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.

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.

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.

Multiple myeloma (MM) is a B cell neoplasia that affects plasma cells located in the bone marrow. Gene expression profile (GEP) analysis through DNA arrays is a powerful tool to dissect the biology of MM. Here we will describe studies that identified new powerful prognostic markers using this technology. In particular, GEP can be used to identify the main chromosomal anomalies, and GEP-based classifications of MM patients into 4 to 7 groups associated with a different prognostic have been reported. We will also describe how GEP analysis of myeloma cells, normal plasma cells and cells of the tumor environment makes it possible to identify of new myeloma growth factors and new potential therapeutic targets for MM.

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.

Le myelome multiple (MM) est une neoplasie B caracterisee par la proliferation d’un clone de cellules plasmocytaires malignes qui s’accumulent dans la moelle osseuse. L’analyse du profil d’expression de genes (GEP, de l’anglais gene expression profiling) par puces a ADN est un outil puissant pour etudier la biologie du MM. Nous decrirons ici comment les etudes utilisant cette technologie ont permis d’identifier de nouveaux marqueurs pronostiques puissants. En particulier, les puces a ADN ont demontre leur efficacite pour identifier les principales anomalies chromosomiques et classer les patients en 4 a 7 groupes associes a un pronostic different. Nous decrirons egalement comment l’analyse du GEP de cellules de myelome, de cellules plasmocytaires normales et de cellules de l’environnement tumoral, a permis d’identifier de nouveaux facteurs de croissance myelomateux et de nouvelles cibles therapeutiques potentielles pour le MM.

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.

La rémission dite « incertaine » selon Cheson, fondée sur des critères morphologiques, atteste des limites de la tomodensitométrie (TDM) dans la caractérisation des masses résiduelles (MR). Le but de ce travail est de rechercher pour chacun des paramètres quantitatifs mesurables sur les images TEP-TDM [Standardizd Uptake Value (SUV) et diamètres des MR], une valeur seuil discriminante qui pourrait permettre de diagnostiquer une maladie résiduelle. Nous avons inclus 19 patients atteints d’un lymphome malin hodgkinien (LH) ou non (LNH) ayant bénéficié d’une TEP-TDM en fin de traitement au CHU de Montpellier et présentant une ou plusieurs MR. Nous avons mesuré pour chaque MR les SUVmax, SUVmoy, SUVmin et les diamètres petit et grand axe, puis nous avons suivi les patients afin de rechercher un lien avec le statut de la maladie à cinq mois. Seuls les SUVmax, SUVmoy et le diamètre petit axe (DPA) sont prédictifs du statut de la maladie à cinq mois. Une valeur seuil de SUVmax supérieure ou égale à 4 est en faveur d’une maladie résiduelle avec une spécificité de 90–100 %. La spécificité est de 92 % pour un DPA supérieur ou égal à 11,5mm. Lorsqu’on compare deux à deux le SUV ou le DPA pris séparément avec la combinaison « SUV–DPA », les courbes ROC (Receiver operating characteristic) montrent une tendance à la supériorité de la combinaison « morphofonctionnelle ». Cependant, il n’y a pas de différence significative entre chacun de ces tests (p>0,05). Une étude complémentaire mettant en jeu un effectif suffisant est donc nécessaire pour confirmer ces résultats préliminaires pour lesquels la combinaison du SUV avec le DPA semble être plus performante dans la caractérisation des MR que le SUV seul, faisant de la TEP-TDM l’examen de choix dans l’évaluation des patients atteints d’un lymphome en fin de traitement.

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 (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.

Except in the subset of patients with localized disease, follicular lymphoma (FL), the most common type of indolent non-Hodgkin's lymphoma, should still be regarded as an incurable malignancy with a relentless relapsing course. New therapeutic approaches have been developed including anti-tumor vaccination strategies with induced humoral and cellular immune responses revealed by sensitive biological analyses but no imaging of the lymphocyte activation developed so far. Because [18F]fluorodeoxyglucose (FDG) is sequestred in metabolically active cells, we prospectively investigated the use of FDG-PET (positron emission tomography) to kinetically monitor cell activation in 7 FL patients vaccinated by repeated intradermal (ID) and intravenous injections of tumor lysate-pulsed autologous dendritic cells (DC-lysate). Patients were administered 5 vaccines and all patients developed a positive delayed-type hypersensitivity reaction at the site of DC-lysate ID injections after several vaccines administration, testifying of the induction of a biological response. Among the 4 patients with measurable disease at time of vaccination, 2 complete remissions (CR) and 2 disease progressions were observed. Three patients with initial normal computed tomographies (CT) were still in CR 12 months after the last vaccine. PETs were performed before vaccination, 14 days and 1 month after the last vaccine. PET revealed initial malignant nodes infracentimetric on concomitant CT, and was very sensitive in documenting disease progression even in patients with no significant variations of CT findings. A transient marked increase in FDG uptake within malignant nodes was observed early after vaccine administration in a patient who achieved a CR (fig 1-3-4-5), while ranges of tracer uptake variations were less important with a continuous increase of FGD uptake, size and number of involved loci in non responding patients (fig 2: follow up of intensity of FDG uptake by 6 tumoral nodes in a non responding pt). In conclusion, FDG-PET is a non invasive imaging procedure which besides being useful for the staging and the evaluation of treatment efficacy in FL, might be of crucial interest to detect cell responses induced by immunotherapies.

The NF-κB pathway is involved in the physiological regulation of cell proliferation in many cell types as well as in the resistance of several malignancies to cell death. 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 IKK/NF-κB. The aim of the present study was to investigate the effect of a pharmaceutical IKK2 inhibitor (IKK2-I), the anilinopyrimidine derivative AS602868 (Serono International SA), on the in vitro growth of human MM cell lines (HMCL) and primary MM cells. We evaluated the effect of AS602868 on the proliferation and the survival of 12 IL-6-dependent HCML and 2 autonomously growing HCML as well as on the survival of total bone marrow mononuclear cells from patients with newly diagnosed MM (n = 6) or with relapsing MM (n = 7). Results show that using HMCL or primary MM cells, AS602868 induces a clear dose-dependent inhibition of MM cell growth (the 50% inhibitory concentration (IC50) ranging from 0.28 to 8.3 μM, mean IC50 = 2.6 μM on HCML). It was shown using HMCL that the growth inhibition induced by AS602868 is the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Importantly, AS602868 does not alter the survival of other bone marrow mononuclear cells (CD138−) co-cultured with primary MM (CD138+) cells except on CD34+ hematopoietic stem cells. Interestingly, using gene expression profiling with Affymetrix microarrays on 13 HMCL, we show that the resistance (high IC50) to AS602868 inhibitor is strongly correlated to APRIL gene expression (r =.7603, p <.01). Thus, an autocrine production of APRIL confers a resistance to the AS602868 IKK2-I. This can be explained since APRIL has been shown to activate the alternative NF-κB pathway which implicates an IKK complex composed of IKK1 homodimers instead of the IKK1/IKK2/NEMO complex involved in the canonical NF-κB pathway. The results demonstrate the important role of NF-κB in maintaining survival of MM cells and suggest that a pharmacological inhibition of the NF-κB pathway by an IKK2-I, AS602868, can efficiently kill myeloma cell lines or primary myeloma cells and might represent an innovative approach for treating MM patients.

7539 Background: Interferons (IFNs), which can offset the Th2 dominance associated with PCL have been successfully used to treat these PCL. Intratumoral (i.t.) injection of TG1042 (a non-replicating recombinant adenovirus with a human IFNγ cDNA insert) induces high local production of IFNγ without severe toxicity associated with systemic delivery. Methods: We undertook a phase I/II multicentric trial of repeated, i.t. injections of TG1042 in patients with advanced primary T cell (CTCL) or B cell (CBCL) CL. One to 3 lesions were injected on day 1, 8, and 15 (a 4-week cycle) and thereafter up to 12 cycles. Immunohistochemistry and quantitative PCR were performed on injected lesions biopsied at baseline and after the 1st cycle. In the phase I, 18 patients were enrolled in 3 successive cohorts at the doses of 3 × 109 viral particles (vp) (n = 3), 3 × 1010 vp (n = 3) and 3 × 1011 vp (n = 12). In the phase II, 18 evaluable patients were planned to be treated at 3 × 1011 vp. Results: To date, enrollment is complete, 39 patients (32 CTCL and 7 CBCL) have been included, 9 of them are still on treatment. Patients received a median of 4 lines of prior therapy. 11 patients were at stage Ib and 16 patients at higher stage. Altogether, 245 injections of TG1042 have been administered. Treatment was well tolerated with 8 grade 3 related adverse events. Injection site reaction and flu like syndrome are the most common adverse events. Histology demonstrates pronounced changes in infiltrate patterns with signs of vasculitis, increased numbers of eosinophils, neutrophils, CD8 and TIA-1+ve cells. CD4/CD8 ratio decreased in most tumors. Transgene-IFNγ mRNA was detected in injected lesions. Gene expression analysis of biopsies and PBMC shows up-regulation of IFNγ genes. Local clinical response has been observed in 17 (including 9 complete responses [CR]) out of 31 evaluable patients. 13 global responses (7 CR) out of 30 evaluable patients have been observed. All 5 evaluable CBCL responded (3 CR). Conclusions: These results demonstrate that TG1042 is well tolerated and presents a potential significant benefit for the treatment of both CTCL and CBCL. A phase II is planned in patients with CBCL to confirm those encouraging results. [Table: see text]

Cancer-testis (CT) antigens are expressed in testis and malignant tumors, but rarely in non-gametogenic tissues. Due to this pattern, they represent attractive targets for cancer vaccination approaches. The aims of the present study were (1) to assess for the first time the expression of CT genes on a pangenomic basis in multiple myeloma (MM), (2) to provide selection strategies of CT antigens for clinical vaccination trials and (3) to assess the impact of CT gene expression on event-free survival. We report here the expression pattern of CT genes in purified MM cells (MMC) of 64 patients with newly-diagnosed MM, 12 patients with monoclonal gammopathy of unknown significance (MGUS), in normal plasma cell and B cell samples and in 20 MMC lines, using gene expression profiling (GEP). Out of 46 CT genes interrogated by the Affymetrix HG U133 Set arrays, 35 were expressed in MMC of at least one patient, according to the Affymetrix “present” call (frequency range: 2% – 66%). Of these, 24 CT genes were expressed in more than 5% of the MMC samples and 25 are located on chromosome X. MMC of 98% of the patients expressed 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 to circumvent tumor escape mechanisms and immune tolerance - could be obtained in MMC of 67% of the patients. Thus, gene expression profiling can be used to select CT antigens as vaccination targets in individual patients. In a series of MMC from 111 patients treated with the same high-dose chemotherapy and autologous peripheral blood stem cell transplantation protocol and having a median two-year follow-up, we found that the expression of six CT genes, i.e. CTAG-1B, CTAG-2, MAGE-A1, MAGE-A2, MAGE-A3 and MAGE-A6 was associated with a shorter event-free survival (EFS). Furthermore, considering only the 25 CT genes encoded by chromosome X, a CT-Xhigh cluster comprising MMC of one third of the patients (35 of 111) could be defined using a binary hierarchical clustering based on Affymetrix call. Patients in the CT-Xhigh cluster had a shorter EFS (median 13 months) compared to patients in the CT-Xlow cluster (median 18 months, P = .003). The CT-Xhigh clsuter included more patients with a stage III disease (P = .004). These results confirm data from previous studies indicating that patients expressing some CT genes located on chromosome X have a poor outcome.

The epidermal growth factor (EGF)/EGF-receptor (ErbB1-4) family is involved in the biology of multiple myeloma (MM). In particular, ErbB-specific inhibitors induce strong apoptosis of myeloma cells (MMC) in vitro. To delineate the contribution of the 10 EGF-family ligands to the pathogenesis of MM, we have assessed their expression and biological activity. Comparing Affymetrix DNA-microarray-expression-profiles of CD138-purified plasma-cells from 65 MM-patients and 7 normal individuals to those of plasmablasts and B-cells, we found 5/10 EGF-family genes to be expressed in MMC. Neuregulin-2 and neuregulin-3 were expressed by MMC only, while neuregulin-1, amphiregulin and transforming growth factor-α were expressed by both MMC and normal plasma-cells. Using real-time polymerase chain reaction, we found HB-EGF, amphiregulin, neuregulin-1 and epiregulin to be expressed by cells from the bone marrow-environment. Only the EGF-members able to bind heparan-sulphate proteoglycans (HSPGs) – neuregulin-1, amphiregulin, HB-EGF – promote the growth of MMC. Those ligands strongly bind MMC through HSPGs. The binding and the MMC growth activity was abrogated by heparitinase, heparin or deletion of the HS-binding domain. The number of HS-binding EGF ligand molecules bound to MMC was higher than 105 molecules/cell and paralleled that of syndecan-1. Syndecan-1, the main HSPG present on MM cells, likely concentrates high levels of HS-binding-EGF-ligands at the cell membrane and facilitates ErbB-activation. Altogether, our data further identify EGF-signalling as promising target for MM-therapy.