Roquinimex (Linomide) Vs Placebo in AML After Autologous Bone Marrow Transplantation

Home , Roquinimex

Bone Marrow Transplantation (2000) 25, 1121–1127  2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt Roquinimex (Linomide) vs placebo in AML after autologous bone marrow transplantation

B Simonsson, T To¨tterman, P Hokland, F Lauria, AM Carella, MN Fernandez, C Rozman, A Ferrant, T de Witte, AR Zander, K Meier, F Hansson and BI Nilsson for the Linomide in AML in Europe Study Group

Summary: ABMT.3–7 DFS is less for patients transplanted at later stages of AML. Roquinimex, Linomide, a quinoline derivative with A role for immunologic mechanisms for eradication of pleiotropic immunomodulatory activity, has previously residual leukemia cells after allogeneic bone marrow trans- been shown to enhance natural killer (NK) cell number plantation has been defined as the GVL phenomenon.8–10 and activity after ABMT in patients with AML. In this GVL is mediated via T lymphocytes, NK cells or other study 278 AML patients in remission were randomized mediators as well.11–16 T lymphocytes with a reactivity to receive Roquinimex 0.2 mg/kg body weight or pla- against leukemic cells distinct from the reactivity against cebo twice weekly for 2 years following ABMT. Out of normal marrow cells have been isolated.13,17 NK cells as 139 patients in each group, 109 Roquinimex patients well as lymphokine-activated killer (LAK) cells have dem- and 108 placebo patients were in their first CR. Median onstrated lysis of allogeneic as well as autologous leukemia age at inclusion was 41 years for Roquinimex patients blast cells.18–20 Furthermore, NK cell activity in AML in and 39 years for placebo patients. Twelve patients in CR has been shown to be inversely correlated with the risk each group had their marrow purged prior to rein- of relapse.21 fusion. Relapse and death were study endpoints. Surviv- NK activity and LAK cell inducibility are more promi- ing patients were followed for 2.6 to 6.9 years. The total nent in complete remission than in frank AML.22–24 These number of relapses was 60 in the Roquinimex group results, together with the observation that tumor cells might and 63 in the placebo group (not significant). Leukemia- inhibit LAK generation,20,25 made it conceivable that the free and overall survivals were similar in the two best prospects for enhancement of NK cell activity were in groups. Recovery of platelet counts was significantly a state of minimal residual disease. delayed in the Roquinimex group as compared to pla- Activated macrophages, like NK and LAK cells, can be cebo. No other significant differences regarding toxicity non-specifically cytotoxic to tumor cells independent of the parameters were recorded. In conclusion, previous fin- MHC.26–30 However, the role of monocytes/macrophages, dings on NK cells could not be confirmed and the study if any, in the eradication of residual AML cells in remission showed no benefit for Roquinimex over placebo regard- remains to be determined. ing relapse or survival following ABMT for AML in Roquinimex (Linomide) has shown broad immunomodu- remission. Bone Marrow Transplantation (2000) 25, latory properties in preclinical studies. NK cell number and 1121–1127. activity were enhanced in mice during the recovery phase Keywords: Roquinimex; Linomide; ABMT; AML after myeloablative therapy.31,32 Antigen-induced T cell responses have been modulated in various models.33,34 Macrophage cytotoxicity has been enhanced in tumor-bear- ing rats.35 Immunomodulatory properties of Linomide in The overall annual incidence of AML is about three per clinical studies have been shown in patients with auto- 100000 population with an increased incidence with age- immmune diseases,36 various cancers,37 and healthy volun- ing. Median age is 62–65 years. Intensive combination teers (Linde A, unpublished). Linomide given intermittent chemotherapy may induce complete remission (CR) in 60– courses to patients with AML in remission after autotrans- 1 80% of patients less than 60 years of age. Older patients plantation, increased the number and activity of NK cells have lower probability of entering CR. A high proportion as well as the number of monocytes.39,40 of patients will relapse, and the ultimate cure rate is The toxicity of Linomide has in general been manageable 2 between 10 and 30% after chemotherapy. Following with symptomatic treatment. Side-effects have mainly con- ABMT for AML in first complete remission (CR1), dis- sisted of musculoskeletal aches, nausea, vomiting, edema, ease-free survival (DFS) is 40–60% at 3 years post skin rash, diarrhea and fatigue.36–40 In ongoing clinical studies a 2% incidence of pericarditis and pleuritis as well as a 0.5% incidence of acute myocardial infarction has been Correspondence: Dr B Simonsson, Department of Medicine, University reported. Hospital, S-751 85 Uppsala, Sweden With AML perceived as a disorder sensitive to manipu- Received 23 August 1999; accepted 30 January 2000 lations of immune function and Roquinimex being an agent Roquinimex (Linomide) vs placebo in AML after BMT B Simonsson et al 1122 with broad immunomodulatory activity and manageable time from ABMT to relapse or death. Relapse rate was toxicity, it was decided to investigate whether Linomide calculated as the percentage of patients with relapse after could prolong the time to relapse or reduce the relapse rate 1 and 2 years. in patients with AML in remission after ABMT. Complete blood counts were obtained at each visit, and bone marrow was aspirated every 3 months and as clini- cally indicated. Patients, materials and methods The effect of Linomide on immunological reconstitution in a subgroup of 97 patients was studied by means of flow Patients with cytochemically verified primary AML in first cytometry. Samples of heparinized blood were drawn 48 h or later CR, and for whom an ABMT was planned, were after the latest dose of study medication, and mononuclear eligible for entry into the trial. Morphologic classification cells were Ficoll separated. Cell numbers were assessed by and CR definition followed the French–American–British FACS, and the following cells were enumerated: NK-like system. The study was carried out at 39 centers in 10 Euro- cells (Leu 11-FITC (CD16), Leu 19-PE (CD56) together pean countries, and was approved by the national health with Leu 4-FITC (CD3)), T cells (Leu 4-FITC (CD3)), B authorities and the local ethics committees. cells (Leu 12-FITC (CD19)) and monocytes (Leu M3-PE (CD14)). Linomide For lymphoid cells CD45 (FITC) always had to be Ͼ90% and Leu M3 (CD14-PE) Ͻ5% in the gate. The pro- During screening for anti-inflammatory agents, Roquinimex portion of CD14ϩ cells was expressed as percent of all (Linomide; Pharmacia & Upjohn, Stockholm, Sweden) was mononuclear cells, not of gated lymphocytes. Results were discovered to have potent immune stimulatory properties. obtained from the Leucogate staining. Its chemical name is N-phenyl-N-methyl-1,2-dehydro-4- In order to permit comparisons between patients at vari- hydroxy-1-methyl-2-oxoquinoline-3-carbo-xamide. Its mole- ous centers, consistency of FACS analysis performance

cular formula is C18H16O3N and its molecular weight is between centers was conﬁrmed. 308.33. Autoantibodies were analyzed at admission, month 6 and month 24 in order to detect possible immunologic compli- Randomization cations of the treatment.

Randomization was made after the patient’s informed con- Statistical methods sent was obtained and before starting the myeloablative treatment. The patients were stratified by center and by first Demographic variables were assessed using descriptive or later CR. Neither the patients nor the investigators nor statistics. The two treatment groups were compared using the sponsor’s study management personnel had access to the normal approximation of the Wilcoxon rank sum test information on treatment assignments. when the variables were continuous and the Pearson’s chi- square test, without continuity correction when the vari- Treatment schedule ables were presented in contingency tables. When the events in the row x column table cells were few, a gen- Linomide or placebo treatment was initiated on day 14 (12– eralization of the Fisher’s exact test was used. 16) after ABMT and the drug was administered twice The following variables were used as prognostic factors weekly. The dose was 0.05 mg/kg body weight (2.5–5 mg) in the analysis of survival, time to relapse and leukemia- twice during the first week and 0.10 mg/kg (5–10 mg) twice free survival: age, number of complete remissions (CR = during the second week. From the third week on, the dose 1 and CR Ͼ 1), FAB classification, extramedullary involve- was 0.20 mg/kg (10–20 mg) twice weekly for 2 years. The ment and country. Data from diagnosis of AML were dose was adjusted downwards to the tablet sizes available incomplete (eg cytogenetics) and could not be evaluated. (2.5, 5 and 10 mg). Dose modifications were made in case Relapse rate at 1 year included all patients who relapsed of suspected toxic side-effects. If chemotherapy for relapse within 1 year of observation. Time to relapse, survival and had to be instituted, the treatment with study medication leukemia-free survival (LFS) used all available information was discontinued. Concomitant use of chemotherapy or about the patients (intent to treat analysis). Patients not other immunomodulating drugs was not allowed. showing relapse or death were censored at the date of latest follow-up visit. Assessments The number of patients required for the efficacy analysis was based on time to relapse and proportion of relapses at Relapse was defined as Ͼ10% blast cells in a normocellular 12 months. With a significance level of 5%, a one-tailed bone marrow aspirate, or as extramedullary leukemia. If the test, a power of 80% and an expected median time to marrow blast percentage was 11–20, a second aspirate had relapse of 12 months for placebo and 18 months for Linom- to be evaluated within 2 weeks; the first one was recorded ide 2 ϫ 109 = 218 patients were needed. With this number as date of relapse. If the blast percentage was above 20, a of patients it was considered possible to detect a true differ- second aspirate was not needed. After discontinuation of ence between a placebo relapse proportion of 50% and a study treatment due to relapse or for any other reason, the Linomide relapse proportion of 33% at 12 months. patients were followed for survival. All models above were analyzed using the Cox Time to relapse as well as survival were defined as the regression model (SAS PHREG). The results from the

Bone Marrow Transplantation Roquinimex (Linomide) vs placebo in AML after BMT B Simonsson et al 1123 chosen models included an estimate of the hazard ratio with Table 2 ABMT data a 95% conﬁdence interval. Linomide Placebo

Ethics ABMT performed 138 139 Purging 12 12 The Study Protocol was reviewed for scientific and ethical Median No. of cells infused (ϫ 108/kg) 7 7 validity and approved by the Ethics Committees or Insti- Conditioning BU-CY 77 68 tutional Review Boards of each of the 39 study centers as CY-TBI 25 35 well as by the Health Authorities of the 10 participating Others 37 36 European countries. Prior to enrollment, each patient was informed, verbally and in writing, about the objectives and procedures of the study, the possible risks involved and the computer pro- Table 2 shows ABMT data. One patient randomized to cessing of individual patient data. Linomide treatment did not undergo ABMT. Twelve Informed consent was obtained from the patient either in patients in each treatment group had the marrow purged writing or orally according to local regulations. before reinfusion. Busulphan plus cyclophosphamide was the most commonly used conditioning regimen. There were no differences between the two treatment groups regarding Results the ABMT procedure. Of 133 patients treated with Linomide, six died and 34 relapsed while on medication. Patients Fifty-seven patients were withdrawn for the following Between February 1991 and May 1994, 278 patients were reasons: refusal (10), adverse event (38), protocol violation randomized. Only 277 patients underwent ABMT as patient (2), other reason (6), unassociated disease (1). Thus, 36 No. 121 (Linomide) relapsed prior to ABMT. Reasons why Linomide patients completed 2 years treatment according 11 patients never received study medication were: death to the protocol. (1), refusal (2), protocol violation (1), other reason (1) in Of 133 placebo patients, one died and 47 relapsed while the Linomide group and death (2), adverse event (1), other on medication. Thirty-four patients were withdrawn for the reason (3) in the placebo group. Thus 266 patients received following reasons: refusal (7), adverse event (24), other rea- at least one dose of study medication after ABMT; 133 son (3), unassociated disease (1). Thus, 51 placebo patients Linomide and 133 placebo. completed 2 years treatment according to the protocol. Patient characteristics are given in Table 1. There were significantly more males in the Linomide group compared Efficacy to placebo (P = 0.012). Sex was not of prognostic significance regarding the study endpoints. Time from diagnosis The number of patients with relapse and the relapse rates to ABMT and time from attainment of CR to ABMT did are shown in Table 3. There was no statistically significant not differ between the arms (data not shown). Thus, the difference between the treatment groups. Figure 1 shows two treatment arms were well balanced. The impact of the overall freedom from relapse. Thirteen late relapses (Ͼ12 cytogenetic risk profiles could not be studied since too few months post ABMT) occurred in the Linomide group as samples were analyzed for cytogenetics. compared with 17 late relapses in the placebo group. Median observation time of surviving patients was 1629 days for placebo and 1586 days for Linomide-treated patients. The observation period was from February 1991 to November 1998. Table 1 Patient characteristics Survival data were analyzed using Kaplan–Meier plots and Cox regression. There were no statistically significant Linomide Placebo

No. of patients (ITT) 139 139 Median age (years) (range) 41 (13–65) 39 (12–60) Table 3 Number and rates of relapses by treatment group Males/Females 85/54 64/75 Extramedullary disease at diagnosis 8 12 Time from ABMT Relapse n (rate) CR1 109 108 months CRϾ13031 FAB Linomide Placebo = = M1 24 24 n 139 n 139 M2 41 35 M3 26 22 0–12 47 (34) 46 (33) M4 26 28 0–24 59 (42) 61 (44) M5 18 26 Total 60 (43) 63 (45) M6/M7/Unclass/missing 4 4 Relapse rate: all patients, and at 1 and 2 years post ABMT, respectively. All features except age are given as number of patients. Distribution by treatment group. All randomized patients, n = 278.

Bone Marrow Transplantation Roquinimex (Linomide) vs placebo in AML after BMT B Simonsson et al 1124 100 100

90 90

80 80

70 70

60 60

50 50

40 40 Alive (%)

Relapse free (%) 30 30

20 20

10 placebo 10 placebo Linomide Linomide 0 0 0123456 0123456 Patients at risk Time (years) Patients at risk Time (years) p 139 84 66 63 43 21 5 p 139 98 74 69 46 24 5 L 139 80 65 61 42 17 2 L 139 102 77 68 47 17 2

Figure 1 Freedom from relapse, by treatment group. n = 278. Solid line, Figure 3 Overall survival, by treatment group. n = 278. Solid line, Lino- Linomide; dashed line, placebo. mide; dashed line, placebo.

100 Table 4 Maximum absolute number of immune cells after ABMT n = 97 90 80 Cell type Treatment n Median Range (cells/␮l) 70

60 CD14+ Linomide 45 356 2–1539 50 Placebo 50 367 7–1625 + 40 CD16 Linomide 45 85 0–2150 Placebo 50 146 0–659 30 CD19+ Linomide 44 37 1–1050 20 Placebo 49 270 1–770 Relapse free and alive (%) 10 placebo CD56+CD3− Linomide 46 92 3–1814 Linomide Placebo 50 162 5–1411 0 0123456 Patients at risk Time (years) CD14ϩ, monocytes; CD16ϩ, NK-like cells; CD56ϩ CD3Ϫ, NK-like cells; p 139 84 66 63 43 21 5 CD19ϩ, B cells. Maximum absolute number obtained at any time after L 139 80 65 61 42 17 2 ABMT. Linomide-treated patients did not reach as high numbers as pla- = ϩ = cebo-treated patients for B cells (CD19, P 0.002) or NK cells (CD56 Figure 2 Leukemia-free survival, by treatment group. n 278. Solid CD3Ϫ, P = 0.04; CD16ϩ, P = 0.05). In the visit by visit analysis, there line, Linomide; dashed line, placebo. were no differences between the two treatment groups regarding B cells, NK cells, or monocytes.

differences between the treatment groups regarding leukemia-free or overall survival (Figures 2 and 3). by visit analysis, there were no differences between the two There were no statistically significant differences treatment groups regarding B cells, NK cells, or monocytes. between Linomide and placebo within any country or com- vs Ͼ plete remission group (CR1 CR 1) regarding freedom Safety from relapse or leukemia-free survival (data not shown). Median time to relapse has not yet been reached for any Two years of treatment according to the protocol was com- of the populations. Median estimated overall survival is pleted by 87 patients (51 placebo, 36 Linomide). The 1126 days for Linomide and 1102 days for placebo-treated median amount of drug administered to the patients was patients. Estimates on leukemia-free survival are 505 days for Linomide 485 mg (range 2.5–3105 mg), and for placebo for Linomide and 520 days for placebo. the corresponding value was 1150 mg (range 2.5– The maximum absolute number of circulating immune 4715 mg). cells has been calculated as the maximum value after treat- Clinical adverse events were similar between the two ment initiation by use of the Wilcoxon test. In Table 4. the treatment groups, both as specific types of events and as maximum absolute value is described. There were signifi- events grouped by body system. All treated patients had cantly lower numbers in the Linomide group than in the recordings of adverse events (Table 5). Twenty-nine placebo group regarding CD19 (B cells) (P = 0.0022) and patients (12 placebo, 17 Linomide) died without a recording CD56 (NK cells) (P = 0.0404), and a borderline signifi- of relapse. Death was associated with infection or bleeding cance regarding CD16 (NK cells) (P = 0.0504). In the visit or leuko- or thrombocytopenia in nine placebo patients who

Bone Marrow Transplantation Roquinimex (Linomide) vs placebo in AML after BMT B Simonsson et al 1125 Table 5 Clinical adverse events of various categories seemed to occur somewhat more frequently in the Linomide group, but there were no Linomide Placebo statistically significant differences. 133 patients 133 patients Recovery of platelet number was significantly delayed in the Linomide treatment group as compared to placebo Nausea/vomiting 76 70 (Figure 4). However, there were no statistically significant Edema 60 47 Musculo-skeletal discomfort 49 42 differences between the two treatment groups regarding Diarrhea 43 35 treatment-related mortality, serious adverse events or Headache 32 32 hemorrhagic complications. Recovery of neutrophils seemed to be delayed for Linomide-treated patients, There were no statistically significant differences between the two treat- although this was not statistically significant. Recovery of ment groups regarding clinical adverse events. Most common events; red cells and hemoglobin was similar between the two treat- from checklist. ment groups. There were no significant differences between Linomide died 16–432 days post ABMT, and in nine Linomide and placebo-treated patients respectively, regarding liver patients who died 5–1025 days post ABMT. function tests, creatinine, ESR, CRP, U-protein or U- Reasons for withdrawal from study treatment are shown glucose. No autoantibodies developed in either of the two in Table 6. Twenty-four placebo and 38 Linomide patients patient groups. were withdrawn due to adverse events, and seven placebo and 12 Linomide patients because of refusal. Of the 24 placebo patients withdrawn due to adverse events, eight Discussion (33%) were alive and in continuing complete remission at the end of the follow-up period, whereas 21 of 38 (55%) The main finding in the final analysis of the present study Linomide patients withdrawn due to adverse events were was the lack of statistically significant differences between alive and relapse-free at the end of the observation period. Linomide and placebo regarding the study endpoints: time Thirty patients in the placebo group and 41 patients in to relapse, relapse rate, leukemia-free survival and overall the Linomide treatment group had recordings of serious survival. However, there were fewer late relapses in the adverse events. Twelve patients in the placebo group had Linomide group than among placebo-treated patients, indi- 18 serious adverse events and 20 patients in the Linomide cating the possibility of an antileukemic effect of Linomide group had 21 serious adverse events that were assessed as in patients with very few residual AML cells after ABMT. possibly or probably related to administration of study Therefore, a continued follow-up of the patients for relapse medication. Of such events, 13 in the placebo group and and survival has been performed till the end of 1998. At 14 in the Linomide group were cytopenia, infection, or this time, the data are considered sufficiently mature for a bleeding. final conclusion to be reached. Pericarditis occurred in four patients in the Linomide Preclinical studies as well as early clinical pilot studies group, two reported as serious. One of these patients showed that Linomide enhanced the number of NK cells 31,32,39,40 developed pericarditis before start of study medication. One in a regenerating bone marrow. Those findings Linomide treated patient had a myocardial infarction. could not be confirmed in the present large randomized The frequency of edema did not differ significantly study. Instead, maximum B cell and NK cell numbers between the groups. However, there were more patients seemed to be decreased by Linomide. A similar decrease with severe edema in the Linomide as compared to the placebo group. A similar finding was made for musculoskeletal discomfort. Moderate vomiting as well as moderate 100 nausea/vomiting was reported somewhat more frequently 90 for Linomide than for placebo patients, but there were no 80 statistically significant differences. Peripheral neuropathy 70

Table 6 Reasons for termination of study treatment (No. of patients) 50

40 Reason Placebo Linomide Total

Recovered (%) 30

Death 3 7 10 20 Patient refusal 7 12 19 Adverse experience 24 38 62 10 placebo Progression of the disease, relapse 47 35 82 Linomide 0 Protocol violation 0 3 3 0 180 360 540 720 900 1080 End of protocol treatment 51 36 87 Patients at risk Time (days) Other reason 6 7 13 p 133 28 8 2 1 0 0 Unassociated disease 1 1 2 L 133 40 16 11 3 0 0

Total 139 139 278 Figure 4 Recovery of platelets to Ͼ50 ϫ 109/l after ABMT, by treatment group. n = 266. Solid line, Linomide; dashed line, placebo.

Bone Marrow Transplantation Roquinimex (Linomide) vs placebo in AML after BMT B Simonsson et al 1126 is also reported in a small subgroup analysis.38 Reasons for allogeneic or autologous bone marrow transplantation and this discrepancy might be the divergence between species chemotherapy in patients with acute myeloid leukemia in first and the small number of patients in the early clinical stud- remission: a prospective controlled trial. Br J Haematol 1989; ies. Treatments in previous studies were of short duration, 72: 57–63. and a different effect of Linomide with a longer treatment 6 Cassileth PA, Anderson J, Lazarus HM et al. Autologous bone marrow transplant in acute myeloid leukemia in first duration cannot be excluded. It is also possible that differ- remission. J Clin Oncol 1993; 11: 314–319. ent results could be obtained with another dose or dose 7 Sierra J, Gran˜ena A, GarcıáJet al. Autologous bone marrow schedule, eg intermittent administration. transplantation for acute leukemia: results and prognostic fac- Delayed regeneration of platelets after the ABMT was tors in 90 consecutive patients. Bone Marrow Transplant the only statistically significant finding in the safety evalu- 1993; 12: 517–523. ation of Linomide. However, this finding was not translated 8 Weiden PL, Keith M, Sullivan KM et al. Antileukemic effect into a higher number of serious or fatal bleedings among of chronic graft-versus-host disease: contribution to improved Linomide-treated patients. Clinical adverse events were not survival after allogeneic marrow transplantation. New Engl J different between the treatment groups. Previous clinical Med 1981; 304: 1529–1533. studies had shown that administration of Linomide had 9 Sullivan KM, Fefer A, Witherspoon R. Graft-versus-leukemia in man: relationship with acute and chronic graft-versus-host been associated with, eg musculoskeletal discomfort, disease to relapse of acute leukemia following allogeneic bone edema, skin rashes, headache, as well as other side-effects. marrow transplantation. In: Truitt RL, Gale RP, Bortin MM The lack of significant differences between the groups (eds). Cellular Immunotherapy of Cancer. Alan R Liss: New could have been due to disturbing side-effects associated York, 1987, pp 391–399. with the ABMT as such, and possibly also to the low num- 10 Horowitz MM, Gale RP, Sondel PM et al. Graft-versus-leuke- ber of patients (87 out of 278) who actually completed the mia reactions after bone marrow transplantation. Blood 1990; 2 year treatment according to the protocol. 75: 555–562. Studies of interleukin-2 in AML in minimal residual dis- 11 Lo¨wenberg B, Wagemarker G, van Bekkum DW et al. Graft- ease have shown encouraging as well as disappointing versus-host disease following transplantation of ‘one log’ vs results in phase II studies,41,42 and randomized studies have ‘two log’ T lymphocyte-depleted bone marrow from HLA- identical donors. Bone Marrow Transplant 1986; 1: 133–140. not shown benefits of IL-2 in AML. One theoretically poss- 12 Sosman JA, Oettel KR, Smith SD et al. Specific recognition ible reason for the apparent lack of benefit from Linomide of human leukemia cells by allogeneic T cells: II. Evidence or IL-2 in AML could be reactive oxygen metabolites, gen- for HLA-D restricted determinants on leukemic cells that are erated by monocytes activated concomitantly with NK cells crossreactive with determinants present on unrelated nonleu- and T cells during treatment with IL-2 or Linomide. Such kemic cells. Blood 1990; 75: 2005–2016. reactive oxygen metabolites have been shown to inhibit 13 Faber LM, van Luxemburg-Heijs SAP, Willemze R, Falken- activation of NK cells and T cells.43,44 burg JHF. Generation of leukemia-reactive cytotoxic T lym- The finding of a lower number of NK cells in the treat- phocyte clones from the HLA-identical bone marrow donor ment group than in the control group was in contrast to of a patient with leukemia. J Exp Med 1992; 176: 1283–1289. earlier findings of increased NK cell numbers upon treat- 14 Dawson MM, Johnston D, Taylor GM, Moore M. Lympho- 31,32,39,40 kine activated killing of fresh human leukaemias. Leukemia ment with Roquinimex. Since Roquinimex has Res 1986; 10: 683–688. been shown to activate monocytes/macrophages, it can be 15 Oshimi K, Oshimi Y, Akutsu M et al. Cytotoxicity of interleu- hypothesized that reactive oxygen metabolites from such kin 2-activated lymphocytes for leukemia and lymphoma cells could have initiated apoptosis in NK cells, as has been cells. Blood 1986; 68: 938–948. shown experimentally.45 16 Lauria F, Raspadori D, Rondelli D et al. In vitro susceptibility In conclusion, Roquinimex did not show superiority as of acute leukemia cells to the cytotoxic activity of allogeneic compared to placebo regarding time to relapse, relapse rate, and autologous lymphokine activated killer (LAK) effectors: leukemia-free survival or overall survival. correlation with the rate and duration of complete remission and with survival. Leukemia 1994; 8: 724–728. 17 Hoffman T, Theobald M, Bunjes D et al. Frequency of bone marrow T cells responding to HLA-identical non-leukemic References and leukemic stimulator cells. Bone Marrow Transplant 1993; 12: 1–8. 1 Volm MD, Tallman MS. Developments in the treatment of 18 Lotzova´ E, Savary CA, Herberman RB. Induction of NK cell acute leukemia in adults. Curr Opin Oncol 1995; 7: 28–35. activity against fresh human leukemia in culture with interleu- 2 Greer JP, Kinney MC. Acute nonlymphocytic leukemia. In: kin-2. J Immunol 1987; 138: 2718–2727. Lee RG, Bithell TC, Foerster J et al (eds). Wintrobe’s Clinical 19 Reittie JE, Gottlieb D, Heslop HE et al. Endogenously gener- Hematology. Lee and Febiger: Philadelphia, 1993, pp 1920– ated activated killer cells circulate after autologous and allo- 1945. geneic marrow transplantation but not after chemotherapy. 3 Zittoun RA, Mandelli F, Willemze R et al. Autologous or allo- Blood 1989; 73: 1351–1358. geneic bone marrow transplantation compared with intensive 20 Archimbaud E, Bailly M, Dore´ J-F. Inducibility of lympho- chemotherapy in acute myelogenous leukemia. New Engl J kine activated killer (LAK) cells in patients with acute myelo- Med 1995; 332: 217–223. genous leukemia in complete remission and its clinical rel- 4Ko¨rbling M, Hunstein W, Fliedner TM et al. Disease-free sur- evance. Br J Haematol 1991; 77: 328–334. vival after autologous bone marrow transplantation in patients 21 Pizzolo G, Trentin L, Vinante F et al. Natural killer cell func- with acute myelogenous leukemia. Blood 1989; 74: 1898– tion in lymphoid cell populations in acute non-lymphoblastic 1904. leukemia in complete remission. Br J Cancer 1988; 58: 5 Reiffers J, Gaspard MH, Maraninchi D et al. Comparison of 268–272.

Bone Marrow Transplantation Roquinimex (Linomide) vs placebo in AML after BMT B Simonsson et al 1127 22 Adler A, Albo V, Blatt J et al. Interleukin-2 induction of lym- Bordetella pertussis in the rat. Immunopharmacol 1986; 11: phokine-activated killer (LAK) activity in the peripheral blood 87–92. and bone marrow of acute leukemia patients: II. Feasibility of 35 Sabzevari H, Koo GC, Szalay J. The effect of Linomide on LAK generation in children with active disease and in growth and metastasis of mammary adenocarcinoma in the remission. Blood 1989; 74: 1690–1697. Fischer 344 rat. J Exp Clin Cancer Res 1994; 13: 21–30. 23 Nasrallah AG, Miale TD. Decreased natural killer cell activity 36 Nived O, Sturfelt G, Nilsson B et al. Clinical pilot study of in children with untreated acute leukemia. Cancer Res 1983; Roquinimex ((Linomide) in patients with autoimmune disease. 43: 5580–5585. Int J Immunother 1994; 10: 49–59. 24 Sørskaar D, Førre Ø, Albrechtsen D, Stavem P. Decreased 37 Bergh JCS, To¨tterman TH, Termander BC et al. A clinical natural killer cell activity versus normal natural killer cell mar- pilot study of Roquinimex (Linomide) in cancer patients with kers in mononuclear cells from patients with smouldering leu- special forms on immunological effects. Cancer Invest 1997; kemia. Scand J Haematol 1986; 37: 154–161. 15: 204–211. 25 Guillou PJ, Sedman PC, Ramsden CW. Inhibition of lympho- 38 Hokland M, Jørgensen H, Holm MS et al. Natural effector kine-activated killer cell generation by cultured tumor cell cells in patients with acute myeloid leukemia treated with the lines in vitro. Cancer Immunol Immunother 1989; 28: 43–53. immunomodulator Linomide after autologous bone marrow 26 Melzer MS, Nacy CA. Delayed-type hypersensitivity and the transplantation. Eur J Haematol 1999; 63: 251–258. induction of activated, cytotoxic macrophages. In: Paul WE 39 Bengtsson M, Simonsson B, Carlsson K et al. Stimulation of (ed). Fundamental Immunology. Raven Press: New York, NK cell, T cell and monocyte functions by the novel immuno- 1989; pp 765–777. modulator Linomide after autologous bone marrow transplan- 27 Nathan CF, Cohn ZA. Cellular components of inflammation: tation. Transplantation 1992; 53: 882–888. monocytes and macrophages. In: Kelly WN, Harris ED Jr, 40 Nilsson BI, Simonsson B, Bengtsson M et al. Immunotherapy Ruddy S, Sledge CB (eds). Textbook of Rheumatology.WB of AML after ABMT – scientific rationale and early experi- Saunders Co: Philadelphia, 1985, pp 144–169. ences with Linomide. In: Dicke KA, Keating A (eds). Autolog- 28 Fidler IJ, Schroit AJ. Recognition and destruction of neoplas- ous Bone Marrow Transplantation – Proceedings of the Sixth tic cells by activated macrophages: discrimination of altered International Symposium. Cancer Treatment Research Edu- self. Biochim Biophys Acta 1988; 948: 151–173. cation Fund: Arlington, Texas, 1993, pp 38–44. 29 Drysdale BE, Agarwal S, Shin HS. Macrophage-mediated 41 Fefer A, Benyunes M, Higuchi C et al. Interleukin-2 Ϯ lym- tumoricidal activity: Mechanisms of activation and cytotoxic- phocytes as consolidative immunotherapy after autologous ity. Prog Allergy 1988; 40: 111–161. bone marrow transplantation for hematologic malignancies. 30 Hamilton TA, Adams DO. Mechanisms of macrophage- Acta Haematol 1993; 89 (Suppl. 1): 2–7. mediated tumor injury. In: Den Otter W, Ruitenberg EJ (eds). 42 MacDonald D, Jiang Y, Gordon AA et al. Recombinant Tumor Immunology: Mechanisms, Diagnosis, Therapy. Else- interleukin 2 for acute myeloid leukaemia in first complete vier: Amsterdam, 1987, pp 89–107. remission: a pilot study. Leukemia Res 1990; 14: 967–973. 31 Kalland T, Alm G, Sta˚lhandske T. Augmentations of mouse 43 Brune M, Hansson M, Mellqvist U-H et al. NK cell-mediated natural killer cell activity by LS 2626, a new immunomodul- killing of AML blasts: role of histamine, monocytes and ator. J Immunol 1985; 134: 3956–3961. reactive oxygen metabolites. Eur J Haematol 1996; 57: 32 Kalland T. Regulation of natural killer progenitors. Studies 312–319. with a novel immunomodulator with distinct effects at the pre- 44 Mielcarek M, Martin PJ, Torok-Storb B. Suppresion of alloan- cursor level. J Immunol 1990; 144: 4472–4476. tigen-induced T-cell proliferation by CD14ϩ cells derived 33 Larsson E-L, Joki A, Sta˚lhandske T. Mechanism of action of from granulocyte colony-stimulating factor-mobilized periph- the immunomodulator LS 2616 on T-cell responses. Int J eral blood mononuclear cells. Blood 1997; 89: 1629–1634. Immunopharmacol 1987; 9: 425–431. 45 Hansson M, Asea A, Ericsson U et al. Induction of apoptosis 34 Sta˚lhandske T, Kalland T. Effects of the novel immunomodul- in natural killer cells by monocyte-derived reactive oxygen ator LS 2616 on the delayed type hypersensitivity reaction to metabolites. J Immunol 1996; 156: 42–47.

Appendix: Study centers/investigators Eppendorf, Hamburg: Zander AR; Charite´ Hospital Berlin: Schultze W. Italy: Ospedale San Martino, Genoa: Carella Sweden: Uppsala University Hospital: Simonsson B, To¨t- AM; Universita´ degli Studi di Parma: Rizzoli V; Universita´ terman T, Carlsson K, O¨ berg G; Lund University Hospital: degli Studi di Bologna: Lauria F, Tura S. Spain: Clinica Lenhoff S; Huddinge Hospital: Bjo¨rkstrand B; Sahlgrenska Puerta de Hierro, Madrid: Fernandez Rodriguez M; Hospi- Hospital: Carneskog J; Karolinska Hospital in Stockholm: tal Reina Sofia, Cordoba: Torres Go´mez A; Hospital Valle Bjo¨rkholm M, Gruber A; O¨ stra Hospital in Gothenburg: Hebroń, Barcelona: Julia Font A; Hospital de la Santa Cruz Lindblad R; Linko¨ping University Hospital: Malm C; Uni- y San Pablo, Barcelona (two centers: adult and pediatric): versity Hospital of Norrland in Umea˚:Lo¨fvenberg E. Nor- Domingo Albos A, Sureda A, Cubells J; Hospital Clinico way: Rikshospitalet Oslo: Brinch L, Evensen S. Denmark: in Barcelona: Rozman C, Sierra J; Hospital Seguridad Arhus Amtssygehus: Hokland P; KAS Herlev: Hansen NE; Social Jerez de la Frontera (Ca´diz): Leoń Lara A; Ciudad Rigshospitalet Copenhagen: Geisler C. United Kingdom: Sanitaria Valle de Hebroń, Barcelona: Ortega Aramburu J, University College Hospital, London: Goldstone AH; Uni- Olive´ T; Hospital de 12 Octubre, Madrid: Ortiz Conde C. versity Hospital of Wales, Cardiff: Poynton CH; John Rad- Belgium: UZ Gasthuisberg, Leuven: Boogaerts MA; Univ- cliffe Hospital, Oxford: Littlewood TJ; The Royal London ersite´ Catholique de Louvain, Brussels: Ferrant A; Institut Hospital: Newland A; Addenbrooke’s Hospital, Cambridge: Jules Bordet, Brussels: Strijckmans P; UZ St Jan Hospital, Marcus R; Leicester Royal Infirmary: Hutchinson RM; East Bruges: Louwagie A, Selleslag D. The Netherlands: Uni- Birmingham Hospital: Milligan D. France: Hopital Hoˆtel versity Hospital, Nijmegen: de Witte T, Dompeling E. Dieu, Paris: Zittoun R. Germany: Univ Krankenhaus Pharmacia & Upjohn: Hansson FP, Meier K, Nilsson BI.

Bone Marrow Transplantation