 induces an antiangiogenic program mediated by IFN-γ–inducible genes and by lymphocyte-endothelial cell cross-talk. The immunomodulating and antiangiogenic functions of IL-12 have provided the rationale for exploiting this cytokine as an anticancer agent. In contrast with the significant antitumor and antimetastatic activity of IL-12, documented in several preclinical studies, clinical trials with IL-12, used as a single agent, or as a vaccine adjuvant, have shown limited efficacy in most instances. More effective application of this cytokine, and of newly identified IL-12 family members (IL-23 and IL-27), should be evaluated as therapeutic agents with considerable potential in cancer patients.

Role of Macrophage Targeting in the Antitumor Activity of Trabectedin

There is widespread interest in macrophages as a therapeutic target in cancer. Here, we demonstrate that trabectedin, a recently approved chemotherapeutic agent, induces rapid apoptosis exclusively in mononuclear phagocytes. In four mouse tumor models, trabectedin caused selective depletion of monocytes/macrophages in blood, spleens, and tumors, with an associated reduction of angiogenesis. By using trabectedin-resistant tumor cells and myeloid cell transfer or depletion experiments, we demonstrate that cytotoxicity on mononuclear phagocytes is a key component of its antitumor activity. Monocyte depletion, including tumor-associated macrophages, was observed in treated tumor patients. Trabectedin activates caspase-8-dependent apoptosis; selectivity for monocytes versus neutrophils and lymphocytes is due to differential expression of signaling and decoy TRAIL receptors. This unexpected property may be exploited in different therapeutic strategies.

Immunity to cancer: attack and escape in T lymphocyte–tumor cell interaction

Summary: Tumor cells may express antigens which are recognized in a form of HLA/peptide complexes by T cells. The frequency at which different antigens are seen by T cells of melanoma patients and healthy donors was evaluated by human leukocyte antigen (HLA)/peptide tetramer technology which stains T cells bearing the specific receptor for a given epitope. By this technique, it was found that the majority of metastatic melanoma patients can recognize differentiation antigens (particularly Melan‐A/MART‐1), whereas such a recognition is scanty in the early phase of the disease and in healthy subjects. Despite the presence of melanoma‐specific T cells infiltrating tumor lesions, tumor rejection rarely occurs. Among the different mechanisms of such inefficient antitumor response, this review discusses the possible anti‐T‐cell counterattack mediated by FasL‐positive tumor cells, and shows that FasL is located in the cytoplasm of melanoma cells and is transported in the tumor microenvironment through the release of melanosomes. Additionally, mechanisms of suboptimal T cell activation through tumor cell expression of peptide analogs with antagonist activity are described, together with the possibility of overcoming such anergy induction by the usage of optimized tumor epitopes. Down‐modulation of HLA expression by target tumor cells and its multiple mechanisms is also considered. Finally, we discuss the role of inducible nitric oxide synthases in determining the inhibition of apoptosis in melanoma cells, which can make such tumor cells resistant to the T‐cell attack.

NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo

NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors.

BRAF alterations are associated with complex mutational profiles in malignant melanoma

To evaluate the mutational profiles associated with BRAF mutations in human melanoma, we have studied BRAF, RAS, PTEN, TP53, CDKN2A and CDK4 genes and their expression in melanoma lesions. Owing to the lack of sufficient material from fresh specimens, we employed short-term cell lines obtained from melanoma biopsies. In all, 41 melanoma obtained from eight primary lesions, 20 nodal, 11 cutaneous and two visceral metastases from patients with sporadic (n=31), familial (n=4) and multiple melanoma (n=2) were analysed. The results revealed novel missense mutations in the BRAF, PTEN, CDKN2A and CDK4 genes. Overall, activating mutations of BRAF and loss of functional p16 and ARF were detected in the majority of melanomas (29/41, 36/41 and 29/41, respectively), while PTEN alterations/loss, NRAS and TP53 mutations occurred less frequently (6/41, 6/41 and 10/41, respectively). In the resulting 12 mutational profiles, p16/ARF loss associated with mutated BRAFV599E was the most represented (n=15). In addition, TP53 and PTEN mutations were always accompanied with BRAF alterations, while PTEN loss was found in association with CDKN2A or TP53 mutations in the absence of BRAF activation. The p16/ARFΔ+BRAF/RAS profile was significantly associated with a longer survival, while complex mutational profiles were detected in highly aggressive disease and poor survival. These data support the existence of several molecularly defined melanoma groups which likely reflect different clinical/biological behaviour, thus suggesting that a more extensive molecular classification of melanoma would significantly impact its clinical management.

Mutation-Independent Anaplastic Lymphoma Kinase Overexpression in Poor Prognosis Neuroblastoma Patients

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase predominantly expressed in the developing nervous system. Recently, mutated ALK has been identified as a major oncogene associated with familial and sporadic neuroblastomas (NBL). Yet, a direct correlation between endogenous expression level of the ALK protein, oncogenic potential, and clinical outcome has not been established. We investigated ALK genetic mutations, protein expression/phosphorylation, and functional inhibition both in NBL-derived cell lines and in 34 localized and 48 advanced/metastatic NBL patients. ALK constitutive phosphorylation/activation was observed in high-ALK expressing cells, harboring either a mutated or a wild-type receptor. No activation was found in cell lines with low expression of wild-type ALK. After 72 hours of treatments, small molecule ALK inhibitor CEP-14083 (60 nmol/L) induced growth arrest and cell death in NBL cells overexpressing wild-type (viability: ALK(high) 12.8%, ALK(low) 73%, P = 0.0035; cell death: ALK(high) 56.4%, ALK(low) 16.2%, P = 0.0001) or mutated ALK. ALK protein expression was significantly up-regulated in advanced/metastatic compared with localized NBLs (ALK overexpressing patients: stage 1-2, 23.5%; stage 3-4, 77%; P < 0.0001). Interestingly, protein levels did not always correlate with ALK genetic alterations and/or mRNA abundance. Both mutated and wild-type ALK receptor can exert oncogenic activity in NBL cells. However, wild-type ALK receptor requires a critical threshold of expression to achieve oncogenic activation. Overexpression of either mutated or wild-type ALK defines poor prognosis patients. Alternative mechanisms other than direct mutations and/or gene amplification regulate the ALK level of expression in NBL cells. Wild-type ALK is a potential therapeutic target for advanced/metastatic NBLs.

Mutually exclusive NRASQ61R and BRAFV600E mutations at the single-cell level in the same human melanoma

Activating BRAF or NRAS mutations have been found in 80% of human sporadic melanomas, but only one of these genetic alterations could be detected in each tumour. This suggests that BRAF and NRAS ‘double mutants’ may not provide advantage for tumour growth, or may even be selected against during tumorigenesis. However, by applying mutant-allele-specific-amplification-PCR method to short-term melanoma lines, one out of 14 tumours was found to harbour both BRAFV600E and the activating NRASQ61R mutations. On the other hand, analysis of 21 melanoma clones isolated by growth in soft agar from this tumour indicated that 16/21 clones harboured a BRAFV600E, but were wild-type for NRAS, whereas the remaining had the opposite genotype (NRASQ61R/wild-type BRAF). When compared to BRAFV600E clones, NRASQ61R clones displayed reduced growth in soft agar, but higher proliferative ability in vitro in liquid medium and even in vivo after grafting into SCID/SCID mice. These data suggest that NRAS and BRAF activating mutations can coexist in the same melanoma, but are mutually exclusive at the single-cell level. Moreover, the presence of NRASQ61R or BRAFV600E is associated with distinct in vitro and in vivo growth properties of neoplastic cells.

Immunotherapy of Metastatic Melanoma Using Genetically Engineered GD2-Specific T cells

Purpose: Genetic engineering of human T lymphocytes to express tumor-directed chimeric antigen receptors (CAR) can produce antitumor effector cells that bypass tumor immune escape mechanisms that are due to abnormalities in protein-antigen processing and presentation. Moreover, these transgenic receptors can be directed to tumor-associated antigens that are not protein-derived, such as the ganglioside GD2, which is expressed in a high proportion of melanoma cells. Experimental Design: We generated chimeric T cells specific for the ganglioside GD2 by joining an extracellular antigen-binding domain derived from the GD2-specific antibody sc14.G2a to cytoplasmic signaling domains derived from the T-cell receptor ζ-chain, with the endodomains of the costimulatory molecules CD28 and OX40. We expressed this CAR in human T cells and assessed the targeting of GD2-positive melanoma tumors in vitro and in a murine xenograft. Results: Upon coincubation with GD2-expressing melanoma cells, CAR-GD2 T lymphocytes incorporating the CD28 and OX40 endodomains secreted significant levels of cytokines in a pattern comparable with the cytokine response obtained by engagement of the native CD3 receptor. These CAR-T cells had antimelanoma activity in vitro and in our xenograft model, increasing the survival of tumor-bearing animals. Conclusion: Redirecting human T lymphocytes to the tumor-associated ganglioside GD2 generates effector cells with antimelanoma activity that should be testable in subjects with disease. (Clin Cancer Res 2009;15(18):5852–60)

Vaccination with autologous tumor-loaded dendritic cells induces clinical and immunologic responses in indolent B-cell lymphoma patients with relapsed and measurable disease: a pilot study

Eighteen relapsed patients with measurable indolent non-Hodgkin lymphoma (NHL) were vaccinated with dendritic cells (DCs) loaded with killed autologous tumor cells. Six patients had objective clinical responses including 3 continuous complete responses (CRs) and 3 partial responses (PRs), with a median follow up of 50.5 months. Eight patients had stable disease, whereas 4 had progressive disease. Clinical responses were significantly associated with a reduction in CD4(+)CD25(+)FOXP3(+) regulatory T cells, an increase in CD3(-)CD56(dim)CD16(+) natural killer (NK) cells, and maturation of lymphocytes to the effector memory stage in either postvaccination peripheral blood or tumor specimen samples. In partial responding patients, vaccination significantly boosted the IFN-gamma-producing T-cell response to autologous tumor challenge. In one HLA-A*0201(+) patient who achieved CR, IL-4 release by circulating T cells in response to tumor-specific IgH-encoded peptides was also documented. Immunohistochemical analysis of tumor biopsies using biotin-conjugated autologous serum samples revealed a tumor-restricted humoral response only in the postvaccination serum from responding patients. Collectively these results demonstrate that vaccination with tumor-loaded DCs may induce both T- and B-cell responses and produces clinical benefits in indolent NHL patients with measurable disease. This study is registered with the Istituto Superiore di Sanità: http://www.iss.it with protocol number 7578-PRE 21-801.

Boosting T Cell-Mediated Immunity to Tyrosinase by Vaccinia Virus-Transduced, CD34+-Derived Dendritic Cell Vaccination: A Phase I Trial in Metastatic Melanoma

Purpose: Six American Joint Committee on Cancer stage IV melanoma patients were enrolled into a Phase I study of vaccination with autologous CD34+-derived dendritic cells transduced with a modified vaccinia Ankara virus encoding human tyrosinase gene (MVA-hTyr). Experimental Design: Patients received a first intravenous injection of 1 × 108 MVA-hTyr–transduced dendritic cells, followed by three s.c. injections at a 14-day interval. Results: Treatment was well tolerated, except for low-grade fever (three of six patients), mild erythema at injection site (five of six), and vitiligo (two of six). A partial response, involving shrinkage of an s.c. nodule, later surgically removed, was observed in 1 patient, who then remained disease-free (>850 days). By human lymphocyte antigen tetramer analysis, significant and often long-lasting increases in frequency of T cells directed to tyrosinase368–376 but not to gp100209–217 were documented in periphery of 4 of 5 HLA-A*0201+ patients, a few days after vaccine administration. In addition, maturation phenotype of tyrosinase-specific T cell shifted toward the T effector memory/T terminally differentiate stages (CCR7−CD45RA−/+) in synchrony with the T-cell frequency peaks. By enzyme-linked immunospot in peripheral blood of five HLA-A*0201+ patients, we found that the vaccine could induce interferon γ-releasing effector cells directed to HLA-A*0201/tyrosinase368–376 and to vaccinia virus HLA-A*0201/H3L184–192 epitopes. Moreover, an interferon γ response after vaccination was elicited even against the HLA-DRB1–1501/tyrosinase386–406 epitope in one out of two HLA-A* DRB1–01501+ patients. Conclusions: These results indicate that vaccination with MVA-hTyr–transduced dendritic cells is well tolerated, can possibly produce clinical responses, and activates tyrosinase- and vaccinia virus-specific T cells in vivo. These data suggest a broad utility of the MVA vector for targeting tumor-associated antigens to dendritic cells for tumor immunotherapy.