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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.

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.

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.

One of the best-characterized resistance mechanisms in acute myeloid leukemia (AML) is the drug extrusion mediated by P-glycoprotein (Pgp). Recently the results of workshops organized by several groups concluded that accurate measurement of low activity of Pgp is a difficult goal in clinical samples. Therefore, highly sensitive and specific assays were developed to assess the functionality of Pgp using JC-1, a fluorescent molecule with the different emission wavelength (green and red fluorescence) according to its concentration in 129 AML samples. It was shown that JC-1 (green and red bands) may define 3 groups of patients: resistant (R) (29% of patients), intermediate (I) (36%), and sensitive (S) (35%). In contrast, rhodamine 123 assay detected only the R group defined by JC-1. Nevertheless, the I group has an intermediate expression of Pgp (0.39, 0.29, and 0.19 for the R, I, and S groups, respectively, P = .002), an intermediate biologic profile (percentage of CD34, 95%, 67%, and 44%, respectively, P

In acute myeloid leukemia (AML) patients, a variety of clinical and biologic parameters, including phenotype, have been examined for potential value in predicting treatment response and survival. The European Group for the Immunological Classification of Leukaemias (EGIL) has proposed that AML be defined immunologically by the expression of 2 or more of the following myeloid markers: myeloperoxidase, CD13, CD33, CDw65, and CD117. With regard to this classification, the prognostic significance of 21 antigens taken separately and with immunophenotypic subgroups were evaluated and compared with other clinical and biological variables in 177 adult AML patients. None of the antigens tested were associated with treatment outcome. In contrast, patients with blasts disclosing a full expression of panmyeloid phenotype (defined by the expression of all 5 myeloid markers) had a higher complete remission rate (P

We reviewed the reports of 784 consecutive patients admitted to our department for newly diagnosed acute myeloid leukemia (AML) over a 16-year period. Median, 5-year and 10-year overall survivals were 9. 5 months, 17.3% and 11.7% respectively. Induction treatment (698 patients) resulted in 50% complete remissions (CR) (from 26.5% in secondary AML to 81.2% in patients60 years with de novo AML). Period of diagnosis (1980-84/85-89/90-95) demonstrated a major significance for CR achievement and OS in multivariate analysis. In patients/=60 years (372), CR rate increased (25% to 36.8%, P = 0. 03), and 5-year OS (3.7% to 10.6%, P = 0.022) improved, probably due to an increase in the proportion of patients administered conventional combined chemotherapy (54.5% to 83.8%, P0.0001). In younger patients CR rate continuously increased (61.5% to 74.8%, P = 0.028) with an associated improvement of 5-year OS (19.2% to 35.4%). No significant change in DFS and CR durations was observed. This large single center study on a large cohort of unselected AML patients reflects the improvement achieved in the management of AML patients, likely due to improvement of supportive care practices, administration of conventional induction to more elderly patients, and intensification of induction and post-remission treatments in patients60 years.

Out of 75 consecutive elderly AML patients who did not receive anti-leukemic treatment (52 pts) or failed to respond to differentiating agent (23 pts), 6 patients had survivals of 13.2 to 98 months with treatment restricted to supportive care. This cut-point is far longer than the median survival of the 235 elderly patients (3.5 mo.), either untreated (med. survival: 1 mo.) or treated (with treatment ranging from conventional induction to palliative chemotherapy) (4 mo.), admitted to our department within the same period of time. These cases of smoldering AML (4 women, 2 men) were all of AML2 FAB subtype (4 de novo, 2 post MDS) and presented with a significantly better performance status, lower WBC and circulating blast counts, higher platelet counts and with lower bone marrow infiltration than AML cases with more rapid progression. Cytogenetical analysis when available (3 pts) showed normal karyotypes and clonogenic assay performed in 3 of these patients showed a lack of (2 pts) or reduced in vitro leukemic cell growth (1 pt). The identification of specific characteristics of smoldering leukemia in the elderly might be an important development in the understanding of the physiopathology of acute leukemia and a tool for helping decision-making when selecting the time and intensity of cytotoxic treatment in these older patients.

Biphenotypic acute leukaemia (BAL) patients represented 8% of the 287 de novo consecutive adult acute leukaemias (23 BAL, 230 acute myeloid leukaemia (AML) and 34 acute lymphoblastic leukaemia (ALL)) referred to our department during the last 4-year period. Of these 23 BAL patients, 14 patients showed myeloid morphology and nine cases lymphoid morphology according to FAB criteria. There were no differences between lymphoid and myeloid BAL according to clinical and biological presentation and treatment outcome. We confirm the poor prognosis of BAL when compared to AML or ALL seen during the same period of time, in terms of complete remission (47%, 62% and 82% respectively, BAL v AML, NS and BAL v ALL, P = 0.006) and 4-year overall survival (8.1%, 25.8% and 23.8% respectively, BAL v AML, P = 0.05 and BAL v ALL, P = 0.003). Comparing adult BAL patients with AML patients, we found an increase in poor prognostic factors: CD34+ phenotype (82% v 60% respectively, P = 0.03), unfavourable karyotype (60% v 20%, P

Recombinant adeno-associated viruses (rAAV) are attractive tools for gene therapy. We designed plasmids in which the human multidrug resistance gene (hMDR1) cDNA was placed downstream from portions of the 5' end of AAV including either a 234-bp cassette or the entire AAV p5 promoter. The drug-resistant phenotype conferred by the P-glycoprotein (Pgp) efflux pump encoded by the hMDR1 cDNA was used to select NIH-3T3 cells transfected with these plasmids. The 234-bp region alone showed promoter activity similar in strength to that of the entire p5 promoter or the retroviral Harvey murine sarcoma virus long terminal repeat (LTR); this result demonstrates that the 234-bp cassette might be used as a small and efficient promoter in rAAV designed to express large genes approaching the packaging limit of AAV particles. After transfection of AAV-MDR1 vectors, the integration of MDR1 sequences into the host cell genome was demonstrated by fluorescent in situ hybridization (FISH). In addition, Southern analysis of low-molecular-weight DNA extracted from drug-resistant cells grown under continuous selection pressure indicated the persistence of nonintegrated AAV-MDR1 plasmids. Coordinate expression of Pgp and human glucocerebrosidase (hGC) was observed in drug-selected NIH-3T3 cells transfected with a bicistronic vector in which MDR1 cDNA was linked to hGC cDNA via the encephalomyocarditis internal ribosome entry site sequence. Moreover, following a single intravenous injection of the bicistronic vector complexed to cationic liposomes into recipient mice, delivery of MDR1 and GC cDNAs was achieved in all the organs we tested. Our results demonstrate that the efficiency of liposomes as vehicles for in vitro and in vivo gene delivery, the advantages of AAV-vectors, and the use of MDR1 as a selectable marker might be successfully combined in gene therapy protocols.

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.

PurposeWe hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF) could potentiate the clinical activity of rituximab given its individual and cooperative effects on FcγRIIa- and FcγRIIIa-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 MethodsThirty three patients with relapsed FL were treated with GM-CSF 5 μg/kg/d on days 1 to 8 and rituximab 375 mg/m2on 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.ResultsOf 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.DiscussionGM-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.

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 HSPEpos 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.

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.

International audience; 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-alpha 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 10(5) 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.

International audience; 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.

Peripheral cytopenias are common in patients with agnogenic myeloid metaplasia (AMM). They are an important cause of morbidity and mortality, and their treatment is difficult. We report on 4 patients with AMM and severe cytopenia treated with danazol (400-600 mg/ day). Three of them became independent of red blood cell (RBC) transfusion, while the other had a slight reduction in RBC requirement. In addition, correction of thrombocytopenia and disappearance of splenomegaly were observed in 1 and 2 patients, respectively. No side effects were observed. In our experience, danazol appears effective and safe in the subset of patients with AMM whose disease is mainly characterized by bone-marrow failure. These data warrant further studies to evaluate this treatment and explore its mechanism of action.

Publisher Summary This chapter discusses the construction of multidrug resistance gene ( MDR1 ) adeno-associated virus vectors (AAV) for gene therapy. Because of the large size of the MDR1 cDNA, the AAV coding sequences must be deleted prior to the insertion of MDR1 between the inverted terminal repeats (ITRs) of recombinant AAV (rAAV) vectors meant for transduction protocols. Whether all rep negative rAAV vectors are capable of stable integration, the mechanisms that contribute to site-specific integration needs to be investigated. The fact that rAAVs frequently remain episomal and may be diluted and lost with cell division may limit the use of AAV vectors when long-term expression is expected. Selectable markers, such as MDR1 may be possible to maintain nonintegrated episomal rAAV in proliferating cells because only cells carrying such episomal AAV-MDR1 survive the selection.

Two successive studies comparing tazocilline (12 g piperacillin + 1.5 g tazobactam/ day) + aminoside (15 mg/k/day amikacin in the first trial, 3 mg/kg/d tobramycin in the second) to ceftazidime (3 g/day) + aminoside included 466 febrile episodes in profoundly neutropenic patients after chemotherapy (or chemo-radiotherapy) for hematological malignancies. Patients were treated for acute leukemia (72%), or autografted for non-Hodgkin lymphoma or myeloma (25%), few patients for solid tumors. The mean duration of neutropenia was 21 days ± 11 (92% > 7 days); 466 febrile episodes were analysed for intention-to-treat, and were attributed to bacteremia in 119 cases, to local infection in 178 cases, and were of unknown origin (FUO) in 169 cases. A majority of Gram positive strains (55.5%) was isolated at day 0. The 72 h apyrexia was obtained more often (51.6% of the cases) with Tazocillin + aminoside than with ceftazidime + aminoside (33.6%, p < 0.0001). This results was due to a better control of bacteremia (p = 0.03) and local infection (p = 0.006) with tazocillin + aminoside, but not FUO (p = 0.19). Identical results of both antibiotic associations were observed during the first course of antitumoral chemotherapy, but tazocillin was significantly superior for 72 h apyrexia when fever occurred during consolidation therapy (p < 0.0001) or relapse treatment (p = 0.006). Superinfections due to Gram positive strains and major infectious events (infectious deaths and retardations of underlying disease treatment due to infection) were less frequent when patients received tazocillin + aminoside (respectively p = 0.002 and p = 0.02). The initial empirical antibiotic association remained unchanged and lead to a persisting apyrexia in only 20.3% of the cases treated with tazocillin + aminoside, but this was significantly better than with the other association (9.5%, p = 0.02).

Primary lymphomas arising in the adrenal gland are extremely rare. The presenting symptoms are nonspecific and may be related to lymphoma or to associated adrenal insufficiency. In this report we describe the case of a 61 year old woman with idiopathic thrombocytopenic purpura and primary bilateral non Hodgkin's lymphoma of the adrenals.

A retrospective analysis was performed on 235 elderly acute myelogenous leukaemia (AML) patients aged 60 years or more, consecutively admitted to a single haematological department during a 10-year period from 1980 to 1989. 46% of patients received only conventional induction chemotherapy. The rate of inclusion in EORTC cooperative clinical trials was significantly lower than for younger patients despite specific protocols proposed for the elderly since 1983, thus confirming the important selection bias of most published series on elderly AML patients. Compared with treatment results in patients60 years, complete remission (CR) rate was lower (33.3% v 65.4%, P0.0001), with a marked drop in patients older than 70, and induction death rate was higher (21.3% v 12.5%, P = 0.04). Intrinsic characteristics of leukaemic cells, especially expression of the MDR1 gene, in vitro growth of the leukaemic clonogenic cells and sensitivity to daunorubicin-(+)cytosine arabinoside, did not differ according to age, except that there was a higher incidence of previous myelodysplastic syndromes and a lower incidence of good prognostic cytogenetics in the elderly patients. Thus, treatment failure in elderly AML patients appears to be mainly due to host-related factors (especially performance status and ageoror = 70 years), and to inadequate treatments. Some elderly patients may have been undertreated because of the planned anthracycline dose reduction, resulting in a higher rate of 'resistant' AML, i.e. patients surviving the induction period without entering into CR, than in younger patients (45.4% v 22.1%, P0.0001). 11 patients (4.7%) with untreated or 'resistant' AML survived more than 1 year, while receiving only supportive care. These slowly progressive AML patients were characterized by a good performance status, and lower circulating blast cells and bone marrow blast counts.

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.

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. Figure Figure

New therapeutic approaches have been developed to treat follicular lymphoma (FL), the most common type of indolent non-Hodgkin’s lymphoma which should still be regarded as an incurable malignancy with a relentless relapsing course, except in the subset of patients with localized disease. For vaccination purposes, dendritic cells (DC) generated and loaded with tumor antigens ex vivo are focus of attention for already 10 years. In 07/04 we started a phase II study to evaluate a vaccination strategy based on multiple injections of autologous DC loaded with tumor lysate (DC-lysate) in FL patients. We reported here the results we observed for the first 7 enrolled patients (3F/4M, age: 44–61; 1 to 8 previous lines of treatment, including autologous PBSC transplant in 2 pts) who completed their treatment consisting in 5 to 7 DC-lysate vaccinations. At time of inclusion an excisional biopsy of a peripheral lymph node was performed in all patients; tumoral B cells were isolated by negative T cell depletion (using immunomagnetic beads and anti-CD3 antibodies) and then lysed by 5 freezing/thawing cycles to produce tumor lysate. Immature DC were obtained from mononuclear cells collected after Cyclophosphamide (2g/m2) and G-CSF (10μg/kg) administration, cultured for 5 days with GM-CSF and IL-4, pulsed with lysate, and then induced to mature with TNFa and PGE2 for 24h. All DC preparation fitted the quality criteria of viability ≥60%, ≤20% CD14+ cells, ≥60% CD83+ cells. For each vaccine 107 DC-lysate were administered, both intravenously [IV] (5×106 cells) and intradermally [ID] (5×106 cells). Vaccinations were safe and well tolerated. All patients developed significant delayed-type hypersensitivity responses to DC-lysate after several vaccines administration, testifying of the induction of a response. Among the 4 patients vaccinated with measurable disease, 2 who presented with low tumor burden (1 stage I, 1 stage II) achieved CR (with normal CT and absolutely no more FDG uptake on PET), so far persistent 3 and 6 months after the last vaccine, while disease progressed in the 2 remaining patients who presented respectively a stage IV and stage II disease at time of vaccination. The 3 patients vaccinated while in CR showed no disease progression at last tumor evaluation (9 to 12 months post last vaccination). This study demonstrated that DC-lysate vaccines might be efficient in FL patients with low tumor burden disease. Because the cause of disease recurrence is the presence of minimal residual disease after conventional treatments, the adjuvant setting might be the most appropriate therapeutic strategy for active specific immunotherapy, when the immunosuppressive effects of bulky disease are virtually absent and when the effector-target ratio is favorable.

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

The heparan sulfate (HS) proteoglycan (PG) syndecan-1 plays a major role in multiple myeloma (MM), in part by concentrating HS-binding growth factors on the surface of MM cells (MMC). The function of HSPG is regulated by extracellular enzymes that modulate the structure of HS-chains. One such enzyme is heparanase (HPSE), an endo-glucuronidase that cleaves HS chains at only a few sites, resulting in fragments of 10–20 sugar units long that are biologically active. Using Affymetrix microarrays and real-time RT-PCR, we showed that the gene encoding HPSE was expressed by 11/19 myeloma cell lines (HMCLs). In HSPE-positive HMCLs, syndecan-1 mRNA expression and production of soluble syndecan-1, unlike expression of membrane syndecan-1, were significantly increased. Downregulation of HPSE by HPSE-siRNA resulted in a decrease of syndecan-1 mRNA expression and soluble syndecan-1 production, without affecting membrane syndecan-1. Contrary to HMCLs, HPSE was expressed in only 4/39 primary MMC samples at a low level, whereas it was highly expressed in 36/39 whole bone marrow (WBM) microenvironment samples. In the latter, HPSE was expressed at a median level in polynuclear cells and T cells, at high level in monocytes and osteoclasts, and it was not expressed in BM stromal cells. In order to investigate the connection of clinical parameters with HPSE gene expression, 30/39 myeloma patients treated with high dose chemotherapy and autologous stem cell transplantation where classified into two groups: 15 patients with the lowest or the highest HPSE expression in the WBM. The HPSEhigh group had an increased percentage of patients with elevated C-reactive protein and β2-microglobulin. According to the international staging system (ISS), the HPSEhigh group included a significant higher frequency of patients with stage III MM and a lower frequency of patients with stage I MM. HPSEhigh patients had a shorter event free survival (EFS) (p=.017) and overall survival (OAS) (p=.023) than HPSElow patients. Classifying these 30 patients into two groups according to HPSE expression in MMC did not yield to any EFS or OAS significant differences between the two groups. Altogether, those data provide evidence that HPSE not only modulate syndecan-1 activity through the cleavage of HS chains but also regulates syndecan-1 mRNA expression and shedding. We report for the first time that the expression of a gene in the BM, i.e. HSPE, is an indicator of poor prognostic for MM patients. Therefore, HPSE inhibitors like PI-88, which is currently in clinical trial, will be of interest for the treatment of patients with MM.

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]

DCs are the most potent antigen-presenting cells and antigen (Ag) delivery by ex vivo Ag-loaded DC cells may be an effective strategy for FL. The relevant source of tumor Ag, vaccine doses, schedules or routes of administration are still a matter of investigation. Tumor cell lysate (TCL) is an interesting source of multiple tumor Ag. After preclinical study and an acceptation of the “dossier de lot” by the french drug agency (afssaps), we started a phase II study evaluating the feasibility and safety of multiple ID and IV injections of autologous DC-TCL, in 07/04. At this time, 5 FL patients (pts) completed their treatment, with 5 to 7 DC-TCL vaccinations every 2–3 weeks, receiving 5.106 cells IV and 5.106 cells ID per vaccination. All the pts (2F/ 3M, age 44–61; 1 to 8 previous lines of therapy, including autologous transplantion in 2 pts) had measurable disease at inclusion. Tumor lymph node biopsy (for preparing TCL) was followed by rituximab administration in pts with no history of previous infusion of the monoclonal antibody. Cyclophosphamide (2g/m2) and G-CSF (5 mg/kg) were administered to mobilize mononuclear cells including CD14 monocytes. Two pts still had measurable disease at time of vaccination (lymph nodes on CT and Pet scans for the 2 pts, cutaneous localizations and molecular analysis evidence of disease in BM and PB, in 1 pt). Immature DC were obtained from thawed leukapheresis cells cultured for 5 days with GM-CSF and IL-4, pulsed with L, and then induced to mature with TNFalpha and PGE2 for 24 hours. All DC preparations fitted the quality criteria defined by the preclinical study (viability≥60%, ≤20% CD14+ cells, ≥60% CD83+ cells). Vaccinations were safe and well tolerated. One of the 2 pts with prevaccination residual disease entered CR (disappearance of Pet-scan positivity after the 7th vaccination, with sequential Pets, showing an increase of the signal during the first vaccinations linked to an initial lymph node activation), while a PR was observed in the 2nd pt with a regression of both lymph nodes and tumoral subcutaneous lesions. Quantitative PCR for bcl-2 signal were performed in both PB and BM. All pts developed significant delayed-type hypersensitivity responses to DC-TCL (as defined by erythema and induration of more than 5 mm of diameter, lasting 48h at least) after the 4th or 5th vaccinations. Analysis of biopsies performed at the site of DC-TCL injections, showed HSR including neutrophils and dense infiltration by T lymphocytes, mainly CD4 cells, as compared to site of injection of unmodified DC. Immunologic tests (Elispot assays) for monitoring vaccine efficacy will be presented. These preliminary results show that DC-TCL might be an efficient immunotherapy in patients with FL and that schedule of administration of the vaccine (especially to perform at least 5 vaccinations in a short period of time) as well as combination of 2 routes of DC administration might be critical points. We are now trying to pursue vaccinations for a longer period of time for every pt, and this study is now a multicenter study from Goelams group.

High-dose chemotherapy (HDC) supported by autograft of hematopoietic progenitors (HP) is a standard therapy for patients with multiple myeloma (MM). High-dose cyclophosphamide (CTX) and G-CSF are widely used to collect HP. As the number of lymphocytes in the autograft is a powerful prognostic factor in patients with MM, our purpose was to study how CTX-G-CSF treatment affects the phenotype and function of T cells, in particular regulatory T cells (Treg), in 15 patients with MM. CTX induced severe T cell immunosuppression with a slow and partial T-cell recovery (a threefold decrease) at the time of HP collection. CTX-G-CSF treatment did not affect the percentages of central memory (CD45RA−, CCR7+), effector memory (CD45RA−, CCR7−), and late effector (CD45RA+, CCR7−) CD4 or CD8 T cells but a decrease of naïve CD4 cells (CD45RA+, CCR7+) was found. The percentages of CD25+ cells increased two- to threefold in CD4 or CD8 T cells, respectively. Post-CTX treatment CD4CD25+ cells included both activated CD4CD25low cells and CD4CD25high T cells. The latter were Treg because they expressed high level of FOXP3 and membrane CTLA-4 mRNA and protein and displayed functional suppressor function. In CTX-G-CSF leukaphereses from 15 patients with MM, the mean Treg number was one fifth that of CD34 and the CD3, CD4 and CD8 numbers respectively 3 fold, 2 fold and equal that of CD34. Post-CTX-G-CSF treatment CD3 cells did not cell cycle in vivo and died in short-term culture in vitro. Adding IL-2 or IL-15 induced their survival and cell cycle, and stimulation with anti-CD3 MoAb led to efficient growth in vitro. These results suggest that following CTX-G-CSF treatment, CD3 cells are preactivated in vivo and do not cell cycle, likely due to a lack of T cell growth factors in vivo. The current data indicate that CTX-G-CSF treatment profoundly affects T cell function without eliminating Treg. The persistence of Treg could be explained by an opposite effect of CTX known to kill Treg and of G-CSF amplifying Treg. Given the major impact of lymphocyte count on patients’ survival post HDC and HP and T cell graft, the present data invite to define novel therapeutic strategies to improve T cell recovery in vivo while limiting Treg expansion.

Aim was to assess the role of the epidermal growth factor (EGF)/EGF receptor (ErbB1 (EGFR), ErbB2, ErbB3 and ErbB4) family in the pathogenesis of multiple myeloma (MM) investigating (i) their expression profile and (ii) their functional activity. Comparing Affymetrix DNA-microarray-expression-profiles of CD138-purified plasma-cells from 65 MM patients and 7 normal individuals to those of plasmablastic- and B-cells, we found 5/10 EGF-family genes to be overexpressed in myeloma cells (MMC). Two of them -neuregulin(NRG)-2 and NRG-3- were expressed by MMC only, while other three -NRG-1, amphiregulin (AREG) and TGF-α- were expressed by both MMC and normal bone marrow (BM) plasma cells (PCs). In addition, using real-time PCR, we found 4/10 EGF-ligands -HB-EGF, AREG, NRG-1 and epiregulin- to be expressed by cells from the BM-environment. ErbB receptors were expressed by normal and malignant plasma cells but were lacking on plasmablastic- and B-cells. ErbB1 and ErbB2 were expressed by MMC and BMPC, whereas ErbB3 and ErbB4 were expressed by MMC exclusively. The growth of myeloma cell lines was promoted by EGF-ligands able to bind heparan sulfate (HS) proteoglycans (PGs), i.e. NRG-1, amphiregulin and HB-EGF, but not by other EGF-ligands, i.e. EGF, TGF-α, epiregulin and betacellulin. Proliferation induced by HS-binding EGF-ligands was completely abrogated when the growth factors where pre-incubated with heparin. A truncated form of NRG-1 that lacks the HS-binding domain but has kept the ErbB binding domain was unable to promote MMC growth. MMC could bind large levels of HS-binding EGF-ligands through syndecan-1, which is expressed on myeloma cells at a high density contrary to other 10 HSPGs -syndecan-2-4, glypican-1-6 and CD44v3-. The binding was abrogated by either pre-treatment with heparitinase (which cleaves HS chains), or pre-incubation of the growth factors with heparin. None of the EGF-ligands could bind or induce proliferation of the XG-10 myeloma cell line which doesn’t express syndecan-1. Thus, EGF/EGF-receptor genes are expressed during plasma cell differentiation and malignant transformation: MMC express ErbB receptors and several HS-binding EGF-ligands. Cells from the BM-environment can also be a source of EGF-family members delivering a paracrine growth signal to MMC. Myeloma cell growth is stimulated by EGF-ligands that bind HS, via binding to syndecan-1, which likely concentrates high levels of HS-binding-EGF-ligands in the proximity of ErbB receptors at the cell membrane and therefore facilitates ErbB-activation. This accumulation of EGF-family members at cell membrane is required to promote their myeloma cell growth activity. We have previously shown that pan-ErbB inhibitors like CI-1033 induce strong apoptosis of primary MMC cultured with their bone-marrow environment. Together with these data, this further recommends EGF-signalling as promising target for MM therapy.

Rituximab, a monoclonal antibody that targets the CD20 antigen, has shown single-agent activity in patients with previously untreated and relapsed follicular lymphoma (FL). Mechanisms of rituximab-mediated antineoplastic activity include complement-mediated cell (CDC) lysis, apoptosis, and the induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Granulocyte macrophage colony stimulating factor (GM-CSF) is a biologic agent capable of enhancing proliferation of cell populations and cell surface receptors necessary for rituximab activity. It was hypothesized that the combination of GM-CSF and rituximab would substantially improve the treatment efficacy of rituximab in relapsed, FL patients. The current study assessed the clinical response, biologic response, and safety of combination rituximab and GM-CSF in patients with relapsed FL. Response and toxicity were defined using previously determined standard criteria (Cheson B, et al. J Clin Oncol.1999;17:1244–1253) and World Health Organization standard criteria. A total of 46 patients, with a median age of 60 years (range, 37–80 years), received rituximab and GM-CSF between January 2000 and October 2000. This analysis focuses on 34 patients with relapsed or progressive FL. GM-CSF was administered at 5 mg/kg/day on Days 1–8 and rituximab was administered at 375 mg/m2/week on Day 5 of each 21-day cycle for a total of 4 cycles. Throughout the treatment and follow-up periods, all patients underwent clinical examinations, bone marrow biopsies, standard biologic assays (eg, lactate dehydrogenase, β2-macroglobulin, creatinine, and liver enzymes), complete blood cell counts, immunophenotyping, and polymerase chain reaction analysis for bcl-2 expression. Response was evaluated at Days 30, 60, 90, and every 4 months until progression. Of the 34 enrolled patients, 1 patient was excluded due to transformation and 4 patients withdrew from the study due to toxicity. The complete response (CR) rate was 45% and the overall response rate was 70% (15 CR + CR unconfirmed and 8 partial responses [PR]) for the 33 intent-to-treat patients with relapsed FL. CR was reached in 63% (7 of 11) patients who received ≥3 lines of previous therapy (range, 3–7 lines). The median progression-free survival was 16.7 months. A significant increase (P

PCL has been successfully treated with interferons (IFNs), which can offset the Th2 dominance associated with PCL. Intratumoral (IT) injection of TG1042 (a non-replicating recombinant adenovirus with a human IFN-γ cDNA insert) allows high local levels of IFN-γ without severe toxicity induced by systemic delivery. We undertook a multicentric, phase I/II, open-label, trial of repeated, IT injection of TG1042 in patients with advanced primary T cell (CTCL) and multilesional B cell (CBCL) cutaneous lymphoma. The designated lesions were injected on day 1, 8 and 15 with no injection in the fourth week (1 cycle) and thereafter up to 4 cycles. Immunohistochemistry and qPCR were performed on injected lesions biopsied at baseline and after each cycle. In the phase I step, undertaken on a monocentric basis, 9 patients were enrolled in 3 successive cohorts at the doses of 3×109 viral particles (vp), 3×1010 and 3×1011 vp injected in only one lesion and 9 additonal patients were treated at the doses of 3×1011 vp divided in up to 3 lesions. In the phase II step, 18 additional patients have been planned to be treated on a multicentric basis at 3×1011 vp injected in up to 3 lesions. To date, the 36 planned patients (30 CTCL and 6 CBCL) have been enrolled and received a total of 280 injections, 26 patients have completed the treatment. Treatment is well tolerated with only 3 grade III toxicity. Injection site reaction and flue like syndrom are the most commonly observed adverse events. Local clinical response has been observed in 13 (7 complete responses) out of 27 evaluable patients. Disappearance of non-injected lesions was also observed and led to 12 overall responses (6 complete responses) out of 26 evaluable patients. Histology demonstrates pronounced differences in infiltrate patterns following treatment, with signs of vasculitis and increased numbers of eosinophils, neutrophils, CD8 and TIA-1+ve cells. CD4/CD8 ratio decreased in most tumors. Transgene-IFN-γ mRNA was detectable in injected lesions. Gene expression analysis of biopsies and PBMC shows up-regulation of IFN-γ and various immune response-associated genes. Final clinical and immunological data will be presented. In conclusion, these data show that the administration of TG1042 is well tolerated and results in clinical responses associated with in situ immunological changes. These encouraging results demonstrate a potential significant benefit of TG1042 for the treatment of primary cutaneous lymphoma.

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.