A phase II study of the cancer vaccine TG4010 alone and in combination with cytokines in patients with metastatic renal clear-cell carcinoma: clinical and immunological findings.

MUC1 over-expression in renal clear-cell carcinoma (RCC) is associated with poor prognosis. This phase II study determined the efficacy and tolerability of TG4010, a cancer vaccine based on a modified vaccinia virus expressing MUC1 and interleukin-2, in combination with cytokines, as first-line therapy in metastatic RCC. Thirty-seven patients with progressive, MUC1-positive RCC received TG4010 10(8) pfu/inj weekly for 6 weeks, then every 3 weeks until progression, when TG4010 was continued in combination with interferon-α2a and interleukin-2. Assessments included clinical response (primary endpoint), safety, time to treatment failure (TTF), overall survival (OS), and immune response. No objective clinical responses occurred. Five of the 27 evaluable patients (18%) had stable disease for >6 months with TG4010 alone and six of 20 patients (30%) had stable disease for >6 months with TG4010 plus cytokines. Median TTF was 4.1, 3.6, and 9.3 months for monotherapy, combination therapy, and overall, respectively. Median OS was 19.3 months for all patients and 22.4 months combination therapy recipients. The most frequent TG4010-related adverse events were minor-to-moderate injection-site reactions, fatigue, and flu-like symptoms. Six of 28 patients showed a MUC1 CD4+ T cell proliferative response during therapy. Anti-MUC1 CD8+ T cells were detected before and after therapy in 3 and 4 patients, respectively. MUC1-specific CD8+ T cell responses were associated with longer survival. Therapy with TG4010 plus cytokines appears to be feasible and well tolerated in patients with metastatic RCC. However, these data should be interpreted with caution, as additional prospective studies are necessary to clarify the clinical efficacy of this therapy.

Phase II clinical trial of intratumoral application of TG1042 (adenovirus-interferon-gamma) in patients with advanced cutaneous T-cell lymphomas and multilesional cutaneous B-cell lymphomas.

Cutaneous lymphomas (CLs) are a heterogeneous group of lymphoproliferative disorders that are manageable by immunotherapy. Twenty-one patients were enrolled in a prospective open-label, dose-escalation multicenter study evaluating the effects of repeated TG1042 [adenovirus-interferon (IFN)-gamma] intralesional injections in patients with primary CLs, of which 18 were of T-cell and 3 of B-cell type. Repeated intralesional therapy using TG1042 consistently results in local tumor regressions in about half of treated patients and one-third of patients also in regressions in noninjected distant lesions, likely reflecting the systemic immune activation after intralesional therapy. Treatment was well tolerated with few adverse events including injection site reactions, chills, lymphopenia, and fever. Immune monitoring in the peripheral blood demonstrated systemic immune activation and the induction of antibodies against tumor antigens in some patients without clear association with clinical responses. CLs, in particular B-cell lymphomas with high objective response rates, seem to be excellent targets for this type of immunotherapy.

Granulocyte-macrophage colony-stimulating factor potentiates rituximab in patients with relapsed follicular lymphoma: results of a phase II study.

PURPOSE: We hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF) could potentiate the clinical activity of rituximab given its individual and cooperative effects on Fc gamma RIIa- and Fc gamma RIIIa-expressing cells. A phase II clinical study combining GM-CSF and rituximab was initiated in patients with relapsed follicular lymphoma (FL) to determine the clinical and biologic responses, as well as safety of the combination. PATIENTS AND METHODS: Thirty three patients with relapsed FL were treated with GM-CSF 5 microg/kg/d on days 1 to 8 and rituximab 375 mg/m(2) on day 5 of each 21-day cycle for four cycles. Clinical response and tolerability were examined according to international criteria. Biologic monitoring included evaluation of immune cells involved in rituximab activity. RESULTS: Of 33 evaluated patients, a 70% overall response rate (complete response plus complete response unconfirmed, 45%) and a median progression-free survival (PFS) of 16.5 months were achieved. Outcome was influenced by the quality of response and the Follicular Lymphoma International Prognostic Index (FLIPI), where low- and intermediate-risk FLIPI groups were associated with significantly better PFS. After treatment there was a significant increase in granulocyte and monocyte counts. Examination of dendritic cell response showed an overall increase in plasmacytoid dendritic cells, especially in non-complete response patients, after treatment. Addition of GM-CSF did not impair tolerance to rituximab, and adverse events were rare and mild. DISCUSSION: GM-CSF plus rituximab results in high response rates, along with a tolerable safety profile in patients with relapsed or progressive FL. The improved efficacy over rituximab monotherapy may be due to increases seen in monocyte, granulocyte, and dendritic cell populations.

Targeting NF-kappaB pathway with an IKK2 inhibitor induces inhibition of multiple myeloma cell growth.

The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IkappaB kinase/nuclear factor-kappaB (IKK/NF-kappaB). The present study aimed at investigating the effect of a pharmaceutical IKK2 inhibitor, the anilinopyrimidine derivative AS602868, on the in vitro growth of 14 human MM cell lines (HMCL) and primary cells from 13 patients. AS602868 induced a clear dose-dependent inhibition of MM cell growth on both HMCL and primary MM cells, which was the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Combination of AS602868 with suboptimal doses of melphalan or Velcade showed an additive effect in growth inhibition of HMCL. AS602868 also induced apoptosis of primary myeloma cells. Importantly, AS602868 did not alter the survival of other bone marrow mononuclear cells (CD138(-)) co-cultured with primary MM (CD138(+)) cells, except for CD34(+) haematopoietic stem cells. The results demonstrate the important role of NF-kappaB in maintaining the survival of MM cells and suggest that a pharmacological inhibition of the NF-kappaB pathway by the IKK2 inhibitor AS602868 can efficiently kill HMCL and primary myeloma cells and therefore might represent an innovative approach for treating MM patients.

Heparanase influences expression and shedding of syndecan-1, and its expression by the bone marrow environment is a bad prognostic factor in multiple myeloma.

The heparan sulfate (HS) proteoglycan, syndecan-1, plays a major role in multiple myeloma (MM) by concentrating heparin-binding growth factors on the surface of MM cells (MMCs). Using Affymetrix microarrays and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we show that the gene encoding heparanase (HPSE), an enzyme that cleaves HS chains, is expressed by 11 of 19 myeloma cell lines (HMCLs). In HSPE(pos) HMCLs, syndecan-1 gene expression and production of soluble syndecan-1, unlike expression of membrane syndecan-1, were significantly increased. Knockdown of HPSE by siRNA resulted in a decrease of syndecan-1 gene expression and soluble syndecan-1 production without affecting membrane syndecan-1 expression. Thus, HPSE influences expression and shedding of syndecan-1. Contrary to HMCLs, HPSE is expressed in only 4 of 39 primary MMC samples, whereas it is expressed in 36 of 39 bone marrow (BM) microenvironment samples. In the latter, HPSE is expressed at a median level in polymorphonuclear cells and T cells; it is highly expressed in monocytes and osteoclasts. Affymetrix data were validated at the protein level, both on HMCLs and patient samples. We report for the first time that a gene's expression mainly in the BM environment (ie, HSPE) is associated with a shorter event-free survival of patients with newly diagnosed myeloma treated with high-dose chemotherapy and stem cell transplantation. Our study suggests that clinical inhibitors of HPSE could be beneficial for patients with MM.

Cancer/testis genes in multiple myeloma: expression patterns and prognosis value determined by microarray analysis.

Cancer-testis (CT) Ags are expressed in testis and malignant tumors but rarely in nongametogenic tissues. Due to this pattern, they represent attractive targets for cancer vaccination approaches. The aims of the present study are: 1) to assess the expression of CT genes on a pangenomic base in multiple myeloma (MM); 2) to assess the prognosis value of CT gene expression; and 3) to provide selection strategies for CT Ags in clinical vaccination trials. We report the expression pattern of CT genes in purified MM cells (MMC) of 64 patients with newly diagnosed MM and12 patients with monoclonal gammopathy of unknown significance, in normal plasma cell and B cell samples, and in 20 MMC lines. Of the 46 CT genes interrogated by the Affymetrix HG-U133 set arrays, 35 are expressed in the MMC of at least one patient. Of these, 25 are located on chromosome X. The expression of six CT genes is associated with a shorter event-free survival. The MMC of 98% of the patients express at least one CT gene, 86% at least two, and 70% at least three CT genes. By using a set of 10 CT genes including KM-HN-1, MAGE-C1, MAGE-A3/6/12, MAGE-A5, MORC, DDX43, SPACA3, SSX-4, GAGE-1-8, and MAGE-C2, a combination of at least three CT genes-desirable for circumventing tumor escape mechanisms-is obtained in the MMC of 67% of the patients. Provided that the immunogenicity of the products of these 10 CT genes is confirmed, gene expression profiling could be useful in identifying which CT Ags could be used to vaccinate a given patient.

Importance of [18F]fluorodeoxyglucose-positron emission tomography scanning for the monitoring of responses to immunotherapy in follicular lymphoma.

We prospectively investigated the use of [18F]fluorodeoxyglucose (FDG)-positron emission tomography (PET) to kinetically monitor cell activation in six follicular lymphoma patients vaccinated with tumor lysate-pulsed autologous dendritic cells. PET revealed additional initial nodes suggestive of lymphoma, that were of less than 10 mm of diameter on computed tomography, and documented disease progression with sensitivity, even within loci with no significant variations of CT findings. Although tracer fixation was not observed in the inguinal nodes draining the dendritic cell intradermal injection sites in the six patients, a transient marked increase in FDG uptake within malignant nodes was observed early after vaccine administration in a patient who achieved complete remission. In non-responding patients, we observed a continuous increase of FDG uptake associated with an increase in the size and number of pathological nodes/locus, or of involved loci. FDG-PET is a non-invasive imaging procedure that might be of crucial interest to detect cell responses induced by immunotherapies.

Functional regulatory T cells are collected in stem cell autografts by mobilization with high-dose cyclophosphamide and granulocyte colony-stimulating factor.

High-dose cyclophosphamide (Cy) and G-CSF are widely used to mobilize hemopoietic stem cells for treating patients with high-dose chemotherapy and autologous stem cell transplantation (ASCT). Because lymphocyte count in the graft collected after Cy-G-CSF treatment is an independent survival factor after ASCT for patients with multiple myeloma, our purpose was to study how Cy-G-CSF treatment affects the phenotype and function of T cells in patients with multiple myeloma. Cy induced a 3-fold decrease of T cell counts with a slow and partial T cell recovery of one-third at the time of hemopoietic stem cell collection. Cy-G-CSF treatment did not affect the relative ratios of central memory, effector memory, and late effector CD4+ or CD8+ T cells, but a decrease in the percentage of naive CD4+ cells was observed. The percentages of CD25+ cells increased 2- to 3-fold in CD4+ and CD8+ T cells, the former including both activated CD25low and CD25high cells. CD4+CD25high cells were regulatory T cells (Treg) that expressed high levels of FOXP3, CTLA-4, and GITR and displayed in vitro suppressive properties. The recovery of Treg absolute counts after Cy-G-CSF treatment was higher than the recovery of other lymphocyte subpopulations. In conclusion, Cy-G-CSF treatment induces a severe T cell count decrease without deleting Treg, which are potent inhibitors of antitumor response. The present data encourage novel therapeutic strategies to improve T cell recovery following ASCT while limiting Treg expansion.

Expression of EGF-family receptors and amphiregulin in multiple myeloma. Amphiregulin is a growth factor for myeloma cells.

A hallmark of plasma cells is the expression of syndecan-1, which has major functions in epithelial cells, in particular as the coreceptor of heparin-binding growth factors. We previously found that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a growth factor for malignant plasma cells. As amphiregulin (AREG) is another heparin-binding factor of the EGF family, we investigated its role in multiple myeloma (MM). Using Affymetrix DNA microarrays, we show here that the AREG gene was expressed by purified primary myeloma cells from 65 patients and that the expression was higher than in normal bone marrow (BM) plasma cells or plasmablastic cells. AREG stimulated IL-6 production and growth of BM stromal cells. Using real-time reverse transcriptase-polymerase chain reaction, we found that MM cells expressed ErbB receptors and that AREG promoted their growth. Furthermore, PD169540 (a pan-ErbB inhibitor) and IRESSA (an ErbB1-specific inhibitor) induced apoptosis of primary myeloma cells from 10/14 and 4/14 patients, respectively, and there was a synergistic effect with dexamethasone. Altogether, our data provide strong evidence that AREG plays an important role in the biology of MM and emphasize the advantages of using ErbB inhibitors, which might target myeloma cells as well as the tumor environment.