PEOPLE (NCT03447678), a first-line phase II pembrolizumab trial, in negative and low PD-L1 advanced NSCLC: clinical outcomes and association with circulating immune biomarkers.

BACKGROUND: The PEOPLE trial aimed to identify new immune biomarkers in negative and low programmed death-ligand 1 (PD-L1) (0%-49%) advanced non-small-cell lung cancer (aNSCLC) patients treated with first-line pembrolizumab. Here we report the main outcomes and the circulating immune biomarkers analysis. PATIENTS AND METHODS: The primary endpoint of this phase II trial was the identification of immune biomarkers associated with progression-free survival (PFS). Overall survival (OS), objective response rate (ORR), disease control rate (DCR), duration of response (DoR) and safety were secondary endpoints. Absolute cell counts for 36 subsets belonging to innate and adaptive immunity were determined by multiparametric flow cytometry in peripheral blood at baseline and at first radiologic evaluation. An orthoblique principal components-based clustering approach and multivariable Cox regression model adjusted for clinical variables were used to analyze immune variables and their correlation with clinical endpoints. RESULTS: From May 2018 to October 2020, 65 patients were enrolled. After a median follow-up of 26.4 months, the median PFS was 2.9 months [95% confidence interval (CI) 1.8-5.6 months] and median OS was 12.1 months (95% CI 8.7-17.1 months). The ORR was 21.5%, DCR was 47.7% and median DoR was 14.5 months (95% CI 6.4-24.9 months). Drug-related grade 3-4 adverse events were 9.2%. Higher T cell and natural killer (NK) cell count at baseline and at the first radiologic evaluation were associated with improved PFS, DCR and OS. On the contrary, higher myeloid cell count at baseline or at the first radiologic evaluation was significantly associated with worse OS and DCR. CONCLUSIONS: Circulating immune biomarkers can contribute to predict outcomes in negative and low PD-L1 aNSCLC patients treated with first-line single-agent pembrolizumab.

Correction to: NFATc2 is an intrinsic regulator of melanoma dedifferentiation.

In Fig. 6e, the authors noticed that wrong blots for MITF, MART-1 expression/modulation, and for ß-actin were presented, due to the similarity with experiments shown in Figure 5c. Correct MITF, MART-1, and ß-actin blots were added to the revised Fig. 6 shown in the associated Correction. The meaning of the results shown in Fig.6e, as well as the conclusions of this paper were not affected, and the authors regret for this error. These errors have not been fixed in the original Article.

NFATc2 is an intrinsic regulator of melanoma dedifferentiation.

Melanoma dedifferentiation, characterized by the loss of MITF and MITF regulated genes and by upregulation of stemness markers as CD271, is implicated in resistance to chemotherapy, target therapy and immunotherapy. The identification of intrinsic mechanisms fostering melanoma dedifferentiation may provide actionable therapeutic targets to improve current treatments. Here, we identify NFATc2 transcription factor as an intrinsic regulator of human melanoma dedifferentiation. In panels of melanoma cell lines, NFATc2 expression correlated inversely with MITF at both mRNA and protein levels. NFATc2(+/Hi) melanoma cell lines were CD271(+) and deficient for expression of melanocyte differentiation antigens (MDAs) MART-1, gp100, tyrosinase and of GPNMB, PGC1-α and Rab27a, all regulated by MITF. Targeting of NFATc2 by small interfering RNA, short hairpin RNA and by an NFATc2 inhibitor upregulated MITF, MDAs, GPNMB, PGC-1α, tyrosinase activity and pigmentation and suppressed CD271. Mechanistically, we found that NFATc2 controls melanoma dedifferentiation by inducing expression in neoplastic cells of membrane-bound tumor necrosis factor-α (mTNF-α) and that melanoma-expressed TNF-α regulates a c-myc-Brn2 axis. Specifically, NFATc2, mTNF-α and expression of TNF receptors were significantly correlated in panels of cell lines. NFATc2 silencing suppressed TNF-α expression, and neutralization of melanoma-expressed TNF-α promoted melanoma differentiation. Moreover, silencing of NFATc2 and TNF-α neutralization downmodulated c-myc and POU3F2/Brn2. Brn2 was strongly expressed in NFATc2(+/Hi) MITF(Lo) cell lines and its silencing upregulated MITF. Targeting of c-myc, by silencing or by a c-myc inhibitor, suppressed Brn2 and upregulated MITF and MART-1 in melanoma cells. The relevance of NFATc2-dependent melanoma dedifferentiation for immune escape was shown by cytolytic T-cell assays. NFATc2(Hi) MITF(Lo) MDA(Lo) HLA-A2.1(+) melanoma cells were poorly recognized by MDA-specific and HLA-A2-restricted CTL lines, but NFATc2 targeting significantly increased CTL-mediated tumor recognition. Taken together, these results suggest that the expression of NFATc2 promotes melanoma dedifferentiation and immune escape.

Synergistic anti-tumor activity and inhibition of angiogenesis by cotargeting of oncogenic and death receptor pathways in human melanoma.

Improving treatment of advanced melanoma may require the development of effective strategies to overcome resistance to different anti-tumor agents and to counteract relevant pro-tumoral mechanisms in the microenvironment. Here we provide preclinical evidence that these goals can be achieved in most melanomas, by co-targeting of oncogenic and death receptor pathways, and independently of their BRAF, NRAS, p53 and PTEN status. In 49 melanoma cell lines, we found independent susceptibility profiles for response to the MEK1/2 inhibitor AZD6244, the PI3K/mTOR inhibitor BEZ235 and the death receptor ligand TRAIL, supporting the rationale for their association. Drug interaction analysis indicated that a strong synergistic anti-tumor activity could be achieved by the three agents and the AZD6244-TRAIL association on 20/21 melanomas, including cell lines resistant to the inhibitors or to TRAIL. Mechanistically, synergy was explained by enhanced induction of caspase-dependent apoptosis, mitochondrial depolarization and modulation of key regulators of extrinsic and intrinsic cell death pathways, including c-FLIP, BIM, BAX, clusterin, Mcl-1 and several IAP family members. Moreover, silencing experiments confirmed the central role of Apollon downmodulation in promoting the apoptotic response of melanoma cells to the combinatorial treatments. In SCID mice, the AZD6244-TRAIL association induced significant growth inhibition of a tumor resistant to TRAIL and poorly responsive to AZD6244, with no detectable adverse events on body weight and tissue histology. Reduction in tumor volume was associated not only with promotion of tumor apoptosis but also with suppression of the pro-angiogenic molecules HIF1α, VEGFα, IL-8 and TGFß1 and with inhibition of tumor angiogenesis. These results suggest that synergistic co-targeting of oncogenic and death receptor pathways can not only overcome melanoma resistance to different anti-tumor agents in vitro but can also promote pro-apoptotic effects and inhibition of tumor angiogenesis in vivo.

Perifosine and sorafenib combination induces mitochondrial cell death and antitumor effects in NOD/SCID mice with Hodgkin lymphoma cell line xenografts.

The effects of the Akt inhibitor perifosine and the RAF/MEK/ERK inhibitor sorafenib were investigated using two CD30(+)Hodgkin lymphoma cell lines (L-540 and HDLM-2) and the CD30(-)HD-MyZ histiocytic cell line. The combined perifosine/sorafenib treatment significantly inhibited mitogen-activated protein kinase and Akt phosphorylation in two of the three cell lines. Profiling of the responsive cell lines revealed that perifosine/sorafenib decreased the amplitude of transcriptional signatures that are associated with the cell cycle, DNA replication and cell death. Tribbles homolog 3 (TRIB3) was identified as the main mediator of the in vitro and in vivo antitumor activity of perifosine/sorafenib. Combined treatment compared with single agents significantly suppressed cell growth (40-80%, P<0.001), induced severe mitochondrial dysfunction and necroptotic cell death (up to 70%, P<0.0001) in a synergistic manner. Furthermore, in vivo xenograft studies demonstrated a significant reduction in tumor burden (P<0.0001), an increased survival time (81 vs 45 days, P<0.0001), an increased apoptosis (2- to 2.5-fold, P<0.0001) and necrosis (2- to 8-fold, P<0.0001) in perifosine/sorafenib-treated animals compared with mice receiving single agents. These data provide a rationale for clinical trials using perifosine/sorafenib combination.

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.

Phases of apoptosis of melanoma cells, but not of normal melanocytes, differently affect maturation of myeloid dendritic cells.

In this study, we investigated whether maturation of monocyte-derived myeloid dendritic cells (DCs) is differentially affected by the uptake of dying human melanoma cells in distinct phases of apoptosis. Maturation of monocyte-derived DCs, as documented by phenotype analysis and T-cell immunostimulatory activity, was inhibited by phagocytosis of dying melanoma cells containing a large fraction of cells in early apoptosis (Annexin-V+ and propidium iodide-) but promoted by the same tumors when in late apoptosis/secondary necrosis (Annexin-V+ and propidium iodide+) or when dying by primary necrosis. These opposite effects on DC maturation were observed after the uptake of early or late apoptotic cells from most vertical growth phase primary tumors and all metastases but not after the uptake of dying cells from a radial growth phase primary tumor or normal adult melanocytes. Inhibition of DC maturation by early apoptotic melanoma cells correlated with expression of interleukin-10 in neoplastic cells and was prevented by preincubating the tumor cells with a neutralizing antibody to interleukin-10 before tumor uptake by DCs. Cross-presentation of the melanoma-associated antigen gp100(209-217) to peptide-specific CTLs by HLA-A*0201+ DCs was achieved 48-72 h after phagocytosis of HLA-A*0201- melanoma cells in apoptosis, or primary necrosis, but only when tumor necrosis factor-alpha was added to DCs 4 h after the initiation of tumor phagocytosis. These results suggest that phases of apoptosis and neoplastic transformation affect maturation of myeloid DCs that take up dying cells of the melanocyte lineage. However, neoplastic cells in late apoptosis, or even in primary necrosis, induce only a partial DC differentiation not sufficient to achieve cross-presentation of tumor antigens to CTLs unless further DC maturation is promoted by additional signals. These results suggest a novel mechanism of tumor escape that may prevent the development of antitumor immunity through the maturation block induced in DCs by neoplastic cells in the early phase of apoptosis.

Large-scale feasibility of gene transduction into human CD34+ cell-derived dendritic cells by adenoviral/polycation complex.

With a view to using multiple injections of anti-cancer dendritic cell (DC)-based vaccines, we evaluated the feasibility of the adenoviral transduction of large amounts of human CD34+ cell-derived DCs, and analysed the persistence of the transgene expression and the integrity of DC functional activity after the transduction/cryopreservation procedures. Mature DCs generated from highly enriched human CD34+ cells were transduced by a recombinant adenovirus (rAd-MFG) that carried a modified, membrane-exposed, alkaline phosphatase (AP) sequence as the reporter gene. Cationic lipids such as LipofectAmine or poly-L-lysine were mixed with the viral particles before the transduction of the target cells. The highest transduction efficiency was obtained at a multiplicity of infection (MOI) rate of 500 (AP + DCs: 50 +/- 2%, viability =95%) under both small- and large-scale conditions. The addition of poly-L-lysine or LipofectAmine increased the percentage of transduced cells at an MOI of 500 (CD1a+/AP+ cells = 85 +/- 3% and 80 +/- 2% respectively). Polycations made it possible to reduce the amounts of viral particles, with high efficiency of transduction being achieved at a MOI of 100 with 10 microg/ml poly-L-lysine (CD1a+/AP+: 68 +/- 9%) or 30 microg/ml LipofectAmine (CD1a+/AP+: 60 +/- 7%). Evaluation of the immunophenotype of the transduced DCs showed that the lack of a DC subpopulation was more susceptible to adenoviral transduction. Cryopreservation of transduced DCs did not modify the viability or percentage of AP+ cells that maintain antigen-presenting cell (APC) functions. These findings indicate the efficacy of this method for the transduction of large amounts of CD34+ cell-derived DCs using small quantities of adenoviral vector mixed with polycations. Cryopreservation of transduced DCs did not damage their viability or APC functions, thus making it possible to plan multiple injections of engineered DC-based vaccines.

The alpha 3 beta 1 integrin is associated with mammary carcinoma cell metastasis, invasion, and gelatinase B (MMP-9) activity.

The alpha 3 beta 1 integrin is elevated in several types of metastatic tumor and has been associated with increased migration and invasion. Our analysis of a series of mammary carcinomas of different histotypes and their corresponding metastases demonstrated significantly increased expression of alpha 3 beta 1 in the tumor metastases. We therefore studied alpha 3 beta 1 expression of several human breast carcinoma cell lines and its association with the invasive phenotype. The MDA-MB-231 cell line expressed high levels of the beta1, alpha 2, alpha 3, alpha 5, and alpha 6 integrin subunits along with moderate levels of the alpha v beta 3 integrin. This line was highly migratory and the most invasive using a chemo-invasion assay. In contrast, the other lines tested, MDA-MB-145, MCF-7, and SK-BR-3, showed lower migratory and invasive activity and reduced alpha 3 integrin subunit expression. Metalloproteases capable of degrading collagen IV are necessary for the invasive process. RT-PCR showed that MDA-MB-231 cells expressed MMP-9, but not MMP-2, gelatinase/collagenase IV. Gelatin zymography demonstrated that invading MDA-MB-231 cells released high levels of MMP-9 gelatinase activity. A direct role for this gelatinase in MDA-MB-231 cell invasion was confirmed by inhibition of invasion using the metalloprotease inhibitor Batimastat. Treatment of MDA-MB-231 cells with a function-blocking anti-alpha 3 antibody strongly inhibited migration and invasion. This correlated with a marked reduction in MMP-9 activity produced by MDA-MB-231 cells, suggesting a role for alpha 3 beta 1 ligand binding in cell signaling and regulation of extracellular matrix degradation.

Peripheral burst of tumor-specific cytotoxic T lymphocytes and infiltration of metastatic lesions by memory CD8+ T cells in melanoma patients receiving interleukin 12.

Systemic effects on T-cell-mediated antitumor immunity, on expression of T-cell adhesion/homing receptors, and on the promotion of T-cell infiltration of neoplastic tissue may represent key steps for the efficacy of immunological therapies of cancer. In this study, we investigated whether these processes can be promoted by s.c. administration of low-dose (0.5 microg/kg) recombinant human interleukin-12 (rHuIL-12) to metastatic melanoma patients. A striking burst of HLA-restricted CTL precursors (CTLp) directed to autologous tumor was documented in peripheral blood by a high-efficiency limiting dilution analysis technique within a few days after rHuIL-12 injection. A similar burst in peripheral CTLp frequency was observed even when looking at response to a single tumor-derived peptide, as documented by an increase in Melan-A/Mart-1(27-35)-specific CTLp in two HLA-A*0201+ patients by limiting dilution analysis and by staining peripheral blood lymphocytes (PBLs) with HLA-A*0201-melanoma antigen-A/melanoma antigen recognized by T cells (Melan-A/Mart)-1 tetrameric complexes. The CTLp burst was associated, in PBLs, with enhanced expression of T-cell adhesion/homing receptors CD11a/CD18, CD49d, CD44, and with increased proportion of cutaneous lymphocyte antigen (CLA)-positive T cells. This was matched by a marked increase, in serum, of soluble forms of the endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecules (VCAM)-1 and intercellular adhesion molecules (ICAM)-1. Infiltration of neoplastic tissue by CDS+ T cells with a memory and cytolytic phenotype was found by immunohistochemistry in eight of eight posttreatment metastatic lesions but not in five of five pretreatment metastatic lesions from three patients. Increased tumor necrosis and/or fibrosis were also found in several posttherapy lesions of two of three patients in comparison with pretherapy metastases. These results provide the first evidence that rHuIL-12 can boost the frequency of circulating antitumor CTLp in tumor patients, enhances expression of ligand receptor pairs contributing to the lymphocyte function-associated antigen-1/ICAM-1, very late antigen-4/VCAM-1, and CLA/E-selectin adhesion pathways, and promotes infiltration of neoplastic lesions by CD8+ memory T cells in a clinical setting.

An expanded peripheral T cell population to a cytotoxic T lymphocyte (CTL)-defined, melanocyte-specific antigen in metastatic melanoma patients impacts on generation of peptide-specific CTLs but does not overcome tumor escape from immune surveillance in metastatic lesions.

It is not known if immune response to T cell-defined human histocompatibility leukocyte antigen (HLA) class I-restricted melanoma antigens leads to an expanded peripheral pool of T cells in all patients, affects cytotoxic T lymphocyte (CTL) generation, and correlates with anti-tumor response in metastatic lesions. To this end, a limiting dilution analysis technique was developed that allowed us to evaluate the same frequency of peptide-specific T cells as by staining T cells with HLA-peptide tetrameric complexes. In four out of nine patients, Melan-A/Mart-1(27-35)-specific CTL precursors (CTLp) were >/=1/2,000 peripheral blood lymphocytes and found mostly or only in the CD45RO(+) memory T cell subset. In the remaining five patients, a low (<1/40,000) peptide-specific CTLp frequency was measured, and the precursors were only in the CD45RA(+) naive T cell subset. Evaluation of CTL effector frequency after bulk culture indicated that peptide-specific CTLs could be activated in all patients by using professional antigen-presenting cells as dendritic cells, but CTLp frequency determined the kinetics of generation of specificity and the final number of effectors as evaluated by both limiting dilution analysis and staining with HLA-A*0201-Melan-A/Mart-1 tetrameric complexes. Immunohistochemical analysis of 26 neoplastic lesions from the nine patients indicated absence of tumor regression in most instances, even in patients with an expanded peripheral T cell pool to Melan-A/Mart-1 and whose neoplastic lesions contained a high frequency of tetramer-positive Melan-A/Mart-1-specific T cells. Furthermore, frequent lack of a "brisk" or "nonbrisk" CD3(+)CD8(+) T cell infiltrate or reduced/absent Melan-A/Mart-1 expression in several lesions and lack of HLA class I antigens were found in some instances. Thus, expansion of peripheral immune repertoire to Melan-A/Mart-1 takes place in some metastatic patients and leads to enhanced CTL induction after antigen-presenting cell-mediated selection, but, in most metastatic lesions, it does not overcome tumor escape from immune surveillance.

High frequency of T cell clonal expansions in primary human melanoma. Involvement of a dominant clonotype in autologous tumor recognition.

It was previously found that primary melenomas from HLA-A2-matched patients display an increased expression of a few T cell receptor (TCR) variable-region beta-chain transcripts (BV) compared with autologous peripheral blood lymphocytes (PBL) and uninvolved skin. In order to see whether expansions of clonal/oligoclonal subsets of T cells occurred, complementarity-determining region 3 (CDR3) of BV transcripts overexpressed in the neoplastic infiltrate were cloned and sequenced. Dominant rearrangements were found for BV14, which were commonly increased in the neoplastic lesions of all analysed HLA-A2 melanoma patients, as well as for other overexpressed BV gene families, but none of them could be identified among autologous PBL. No identical CDR3 sequences could be detected in the dominant BV14 rearrangements obtained from the different patients. In one patient a single clonally expanded SLSGTGVNEQF CDR3 clonotype accounted for the entire BV14 relative frequency of expression (24%) in tumor-infiltrating lymphocytes (TIL). Two independent mixed lymphocyte/tumor cultures (MLTC) could be successfully established from TIL of the patient and were found to exert HLA-class-I-restricted cytotoxicity for the autologous melanoma line. BV14 T cells that constituted from 22% to 32% of all T cells present in both MLTC lines, as assessed by flow cytometry, all displayed the same CDR3 clonotype found in vivo and could be shown, by TCR down-modulation experiments, to be involved in autologous tumor recognition. These results support the hypothesis of a tumor-antigen-driven origin of clonally amplified T cells present at high frequency in the in situ neoplastic infiltrate.

Pilot study of subcutaneous recombinant human interleukin 12 in metastatic melanoma.

The aim of this study was to evaluate the safety profile of s.c. administered recombinant human interleukin 12 (rHuIL-12). Pharmacokinetics and pharmacodynamics of rHuIL-12 and any evidence of antitumor effect were also considered. Ten pretreated patients with progressive metastatic melanoma were enrolled in this pilot study. Patients received a fixed dose of rHuIL-12 (0.5 microgram/kg) for two identical 28-day cycles, with injections given on days 1, 8, and 15 of each cycle. In case of any evidence of response or disease stabilization, the treatment was continued for two further 28-day cycles. Toxicity mainly consisted of a flu-like syndrome. Transient increases in transaminasemia (6 of 10 patients) and triglyceridemia (8 of 10 patients) were observed. Peak serum IL-12 levels were reached 8-12 h after the first injection in all patients; no serum IL-12 was detectable in 6 of 9 evaluable patients after the last injection of the second cycle. No antibody response to rHuIL-12 could be detected in any of the patients. A marked, transient reduction in circulating CD8+ and CD16+ lymphocytes and neutrophils was observed after the first administration and high levels of serum IFN-gamma and IL-10 were detected in all patients within 24-48 h. Tumor shrinkage, not reaching partial or complete remission, involved the regression of s.c. nodules (2 of 3 patients), superficial adenopathies (1 of 3 patients), and hepatic metastases (1 of 3 patients); regressions were detected after the first cycle of treatment and were maintained in spite of progression at different sites. s.c. rHuIL-12 treatment was well tolerated and had marked effects on immune parameters and potential antitumor activity.

Monocyte-derived dendritic cells and monocytes migrate to HIV-Tat RGD and basic peptides.

OBJECTIVE AND DESIGN: Extracellular Tat released from HIV-1-infected cells is a mitogenic and motogenic factor for endothelial and Kaposi's sarcoma (KS)-derived cells and is angiogenic in vivo. Here we show for the first time that Tat induces migration of human dendritic cells in a concentration-dependent manner and that the Arg-Gly-Asp (RGD) and basic Tat peptides contribute to dendritic and monocyte cell migration. In vivo, Tat stimulates invasion of macrophages into a matrigel sponge. METHODS: Monocyte and dendritic cell chemotaxis was assessed using the Boyden chamber assay. RESULTS: Tat induced migration of monocyte-derived dendritic cells at the same levels as the N-formyl-Met-Leu-Phe peptide, and of monocytes at levels comparable to RANTES. Peptide mapping of the chemotactic activity of Tat showed that the RGD domain, which has been shown to support integrin-mediated cell migration, and the basic domain which binds and activates the tyrosine kinase receptor KDR on endothelial cells, both had activity. Antibody-blocking experiments indicate that responses to the RGD domain was inhibited by beta1 and alpha vbeta3 integrin blocking antibodies. Combination of the Tat RGD and basic peptides did not show additive effects; however, Tat co-operated with macrophage-chemotactic protein or RANTES in inducing monocyte migration. CONCLUSIONS: Our results show that Tat can act as a chemoattractant for dendritic cells, and that both the RGD and basic domains are involved in this response. These same domains attract monocytes. The alpha vbeta3 and beta1 integrins are equally involved in Tat-induced monocyte migration, while the alpha vbeta3 integrin largely mediates the dendritic cell response to Tat.

Human dendritic cells: natural adjuvants in antitumor immunotherapy.

Although T-cell-defined tumor antigens have recently been identified in several tumors, human neoplastic cells are considered to be poorly immunogenic. The development of clinical approaches to the immunotherapy of human tumors thus requires the identification of effective adjuvants. Dendritic cells (DC) are a specialized system of antigen-presenting cells (APC) that could be utilized as natural adjuvants to elicit antitumor immune responses. In an attempt to overcome the problem of the low frequency of mature DC in peripheral blood, several methods have been applied for the ex vivo generation of human DC by culturing mobilized CD34+ cells or monocytes with combinations of cytokines. As shown in murine models as well as in the human system, after loading with peptides or tumor lysates or infection with recombinant viral vectors, DC expressing tumor antigens are able to elicit specific antitumor T cells and to mediate tumor regression. These initial results suggest that this new approach may lead to effective antitumor responses even in heavily pretreated patients bearing advanced cancers, but further clinical trials are required to validate the efficacy of vaccination with tumor-antigen-loaded DC.

Gene transfer into human dendritic antigen-presenting cells by vaccinia virus and adenovirus vectors.

In a search for means to deliver exogenous gene(s) into human dendritic cells (DCs) from the perspective of tumor-specific vaccination, we have evaluated two recombinant viruses, both of which carry a reporter gene which is namely a modified vaccinia virus Ankara (MVA) and an adenovirus, as possible expression vectors. The recombinant MVA-P11 LZ vector carries the Escherichia coli lacZ gene coding for the enzyme beta-galactosidase, and the recombinant Ad-MFG-AP vector carries a modified membrane-exposed alkaline phosphatase (AP) gene. DCs were generated ex vivo in the presence of tumor necrosis factor-alpha, granulocyte macrophage colony-stimulating factor, stem cell factor, and flk-2/flt-3 ligand taken from CD34+ hematopoietic progenitors that were mobilized into the peripheral blood of cancer patients treated with high-dose cyclophosphamide and filgrastim. The target cells used for gene delivery were either CD34+ cells that had been subsequently induced to differentiate into mature DCs or DCs transduced after ex vivo generation from CD34+ cells. The results showed that: (a) infection of CD34+ cell derived-DCs (mature DCs) with either viral vector resulted in the efficient synthesis of recombinant protein, and (b) CD34+ cells were permissive for the expression of the recombinant reporter gene after infection with Ad-MFG-AP but not after infection with MVA-P11 LZ. In conclusion, these results suggest that vaccinia and adenovirus vectors are candidate to act as vehicles in genetically engineering human DCs.

Expansion of Immunostimulatory Dendritic Cells from Peripheral Blood of Patients with Cancer.

Clinical investigators are keenly interested in the role of antigen-presenting cells (APCs) in the initiation of immune responses because of potential use of APCs in immune cell therapy. Pioneer studies in mammals by Steinman and coworkers have demonstrated that the specialized system of APCs is constituted by bone marrow-derived dendritic cells. Dendritic cells are distinguished by their unique ability to capture, process, and present antigens into peptide-HLA complexes to naive T lymphocytes and to deliver the costimulatory signal necessary for T-lymphocyte activation. In this article, we summarize the main experimental evidence supporting the hypothesis that individuals vaccinated with manipulated dendritic cells can mount tumor-specific humoral and cellular responses. This can lead to tumor regression as well as protective immunity to tumor growth in vivo.

Autologous dendritic cells derived from CD34+ progenitors and from monocytes are not functionally equivalent antigen-presenting cells in the induction of melan-A/Mart-1(27-35)-specific CTLs from peripheral blood lymphocytes of melanoma patients with low frequency of CTL precursors.

Peptide presentation by autologous dendritic cells (DCs) is a new tool to activate tumor antigen-specific T cells in melanoma patients. However, it is not known whether autologous DCs, differentiated by two of the most efficient protocols (from CD34+ progenitors or from monocytes), are equally effective as professional antigen-presenting cells (APCs) when the patients have a low frequency of peptide-specific precursors. To this end, a limiting dilution assay was applied to evaluate the frequency of antigen-specific CTL precursors (CTLps) in peripheral blood of HLA-A*0201+ melanoma patients. Then, from two melanoma patients showing low frequency of CTLps to melanoma antigen-A/melanoma antigen recognized by T cell (Melan-A/Mart-1)(27-35) peptide, autologous DCs were differentiated from granulocyte colony-stimulating factor-mobilized CD34+ progenitors or from monocytes. CD34+- and monocyte-derived DCs were characterized by a similar proportion of CD1a+ cells expressing HLA class II antigens and CD54, CD80, and CD86 molecules. Both types of DC presented Melan-A/Mart-1(27-35) and tyrosinase(369-377) peptides to melanoma-specific CTL clones and were equally effective as peptide-pulsed APCs in the activation of influenza A matrix(58-66)-specific CTLs from high-frequency precursors (1294/10(6) and 1789/10(6) lymphocytes in the two patients). However, efficient activation of Melan-A/Mart-1(27-35)-specific CTLs from low-frequency precursors (158/10(6) and 77/10(6) lymphocytes) of the two patients was markedly dependent on the use of peptide-loaded CD34+-derived DCs. These results suggest that CD34+- and monocyte-derived DCs are not functionally equivalent APCs for the activation of low-frequency peptide-specific CTLps.

Differential patterns of HOX gene expression are associated with specific integrin and ICAM profiles in clonal populations isolated from a single human melanoma metastasis.

Homeobox-containing genes comprise a gene family coding for transcription factors involved in normal development. Class I human homeobox (HOX) genes display a peculiar chromosomal organization, perhaps directly related to their function. Aberrant expression of homeobox genes has been associated with both morphological abnormalities and oncogenesis. We have reported that HOX gene expression is (i) specific for normal adult human organs (kidney, colon, lung) and (ii) altered in cancer specimens according to their histological type and stage of tumor progression. Here, we have investigated whether patterns of HOX gene expression are associated with tumor heterogeneity by analyzing the expression of the entire panel of 38 HOX genes in clones isolated from a single human metastatic melanoma call line (Me 665/2). The differential expression of a block of genes located at the 5' end of the HOX C locus allows melanoma clones to be classified into 2 major groups. The 2 patterns of HOX gene expression are inversely associated with 2 distinct surface phenotypes for integrins (VLA-2, VLA-5 and VLA-6) and the adhesion molecule ICAM-1. The genes of the HOX C locus are silent in the clones with high levels of integrins VLA-2, VLA-5 and VLA-6 and of the adhesion molecule ICAM-1 but actively expressed in the clones with low levels of ICAM-1 and lacking VLA-2, VLA-5 and VLA-6. Our results indicate that HOX gene expression reflects the intra-tumor heterogeneity of melanoma clones and suggest that the expression of surface molecules involved in cell-cell and cell-matrix interactions may be related to the patterns of HOX gene expression.

Interaction with fibronectin regulates cytokine gene expression in human melanoma cells.

Our study was aimed at investigating whether interaction of human melanoma cells with the extracellular matrix (ECM) protein fibronectin (FN) could regulate lymphokine gene expression. Serum-deprived cells (quiescent condition) of a metastatic melanoma cloned line were cultured either on uncoated or on FN- or BSA-coated surfaces. By means of reverse transcriptase- polymerase chain reaction (RT-PCR), we analyzed mRNA expression of 4 cytokines interleukin (IL)-1alpha, IL, 1beta, IL-6 and IL-8-and 9 growth factors-endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), fibroblast growth factor (FGF)-5, HST, keratinocyte growth factor (KGF), transforming growth factor (TGF)-alpha TGF-beta1, TGF-beta2 and TGF-beta3. When cultured on FN, melanoma cells expressed IL-1beta and IL-6 transcripts in addition to IL-1beta, IL-8, ECGF, TGF-beta1, TGF-beta2 and TGF-beta3, already present in quiescent cells. Amplification parameters to achieve semi-quantitative RT-PCR were then determined for each detectable factor, thus allowing us to measure a selective enhancement of mRNA levels for IL-1alpha, IL-6, IL-8 and TGF-beta2 upon interaction with FN by quiescent melanoma cells. This augmented expression was inhibited by an anti-integrin beta1 chain monoclonal antibody (MAb). Moreover, the amounts of IL-6, IL-8 and IL-beta produced in the supernatants, as assessed by ELISA, correlated with the corresponding mRNA expression. Extension of this analysis to the other 5 human primary and metastatic melanoma lines confirmed the ability of FN to selectively up-regulate only IL-6 and IL-8 secretion. Our data indicate that FN is able to modulate expression and secretion of a defined subset of lymphokines in human melanoma.